Asymmetric synthesis of new β-lactam lipopeptides as bacterial signal peptidase i inhibitors

Article abstract of DOI:10.1002/ejoc.201100148

The transmembrane bacterial enzyme, signal peptidase I, is recognized as being a promising target for reducing the emergence of drug resistance. The asymmetric synthesis and the biological evaluation of original β-lactam lipopeptides have been performed t

Full text of DOI:10.1002/ejoc.201100148

FULL PAPER
DOI: 10.1002/ejoc.201100148
Asymmetric Synthesis of New β-Lactam Lipopeptides as Bacterial Signal
Peptidase I Inhibitors
Céline Crauste,[a] Matheus Froeyen,[a] Jozef Anné,[b] and Piet Herdewijn*[a]
Keywords: Medicinal chemistry / Antibiotics / Enzymes / Peptides / Lactams / Asymmetric synthesis
The transmembrane bacterial enzyme, signal peptidase I, is
recognized as being a promising target for reducing the
emergence of drug resistance. The asymmetric synthesis and
the biological evaluation of original β-lactam lipopeptides
have been performed to discover potent signal peptidase in-
hibitors. The importance of the azetidinone motif of these
lipopeptides has been demonstrated and can serve as a start-
ing point to exploit and improve the reactivity of the β-lactam
in peptidomimetics.
catalytic domain of the enzyme in which both alanine resi-
dues (AXA) of natural peptide substrates are oriented.[7]
Various classes of molecules have been described as po-
tential inhibitors of SPase I of E. coli: β-Lactam com-
pounds such as the allyl (5S,6S)-6-[(R)-acetoxyethyl]penem-
3-carboxylate (Penem A, see part A of Figure 1 and Figure
2),[8,9] peptides (Arylomycin A2),[10] and lipoglycopeptide
derivatives.[11] For peptide derivatives, N-terminal acylation
with fatty acids like decanoyl acid seem to enhance the po-
tency of the inhibitors.[12,13] The decanoyl chain is believed
to interact with the membrane or the detergent used for
modeling the membrane bilayer in in vitro assays.[7]
Up to now, small peptides that mimic the protein cleav-
age site have proven to be inefficient as SPase I inhibi-
tors.[14] However, we first investigated the possibility of de-
veloping small peptidomimetics as potential SPase I inhibi-
tors based on the presence of the N-decanoyl side-chain and
the AXA moiety. Our work[13] led to the discovery of a
decanoyl-PTANA-COH peptide aldehyde (Figure 2) with
an IC50 value of 0.09 μm when tested against SPsB, the
SPase I of S. aureus. This lipopeptide inhibitor has a C-
terminal aldehyde function that allows nucleophilic attack
of the serine amino acid involved in the peptide cleavage
mechanism.[15,16] Despite its “serine trap” efficiency, the al-
dehyde function is often metabolically unstable and hence
its replacement by other classes of electrophiles would be a
promising optimization. Several chemically reactive groups
have been introduced into protease inhibitor structures to
act as “serine traps”, like trifluoromethyl ketones,[17] α-keto
amides,[18] β-lactams,[19,20] or boronic acids.[21,22] Among
them, the β-lactam residue is the only one previously de-
scribed in non-peptidic SPase I inhibitors.[8,9] Therefore, to
improve the activity of the decanoyl-PTANA-COH lipo-
peptide and to obtain more information on the binding of
this kind of inhibitor in the active site of the enzyme, we
replaced the aldehyde function by β-lactam residues, com-
monly used in other protease inhibitors[20,23,24] (compounds
Introduction
Faced with the emergence of drug-resistant bacteria, the
discovery of antibiotics that act by novel and various
mechanisms is of crucial importance. The bacterial enzyme,
signal peptidase I (SPase I), has received increasing interest
as a potential new target for the discovery of antibacterial
agents.[1] This transmenbrane enzyme appears to be essen-
tial for the viability of both Gram-positive and -negative
bacteria (i.e., Escherichia coli and Staphylococcus au-
reus).[2,3] Its physiological function is to ensure the release
of secreted bacterial proteins by cleaving off the signal pep-
tide from translocated pre-proteins. The bacterial signal
peptidases use a unique dyad mechanism that involves ser-
ine and lysine amino acids, whereas eukaryotic signal pep-
tidases use a different dyad mechanism based on serine and
histidine amino acids.[4] As a consequence, it should be pos-
sible to develop specific SPase I inhibitors with high selec-
tivity and low toxicity.
The natural substrates of SPase I contain a C-terminal
domain (c-region) that is responsible for substrate recogni-
tion. In most cases alanines are found in proximity to the
cleavage site in the P1 and P3 positions.[5,6] Therefore the
substrate recognition rule is often called the Ala-X-Ala
(AXA) rule. This observation was confirmed by the crystal
structure of E. coli SPase I LepB, with two small hydro-
phobic pockets (S1 and S3) identified on the surface of the
[a] Laboratory for Medicinal Chemistry, Rega Institute for
Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium
Fax: +32-16-337340
E-mail: Piet.Herdewijn@rega.kuleuven.be
[b] Department of Microbiology and Immunology, Rega Institute
for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium
E-mail: Jozef.anne@rega.kuleuven.be
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900148.
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C. Crauste, M. Froeyen, J. Anné, P. Herdewijn
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Figure 1. A: Structure of Penem A in E. coli SPase I covalently bound to Ser90.[7] B: Model of decanoyl-PTAN-aminoethyl-azetidinone
1a (without decanoyl) in the E. coli SPase I active site. The S1 site is printed in yellow, the S3 site in red, and common residues in orange.
The Ser90 and Lys145 active site residues are also shown (C atoms: grey). The two alanine side-chains in the AXA motif are printed in
cyan.
we focused our work on the synthesis of several isomers of
this new class of β-lactam lipopeptides. We report herein
the design, synthesis, and biological evaluation of these new
β-lactam-lipopeptides.
Results and Discussion
The research started with modeling experiments by dock-
ing potential peptidomimetics in the catalytic site of the en-
zyme and selecting the best-fitting molecules for future syn-
thesis. The model of the new lipopeptides was created start-
ing from the X-ray structure of the E. coli Signal peptidase
I acyl-enzyme[7] covalently bound to the 5S,6S β-lactam in-
hibitor Penem A (Figure 2). This crystal structure (Figure 1,
A) shows that the binding to E. coli SPase I can be influ-
enced by the properties of the C6 substituent of such
penems oriented towards the S1 hydrophobic pocket,
whereas modification at the C2 and C3 positions offers little
or no opportunity for binding optimization.[26] In fact, co-
crystallization reveals that the 16-methyl group of Penem A
is oriented toward the S1 pocket of the active site of the
enzyme (like the alanine residue in the P1 position of natu-
ral substrates). However, no access to the S3 pocket is re-
vealed with this penem inhibitor.
As the modeling experiments[7] predicted that the methyl
moiety of a β-lactam alkyl ester residue could mimic the
last amino acid of the AXA sequence oriented towards the
S1 pocket, we investigated the design of a new class of pep-
tide inhibitors to optimize the orientation towards both the
S1 and S3 pockets. We planned to combine the structures
and properties of the decanoyl-peptide and a β-lactam resi-
due. Therefore we decided to replace the last alanine residue
of the decanoyl-PTANA-COH peptide by the aminoethyl-
azetidinone 9 (Figure 2) to create lipopeptide of type 1 with
a monocyclic β-lactam moiety. We initially chose a monocy-
Figure 2. Chemical structures of previously described SPase I in- clic β-lactam (mono-bactam) residue because no interac-
hibitors (decanoic-peptide-aldehyde[13] and Penem A[8,9]), potential
new lipopeptide inhibitors (1 and 2), and their synthetic precursors.
tions were observed between the enzyme and the five-mem-
bered ring of Penem A.[26] By introducing the decanoyl-
PTAN moiety onto the amino group of the azetidinone 9,
1 and 2). Because an appropriate stereochemistry of the β- we expected to optimize the interactions with the S3 pocket
lactam structure is essential for its biological activity,[8,25] to increase the affinity for the active site.
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β-Lactam Lipopeptides as Bacterial Signal Peptidase I Inhibitors
To choose the appropriate stereochemistry for the syn-
thesis of the lipopeptide 1, we first focused on the results
published by Allsop et al.[8] According to them, the 15R
configuration of Penem A seems to be essential for activity.
Indeed by modeling both lipopeptides 1a and 1b inside the
E. coli SPase I active site, a better fit was observed for the
5R lipopeptide 1a (Figure 1, B).[27] In this theoretical
model, the two methyl functions (the methyl group of ala-
nine and the 6-methyl group) colored in cyan appear to fit
into both hydrophobic pockets S1 and S3. However, the C5
carbon of the lipopeptide 1a, which mimics the α carbon of
the alanine residue, has an R configuration (d), in contrast
to the natural S configuration (l) seen for all natural amino
acids. Because of this observation both the 5R and 5S iso-
mers of molecule 1 were synthesized and evaluated.
Based on the structures of the two SPase I inhibitors de-
scribed (Figure 2), we also explored the direct coupling of
the decanoic peptide with the commercially available and
inexpensive 6-aminopenicillanic acid (6-APA, compounds
2a–d). In this case, substrate recognition was based only on
the decanoyl-PTAN moiety and the “serine trap” reactivity
was introduced through a bicyclic penam moiety. The bio-
logical activity of the Penem A has been described only for
its cis-5S,6S isomer, which has the opposite stereochemistry
to most β-lactam antibiotics, and the starting material 6-
APA. Thus, synthetic schemes had to be elaborated to in-
vert both stereocenters of the 6-APA to obtain the four dia-
stereoisomers of compound 2 and explore the importance
of the stereochemistry for the activity of this new class of
potential SPase I inhibitors. Indeed, synthetic schemes to
invert the configuration and obtain the amine-free penam
of the 6-APA derivative are poorly described due to the
instability of the unprotected penam β-lactam ring.
Scheme 1. Reagents and conditions: (a) NaBH4, EtOHabs, 10 °C,
1 h 30 min (81%); (b) 1 n HCl, CH3CN, room temp., 1 h 30 min
(77%); (c) i. PhCOOH, PBu3, TMAD, 60 °C, 1 d, (35%); ii. 5 m
NaOH, MeOH, room temp., overnight (70%); (d) MsCl, pyridine,
0 °C, 2 h 30 min (7a: 99%; 7b: 60%); (e) LiN3, DMF, 60 °C, over-
night (8a: 81%; 8b: 80%); (f) H2 10% Pd/C, MeOH, room temp.,
3 h (9a: 80%; 9b: 84%); (g) Dec-PTAN-COOH (4), HOBt, DIPEA,
EDCI, DMF/DCM, room temp., 6 h (1a: 62%; 1b: 45%).
The synthesis of the lipopeptides 1 and 2 first required duction processes. Unexpectedly, the trivial conversion of
the solid-phase synthesis of decanoyl-PTAN-COOH pep- the hydroxy function into an activated mesylate 7a pre-
tide 4, which was then coupled to the appropriate residues sented some difficulties.[32] Of the solvents tested (DMF,
9 or 14. The supported synthesis was performed on a Wang DCM, and pyridine), only pyridine was found to be appro-
resin using classical coupling reagents (HOBt/DIPEA/DIC) priate and several additions of MsCl during the reaction
for peptide bond formation and Fmoc-protected amino ac- were needed to achieve completion of the reaction. Nucleo-
ids.[28] The last steps involved N-terminal decanoylation fol- philic displacement of the mesylate 7a with lithium azide
lowed by side-chain deprotection and cleavage from the so- was performed at 60 °C to provide the (5S)-azidoazetidin-
lid support using a TFA/H2O/thioanisole solution.
one 8a (81% yield), which, after hydrogenolysis, afforded
The azetidinone-peptides 1a,b were synthesized by con- the desired aminoazetidinone 9a.
densation between decanoyl-PTAN-COOH and the (5R)-
To obtain the 5R isomer 9b from the hydroxyazetidinone
or (5S)-(1-aminoethyl)azetidinone 9a or 9b (for atom num- 6a, an inversion of the absolute configuration at the C5 po-
bering see Scheme 1). We used the commercially available sition was performed by Mitsunobu esterification followed
silylated azetidinone as the starting material to give the by saponification.[33,34] Under the usual Mitsunobu condi-
(5R)-hydroxyazetidinone 6a (Scheme 1). Reduction of the tions of DEAD (diethyl azodicarboxylate), PPh3 (tri-
commercially protected β-lactam with NaBH4 in ethanol at phenylphosphane), and benzoic acid, the reaction was low
10 °C[29] led to the C4-unsubstituted compound 5 in 81% yielding and most of the starting material was recovered.
yield. Deprotection of the silyl group was performed by Several reaction methods were investigated in an attempt to
using HCl solution[30] to afford 6a in 77% yield whereas improve its efficiency.[35] As this reaction is known to be
TBAF deprotection[31] gave only 40% yield due to some highly sensitive to the steric hindrance of the starting
degradation. The hydroxyazetidinone 6a obtained was then alcohol, several methods were developed to overcome this
used as the starting material to synthesize both the (5R)- drawback. We tried to perform the nucleophilic substitution
and (5S)-aminoazetidinones 9a and 9b.
by replacing benzoic acid by a stronger acid (nitrobenzoic
The (5S)-(1-aminoethyl)azetidinone 9a was synthesized acid). However, additional degradation reactions and the
following a sequence of mesylation, aziridination, and re- formation of side-products were observed. The activation
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of the hydroxy function was then tested by using a mixture APA, seems to be improved when a phthalimido-protected
of DIAD (diisopropyl azodicarboxylate)/PPh3, ADDP strategy is used.[39]
(azodicarbonyldipiperidine)/PBu3 (tributylphosphane),[36]
To perform the epimerization steps, the sodium salt of
or TMAD (N,N,NЈ,NЈ-tetramethylazodicarboxamide)/ commercial 6-APA was treated with N-ethoxycarbonyl-
PBu3.[37] The use of ADDP or TMAD as activating reagent phthalimide and then esterified in the presence of benzyl
coupled with the more nucleophilic phosphane PBu3 in- bromide and triethylamine to obtain the protected cis-
creased the yield up to 35%. These conditions also facili- 5R,6R isomer 11a in 42% yield (for the two steps;
tated the purification step. (5S)-Hydroxyazetidinone 6b was Scheme 2). From compound 11a, epimerization at the 6-
then obtained after saponification. Finally, the desired position was accomplished by using a catalytic amount of
(5R)-aminoazetidinone 9b was obtained from the hydroxy the strong base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
derivative 6b after mesylation, aziridination, and reduction to give the trans-5R,6S isomer 11b in good yield (80%).
processes, as described above.
Then the Kukolja protocol[40] was employed to perform the
Both (5R)- and (5S)-aminoazetidinones 9 were then cou- epimerization at the 5-position. From the trans isomer 11b,
pled to the previously synthesized decanoyl-PTAN-COOH 1 equiv. of sulfuryl chloride was used to cleave the C5–S
(4) by using the usual coupling reagents (HOBt/EDCI). The bond of the penicillin thiazolidine ring. The trans and cis
5S isomer of the lipopeptide 1 was obtained in dia- chloro derivatives were obtained in a ratio of 9:1. The mix-
stereomerically pure form. However, after purification by ture was then treated with anhydrous tin(II) chloride to
chromatography and semi-preparative reversed-phase achieve the highly trans-selective recyclization of the second
1
HPLC, H NMR analysis of the 5R isomer 1a showed a cycle of the penam system. Thus, a mixture of both cis-
small proportion of a second product, probably formed by (5S,6S)-11d (31% in two steps) and trans-(5R,6S)-11b iso-
epimerization at the C3 position.
mers were obtained and separated by silica gel chromatog-
In the second part of our work, we focused on the syn- raphy. Application of the two-step Kukolja protocol to the
thesis of lipopeptides 2a–d, directly conjugated to penam cis-5R,6R isomer 11a yielded the trans-5S,6R isomer 11c
residues. This synthetic pathway started with the synthesis without any recovery of the starting isomer.
of the four 5,6 diastereoisomers of the commercially avail-
A last deprotection step had to be performed to realize
able 6-APA (14a–d; Scheme 2). Epimerization at the 6- and/ the coupling to the decanoic peptide. Several examples of
or 5-positions has already been reported for penicillin bear- successful dephthaloylation in one step using hydrazine de-
ing an imido substituent at the 6-position and ester protec- rivatives have been reported for monocyclic β-lactam, but
tion of the carboxylic function.[38–41] Indeed, it has been only a few examples are described for bicyclic derivatives
recognized that phthalimido-substituted β-lactam is more like cephalosporins,[39] carbacephems,[42] and isoce-
stable to a wide variety of reaction conditions than the cor- phems.[43] As previously reported,[44] we observed that it
responding amido derivatives. Moreover, the synthetic yield was not possible to carry out efficient hydrazinolysis on
of the cis-5S,6S isomer 14d, starting from the (5R,6R)-6- penicillin derivatives without β-lactam ring-opening. The
reaction performed at a lower temperature (–25 to –78 °C)
or by using a less reactive hydrazine (N-methylhydrazine)
preserved the β-lactam ring but decreased the efficiency of
the deprotection. To overcome this instability of the β-lac-
tam, the phthalimido group had to be converted into a
more unstable phthalisoimido group prior to hydrazinoly-
sis. For the first time this “three-step” method[44] was ap-
plied separately to the deprotection of the four diastereoiso-
mers 11a–d (Scheme 3).
Compounds 11a–c were hydrolyzed to give the phthal-
imic acids by slow addition of 1 equiv. of aqueous sodium
sulfide at 0–5 °C over 15 min (48–67%; Scheme 3). Under
the same basic conditions, cis-(5S,6S)-11d appeared to be
much more sensitive and unstable compared with the other
isomers, probably because of a bigger strain in the bicyclic
ring. To limit the degradation of the β-lactam ring, less than
1 equiv. of sodium sulfide was used as well as a careful neu-
tralization. The phthalimic acids 12 were cyclized to phthal-
isoimides 13 by using N,N-dicyclohexylcarbodiimide as the
activating reagent. Only the phthalisoimide form (13a–c)
was observed (by 1H NMR analysis) in both the crude ma-
Scheme 2. Reagents and conditions: (a) i. N-(ethoxycarbonyl)-
terials and the purified compounds (45–70%) without any
phthalimide, Na2CO3, H2O, room temp., 3 h (70%); ii) BnBr, NEt3,
traces of the phthalimide form. However, during the synthe-
DMF, room temp., 6 h (61%); (b) DBU, DCM, room temp., 1 h
sis of the cis-(5S,6S)-13d, purification by silica gel
(80%); (c) i. SO2Cl2, CCl4, DCM, room temp., 1 h; ii) SnCl2, THF,
room temp., 2 h (11c: 80%; 11d: 31%).
chromatography led to the total conversion to the starting
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β-Lactam Lipopeptides as Bacterial Signal Peptidase I Inhibitors
sized compounds was evaluated by a continuous fluorimet-
ric assay.[13,46] This quantitative in vitro assay is based on
the processing of an internally quenched fluorescent syn-
thetic peptide substrate (FRET-based assay). After cleavage
of the peptide by the SPase, an increase in the fluorescence
was measured. Therefore the inhibition of SPase I can be
analyzed and quantified in the presence and absence of the
synthetic lipopeptides. The results of the in vitro assays on
the isolated enzyme are summarized in Table 1.
Table 1. Results of the inhibitory activity assay of the synthesized
lipopeptides on S. aureus.[a]
Scheme 3. Reagents and conditions: (a) Na2S·9H2O, THF/H2O,
0 °C, 15 min (12a: 67%; 12b: 51%; 12c: 48%); (b) DCC, DCM,
0 °C, 1 h, room temp., 1 h (13a: 70%; 13b: 57%; 13c: 45%); (c) N-
methylhydrazine, THF, –20 °C, 20 min (14a: quant.; 14b: quant.;
14c: 94%; 14d: 18% from 11d); (d) Dec-PTAN-COOH (4), HOBt,
EDCI, room temp., 6 h (2a: 20%; 2b: 46%; 2c: 70%; 2d: 68%).
[a] Concentration of the SPsB Spase I: 0.25 μm; concentration of
the quenched peptide: 5 μm. Experiments were performed in dupli-
cate. [b] Ref.[47] [c] Deprotected form of 2a.
The biological evaluation showed the lipopeptides 1 and
2 to be moderately-to-weakly active. In each case we found
that the modifications introduced into the lipopeptide did
not reach the inhibition observed for the lipopeptide alde-
hyde (decanoyl-PTANA-COH). However, the high activity
observed for the new lipopeptides 1 and 2 compared with
the previously studied hydroxyethyl isoster B[47] (entry 1,
Table 1 and Figure 3) confirms that the introduction of a
β-lactam moiety at the carboxylate end seems to be a better
approach to obtaining potent SPase I inhibitors. The results
obtained for the β-lactam inhibitors show slight differences
depending upon the compound. Indeed, the percentage in-
hibition obtained for the mono-bactam compounds (en-
tries 2 and 3, 57 and 47%) appears to be two-fold higher
than those obtained for the penicillin peptides (entries 4–8;
25, 21, 36, 12, and 26%). This result does not correspond
to the chemical reactivity of either the mono-bactam or
penam as the amide function of the bicyclic derivative is
usually more reactive. However, we did not consider the
time frame of the inhibition experiments during the first
screening assay and this study may be the subject of further
investigation.
phthalimide form 12d, whereas only the phthalisoimide
form was present in the crude material. Thus, the crude
phthalisoimide cis isomer 13d was subjected to hydrazinoly-
sis without any purification. According to the procedure
described by Kukolja, the final hydrazinolysis requires a
very low temperature and was carried out at –25 °C. To
avoid degradation of the β-lactam ring, N-methylhydrazine
was used instead of hydrazine, leading to the presumed
methylhydrazine adduct intermediate,[45] which was con-
verted into the free aminopenicillins 14a–d by intramolecu-
lar nucleophilic attack on the amido-carbonyl group.
The coupling step with the decanoyl-PTAN-COOH pep-
tide 4 was then performed by using HOBt/EDC and af-
forded the four lipopeptides 2a–d in moderate yields. Dur-
ing this synthetic work we observed the β-lactam ring of
the two cis isomers to be very reactive, as based on the
following observations. First, the MeOH used as eluent
during the silica gel purification was able to react and open
the β-lactam ring of the cis isomers. Moreover, when more
than 1 equiv. of the cis amines 14a and 14d was used during
the coupling step, the formation of an additional compound
was observed by mass spectrometry. This side-product re-
sulted from nucleophilic attack of the hydroxy function of
the amino acid threonine on the amide function of the β-
lactam.
The inhibitory activities of the synthesized compounds
were tested in vitro with the isolated signal peptidase en-
zyme. SPsB, the SPase I from S. aureus, was selected be-
cause the original decanoic peptide aldehyde sequence was
based and designed on a consensus sequence of pre-proteins
Figure 3. Additional compounds tested in the FRET-based assay
from S. aureus.[12,13] The inhibitory activity of the synthe- on S. aureus sPsB.
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To eliminate the influence of solubility problems on the due, opening the way for further asymmetric synthesis of
small differences of inhibitory activity, the deprotected car- penam compounds. In vitro studies showed moderate SPase
boxylic acid of 2a was synthesized (by hydrogenation) and I inhibition, which could be due to inappropriate position-
evaluated (2Јa, entry 5). The same percentage inhibition was ing of the β-lactam in the active site. However, because the
obtained for both the protected and the deprotected com- β-lactam motif seems to be involved in the inhibitory ac-
pounds. The methyl group used as a mimic of the last ala- tivity, other peptide sequences bearing the PTANA motif
nine of the AXA sequence (present only in compounds 1a should be envisaged, as well as modifications of the β-lac-
and 1b) might play a role in the recognition of the active tam moiety, to improve the binding and reactivity with the
site. To demonstrate the importance of the β-lactam moiety enzyme SPsB and for the design of new potential peptido-
in the inhibition observed, decanoyl-PTAN-NH2 (3; Fig- mimetic β-lactam lipopeptides.
ure 3) was also synthesized from peptide 4 (by treatment
with ammonium chloride) and tested on SPase I SpsB. To
Experimental Section
reach the same level of inhibition as compounds 1, dec-
anoyl-PTAN-NH2 (3; devoid of the β-lactam moiety) had
to be tested at 100 mm, a concentration 103-fold higher than
that used for the lipopeptides 1. As the removal of the β-
lactam moiety resulted in a complete loss of activity, this
function might be implicated in the binding to the active
site. However, regarding the influence of the stereochemis-
try of compounds 1 and 2, no significant differences were
observed between the activities of the several isomers syn-
thesized. As a consequence, the penam or the mono-bactam
residue might not be ideally accommodated in the active
site and for this reason only modest activities were observed
with compounds 1 and 2.
Inhibition tests were also performed on the SPase I en-
zyme best characterized until now, the LepB of E. coli, the
crystal structure of which was used in the modeling experi-
ment (as the S. aureus SPase I has not yet been crystallized).
No significant activities were observed at 10 mm. Indeed the
decanoyl-PTANA sequences appear less suitable for bind-
ing to E. coli SPase I than to the enzyme of Gram-positive
species. However, a structural study should be performed to
find the appropriate peptide sequences that would allow
both to adopt a β-sheet-like conformation and to fit cor-
rectly the β-lactam ring into the active site of the SpsB en-
zyme. Furthermore, given the large differences in the bio-
logical activity of the peptide aldehyde[13] and the present
β-lactam conjugates, it might be hypothesized that, in ad-
dition to the non-optimal positioning in the active site, the
reactivities of the β-lactam moieties of compounds of type
1 and 2 are not high enough to allow covalent bond forma-
tion. A β-lactam lipopeptide obtained from coupling be-
tween the decanoic-peptide and a penem moiety like Penem
A could be a better alternative to achieve higher reactivity.
General: NMR spectra were recorded with a Bruker UltraShield
300 (1H: 300 MHz; 13C: 75 MHz; 31P 121 MHz), 500 MHz (1H:
500 MHz) or 600 MHz (1H: 600 MHz) spectrometer using tet-
ramethylsilane as internal standard. Chemical shifts are reported
in δ units and coupling constants (J values) in Hz. Reagents and
solvents were obtained from commercial sources (Sigma–Aldrich,
Acros-Organics, NovaBiochem, Alfa-Aesar and TCI Europe) and
used as received (THF and DCM were used after distillation over
Na/benzophenone and CaH2, respectively). The progress of the re-
action was monitored either by TLC, performed on Alugram SIL
G/UV254 plates, or by mass analysis performed with a TSQ Quan-
tum Thermo Scientific instrument. Column chromatography was
performed with Grace Silicagel DAVISIL LCA (0.040–0.063 or
0.070–0.200 mm). Purification on HPLC was realized with a
semi-preparative Polymer Lab column, PRLPS 10 μm
(21.2 cmϫ150 mm ID; mobile phase flow, 2 mL/min, gradient
H2O/CH3CN or H2O/CH3CN/0.1% TFA, UV detection 214/
254 nm). Optical rotations were evaluated with a Perkin–Elmer po-
larimeter (model 341) in chloroform. All final lipopeptide purities
were checked by HRMS and HPLC. HRMS spectra were recorded
with a Q-Tof-2 (Micromass, Manchester, UK) mass spectrometer.
Accurate masses were determined by co-infusion of the samples
with 2 μm leucine enkephalin (YGGFL) and recalibration of the
spectrum using the peak at m/z = 556.2771 as the lock mass. At
least 10 spectra were recorded and averaged. HPLC was performed
with a Shimadzu SPD-M20A HPLC System using an intersil ODS-
3 4 μm (4.6 cm ϫ100 mm ID) column. The compounds were ana-
lyzed by using H2O/CH3CN/0.1% TFA or H2O/CH3CN solvent
systems with a 30 or 35 min gradient: 5–100% CH3CN with a flow
rate of 1.0 mL/min.
Abbreviations: HOBt: hydroxybenzotriazole; DIPEA: N-ethyldi-
isopropylamine; DIC: NЈ,NЈ-diisopropylcarbodiimide; DMF: di-
methylformamide; TFA: trifluoroacetic acid; EDCI: 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; DCM: dichloromethane; 6-
APA: 6-aminopenicillanic acid; PTAN: l-prolyl-l-threonyl-l-ala-
nyl-l-asparaginyl.
Peptide Synthesis
Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-asparagine (4): The solid-
Conclusions
phase synthesis of this peptide was performed on Wang resin
(0.80 mg, 0.52 mmol) following a Fmoc strategy. DMF was used
as solvent, HOBt (4 equiv.), DIPEA (2 equiv.), and DIC (4 equiv.)
as coupling reagents, and all the amino acids coupled were Fmoc-
protected. First, Fmoc-asparagine(trt) (4 equiv.) was coupled to the
resin over 5 h. After capping in the presence of acetic anhydride
(20% in pyridine) and Fmoc deprotection in the presence of piperi-
dine (20% in DMF), the next amino acid was coupled. The same
process was performed for the coupling of Fmoc-alanine (4 equiv.),
A new class of lipopeptides bearing a β-lactam moiety
has been synthesized. This class of compounds has been
poorly described and usually only the racemic form of the
penicillin moiety is represented.[48] An effort has been made
to use stereoselective procedures and to obtain several dia-
stereoisomers of the lipopeptides 1 and 2. The deprotection
of the phthalimide group was performed for the first time
on the four diastereoisomers of the protected 6-APA resi- Fmoc-threonine(tbu) (4 equiv.), Fmoc-proline (4 equiv.), and then
3442
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3437–3449

β-Lactam Lipopeptides as Bacterial Signal Peptidase I Inhibitors
decanoic acid (4 equiv.). The deprotection of the lateral protecting
groups and the cleavage of the resin were performed with a solution
of TFA/thianisole/H2O (9.5:0.25:0.25) over 3 h. The desired peptide
4 (62 mg, 21% in six steps) was then obtained after precipitation
in diisopropyl ether and purification by preparative reversed-phase
HPLC (A: H2O/0.1% TFA/B: CH3CN/0.1% TFA). 1H NMR
(300 MHz, CDCl3/CD3OD): δH = 4.48 [m, 1 H, Hα(Asn)], 4.23–
addition of a 1 n NaOH solution. The solvents were removed under
reduced pressure and the residue obtained was purified by
chromatography on silica gel using DCM/ethyl acetate/MeOH
(10:10:0.3) as eluent to give 6a (194 mg, 77%) as a white solid. Rf
= 0.2 (DCM/MeOH, 9.5:0.5). 1H NMR (300 MHz, [D6]DMSO):
δH = 7.68 (br., 1 H, NH), 4.78 (d, J = 5.1 Hz, 1 H, OH), 3.80 (dq,
J = 5.4, J = 6.3 Hz, 1 H, 5-H), 3.14 (t, J = 5.4 Hz, 1 H, 4-Hb),
4.10 [m, 4 H, Hα(Ala, Pro, Thr), Hβ(Thr)], 3.48, 3.39 [m, 2 H, 3.06–3.00 (m, 2 H, 4-Ha, 3-H), 1.11 (d, J = 6.3 Hz, 3 H, 6-H) ppm.
CH2δ(Pro)], 2.52 [m, 2 H, CH2β(Asn)], 2.17 [m, 2 H, CH2β(Pro)],
13C NMR (75 MHz, [D6]DMSO): δC = 168.9, 64.5, 58.6, 37.6,
1.99–1.84 [m, 4 H, CH2γ(Pro), CH2deca], 1.41 (m, 2 H, CH2deca), 22.1 ppm. HRMS (EI): calcd. for C5H10NO2 [M + H]+ 116.0711;
1.23 [d, J = 7.2 Hz, 3 H, CH3(Ala)], 1.14–1.05 (m, 12 H, 6 CH2deca),
1.01 [d, J = 6.3 Hz, 3 H, CH3(Thr)], 0.67 (m, 3 H, CH3deca) ppm.
13C NMR (75 MHz, CDCl3/CD3OD): δC = 173.2, 172.6, 172.5,
172.4, 172.3, 170.2, 66.7, 60.1, 57.8, 48.8, 47.4, 36.5, 34.3, 31.4,
29.1, 29.0, 28.8, 28.8, 24.5, 24.3, 22.2, 18.6, 16.5, 13.6 ppm. HRMS
(EI): calcd. for C26H46N5O8 [M + H]+ 556.3346; found 556,3354.
found 116.0704.
(3S)-3-[(1S)-1-(Benzoyloxy)ethyl]azetidin-2-one (6Ј): Tributylphos-
phane (0.16 mL, 0.65 mmol) and benzoic acid (53 mg, 0.43 mmol)
were added at 0 °C to a solution of the azetidinone 6a (50 mg,
0.43 mmol) in dry toluene (3 mL). Then NЈ,NЈ,NЈ,NЈ-tetrameth-
ylazodicarboxamide (TMAD; 112 mg, 0.65 mmol) was added in
one portion. The reaction mixture was stirred at 0 °C for 10 min
and then at 60 °C for 1 d. Dihydro-TMAD was precipitated and
filtered off after the addition of hexane to the reaction mixture.
The solvents were removed under reduced pressure and the re-
sulting residue was purified by chromatography on silica gel using
hexane/ethyl acetate (8.5:1.5) as eluent to give 6Ј (35 mg, 35%) as
Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-asparaginylcarboxamide
(3): HOBt (3.60 mg, 0.02 mmol), EDCI (5.10 mg, 0.02 mmol), and
then DIPEA (12 μL, 0.07 mmol) and NH4Cl (1.92 mg, 0.03 mmol)
were added to a solution of decanoyl-PTAN-COOH (4; 10 mg,
0.02 mmol) in DMF/DCM (1 mL/1 mL) at 0 °C. The mixture was
stirred for 3 h at room temperature and then the solvent was evapo-
rated under reduced pressure. The crude residue was purified by
preparative reversed-phase HPLC (A: H2O/0.1% TFA/B: CH3CN/
0.1% TFA) to afford 3 (5 mg, 50%) as a white solid. 1H NMR
(300 MHz, CDCl3/CD3OD): δH = 4.64 [m, 1 H, Hα(Asn)], 4.41–
4.21 [m, 4 H, Hα(Ala, Pro, Thr), Hβ(Thr)], 3.64, 3.49 [m, 2 H,
CH2δ(Pro)], 2.75 [m, 2 H, CH2β(Asn)], 2.32–2.26 [m, 2 H,
CH2β(Pro)], 2.15–1.90 [m, 4 H, CH2γ(Pro), CH2deca], 1.56 (m, 2 H,
CH2deca), 1.35 [d, J = 7.2 Hz, 3 H, CH3(Ala)], 1.25–1.18 (m, 12 H,
6 CH2deca), 1.12 [d, J = 6.3 Hz, 3 H, CH3(Thr)], 0.83 (m, 3 H,
CH3deca) ppm. 13C NMR (75 MHz, CDCl3/CD3OD): δC = 173.6,
173.3, 173.1, 172.9, 172.5, 171.2, 66.3, 60.1, 58.1, 49.7, 49.5, 35.9,
34.0, 31.2, 28.8, 28.7, 28.6, 24.3, 24.1, 21.9, 18.6, 15.7, 13.2 ppm.
HRMS (EI): calcd. for C26H47N6O7 [M + H]+ 555.3505; found 555.
3506.
1
a white amorphous solid. Rf = 0.3 (hexane/ethyl acetate, 3:7). H
NMR (300 MHz, CDCl3): δH = 8.06 (d, J = 7.2 Hz, 2 H, HoBz),
7.58 (t, J = 7.2 Hz, 2 H, HpBz), 7.45 (t, J = 7.2 Hz, 2 H, HmBz),
5.92 (br., 1 H, NH), 4.07 (qd, J = 4.0, J = 6.3 Hz, 1 H, 5-H), 3.61
(m, 1 H, 3-H), 3.42 (t, J = 5.7 Hz, 1 H, 4-Hb), 3.23 (dd, J = 2.7, J
= 5.7 Hz, 1 H, 4-Ha), 1.57 (d, J = 6.3 Hz, 3 H, 6-H) ppm. 13C
NMR (75 MHz, CDCl3): δC = 167.5, 165.6, 132.6, 129.7, 129.3,
128.1, 67.5, 55.3, 37.9, 17.2 ppm. HRMS (EI): calcd. for
C12H14NO3 [M + H]+ 220.0973; found 220.0978.
(3S)-3-[(1S)-1-Hydroxyethyl]azetidin-2-one (6b): A 0.5 m solution of
NaOH (0.05 mL, 0.09 mmol) was added at room temperature to a
solution of 6Ј (20 mg, 0.09 mmol) in MeOH (4 mL). The reaction
mixture was stirred overnight and a further 1 equiv of NaOH
(0.05 mL, 0.09 mmol) was added. The reaction mixture was stirred
for 40 min and then neutralized with an excess of acetic acid. The
solvents were removed under reduced pressure and the residue ob-
tained was purified by chromatography on silica gel using ethyl
acetate/DCM/MeOH (50:50:0.4) as eluent to give 6b (7.50 mg,
Synthesis of Lipopeptide Type 1
(3S)-3-[(1R)-1-(tert-Butyldimethylsilyloxy)ethyl]azetidin-2-one (5):
NaBH4 (0.72 g, 16.10 mmol) was added to a solution of the com-
mercial silylated azetidinone (5 g, 17.40 mmol) in absolute ethanol
(25 mL) at 10 °C and the resulting suspension was kept at 10 °C
for 1 h 30 min. Amberlite acid resin (15 g) was added to the suspen-
sion and the reaction mixture was stirred for a further 30 min. The
mixture was then filtered and the solvent was evaporated. The re-
sulting residue was washed with ethyl acetate and several times with
DCM. The filtrates were combined and the solvents were removed
under reduced pressure. The residue obtained was purified by
chromatography on silica gel using DCM/ethyl acetate (9.5:0.5) as
eluent to give 5 (3.23 g, 81%) as a white solid. Rf = 0.3 (DCM/
ethyl acetate, 9.5:0.5). 1H NMR (300 MHz, CDCl3): δH = 5.67 (br.,
1 H, NH), 4.22 (qd, J = 4.7, J = 6.2 Hz, 1 H, 5-H), 3.36 (dd, J =
2.7, J = 5.0 Hz, 1 H, 4-Ha), 3.29 (t, J = 5.0 Hz, 1 H, 4-Hb), 3.29
(m, 1 H, 3-H), 1.20 (d, J = 6.2 Hz, 3 H, 6-H), 0.88 [s, 9 H,
(CH3)3C], 0.08 [s, 6 H, (CH3)2Si] ppm. 13C NMR (75 MHz,
CDCl3): δC = 169.2, 64.9, 59.0, 37.3, 25.4, 22.2, 17.6, –4.6,
1
70%) as a white solid. Rf = 0.3 (ethyl acetate/MeOH, 9.5:0.5). H
NMR (300 MHz, CDCl3): δH = 6.43 (br., 1 H, NH), 4.07 (quint.,
J = 6.3 Hz, 1 H, 5-H), 3.37 (t, J = 5.4 Hz, 1 H, 4-Hb), 3.30–3.25
(m, 1 H, 3-H), 3.18 (dd, J = 2.4, J = 5.4 Hz, 1 H, 4-Ha), 2.95 (br.,
1 H, OH), 1.31 (d, J = 6.3 Hz, 3 H, 6-H) ppm. 13C NMR (75 MHz,
CDCl3): δC = 170.1, 65.6, 57.4, 38.2, 20.6 ppm. HRMS (EI): calcd.
for C5H10NO2 [M + H]+ 116.0711; found 116.0715.
(3S)-3-[(1R)-1-(Methylsulfonyloxy)ethyl]azetidin-2-one (7a): Meth-
anesulfonyl chloride (0.15 mL, 1.90 mmol) was added to a solution
of the alcohol 6a (150 mg, 1.30 mmol) in dry pyridine at 0 °C. The
reaction mixture was stirred at 0 °C for 1 h 30 min and an ad-
ditional 1.5 equiv. of methanesulfonyl chloride was added. The re-
action mixture was stirred for 1 h to reach completion of the reac-
tion. After evaporation of the solvent, the residue obtained was
dissolved in DCM and washed with water and brine. The aqueous
phase was extracted several times with DCM. The organic phases
were combined, dried (MgSO4), and concentrated in vacuo. The
residue obtained was purified by chromatography on silica gel
using hexane/ethyl acetate (2:8) as eluent to give 7a (250 mg, 99%)
as a white solid. Rf = 0.4 (ethyl acetate). 1H NMR (300 MHz,
CDCl3): δH = 5.81 (br., 1 H, NH), 5.08 (quint., J = 6.3 Hz, 1 H,
5-H), 3.50–3.43 (m, 3 H, 4-H, 3-H), 3.06 (s, 3 H, CH3SO2), 1.57
–5.3 ppm. HRMS (EI): calcd. for C11H24NO2Si [M
230.1576; found 230.1567.
+
H]+
(3S)-3-[(1R)-1-Hydroxyethyl]azetidin-2-one (6a): Compound
5
(0.50 g, 2.20 mmol) was dissolved in CH3CN (12 mL), and 1 n HCl
(2 mL) was added. The reaction was monitored by TLC and after
1 h 30 min another 1 n HCl (0.50 mL) was added to afford total
deprotection. After 30 min, the mixture was neutralized by the slow
Eur. J. Org. Chem. 2011, 3437–3449
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3443

C. Crauste, M. Froeyen, J. Anné, P. Herdewijn
(3S)-3-{(1R)-1-[(N-Decanoyl- -prolyl- -threonyl- -alanyl- -aspara-
FULL PAPER
(d, J = 6.3 Hz, 3 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δC
= 166.1, 76.2, 56.4, 38.5, 38.2, 19.7 ppm. HRMS (EI): calcd. for
C6H12NO4S [M + H]+ 194.0486; found 194.0494.
L
L
L
L
ginyl)amino]ethyl}azetidin-2-one (1a): Decanoyl-PTAN-COOH (4;
15 mg, 0.03 mmol) and the aminoazetidinone 9a (3 mg, 0.03 mmol)
were dissolved in dry DMF (1.5 mL) and the solution was cooled
to 0 °C. HOBt (5.40 mg, 0.04 mmol), EDCI (10.20 mg, 0.06 mmol),
and then DIPEA (12 μL, 0.06 mmol) were added to the solution
and the reaction was stirred at room temperature for 6 h. The sol-
vent was then evaporated to dryness under vacuum. The residue
obtained was dissolved in DCM and washed successively with citric
acid solution (1 m) and brine. The aqueous phases were extracted
several times with DCM and the resulting organic phases were
combined, dried, and concentrated under vacuum. The residue ob-
tained was purified by chromatography on silica gel using DCM/
MeOH (9.5:0.5 to 8:2) as eluent to give 1a (11 mg, 62%) as a white
solid contaminated by the 3R isomer. Major product: Rf = 0.5
(DCM/MeOH, 9:1). 1H NMR (300 MHz, CDCl3): δH = 4.62 [m, 1
H, Hα(Asn)], 4.46–4.20 [m, 4 H, Hα(Ala, Pro, Thr), Hβ(Thr)], 3.69,
3.54 [m, 2 H, CH2δ(Pro)], 3.45–3.15 (m, 4 H, 5-H, 4-H, 3-H), 2.68
[m, 2 H, CH2β(Asn)], 2.40–2.00 [m, 6 H, CH2β(Pro), CH2γ(Pro),
CH2deca], 1.60 (m, 2 H, CH2deca), 1.44 [d, J = 7.2 Hz, 3 H,
CH3(Ala)], 1.30 (d, J = 7.2 Hz, 3 H, 6-H), 1.28–1.17 (m, 12 H,
6 CH2deca), 1.21 [d, J = 6.0 Hz, 3 H, CH3(Thr)], 0.87 (m, 3 H,
CH3deca) ppm. 13C NMR (75 MHz, CDCl3): δC = 173.5, 172.7,
172.4, 171.5, 171.0, 169.7, 167.0, 66.9, 60.2, 58.1, 55.7, 50.2, 50.1,
47.3, 43.6, 38.5, 36.0, 34.3, 31.4, 29.0, 28.9, 28.8, 28.7, 24.5, 24.3,
22.2, 19.2, 17.1, 16.1, 13.6 ppm. HRMS (EI): calcd. for
C31H54N7O8 [M + H]+ 652.4033; found 652.4017.
(3S)-3-[(1S)-1-(Methylsulfonyloxy)ethyl]azetidin-2-one (7b): The
same process was used starting from the alcohol 6b (30 mg,
0.26 mmol) and afforded the mesylate 7b (30 mg, 60%) as a white
solid after purification by chromatography on silica gel using hex-
1
ane/ethyl acetate (2:8) as eluent. Rf = 0.3 (ethyl acetate). H NMR
(500 MHz, CDCl3): δH = 5.98 (br., 1 H, NH), 5.12 (quint., J =
6.5 Hz, 1 H, 5-H), 3.57 (m, 1 H, 3-H), 3.45 (t, J = 6.0 Hz, 1 H, 4-
Hb), 3.35 (dd, J = 2.5, J = 6.0 Hz, 4-Ha), 3.10 (s, 3 H, CH3SO2),
1.62 (d, J = 6.5 Hz, 3 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3):
δC = 166.3, 74.9, 55.3, 38.6, 37.7, 18.4 ppm. HRMS (EI): calcd. for
C6H12NO4S [M + H]+ 194.0486; found 194.0492.
(3S)-3-[(1S)-1-Azidoethyl]azetidin-2-one (8a): LiN3 (8.07 mg,
0.16 mmol) was added to a solution of the mesylate 7a (22 mg,
0.11 mmol) in dry DMF at room temperature. The reaction mix-
ture was stirred overnight at 60 °C and the solvent was evaporated
under reduced pressure. The residue obtained was dissolved in
DCM and washed with water and brine. The aqueous phase was
extracted several times with DCM. The organic phases were com-
bined, dried (MgSO4), and concentrated in vacuo. The residue ob-
tained was purified by chromatography on silica gel using hexane/
ethyl acetate (3:7) as eluent to give 8a (13 mg, 81%) as an amorph-
ous solid. Rf = 0.5 (ethyl acetate). 1H NMR (300 MHz, CDCl3):
δH = 6.38 (br., 1 H, NH), 3.87 (qd, J = 6.6, J = 5.1 Hz, 1 H, 5-H),
3.42–3.38 (m, 1 H, 3-H), 3.37 (t, J = 5.6 Hz, 1 H, 4-Hb), 3.21 (dd,
J = 2.1, J = 5.6 Hz, 4-Ha), 1.43 (d, J = 6.6 Hz, 3 H, 6-H) ppm.
13C NMR (75 MHz, CDCl3): δC = 167.9, 55.2, 54.7, 37.8,
16.2 ppm. HRMS (EI): calcd. for C5H9N4O [M + H]+ 141.0776;
found 141.0787.
(3S)-3-{(1S)-1-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-
asparaginyl)amino]ethyl}azetidin-2-one (1b): Decanoyl-PTAN-
COOH (4; 15 mg, 0.03 mmol), and the aminoazetidinone 9b (3 mg,
0.03 mmol) were dissolved in dry DMF (1.50 mL) and the solution
was cooled to 0 °C. HOBt (5.40 mg, 0.04 mmol), EDCI (10.20 mg,
0.06 mmol), and then DIPEA (12 μL, 0.06 mmol) were added to
the solution and the reaction mixture was stirred at room tempera-
ture for 6 h. The solvent was then evaporated to dryness under
vacuum. The residue obtained was dissolved in DCM and washed
successively with citric acid solution (1 m) and brine. The aqueous
phases were extracted several times with DCM and the resulting
organic phases were combined, dried, and concentrated under vac-
uum. The residue obtained was purified by chromatography on sil-
ica gel using DCM/MeOH (9.5:0.5 to 8:2) as eluent to give 1b
(8 mg, 45%) as a white solid. Rf = 0.5 (DCM/MeOH, 9:1). 1H
NMR (300 MHz, CDCl3/CD3OD): δH = 4.58 [m, 1 H, Hα(Asn)],
4.38–4.17 [m, 4 H, Hα(Ala, Pro, Thr), Hβ(Thr)], 3.65, 3.51 [m, 2
H, CH2δ(Pro)], 3.25 (m, 1 H, 5-H), 3.15–2.64 (m, 3 H, 4-H, 3-H),
2.66 [m, 2 H, CH2β(Asn)], 2.22 [m, 2 H, CH2β(Pro)], 2.20–1.90 [m,
4 H, CH2γ(Pro), CH2deca], 1.56 (m, 2 H, CH2deca), 1.39 [d, J =
6.0 Hz, 3 H, CH3(Ala)], 1.34–1.17 (m, 15 H, 6-H, 6 CH2deca), 1.14
[d, J = 6.0 Hz, 3 H, CH3(Thr)], 0.80 (t, J = 6.9 Hz, 3 H,
CH3deca) ppm. 13C NMR (75 MHz, CDCl3): δC = 173.8, 173.2,
172.8, 172.4, 171.1, 170.3, 166.8, 66.1, 60.5, 58.1, 55.0, 50.4, 50.1,
47.4, 42.9, 38.0, 36.4, 34.3, 31.4, 29.1, 29.0, 28.9, 24.6, 24.3, 22.2,
19.1, 17.2, 16.2, 13.6 ppm. HRMS (EI): calcd. for C31H54N7O8 [M
+ H]+ 652.4033; found 652.4019.
(3S)-3-[(1R)-1-Azidoethyl]azetidin-2-one (8b): The same process was
used starting from the mesylate 7b (24 mg, 0.12 mmol) and af-
forded compound 8b (14 mg, 80%) as an amorphous solid after
purification by chromatography on silica gel using hexane/ethyl
acetate (4:6) as eluent. Rf = 0.5 (ethyl acetate). 1H NMR (300 MHz,
CDCl3): δH = 5.99 (br., 1 H, NH), 3.90 (quint., J = 6.6 Hz, 1 H,
5-H), 3.43 (m, 1 H, 4-H), 3.30–3.24 (m, 2 H, 4-H, 3-H), 1.43 (d, J
= 6.6 Hz, 3 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δC = 167.6,
56.2, 55.8, 38.8, 17.4 ppm. HRMS (EI): calcd. for C5H8N4ONa [M
+ Na]+ 163.0596; found 163.0582.
(3S)-3-[(1S)-1-Aminoethyl]azetidin-2-one (9a): Compound 8a
(43 mg, 0.30 mmol) was hydrogenated in MeOH (4 mL) under at-
mospheric pressure on 10% palladium on carbon (15 mg) for 3 h.
The catalyst was filtered and washed with MeOH. The filtrate was
concentrated under reduced pressure to afford compound 9a
(28 mg, 80%) as a white solid. Rf = 0.2 (DCM/MeOH, 8:2). 1H
NMR (300 MHz, [D6]DMSO): δH = 7.70 (br., 1 H, NH), 3.32 (br.,
2 H, NH2), 3.14–2.92 (m, 4 H, 5-H, 4-H, 3-H), 0.99 (d, J = 6.3 Hz,
3 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δC = 170.1, 58.3,
46.2, 37.5, 21.3 ppm. HRMS (EI): calcd. for C5H10N2O [M + H]+
115.0871; found 115.0876.
Synthesis of Lipopeptides Type 2
(3S)-3-[(1R)-1-Aminoethyl]azetidin-2-one (9b): The same process as
described for the synthesis of 9a was used to obtain the amine 9b
(20 mg, 84%) as a white solid from compound 8b (29 mg,
(2S,5R,6R)-3,3-Dimethyl-7-oxo-6-phthalimido-4-thia-1-azabicyclo-
[3.2.0]heptane-2-carboxylic Acid (10): N-(Ethoxycarbonyl)phthal-
0.20 mmol). Rf = 0.2 (DCM/MeOH, 8:2). 1H NMR (300 MHz, imide (5.07 g, 23 mmol) was added to a vigorously stirred solution
D2O): δH = 3.37–3.43 (m, 1 H, 5-H), 3.20–3.12 (m, 3 H, 4-H, 3-
H), 1.08 (d, J = 6.0 Hz, 3 H, 6-H) ppm. 13C NMR (75 MHz,
CDCl3): δC = 172.8, 56.6, 44.8, 38.7, 19.3 ppm. HRMS (EI): calcd.
for C5H10N2O [M + H]+ 115.0871; found 115.0869.
of 6-aminopenicillanic acid (6-APA; 5 g, 23 mmol) and Na2CO3
(2.45 g, 23 mmol) in water (35 mL). The mixture was stirred at
room temperature for 3 h and then extracted with DCM. The aque-
ous layer was mixed with a fresh portion of DCM and acidified
3444
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3437–3449

β-Lactam Lipopeptides as Bacterial Signal Peptidase I Inhibitors
during vigorous stirring with 1 m HCl (46 mL, 46 mmol). The
phases were separated and the extraction was completed with two
additional portions of DCM. The combined organic extracts were
washed with water and brine. The organic layer was dried (MgSO4)
and concentrated under reduced pressure to afford compound 10
(5.64 g, 70%) as a white solid, which was used in the next step
without further purification. An analytical sample was obtained as
CH3) ppm. The foam obtained was dissolved in THF (150 mL) and
treated with anhydrous SnCl2 (1.08 g, 5.73 mmol). The mixture was
stirred at room temperature for 3 h and then the solvent was evapo-
rated under reduced pressure. The residual oil was dissolved in
EtOAc and washed with water (ϫ3) and brine. The organic phase
was dried (MgSO4) and the solvents evaporated under vacuum. The
residue obtained was purified by chromatography on silica gel
using toluene/ethyl acetate (9.5:0.5) as eluent and afforded the de-
a white crystalline solid by crystallization from acetone. Rf = 0.5
1
(DCM/MeOH, 8:2). H NMR (300 MHz, CDCl3): δH = 7.92–7.77 sired isomer 11c (2 g, 80% for two steps) as white solids. Rf = 0.3
1
(m, 4 H, Hpht), 5.70 and 5.61 (2ϫ d, J = 3.9 Hz, 2ϫ 1 H, 5-H, 6- (toluene/ethyl acetate, 9.5:0.5). H NMR (300 MHz, CDCl3): δH
=
H), 4.71 (s, 1 H, 2-H), 1.85 (s, 3 H, CH3), 1.62 (s, 3 H, CH3) ppm.
13C NMR (75 MHz, CDCl3): δC = 171.1, 168.5, 166.2, 134.2, 131.1,
123.6, 70.6, 66.3, 65.3, 57.9, 30.1, 27.7 ppm. HRMS (EI): calcd. for
C16H15N2O5S [M + H]+ 347.0701; found 347.0706.
7.95–7.85 (m, 4 H, Hpht), 7.45–7.34 (m, 5 H, HPh), 5.57 and 5.45
(2ϫ d, J = 2.1 Hz, 2ϫ 1 H, 5-H, 6-H), 5.27 (d, J = 4.2 Hz, 2 H,
CH2Ph), 3.93 (s, 1 H, 2-H), 1.67 (s, 3 H, CH3), 1.41 (s, 3 H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 166.2, 166.1, 165.3,
134.5, 134.2, 131.2, 128.5, 128.3, 128.3, 123.5, 69.8, 67.5, 66.0, 65.7,
59.5, 31.0, 24.3 ppm. HRMS (EI): calcd. for C23H20N2O5SK [M +
K]+ 475.0729; found 475.0708.
Benzyl (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-phthalimido-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylate (11a): Triethylamine (1.30 mL,
9.39 mmol) followed by benzyl bromide (1.46 mL, 12.20 mmol)
were added to a stirred solution of acid 10 (3.25 g, 9.39 mmol) in
dry DMF (20 mL). The mixture was stirred at room temperature
for 6 h and then poured into vigorously stirred ice–water (150 mL).
The resulting suspension was extracted three times with chloroform
and the combined organic layers were successively washed with a
saturated solution of NaHCO3 (ϫ3), water (ϫ3), and brine. The
organic phases were dried (MgSO4) and concentrated under re-
duced pressure to give a light-yellow oil, which was crystallized in
Et2O to afford 11a (2.50 g, 61%) as a white solid. Rf = 0.5 (hexane/
Benzyl (2S,5S,6S)-3,3-Dimethyl-7-oxo-6-phthalimido-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylate (11d): A solution of ester 11b
(0.50 g, 1.14 mmol) in DCM (13 mL) and CCl4 (6.40 mL) was
treated with an equimolar amount of sulfuryl chloride (92 μL,
1.14 mmol, solution in 1 mL of CCl4) and stirred at room tempera-
ture for 1 h. The solvents were evaporated under vacuum to give a
mixture of two chlorinated compounds, the trans and cis isomers,
in a ratio of around 9:1 as a yellow foam. Major product, the trans
isomer: 1H NMR (300 MHz, CDCl3): δH = 7.94–7.78 (m, 4H,
ethyl acetate, 7:3). 1H NMR (300 MHz, CDCl3): δH = 7.91–7.76 Hpht), 7.44–7.35 (m, 5H, HPh), 6.12 and 5.49 (2ϫ d, J = 1.5 Hz,
(m, 4 H, Hpht), 7.40 (m, 5 H, HPh), 5.69 and 5.61 (2ϫ d, J = 2ϫ 1 H, 5-H, 6-H), 5.30 and 5.26 (2ϫ d, J = 20 Hz, 2ϫ 1 H,
4.0 Hz, 2ϫ1 H, 5-H, 6-H), 5.23 (s, 2 H, CH2Ph), 4.71 (s, 1 H, 2- CH2Ph), 4.41 (s, 1 H, 2-H), 1.72 (s, 3H, CH3), 1.72 (s, 3H,
H), 1.81 (s, 3 H, CH3), 1.45 (s, 3 H, CH3) ppm. 13C NMR (75 MHz,
CDCl3): δC = 167.9, 167.5, 166.2, 134.4, 134.2, 131.1, 128.4, 128.3,
123.5, 70.5, 67.1, 66.6, 65.7, 58.2, 30.7, 27.5 ppm. HRMS (EI):
calcd. for C23H20N2O5SK [M + K]+ 475.0729; found 475.0753.
CH3) ppm. The foam obtained was dissolved in THF (26 mL) and
treated with anhydrous SnCl2 (0.21 g, 1.14 mmol). The mixture was
stirred at room temperature for 2 h and then the solvent was evapo-
rated under reduced pressure. The residual oil was dissolved in
EtOAc and washed with water (ϫ3) and brine. The organic phase
was dried (MgSO4) and concentrated under vacuum. The residue
obtained was purified by chromatography on silica gel using tolu-
ene/ethyl acetate (9.5:0.5) as eluent to give the starting 11b (0.26 g,
52% for two steps) and the desired isomer 11d (0.15 g, 31% for
two steps) as white solids. Rf (11d) = 0.3 (toluene/ethyl acetate,
9.5:0.5). 1H NMR (300 MHz, CDCl3): δH = 7.92–7.76 (m, 4 H,
Hpht), 7.49–7.37 (m, 5 H, HPh), 5.64 and 5.28 (2ϫ d, J = 4.0 Hz,
2ϫ 1 H, 5-H, 6-H), 5.33 (m, 2 H, CH2Ph), 4.03 (s, 1 H, 2-H), 1.71
(s, 3 H, CH3), 1.70 (s, 3 H, CH3) ppm. 13C NMR (75 MHz,
CDCl3): δC = 166.2, 164.7, 164.4, 134.6, 134.1, 131.2, 128.5, 128.3,
123.5, 72.6, 67.4, 64.6, 62.9, 58.7, 29.8, 27.0 ppm. HRMS (EI):
calcd. for C23H20N2O5SK [M + K]+ 475.0729; found 475.0755.
Benzyl (2S,5R,6S)-3,3-Dimethyl-7-oxo-6-phthalimido-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylate (11b): 1,8-Diazabicyclo[5.4.0]-
undec-7-ene (0.05 mL, 0.04 mmol) was added to a solution of ester
11a (2.50 g, 7.22 mmol) in dry DCM (25 mL) and the mixture was
stirred at room temperature for 1 h. The solution was washed with
1 m NH4Cl (ϫ2), water, and brine. The organic phase was dried
(MgSO4) and concentrated under vacuum. The residue obtained
was purified by chromatography on silica gel using toluene/ethyl
acetate (9.5:0.5) as eluent to give 11b (2 g, 80%) as a white solid.
Rf = 0.4 (toluene/ethyl acetate, 9.5:0.5). 1H NMR (300 MHz,
CDCl3): δH = 7.93–7.77 (m, 4 H, Hpht), 7.43–7.37 (m, 5 H, HPh),
5.59 and 5.42 (2ϫ d, J = 2.0 Hz, 2ϫ 1 H, 5-H, 6-H), 5.24 (s, 2 H,
CH2Ph), 4.68 (s, 1 H, 2-H), 1.65 (s, 3 H, CH3), 1.44 (s, 3 H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 166.8, 166.6, 166.0, General Procedure 1. Synthesis of Benzoic Acid Derivatives from the
134.5, 134.3, 131.2, 128.3, 128.2, 128.1, 123.5, 68.8, 68.7, 67.0, 64.0,
63.9, 34.2, 24.9 ppm. HRMS (EI): calcd. for C23H20N2O5SK [M +
K]+ 475.0729; found 475.0737.
Protected Phthalimide Penicillin (Example: Compound 12a)
2-[(2S,5R,6R)-2-(Benzyloxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-6-ylcarbamoyl]benzoic Acid (12a): Sodium
Benzyl (2S,5S,6R)-3,3-Dimethyl-7-oxo-6-phthalimido-4-thia-1-aza- disulfide (0.27 g, 1.14 mmol) was added at 0–5 °C to a solution of
bicyclo[3.2.0]heptane-2-carboxylate (11c): A solution of ester 11a the protected phthalimide derivative 11a (0.50 g, 1.14 mmol) in a
(2.50 g, 5.73 mmol) in DCM (65 mL) and CCl4 (32 mL) was treated THF/H2O mixture (13 mL/13 mL). The solution was stirred at 0–
with an equimolar amount of sulfuryl chloride (0.46 mL,
5.73 mmol, solution in 2 mL of CCl4) and stirred at room tempera-
ture for 2 h. The solvents were evaporated under reduced pressure
to give a mixture of two chlorinated compounds, the trans and cis
isomers, in a ratio of around 8:2 as a yellow foam. Major product,
5 °C for 15 min and then acidified with citric acid (0.5 m) to pH 4.
DCM was added and the aqueous phase was extracted three times
with DCM. The organic phases were combined and washed suc-
cessively with water and brine, dried (MgSO4), and concentrated
under vacuum. The residue obtained was purified by chromatog-
the trans isomer: 1H NMR (300 MHz, CDCl3): δH = 7.93–7.79 (m, raphy on silica gel using DCM/ethyl acetate (9:1 to 8:2) as eluent
4 H, Hpht), 7.47–7.36 (m, 5 H, HPh), 6.04 and 5.54 (2ϫ d, J =
1.5 Hz, 2ϫ 1 H, 5-H, 6-H), 5.33 and 5.29 (2ϫ d, J = 25 Hz, 2ϫ
1 H, CH2Ph), 4.65 (s, 1 H, 2-H), 1.74 (s, 3 H, CH3), 1.72 (s, 3 H,
to give 12a (0.35 g, 67%) as a white solid. Rf = 0.2 (DCM/ethyl
acetate, 4:6). 1H NMR (300 MHz, CDCl3): δH = 8.00 (d, J =
7.2 Hz, 1 H, Hph), 7.62–7.44 (m, 3 H, Hph), 7.44 (s, 5 H, Har),
Eur. J. Org. Chem. 2011, 3437–3449
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3445

C. Crauste, M. Froeyen, J. Anné, P. Herdewijn
6.88 (d, J = 8.7 Hz, 1 H, NH), 5.88 (dd, J = 4.0, J = 8.7 Hz, 1 H, General Procedure 2. Synthesis of the Phthalisoimido Derivative
FULL PAPER
6-H), 5.67 (d, J = 4.0 Hz, 1 H, 5-H), 5.20 (s, 2 H, CH2Ph), 4.46 (s,
1 H, 2-H), 1.56 (s, 3 H, CH3), 1.41 (s, 3 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3): δC = 173.2, 169.1, 168.9, 167.2, 136.7, 134.4,
132.4, 130.8, 129.9, 128.4, 127.9, 127.6, 70.2, 67.7, 67.2, 64.5, 58.8,
31.2, 26.6 ppm. HRMS (EI): calcd. for C23H22N2O6SNa
[M + Na]+ 477.1096; found 477.1107.
from the Benzoic Acid Derivative (Example: Compound 13a)
Benzyl (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-phthalisoimido-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate (13a): DCC (0.12 g,
0.60 mmol) was added to a stirred solution of the acid 12a (0.27 g,
0.60 mmol) in dry DCM (10 mL) at 0 °C. The reaction mixture was
stirred for 1 h at 0 °C and for an additional 1 h at room tempera-
ture. Then the reaction mixture was again cooled to 0 °C and the
precipitated dicyclohexylurea (DCU) was removed by filtration.
The residue obtained was purified by chromatography on silica gel
using DCM/ethyl acetate (9:1) as eluent to give 13a (0.18 g, 70%)
as a white foam. Rf = 0.5 (hexane/ethyl acetate, 7:4). 1H NMR
(300 MHz, CDCl3): δH = 8.04 (d, J = 7.4 Hz, 1 H, Hpht), 7.96 (d,
J = 7.2 Hz, 1 H, Hpht), 7.83 (td, J = 7.2, J = 1.2 Hz, 1 H, Hpht),
7.76 (td, J = 7.4, J = 1.2 Hz, 1 H, Hpht), 7.42–7.35 (m, 5 H, HPh),
5.71 and 5.63 (2ϫ d, J = 4.2 Hz, 2ϫ1 H, 6-H, 5-H), 5.23 (d, J =
2.1 Hz, 2 H, CH2Ph), 4.55 (s, 1 H, 2-H), 1.66 (s, 3 H, CH3), 1.43
(s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 172.2, 167.4,
163.2, 151.1, 135.4, 135.2, 134.5, 133.1, 128.3, 127.7, 125.1, 123.5,
70.4, 68.0, 67.3, 67.1, 63.3, 30.6, 26.2 ppm. HRMS (EI): calcd. for
C23H20N2O5SNa [M + Na]+ 459.0990; found 459.0983.
2-[(2S,5R,6S)-2-(Benzyloxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-6-ylcarbamoyl]benzoic Acid (12b): General
procedure 1 was carried out by using compound 11b (0.50 g,
1.14 mmol) as the starting material. The residue obtained was puri-
fied by chromatography on silica gel using DCM/ethyl acetate (10:0
to 9:1) as eluent to give 12b (0.27 g, 51%) as a white solid. Rf =
0.4 (DCM/MeOH, 9:1). 1H NMR (300 MHz, CDCl3): δH = 8.00
(d, J = 7.6 Hz, 1 H, Hph), 7.54–7.49 (m, 3 H, Hph), 7.37–7.29 (m,
5 H, Har), 5.39 (d, J = 1.4 Hz, 1 H, 5-H), 5.26 (dd, J = 9.0, J =
1.4 Hz, 1 H, 6-H), 5.09 (s, 2 H, CH2Ph), 4.50 (s, 1 H, 2-H), 1.57
(s, 3 H, CH3), 1.32 (s, 3 H, CH3) ppm. 13C NMR (75 MHz,
CDCl3): δC = 172.6, 169.2, 168.7, 167.4, 136.5, 134.2, 132.3, 130.7,
129.8, 128.3, 128.2, 127.7, 69.2, 68.8, 67.3, 66.3, 63.9, 32.8,
25.4 ppm. HRMS (EI): calcd. for C23H22N2O6SNa [M + Na]+
477.1096; found 477.1082.
Benzyl (2S,5R,6S)-3,3-Dimethyl-7-oxo-6-phthalisoimido-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate (13b): General procedure 2
was carried out by using compound 12b (0.14 g, 0.32 mmol) as the
starting material. The residue obtained was purified by chromatog-
raphy on silica gel using DCM/ethyl acetate (9:1) as eluent to give
13b (0.07 g, 57%) as a white foam. Rf = 0.4 (hexane/ethyl acetate,
2-[(2S,5S,6R)-2-(Benzyloxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-6-ylcarbamoyl]benzoic Acid (12c): General
procedure 1 was carried out by using compound 11c (0.50 g,
1.12 mmol) as the starting material. The residue obtained was puri-
fied by chromatography on silica gel using DCM/ethyl acetate (9:1
to 8.5:1.5) as eluent to give 11c (0.25 g, 48%) as a white solid. Rf
= 0.4 (DCM/MeOH, 9:1). 1H NMR (300 MHz, CDCl3): δH = 7.93
(d, J = 7.6 Hz, 1 H, Hph), 7.53–7.44 (m, 3 H, Hph), 7.38–7.34 (m,
5 H, Har), 5.32 (d, J = 1.8 Hz, 1 H, 5-H), 5.17 (s, 2 H, CH2Ph),
5.10 (dd, J = 7.2, J = 1.8 Hz, 1 H, 6-H), 3.79 (s, 1 H, 2-H), 1.60
(s, 3 H, CH3), 1.35 (s, 3 H, CH3) ppm. 13C NMR (75 MHz,
CDCl3): δC = 170.1, 168.6, 167.8, 166.0, 136.1, 134.5, 132.1, 130.7,
129.9, 128.5, 128.3, 127.9, 69.8, 67.4, 66.7, 64.9, 63.5, 30.5,
24.7 ppm. HRMS (EI): calcd. for C23H22N2O6SNa [M + Na]+
477.1096; found 477.1105.
1
7:4). H NMR (300 MHz, CDCl3): δH = 7.98 (t, J = 6.5 Hz, 2 H,
Hpht), 7.84 (dt, J = 7.2, J = 1.2 Hz, 1 H, Hpht), 7.70 (dt, J = 7.2,
J = 0.9 Hz, 1 H, Hpht), 7.42–7.36 (m, 5 H, HPh), 5.53 and 5.40
(2ϫ d, J = 1.5 Hz, 2ϫ 1 H, 5-H, 6-H), 5.23 (d, J = 4.8 Hz, 2 H,
CH2Ph), 4.62 (s, 1 H, 2-H), 1.66 (s, 3 H, CH3), 1.44 (s, 3 H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 168.3, 167.0, 163.3,
151.4, 135.5, 135.2, 134.5, 133.1, 128.4, 128.3, 125.2, 123.3, 74.6,
69.5, 69.3, 67.0, 64.3, 33.2, 25.5 ppm. HRMS (EI): calcd. for
C23H20N2O5SNa [M + Na]+ 459.0990; found 459.0998.
Benzyl (2S,5S,6R)-3,3-Dimethyl-7-oxo-6-phthalisoimido-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate (13c): General procedure 2
was carried out by using compound 12c (0.30 g, 0.66 mmol) as the
starting material. The residue obtained was purified by chromatog-
raphy on silica gel using DCM/ethyl acetate (9:1) as eluent to give
13c (0.12 g, 45%) as a white foam. Rf = 0.4 (hexane/ethyl acetate,
2-[(2S,5S,6S)-2-(Benzyloxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-6-ylcarbamoyl]benzoic Acid (12d): Sodium
disulfide (0.26 g, 1.08 mmol) was dissolved in water (1 mL) and
added dropwise to a solution of the protected phthalimide deriva-
tive 11d (0.56 g, 1.28 mmol) in a THF/H2O mixture (13 mL/13 mL)
at 0–5 °C. The solution was stirred at 0–5 °C for 15 min and then
acidified with citric acid (0.5 m) to pH 4. DCM was added and the
aqueous phase was extracted three times with DCM. The organic
phases were combined and washed successively with water and
brine, dried (MgSO4), and concentrated under vacuum. A mixture
of 12d and the starting phthalimide 11d in a ratio of 70:30 (0.59 g)
was obtained as a yellow foam and used in the next step without
further purification. A sample of the compound was purified by
chromatography on silica gel using DCM/MeOH (9.8:0.2) as eluent
to give 12d as a white solid. Rf = 0.3 (DCM/MeOH, 9:1). 1H NMR
1
7:4). H NMR (300 MHz, CDCl3): δH = 7.98 (t, J = 6.5 Hz, 2 H,
Hpht), 7.84 (td, J = 7.2, J = 1.2 Hz, 1 H, Hpht), 7.70 (td, J = 7.2,
J = 0.9 Hz, 1 H, Hpht), 7.42–7.36 (m, 5 H, HPh), 5.53 and 5.40
(2ϫ d, J = 1.5 Hz, 2ϫ 1 H, 5-H, 6-H), 5.23 (d, J = 4.8 Hz, 2 H,
CH2Ph), 4.62 (s, 1 H, 2-H), 1.66 (s, 3 H, CH3), 1.44 (s, 3 H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 166.6, 165.8, 163.2,
151.5, 135.5, 135.2, 134.5, 133.1, 128.6, 128.3, 128.0, 125.2, 123.3,
71.3, 70.4, 67.5, 67.0, 64.8, 30.3, 24.9 ppm. HRMS (EI): calcd. for
C23H20N2O5SNa [M + Na]+ 459.0990; found 459.0996.
Benzyl (2S,5S,6S)-3,3-Dimethyl-7-oxo-6-phthalisoimido-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate (13d): DCC (0.20 g,
(300 MHz, CDCl3): δH = 8.03 (d, J = 7.5 Hz, 1 H, Hph), 7.65–7.50 0.97 mmol) was added to a stirred solution of the crude acid 12d
(m, 3 H, Hph), 7.38–7.21 (m, 5 H, Har), 6.79 (d, J = 8.5 Hz, 1 H,
NH), 5.74 (dd, J = 4.0, J = 8.5 Hz, 1 H, 6-H), 5.42 (d, J = 4.0 Hz,
1 H, 5-H), 5.20 (s, 2 H, CH2Ph), 3.90 (s, 1 H, 2-H), 1.65 (s, 3 H,
(0.59 g; contaminated by the starting phthalimide) in dry DCM
(25 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C
and for an additional 1 h at room temperature. Then the reaction
mixture was again cooled to 0 °C and the precipitated dicyclohex-
ylurea (DCU) was removed by filtration. The filtrate was concen-
trated and afforded the crude phthalisoimide 13d contaminated by
a small proportion of DCU and the starting phthalimide 11d. The
yellow foam (0.56 g) was used in the next step without further puri-
CH3), 1.43 (s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δC
=
170.5, 169.3, 167.5, 165.7, 135.9, 134.2, 132.3, 131.3, 130.2, 128.4,
128.3, 127.7, 127.5, 71.3, 67.6, 65.0, 63.7, 58.2, 30.3, 25.2 ppm.
HRMS (EI): calcd. for C23H22N2O6SNa [M + Na]+ 477.1096;
found 477.1090.
3446
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Eur. J. Org. Chem. 2011, 3437–3449

β-Lactam Lipopeptides as Bacterial Signal Peptidase I Inhibitors
fication because of the total degradation of the compound on silica
gel chromatography. Rf = 0.4 (DCM/ethyl acetate, 9.8:0.2). Major
product 13d: 1H NMR (300 MHz, CDCl3): δH = 8.04 (d, J =
7.5 Hz, 1 H, Hpht), 7.95 (d, J = 7.5 Hz, 1 H, Hpht), 7.85–7.73 (m,
2 H, Hpht), 7.44–7.34 (m, 5 H, HPh), 5.52 and 5.46 (2ϫ d, J =
acetate (9:1) as eluent to give 14d (0.07 g, 18% for three steps) as
a white solid. Rf = 0.4 (DCM/ethyl acetate, 6:4). [α]2D0 = +0.10 (c =
3.5, chloroform). 1H NMR (300 MHz, CDCl3): δH = 7.39–7.30 (m,
5 H, HPh), 5.25–5.23 (m, 3 H, 6-H, CH2Ph), 4.41 (dd, J = 0.9, J
= 4.2 Hz, 1 H, 5-H), 3.85 (s, 1 H, 2-H), 1.67 (s, 3 H, CH3), 1.48 (s,
4.1 Hz, 2ϫ 1 H, 6-H, 5-H), 5.27 (s, 2 H, CH2Ph), 3.91 (s, 1 H, 2- 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 175.2, 165.6,
H), 1.64 (s, 3 H, CH3), 1.54 (s, 3 H, CH3) ppm. HRMS (EI): calcd.
134.5, 128.3, 70.6, 67.3, 66.1, 62.4, 61.8, 29.8, 26.0 ppm. HRMS
for C23H20N2O5SNa [M + Na]+ 459.0990; found 459.0985.
(EI): calcd. for C15H19N2O3S [M + H]+ 307.1116; found 307.1114.
General Procedure 3. Deprotection of the Phthalisoimide Group (Ex-
ample: Compound 14a)
Benzyl (2S,5R,6R)-6-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-
asparaginyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate (2a): Decanoyl-PTAN-COOH (4; 25 mg,
0.04 mmol) and the amine 14a (15 mg, 0.04 mmol) were dissolved
in dry DMF (3 mL) and the solution was cooled to 0 °C. HOBt
(9.10 mg, 0.06 mmol) and EDCI (17 mg, 0.08 mmol) were added
to the solution and the reaction mixture was stirred at room tem-
perature for 6 h. The solvent was then evaporated to dryness under
vacuum. The residue obtained was dissolved in DCM and washed
successively with water, saturated NaHCO3 solution, and brine.
The aqueous phases were extracted several times with DCM and
the resulting organic phases were combined, dried, and concen-
trated under vacuum. The residue obtained was purified by
chromatography on silica gel using DCM/iPrOH (9:1 to 8:2) as
eluent to give 2a (8 mg, 20%) as a white solid. Rf = 0.3 (DCM/
MeOH, 9:1). 1H NMR (600 MHz, CDCl3/CD3OD): δH = 7.93,
7.80, 7.50, 6.28, 5.71 (5ϫ br., 5ϫ 1 H, 4NH, OH), 7.37 (m, 5 H,
HPh), 5.55 (dd, J = 4.2, J = 8.4 Hz, 1 H, 6-H), 5.52 (d, J = 4.2 Hz,
1 H, 5-H), 5.18 (d, J = 2.4 Hz, 2 H, CH2Ph), 4.78 [m, 1 H,
Hα(Asn)], 4.46 (s, 1 H, 2-H), 4.46–4.31 [m, 4 H, Hα(Ala, Pro, Thr),
Hβ(Thr)], 3.62, 3.53 [m, 2 H, CH2δ(Pro)], 2.83 [dd, J = 15, J =
5.4 Hz, 1 H, CH2aβ(Asn)], 2.67 [dd, J = 15, J = 6 Hz, 1 H,
CH2bβ(Asn)], 2.36–2.29 [m, 2 H, CH2β(Pro)], 2.22–2.01 [m, 4 H,
CH2γ(Pro), CH2deca], 1.62 (s, 3 H, CH3), 1.60 (m, 2 H, CH2deca),
1.46 [d, J = 7.2 Hz, 3 H, CH3(Ala)], 1.39 (s, 3 H, CH3), 1.27–1.25
(m, 12 H, 6 CH2deca), 1.16 [d, J = 6.6 Hz, 3 H, CH3(Thr)], 0.87 (t,
J = 7.2 Hz, 3 H, CH3deca) ppm. 13C NMR (75 MHz, CDCl3/
CD3OD): δC = 173.7, 172.9, 172.7, 172.5, 172.2, 170.5, 170.2, 167.3,
134.4, 128.3, 70.1, 67.5, 67.1, 66.2, 64.3, 60.5, 58.9, 58.1, 49.7, 49.6,
47.4, 36.1, 34.4, 31.5, 31.4, 29.1, 29.0, 28.9, 28.6, 26.5, 24.8, 24.4,
2 2 . 3 , 1 9 . 1 , 1 6 . 6 , 1 3 . 7 p p m . H R M S ( E I ) : c a l c d . f o r
C41H61N7O10SNa [M + Na]+ 866.4098; found 866.4114.
Benzyl (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicy-
clo[3.2.0]heptane-2-carboxylate (14a): The phthalisoimide 13a
(0.17 g, 0.38 mmol) was dissolved in dry THF (8.50 mL) and the
solution was cooled to –20 °C. A 2 m solution of N-methylhydraz-
ine (0.19 mL, 0.38 mmol) in THF was added dropwise and the re-
action mixture was stirred for 20 min at –20 °C. The solvent was
evaporated under reduced pressure and the residue obtained was
co-evaporated several times with chloroform until precipitation of
the byproduct hydrazide. The byproduct was removed by filtration,
and the filtrate was concentrated to afford the crude amine 14a
(0.12 mg, quant.) as a white amorphous solid. No further purifica-
tion was necessary. Rf = 0.4 (DCM/ethyl acetate, 6:4). [α]2D0 = –0.17
1
(c = 4, chloroform). H NMR (300 MHz, CDCl3): δH = 7.39 (s, 5
H, HPh), 5.53 and 4.58 (2ϫ d, J = 4.2 Hz, 2ϫ 1 H, 6-H, 5-H),
5.20 (s, 2 H, CH2Ph), 4.44 (s, 1 H, 2-H), 1.63 (s, 3 H, CH3), 1.44
(s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δC = 177.4, 167.6,
134.5, 128.3, 69.7, 69.6, 67.0, 63.6, 62.5, 31.4, 26.7 ppm. HRMS
(EI): calcd. for C15H18N2O3SNa [M + Na]+ 329.0936; found
329.0932.
Benzyl (2S,5R,6S)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicy-
clo[3.2.0]heptane-2-carboxylate (14b): General procedure 3 was ap-
plied to the phthalisoimide 13b (70 mg, 0.16 mmol) and afforded
the amine 14b (49 mg, quant.) as a white solid. Rf = 0.3 (DCM/
ethyl acetate, 6:4). [α]2D0 = +0.22 (c = 2.2, chloroform). 1H NMR
(300 MHz, CDCl3): δH = 7.37 (s, 5 H, HPh), 5.19 (s, 2 H, CH2Ph),
5.11 and 4.23 (2ϫ d, J = 1.4 Hz, 2ϫ 1 H, 5-H, 6-H), 4.50 (s, 1 H, 2-
H), 1.56 (s, 3 H, CH3), 1.39 (s, 3 H, CH3) ppm. 13C NMR (75 MHz,
CDCl3): δC = 172.8, 167.3, 134.5, 128.3, 71.6, 71.1, 68.2, 67.4, 64.1,
33.0, 25.6 ppm. HRMS (EI): calcd. for C15H19N2O3S [M + H]+
307.1116; found 307.1100.
(2S,5R,6R)-6-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-aspara-
Benzyl (2S,5S,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicy-
clo[3.2.0]heptane-2-carboxylate (14c): General procedure 3 was ap-
plied to the phthalisoimide 12c (0.12 g, 0.27 mmol) and afforded
the amine 14c (80 mg, 94%) as a white solid. Rf = 0.3 (DCM/ethyl
acetate, 6:4). [α]2D0 = –0.09 (c = 3, chloroform). 1H NMR (300 MHz,
CDCl3): δH = 7.39–7.35 (m, 5 H, HPh), 5.22 (d, J = 1.5 Hz, 2 H,
CH2Ph), 4.87 and 4.43 (2ϫ d, J = 1.7 Hz, 2ϫ 1 H, 5-H, 6-H), 3.76
(s, 1 H, 2-H), 1.60 (s, 3 H, CH3), 1.37 (s, 3 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3): δC = 171.5, 166.2, 134.5, 128.3, 69.7, 69.6, 67.5,
67.3, 64.7, 30.4, 24.7 ppm. HRMS (EI): calcd. for C15H19N2O3S
[M + H]+ 307.1116; found 307.1095.
ginyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic Acid (2Јa): Decanoyl-PTAN-COOH (4; 40 mg,
0.07 mmol) and the free amine, N-(p-nitrobenzyloxycarbonyl)-6-
aminopimelic acid[1] (55 mg, 0.15 mmol), were dissolved in dry
DMF (4 mL) and the solution was cooled to 0 °C. HOBt (14 mg,
0.10 mmol) and EDCI (27 mg, 0.14 mmol) were added to the solu-
tion and the reaction mixture was stirred at room temperature for
6 h. The solvent was then evaporated to dryness under vacuum.
The residue obtained was dissolved in DCM and washed with
water. The aqueous phase was extracted several times with DCM
and the resulting organic phases were combined, dried, and con-
Benzyl (2S,5S,6S)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicy- centrated under vacuum. The residue obtained was dissolved in a
clo[3.2.0]heptane-2-carboxylate (14d): The crude phthalisoimide
13d (0.56 g) was dissolved in dry THF (27 mL) and the solution
was cooled to –20 °C. A 2 m solution of N-methylhydrazine
(0.51 mL, 1.02 mmol) in THF was added dropwise and the reaction
mixture was stirred for 20 min at –20 °C. The solvent was evapo-
rated under reduced pressure and the residue obtained was co-evap-
orated several times with chloroform until the precipitation of the
byproduct hydrazide. The byproduct was removed by filtration and
the filtrate was concentrated under vacuum. The residue obtained
was purified by chromatography on silica gel using DCM/ethyl
mixture of THF/dioxane (9:1, 3 mL) and hydrogenated under at-
mospheric pressure on 10% palladium on carbon (25 mg) for 3 h.
The catalyst was filtered and washed with MeOH. The filtrate was
concentrated under reduced pressure and purified by reversed-
phase semi-prep. HPLC (H2O to CH3CN) to give 2Јa (7 mg, 13%)
1
as a white solid. H NMR (300 MHz, D2O): δH = 5.72–5.62 (m, 2
H, 6-H, 5-H), 4.85–4.35 [m, 6 H, Hα(Asn, Ala, Pro, Thr), Hβ(Thr),
2-H], 3.85–3.71 [m, 2 H, CH2δ(Pro)], 3.05–2.83 [m, 2 H,
CH2β(Asn)], 2.60–2.15 [m, 4 H, CH2β(Pro), CH2γ(Pro)], 2.17 (m,
3 H, CH2deca), 1.79 (s, 3 H, CH3), 1.75 (m, 2 H, CH2deca), 1.69 (s,
Eur. J. Org. Chem. 2011, 3437–3449
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3447

C. Crauste, M. Froeyen, J. Anné, P. Herdewijn
FULL PAPER
3 H, CH3), 1.56 [m, 3 H, CH3(Ala)], 1.55–1.40 (m, 12 H, 6
H, CH3), 1.27–1.24 (m, 12 H, 6 CH2deca), 1.17 [d, J = 6.3 Hz, 3
CH2deca), 1.37 [m, 3 H, CH3(Thr)], 1.07 (m, 3 H, CH3deca) ppm. H, CH3(Thr)], 0.84 (t, J = 7.2 Hz, 3 H, CH3deca) ppm. 13C NMR
13C NMR (75 MHz, D2O): δC = 176.5, 175.8, 175.3, 175.2, 175.1,
172.8, 172.4, 171. 3, 74.0, 67.9, 67.7, 65.6, 61.7, 59.6, 59.1, 51.2,
50.8, 49.1, 37.3, 35.3, 32.5, 31.9, 30.7, 29.8, 27.7, 25.6, 25.5, 23.4,
20.1, 18.0, 14.6 ppm. HRMS (EI): calcd. for C34H55N7O10SNa [M
+ Na]+ 776.3628; found 776.3614.
(75 MHz, CDCl3/CD3OD): δC = 173.8, 173.0, 172.9, 172.3, 171.4,
171.1, 167.9, 166.2, 134.5, 128.4, 69.6, 67.3, 67.0, 66.3, 65.1, 62.8,
60.5, 58.3, 49.8, 49.5, 47.4, 36.7, 34.3, 31.4, 30.7, 29.6, 29.3, 29.1,
29.0, 28.8, 24.6, 24.3, 22.3, 19.2, 16.1, 13.7 ppm. HRMS (EI): calcd.
for C41H61N7O10SNa [M + Na]+ 866.4098; found 866.4103.
Benzyl (2S,5R,6S)-6-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-
Benzyl (2S,5S,6S)-6-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-
asparaginyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate (2b): Decanoyl-PTAN-COOH (4; 20 mg,
0.03 mmol) and the amine 14b (16.50 mg, 0.05 mmol) were dis-
solved in dry DMF (1.5 mL) and the solution was cooled to 0 °C.
HOBt (7.20 mg, 0.05 mmol) and EDCI (13.80 mg, 0.06 mmol) were
added to the solution and the reaction mixture was stirred at room
temperature for 6 h. The solvent was then evaporated to dryness
under vacuum. The residue obtained was dissolved in DCM and
washed successively with citric acid solution (1%), water, saturated
NaHCO3 solution, and brine. The aqueous phases were extracted
several times with DCM and the resulting organic phases were
combined, dried, and concentrated under vacuum. The residue ob-
tained was purified by chromatography on silica gel using DCM/
MeOH (9.5:0.5 to 9:1) as eluent to give 2b (14 mg, 46%) as a white
solid. Rf = 0.4 (DCM/MeOH, 9:1). 1H NMR (300 MHz, CDCl3/
CD3OD): δH = 8.28, 7.87, 7.82, 6.75, 6.24 (5ϫ br., 5ϫ 1 H, 4NH,
OH), 7.37 (m, 5 H, HPh), 5.28 (d, J = 1.2 Hz, 1 H, 5-H), 5.18 (s,
2 H, CH2Ph), 5.02 (dd, J = 1.2, J = 7.8 Hz, 1 H, 6-H), 4.83 [m, 1
H, Hα(Asn)], 4.51 (s, 1 H, 2-H), 4.51–4.27 [m, 4 H, Hα(Ala, Pro,
Thr), Hβ(Thr)], 3.69, 3.52 [m, 2 H, CH2δ(Pro)], 2.87 [dd, J = 15.5,
J = 6.6 Hz, 1 H, CH2aβ(Asn)], 2.70 [dd, J = 15.5, J = 5.1 Hz, 1 H,
CH2bβ(Asn)], 2.38–2.29 [m, 2 H, CH2β(Pro)], 2.25–1.97 [m, 4 H,
CH2γ(Pro), CH2deca], 1.61 (m, 2 H, CH2deca), 1.56 (s, 3 H, CH3),
1.44 [d, J = 7.3 Hz, 3 H, CH3(Ala)], 1.36 (s, 3 H, CH3), 1.31–1.25
(m, 12 H, 6 CH2deca), 1.17 [d, J = 6.3 Hz, 3 H, CH3(Thr)], 0.88 (t,
J = 6.9 Hz, 3 H, CH3deca) ppm. 13C NMR (75 MHz, CDCl3/
CD3OD): δC = 173.3, 173.1, 172.8, 172.4, 171.6, 170.8, 169.6, 167.1,
134.5, 128.3, 69.4, 68.8, 67.0, 66.7, 66.2, 63.7, 60.4, 58.2, 50.5, 49.7,
47.4, 36.3, 34.4, 33.0, 31.5, 29.3, 29.2, 29.1, 28.9, 25.4, 24.7, 24.4,
2 2 . 3 , 1 9 . 3 , 1 6 . 4 , 1 3 . 8 p p m . H R M S ( E I ) : c a l c d . f o r
C41H61N7O10SNa [M + Na]+ 866.4098; found 866.4094.
asparaginyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate (2d): Decanoyl-PTAN-COOH (4; 18 mg,
0.03 mmol) and the amine 14d (15 mg, 0.05 mmol) were dissolved
in dry DMF (4 mL) and the solution was cooled to 0 °C. HOBt
(6.5 mg, 0.05 mmol) and EDCI (12.4 mg, 0.06 mmol) were added
to the solution and the reaction mixture was stirred at room tem-
perature for 6 h. The solvent was then evaporated to dryness under
vacuum. The residue obtained was dissolved in DCM and washed
successively with water, saturated NaHCO3 solution, and brine.
The aqueous phases were extracted several times with DCM and
the resulting organic phases were combined, dried, and concen-
trated under vacuum. The residue obtained was purified by
chromatography on silica gel using DCM/IPrOH (9.5:0.5 to 8:2) as
eluent to give 2d (19 mg, 68%) as a white solid. Rf = 0.3 (DCM/
MeOH, 9:1). 1H NMR (500 MHz, CDCl3/CD3OD): δH = 8.84,
7.50, 7.45, 6.54, 6.15 (5ϫ br., 5ϫ1 H, 4NH, OH), 7.39–7.32 (m, 5
H, HPh), 5.41 (dd, J = 4.1, J = 7.8 Hz, 1 H, 6-H), 5.23 (d, J =
4.1 Hz, 1 H, 5-H), 5.22 (d, J = 3.6 Hz, 2 H, CH2Ph), 4.88 [m, 1 H,
Hα(Asn)], 4.52–4.32 [m, 4 H, Hα(Ala, Pro, Thr), Hβ(Thr)], 3.84 (s,
1 H, 2-H), 3.64, 3.48 [m, 2 H, CH2δ(Pro)], 2.82 [dd, J = 15.5, J
= 6 Hz, 1 H, CH2aβ(Asn)], 2.71 [dd, J = 15.5, J = 5.5 Hz, 1 H,
CH2bβ(Asn)], 2.34–2.28 [m, 2 H, CH2β(Pro)], 2.15–1.80 [m, 4 H,
CH2γ(Pro), CH2deca], 1.62 (s, 3 H, CH3), 1.59 (m, 2 H, CH2deca),
1.48 (s, 3 H, CH3), 1.43 [d, J = 7.0 Hz, 3 H, CH3(Ala)], 1.30–1.22
(m, 12 H, 6 CH2deca), 1.16 [d, J = 6.0 Hz, 3 H, CH3(Thr)], 0.87 (t,
J = 7.0 Hz, 3 H, CH3deca) ppm. 13C NMR (75 MHz, CDCl3/
CD3OD): δC = 173.2, 172.9, 172.5, 172.4, 170.6, 170.5, 170.4, 165.1,
134.4, 128.3, 71.4, 67.3, 66.9, 63.8, 62.6, 60.2, 59.1, 58.0, 49.6, 49.5,
47.4, 36.5, 34.4, 31.5, 29.3, 29.1, 29.0, 28.9, 28.7, 26.6, 24.7, 24.4,
22.3, 19.0, 17.1, 13.7 ppm. HRMS (EI): calcd. for
C41H61N7O10SNa [M + Na]+ 866.4098; found 866.4109.
Benzyl (2S,5S,6R)-6-[(N-Decanoyl-L-prolyl-L-threonyl-L-alanyl-L-
Supporting Information (see footnote on the first page of this arti-
cle): Experimental characterization data (1H and 13C NMR,
HRMS) for all the compounds synthesized and procedures for the
biological in vitro evaluation.
asparaginyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate (2c): Decanoyl-PTAN-COOH (4; 25 mg,
0.04 mmol) and the amine 14c (27 mg, 0.09 mmol) were dissolved
in dry DMF (2 mL) and the solution was cooled to 0 °C. HOBt
(9.1 mg, 0.07 mmol) and EDCI (17 mg, 0.09 mmol) were added to
the solution and the reaction was stirred at room temperature for
6 h. The solvent was then evaporated to dryness under vacuum.
The residue obtained was dissolved in DCM and washed success-
ively with citric acid solution (1%), water, saturated NaHCO3 solu-
tion, and brine. The aqueous phases were extracted several times
with DCM and the resulting organic phases were combined, dried,
and concentrated under vacuum. The residue obtained was purified
by chromatography on silica gel using DCM/MeOH (9.5:0.5 to 9:1)
as eluent to give 2c (30 mg, 70%) as a white solid. Rf = 0.3 (DCM/
MeOH, 9:1). 1H NMR (300 MHz, CDCl3/CD3OD): δH = 8.19,
7.90, 7.83, 6.83, 5.91 (5ϫ br., 5ϫ 1 H, 4NH, OH), 7.37–7.33 (m,
5 H, HPh), 5.18 (m, 3 H, 5-H, CH2Ph), 4.84 (d, J = 2.1 Hz, 1 H,
6-H), 4.72 [m, 1 H, Hα(Asn)], 4.43–4.23 [m, 4 H, Hα(Ala, Pro,
Thr), Hβ(Thr)], 3.80 (s, 1 H, 2-H), 3.66, 3.52 [m, 2 H, CH2δ(Pro)],
2.78 [dd, J = 15.0, J = 6.3 Hz, 1 H, CH2aβ(Asn)], 2.69 [dd, J =
15.0, J = 5.4 Hz, 1 H, CH2bβ(Asn)], 2.38–2.27 [m, 2 H, CH2β(Pro)],
2.20–1.90 [m, 4 H, CH2γ(Pro), CH2deca], 1.59 (s, 3 H, CH3), 1.58
(m, 2 H, CH2deca), 1.43 [d, J = 7.5 Hz, 3 H, CH3(Ala)], 1.33 (s, 3
Acknowledgments
The authors are grateful for financial support from the Agentschap
voor Innovatie door Wetenschap en Technologie (Rational Drug
Design and Innovative High Throughput Screening Approach to
Combat Antibiotic Resistant Bacteria, grant number 050146) and
to Dr. J. Rozenski for performing the MS analysis
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Received: February 1, 2011
Published Online: May 20, 2011
Eur. J. Org. Chem. 2011, 3437–3449
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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