A structural insight into the P1 S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases

Article abstract of DOI:10.1016/j.ejmech.2016.04.018

N′-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., K_i Combining double low line 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region.

Full text of DOI:10.1016/j.ejmech.2016.04.018

European Journal of Medicinal Chemistry 117 (2016) 187e196
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
A structural insight into the P1eS1 binding mode of
diaminoethylphosphonic and phosphinic acids, selective inhibitors of
alanine aminopeptidases
Ewelina We˛glarz-Tomczak a, Łukasz Berlicki a, Małgorzata Pawełczak b, Bogusław Nocek c,
,
*
Andrzej Joachimiak c, Artur Mucha a
a
_
ꢀ
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
b Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
c Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue,
Argonne, Illinois 60439, USA
a r t i c l e i n f o
a b s t r a c t
N0-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were
explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine
aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were
obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic
pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metal-
loaminopeptidases of different sources (bacterial, human and porcine), revealed several potent com-
pounds (e.g., Ki ¼ 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria
meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-
diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these
structures and the models of the phosphonic acid complexes of the human ortholog provided an insight
into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1
active site region.
Article history:
Received 15 February 2016
Received in revised form
4 April 2016
Accepted 6 April 2016
Available online 9 April 2016
Keywords:
Metalloaminopeptidases
Aminopeptidase N
Neisseria meningitidis
Phosphonic and phosphinic acids
APN-inhibitor complex structures
S1 binding mode
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
[8e10]. The privileged features of the amino acid substrates can be
readily translated into the structure of potential inhibitors. The a-
A detailed substrate fingerprint study [1] has recently revealed a
broad substrate tolerance of the S1 pocket of the M1 alanine met-
alloaminopeptidase from Neisseria meningitidis (NmAPN) e a gram-
negative diplococcus bacterium that is considered the major
causative agent of meningitis and other meningococcal diseases
aminoalkylphosphonic acids provide an opportunity to install the
preferred P1 substituents on the NeCeP scaffold and are
commonly recognized as transition state analogue inhibitors of zinc
metalloaminopeptidases [11]. However, the insertion of an addi-
tional heteroatom-based group into the substituent structure is a
separate and not trivial task, in particular to be performed in a
parallel manner. One such convenient modification is azir-
idinephosphonate ring opening to yield N0-substituted 1,2-
diaminoethylphosphonic acids, which was originally proposed to
provide inhibitors of metalloaminopeptidases from the porcine
[2e4]. Fluorogenic substrates, derived from unnatural
a-amino
acids that comprise bulky hydrophobic P1 side-chain residues
bearing a fragment of a basic character, were ranked among the
most favored ligands. A similar hydrophobic and base-oriented P1
specificity profile was observed for mammalian (human and
porcine) [5] and protozoan (Plasmodium falciparum [6] and Eimeria
tenella [7]) alanine aminopeptidases, the abundantly spread met-
allohydrolases of multiple functions and medical implications
kidney [12]. The compounds contain an extra b-amino group that
modifies the character of the P1 substituent to basic. Indeed, several
compounds were found to be good inhibitors of mono-zinc alanyl
aminopeptidase and discriminate versus two zinc atom-containing
leucine aminopeptidase (LAP), for which they exhibited poor or no
inhibition [12]. This was a quite unique observation, as the struc-
tural fragment H2NeCePO2 typically provides much more effective
* Corresponding author.
E-mail address: artur.mucha@pwr.edu.pl (A. Mucha).
http://dx.doi.org/10.1016/j.ejmech.2016.04.018
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

188
E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
complexation systems for the two zinc ions in LAP than for the
single one in APNs [11,13]. Apparently, the additional -amino
nucleophilic substitution than their non-modified counterparts.
Accordingly, our first alternative approach involved the verification
of N-tosylaziridinephosphonates 7 as convenient intermediates
(Scheme 1, pathway B). Indeed, we managed to open a tosylated
phosphonate diester with benzylamine in a simple and clean
manner. However, obtaining such a material (7) by nitrene-
mediated aziridination appears to be far more problematic. Con-
trary to the literature data, different versions of both metal and
non-metal assisted oxidative additions of Chloramine T (N-chlor-
otosylamide sodium salt) to the double bond of 3 are ineffective
[17e19]. Extensive optimization of the reaction conditions with
other tosyl precursors and hypervalent iodobenzene-based re-
agents [20e22] finally led to the desired products under copper(I)
catalysis [21], but in a very poor yield (<10% after column chro-
matography). Although the subsequent substitution to compounds
8 looked promising, we decided to abandon the approach.
b
group does not allow convenient P1eS1 side-chain docking (hy-
drophobic residues are strongly preferred) and distorts the overall
binding mode to this particular aminopeptidase. The precise rea-
sons for the good affinity to the porcine APN remain elusive. For
NmAPN, we recently postulated that an additional heteroatom
group located in the proximity of the side chain is able to enhance
interactions with the glutamate-rich anionic site that is formed by
Glu117, Glu260 and Glu316 and typically dedicated to the binding
the N-terminus of peptidic substrates (Fig. 1, panel A) [1].
As N0-substituted 1,2-diaminoethylphosphonic acids (1) are also
promising lead compounds to obtain specific and potent inhibitors
of alanyl aminopeptidase by the P10 elongation to phosphinic di-
peptides (2, Fig. 1, panel B), we have decided to revisit the issue. An
extensive series of novel compounds was obtained in an improved
synthetic approach. Their inhibitory activity was tested towards
four enzymes: NmAPN, human (HsAPN) and porcine APN (SsAPN),
and LAP. Finally, the crystal structures of two inhibitor complexes
(NmAPN-1h, and NmAPN-1n) were determined by X-ray crystal-
lography and compared against modeled NmAPN-inhibitor and
HsAPN-inhibitor complexes, providing structural insight of the
ligandeprotein interactions and inhibition modes. The potential of
the P10 extension was evaluated for selected N0 substituents.
In such circumstances, the elimination option was reconsidered,
but ammonia was replaced by Et3N (the first stage) and subse-
quently by a benzylamine derivative (the second step) [23]. The use
of benzyl or
a-methylbenzylamine gave N-substituted azir-
idinephosphones 9 in a reasonable yield (Scheme 1, pathway C).
The benzylamine derivatives were chosen as they could be readily
hydrogenated to free aziridine 5. Diphenylmethylamine (benzhy-
drylamine) did not work to the same extent, apparently because of
the decreased nucleophilicity and steric hindrance of the amino
group. Products 9 were easily purified by flash chromatography on
silica. The subsequent catalytic hydrogenation was performed in
standard conditions prior to the ring opening with selected amines.
The two-step procedure appeared more practical to obtain 5 than
the direct use of ammonia.
2. Results and discussion
2.1. Synthesis of 1,2-diaminoethylphosphonic acids
The
(Scheme 1) is the usual substrate for the synthesis of 1,2-
diaminoethyl derivatives in pathway via azir-
a
,
b
-unsaturated system of dialkyl vinylphosphonates 3
An excess of the reacting amine, harsh reaction conditions and
the use of a water medium in place of an organic solvent ensured
the substitution without the prerequisite hydrolysis of the ester
groups. As a matter of fact, both transformations proceeded
simultaneously, and the 1,2-diaminoethylphosphonate products
were in the form of ammonium salts of phosphonate monoesters
10 and preparatively separated after HPLC chromatography to
afford 11. Apparently, the monohydrolysis occurring in basic
aqueous conditions sufficiently increased the susceptibility of the
aziridine to substitution for it to be opened without requiring
exhaustive hydrolysis. Different primary and secondary structurally
diversified (linear, branched, cyclic and benzyl, including
heteroatom-substituted, listed in Scheme 1 and Table 1) amines
reacted readily. In the cases of difunctional amines, the high excess
of the nucleophilic component ensured mono-substitution,
including evident regioselectivity. For example, the aromatic
amino function of 4-aminobenzylamine remained non-modified, as
evidenced in products 11r.
1
a
idinephosphonates. In the classical Gabriel-Cromwell method,
bromine is readily added to the unsaturated bond to produce 1,2-
dibromophosphonate esters 4 [14]. Then, consecutive elimina-
tions of HBr with gaseous and liquid ammonia yield the hetero-
cyclic system 5 in the historical literature method (Scheme 1,
pathway A) [14]. Nucleophilic substitution with a primary or sec-
ondary amine enables the aziridine ring opening [15]. However, the
approach is characterized by at least two shortcomings. First,
ammonia is the reagent of choice to obtain the non-substituted
aziridine, but the elimination is capricious and not pure [14,16].
Second, for not fully clear reasons, esters of aziridinephosphonate 5
are very resistant to the nucleophilic attack of an amine and ring
opening. As a consequence, they demand hydrolysis to free acids 6
prior to substitution [15,16]. These complications were associated
with problematic purification, as a careful ion-exchange chroma-
tography was applied after each of those steps.
Acidic conditions, applied to remove the remaining alkyl ester
N-Tosylaziridines are considered much more susceptible to
Fig. 1. Structure of N0-substituted 1,2-diaminoethylphosphonic acids (1) and 1,2-diaminoethylphosphinic pseudodipeptides (2) and the presumed role of the amino groups in
binding to NmAPN.

E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
189
Scheme 1. Reagents and conditions: a) Br2, CH2Cl2; b) gaseous NH3 then liquid NH3 (or NH3/H2O followed by NaOH/H2O); c) HCl/H2O; d) R1(R2)NH, EtOH; e) TsNH2, PhI]O, CuCl,
MS 4 Å, MeCN/MeNO2 (4:1, vv); f) BzlNH2; g) Et3N then Ph(R)CHNH2; h) H2, 10% Pd/C, MeOH; i) R1(R2)NH, H2O/dioxan; j) Hþ/RP-HPLC. For the expanded structures of R1 and R2, see
Table 1.
group, completed the reaction sequence. Hydrogenation, ring
opening/monohydrolysis and the final hydrolysis could be also
performed in a convenient one-pot manner. Each step proceeded
efficiently and can be easily controlled by 31P NMR, so that the
removal of solvents is the only operation demanded before the
following stage. The separation of the target compounds 1 involves
standard treatment with propylene oxide. Analytical samples could
be conveniently recrystallized from water. In summary, even
though the overall procedure is extended by one step, it seems to be
advantageous to the approach described previously. The detailed
synthetic procedure together with the spectroscopic and analytical
data for novel compounds is given in the Supporting Materials.
The studied compounds were found to be good, generally low-
micromolar-range competitive and fast-binding inhibitors of
NmAPN. Unsubstituted 1,2-diaminoethylphosphonic acids (1a)
appeared to be the most potent inhibitor, with a submicromolar Ki
value (0.6 mM). The binding mode of 1a with NmAPN obtained by
molecular modeling (Fig. S1) principally confirmed the assumed
interactions of the amino groups with the active site glutamate
residues. Despite unfavorable electrostatic repulsions both posi-
tively charged nitrogen atoms are aimed at Glu117 and Glu260
(compare also Fig. 1, panel A). Compounds derived with bulky,
extended alkyl, cycloalkyl and arylalkyl fragments (1e,1h,1i,1p and
1q) were only slightly less potent (Ki ¼ 1.1e3.0
mM). The presence of
a smaller alkyl chain (1b-d), a heterocyclic ring (k-m), a benzyl
portion (1n and 1o) and cyclic piperazine or morpholine (1v and
1w) diminished the activity, typically by 1e2 orders of magnitude.
Interestingly, an additional terminal amino or methoxy group
located at the benzyl residue (compounds 1r and 1s) improved the
affinity by 3-to-4-fold compared with the non-modified analogue
2.2. SAR studies and binding modes of 1,2-diaminoethylphosphonic
acids
Amines of a different structure were used to explore the effi-
ciency of binding of
the N0
substituent of 1,2-
1n (4.2 and 4.8 mM versus 16.6 mM). A similar effect was achieved
diaminoethylphosphonic acids to the NmAPN and the reference
aminopeptidases (Table 1). The collection included the unsub-
stituted compound 1a, being the product of the reaction of azir-
idine with ammonia, alkyl (1b-e), cycloalkyl (1h and 1i) and
phenylalkyl derivatives (1n-q). The three series of compounds were
further diversified with representatives containing an additional
functionalization: 1f and 1g for alkyls, 1j-m for cycloalkyls and 1r-u
for arylalkyls. The functionalization mainly involved an extra ter-
minal amino group or an ether function, which were dedicated to
forming interactions with the distal part lining the S1 binding re-
gion of NmAPN. Finally, piperazine (1v) and morpholine analogues
(1w) were derived from cyclic secondary amines.
for a more flexible and basic 4-aminomethyl functionalization of
benzylic R1 (1t, Ki ¼ 4.2
mM). Twice as potent as the heteroatom-
modified benzyls was the elongated 4-methoxy-functionalized
phenylethyl fragment of compound 1u (Ki ¼ 2.3
m
M).
selected
The actual binding mode of
1,2-
diaminoalkylphosphonic acids to the active site of NmAPN was
revealed by crystallographic studies (Fig. 2, panel A for N0-cyclo-
hexyl and panel B for N0-benzyl derivative). The crystals of the re-
combinant enzyme soaked with an inhibitor do not show
significant changes in the overall binding site architecture
compared with the ligand-unbound protein [24], nor to those

190
E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
Table 1
Structure-activity relationship for 1,2-diaminoethylphosphonic acid inhibitors of N. meningitides APN and mammalian aminopeptidases: porcine and human APNs and porcine
LAP (NI e no inhibition up to 0.8 mM inhibitor concentration). In the cases of compounds previously tested toward SsAPN and LAP [12] the originally measured activity is
presented, unless the difference exceeds 25%.
No.
Ki [mM]
R1
R2
NmAPN
SsAPN
HsAPN
LAP
1a
1b
1c
H
Me
H
H
H
0.589 0.17
29.6 4.4
6.72 1.1
2.88 0.43 (19.0 [12])
46.0 [12]
1.91 [12]
10.6 2.2
7.42 0.96
1.66 0.13
NI [12]
NI [12]
NI [12]
1d
H
23.3 0.6
1.71 [12]
2.51 0.29
NI [12]
1e
1f
H
H
1.11 0.11
43.1 5.4
1.21 0.22
32.4 3.9
0.297 0.093
41.1 2.5
119 14
289 23
1g
1h
H
H
26.0 1.4
19.3 0.46
0.87 [12]
5.74 0.26
72.6 6.0
NI [12]
2.38 0.28
0.691 0.046
1i
1j
H
H
1.68 0.03
26.5 3.7
0.520 0.040
27.7 1.6
0.397 0.077
13.5 0.70
20.8 2.7
240 8.6
1k
1l
H
H
9.67 0.49
11.4 1.7
3.64 0.38
9.39 0.80
0.782 0.015
3.29 0.52
69.1 6.8
68.4 6.8
1m
1n
1o
H
H
H
27.7 0.46
16.6 0.71
15.4 0.97
7.00 0.89
3.22 0.15
0.341 0.091
0.391 0.11
55.8 7.6
350 [12]
600 [12]
2.30 [12]
14.5 2.4 (2.80 [12])
1p
H
1.42 0.050
6.58 0.25
2.55 0.37
71.1 1.2
1q
1r
H
H
3.00 0.035
4.79 0.18
6.04 0.81
1.22 0.18
1.49 0.19
108 7.8
2.49 0.057
7.02 0.42
1s
H
4.32 0.52
2.29 0.046
0.357 0.036
26.2 2.0
1t
H
H
4.19 1.2
5.42 0.28
2.89 0.43
26.5 2.1
19.5 2.4
1u
2.33 0.14
0.723 0.040
0.065 0.009
1v
12.5 2.1
197 8.9
14.7 [12]
94.2 11
6.04 0.38
37.7 1.6
1410 [12]
NI
1w

E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
191
Fig. 2. Close-up views showing interactions of N0-cyclohexyl- (1h, panel A) and N0-benzyl-1,2-diaminoalkylphosphonic acid (1n, panel B) ligands with NmAPN as observed in the
crystal structures. The hydrogen bonds and ligand-metal interactions are marked as green lines, distances are shown in Angstroms. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
visualized for NmAPN complexed with 1-aminoalkylphosphonic
acids [25]. Two negatively charged phosphonic oxygen atoms co-
ordinate the catalytic zinc ion (2.41 and 2.23 Å for 1h and 2.07 and
2.20 Å for 1n), mimicking the gem-diolate transition state of the
scissile peptide bond. One of them also interacts with the hydroxyl
group of Tyr377 (2.55 Å for 1h and 2.52 Å for 1n), the key residue
for the hydrolytic mechanism. Nevertheless, the phosphonate of
1,2-diaminophosphonic acids seems to be buried somewhat shal-
lower than in the 1-amino analogues for which the corresponding
distances are slightly shorter, typically, OeZn below 2.1 Å and O-
Tyr377 below 2.5 Å [25]. The fundamental contacts (the salt
plays a variable role in binding. For the N0-cyclohexyl residue of 1h,
it is aimed at the interior of the pocket, and its hydrogen-bonding
potential is somewhat shielded by the intramolecular alkyl and
cycloalkyl surroundings. Nevertheless, it is directed straight to the
negatively charged surface (compare also Fig. S1), although beyond
the hydrogen bond distance (3.43 Å to carboxylate oxygen atom of
Glu117). For the N0-benzyl, the
b-amino functionality is located in
the opposite conformation. It is also at the limit of the interatomic
distances of hydrogen bonding to C]O of Ala258 and to the third,
solvent-exposed, phosphonate oxygen atom (both distances
3.14 Å). Most probably these plausible interactions are not essential
effects, as 1-amino-3-phenylpropylphosphonic acid (the homo-
bridges) of the a-amino group with the glutamates responsible for
the binding of the N-terminus are also well reproduced, in partic-
ular for the N0-benzyl derivative 1n, with distances 2.65 Å to Glu117
and Glu260. In general, the NCP fragment of compounds 1 binds to
the NmAPN in a fairly predictable way with some minor distortions,
apparently caused by the bulkiness of the N0 substituent.
phenylalanine analogue), the compound missing the
group, is bound in a quite similar manner (Fig. S2) but exhibits an
M [1]).
The range of inhibition obtained for porcine kidney SsAPN is
quite similar to that measured for the N. meningitidis ortholog. In
Table 1, the results obtained for novel compounds 1e, 1g, 1j-l and
1n are compiled with the data acquired previously (if currently
b-amino
improved activity (Ki ¼ 1.26
m
Large-size P1 residues fill the hydrophobic S1 cleft well. To our
surprise, the
b-amino group is indiscriminately positioned and

192
E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
measured Ki values are consistent with the literature data for
compounds 1a-d, 1f, 1h, 1i and 1m [12] and do not differ by more
than 25%, the original number is given; otherwise, both are
included). For SsAPN, a much broader tolerance to different sub-
stitutions is visible. The majority of the studied inhibitors show a
dissociation constant below 10
mM, with the newly synthesized
cycloheptyl derivative 1g being the most potent (Ki ¼ 0.5
m
M).
Far more impressive data in terms of the inhibition constant
value were collected for human alanine aminopeptidase. Several
aliphatic compounds display Ki below 1 m
M, being equally (N0-n-
propyl, 1c, N0-cyclohexyl, 1h, and N0-cycloheptyl, 1i) or more active
(N0-n-hexyl, 1e, Ki ¼ 0.3
mM) than for the highly homologic porcine
APN. Significantly, arylalkyl residues are much better accepted for
human than for pig APN. N0-Benzyl, 1n, N0-(
and N0-(4-methoxybenzyl), 1s, displayed strong inhibition, with Ki
values falling in a submicromolar range (all values below 0.4 M).
a-methylbenzyl), 1o,
m
The higher homolog of the last compound (N0-2-(4-
methoxyphenyl)ethyl, 1u) appeared to be the most potent com-
pound found in this study (Ki ¼ 65 nM). To the best of our knowl-
edge, it is the most active amino acid analogue inhibitor of HsAPN
reported to date. To discuss the presumed binding mode and the
reasons for the potency of selected compounds, molecular
modeling studies were performed. The crystal structure of alanyl
aminopeptidase from H. sapiens [26] was used to dock the ligand
and analyze the interactions. The HsAPN-1u complex (Fig. 3) shows
a typical pattern of contacts of the ligand NeCeP fragment with the
enzyme, similar to those described above for NmAPN, namely, the
involvement of two PeO oxygen atoms in Zn complexation and the
Fig. 3. A model of the complex of N0-[2-(4-methoxyphenyl)ethyl]-1,2-
diaminoalkylphosphonic acid (1u) with HsAPN showing P1eS1 interactions. Intra-
and intermolecular hydrogen bonds and ligand-metal interactions are marked in
green.
for the other two APNs. The much more potent N0-[2-(4-
methoxyphenyl)-ethyl]-1,2-diaminoalkylphosphonic acid (1u) is
also at least 10-fold more active for HsAPN than for the alanyl
aminopeptidase of other organisms.
The selectivity ratio between the APNs and LAP is indeed sig-
nificant.
In
general,
N0-alkyl-
and
N0-cyclo-
alkyldiaminoethylphosphonic acids do not inhibit (up to 800
inhibitor concentration) or poorly inhibit porcine kidney leucine
aminopeptidase, whereas they appear to be quite active for APNs
m
M
ion pairing/hydrogen bonding of the
glutamine (Glu355, Glu414 and Gln213). Interestingly, the
a
-amino with glutamates/
-amino
b
group adapt a conformation similar to that evidenced in the crystal
structure of the N0-benzyl-1,2-diaminoalkylphosphonic acid-
NmAPN complex. The group is exposed externally out of the S1
enzyme cavity and tentatively linked with the solved-aimed oxy-
gen atom via an intramolecular hydrogen bond. The (4-
methoxyphenyl)ethyl fragment fits particularly well to the S1
binding site, filling it very tightly (Fig. 3 and Graphical Abstract).
The aromatic ring is surrounded by the phenyl of Phe348 (edge to
face) and the amide groups of Gln211 and Asn350. The electron-rich
character of the aromatic ring definitely improves the contacts with
the neighboring residues. The ether oxygen atom is in proximity to
the N-terminal amide NeH of Asn350, but the potential hydrogen
bonding can be a vague suggestion because of a not favored ge-
ometry. Suggestion of the interaction between the inhibitor oxygen
atom of OMe and the side-chain amide NH2 group of Asn350 seems
to be more justified for compound 1s, a methylene group shorter
homolog of 1u. The high activity of inhibitor 1s (Ki ¼ 357 nM) can
be attributed to this specific bond (Fig. S3) as the overall P1eS1 fit is
not so perfectly tight as modeled for 1u. The latter statement is also
valid for the N0-2-phenylethyl substituent of 1q, lacking OMe group
compared to 1u (Fig. S4), which gives rise to a moderate inhibitory
potency. To conclude, the length and character of the N0-2-(4-
methoxyphenyl)ethyl portion can be considered optimal to bind
to the S1 pocket of HsAPN.
(Ki ~ 1 mM). The cyclohexyl derivative 1h is the most significant
example. It is a low micromolar inhibitor of three alanyl amino-
peptidases and does not influence the LAP activity at all. In this and
similar instances, the selectivity ratio can be estimated to be three
orders of magnitude. When the effect of LAP inhibition is measur-
able, the ratio is typically 1.5e2 orders of magnitude (compare, for
example, the Ki values found for compound 1e: 1.1
mM for NmAPN,
1.2 M for SsAPN and 0.3 M for HsAPN versus 119 m
m
m
M for LAP).
Only individual compounds (arylalkyl) show somewhat significant
affinity toward LAP. Among them, the N0-4-aminobenzyl analogue
(1k) can be considered a universal inhibitor of aminopeptidases,
with all the Ki values between 2.5 and 7.0 mM.
2.3. Synthesis and activity of 1,2-diaminoethylphosphinic-derived
dipeptides
In continuation, we undertook an effort to adapt the optimized
protocols of obtaining and opening the corresponding aziridine
system for a suitable phosphinic dipeptide precursor. Such a
building block should contain an appropriate P10 fragment. The
aromatic ring of the benzyl-derived portion is favorably bound in a
shallow and hydrophobic S10 cleft [27], so we selected unsaturated
compound 16 and aziridine 18 (Scheme 2) as the target scaffolds.
The synthetic challenge to prepare vinylphosphinate 16 involves a
Discussing the selectivity issues between APNs from different
sources, several interesting discrimination cases can be observed.
For example, unsubstituted derivative 1a clearly favors binding to
NmAPN. The difference in inhibition constants is approximately 5-
fold to porcine and 20-fold to human enzyme. The opposite cases
(mammalian APNs versus the bacterial one) are even more pro-
nounced. For example, compound 1d (N0-isopropyl) exhibits over
an order of magnitude higher potency in favor of SsAPN and HsAPN
than NmAPN. Certain inhibitors preferentially bind to only the
multistep approach starting from methyl
a-benzylacrylate 12
[28,29]. Diethyl phosphite was readily added to the acrylate in a
phospha-Michael reaction under strong base catalysis [30] to
obtain 13. The triester was selectively monodealkylated with NaI in
acetone [31] to the mixed diester 14. Subsequently, the phosphonic
monoacid function was converted into the corresponding chlor-
idate 15 by thionyl chloride [32]. Without preparative separation,
phosphonochloridate was reacted with vinylmagnesium chloride
[33], the Grignard compound, to form the demanded vinyl-to-
phosphorus portion. The following steps of the procedure were
human ortholog. The N0-
guishes human aminopeptidase, with Ki ¼ 0.4
a
-methylbenzyl (1o) derivative distin-
M versus Ki ¼ 15
m
mM

E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
193
analogous to those elaborated for the amino acid analogs. They
involved the addition of bromine to yield 17 and ring closure to
aziridine 18. The last compound was heated in parallel with
selected amines (or ammonia) to obtain the N0-substituted dia-
minoethyl fragment of 19. Catalytic hydrogenation and a final hy-
drolysis led to the target products 2.
incorporation, a favorable residue accommodated in the S10 bind-
ing sub-site. Participation of the P10 part of the molecules in in-
teractions with LAP seems to be decisive in determining the
compound activity, which does not depend on the P1 portion
structure.
As evidenced by previous studies on organophosphorus in-
hibitors, more potent inhibitors of aminopeptidases are those
extended to the P10 fragment rather than simple amino acid ana-
logues designed to bind only within the S1 region [11,25,27,34].
This is not the case in the current study. The phosphinic dipeptides
2, although containing selected favorable N0-substituents, appeared
to be less potent inhibitors of alanyl aminopeptidases than the
corresponding phosphonic amino acids 1 (Table 2). A particular
drop in activity was evidenced for NmAPN. The Ki values increased
2.4. Discussion and conclusions
A careful and thorough optimization of the P1 and P10 substit-
uent structure (modification/extension) in phosphorusebased
transition state inhibitors and the examination of their interactions
with the corresponding sub-sites has led to the discovery of very
potent and selective inhibitors of metallo-dependent aminopepti-
dases, including APNs [25,27,34]. Although synthetically chal-
lenging, this method seems to be a rational and attractive approach
for the construction of new ligands and an alternative to the
elongation of pseudopeptidic sequences [35]. The selectivity issues
are not trivial aspects, as the amino acid content and architecture of
the APN active sites are well conserved between species. Never-
theless, certain structural nuances can be explored by systematic
guest-host matching, which in turn demands access to extensive
libraries of compounds. The parallel opening of aziridine phos-
phonates/phosphinates with amines fulfills these requirements.
We have presented here an improved and versatile version of the
reaction, validated for both amino acid and dipeptide analogues. It
enables the preparation of a vast collection of diaminoethyl com-
by 1e2 orders of magnitude and exceeded 100 mM for the majority
of compounds. A minor loss of activity was evidenced for the
porcine APN, typically a 2e5-fold decrease. For the human enzyme,
the results obtained for dipeptides are comparable with those ac-
quired for the amino acid analogues. N0-Alkyl and cycloalkyl de-
rivatives 2b-e showed good affinity, with Ki values between 0.2 and
1.1
improved when compared with corresponding compounds 1c, 1d,
M). The
mM (the bulkier the better). Each of these results was slightly
1i and 1j comprising only the P1 substituent (Ki ¼ 0.4e2.5
m
affinity of the most potent amino acid compound 1u did not change
(within the experimental error) upon the structure elongation and
remained at a very good nanomolar level for 2f.
pounds, substituted at the
b-amino group. The substitution is
The affinity of pseudodipeptidic compounds 2 towards LAP
flattened. Those amino acids that exhibited a moderate potency
(Ki ~ 20 mM, 1i and 1u) retained the activity upon the structure
elongation (2e and 2f, respectively). Those that were inactive (1c,
1d and 1h) gained affinity after the P10 addition (2b-d) to the
aforementioned level. Only the N0-unsubstituted derivative 2a
displayed three-fold worse inhibition parameters than the other
dipeptides. This improvement is definitely associated with benzyl
defined by the structure of the amine substrates, which include
alkyls, cycloalkyls and arylalkyls, also variously modified. Very
potent inhibitors were identified among the N0-derived 1,2-
diaminoethylphosphonic acids, and what is equally important,
the compounds showed satisfactory selectivity. In particular, the
studied bacterial ortholog, NmAPN, could be exclusively inhibited
by the non-substituted compound 1a. N0-Arylalkyls (e.g.,
ylbenzyl in compound 1o) are specific residues that give rise to Ki
a-meth-
Scheme 2. Reagents and conditions: a) t-BuOK, HP(O) (OEt)2, MeOH; b) NaI, acetone; c) SOCl2, CH2Cl2; d) CH2]CHMgCl, CH2Cl2; e) Br2, CH2Cl2; f) Et3N then Ph(R)CHNH2; g) R1NH2,
H2O/dioxan; h) H2, 10% Pd/C, MeOH; i) HCl/H2O.

194
E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
Table 2
Structure-activity relationship for 1,2-diaminoethylphosphinic dipeptide inhibitors of N. meningitidis APN and mammalian aminopeptidases.
No.
Ki [mM]
R1
H
NmAPN
SsAPN
HsAPN
LAP
2a
2b
171 5.5
131 16
29.0 2.3
6.74 0.54
4.45 0.50
1.11 0.057
64.4 2.6
21.0 2.5
2c
101 13
4.54 0.26
3.83 0.43
0.499 0.031
0.275 0.040
15.2 2.6
2d
114 14
28.3 2.5
2e
2f
109 13
3.24 0.41
1.48 0.18
0.207 0.019
0.078 0.009
23.8 2.4
21.1 2.6
39.5 9.6
constants at least one order of magnitude lower for human APN
than for the remaining two enzymes. One member of this group
(N0-[2-(4-methoxyphenyl)ethyl]-substituted phosphonic acid 1u)
appeared to be the most potent inhibitor revealed in the study
(Ki ¼ 65 nM). N0-substitution with an extended alkyl and cycloalkyl
(e.g., 1e and 1i) produced universal ligands with good affinity to all
three bacterial and mammalian APNs. Less bulky derivatives (1c, 1d
and 1h) of an aliphatic character retain their activity towards APNs
and do not inhibit LAP at all, even though both aminopeptidases
show a quite similar substrate specificity [36,37]. The SAR results of
new weak contacts is only moderately profitable.
The lack of potency of diaminoalkylphosponates towards LAP is
easier to explain. The overall binding mode of the NeCeP ligands to
the two-zinc containing aminopeptidase is quite different. LAP does
not contain such a precisely defined acidic region. Instead, Asp273
and one of the metal ions are involved in the binding of the N-
terminal amino group. This is the supplementary role of the zinc
apart from participation in the complexation of the gemediolate
transition state product of the scissile bond hydrolysis. This was
confirmed by the crystal structure of transition state inhibitor, the
phosphonic analogues of leucine bound to this aminopeptidase
analogous compounds, alkyl and cycloalkyl-substituted
a-amino-
phosphonic acids (missing the -amino group), are mirror-
b
[40]. An additional b-amino functionality would definitely alter the
reflected: they display a low micromolar and submicromolar in-
hibition of LAP and poor inhibition of APN (porcine kidney) (Fig. 4)
[12,38].
Accordingly, the motivation of our study on dia-
minoethylphosphonic/phosphinic compounds was also focused on
addressing the basic question of the structural reasons for such an
unusual behavior. The crystal structures of a few ligands complexed
favorable and fragile interaction network.
The decrease in potency, in particular towards NmAPN, observed
for phosphinic dipeptides
2 upon extension of the dia-
minoethylphosphonate structure 1 by the P10 fragment seems to be
the most unexpected result revealed in this study. Typically,
tightening organophosphorus ligand-APN interactions by P10-S10
contacts provides at least a one to two order of magnitude gain in
the binding constants [11,25]. Molecular modeling has somewhat
clarified this issue. The steric fit of diaminoalkylphosphinic dipep-
with NmAPN did not provide a definite answer on the role of the b-
amino group in binding. Instead, two possible conformations were
envisaged (Fig. 2 and S1). For the N0-cyclohexyl derivative 1h
tide possessing the additional b
-amino group within the S10 subsite
(Fig. 2A), the
b
-amino group is buried in the S1 pocket without any
has to be virtually identical to that evidenced for the phosphinic
analogue of hPhePhe, a potent inhibitor of NmAPN (Ki ¼ 302 nM)
[25]. Most probably the difference concerns the S1 region. Super-
imposition of two enzyme-inhibitor structures containing either
N0-benzyl-1,2-diaminoalkylphosphonic acids (Fig. 2B) or the
close contacts, yet it is clearly pointed at the negatively polarized
glutamate region, in particular at Glu117. This is in general accor-
dance with our hypothesis depicted in Fig. 1A, confirmed by mo-
lecular modeling for unsubstituted analogue 1a (Fig. S1), and
interactions displayed by certain disubstituted ligands, including
diaminocarboxylic compounds complexed with Escherichia coli
APN [39].
In the structure of N0-benzyl compound 1n (Fig. 2B), the
b-
amino group is exposed outside of the S1 cavity of NmAPN. In this
case, the ligand conformation is the most likely to be stabilized by
an intramolecular interaction with the phosphonic acid and a
hydrogen bond with a neighboring residue (Ala258). Similar ar-
rangements could be observed in three modeled inhibitor-HsAPN
complexes (Fig. 3, S3, and S4). These interactions might produce a
certain energetic gain upon complexation. However, taking into
account the number of hydrogen bonds with water that need to be
released upon the inhibitor entry to the active site, the formation of
Fig. 4. Preference of the P1 substituents in aminophosphonic acid inhibitors of neutral
aminopeptidases, a heteroatom-modified cycloalkyl of 1h favored by APNs and a
typically lipophilic cycloalkyl favored by LAP [12,38].

E. We˛glarz-Tomczak et al. / European Journal of Medicinal Chemistry 117 (2016) 187e196
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Acknowledgments
The work was financed by a statutory activity subsidy from the
Polish Ministry of Science and Higher Education for the Faculty of
Chemistry of Wrocław University of Technology, and by Wrocław
Centre of Biotechnology, program The Leading National Research
Centre (KNOW) for years 2014e2018. Ewelina We˛glarz-Tomczak
was supported by a grant from the Polish National Science Centre
(Grant UMO-2012/05/N/ST5/01145). The Biovia Discovery Studio
package was used under a Polish country-wide license. The use of
software resources (Biovia 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 the U.S. Department of Energy Office of Bio-
logical 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. We gratefully acknowledge
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