Structure-guided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases

Article abstract of DOI:10.1021/jm501071f

Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1′-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH₂]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1′ residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π-π stacking interaction between a pyridine ring and Tyr372.

Full text of DOI:10.1021/jm501071f

Article
pubs.acs.org/jmc
Structure-Guided, Single-Point Modifications in the Phosphinic
Dipeptide Structure Yield Highly Potent and Selective Inhibitors of
Neutral Aminopeptidases
,
†
‡
‡
§
Stamatia Vassiliou,* Ewelina Węglarz-Tomczak, Łukasz Berlicki, Małgorzata Pawełczak,
∥
∥
,∥
,‡
Bogusław Nocek, Rory Mulligan, Andrzej Joachimiak,* and Artur Mucha*
†Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Zografou, 15701 Athens,
Greece
‡
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze
0-370 Wrocław, Poland
̇
́
Wyspianskiego 27,
5
§
Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
∥
Midwest Center for Structural Genomics, and Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700
South Cass Avenue, Argonne, Illinois 60439, United States
*
S Supporting Information
ABSTRACT: Seven crystal structures of alanyl amino-
peptidase from Neisseria meningitides (the etiological agent of
meningitis, NmAPN) complexed with organophosphorus
compounds were resolved to determine the optimal
inhibitor−enzyme interactions. The enantiomeric phosphonic
acid analogs of Leu and hPhe, which correspond to the P1
amino acid residues of well-processed substrates, were used to
assess the impact of the absolute configuration and the
stereospecific hydrogen bond network formed between the
aminophosphonate polar head and the active site residues on
the binding affinity. For the hPhe analog, an imperfect
stereochemical complementarity could be overcome by
incorporating an appropriate P1 side chain. The constitution of P1′-extended structures was rationally designed and the lead,
phosphinic dipeptide hPhePψ[CH ]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based
2
fragment to either the P1 or P1′ residue required new synthetic pathways. The compounds in the refined structure were low
nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase
(
LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable
selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to
confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were
identified, including a π−π stacking interaction between a pyridine ring and Tyr372.
INTRODUCTION
which cleave internal peptide bonds, by peptide elongating at
the N- and C-termini. A fundamental P1−P1′ pseudodipeptide
building block is typically incorporated into a sequence that
corresponds to the privileged structures of well-studied
substrates. Alternatively, the P2−Pn and P2′−Pn′ amino acid
■
Phosphinic peptide analogues are potent, reversible, and
competitive inhibitors of metalloproteases, the enzymes that
catalyze the cleavage of an amide bond in the biologically active
1
−3
peptide through a metal-ion-assisted process.
The tetrahe-
4
,5
sequence is refined through combinatorial techniques. For
exopeptidases, any modifications are limited to one side of the
central ligand core. Therefore, the basic P1−P1′ scaffold must
be more carefully optimized, which creates a challenging
synthetic problem. Dedicated multistep preparations are
required when residues designed to complement the most
discriminating S1 and S1′ subsites are more complex than
drally shaped phosphinate group mimics the structural and
electronic features of the gem-diolate transition state
intermediate from the hydrolysis process. After binding, the
group is coordinated to the central metal ion, blocking its
catalytic activity (Figure 1).
The selectivity issues with the phosphinic peptide inhibitors
are primarily attributed to the structural features of the Pn and
Pn′ residues, which must access the corresponding (Sn and Sn′)
enzyme binding pockets. These structural attributes can be
optimized further for the inhibition of metalloendopeptidases,
Received: July 17, 2014
©
XXXX American Chemical Society
A
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Figure 1. Schematic representation showing the binding mode of phosphinic peptide inhibitors and zinc metalloproteases. The P1 and P1′ side
chains specific to the S1 and S1′ binding pocket are colored in pink (left panel). Shown are the structure of hPhePψ[CH ]Phe 1, which is a
2
recognized inhibitor of aminopeptidases and the lead compound for this study, and the structure of hPhePψ[CH ]Tyr 2, which is a unique P1′-
2
2
5,26
modified compound with improved activity (right panel).
simple alkyl or aryl substituents and contain a functional
group. Parallel approaches that provide access to diverse
of the latter to APNs (of the porcine kidney and N.
meningitidis) increased 6- to 8-fold compared to the lead.
6
25,26
phosphinic dipeptides are valuable but still limited by certain
starting materials and reaction types. This scenario is
adequately illustrated by the successful parallel addition of C-,
The preference for the modified side chain residue was
explained using a mammalian homology model and involved
formation of one specific hydrogen bond to the γ-carboxylate of
7
−9
25
N-, and S-nucleophiles to P1′ dehydroalanine,
the synthesis
Glu413.
of P1′ isoxazole and isoxazoline derivatives through a 1,3-
Consequently, we have planned to develop a series of single-
dipolar cycloaddition starting from the appropriate unsaturated
point modified hPhePψ[CH ]Phe 1 derivatives, including both
2
1
0,11
systems,
and the alkylation/arylation of an amino-modified
P1 and P1′ unnatural analogs. We have hypothesized that
improving the activity and selectivity of the dipeptidic
derivatives may be rationally controlled with single modifica-
tions of the side chain substituent structure. The huge
advantage of using this approach is that neither an overall
sequence expansion nor the production of large combinatorial
libraries is required.
Here, we show studies on phosphinic compounds of the
designed structures, which were synthesized using dedicated
multistep approaches, and their structure−activity relationships
against four aminopeptidases: N. meningitides APN, human
1
2
P1′ side chain. Usually, a single heteroatom-containing
structural modification requires a unique synthetic approach.
The function and substrate specificity of zinc-dependent
aminopeptidases make them excellent model enzymes for
validating the effectiveness of P1/P1′ modifications in
phosphinic dipeptides. The most recognized representatives
of these hydrolases are microsomal alanyl aminopeptidase (EC
3.4.11.2, APN/CD13) and cytosolic leucine aminopeptidase
(
EC 3.4.11.1, LAP), ubiquitous enzymes among kingdoms and
1
3−18
species.
They are the members of the metalloprotease
(
Homo sapiens) APN, porcine (Sous scrofa) APN, and porcine
superfamily (M) and are formally referred to as M1 and M17
1
3,14
LAP. We have also investigated the binding mode of seven
unique ligands to NmAPN by using the X-ray crystallography,
which illustrated optimization of the inhibitor structure and
gave insights into transition state analogue interactions with a
monozinc aminopeptidase.
aminopeptidases, respectively.
Both peptidases preferen-
tially release N-terminal hydrophobic amino acids from
peptides or proteins; the substrate specificity of APN also
includes basic residues. In mammals, this activity is involved in
physiological metabolism of regulatory and bioactive peptides,
15−18
antigen presentation, and angiogenesis control (APN).
RESULTS AND DISCUSSION
The medical potential of APN and LAP is connected with their
functions in tumorigenesis and metastasis and with patho-
■
P1 Amino Acid Analogs. The phosphonic analogues of
amino acids are among the simplest transition state inhibitors
of aminopeptidases. The acidic group holds the shape and
charge necessary to mimic the high-energy gem-diolate
structure, while the constituents of the P1 side chain typically
correspond to the preferentially bound N-terminal residues of
the peptidic substrates. Accordingly, the substrate specificity of
these enzymes, which is expressed using the lowest values of
the Michaelis constant, can be translated into the structure of
phosphonic ligands after designing an appropriate P1 frag-
1
6,19−22
genesis of hypertension (APN).
M1 and M17 amino-
peptidases found in human pathogens, e.g., Plasmodium
falciparum, are responsible for digestive proteolysis and
nutrition delivery. Since hemoglobin degradation in the host
erythrocytes is directly responsible for the clinical symptoms of
malaria, the proteases have been identified as molecular targets
23
to treat this prevalent disease. Importantly, a comprehensive
structure−activity relationship for the inhibition of APN and
LAP from different sources with phosphorus-containing
3
,24
27
inhibitors is currently available.
However, only one case
ment. For alanyl aminopeptidases, the hydrophobic and basic
13
illustrates the significance of a rational, heteroatom-involved
modification in the phosphinic dipeptides structure. When a
canonical inhibitor of aminopeptidases, specifically hPhePψ-
substituents of Leu, Phe, Arg, and Lys are clearly favorable.
Nevertheless, it has been found that fluorogenic substrates
comprising extended substituents of noncoded amino acids are
bound tighter than the derivatives of naturally occurring
[
CH ]Phe 1, was substituted with a p-hydroxyl group in the P1′
2
2
7,28
portion, generating hPhePψ[CH ]Tyr 2 (Figure 1), the affinity
counerparts.
The α-aminoalkylphosphonic acids based on
2
B
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Table 1. Inhibitory Activity of Enantiomeric and Racemic Phosphorus-Containing Analogs of Amino Acids: Phosphonic Acid
Analog of Leucine (3), Phosphonic (4) and H-Phosphinic (5) Acid Analogs of Homophenylalanine toward N. meningitides APN,
Human (H. sapiens) APN, and Porcine (S. scrofa) APN and Porcine LAP
these bulkier residues exhibit inhibition constants within the
low micromolar or submicromolar range, making them an
attractive scaffold for further elongation at the P1′ posi-
more active toward NmAPN than LeuP, and its potency does
not depend on the stereochemical arrangement. K is 0.97 μM
i
for the L enantiomer, and the same value, within the
experimental error, is displayed for the racemic mixture 4.
This observation is valuable because the optically pure forms of
hPheP are not readily available. The hPheP enantiomers cannot
be obtained/separated through typical 1-phenylethylamine-
3
,24,26,29,30
tion.
To analyze the binding of the P1 phosphorus ligands to the
APNs, we selected two prototypical aminophosphonic acids:
analogues of Leu (3) and hPhe (4) that exhibited promising
kinetic parameters (Table 1). These parameters were correlated
with the stereochemistry of the ligand and the formation of a
specific hydrogen bond network between the polar head of the
compounds and the NmAPN active site, as revealed by the
crystal structures. The activity of LeuP toward meningococcal
and human APNs depended strongly on the absolute
configuration of the α-carbon atom. The natural L analog (R-
33
mediated approaches: convenient stereoselective syntheses or
recrystallization of the diastereomeric salt of their phosphinic
34
precursors (Cbz-5). The only reported enantio-enriching
method for R-4 and S-4 involves a careful stereoselective
3
5
separation of Cbz-5 using chiral chromatography, followed by
34
N-deprotection and P-oxidation. Apparently, the flexible 2-
phenylethyl side chain of hPhe is particularly well accom-
modated inside the S1 cavity, making the absolute configuration
less critical. A similar situation is revealed for three peptidases
tested in this work (NmAPN, SsAPN, and SsLAP): the activity
of the enantiomeric and racemic hPhe is practically equal,
3
) was a low micromolar inhibitor (K = 1.92 μM for NmAPN
i
and K = 2.62 μM for HsAPN), approximately 2 orders of
i
magnitude more potent than the D enantiomer (S-3). An even
more pronounced difference was found previously for
3
1,32
mammalian leucine aminopeptidase.
Unpredictably, the
remaining at a good level (K < 5 μM). For the human APN,
i
absolute configuration of the ligand was not critical for SsAPN.
the inhibition reaches a nanomolar level and stereodiscrimina-
However, the binding to that enzyme was clearly weaker (K =
tion of hPheP becomes slightly more pronounced (K = 230
i
i
3
2
53 μM) possibly because of a shortage of stereospecific
nM for the enantiomeric inhibitor and K = 790 nM for the
i
contacts. The phosphonic acid analog of hPhe (R-4) is slightly
racemic one). Interestingly, H-phosphinic acid (5) reproduces
C
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the potency of its oxidized counterpart, which contains a three-
oxygen-atom phosphorus group. This observation reveals that
the presence of one oxygen atom in the inhibitors was not
critical for binding; therefore, this position could be used to
elongate the P1′ fragment further.
S-3, the heteroatoms are 3.15 Å apart. This structural disparity
may explain the weaker interaction between S-3 and the
enzyme and its inhibitory constant, which is 2 orders of
magnitude higher. The inclusion of the hydrophobic isobutyl
fragment in the cavity is facilitated by the Met256 residue,
which adopts a conformation suitable for sticking to the
inhibitor portion through lipophilic interactions.
The binding mode of 1-aminoalkylphosphonic acids LeuP
(3) and hPheP (4) to the active site of NmAPN was revealed
through crystallographic studies and molecular modeling. The
crystals containing the recombinant selenomethionine-labeled
protein soaked with an inhibitor do not significantly change the
overall arrangement of the binding site architecture compared
The strength of binding for inhibitor R-3 is 1 order of
magnitude higher for LAP than for NmAPNs. Indeed, the mode
of interaction with (R)-LeuP, which was revealed using the
crystal structure of the monozinc NmAPN complex, is quite
36
to the native protein and the corresponding Escherichia coli
different from that observed for dinuclear aminopepti-
3
7−41
42−44
enzyme.
The inhibitors dock to the S1 cleft, mimicking the
dases.
When the bovine lens LAP is cocrystallized with
42
P1 fragment of a substrate in its transition state (Figure 2). The
R-3, the phosphonic acid is a tridentate ligand for zinc ions.
One zinc ion is bound by two phosphonic oxygen atoms, while
the other is bound by one of the oxygens (the metal-bridging O
atom) and an amino group. Therefore, one metal site binds the
N-terminal NH group on the inhibitors and substrates. In
2
APN, this role is entirely fulfilled by the glutamate-rich region
(
Glu117, Glu260, and Glu316) of the S1 pocket. When the
amount of metal−inhibitor contact between aminophosphonic
acids and a two-metal system (LAP) is increased relative to a
one-metal site (APN), the development of selective APN
inhibitors versus LAP seems difficult. This challenge can be
addressed by optimizing the structure of the side chain to
explore the specificity of the binding pocket.
Although phosphonic acid analogs of homophenylalanine
have arisen as important inhibitors of aminopepti-
3
,24,26,29,30
dases,
the structural and stereochemical context and
the nature of favorable binding to these proteins have not been
disclosed. The crystal structure of the R-4-NmAPN complex
(
2
Figure 2C) and the molecular model of S-4-NmAPN (Figure
D) imply that the binding mode between the amino-
phosphonate head of the enantiomeric phosphonic acid analogs
of homophenylalanine and the protein is identical to that with
LeuP. However, the aromatic portion binds to the S1 cavity
slightly differently, depending on the configuration of the α
carbon atom. The crystal structure of the R-4 complex reveals
the free arrangement of 2-phenylethyl without any of the
characteristic hydrophobic contacts between the aromatic ring
and the surrounding residues (Figure 2C). In contrast, the
docking model of the S enantiomer implies that a defined
pointing direction, specifically toward the position needed for
π−π stacking with the aromatic ring of Tyr372 (Figure 2D), is
necessary. This favorable interaction may decrease the
difference between the free energies of binding that arise
from the different hydrogen bond networks formed by the
enantiomers. The sum of these contradictory effects makes the
measured inhibition constants equal.
Figure 2. Crystal structures of the complexes containing enantiomeric
phosphonic acid analogs of leucine and homophenylalanine with
NmAPN: (R)-LeuP-NmAPN (A), (S)-LeuP-NmAPN (B), and (R)-
hPheP-NmAPN (C), and a modeled complex of (S)-hPheP-NmAPN
(
D). The hydrogen bonds and ligand−metal interactions are marked
as green lines.
two negatively charged oxygen atoms of the phosphonate
moiety bidentately coordinate with the central zinc cation. The
O1−Zn and O2−Zn distances are almost equal for both
enantiomers of leucine analog 3, specifically 2.10 and 2.04 Å for
the R enantiomer and 2.10 and 2.06 Å for the S enantiomer
Phosphinic Dipeptides Design. The novel inhibitors
were rationally designed based on the structures of NmAPN,
porcine kidney APN, and human APN complexed with a
canonical phosphinate ligand that showed a high affinity for M1
(
Figure 2A and Figure 2B, respectively). Each of these oxygen
atoms forms an additional hydrogen bond: one with Tyr377
2.42 and 2.48 Å for R-3 and S-3, respectively) and the other
2
5,26,45,46
and M17 aminopeptidases.
Diastereomerically pure
47
(
hPhePψ[CH ]Phe (1) of an LL (R,S) stereochemistry, which
2
with Glu294 (2.97 and 2.80 Å for R-3 and S-3, respectively).
The third oxygen atom is exposed to the solvent. The amino
moiety forms three hydrogen bonds with the carboxylates of
Glu117, Glu260, and Glu316. The N−H···O distances for
Glu117 and Glu260 are similar for both enantiomers (2.73 and
corresponds to the natural amino acid configuration, was
cocrystallized with the bacterial protein. An analogous complex
containing the porcine and human orthologs were modeled
48
based on the crystal structures of bestatin-bound SsAPN and
49
amastatin-bound HsAPN. The overall mode of the inhibitor
binding was similar between the complexes (Figure 3 and
Supporting Information Figure S1A and Figure S1B,
respectively) and comparable to that of the previously
2
.89 Å for R-3 and 2.73 and 2.86 Å for S-3). The corresponding
distance to Glu316 differs significantly between the enan-
tiomers. For natural analog R-3, the length is 2.92 Å, while for
D
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Figure 3. Crystal structure of the complex containing R,S-1 with NmAPN showing the potential interactions between a modified or substituted P1
and P1′ phenyl rings and the active site residues (those involved are highlighted in bold). Intermolecular hydrogen bonds and ligand−metal
interactions are marked in green. The potential contact sites are marked as blue-gray, red-dark gray, or gray spheres, indicating hydrogen bond
donors, acceptors, or lipophilic fragments, respectively.
a
Scheme 1. Synthesis of the P1-Modified Phosphinic Pseudodipeptides with the 3-Pyridyl and 4-Pirydyl-3-propanal Substrates
a
Reagents and conditions: (a) EDC·HCl, EtSH, CH Cl , 2 h; (b) Et Si, 10% Pd/C, CH Cl , 1 h; (c) malonic acid, pyridine, piperidine, 100 °C, 30
2
2
3
2
2
min; (d) MeOH, conc H SO , reflux, 3 h; (e) H , 10% Pd/C, MeOH, 24 h; (f) DIBAL-H, −78 °C, Et O, 1 h; (g) AcOH/AcCl 5/1, 48 h; (h) 6 M
2
4
2
2
+
HCl, reflux, 12 h; (i) Dowex AG 50W-X4 (H ).
determined complex structure of tripeptide LLL (R,S,S)-
hydrophobic side chains within the binding pockets would
require the appropriate adjustments.
40
AlaPψ[CH ]PhePhe and E. coli APN. This binding mode
2
involved the bidentate coordination of negatively charged
phosphinate oxygens to the zinc metal ion, P−O···H bonds to
the hydroxylate of tyrosine (Tyr477 for HsAPN and Tyr472 for
SsAPN) and the carboxylate of glutamic acid (Glu389 for
HsAPN and Glu350 for SsAPN), and a hydrogen network
formed between the amino group and the neighboring
carboxylates/carboxyamide of the glutamic acids/glutamine
An analysis of the potential interactions, which was
performed using the Pharmacophore module of the Discovery
Studio software package (Accelrys, Inc., San Diego, CA, USA),
revealed several options for modifying the core structure of
compound 1, particularly the phenyl fragments located in both
the S1 and S1′ cavities of the meningococcal and mammalian
(human and porcine) APNs (Figure 3 and Supporting
Information Figure S1A and Figure S1B, respectively). The
(
Glu355, Glu411, and Gln213 for HsAPN and Glu350, Glu406,
and Gln208 for SsAPN). These interactions corresponded to
those defined for phosphonic acids and NmAPN (Figure 2).
Additionally, the C-terminal carboxylate of the inhibitor fell
within the hydrogen bond distance to the N−H of Gly257 in
the structures of NmAPN (Gly352 and Arg381 for HsAPN, and
Ala348 for SsAPN). These fundamental contacts between the
inhibitor backbone and the enzyme residues should have been
reproduced in the modified target structures, while the fit of the
N-terminal phenyl ring of (R,S)-hPhePψ[CH ]Phe is located
2
close to several hydrogen bond-accepting carbonyl groups
(Gln115, Glu117, Gln818, and Ala258 for NmAPN, Gln211,
Asn350, and Ala351 for HsAPN, and Leu185, Gln208, and
Ala346 for SsAPN). Most of the proposed N−H structural
fragments (to be introduced), which complement the carbon-
yls, either overlap with the phenyl ring (in particular for
NmAPN) or hold a position in proximity to the ring. Therefore,
E
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Scheme 2. Synthesis of P1′-Modified Phosphinic Pseudodipeptides with the Detailed Preparation of α-
a
(
Aminomethylbenzyl)acrylate Substrates
a
Reagents and conditions: (a) BaCO , H O, 4 h, reflux; (b) H , 10% Pd/C, MeOH, 2 days; (c) 46% HBr/H O, 3.5 h, reflux; (d) (Boc) O,
3
2
2
2
2
NaHCO , H O, dioxane, 18 h; (e) NaH, CH (CO Et) , DMF, 20 min, then 49 or 50, 60 °C, 19 h; (f) KOH, EtOH, 18 h, then HCl 0.5 N; (g)
3
2
2
2
2
+
Et NH, HCHO, CH Cl , 18 h, (h) BSA, CH Cl , 48 h; (i) 6 M HCl, reflux, 18 h; (j) Dowex AG 50W-X4 (H ).
2
2
2
2
2
a pyridyl ring seemed to be a consensus structure comprising
the favorable changes of the phenyl on the lead. In addition,
other hydrogen bond donors (Asn255, Met256, and Asn369 for
NmAPN, Ser895 for HsAPN, and Gln206, Ser209, and Ser464
for SsAPN) are positioned near the distal portion of the
Therefore, acid 20 was converted to the corresponding
thioester (21) through an EDC-mediated esterification with
thioethanol. The reduction using triethylsilane and Pd/C
proceeded smoothly, providing aldehyde 18. The preparation
of another isomer began by elongating 4-pyridinecarboxalde-
hyde 22 in a Knoevenagel condensation/decarboxylation.
Acrylic acid 23 was esterified to 24 quantitatively in refluxing
methanol. Afterward, the double bond was reduced using a
standard hydrogenation over Pd/C to give 25. The reduction of
aldehyde 19 proceeded smoothly when using DIBAL-H. The
product was not stable; therefore, it was purified and used
immediately in the amidoalkylation reaction. Detailed proce-
dures are included in the Supporting Information.
The aldehydes were used in the subsequent amidoalkylation
reaction with benzyl carbamate and H-phosphinic acid 26 in
acetic acid/acetyl chloride for 48 h, generating the desired
phosphinic dipeptides 27−33. Free phosphinic compounds 6−
12 were isolated in their pure forms after hydrolysis and
reversed phase silica gel column chromatography.
aromatic fragment. The OH and CH OH groups used to
2
replace the phenyl meta or para hydrogen atom should have
potential contacts with these groups. When modifying the C-
terminal phenyl ring, the proximity of carboxylates (Glu378 and
Asp323 for NmAPN, Glu418 for HsAPN, and Glu413 and
Asp434 for SsAPN) seemed the most beneficial. A positively
charged methylamino substituent introduced in position 3 or 4
of the P1′ aromatic ring of an inhibitor should form a salt
bridge with one of these acidic groups. Finally, both the S1 and
S1′ cavities are spacious, indicating that a phenyl substituent
extension may increase the surface area available for lipophilic
interactions.
Synthesis. The α,α′-phosphinic pseudodipeptides were
obtained through alternative synthetic approaches that involved
formation of two C−P bonds starting from hypophosphorus
The N → C approach is a typical P1′ divergent route
(Scheme 2) involving a phospha-Michael addition of a
protected H-phosphinic analog of an amino acid (which is
easily obtained from a Kabachnik−Fields reaction with H PO )
6
acid (H PO ). To prepare the P1-diversified set of phosphinic
3
2
pseudodipeptides (6−12), the C → N strategy seemed more
convenient (Scheme 1). This route usually involves a three-
component amidoalkylation reaction between a carbamate, an
3
2
to the appropriate acrylates (α-substituted α,β-unsaturated
esters). Accordingly, to synthesize compounds 34−36, we
utilized the P−H analog of homophenylalanine 37 as the
phosphorus-containing component. Obtaining the acrylates
from commercially available materials was a separate and
demanding challenge, as illustrated by the multistep preparation
of α-(aminomethylbenzyl)acrylate precursors of the P1′
fragments (39 and 40, Scheme 2, insert). Starting from
bromomethylbenzonitriles 41 and 42, the corresponding amino
alcohols 45 and 46 were obtained after substituting the
50
aldehyde and an H-phosphinic acid, such as 26 (which is
obtained through a phospha-Michael addition of α-benzylacry-
51
late to H PO , as described elsewhere ). In our case, the
3
2
synthetic challenge involved preparing aldehydes 13−19, which
are key substrates for the condensation. To achieve this goal,
two types of ester reductions were used: a Fukuyama reduction
with a thioester using a silyl hydride in the presence of a
52
palladium catalyst and a DIBAL-H reduction of a carboxylic
5
3
ester. The utility of both versions can be exemplified by
obtaining 3-pyridyl (18) and 4-pirydyl-3-propanal (19) from
commercially available starting materials (Scheme 1, insert).
bromides using aqueous BaCO , followed by Pd-mediated
3
catalytic hydrogenation of intermediate nitriles 43 and 44. A
F
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Table 2. Inhibitory Activity of the Newly Designed and Obtained Phosphinic Dipeptide Analogs toward N. meningitides APN
and Mammalian Aminopeptidases: Human APN, Porcine APN, and Porcine LAP, Compared to Lead Compounds
a
hPhePψ[CH ]Phe (1) and hPhePψ[CH ]Tyr (2)
2
2
G
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Article
Table 2. continued
a
The most significant inhibition is highlighted in bold. Measurements were made after 30−60 min of incubation and calculated using Morrison’s
equation for tight binding inhibitors (for details see the Supporting Information).
significant improvement in the reaction yield was evident
single-point options, the biological activity is supplemented
with the crystal structures, which show the binding mode of
theses ligands to the NmAPN active site.
12
compared to the previously described LiAlH reduction. The
4
bromide was reintroduced by heating the amino alcohols with
4
6% aqueous HBr. The amino group was Boc-protected
All of the studied compounds exhibited strong, frequently
slow-binding (for NmAPN and HsAPN exclusively), compet-
without separating 47 and 48. Bromides 49 and 50 were used
for the C-alkylation of diethyl malonate in the presence of
NaH, which proceeded in very good yield. Finally, monoacids
itive inhibition of the aminopeptidases (K values given in Table
i
2; for further details see Supporting Information). Nearly all of
53 and 54, which were obtained from substituted malonates 51
the K constants were in a nanomolar range. Tested against
i
and 52 after monosaponification, were transformed to the
desired acrylates (39 and 40) through a consecutive
decarboxylation/Mannich reaction using diethylamine and
human APN, several phosphinic pseudodipeptides (10−12, 35,
36, 58, and 59) showed an exceptional inhibition level (0.2−2.0
nM), falling into a picomolar range in two particular cases. The
kinetic studies and the crystal structures of selected ligand−
NmAPN complexes show that all of the phosphinates interact
with the enzymes in a manner analogous to the lead compound.
Consequently, the backbone of the novel pseudopeptide
reproduces the contacts shown in the skeleton of hPhePψ-
54
formaldehyde at room temperature.
Acrylates 38−40 were subsequently added to 37, which was
activated to more nucleophilic tervalent ester with N,O-
bistrimethylsilylacetamide (BSA) as the silylating agent. The
BSA-mediated phospha-Michael reaction proceeded under mild
conditions and was fully compatible with the relatively sensitive
Boc group, generating N-protected phosphinic dipeptide
analogs 55−57 in good yield after purification. Global
deprotection was achieved easily by heating the phosphinates
with 6 M HCl, followed by ion exchange chromatography
purification to obtain compounds 34−36. For further details,
see the Supporting Information.
[CH ]Phe 1 (Figure 3 and Figure S1, and Figure 4A):
2
bidentate Zn complexation with the phosphinate group and a
set of hydrogen bonds between the ligand functional groups
and the enzyme. Thus, the observed difference in the inhibitory
activity should likely result from the mode and free energy of
binding of the modified P1 and P1′ side chain residues.
Increasing the size of the P1 and P1′ hydrophobic fragments
of the inhibitors by substituting a 3,5-dimethylphenyl group
was not beneficial. The affinity of compounds 6 and 34 is
particularly diminished for LAP. For APNs, the inhibition
constant values do not differ much from the data obtained for
nonsubstituted phosphinate 1. Therefore, more spatial and less
sterically restrictive S1 and S1′ cavities are present in the latter
enzymes compared to LAP. The strongly electron-withdrawing
p-nitro substitution at P1 exerts an ambiguous influence on the
affinity of the ligand 7. No change in potency was observed
with NmAPN and HsAPN. Pig APN accommodates well
Structure−Activity Study. The newly synthesized phos-
phinic dipeptide analogs were tested for inhibition against the
four aminopeptidases (one bacterial and three mammalian) and
compared to the reference compounds: hPhePψ[CH ]Phe (1)
2
and hPhePψ[CH ]Tyr (2). Pseudodipeptides 6−12 contain a
2
modification of the P1 portion, and 34−36 are P1′-modified.
Finally, two refined structures (58 and 59) (synthesized
according to the general procedure depicted in Scheme 1 and
described in detail in the Supporting Information) combine
both privileged optimization variants. For the best P1 and P1′
H
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Figure 4. Crystal structures of NmAPN complexes with phosphinic dipeptides. Hydrogen bonds and ligand−metal interactions are marked in green.
All of the interactions with the pseudopeptidic backbone are marked for (R,S)-hPhePψ[CH ]Phe (A). For P1 and P1′-modified compounds only the
2
zinc coordination and specific contacts are shown: NmAPN-11 B (B), NmAPN-35 (C), and NmAPN-36 (D).
enlarged hydrophobic fragments and altered aromatic systems.
aromatic ring was suggested in the model of the (S)-hPheP-
NmAPN complex (Figure 3D). Therefore, Tyr372 emerged as
a primary target for optimizing the inhibitor structure.
This behavior manifests as a 4-fold improvement in the K value
i
to 69 nM for 7 (compared to 276 nM measured for 1).
However, the opposite effect was measured for LAP (3-fold
drop).
Impressive data were obtained when aminomethylene was
substituted at the P1′ position (compounds 35 and 36). Para
derivative 36 exhibited an inhibition constant below 10 nM for
all APN orthologs, specifically 9.0 nM for NmAPN, 0.69 nM for
the human enzyme, and 4.0 nM for the porcine one. Meta-
substituted compound 35 was only slightly less active (10−20
nM for NmAPN and SsAPN). To illustrate the structural
context, complexes involving NmAPN and both of the active
inhibitors were resolved (Figure 4C and Figure 4D). The
predicted significance of the salt bridge between the added
amino group of the inhibitor and the carboxylate of Asp323 was
confirmed for compound 36. The distance between the
heteroatoms is 2.65 Å, revealing a tight hydrogen bonding
interaction (Figure 4D). For phosphinate 35, the P1′
aminomethylbenzyl residue is partially disordered. Two equally
populated conformations are visible (Figure 4C). The predicted
conformation shows that the amino group was directed toward
Asp323, while the symmetrically rotated molecule displays the
same group exposed to the solvent. Both 35 and 36 are
reasonably selective versus LAP by at least a factor of 15, which
is difficult to achieve for organophosphorus inhibitors. LAP is a
prototypical two-zinc aminopeptidase, and the interactions of
the aminophosphonate fragment with both metals are typically
Substituting the P1 phenyl ring with meta and para hydroxyl
groups (compounds 8 and 9) decreased the inhibitory activity
compared to 1. Therefore, electron-donating functionalities are
not well tolerated; the expected hydrogen bonds were not
formed with Asn255, Met256, and Asn369 of NmAPN (or
corresponding residues of the human and porcine orthologs).
However, when the same functional group (OH) is not
conjugated with the aromatic system (instead, it is separated
with a methylene linker (CH OH)), a highly active inhibitor
2
(
10) of porcine enzymes was obtained. The K improved 7-fold
i
compared to 1 for SsAPN and 3-fold for LAP. Therefore,
compound 10 is the most active organophosphorus inhibitor of
leucine aminopeptidase reported so far.
In accordance with the molecular models, replacing the
phenyl ring with a pyridyl moiety (compounds 11 and 12)
appeared the most advantageous when optimizing the P1
position. The affinity of these phosphinates toward all four
aminopeptidases is enhanced, particularly toward porcine APN
(
K = 19 nM for 3-pyridyl and K = 23 nM for 4-pirydyl). The
i
i
reason for this effectiveness was unexpected. As revealed by the
crystal structure of NmAPN-11 (Figure 4B), the pyridyl group
does not form any of the predicted hydrogen bonds with the
residues lining the proximal portion of the S1 pocket (Gln115,
Glu117, Gln818, Ala258); instead, it is favorably stacked against
the phenol group of Tyr372. The π−π stacking, which is
mediated by the reversed polarization of the interacting rings,
produced high inhibition levels, although the optimal binding of
the α-amino group was visibly distorted. The distance covered
by one of the hydrogen bonds formed by NH3+ is extended to
much stronger for these types of molecules (N-C-PO -; see
2
discussion of (R)-LeuP binding).
Finally, two combinations (compounds 58 and 59)
containing privileged substituents at P1 (pyridylethyl) and
P1′ (aminomethylbenzyl) were verified and high anti-amino-
peptidase activity was revealed. In particular, the top potency
was achieved with the human APN (K = 0.21 nM for 58 and K
i
i
= 1.1 nM for 59) and the porcine kidney APN (K = 1.5 nM for
i
3.2 Å. A similar effect of compensation of the amino group
58 and K = 3.9 nM for 59). To our knowledge compound 58 is
i
distortion by favorable π−π stacking provided by the P1
the most active inhibitor of HsAPN described to date. For
I
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Journal of Medicinal Chemistry
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SsAPN, additional extended phosphinic analogs, which were at
ACKNOWLEDGMENTS
■
least tripeptides, reportedly exhibited similar levels of
The work was supported by a grant from the Polish National
Science Centre (Grant 2013/09/B/ST5/00090). The Discov-
ery Studio package was used under a Polish country-wide
license. The use of software resources (Discovery Studio
program package) of the Wrocław Centre for Networking and
Supercomputing is also kindly acknowledged. The Structural
Biology Center beamlines at APS are supported by U.S.
Department of Energy Office of Biological and Environmental
Research program under Contract DE-AC02-06CH11357. The
structural studies were performed at the Midwest Center for
Structural Genomics supported by the National Institutes of
Health Grant GM094585.
5
5,56
inhibition.
Doubled modifications were less favorable for
NmAPN than for SsAPN, possibly indicating that the adverse
constraints were caused by two strong interactions.
CONCLUSIONS
■
The phosphinic moiety is commonly believed to imitate the
geometry, electron distribution, and metal complexation ability
of the gem-diolate transition state formed during peptide bond
hydrolysis. Accordingly, phosphinic-based pseudopeptide ana-
logues are effective inhibitors of zinc-containing biomedically
significant proteases, including representatives of the M1 alanyl
and M17 leucine families of aminopeptidases. In this work, we
showed that a significant improvement in the activity and
selectivity of the dipeptidic phosphinate inhibitors could be
achieved after optimizing the P1 and P1′ substituent rationally;
however, this process required the development of synthetically
challenging, multistep procedures. The derivatives of the
refined structure were low nanomolar inhibitors of NmAPN,
human APN, and aminopeptidases APN and LAP from porcine
kidney, which are the most potent compounds of this type
reported to date. Merging the favorable P1 and P1′
modifications was particularly effective for the mammalian
ABBREVIATIONS USED
■
AaP, phosphonic analog of an amino acid; AaPH, H-phosphinic
analog of an amino acid; Aa Pψ[CH ]Aa , a phosphinic
dipeptide analogue; DIBAL-H, diisobutylaluminum hydride;
Boc, tert-butyloxycarbonyl; BSA, N,O-bis(trimethylsilyl)-
acetamide, Cbz: benzyloxycarbonyl, EDC, 1-ethyl-3-(3′-
dimethylaminopropyl)carbodiimide; hPhe, homophenylalanine
1 2 2
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Diastereoselective Solution and Multipin-Based Combinatorial Array
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