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DOI: 10.1002/cmdc.201402140

Combinatorial Multicomponent Access to NaturalProducts-Inspired Peptidomimetics: Discovery of Selective

Inhibitors of Microbial Metallo-aminopeptidases
Yanira M!ndez,[a] Karell P!rez-Labrada,[a] Jorge Gonz"lez-Bacerio,[b] Gilberto Vald!s,[b] Mar#a $.
de los Ch"vez,[b] Joel Osuna,[c] Jean-Louis Charli,[c] Isel Pascual,[b] and Daniel G. Rivera*[a]
The development of selective inhibitors of microbial metalloaminopeptidases is an important goal in the pursuit of antimicrobials for therapeutic applications. Herein, we disclose a combinatorial approach relying on two Ugi reactions for the generation of peptidomimetics inspired by natural metallo-aminopeptidase inhibitors. The library was screened for inhibitory activity against the neutral metallo-aminopeptidase of Escherichia
coli (ePepN) and the porcine kidney cortex metallo-aminopeptidase (pAPN), which was used as a model of the M1-amino-

peptidases of mammals. Six compounds showed typical dose

response inhibition profiles toward recombinant ePepN, with
two of them being very potent and highly selective for ePepN
over pAPN. Another compound showed moderate ePepN inhibition but total selectivity for this bacterial enzyme over its
mammalian orthologue at concentrations of physiological relevance. This strategy proved to be useful for the identification
of lead compounds for further optimization and development.

Introduction
Aminopeptidases are proteolytic enzymes that hydrolyze peptide bonds from the amino termini of polypeptide chains.
Among the dissimilar families of these enzymes, metallo-aminopeptidases (MAPs) comprise a distinctive group that utilizes
one or two metal ions in the active sites to carry out the catalytic process.[1] These enzymes are involved in crucial physiological processes and, therefore, constitute attractive targets
for the treatment of several diseases. They are known to participate in protein degradation, nutrient utilization,[2] downstream
processing of cytosolic proteins,[3] recycling of amino acids,[4]
and regulation during chemical-induced stress,[5] among other
functions. In the last few years, microbial MAPs have been recognized as targets to combat bacterial and parasite protozoan
diseases. Thus, microbial MAP inhibitors have been designed
as second-generation antibiotics,[6] vaccine candidates, and
drugs for parasite infections.[7]
The neutral aminopeptidase from the bacterium Escherichia
coli (ePepN; EC 3.4.11.2) is a broad-specificity Zn2 + -dependent
aminopeptidase belonging to clan MA, family M1. This cytosolic enzyme contains the consensus Zn2 + -binding motif HEXXHX18-E and the exopeptidase motif GXMEN in the active site.[8]
This enzyme is the major aminopeptidase in E. coli[9] and
[a] Y. M!ndez, K. P!rez-Labrada, D. G. Rivera
Center for Natural Products Research, Faculty of Chemistry
University of Havana, Zapata y G, 10400, La Habana (Cuba)
E-mail: dgr@fq.uh.cu
[b] J. Gonz"lez-Bacerio, G. Vald!s, M. A. de los Ch"vez, I. Pascual
Center for Protein Studies, Faculty of Biology
University of Havana, 25 y J, 10400, La Habana (Cuba)
[c] J. Osuna, J.-L. Charli
Institute of Biotechnology, Autonomous National University of Mexico
Av. Universidad 2001, 62210, Cuernavaca, Morelos (M!xico)

% 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

shows substrate specificity for basic and hydrophobic (including aromatic and branched) residues at the P1 position[3, 5] (that
is, the N-terminal amino acid). An important feature of ePepN
is its high similarity in both sequence and kinetic characteristics with other microbial MAPs from the M1 family,[3, 10] including important targets in the treatment of human diseases like
the M1 aminopeptidase of Plasmodium falciparum (PfA-M1),
a causative agent of malaria. From consideration of this latter
fact and the high availability of ePepN by recombinant means,
this enzyme is considered as a model for the rapid identification of microbial MAP inhibitors by the screening of compound libraries. ePepN has been useful for understanding the
structural basis of enzymeligand interactions through assessment of the tridimensional structures of enzymeligand complexes, including complexes with different l-amino acids[10c]
and the prototypal inhibitor bestatin (1, Figure 1 A).[10b, 11] This
latter antimicrobial agent[12] is known to be a potent inhibitor
of MAPs belonging to the M1 family.[13] The catalytic mechanism of M1-family MAPs combines the critical involvement of
Zn2 + ions in both activating a water molecule as a nucleophile
and stabilizing the reaction intermediates.[14] The specific recognition of a peptide with a free terminal amino group[15] and
with either a hydrophobic or basic side chain in the amino
acid at the P1 position[15, 16] is also a key factor in the catalytic
process. Small-molecule inhibitors may be classified according
to different structural classes that depend upon the structural
elements of the inhibitors and on the zinc-binding group.
Nature provides a variety of effective inhibitors of MAPs, which
are usually peptide mimics with either the a-hydroxyamide
(for example, bestatin (1) and related peptides) or the hydroxamic moiety (for example, actinonin (2); Figure 1 A).[17] Accordingly, the most prominent M1-MAP inhibitors incorporate
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Figure 1. A) Natural and synthetic MAP inhibitors. B) The Ugi four-component reaction (Ugi-4CR). C) The Ugi five-center four-component reaction
(Ugi-5C-4CR).

a metal-binding group into pseudopeptide scaffolds with hydrophobic substituents.[18] Among the synthetic structures designed to target M1-MAPs are phosphinate dipeptide analogues,[19] hydroxamic acids,[20] a-aminoalkylphosphonates,[21]
and bestatin analogues.[22]
Isocyanide-based multicomponent reactions (I-MCRs) have
proven to be effective tools for the parallel preparation and
screening of peptidomimetics based on protease inhibitor
pharmacophores.[23] Among such multicomponent strategies
to generate protease inhibitors, a very successful one is the
PADAM approach (that is, Passerini reactionamine deprotectionacyl migration), which was independently reported by
two groups in 2000 as an efficient way to access a-hydroxyamide- and a-ketoamide-containing compounds, including bestatin (1).[24]
Ugi reactions have also shown great success in the combinatorial preparation of peptidomimetics incorporating a hydroxamic moiety. A small library of marimastat analogues (Figure 1 A) bearing a terminal hydroxamate was prepared by
means of a one-pot, two-step process consisting of the Ugi
four-component reaction (Ugi-4CR; Figure 1 B) followed by
aminolysis with hydroxylamine.[20d] Similarly, a library of actinonin mimics (Figure 1 A) was prepared by means of the Ugi fivecenter four-component reaction (Ugi-5C-4CR; Figure 1 C) followed by reaction with hydroxylamine.[20c] Nevertheless, these
reports focused on the design of hydroxamate-containing pep% 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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tidic scaffolds to act as inhibitors of matrix metalloproteases,
a class of zinc-dependent enzymes, the overexpression of
which is involved in several human diseases. An important example of the versatility of the Ugi reaction in the preparation
of microbial MAP inhibitors is the preparation of a compound
library based on the quinoline scaffold. This approach rendered
4-aminoquinoline-based peptidomimetics with high inhibitory
activity against an aminopeptidase of P. falciparum, as well as
potent in vivo antimalarial activity.[25]
Whereas bestatin (1) and actinonin (2) have proven to be
potent MAP inhibitors, an important drawback is their low selectivity between microbial and mammalian M1-family MAPs,
which limits the therapeutic applications of these compounds.
This is due to the presence of the a-hydroxyamide and hydroxamate moieties, which provide strong binding to the metal ion
found in MAP active sites. In the pursuit of compounds showing selective inhibitory activity against microbial MAPs,
a useful strategy may be the removal of such potent metalbinding groups with retention of other structural motifs that
are also known to enable recognition by the enzyme, for example, the hydrophobic amino acid side chain and terminal
free amino group.
Herein, we report on the utilization of two distinctive Ugi
multicomponent reactions for the combinatorial generation of
peptidomimetics that are structurally inspired by natural products 1 and 2, although lacking the key metal-binding groups.
Furthermore, we communicate the results obtained in the
screening of the compound collection in microbial and mammalian MAP inhibition assays and, thus, provide new insights
into the structureactivity relationship (SAR) related to the potency and selectivity (regarding mammalian M1-MAPs) of such
enzyme inhibition processes.

Results and Discussion

Combinatorial synthesis by Ugi reactions
Bestatin (1) is a natural pseudopeptide resembling the structure of the dipeptide PheLeu but incorporating a-hydroxy-bphenylalanine at the N terminus instead of Phe. As pointed
out above, we focused on the removal of the a-hydroxyamide
moiety with the aim of reducing the potent Zn2 + -coordination
capability. We reasoned that compounds lacking such a transition-state-analogue motif should exhibit poorer inhibitory activity against MAPs but probably greater selectivity between
microbial and mammalian MAPs. Previously, a bestatin-based library of microbial MAP (that is, PfA-M1) inhibitors provided
useful information to address the selectivity issue in MAP inhibitors.[22] Based on that study, we designed a compound collection with the terminal free amine of bestatin (1), which is
known to interact by hydrogen bonding with one or more glutamate residues in the M1 MAP active site, and with hydrophobic side chains capable of interacting with the S1 and S1
enzyme pockets.[22] To achieve this, we focused on the utilization of the Ugi-4CR for the combinatorial preparation of N-alkylated branched peptides based on bestatin and related hydrophobic sequences. The classic Ugi-4CR comprises the conChemMedChem 0000, 00, 1 10

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Table 1. Synthesis of bestatin-inspired N-alkylated peptidomimetics by

using the Ugi-4CR.[a]

Table 2. Synthesis of imino-based peptidomimetics by using the Ugi-5C4CR.[a]

Entry
Entry
1
2
3
4
5
6
7
8
9

Acid
(R1)

Amine
(R2)

Boc-Phe-OH
Boc-Phe-OH
Boc-Phe-OH
Boc-Phe-OH
Boc-Phe-OH
Boc-Phe-OH
Boc-Phe-OH
Boc-Leu-OH
Boc-Leu-OH

Leu-OMe
Leu-OMe
Leu-OMe
Leu-OMe
Leu-OMe
Leu-OMe
Leu-OMe
Phe-OMe
Val-OMe

R3

R4

H
Ph
H
Furyl
Furyl
Ph
Ph
H
H

tBu
tBu
Bn
tBu
Bn
Bn
CH2CO2Me
tBu
Bn

Peptido- Yield d.r.[d]

mimetic[b] [%][c]
3
4
5
6
7
8
9
10
11

81
75
76
70
79
76
80
80
65

1.3:1

2:1
1.1:1
1.6:1
1.4.1

[a] Reaction was conducted at room temperature in MeOH for 24 h, by

using Et3N (1 equiv) and the hydrochloride salt of amino acid methyl
ester. Boc: tert-butoxycarbonyl; TFA: trifluoroacetic acid; Bn: benzyl.
[b] The intermediate Ugi products were purified and characterized by
NMR spectroscopy. The final peptidomimetics were characterized by ESIMS. [c] Overall yield over three steps for the trifluoroacetate salts. [d] Determined by 1H NMR spectroscopy.

densation of a primary amine, an oxo compound (that is,

a ketone or aldehyde), a carboxylic acid, and an isocyanide to
produce an N-substituted dipeptide backbone.[26] Owing to its
multicomponent nature, this reaction enables the installation
of additional elements of diversity, that is, those arising from
the oxo and isocyano components of the Ugi-4CR, around
a specific dipeptide sequence.
As shown in Table 1 (entries 17), the one-pot process consists of the Ugi-4CR-based ligation of Boc-l-Phe-OH and HCl-lLeu-OMe with variation in the remaining two components, followed by deprotection of both peptide termini through standard Boc and methyl ester removal protocols. The Ugi-4CRs
proceeded efficiently to furnish the Ugi products in high yields
after column chromatography. The products were characterized by NMR spectroscopy and next subjected to C- and N-terminal deprotection. Except when formaldehyde was used, the
N-alkylated peptides were obtained as mixtures of diastereomers in variable ratios and were used as so in the biological
assays.
It must be noticed that peptidomimetics 39 are branched
tripeptides of the type PheN(AA)Leu, with AA being the
amino acid portion formed during the Ugi-4CR. Thus, an important issue was the selection of the oxo and isocyano components that result in the N-substitution of the common Phe
Leu sequence. Based on previous SAR studies on bestatin, we
focused on the installation of lipophilic moieties that might
provide effective hydrophobic contacts with the S1 and S1
pockets of the target enzyme. The incorporation of an additional carboxylic acid was accomplished in compound 9
(Table 1, entry 7) with the use of methyl isocyanoacetate. Entries 8 and 9 in Table 1 report the preparation of N-alkylated
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1
2
3
4
5
6

Amino acid
(R1)
Phe
Leu
Ile
Ile
Leu
Trp

R2
H
H
Ph
H
Ph
H

Peptidomimetic[b]

Yield
[%][c]

d.r.[d]

12
13
14
15
16
17

80
84
85
79
85
79

4.5:1

5:1

[a] Reactions were conducted at room temperature in MeOH for 24 h.

[b] The intermediate Ugi products were purified and characterized by
NMR spectroscopy. The final peptidomimetics were characterized by ESIMS. [c] Overall yield over two steps. [d] Determined by 1H NMR spectroscopy.

peptides with sequences that are different from that of bestatin, so that the effect of this change on the biological activity
could be evaluated. It is important to mention that the N-alkylation of peptides provides interesting features, such as higher
membrane permeability, improved metabolic stability, and the
frequent occurrence of both the cis and trans rotamers of the
N-substituted amide bond.[27]
Table 2 depicts the strategy towards imino-based peptidomimetics derived from the Ugi-5C-4CR, an impressive variation of
the classic Ugi-4CR in which a-amino acids are used as bifunctional building blocks.[28] This diastereoselective reaction was
previously employed for the preparation of actinonin mimics
(Figure 1 A), through variation of the isocyanide and aldehyde
components in the Ugi-5C-4CR followed by the installation of
the hydroxamic moiety.[20d] As an alternative, the focus of our
design was the variation of the a-amino acids and the aldehyde, whereas the hydroxamic moiety was substituted by a terminal carboxylic acid. Cyclohexylisonitrile was kept as a fixed
component, with the aim of mimicking the western part of the
actinonin scaffold. As mentioned before, the lack of the hydroxamate functionality in this type of skeleton should lead to
a lower Zn-coordination capability and is likely to increase the
selectivity between microbial and mammalian MAPs. As shown
in Table 2, the Ugi-5C-4CR with hydrophobic amino acids and
either formaldehyde or benzaldehyde furnished the iminodicarboxylic acid peptidomimetics in excellent yields and reasonable diastereoselectivities. The Ugi products were purified by
flash chromatography and next subjected to C-deprotection to
afford peptidomimetics 1217 in good overall yields.
Screening of the compound library in MAP inhibition assays
Table 3 shows the results of the inhibitory activity of peptidomimetics 317 against recombinant ePepN. Eight compounds
(6, 9, 11, 12, 13, 14, 16, and 17) caused a reduction of the enChemMedChem 0000, 00, 1 10

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Table 3. Inhibitory activity of Ugi derived peptidomimetics against microbial and mammalian M1 MAPs.[a]
Compd

Bestatin
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

rePepN
Residual activity
[20 mm inhibitor][b]

Ki [mm]

pAPN
Ki [mm]

Selectivity ratio
Ki (pAPN)/
Ki (rePepN)

0.443 ! 0.043
1.399 ! 0.034
0.825 ! 0.041
1.251 ! 0.009
0.687 ! 0.069
0.989 ! 0.115
1.016 ! 0.115
0.508 ! 0.068
1.276 ! 0.167
0.734 ! 0.139
0.706 ! 0.153
0.652 ! 0.104
0.561 ! 0.028
1.123 ! 0.032
0.733 ! 0.088
0.182 ! 0.019

3.03[c]
NI
NI
NI
27.9 ! 14.7
NI
NI
18.6 ! 1.8
NI
18.8 ! 0.9
8.1 ! 2.9
NI
NI
NI
19.0 ! 8.3
3.9 ! 0.9

0.3 ! 0.1

NI

25.8 ! 15.9

24.5 ! 1.3
21.0 ! 10.3

19.7 ! 6.5
23.6 ! 15.4

0.1

1.4

1.3
2.6

1.0
6.1

[a] rePepN: Recombinant M1-aminopeptidase from E. coli; pAPN: porcine

kidney cortex aminopeptidase as a model of the M1 family of aminopeptidases from mammals; Ki : inhibition constant; NI: not dose-dependent
inhibition; 1: total selectivity for rePepN at an inhibitor concentration
range of physiological relevance. [b] The results are expressed as the
mean ! standard deviation of three parallel assays. [c] According to
Mucha et al.[18a]

zymatic activity to 75 % or less, relative to the control, at the

sole dose of 20 mm. This is a concentration value that has been
reported to be suitable for the screening of enzyme inhibitors
with therapeutic potential.[20b] The remaining compounds
either did not cause inhibition at this concentration, behaved
as enzyme activators (that is, the residual activity was higher
than 1), or proved to be weak rePepN inhibitors. For this
reason, they were not selected for Ki determination. In addition, the Ki values of compounds 13 and 14 could not be assessed because of their non-dose-dependent profile. In terms
of inhibitory activity against rePepN, the peptidomimetics derived from the Ugi-5C-4CR reaction (Table 2) were generally
more potent than those derived from the Ugi-4CR (Table 1). In
order to address selectivity, those compounds active against
rePepN were also screened against the porcine neutral aminopeptidase (a model for M1 aminopeptidases from mammals).
Thus, whereas bestatin mimic 6 showed a moderate efficacy in
the inhibition of rePepN at 20 mm, it proved to be completely
inactive against pAPN. Such a complete selectivity of compound 6 for microbial over mammalian MAPs at a concentration range of physiological significance can be considered as
a promising result in the pursuit of effective therapeutic
agents.
Compound 17 was the most potent rePepN inhibitor (Ki =
3.9 ! 0.9 mm) among all of the assayed peptidomimetics and,
interestingly, was the sole structure containing an indole
moiety. As reported by Addlagatta and co-workers,[10c] the S1
subsite (that is, the active-site pocket that recognizes the P1
residue) in the active site of ePepN has a cylinder form, with
a hydrophobic wall that is capable of accommodating the
% 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

large and lipophilic side chains of phenylalanine, tyrosine, and

tryptophan. These authors resolved the tridimensional structures of ePepN in complexes with several l-amino acids and
corroborated that tryptophan fills the entire S1 pocket, with
tight contacts between the indole moiety and the hydrophobic
walls of the S1 subsite. Differently from other enzymeamino
acid complexes, significant differences between the free-ligand
structure and the enzymetryptophan complex were not observed in that study, with the exception of the Met260 residue.
Interestingly, the only residue that undergoes a significant displacement in the ligand-bound structures, Met260, was ordered
in ePepNbestatin and ePepNtryptophan complexes but not
in the other ePepNhydrophobic amino acids complexes.
These data help to explain that the inhibitory potency of compound 17 against rePepN is similar to that of bestatin and suggest a close reversible interaction between the indole group of
17 and the ePepN S1 subsite. Hence, the indole group at the
P1 position of 17 could act as a substrate analogue and
bind the S1 pocket of rePepN in a competitive fashion, to produce an effect that can be classified in the limit between classical and tight-binding enzymatic inhibitors.
Compound 12 was the second most potent rePepN inhibitor
of the whole compound library (Ki = 8.1 ! 2.9 mm). The only
structural difference between 12 and 17 is the replacement of
the indole group in 17 by the smaller phenyl ring in 12. The
phenyl group, also present at the P1 position of bestatin, is enclosed within the hydrophobic ePepN S1 subsite, as confirmed
by crystallographic structures of ePepNbestatin and ePepN
phenylalanine complexes.[10c] Similarly to 17, the phenyl group
at the P1 position of 12 could interact with the enzyme S1
pocket as a competitive inhibitor. Consistently, the phenyl substituent at the P1 position of a hydroxamate of malonic acid
(a potent PfA-M1 inhibitor; half-maximal inhibitory concentration, IC50 = 6 nm) can be accommodated by the S1 pocket of
PfA-M1,[29] an enzyme that has 85 % sequence identity with
ePepN in the active site.[10a] Another possibility is that the interaction could take place between the aromatic side chain of
either 17 or 12 and the ePepN S1 pocket (that is, the activesite pocket that recognizes the P1 residue), but this hypothesis
is not supported by experimental evidence due to the lack of
detailed structure information about this subsite. However, it
has been reported that the nature of the P1 residue determines, to a large extent, the affinity of peptidic inhibitors for
the active site of ePepN.[30] Compounds 13, 14, 15, and 16
were weaker or inactive rePepN inhibitors, probably owing to
the lack of aromatic hydrophobic side chains.
Importantly, compounds 12 and 17 could be also considered
as selective inhibitors of microbial MAPs, because their Ki
values toward the mammalian enzyme were 2.6 and 6.1 times
higher, respectively, than their Ki values against rePepN. The
rest of the rePepN inhibitors assayed were less or nonselective
for the microbial aminopeptidase. These results point to 12
and 17 as two potent and relatively selective ePepN inhibitors,
as well as to 6 as a fully selective ePepN inhibitor.

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Conclusions
We have implemented a combinatorial approach based on
two related Ugi multicomponent reactions for the generation
of peptidomimetics that were structurally inspired by natural
inhibitors of metallo-aminopeptidases. The approach proved
to be versatile and efficient in the creation of a compound collection for screening of the inhibitory activity against microbial
and mammalian MAPs. From the library of 15 peptidomimetics,
compounds 12 and 17, derived from the Ugi-5C-4CR, proved
to be as potent ePepN inhibitors as the natural pseudopeptide
bestatin and they showed good selectivity for the microbial
enzyme over the mammalian one. In addition, compound 6,
derived from the Ugi-4CR library, exhibited a moderate inhibitory profile but was fully selective for ePepN over pAPN. Based
on these results, such compounds can be considered as lead
structures in the rational design of more potent and selective
inhibitors of microbial MAPs.

Experimental Section
Chemical synthesis
General: 1H NMR and 13C NMR spectra were recorded at 400 MHz
and 100 MHz, respectively. Chemical shifts (d) are reported in parts
per million relative to the residual solvent signals, and coupling
constants (J) are reported in Hertz. High-resolution ESI mass spectra were obtained from a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, with an RF-only hexapole ion
guide and an external electrospray ion source. Flash column chromatography was carried out by using silica gel 60 (70230 mesh)
and analytical thin layer chromatography (TLC) was performed by
using silica gel aluminum sheets. All commercially available chemicals were used without further purification. Because of the cleanness of the deprotection steps, the final peptidomimetics were not
further purified by preparative chromatography but were instead
analyzed by analytical reversed-phase HPLC and ESI-MS to ensure
their identity and suitable purity (95 %) for the biological assays.
General Ugi-4CR-based procedure: A suspension of the a-amino
acid methyl ester hydrochloride (1.0 mmol), Et3N (1.0 mmol), and
the aldehyde (1.0 mmol) in MeOH (5 mL) was stirred for 2 h at
room temperature. The Boc-protected a-amino acid (1.0 mmol)
and the isocyanide (1.0 mmol) were then added and the reaction
mixture was stirred at room temperature for 24 h. The volatiles
were removed under reduced pressure and the resulting crude
product was dissolved in CH2Cl2 (60 mL). The organic phase was
washed sequentially with an aqueous saturated solution of citric
acid (50 mL), aqueous 10 % NaHCO3 (50 mL), and brine (50 mL) and
then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column
chromatography (n-hexane/EtOAc) to afford the Ugi product.
General methyl ester removal procedure: The Ugi product
(1.0 mmol) was dissolved in THF/H2O (2:1, 3 mL) and LiOH (105 mg,
2.5 mmol) was added at 0 8C. The mixture was stirred at 0 8C for
3 h and then acidified with aqueous 10 % NaHSO4 to pH 3. The resulting phases were separated and the aqueous phase was extracted with EtOAc (2 & 50 mL). The combined organic phases were
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to yield the C-deprotected peptidomimetic.
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General Boc removal procedure: The product was dissolved in
a 30 % solution of TFA in CH2Cl2 (3 mL) and the reaction mixture
was stirred for 2 h. The solvent was evaporated to dryness to
afford the corresponding peptidomimetic as the trifluoroacetate
salt.
Peptidomimetic 3: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), paraformaldehyde (34 mg, 1.13 mmol), BocPhe
(300 mg, 1.13 mmol), and tert-butylisocyanide (130 mL, 1.13 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
4:1) afforded the fully protected compound 3 (462 mg). Rf = 0.25
(n-hexane/EtOAc, 4:1); 1H NMR (400 MHz, CDCl3): d = 0.89 (d, 6 H,
J = 6.4 Hz, 2 & CH3), 1.36 (s, 9 H, (CH3)3C), 1.39 (s, 9 H, (CH3)3C), 1.79
1.96 (m, 1 H), 2.93 (m, 2 H), 3.52 (d, 1 H, J = 18 Hz), 3.74 (s, 3 H,
OCH3), 3.81 (d, 1 H, J = 18 Hz), 4.07 (t, 1 H, J = 6.9 Hz), 4.53 (dd, 1 H,
J = 7.1, 14.6 Hz), 5.10 (d, 1 H, J = 7.3 Hz, NH), 7.167.35 (m, 5 H, Ar),
7.46 ppm (br s, 1 H, NH); 13C NMR (100 MHz, CDCl3): d = 22.2, 23.1
(CH3), 24.9 (CH), 28.4, 28.6 (CH3), 38.1, 39.0, 51.8 (CH2), 52.2 (CH3),
52.7, 59.3 (CH), 80.4 (C), 127.1, 128.8, 129.5 (CH), 135.8 (C), 155.5,
167.0, 171.9, 173.2 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for
C27H43N3O6Na [M + Na] + : 528.3050; found: 528.3060. The Ugi product (420 mg, 0.83 mmol) was subjected to consecutive removal of
the methyl ester and the Boc group according to the general deprotection procedures. The resulting product was dried under high
vacuum for 2 h, suspended in water/acetonitrile (1:1, 5 mL) and
lyophilized to furnish compound 3 as the trifluoroacetate salt
(420 mg, 81 %). HRMS (ESI-FT-ICR): m/z calcd for C21H33N3O4Na [M +
Na] + : 414.2369; found: 414.2373.
Peptidomimetic 4: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), benzaldehyde (115 mL, 1.13 mmol), BocPhe (300 mg,
1.13 mmol), and tert-butylisocyanide (130 mL, 1.13 mmol) were
mixed in MeOH (3 mL) according to the general procedure for the
Ugi-4CR. Flash column chromatography (n-hexane/EtOAc, 6:1) afforded the fully protected compound 4 (493 mg). Rf = 0.37 (nhexane/EtOAc, 6:1); a mixture of diastereomers in a 1.3:1 ratio was
observed; 1H NMR (400 MHz, CDCl3): d = 0.810.92 (m, 6 H, 2 & CH3),
1.29, 1.38 (2 & s, 9 H, (CH3)3C), 1.42, 1.44 (2 & s, 9 H, (CH3)3C), 1.64
1.86 (m, 1 H), 2.10 (m, 1 H), 2.182.32 (m, 1 H), 2.752.92 (m, 1 H),
2.943.03 (m, 1 H), 3.173.25 (m, 1 H), 3.65, 3.76 (2 & s, 3 H, OCH3),
4.64 (m, 1 H), 4.72 (m, 1 H), 4.82 (m, 1 H), 4.895.14 (m, 1 H), 5.20 (t,
1 H, J = 8.5 Hz), 6.57 (br s, 1 H, NH), 7.137.32 ppm (m, 5 H, Ar);
13
C NMR (100 MHz, CDCl3): d = 22.2 (CH3), 24.5 (CH), 28.1, 28.2 (CH3),
37.9, 39.2 (CH2), 51.5 (CH), 52.3 (CH3), 52.9 (C), 56.9, 58.7 (CH), 79.9
(C), 127.1, 126.6, 128.0, 129.2, 129.8, 130.7 (CH), 135.5, 137.7 (C),
156.5, 168.0, 170.8, 173.2 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd
for C33H47N3O6Na [M + Na] + : 604.3363; found: 604.3382. The Ugi
product (450 mg, 0.77 mmol) was subjected to consecutive removal of the methyl ester and the Boc group according to the general
deprotection procedures. The resulting product was dried under
high vacuum for 2 h, suspended in water/acetonitrile (1:1, 5 mL),
and lyophilized to furnish compound 4 as the trifluoroacetate salt
(450 mg, 75 %). HRMS (ESI-FT-ICR) m/z calcd for C27H37N3O4Na [M +
Na] + : 490.2682; found: 490.2677.
Peptidomimetic 5: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), paraformaldehyde (34 mg, 1.13 mmol), BocPhe
(300 mg, 1.13 mmol), and benzylisocyanide (140 mL, 1.13 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
6:1) afforded the fully protected compound 5 (463 mg). Rf = 0.68
(n-hexane/EtOAc, 6:1); 1H NMR (400 MHz, CDCl3): d = 0.800.94 (m,
6 H, 2 & CH3), 1.26 (s, 9 H, (CH3)3C), 1.60 (m, 1 H), 1.761.91 (m, 2 H),
2.852.96 (m, 1 H), 3.13 (m, 1 H), 3.67 (s, 3 H, OCH3), 3.693.76 (m,

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1 H), 3.924.03 (m, 1 H), 4.334.45 (m, 1 H), 4.464.58 (m, 1 H), 4.84
(m, 1 H), 5.26 (d, 1 H, J = 8.7 Hz, NH), 7.187.36 (m, 5 H, Ar),
7.94 ppm (br s, 1 H, NH); 13C NMR (100 MHz, CDCl3): d = 21.9, 22.9
(CH3), 24.6 (CH), 28.1, 28.2 (CH3), 36.9, 38.3, 38.9, 43.5 (CH2), 50.7
(CH3), 52.2, 58.1 (CH), 79.9 (C), 126.8, 127.2, 127.6, 128.5, 128.8,
129.4 (CH), 136.5, 140.1 (C), 156.1, 169.0, 170.3, 173.9 ppm (C=O);
HRMS (ESI-FT-ICR) m/z calcd for C30H41N3O6Na [M + Na] + : 562.2893;
found: 562.2889. The Ugi product (420 mg, 0.78 mmol) was subjected to consecutive removal of the methyl ester and the Boc
group according to the general deprotection procedures. The resulting product was dried under high vacuum for 2 h, suspended
in water/acetonitrile (1:1, 5 mL), and lyophilized to furnish compound 5 as the trifluoroacetate salt (420 mg, 76 %). HRMS (ESI-FTICR): m/z calcd for C24H31N3O4Na [M + Na] + : 448.2212; found:
448.2230.
Peptidomimetic 6: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), furfuraldehyde (141 mg, 1.13 mmol), BocPhe
(300 mg, 1.13 mmol), and tert-butylisocyanide (130 mL, 1.13 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
8:1) afforded the fully protected compound 6 (452 mg). Rf = 0.52
(n-hexane/EtOAc, 6:1); a mixture of diastereomers in a 2:1 ratio
was observed by NMR spectroscopy; 1H NMR (400 MHz, CDCl3): d =
0.89, 0.94 (2 & d, 6 H, J = 6.6 Hz, 2 & CH3), 1.33, 1.34 (2 & s, 9 H,
(CH3)3C), 1.39, 1.41 (2 & s, 9 H, (CH3)3C), 1.771.90 (m, 2 H), 2.142.28
(m, 1 H), 2.762.88 (m, 1 H), 3.06 (m, 1 H), 3.72, 3.73 (2 & s, 3 H,
OCH3), 3.743.87 (m, 3 H), 4.03 (dd, 1 H, J = 5.6, 8.5 Hz), 4.8 (dt, 1 H,
J = 8.1, 7.5 Hz), 5.21 (d, 1 H, J = 8.1 Hz, NH), 5.46 (s, 1 H), 6.256.42
(m, 1 H), 6.456.64 (m, 2 H), 6.84 (d, 1 H, J = 3.3 Hz), 7.187.43 (m,
11 H), 7.99 ppm (br s, 1 H, NH); 13C NMR (100 MHz, CDCl3): d = 21.5,
21.7 (CH3), 22.9 (CH), 28.3, 28.5 (CH3), 38.8, 39.4 (CH2), 51.8, 51.9
(CH), 52.6 (CH3), 53.1 (C), 57.1 (CH), 80.4 (C), 110.0, 111.0, 127.1,
128.8, 129.8 (CH), 136.1, 143.0 (C), 145.2, 148 (C=O), 151.6 (CH),
165.4, 172.4 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for
C31H45N3O7Na [M + Na] + : 594.3155; found: 594.3158. The Ugi product (410 mg, 0.72 mmol) was subjected to consecutive removal of
the methyl ester and the Boc group according to the general deprotection procedures. The resulting product was dried under high
vacuum for 2 h, suspended in water/acetonitrile (1:1, 5 mL), and
lyophilized to furnish compound 6 as the trifluoroacetate salt
(410 mg, 70 %). HRMS (ESI-FT-ICR): m/z calcd for C25H35N3O5Na [M +
Na] + : 480.2474; found: 480.2472.
Peptidomimetic 7: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), furfuraldehyde (141 mg, 1.13 mmol), BocPhe
(300 mg, 1.13 mmol), and benzylisocyanide (140 mL, 1.13 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
8:1) afforded the fully protected compound 7 (540 mg). Rf = 0.66
(n-hexane/EtOAc, 6:1); a mixture of diastereomers in a 1:1 ratio
was observed; 1H NMR (400 MHz, CDCl3): d = 0.92 (2 & d, 6 H, J =
6.7 Hz, 2 & CH3,), 1.26, 1.29 (2 & s, 9 H, (CH3)3C), 1.33, 1.39 (2 & s, 9 H,
(CH3)3C), 1.591.66 (m, 1 H), 2.16 (m, 2 H), 2.923.19 (m, 2 H), 2.88
3.57 (d, 1 H, J = 9 Hz), 3.69, 3.80 (2 & s, 3 H, OCH3), 4.304.70 (m, 8 H),
4.90, 5.02 (2 & s, 1 H, NH), 6.296.54 (m, 4 H), 7.207.37 (m, 5 H, Ar),
7.38 (d, 1 H, J = 9.1 Hz), 8.55 ppm (br s, 1 H, NH); 13C NMR (100 MHz,
CDCl3): d = 23.1 (CH3), 24.9 (CH), 28.3 (CH3), 37.4, 43.5 (CH2), 50.7,
51.8 (CH), 52.0 (CH3), 55.3 (CH), 80.8 (C), 110.6, 111.7 (CH), 127.2,
127.4, 127.7, 128.5, 129.2, 129.8 (CH), 135.7, 137.9 (C), 143.7 (CH),
148.0 (C), 155.8, 165.9, 166.3 ppm (C=O); HRMS (ESI-FT-ICR): m/z
calcd for C34H43N3O7Na [M + Na] + : 628.2999; found: 628.2990. The
Ugi product (500 mg, 0.83 mmol) was subjected to consecutive removal of the methyl ester and the Boc group according to the

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general deprotection procedures. The resulting product was dried
under high vacuum for 2 h, suspended in water/acetonitrile (1:1,
5 mL), and lyophilized to furnish compound 7 as the trifluoroacetate salt (500 mg, 79 %). HRMS (ESI-FT-ICR): m/z calcd for
C28H33N3O5Na [M + Na] + : 514.2318; found: 514.2320.
Peptidomimetic 8: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), benzaldehyde (115 mL, 1.13 mmol), BocPhe (300 mg,
1.13 mmol), and benzylisocyanide (140 mL, 1.13 mmol) were mixed
in MeOH (3 mL) according to the general procedure for the Ugi4CR. Flash column chromatography (n-hexane/EtOAc, 10:1) afforded the fully protected compound 8 (528 mg). Rf = 0.70 (n-hexane/
EtOAc, 6:1); a mixture of diastereomers in a 1.6:1 ratio was observed; 1H NMR (400 MHz, CDCl3): d = 0.790.99 (m, 6 H, 2 & CH3),
1.37, 1.40 (2 & s, 9 H, (CH3)3C), 1.431.60 (m, 1 H), 1.80 (m, 1 H), 2.24
(m, 1 H), 2.782.97 (m, 2 H), 3.08 (m, 1 H), 3.69, 3.71 (2 & s, 3 H,
OCH3), 4.314,60 (m, 1 H), 4.89 (d, J = 6.5 Hz, 2 H), 4.99 (d, J = 6.0 Hz,
2 H), 5.045.23 (m, 1 H), 6.11, 6.12 (2 & br s, 1 H), 6.41 (br s, 1 H, NH),
7.127.38 (m, 5 H, Ar), 8.08 ppm (m, 1 H, NH); 13C NMR (100 MHz,
CDCl3): d = 22.0, 22.9 (CH3), 25.3 (CH), 28.5 (CH3), 38.9, 39.2, 41.8
(CH2), 50.8 (CH), 52.3 (CH3), 55.6, 58.7 (CH), 76.3 (C), 126.8, 127.4,
127.5, 127.6, 127.8, 128.7, 128.8, 129.0, 129.2, 129.5, 129.9 (CH),
135.1, 135.2, 135.8 (C), 155.5, 156.2, 162.2, 170.6 ppm (C=O); HRMS
(ESI-FT-ICR): m/z calcd for C36H45N3O6Na [M + Na] + : 638.3206;
found: 638.3210. The Ugi product (490 mg, 0.80 mmol) was subjected to consecutive removal of the methyl ester and the Boc
group according to the general deprotection procedures. The resulting product was dried under high vacuum for 2 h, suspended
in water/acetonitrile (1:1, 5 mL), and lyophilized to furnish compound 8 as the trifluoroacetate salt (490 mg, 76 %). HRMS (ESI-FTICR): m/z calcd for C30H35N3O4Na [M + Na] + : 524.2525; found:
524.2530.
Peptidomimetic 9: Leu-OMe hydrochloride (205 mg, 1.13 mmol),
Et3N (160 mL), benzaldehyde (115 mL, 1.13 mmol), BocPhe (300 mg,
1.13 mmol), and methyl isocyanoacetate (110 mL, 1.13 mmol) were
mixed in MeOH (3 mL) according to the general procedure for the
Ugi-4CR. Flash column chromatography (n-hexane/EtOAc, 6:1) afforded the fully protected compound 9 (539 mg). Rf = 0.30 (nhexane/EtOAc, 6:1); a mixture of diastereomers in a 1.4:1 ratio was
observed; 1H NMR (400 MHz, CDCl3): d = 0.760.94 (m, 6 H, 2 & CH3),
1.39, 1.41 (2 & s, 9 H, (CH3)3C), 1.441.52 (m, 1 H), 1.551.62 (m, 1 H),
1.701.79 (m, 1 H), 2.813.05 (m, 2 H), 3.69, 3.71 (2 & s, 3 H, OCH3),
3.74, 3.76 (2 & s, 3 H, OCH3), 4.024.06 (m, 1 H), 4.754.82 (m, 2 H),
6.14, 6.22 (2 & s, 1 H), 6.41 (s, 1 H, NH), 7.137.34 (m, 5 H, Ar),
7.65 ppm (m, 1 H, NH); 13C NMR (100 MHz, CDCl3): d = 22.2 (CH3),
24.8 (CH), 28.2 (CH3), 35.9, 38.2, 44.9 (CH2), 51.7 (CH), 52.2, 52.9
(CH3), 56.9, 58.7 (CH), 80.1 (C), 126.3, 126.9, 127.4, 127.9, 128.2,
128.8, 129.3, 129.8 (CH), 136.1, 137.2 (C), 156.5, 157.2, 163.2, 170.6,
175.4 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for C32H48N3O8Na
[M + Na] + : 620.2948; found: 620.2945. The Ugi product (500 mg,
0.84 mmol) was subjected to consecutive removal of the methyl
ester and the Boc group according to the general deprotection
procedures. The resulting product was dried under high vacuum
for 2 h, suspended in water/acetonitrile (1:1, 5 mL), and lyophilized
to furnish compound 9 as the trifluoroacetate salt (488 mg, 80 %).
HRMS (ESI-FT-ICR): m/z calcd for C25H31N3O6Na [M + Na] + : 492.2111;
found: 492.2116.
Peptidomimetic 10: Phe-OMe hydrochloride (280 mg, 1.29 mmol),
Et3N (180 mL), paraformaldehyde (40 mg, 1.29 mmol), BocLeu
(300 mg, 1.29 mmol), and tert-butylisocyanide (150 mL, 1.29 mmol)
were mixed in CH3OH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
4:1) afforded the fully protected compound 10 (521 mg). Rf = 0.30

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(n-hexane/EtOAc, 6:1); 1H NMR (400 MHz, CDCl3): d = 0.89 (d, 3 H,
J = 6.5 Hz, CH3), 0.95 (dd, 3 H, J = 9.4, 6.6 Hz, CH3), 1.35, 1.42 (2 & s,
9 H, (CH3)3C), 1.391.49 (m, 1 H), 3.073.15 (m, 2 H), 3.75 (d, 1 H, J =
15.8 Hz), 3.78 (s, 3 H, OCH3), 3.82379 (m, 1 H), 3.92 (dd, 1 H, J = 4.8,
10.7 Hz), 4.13 (t, 1 H, J = 8.5 Hz), 5.91 (d, 1 H, J = 8.7 Hz, NH), 7.12 (d,
2 H, J = 6.2 Hz, Ar), 7.257.31 (m, 3 H, Ar), 7.68 ppm (br s, 1 H, NH);
13
C NMR (100 MHz, CDCl3): d = 23.1 (CH3), 24.8 (CH), 28.4 (CH3), 34.6,
41.4, 46.0 (CH2), 49.9 (CH3), 52.9, 53.8 (CH), 79.8 (C), 127.5, 129.0,
129.3 (CH), 136.9 (C), 155.7, 167.3, 170.6, 173.9 ppm (C=O); HRMS
(ESI-FT-ICR): m/z calcd for C27H43N3O6Na [M + Na] + : 528.3050;
found: 528.3045. The Ugi product (480 mg, 0.95 mmol) was subjected to consecutive removal of the methyl ester and the Boc
group according to the general deprotection procedures. The resulting product was dried under high vacuum for 2 h, suspended
in water/acetonitrile (1:1, 5 mL), and lyophilized to furnish compound 10 as the trifluoroacetate salt (480 mg, 80 %). HRMS (ESI-FTICR): m/z calcd for C21H33N3O4Na [M + Na] + : 414.2369; found:
414.2350.
Peptidomimetic 11: Val-OMe hydrochloride (218 mg, 1.30 mmol),
Et3N (185 mL), paraformaldehyde (39 mg, 1.30 mmol), BocLeu
(300 mg, 1.30 mmol), and benzylisocyanide (160 mL, 1.30 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-4CR. Flash column chromatography (n-hexane/EtOAc,
2:1) afforded the fully protected compound 11 (415 mg). Rf = 0.50
(n-hexane/EtOAc, 1:1); 1H NMR (400 MHz, CDCl3): d = 0.86 (m, 6 H,
2 & CH3), 0.880.95 (m, 3 H, CH3), 1.001.05 (m, 3 H, CH3), 1.38 (s, 9 H,
(CH3)3C), 1.451.55 (m, 1 H), 1.601.73 (m, 3 H), 2.132.20 (m, 1 H),
3.76 (s, 3 H, OCH3), 4.05 (s, 1 H), 4.16 (d, 1 H, J = 5.8 Hz), 4.40 (t, 1 H,
J = 7.0 Hz), 4.72 (s, 1 H), 4.87 (d, 2 H, J = 6.3 Hz), 5.30 (d, 1 H, J =
7.0 Hz, NH), 7.207.30 (m, 5 H, Ar), 7.50 ppm (br s, 1 H, NH); 13C NMR
(100 MHz, CDCl3): d = 17.5, 18.9, 22.5 (CH3), 25.0 (CH), 28.3 (CH3),
28.9 (CH), 40.9, 42.7 (CH2), 51.9 (CH3), 52.8 (CH2), 53.2, 62.9 (CH),
79.6 (C), 126.9, 127.5, 128.5 (CH), 136.8 (C), 156.0, 169.5, 172.9,
174.0 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for C26H41N3O6Na
[M + Na] + : 514.2893; found: 514.2880. The Ugi product
(415 mg,1.2 mmol) was subjected to consecutive removal of the
methyl ester and the Boc group according to the general deprotection procedures. The resulting product was dried under high
vacuum for 2 h, suspended in water/acetonitrile (1:1, 5 mL), and
lyophilized to furnish compound 11 as the trifluoroacetate salt
(390 mg, 65 %). HRMS (ESI-FT-ICR): m/z calcd for C20H31N3O4Na [M +
Na] + : 400.2212; found: 400.2215.
General Ugi-5C-4CR-based procedure: A suspension of the aamino acid (1.0 mmol) and the aldehyde (1.0 mmol) in MeOH
(5 mL) was stirred for 2 h at room temperature. The isocyanide
(1.0 mmol) was then added and the reaction mixture was stirred at
room temperature for 24 h. The volatiles were removed under reduced pressure and the resulting crude product was purified by
flash column chromatography (n-hexane/EtOAc) to afford the Ugi
product.
Peptidomimetic 12: l-Phe (500 mg, 3.03 mmol), paraformaldehyde
(92 mg, 3.03 mmol), and cyclohexylisocyanide (380 mL, 3.03 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 3:1) afforded the protected Ugi product 12 (771 mg). Rf =
0.20 (n-hexane/EtOAc, 3:1); 1H NMR (400 MHz, CDCl3): d = 1.211.29
(m, 1 H), 1.321.42 (m, 2 H), 1.491.54 (m, 1 H), 1.591.66 (m, 2 H),
1.932.01 (m, 4 H), 2.782.83 (m, 1 H), 2.983.02 (m, 1 H), 3.193.30
(m, 2 H), 3.33, 3.40 (m, 1 H), 3.573.60 (m, 1 H), 3.74 (s, 3 H, OCH3),
4.10 (s, 1 H, NH), 5.20 (s, 1 H, NH), 7.187.36 ppm (m, 5 H, Ar);
13
C NMR (100 MHz, CDCl3): d = 23.3, 25.5, 33.4, 40.1 (CH2), 50.0 (CH),
51.5 (CH3), 52.3 (CH2), 59.8, 127.1, 128.6, 129.0 (CH), 132.9 (C), 170.2,

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175.4 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for C18H26N2O3Na
[M + Na] + : 341.1841; found: 341.1843. The protected Ugi product
12 (247 mg, 0.77 mmol) and LiOH (97 mg, 2.3 mmol) were mixed
in THF/H2O (3 mL, 2:1) according to the methyl ester removal procedure to give compound 12 (234 mg, 80 %). HRMS (ESI-FT-ICR): m/
z calcd for C17H24N2O3Na [M + Na] + : 327.1685; found: 327.1679.
Peptidomimetic 13: l-Leu (200 mg, 1.52 mmol), paraformaldehyde
(46 mg, 1.52 mmol), and cyclohexylisocyanide (190 mL, 1.52 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 3:1) afforded the protected Ugi product 13 (363 mg). Rf =
0.26 (n-hexane/EtOAc, 3:1); 1H NMR (400 MHz, CDCl3): d = 0.830.87
(m, 3 H, CH3), 1.191.30 (m, 2 H), 1.331.50 (m, 3 H), 1.511.56 (m,
2 H), 1.561.67 (m, 2 H), 1.962.03 (m, 4 H), 3.133.18 (m, 1 H), 3.21
3.30 (m, 2 H), 3.633.69 (m, 1 H), 3.73 (s, 3 H, OCH3), 3.753.80 (m,
1 H), 4.01 (s, 1 H, NH), 5.03 ppm (s, 1 H, NH); 13C NMR (100 MHz,
CDCl3): d = 22.1 (CH3), 23.5 (CH2), 24.4 (CH), 25.5, 34.9, 41.2 (CH2),
50.2 (CH), 51.4 (CH3), 52.8 (CH2), 55.5 (CH), 170.1, 173.5 ppm (C=O);
HRMS (ESI-FT-ICR): m/z calculated for C15H28N2O3Na [M + Na] + :
307.1998; found: 307.1996. The protected Ugi product 13 (200 mg,
0.70 mmol) and LiOH (89 mg, 2.11 mmol) were mixed in THF/H2O
(3 mL, 2:1) according to the methyl ester removal procedure to
give compound 13 (189 mg, 84 %). HRMS (ESI-FT-ICR): m/z calcd for
C14H26N2O3Na [M + Na] + : 293.1841; found: 293.1821.
Peptidomimetic 14: l-Ile (200 mg, 1.52 mmol), benzaldehyde
(155 mL, 1.52 mmol), and cyclohexylisocyanide (190 mL, 1.52 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 3:1) afforded protected Ugi product 14 (465 mg). Rf = 0.38
(n-hexane/EtOAc, 3:1); a mixture of diastereomers in a 4.5:1 ratio
was observed; 1H NMR (400 MHz, CDCl3): d = 0.84 (t, 3 H, J =
0.74 Hz, CH3), 0.86 (d, 3 H, J = 6.8 Hz, CH3), 0.890.95 (m, 1 H), 0.99
(m, 1 H), 1.071.21 (m, 3 H), 1.281.40 (m, 2 H), 1.451.69 (m, 2 H),
1.74 (m, 2 H), 1.81 (m, 3 H), 2.97 (d, 1 H, J = 5.9 Hz), 3.70 (s, 3 H,
OCH3), 3.73 (s, 1 H), 4.18 (br s, 1 H, NH), 6.41 (d, 1 H, J = 7.9 Hz, NH),
7.277.39 ppm (m, 5 H, Ar); 13C NMR (100 MHz, CDCl3): d = 11.5,
15.5 (CH3), 24.8, 25.5, 25.7, 32.9 (CH2), 38.1, 48.0 (CH), 51.8 (CH3),
63.6, 66.2, 128.2, 128.6, 129.0 (CH), 140.4 (C), 172.3; 175.3 ppm (C=
O); HRMS (ESI-FT-ICR): m/z calcd for C21H32N2O3Na [M + Na] + :
383.2311; found: 383.2317. The protected Ugi product 14 (200 mg,
0.55 mmol) and LiOH (70 mg, 1.67 mmol) were mixed in THF/H2O
(3 mL, 2:1) according to the methyl ester removal procedure to
give compound 14 (190 mg, 85 %). HRMS (ESI-FT-ICR): m/z calcd for
C20H30N2O3Na [M + Na] + : 369.2154; found: 369.2155.
Peptidomimetic 15: l-Ile (200 mg, 1.52 mmol), paraformaldehyde
(46 mg, 1.52 mmol), and cyclohexylisocyanide (190 mL, 1.52 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 4:1) afforded the protected Ugi product 15 (341 mg). Rf =
0.40 (n-hexane/EtOAc, 3:1); 1H NMR (400 MHz, CDCl3): d = 0.92 (t,
3 H, J = 7.4 Hz, CH3), 0.95 (d, 3 H, J = 6.8 Hz, CH3), 1.101.21 (m, 3 H),
1.321.35 (m, 1 H), 1.351.41 (m, 1 H), 1.411.44 (m, 1 H), 1.471.55
(m, 1 H), 1.61 (m, 1 H), 1.671.74 (m, 3 H), 1.861.93 (m, 2 H), 2.93
(br s, 1 H, NH), 2.99 (s, 1 H), 3.05 (d, 1 H, J = 5.6 Hz), 3.38 (s, 1 H), 3.42
(s, 1 H), 3.72 (s, 3 H, OCH3), 3.693.74 (m, 1 H), 7.15 ppm (d, J =
7.3 Hz, NH); 13C NMR (100 MHz, CDCl3): d = 11.7, 16.9 (CH3), 24.9,
25.3, 25.7, 33.3 (CH2), 38.3, 47.7 (CH), 51.5 (CH3), 51.9 (CH2), 66.6
(CH), 170.2, 174.9 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for
C15H28N2O3Na [M + Na] + : 307.1998; found: 307.1992. The protected
Ugi product 15 (200 mg, 0.70 mmol) and LiOH (89 mg, 2.11 mmol)
were mixed in THF/H2O (3 mL, 2:1) according to the methyl ester
removal procedure to give compound 15 (189 mg, 79 %). HRMS

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(ESI-FT-ICR): m/z calcd for C17H24N2O3Na [M + Na] + : 293.1841;
found: 293.1848.
Peptidomimetic 16: l-Leu (200 mg, 1.52 mmol), benzaldehyde
(155 mL, 1.52 mmol), and cyclohexylisocyanide (190 mL, 1.52 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 3:1) afforded the protected Ugi product 16 (465 mg). Rf =
0.33 (n-hexane/EtOAc, 3:1); a mixture of diastereomers in a 5:1
ratio was observed; 1H NMR (400 MHz, CDCl3): d = 0.820.86 (m,
3 H, CH3), 1.211.32 (m, 4 H), 1.341.41 (m, 3 H), 1.511.56 (m, 2 H),
1.581.68 (m, 2 H), 1.992.05 (m, 4 H), 3.153.20 (m, 1 H), 3.73, 3.74
(2 & s, 3 H, OCH3), 3.753.80 (m, 1 H), 4.20 (br s, 1 H, NH), 5.03 (br s,
1 H, NH), 7.107.39 ppm (m, 5 H, Ar); 13C NMR (100 MHz, CDCl3): d =
22.0 (CH3), 23.3 (CH), 24.8, 25.5, 32.9, 40.0 (CH2), 48.2 (CH), 51.7
(CH3), 55.8, 64.5, 128.4, 129.6, 131.0 (CH), 142.7 (C), 173.0,
174.3 ppm (C=O); HRMS (ESI-FT-ICR): m/z calcd for C21H32N2O3Na
[M + Na] + : 383.2311; found: 383.2317. The protected Ugi product
16 (210 mg, 0.58 mmol) and LiOH (74 mg, 1.75 mmol) were mixed
in THF/H2O (3 mL, 2:1) according to the methyl ester removal procedure to give compound 16 (201 mg, 85 %). HRMS (ESI-FT-ICR): m/
z calcd for C20H30N2O3Na [M + Na] + : 369.2154; found: 369.2150.
Peptidomimetic 17: l-Trp (200 mg, 0.98 mmol), paraformaldehyde
(29 mg, 0.98 mmol), and cyclohexylisocyanide (125 mL, 0.98 mmol)
were mixed in MeOH (3 mL) according to the general procedure
for the Ugi-5C-4CR. Flash column chromatography (n-hexane/
EtOAc, 5:1) afforded the protected Ugi product 17 (276 mg). Rf =
0.65 (n-hexane/EtOAc, 3:1); 1H NMR (400 MHz, CDCl3): d = 1.201.31
(m, 1 H), 1.321.43 (m, 2 H), 1.521.58 (m, 1 H), 1.601.66 (m, 2 H),
1.901.98 (m, 2 H), 2.893.06 (m, 2 H), 3.203.33 (m, 2 H), 3.603.65
(m, 1 H), 3.74 (s, 3 H, OCH3), 3.753.80 (m, 1 H), 5.50 (br s, 1 H, NH),
6.25 (br s, 1 H, NH), 6.50 (s, 1 H, NH), 6.906.99 (m, 1 H, Ar), 7.117.15
(m, 2 H, Ar), 7.257.32 (m, 1 H, Ar), 7.507.54 ppm (m, 1 H, Ar);
13
C NMR (100 MHz, CDCl3): d = 23.4, 26.2, 30.1, 33.0 (CH2), 48.5 (CH),
51.50 (CH3), 52.3 (CH2), 66.2, 110.9 (CH), 111.40 (C), 117.3, 119.5,
119.8, 123.2 (CH), 128.0, 135.0 (C), 172.9, 175.2 ppm (C=O); HRMS
(ESI-FT-ICR): m/z calcd for C20H27N3O3Na [M + Na] + : 380.1950;
found: 380.1954. The protected Ugi product 17 (220 mg,
0.62 mmol) and LiOH (78 mg, 1.86 mmol) were mixed in THF/H2O
(3 mL, 2:1) according to the methyl ester removal procedure to
give compound 17 (213 mg, 79 %). HRMS (ESI-FT-ICR): m/z calcd for
C19H25N3O3Na [M + Na] + : 366.1794; found: 366.1796.

Biological activity
Obtainment of rePepN: Overexpression of rePepN was performed in
E. coli BL-21 (Gold) DE3 strain transformed with the pET15b-ePepN
construct, which was kindly donated by Dr. Anthony Addlagatta
(Indian Institute of Chemical Technology). The bacterium was cultured in TB (12 g L"1 tryptone, 24 g L"1 yeast extract, 0.4 % glycerol,
2.31 g L"1 KH2PO4, and 12.54 g L"1 K2HPO4) supplemented with
100 mg mL"1 ampicillin at the 5 L fermenter scale and 37 8C until
the late exponential phase of growth was reached (optical density,
OD600nm = 0.40.8). At this time, expression of rePepN was induced
by addition of 0.5 mm isopropyl-b-d-1-thiogalactopyranoside (IPTG,
Sigma) and incubation for another 18 h under the above-mentioned conditions. Bacterial cells were collected by centrifugation,
resuspended in 50 mm tris(hydroxymethyl)aminomethane-HCl (TrisHCl) buffer (pH 8.0; 200 mL), and lyzed in a French press. The
rePepN-enriched cell-free protein extract was prepared by centrifugation at 9000 g and 4 8C. It was then exhaustively dialyzed against
the same buffer and the rePepN was purified by ionic exchange
chromatography on a Q-Sepharose Fast Flow column (Sigma).
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rePepN was eluted with 300 mm NaCl and was obtained with
a final volumetric yield > 50 mg of protein per liter of culture and
with > 90 % purity, as verified by densitometric analysis of the gel
derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (8 % acrylamide).[31] The protein concentration was determined by the Bradford method.[32]
Purification of pAPN: The intact membrane-bound form of pAPN
was purified from porcine kidney cortex. All purification steps were
performed at 4 8C. The kidney cortex was dissected from fresh or
recently thawed porcine kidneys, washed with cold distilled water,
weighed, cut into small pieces with scissors, and homogenized in
distilled water (2 mL g"1). The homogenate was treated with 0.1 %
Triton X-100 to solubilize the membrane-bound proteins and centrifuged (10 000 g for 1 h at 4 8C). pAPN was purified from the previously dialyzed supernatant by ionic exchange chromatography
on a Q-Sepharose Fast Flow column (Sigma) as for rePepN.
Screening of inhibitory activity toward rePepN: Aminopeptidase activity was determined by a continuous method, by using 300 mm
chromogenic substrate leucine-p-nitroanilide (Bachem) and monitoring the absorbance at l = 405 nm and 15 s intervals during
3 min. Kinetic assays were conducted with volumes of purified enzymes (rePepN or pAPN) that were linearly related to the initial
rates (v0) in 50 mm Tris-HCl buffer (pH 8.0) at 37 8C on 96-well
plates (200 mL final volume) by using a microplate spectrophotometer (Multiscan FC, Thermo Scientific). Before addition of the substrate, the purified enzymes were preincubated with the inhibitors
solubilized in DMSO (1 % of final reaction volume) for 30 min,
except the controls, which were preincubated with the same
volume of DMSO. Screening was performed at a sole dose of
20 mm, but doseresponse curves were constructed with variable
concentrations of inhibitors. Residual activity was defined as the
ratio between the enzymatic activity in the presence of the inhibitor and the activity of the control (without inhibitor). The results
are expressed as the mean of three parallel assays. Only bestatin
derivatives that caused inhibition of rePepN higher than 25 % at
20 mm, and in a dose-dependent manner, were selected for Ki determinations toward both enzymes. The Ki values were determined
from the IC50 values (calculated by nonlinear regression of the
doseresponse curves) by using Copelands equation for classical
reversible inhibitors: Ki = IC50/(1+[S]/Km), in which Km is the Michaelis constant. For the tight-binding inhibitor bestatin, the Ki value
was determined by using the Morrison quadratic equation as previously described.[33] The Ki value was corrected according to the
equation Ki = Ki/(1+[S]/Km) to take into account the substrate concentration. The selectivity ratio of inhibition was calculated as the
ratio between the Ki value toward pAPN and the Ki value toward
rePepN.

Acknowledgements
This research was partially supported by the International Foundation for Sciences (IFS) grant F/4730-1, the IFS and the Organisation for the Prohibition of Chemical Weapons (OPCW) grant
3276-3, and the Mexican National Council for Science and Technology (CONACyT) grant MexicoCuba 2009. The authors thank
Dr. Anthony Addlagatta (Indian Institute of Chemical Technology)
for the pET15b-ePepN vector, and Fidelia Romero (Institute of Biotechnology, National Autonomous University of Mexico) for assistance in the molecular biology techniques. The authors also
thank Jorge Vald!s and Victoria Lugo (Technological DevelopChemMedChem 0000, 00, 1 10

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ment Unit, Center for Genetic Engineering and Biotechnology,
Havana, Cuba) for technical assistance in the production of
rePepN and the HPLC analysis of the peptidomimetics. Finally,
the authors are indebted to Dagmara D#az (Center for Protein
Studies, Faculty of Biology, University of Havana, Cuba) for technical assistance in the purification of rePepN and pAPN.
Keywords: aminopeptidases combinatorial chemistry
medicinal chemistry multicomponent reactions protease
inhibitors

[1] T. S. Skinner-Adams, C. M. Stack, K. R. Trenholme, C. L. Brown, J. Grembecka, J. Lowther, A. Mucha, M. Drag, P. Kafarski, S. McGowan, J. C.
Whisstock, D. L. Gardiner, J. P. Dalton, Trends Biochem. Sci. 2010, 35, 53.
[2] A. Taylor, Trends Biochem. Sci. 1993, 18, 167.
[3] D. Chandu, A. Kumar, D. Nandi, J. Biol. Chem. 2003, 278, 5548.
[4] N. Tamura, F. Lottspeich, W. Baumeister, T. Tamura, Cell 1998, 95, 637.
[5] D. Chandu, D. Nandi, Microbiology 2003, 149, 3437.
[6] J. Travis, J. Potempa, Biochim. Biophys. Acta Protein Struct. Mol. Enzymol.
2000, 1477, 35.
[7] a) G. Knowles, J. Antimicrob. Chemother. 1993, 32, 172; b) L. Piacenza, D.
Acosta, I. Basmadjian, J. P. Dalton, C. Carmona, Infect. Immun. 1999, 67,
1954; c) M. F. Nankya-Kitaka, G. P. Curley, C. S. Gavigan, A. Bell, J. P.
Dalton, Parasitol. Res. 1998, 84, 552; d) R. E. Morty, J. Morehead, J. Biol.
Chem. 2002, 277, 26057; e) G. Cadavid-Restrepo, T. S. Gastardelo, E.
Faudry, H. deAlmeida, I. M. D. Bastos, R. S. Negreiros, M. M. Lima, T. C.
Assumpo, K. C. Almeida, M. Ragno, C. Ebel, B. M. Ribeiro, C. R. Felix,
J. M. Santana, BMC. Biochem. 2011, 12, 46.
[8] N. M. Hooper, FEBS Lett. 1994, 354, 1.
[9] C. Lazdunski, J. Busuttil, A. Lazdunski, Eur. J. Biochem. 1975, 60, 363.
[10] a) I. Florent, Z. Derhy, M. Allary, M. Monsigny, R. Mayer, J. Schr!vel, Mol.
Biochem. Parasitol. 1998, 97, 149; b) K. Ito, Y. Nakajima, Y. Onohara, M.
Takeo, K. Nakashima, F. Matsubara, T. Ito, T. Yoshimoto, J. Biol. Chem.
2006, 281, 33664; c) A. Addlagatta, L. Gay, B. W. Matthews, Biochemistry
2008, 47, 5303.
[11] A. Addlagatta, L. Gay, B. W. Matthews, Proc. Natl. Acad. Sci. USA 2006,
103, 13339.
[12] H. Umezawa, T. Aoyagi, H. Suda, M. Hamada, T. Takeuchi, J. Antibiot.
1976, 29, 97.
[13] a) H. Suda, T. Aoyagi, T. Takeuchi, H. Umezawa, Arch. Biochem. Biophys.
1976, 177, 196; b) S. K. Burley, P. R. David, W. N. Lipscomb, Proc. Natl.
Acad. Sci. USA 1991, 88, 6916; c) H. Tsuge, H. Ago, M. Aoki, M. Furuno,
M. Noma, M. Miyano, M. Minami, T. Izumi, T. Shimizu, J. Mol. Biol. 1994,
238, 854; d) O. A. Scornik, V. Botbol, Curr. Drug Metab. 2001, 2, 67.
[14] P. M. Jones, M. W. Robinson, J. P. Dalton, A. M. George, PLoS One 2011,
6, e28589.
[15] S. McGowan, C. J. Porter, J. Lowther, C. M. Stack, S. J. Golding, T. S. Skinner-Adams, K. R. Trenholme, F. Teuscher, S. M. Donnelly, J. Grembecka,
A. Mucha, P. Kafarski, R. DeGori, A. M. Bucle, D. L. Gardiner, J. C. Whisstock, J. P. Dalton, Proc. Natl. Acad. Sci. USA 2009, 106, 2537.
[16] M. Allary, J. Schr!vel, I. Florent, Parasitology 2002, 125, 1.
[17] a) J. J. Gordon, B. K. Kelly, G. A. Miller, Nature 1962, 195, 701; b) J. J.
Gordon, T. P. Delvin, A. J. East, W. D. Ollis, I. O. Sutherland, D. E. Wrisly, L.
Ninet, J. Chem. Soc. Perkin Trans. 1 1975, 819.

% 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org
[18] a) A. Mucha, M. Drag, J. P. Dalton, P. Kafarski, Biochimie 2010, 92, 1509;
b) R. J. White, P. S. Margolis, J. Trias, Z. Yuan, Curr. Opin. Pharmacol.
2003, 3, 502.
[19] a) J. Grembecka, A. Mucha, T. Cierpicki, P. Kafarski, J. Med. Chem. 2003,
46, 2641; b) T. S. Skinner-Adams, J. Lowther, F. Teuscher, C. M. Stack, J.
Grembecka, A. Mucha, P. Kafarski, K. R. Trenholme, J. P. Dalton, D. L.
Gardiner, J. Med. Chem. 2007, 50, 6024.
[20] a) M. Flipo, T. Beghyn, J. Charton, V. A.; Leroux, B. P. Deprez, R. F.
Deprez-Poulain, Bioorg. Med. Chem. 2007, 15, 63; b) M. Flipo, T. Beghyn,
V. Leroux, I. Florent, B. P. Deprez, R. F. Deprez-Poulain, J. Med. Chem.
2007, 50, 1322; c) M. Thormann, M. Almstetter, Method of preparation of
bioisosteres of actinonin of interest as metalloproteinase inhibitors, WO
2004099124, 2004; d) S. Patel, L. Saroglou, C. D. Floyd, A. Miller, M.
Whittaker, Tetrahedron Lett. 1998, 39, 8333.
[21] E. Cunningham, M. Drag, P. Kafarski, A. Bell, Antimicrob. Agents Chemother. 2008, 52, 3221.
[22] G. Velmourougane, M. B. Harbut, S. Dalal, S. McGowan, C. A. Oellig, N.
Meinhardt, J. C. Whisstock, M. Klemba, D. C. Greenbaum, J. Med. Chem.
2011, 54, 1655.
[23] For reviews, see: a) P. Slobbe, E. Ruijter, R. V. A. Orru, Med. Chem.
Commun. 2012, 3, 1189; b) A. Dmling, W. Wang, K. Wang, Chem. Rev.
2012, 112, 3083; c) I. Akritopoulou-Zanze, Curr. Opin. Chem. Biol. 2008,
12, 324; d) C. Hulme in Multicomponent Reactions (Eds.: J. Zhu, H. Bienyam!), Wiley-VCH, Weinheim, 2005, pp. 311 341; e) C. Hulme, V. Gore,
Curr. Med. Chem. 2003, 10, 51.
[24] a) J. E. Semple, T. D. Owens, K. Nguyen, O. E. Levy, Org. Lett. 2000, 2,
2769; b) L. Banfi, G. Guanti, R. Riva, Chem. Commun. 2000, 985. For further examples, see: c) T. D. Owens, J. E. Semple, Org. Lett. 2001, 3, 3301;
d) L. Banfi, G. Guanti, R. Riva, A. Basso, E. Calcagno, Tetrahedron Lett.
2002, 43, 4067; e) S. Faure, T. Hjelmgaard, S. P. Roche, D. J. Aitken, Org.
Lett. 2009, 11, 1167.
[25] a) C. C. Musonda, J. Gut, P. J. Rosenthal, V. Yardley, R. C. Carvalho de Sousa, K. Chibale, Bioorg. Med. Chem. 2006, 14, 5605; b) C. C. Musonda, D.
Taylor, J. Lehman, J. Gut, P. J. Rosenthal, K. Chibale, Bioorg. Med. Chem.
2004, 14, 3901.
[26] a) A. Dmling, I. Ugi, Angew. Chem. 2000, 112, 3300; Angew. Chem. Int.
Ed. 2000, 39, 3168; b) S. Marcaccini, T. Torroba, Nat. Protoc. 2007, 2,
632 639; c) I. Ugi, R. Meyr, U. Fetzer, C. Steinbr(cker, Angew. Chem.
1959, 71, 386.
[27] a) J. Chatterjee, C. Gilon, A. Hoffman, H. Kessler, Acc. Chem. Res. 2008,
41, 1331; b) J. A. Patch, K. Kirshenbaum, S. L. Seurynck, R. N. Zuckermann, A. E Barron, Pseudo-Peptides in Drug Discovery (Ed.: P. E. Nielsen),
Wiley-VCH, Weinheim, 2004, pp. 1 31.
[28] a) A. Demharter, W. Hrl, E. Herdtweck, I. Ugi, Angew. Chem. 1996, 108,
185; Angew. Chem. Int. Ed. Engl. 1996, 35, 173; b) I. Ugi, A. Demharter,
W. Hrl, T. Shmid, Tetrahedron 1996, 52, 11657; c) G. Dyker, K. Breitenstein, G. Henkel, Tetrahedron: Asymmetry 2002, 13, 1929; d) W. Wang, A.
Dmling, J. Comb. Chem. 2009, 11, 403.
[29] R. Deprez-Poulain, M. Flipo, C. Piveteau, F. Leroux, S. Dassonneville, I.
Florent, L. Maes, P. Cos, B. Deprez, J. Med. Chem. 2012, 55, 10909.
[30] D. Chappelet-Tordo, C. Lazdunski, M. Murgier, A. Lazdunski, Eur. J. Biochem. 1977, 81, 299.
[31] U. K. Laemmli, Nature 1970, 227, 680.
[32] M. M. Bradford, Anal. Biochem. 1976, 72, 248.
[33] J. G. Bieth, Methods Enzymol. 1995, 248, 59.
Received: April 22, 2014
Published online on && &&, 0000

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Y. M!ndez, K. P!rez-Labrada,
J. Gonz"lez-Bacerio, G. Vald!s,
M. A. de los Ch"vez, J. Osuna, J.-L. Charli,
I. Pascual, D. G. Rivera*
&& &&
Combinatorial Multicomponent Access
to Natural-Products-Inspired
Peptidomimetics: Discovery of
Selective Inhibitors of Microbial
Metallo-aminopeptidases

% 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Muticomponent facilitation: Inspired

by natural nonselective metallo-aminopeptidase inhibitors, a collection of peptidomimetics derived from Ugi reactions
was built and biologically screened to
discover potent and selective inhibitors
of microbial metallo-aminopeptidases
(Ki : inhibition constant).

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