The most potent organophosphorus inhibitors of leucine aminopeptidase. Structure-based design, chemistry, and activity

Article abstract of DOI:10.1021/jm030795v

A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase complexed with the phosphonic acid analogue of leucine (LeuP) wa

Full text of DOI:10.1021/jm030795v

J . Med. Chem. 2003, 46, 2641-2655
2641
Th e Most P oten t Or ga n op h osp h or u s In h ibitor s of Leu cin e Am in op ep tid a se.
Str u ctu r e-Ba sed Design , Ch em istr y, a n d Activity
J olanta Grembecka,*,† Artur Mucha,† Tomasz Cierpicki,‡ and Paweł Kafarski†
Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocław University of Technology,
Wybrzez˘ e Wyspian´skiego 27, 50-370 Wrocław, and Institute of Biochemistry and Molecular Biology, University of Wrocław,
Tamka 2, 50-137 Wrocław, Poland
Received J anuary 31, 2003
A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the
metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase
complexed with the phosphonic acid analogue of leucine (LeuP) was used for structure-based
design of novel LAP inhibitors and for the analysis of their interactions with the enzyme binding
site. The inhibitors were designed by modification of phosphonic group in the LeuP structure
toward finding the substituents bound at the S′ side of the enzyme. This resulted in two classes
of compounds, the phosphonamidate and phosphinate dipeptide analogues, which were
synthesized and evaluated as inhibitors of the enzyme. The in vitro kinetic studies for the
phosphinate dipeptide analogues revealed that these compounds belong to the group of the
most effective LAP inhibitors found so far. Their further modification at the P1 position resulted
in more active inhibitors, hPheP[CH2]Phe and hPheP[CH2]Tyr (Ki values 66 nM and 67 nM,
respectively, for the mixture of four diastereomers). The binding affinities of these inhibitors
toward the enzyme are the highest, if considering all compounds containing a phosphorus atom
that mimick the transition state of the reaction catalyzed by LAP. To evaluate selectivity of
the designed LAP inhibitors, additional tests toward aminopeptidase N (APN) were performed.
The key feature, which determines their selectivity, is structure at the P1′ position. Aromatic
and aliphatic substituents placed at this position strongly interact with the LAP S1′ binding
pocket, while a significant increase in binding affinity toward APN was observed for compounds
containing aromatic versus leucine side chains at the P1′ position. The most selective inhibitor,
hPheP[CH2]Leu, binds to LAP with 15 times higher affinity than to APN. One of the studied
compounds, hPheP[CH2]Tyr, appeared to be very potent inhibitor of APN (Ki ) 36 nM for the
mixture of four diastereomers). The most promising LAP inhibitors designed by computer-
aided approach, the phosphonamidate dipeptide analogues, were unstable at pH below 12,
because of the P-N bond decomposition, which excluded the possibility of determination of
their binding affinities toward LAP.
In tr od u ction
in protein modification, activation, and degradation as
well as in the metabolism of biologically active peptides
and activity regulation of hormonal and nonhormonal
peptides.5 Altered activity of human leucine aminopep-
tidase has been associated with several pathological
disorders, such as cancer,13,14 eye lens aging and
cataracts,15-17 and myeloid leukemia and blood cell
counts.18 This protease may also be important in the
processing of antigenic peptides and in the determina-
tion of the immunodominance of various peptides.19
Moreover, LAP seems to play an important role in the
early stages of HIV infection, and thus serum activity
of this enzyme may be useful as a surrogate marker for
HIV infection and as a response to chemotherapy.20
LAPs from different sources (beef, hog, human) are
very similar in terms of the amino acid sequences and
kinetics,4,17,21 and they probably share the same active
site architecture.5 This makes the structure of the
enzyme from bovine eye lens (blLAP), the only one for
which 3D structure is available in Protein Data Bank,
applicable to design inhibitors of the enzyme from
porcine and human tissues.21,22
A structure-based approach to design potent and
selective inhibitors is an important component of the
modern drug development process.1-3 The design and
synthesis of inhibitors for aminopeptidases may result
in potential therapeutic agents, as the altered activity
of these enzymes is associated with variety pathologies,
including cancer, leukemia, and cystic fibrosis.4-6 Pro-
teases of this family have broad substrate specificity and
are widely distributed in many tissues and cells in
plants, animals, bacteria, and viruses, which indicate
their significant role in various biological processes.4,7,8
One of the first discovered and the most widely studied
aminopeptidases with respect to sequence, structure,
and mechanism of action is cytosolic leucine aminopep-
tidase (LAP, EC 3.4.11.1).4,9-12 LAP is a zinc-containing
exopeptidase that catalyzes the removal of amino acids
from the N-terminus of peptides or proteins. Similar to
other aminopeptidases, this enzyme is of significant
biological and medical importance because of its key role
* Corresponding author. Tel +48 71 320 2137, fax +48 +71 328
4064, e-mail jola@neon.ch.pwr.wroc.pl.
A number of various inhibitors have been reported
to bind to LAP.23 Among the amino acid analogues, the
† Institute of Organic Chemistry, Biochemistry and Biotechnology.
‡ Institute of Biochemistry and Molecular Biology.
10.1021/jm030795v CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/20/2003

2642 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
phosphonic analogue of L-leucine, (R)-LeuP, plays an
important role in leucine aminopeptidase inhibition, as
it is strong (Ki ) 0.23 µM)24 and selective toward the
enzyme. Because of the presence of the aminophospho-
nic fragment in the structure, this compound mimics
the tetrahedral gem-diolate transition state of the
peptide bond hydrolysis by zinc peptidases. The binding
mode of LeuP, revealed by the X-ray structure of its
complex with blLAP, showed the interactions of the
aminophosphonate fragment of the molecule with two
zinc ions and formation of three hydrogen bonds with
the LAP active site as well as the interactions of LeuP
side chain with the S1 pocket.25 However, strong LAP
inhibition by dipeptide analogue bestatin (Ki ) 0.6 nM)26
demonstrates the significance of the interactions with
the S1′ pocket as well. The availability of the crystal
structures of both the free enzyme10 and its complexes
with the inhibitors10,27-29 including LeuP25 enable the
application of molecular modeling methods for rational
design of LAP inhibitors.
In this paper we report the use of the crystal structure
of complex LAP-LeuP for the design of new, potent
inhibitors of leucine aminopeptidase by application of
the method implemented in the Ludi program, which
had been used successfully to generate several sets of
the active compounds.30-34 We have focused on finding
new fragments bound in the S1′ pocket of the enzyme
in order to extend the inhibitor-protein interaction
area, compared to LeuP and on replacement of leucine
side chain by a substituent, which should fit better to
the S1 pocket of LAP. The design, analysis of the
interactions with the enzyme, and synthesis of the
inhibitors are reported in this paper. The experimentally
measured binding affinities toward LAP are presented,
which well evaluate the practical applicability of the
theoretical approach used. Finally, the binding affinities
of the same compounds toward a monozinc exopepti-
dase, aminopeptidase N (APN, EC 3.4.11.2), an en-
kephalin-degrading enzyme, are reported, to compare
the potency of the same set of inhibitors toward the two
enzymes and evaluate their selectivity to LAP versus
APN.
F igu r e 1. The strategy applied to design LAP inhibitors. The
replacement of hydroxy group (in the circle) of LeuP (depicted
as a starting molecule) by new substituents from Ludi link
library resulted in compounds 1, 2, 3, 4, containing additional
residue at the P1′ position. Simultaneous replacement of
leucine at the P1 position by homophenylalanine side chain,
resulted in compounds 5, 6, 7.
Resu lts a n d Discu ssion
Design ed In h ibitor s. The extensive search of new
substituents interacting with the S1′ side of leucine
aminopeptidase resulted in the selection of two classes
of pseudodipeptides, namely phosphonamidates (con-
taining the P-N bond in place of the substrate peptide
bond) and phosphinates (containing the P-CH2 moiety)
(Figure 1).
Phosphonamidate and phosphinate peptide analogues
have not been studied as leucine aminopeptidase inhibi-
tors so far. In fact, the literature reports only two groups
of structurally related dipeptide analogues containing
a phosphorus atom in the structure, namely the phos-
phonate analogues of LAP substrates (with a P-O bond)
and phosphorus derivatives similar to bestatin.24,35
Their moderate activity toward LAP (the binding af-
finities about 2 orders of magnitude lower than for
LeuP) strongly disfavored further studies on this class
of compounds.
F igu r e 2. The modeled binding mode of LeuP[NH]Leu by
leucine aminopeptidase. The S1′ pocket and active site residues
of LAP, interacting with dipeptide analogues, are presented.
Hydrogen bonds and interactions of the inhibitor with zinc ions
are shown as black dashed lines.
group of Leu360 of the enzyme (Figure 2), in a way
similar to that for LAP peptide substrates and LeuP (in
which a hydroxy group interacts with Leu360 of LAP).
This makes the compounds with a P-N bond more
The phosphonamidate dipeptide analogues have the
potential to form a hydrogen bond with the carbonyl

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2643
Sch em e 1a
a
(a) AcOH; (b) KF/MeOH; (c) NaOH/MeOH then HCl; (d) SOCl2; (e) HCl‚H2NCH(R)COOMe, NEt3; (f) H2, Pd/C; (g) LiOH/MeOH; (h)
NEt3; (i) BzlBr, Ag2O; (j) NaOH/EtOH.
interesting as potential LAP inhibitors than the phos-
phinates, which lack this ability, because of the presence
of a CH2 group at the corresponding position. Moreover,
both classes of inhibitors contain a C-terminal carboxyl
group, which is able to form a hydrogen bond with
Gly362, while its second oxygen is probably involved in
the interactions with the solvent. The presence of the
side chain at the P1′ position of these inhibitors opens
the possibility for additional interactions with the S1′
pocket of LAP, formed by Ile421, Ala333, Asn330, and
Asp332 (Figure 2).
Among the phosphonamidate compounds, designed as
complementary to the binding site of the enzyme, there
are some analogues of LAP dipeptide substrates as well
as compounds that are not analogues but with quite
unexpected structural features, which would not be
possible to find without the use of computer-aided
methods (data not shown). Since the S1′ pocket of
leucine aminopeptidase is rather hydrophobic in nature
(Figure 2), mainly nonpolar side chains are favored
because they can interact well with this cleft. To
evaluate the influence of incorporation of phosphona-
midate moiety and the presence of a side chain at the
P1′ position, we have chosen two dipeptide mimetics,
the analogues of leucyl-glycine (LeuP[NH]Gly, 1) and
leucyl-leucine (LeuP[NH]Leu, 2) for synthesis and fur-
ther studies. The modeled binding mode of the latter is
similar to bestatin, which is known from the crystal
structure of its complex with LAP.29 The analogous
phosphinate dipeptides (LeuP[CH2]Gly, 3, and LeuP-
[CH2]Leu, 4), were also selected for synthesis and
activity measurements. Although these compounds are
straightforward analogues of LAP natural substrates,
their design by Ludi confirms the usefulness of the
method applied and allows detailed analysis of the
interactions of docked inhibitors with the enzyme.
Additionally, three other phosphinate dipeptide ana-
logues, containing the phenylethyl side chain at the P1
position (and thus being analogues of homophenylala-
nine, designed to replace isobutyl side chain of LeuP)
and three different amino acid residues at P1′: Leu
(hPheP[CH2]Leu, 5), Phe (hPheP[CH2]Phe, 6), and Tyr
(hPheP[CH2]Tyr, 7) were also synthesized and studied
as LAP inhibitors.
Ch em istr y of th e P ep tid es Con ta in in g a P h os-
ph on am idate Bon d. Although phosphonamidates have
been quite frequently used as peptide isosteres of strong
inhibitory activity against metalloproteases, their syn-
thesis is still not trivial. This is not only because of the
presence of the diverse functionalities in one molecule,
and thus the necessity of their selective protection and
deprotection, but more importantly the side reactions
accompanying P-N bond formation via phosphono-
chloridates36-39 and instability of this bond.40-42 There
are only very few reports of the synthesis of fully
deprotected phosphonamidate dipeptides with free an
R-amino group adjacent to the P-N bond and their
application as enzyme inhibitors.43,44
The applied synthetic strategy in this work is il-
lustrated in Scheme 1 (for R-aminophosphonamidates,
the synthetic pathway is similar to those described in
the literature44,45). The protocol of P-N bond formation
starts from the synthesis of suitably protected R-ami-
nophosphonate 10 or R-hydroxyphosphonate alkyl mo-
noester 15 bearing leucine side chain residue. The latter
substrate was used in order to obtain LeuOHP[NH]Gly
(17) (LeuOHP denotes phosphonic acid analogue of
hydroxyleucine, containing a hydroxy group instead of
an R-amino group). Compounds 10 and 15 were then
converted into P-N peptides via the standard phospho-
nochloridate approach followed by aminolysis with the
appropriate amino ester.36-38 The moderate yield ob-
served when using the phosphonochloridate procedure
is caused by side reactions, particularly by the formation
of pyrophosphonate.37 However, pyrophosphonate may
react slowly with the amino nucleophile and can be

2644 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
Ta ble 1. Measured Binding Affinities of Phosphinate
Dipeptide Analogues toward LAP and APN
partially transformed into phosphonamidate under
these reaction conditions as well. Thus, to increase the
product yield we have significantly prolonged the time
of the reaction. This methodology gives at least satisfac-
tory results in contrast to the unsuccessful application
of the standard peptide coupling agents.46
Ki [nM]
inhibitor
LAP
APN
LeuP[CH2]Leua (27)
LeuP[CH2]Leub (4)
hPheP[CH2]Leuc (5)
hPheP[CH2]Phed (6)
hPheP[CH2]Tyre (7)
LeuP[CH2]Glyf (3)
LeuOHP[NH]Glyf (17)
LeuP
65
110
1015
ND
1006
276
74
In our experiments SOCl2 was applied for the prepa-
ration of phosphonochloridates, and the reaction was
monitored by 31P NMR. In the case of compound 10, the
obtained phosphonochloridate exhibited two signals at
42.8 and 43.7 ppm corresponding to the equimolar ratio
of two pairs of diastereoisomers. The complex signal at
17.7 ppm belongs presumably to the pyrophosphonate.
The obtained reaction mixture was then treated with
the appropriate amino ester in the presence of triethy-
lamine as long as only two 31P NMR signals were
observed: one, a multiplet for the product, and the
second, a singlet, for the recovered starting material.
Final, fully protected, P-N peptide analogues (11, 12,
and 16) have been purified chromatografically after
standard workup. The overall yield of the procedure was
50-60%.
In the next two steps the peptide protecting groups
were removed under mild conditions. Benzyloxycarbonyl
or benzyl groups were removed by hydrogenolysis using
10% Pd/C catalyst. The hydrolysis of the ester groups
required basic conditions in order to remain the phos-
phonamidate moiety unchanged. It proceeded gently
with the use of 3 equiv of LiOH. The deprotection
process was followed by 31P NMR, which had shown that
both reactions proceeded practically quantitatively (with
yields exceeding 95%). It is worth mentioning that at
strongly basic pH we have not observed cyclization of
the products to analogues of diketopiperazine, a side-
reaction present during saponification of phosphinic
peptide methyl esters.
66
67
330
4880
230 (R)h
58000i
36
1415
NDg
55000 (R)h
LeuP[O]Leu
ND
a
b
Mixture of two diastereomers(1:1). Mixture of four diaster-
eomers, the proportion of the pairs of enantiomers (1:1.3). c Mix-
ture of four diastereomers, the proportion of the pairs of enanti-
omers 1:1.5. dMixture of four diastereomers, the proportion of the
pairs of enantiomers 1:2. e Mixture of four diastereomers, the
proportion of the pairs of enantiomers 1:1.7. f Racemic mixture.
g
Ki value not determined due to instability of the compound at
h
i
pH 7.5. Reference 54. Reference 24. ND: Ki value not deter-
mined.
only in strongly basic conditions. The decrease in pH
below 12 resulted in P-N bond hydrolysis yielding LeuP
and the appropriate amino acid, Gly or Leu. At pH 8.5,
which is optimal for LAP activity measurements, all the
phosphonamidate was hydrolyzed very fast (after 5 min
only products of P-N bond decomposition were observed
in NMR spectra). This excludes the possibility to
determine the binding affinities of phosphonamidates
with a free R-amino group toward leucine aminopepti-
dase. LAP exhibits aminoexopeptidase activity, and the
presence of a free R-amino group is crucial for inhibition
because there is no space in the active site to bind bulky
protecting groups. Additionally, the presence of an
unsubstituted phosphonic group is essential for strong
interaction with the LAP active site.24 These features
require the application of fully deprotected dipeptide
analogues containing a phosphonamidate bond, which
are unfortunately unstable under test conditions and
thus totally inapplicable as LAP inhibitors.
Quite interestingly, stability studies of the phospho-
namidate containing the hydroxy instead the of R-amino
group, LeuOHP[NH]Gly (17), showed significantly higher
resistance to hydrolysis compared to LeuP[NH]Gly. The
hydroxy analogue 17 appeared to be stable enough at
pH 8.5 to check its inhibitory activity toward LAP.
Th e Bin d in g Affin ity of Leu OHP [NH]Gly tow a r d
LAP . This compound is a reversible and competitive
LAP inhibitor, with an inhibition constant 4.88 µM for
the racemic mixture (Table 1). This means that the
replacement of the hydroxy group in the phosphonic
moiety of LeuOHP (Ki ) 28.0 µM for the racemic mixture)
by a NHCH2COO- fragment results in a 6-fold increase
in activity. This is probably because of the existence of
the additional hydrogen bond formed by this fragment
with the amide group of Gly362. The interactions with
Leu360 are possible both for LeuOHP and LeuOHP[NH]-
Gly and are of similar nature as for LeuP25 and as
proposed for LeuP[NH]Leu (Figure 2). If an analogous
effect of enhancement of binding affinity was to be
observed in the case of LeuP[NH]Gly versus LeuP, the
Ki value for the former should lay in nanomolar range.
This arises from the fact that L-LeuP (Ki ) 0.23 µM)
inhibits LAP about 2 orders of magnitude stronger than
LeuOHP54 and results from differences in both steric and
electronic properties of these amino acid analogues and
Both protected and deprotected phosphonamidates
were obtained as their enantiomeric or diastereomeric
mixtures. Because of the discovered liability of the final
products which excludes their practical application, the
attempts to separate or synthesize compounds with (R)
configuration at the R-carbon were not undertaken.
Sta bility Stu d ies of P h osp h on a m id a te Dip ep tid e
An a logu es. The results concerning the stability of
phosphonamidate moiety obtained by various authors
are quite contradictory. Although phosphonamidate
pseudopeptides are known as very potent enzyme
inhibitors,47-49 the P-N bond is considered to be
unstable.40-42,50,51 An example of pH dependent cleavage
of the P-N dipeptide was presented in crystallographic
studies of carboxypeptidase A complexed with Cbz-Gly-
[NH]Phe by Christianson and Lipscomb.52,53 The pres-
ence of a free R-amino group at proximity to the
phosphonamidate moiety was also mentioned to en-
hance the susceptibility of such analogues to hydroly-
sis.35 On the other hand, such compounds were designed
and studied as inhibitors of numerous enzymes.43,44
These incompatible literature data, particularly con-
cerning the pseudodipeptides with a free R-amino group,
induced us to more detailed studies on the phosphona-
midate stability in terms of the possibilities of measure-
ment their binding affinities toward leucine aminopep-
tidase (they will be a subject of separate paper). As a
result we have found that compounds 1 and 2 are stable

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2645
Sch em e 2a
Even though it could be easily removed by extraction
with CHCl3, the usefulness of acid hydrolysis was also
investigated. The carboxylate methyl ester showed
unexpected resistance against the action of strong acids.
Reflux for 24 h in 20% HCl yielded only 40% of LeuP-
[CH2]GlyOMe cleavage, whereas complete hydrolysis
was achieved only upon prolonged heating in 40% HBr
(48 h at 100 °C). Because of these limitations associated
with solubility problems, the reverse order of deprotec-
tion was studied and applied for homophenylalanine
derivatives (24-26). Saponification of the ester preced-
ing Cbz removal allowed us to obtain the final products
under milder conditions. All of them were obtained as
a mixture of two isomers (LeuP[CH2]Gly, 3) or four
isomers (4-7).
The efforts to obtain a single stereoisomer of LeuP-
[CH2]Leu corresponding to L,L configuration was also
undertaken. The enantiomer of the starting phosphinic
acid of absolute configuration (R) (19), corresponding
to the L form, was separated from its racemic mixture
using optically active R-methylbenzylamine similarly to
that reported earlier.62 The use of the enantiomerically
pure 19 afforded final (R)-LeuP[CH2]-(R,S)-Leu (27)
which consisted of only two diastereoisomers. However,
they appeared to be not separable by means of HPLC.
The same limitations are described in the literature
even for larger peptides bearing bulky side chain
substituents.63
a
(a) Hexamethyldisilazane then H2CdCH(R′)COOMe; (b, c)
HBr/AcOH then HBr/H2O or NaOH/MeOH then HBr/AcOH.
is predictable by our theoretical investigations.55 These
observations suggest altogether that the phosphonami-
date dipeptide analogues, containing a free R-amino
group, should be the most active LAP inhibitors studied
so far among the compounds containing a phosphorus
atom that mimicks the transition state of the enzymatic
reaction.
Ch em istr y of P ep tid es Con ta in in g a P h osp h i-
n a te Bon d . Phosphinate analogues of dipeptides make
the second group of designed LAP inhibitors. Phosphinic
acid compounds are known to inhibit different metal-
loproteases, and their inhibitory effects have been
reviewed quite recently.36,56 However, they have not
been studied as leucine aminopeptidase inhibitors so far.
Thus, five of such analogues, LeuP[CH2]Gly (3), LeuP-
[CH2]Leu (4), hPheP[CH2]Leu (5), hPheP[CH2]Phe (6),
and hPheP[CH2]Tyr (7), were synthesized and studied
as LAP inhibitors.
Activity of P h osp h in a tes tow a r d LAP . The phos-
phinate analogues of dipeptides 3-7 are exceptionally
stable in aqueous solutions at the whole range of pH.
They appeared to be reversible, competitive, and slow
binding inhibitors of LAP. The equilibrium in the
inhibitor binding was reached within several minutes
after addition of LAP to the mixture of inhibitor and
substrate. Such an inhibition type was observed previ-
ously for the most active phosphonic analogues of amino
acids.24,54
Phosphinic pseudodipeptides are usually synthesized
by addition of an appropriate acrylate to an aminophos-
phinic acid preactivated into the form of trivalent silyl
ester57-59 or by addition to aminophosphinate ester in
the presence of a strong base.60,61 In this work the first
procedure has been chosen (Scheme 2).
The most active among studied compounds were
hPheP[CH2]Phe (6) and hPheP[CH2]Tyr (7) analogues.
The binding affinities found for the mixtures of four
diastereomers of 6 and 7 (Ki ) 66 nM and 67 nM,
respectively) are almost 4 times higher than this for (R)-
LeuP (Table 1). The corresponding analogue with Leu
at the P1′ position, compound 5, also exhibits very
similar affinity (Ki ) 74 nM for the mixture of four
isomers). The mixture of four isomers of phosphinic
analogue of leucyl-leucine (4) (with the contribution of
1:1.3 for the pairs of enantiomers) was two times more
active (Ki ) 110nM) than (R)-LeuP, while the mixture
of two diastereomers of this compound, (R)-LeuP[CH2]-
(R,S)-Leu, has the same binding affinity (Ki ) 65nM)
as 6 and 7 (Table 1). Therefore compounds 6, 7, and 4
are the most active leucine aminopeptidase inhibitors
among those which contain a phosphorus atom that
mimicks the transition state of the reaction catalyzed
by the enzyme. Considering all the inhibitors of leucine
aminopeptidase studied so far, these analogues are
placed after bestatin (Ki ) 0.6 nM)64 and amastatin (Ki
) 30 nM)65 and are equipotent to leucinal (Ki ) 60
nM).66
N-Benzyloxycarbonylaminophosphinic acid (18, 20)62
was activated by heating with hexamethyldisilazane,
and then an appropriate acrylate was added (Scheme
2). After workup and purification, the protected pseudo-
peptides were separated. The benzyloxycarbonyl group
might be removed by hydrogenolysis as for phosphona-
midates or by action of HBr in AcOH, as there was no
danger of labile pseudopeptide bond cleavage. We have
chosen the second procedure. For the ester hydrolysis,
initially we used the same basic conditions as for
phosphonamidate derivatives, unfortunately observing
the formation of a cyclic peptide similarly as described
in the literature.58 The ratio of products was dependent
on the structure of the R′ substituents. Thus, in the case
of LeuP[CH2]LeuOMe, diketopiperazine analogue 28
However, it is necessary to consider the stereochem-
istry of dipeptide analogues which interact favorably
with LAP. It is known that bestatin isomer (2S,3R)-
appeared to be the main reaction product (70% of yield).

2646 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
AHPA-(R)-Leu is at least 10 times more active toward
LAP than remaining stereoisomers.26 Thus, in an analo-
gous way, one designed isomer of 6, 7, and 4, which
corresponds to L,L stereoisomer of dipeptide substrate,
is expected to be much more active than the remaining
ones and probably mostly responsible for the measured
activities of the mixtures. This might suggest that the
activity of (R)-hPheP[CH2]-(R)-Phe and (R)-hPheP[CH2]-
(R)Tyr should be comparable or even higher than this
found for amastatin.
resembles the tetrahedral transition state of the amide
bond hydrolysis.68-72 These moieties are able to coor-
dinate zinc ions present in the active sites of metallo-
proteases and to block their function in the process of
hydrolysis. Thus, their chemistry continues to attract
considerable interest36-38,73 and provides a wide variety
of potent inhibitors of proteases.36,56,74
There is a striking analogy between the inhibition of
LAP and another zinc protease, thermolysin, by phos-
phonate, phosphinate, and phosphonamidate peptide
mimetics. The activities of phosphonamidates toward
thermolysin (e.g., Cbz-GlyP[NH]Leu-Leu) are about
1000 times higher than those observed for the respective
phosphonate esters (e.g., Cbz-GlyP[O]Leu-Leu), whereas
the corresponding phosphinate analogues (e.g., Cbz-
GlyP[CH2]Leu-Leu) are only 10 times less active than
the phosphonamidates.47,48 The X-ray structure of the
phosphonate-enzyme complex revealed that this in-
hibitor indeed binds to thermolysin in the same way as
do the phosphonamidate.69 This suggests that the
appropriate phosphinate derivative, for which the crys-
tal structure is not available, should be bound in a
nearly identical way. Thus, it is not surprising that
special attention was paid to explain such a large
activity differences of these compounds toward ther-
molysin. It is postulated that the solvation effects,
counted by comparison of the number of hydrogen bonds
they form with solvent before binding to the protein and
formed with the enzyme after formation of the complex,
may explain these activity differences. Moreover, the
poor effectiveness of phosphonates might additionally
result from the repulsive electrostatic interaction be-
tween the oxygen atom of the ligand and carbonyl group
of Ala113 which both carry a negative partial charge.
The influence of the different basicity of phosphonate,
phosphinate, and phosphonamidate moieties on the
coordination energy of these inhibitors to zinc in the
thermolysin binding site was also suggested.57
The comparison of the binding affinities of 6 and 7
indicate that hydroxy group of the side chain of Tyr at
P1′ is most likely not involved in any hydrogen bond
with the enzyme, as no enhancement of the binding
affinity is observed compared to the derivative with Phe
at that position. The modeled binding mode of 7 by LAP
suggests the existence of only hydrophobic interactions
with the S1′ pocket, in the same way as for inhibitors
with hydrophobic side chains at that position (Phe or
Leu). There is an amide group of Asn330 at the bottom
of the S1′ pocket of LAP (Figure 2); however, the
geometry for hydrogen bond formation between this
residue and Tyr at P1′ would be unfavorable (in terms
of hydrogen bond distance and angle). The S1′ pocket
is open to solvent (the area between Ile421 and Asn330,
Figure 2), and it is most likely that the amide group of
Asn330 interacts with some water molecules (as seen
in the crystal structure of LAP-LeuP complex25) and
also that the hydroxy group of Tyr of 7 interacts with
the solvent molecules after binding.
The presence of the aromatic ring at the P1′ position
(6) does not introduce additional or stronger interactions
with LAP compared to Leu (5), as almost no improve-
ment of the binding affinity for such a replacement was
observed. On the other hand the replacement of the Leu
side chain (4) by hPheP (5) at the P1 position increases
the activity almost two-fold. This results probably from
additional hydrophobic interactions of the side chain of
hPheP with the strongly hydrophobic S1 pocket, which
is big enough to bind such a large side chain of the
inhibitor.
The same may refer to the analogous group of leucine
aminopeptidase inhibitors and suggest that a similar
binding mode of these ligands is very probable. Phos-
phonate derivative of leucyl-leucine (LeuP[O]Leu, mix-
ture of two isomers)24 is about 1000 times less active
compared to LeuP[CH2]Leu. The binding affinity of the
respective phosphonamidate (LeuP[NH]Leu) was not
determined because of its instability at pH 8.5. However,
both experimental results obtained for LeuOHP[NH]Gly
(the energetic gain arising from the incorporation of
glycine residue instead of hydroxyl group of LeuOHP)
and phosphinates (the increase in the binding affinity
after replacement of Gly by Leu at the P1′ position), as
well as the Ki values predicted by the Ludi scoring
function for 1 (46 nM) and 2 (5 nM), suggest that the
compound with a P-N bond should be several times
more active than LeuP[CH2]Leu (Table 1). Thus, it is
highly possible that its binding affinity toward LAP
should be the highest among the enzyme inhibitors
considered in this work. The binding affinity differences
obtained for phosphonate and phosphinate analogues
toward LAP probably result from similar reasons as
described above for thermolysin and should be discussed
in terms of hydrogen bond formation with solvent and
with the enzyme upon binding. Although crystal struc-
tures of LAP with phosphinate, phosphonamidate, and
The racemic mixture of 3 exhibits slightly weaker
binding affinity compared to (R)-LeuP (Table 1). How-
ever, it should be noticed that only the R isomer of this
phosphinate is expected to be active toward LAP, similar
to that for LeuP where (S)-LeuP is about 1000 times
less active than the R isomer.54 This suggests that the
Ki values for (R)-LeuP[CH2]Gly should be close to that
observed for (R)-LeuP. The lack of a hydrogen bond by
phosphinates with Leu360, which is present when LeuP
is bound, is most probably balanced by the energetic
gain resulting from the hydrogen bond formation be-
tween the C-terminal carboxyl group of 3 and the amide
group of Gly362, in a manner similar to that for bestatin
and amastatin.27,29 The activity predicted by application
of Ludi scoring function67 (Ki,calc ) 250 nM and Ki,calc
)
48 nM for (R)-LeuP[CH2]Gly and (R,R)-LeuP[CH2]Leu,
respectively) seems to be in very good agreement with
the experimental results.
Com p a r ison of Activity of P h osp h on a tes, P h os-
p h in a tes, a n d P h osp h on a m id a tes tow a r d LAP .
Phosphonamidates, phosphinates, and phosphonates
form an important family of phosphorus analogues of
peptides, in which the organophosphorus fragment

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2647
phosphonate dipepeptide analogues are not available,
the similar binding mode of these compounds to LAP,
comparable to this observed for LeuP,25 seems to be
highly possible. The NH group of phosphonamidates has
a possibility to form a hydrogen bond with the carbonyl
group of Leu360 after binding (Figure 2) and with a
water molecule before binding. This would keep the total
number of hydrogen bonds unchanged. On the other
hand, the CH2 group of the phosphinate is not able to
form regular hydrogen bonds neither before nor after
formation of the complex with the enzyme. Thus, in this
case the number of hydrogen bonds is also balanced.
However, an additional specific interaction might ap-
pear after binding to LAP, similar to that observed in
the crystal structures of astacin70 and stromelysin-371
complexed with phosphinic pseudopeptide inhibitors.
The methylene group of the phosphinic moiety was
located within hydrogen bond range to carbonyl oxygens
of the appropriate enzyme residues, thus mimicking the
interaction characteristic for the amide group of a
cleaved substrate.70,71 Such good analogy to the tetra-
hedral intermediate might explain the origin of potency
of phosphinic compounds, including LAP inhibitors
described in this paper. In contrast, phosphonate is not
able to form a hydrogen bond with Leu360, while such
a bond is presumably formed with the solvent before
binding. Thus, the complexation with the enzyme is
accompanied by a net loss of one hydrogen bond, and it
is considered as a reason of a weak binding affinity of
phosphonates compared to phosphinates. Additionally,
the presence of a CH2 group does not result in such a
strong electrostatic repulsion, as in the case of phos-
phonate esters, where the oxygen atom of the ligand is
close to the carbonyl oxygen of Leu360. Moreover, the
differences in the basicity of these analogues may
influence their interactions with two zinc ions, Zn488
and Zn489, in the LAP active site. In conclusion, the
significant differences in the activity between these
groups of LAP inhibitors probably result from their
distinct potential for hydrogen bond formation, electro-
static repulsion, solvent effects, and differences in
coordination energy of zinc ions.
reported in the literature for similar compounds.75
However, the replacement of the side chain of leucine
(5) by this of phenylalanine (6) at P1′ increases the
binding affinity almost 4 times (Ki ) 276 nM for hPheP-
[CH2]Phe). The incorporation of tyrosine at that position
has an even stronger effect, and hPheP[CH2]Tyr is a
much more efficient APN inhibitor (Ki ) 36 nM) than
other compounds studied in this paper, as well as the
dipeptide analogues described in the literature.75 Thus,
the presence of the hydroxy group in the C-terminal
aromatic fragment of 7 makes it the most active
compound among phosphinate dipeptide analogues stud-
ied so far. To the best of our knowledge, such analogues
containing a polar group at the P1′ position were not
reported as APN inhibitors. There is a literature report
on even more potent phosphinate inhibitors of APN
(with the binding affinity around 10-9 M),63,75 but these
are tripeptide analogues with additional substituent at
the P2′ position. Thus, the introduction of additional
structural features to the latter compounds, which
results in potential formation of additional contacts with
the enzyme, resulted in the increase of binding affinity
toward APN of 2 orders of magnitude. As the crystal
structure of APN is not available, we built the model of
the enzyme active site (Figure 3), to explain the differ-
ences in activity between compounds 5, 6, and 7. The
model was built based on homology of APN to human
leukotriene A4 hydrolase/aminopeptidase (LTA4H).76
This protein is a bifunctional zinc enzyme, which
exhibits both epoxide hydrolase activity and aminopep-
tidase activity in a common center.76 LTA4H and APN
belong to the same family (peptidase M1 family), and
the former is the only protein with reasonable sequence
identity to APN (27% for most of APN catalytic domain),
the structure of which was solved and deposited in
Protein Data Bank (1hs6 code in PDB). The similarity
is even much higher at the binding site area, particu-
larly at the zinc binding site and at the S1′ pocket
(Figure 3), which makes our model of APN binding site
reliable. According to this model, the S1′ pocket of APN
comprises tyrosine and histidine residues (Figure 3).
This gives more favorable interactions with Phe (in
compound 6) than with Leu (in compound 5) side chains
because of the increase of hydrophobic contact area and
possible interactions between the aromatic rings of 6
and the S1′ pocket residues. Such interactions well
explain the 4-fold activity increase of 6 versus 5. The
APN active site model also predicts the existence of a
hydrogen bond between the hydroxy group of Tyr
present at the P1′ position and the carboxyl group of
Glu413 placed at the bottom of the S1′ enzyme pocket
(Figure 3). This might explain the 8-fold binding affinity
increase of 7 versus hPheP[CH2]Phe. The consistency
of the results arising from kinetic studies with the
proposed interaction mode of studied phosphinate ana-
logues with APN confirms the reliability of the con-
structed enzyme binding site model.
Activity of P h osp h in a tes tow a r d AP N. APN was
selected intentionally for the inhibitory studies of phos-
phinates designed to inhibit LAP, as similar compounds
have been reported as effective and selective APN
inhibitors versus other Zn2+ metalloenzymes.63,75 Dipep-
tide phosphinate analogues exhibited only moderate
inhibitory activity (Ki values at micromolar range).75 For
strong inhibitory activity toward this enzyme, interac-
tion with its S2′ pocket is required. The compounds
studied toward LAP are dipeptide mimetics and thus
are expected to be less active toward APN. Therefore,
they were studied to compare their potency toward the
two enzymes and thus to evaluate their selectivity.
The binding affinities of phosphinate inhibitors to-
ward APN were strongly dependent on the nature of
the side chain at the P1′ position (Table 1). The
potencies of inhibitors containing Leu (27 and 5) and
Gly (3) at P1′ are very similar to each other and
practically independent on the character of the sub-
stituent at the P1 position (Table 1). The Ki values for
these APN inhibitors range between 1.0 and 1.4 µM,
which remains in a good agreement with the data
Differ en ce in Affin ities of P h osp h in a te Dip ep -
tid e An a logu es tow a r d LAP a n d AP N. Our studies
emphasize the significance of P1 and P1′ substituents
while designing potent phosphinate inhibitors toward
LAP and APN. However, the key feature discriminating
their selectivity concerns the P1′ side chain. Both,
aromatic and aliphatic side chains can interact strongly

2648 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
F igu r e 3. The proposed binding mode of hPheP[CH2]Tyr to APN binding site model, built based on the structure of human
leukotriene A4 hydrolase (LTA4H) in complex with bestatin (1hs6 in PDB). The binding mode of hPheP[CH2]Tyr to APN is similar
to the interactions of bestatin with LTA4H.76 Hydrogen bonds and interactions with zinc ion are shown as black dashed lines.
Enzyme residues within 5Å radius around the inhibitor are shown as dark gray sticks. Because of clarity of the picture, only the
residues involved in hydrogen bonds with hPheP[CH2]Tyr (Tyr472, Glu350, Glu384, Gly347, Ala348, Glu413) are labeled, together
with His383, which interacts with the P1′ fragment of the inhibitor. One of the oxygen atoms and the nitrogen atom of
aminophosphinic fragment of the inhibitor donate electron lone pairs to zinc ion at APN active site. The active site residues in
APN: His383, His387, Glu406 (interacting with zinc ion) and Glu384, Glu350, Tyr472 (involved in hydrogen bonds with inhibitor)
are strongly conserved in LTA4H (corresponding residues in LTA4H are: His295, His299, Glu318, Glu296, Glu271, Tyr383,
respectively).76
with LAP S1′ pocket, while a significant increase in
binding affinity to APN is observed for peptide ana-
logues containing aromatic versus leucine side chain at
the P1′ position (Table 1). The most pronounced differ-
ence in the binding affinity of the studied phosphinate
dipeptide analogues between LAP and APN was ob-
tained for two inhibitors containing Leu at the P1′
position (R-LeuP[CH2]Leu and hPheP[CH2]Leu), which
are around 15 times more active toward LAP than APN.
Incorporation of an aromatic residue at the P1′ site
increases the binding affinity toward APN with a
simultaneous decrease of specificity toward these two
enzymes. Comparison of two of the strongest LAP
inhibitors (6, 7) containing Phe and Tyr at P1′ shows
that 6 is around four times more active toward LAP
versus APN, while 7 is 2 times more potent to APN.
F u tu r e P r osp ects. Our studies have shown that the
presence of a free R-amino group in presumably the
most active LAP inhibitors, phosphonamidate dipeptide
analogues, makes the P-N bond vulnerable at pH <
12.0. The hydrolysis of this bond is strongly dependent
on the protonation of the R-amino group (to be pub-
lished). There are two possible ways to increase the
stability of phosphonamidates in order to enable studies
on their inhibitory activity: (1) the replacement of
amino group by other moieties; (2) lowering the pKa of
the amino group. The first solution is less possible since
the presence of a free R-amino group is crucial for strong
interactions with LAP as was shown by its replacement
by a hydroxy group, which resulted in strong decrease
of the activity (see above). To lower the pKa value of
the amino group, we propose to consider the phospho-
namidate containing 2-aminophenylphosphonic acid at
the P1 position. The influence of the aromatic ring
significantly lowers the pKa of the amino group, and this
compound should at least be as stable as CbzLeuP[NH]-
Gly and LeuOHP[NH]Gly (17) in test conditions. 2-Ami-
nophenylphosphonic acid was docked to the enzyme
binding site, and the relative orientation of the amino
and phosphonic groups in this compound is similar to
this present for LeuP. This enables the favorable
interactions with Zn489 and Asp273, while the aromatic
ring may be involved in the hydrophobic interactions
with the S1 pocket of LAP. Thus, this compound seems
to be a new promising lead for further development of
phosphonamidates containing a free amino group as
inhibitors of leucine aminopeptidase and similar en-
zymes.
Con clu sion s
The structural information available for leucine ami-
nopeptidase and the application of the molecular model-
ing techniques provided an opportunity for designing a
new class of low-molecular, very potent inhibitors of the
enzyme. The replacement of the hydroxyl group in the
phosphonate fragment of LeuP by new structural frag-
ments bound at the S1′ pocket of the enzyme led to the
phosphinate dipeptide analogues. To the best of our
knowledge, they are the most active LAP inhibitors
among the compounds containing a phosphorus atom
that mimicks the tetrahedral transition state of the
reaction catalyzed by LAP, with the binding affinities
in nanomolar range. Simultaneously, the modification
at the P1 position of such analogues resulted in ad-
ditional enhancement of the activity (Ki values 66 and
67 nM for (R,S)-hPheP[CH2]-(R,S)-Phe and (R,S)-hPheP-

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2649
Melting points were determined on Boetius apparatus and
were not corrected. IR spectra were recorded in KBr pellets
or in a film on a Perkin-Elmer System 2000 FT IR spectrom-
eter. Proton and phosphorus NMR spectra were recorded on
a Bruker DRX spectrometer operating at 300.13 MHz for 1H
and 121.50 MHz for 31P. Measurements were made in CDCl3,
DMSO-d6, or D2O solutions. Proton chemical shifts are re-
ported in relation to tetramethylsilane used as internal
standard. 31P NMR spectra were obtained with use of broad-
band 1H decoupling; chemical shifts are reported in relation
to 85% H3PO4 (sealed capillary). Microanalyses were performed
by Instrumental Analysis Unit of the Institute of Organic
Chemistry, Biochemistry and Biotechnology, Wrocław Uni-
versity of Technology.
[CH2]-(R,S)-Tyr, respectively). Their increased activities
compared to LeuP arise possibly both from formation
of the additional hydrogen bond with Gly362 LAP
(Figure 2) and from the interactions with the S1′ pocket.
The replacement of leucine by a homophenylalanine side
chain at the P1 position results in a better fit to the
hydrophobic S1 pocket of LAP. The importance of
absolute configuration of the asymmetric carbon atoms
in didepetide analogues as LAP inhibitors (for example
compounds 27 and 4 as well as bestatin isomers)
suggests that the activities of the appropriate diaster-
eomers of tested compounds should be even higher. This
new class of the tight-binding, competitive inhibitors of
leucine aminopeptidase may be considered as new leads
and offer the possibility of further modifications at P1
and P1′ positions, which should result in even more
active and selective compounds, with the possibility of
their medical application. Thus, in addition to monozinc
metallopeptidases, for which phosphinic peptides were
shown in many studies to behave as potent inhibitors,
our results suggest to consider also bizinc aminopepti-
dases as other possible targets for this class of com-
pounds.
Most of the tested compounds exhibited several times
stronger inhibitory effect toward LAP than to APN, with
the biggest difference for LeuP[CH2]Leu, which is
around 15 times more active for LAP. The only exception
was hPheP[CH2]Tyr, which was found to be the most
potent inhibitor of both LAP and APN, with the affinity
almost 2 times higher for APN. hPheP[CH2]Tyr is
almost 1 order in magnitude more potent toward APN
than similar analogues bearing hydrophobic groups at
the P1′ position. It is a quite unexpected result and
might be explained by formation of an additional
hydrogen bond between the hydroxy group of 7 and the
enzyme, as revealed by the modeled binding mode of
hPheP[CH2]Tyr to APN. Finding a strong inhibitory
effect of this compound toward APN opens the possibil-
ity of further modifications at the P1′ position and
design of even more active and selective enzyme inhibi-
tors.
Dip h en yl [1-(N-Ben zyloxyca r bon yla m in o)-3-m eth yl-
bu tyl]p h osp h on a te (8). The compound was synthesized by
condensation of benzyl carbamate, isovaleryl aldehyde and
triphenyl phosphite as described by Oleksyszyn.77 Yield: 38%;
mp 120-123 °C (lit. mp 122-123 °C).
Dim eth yl [1-(N-Ben zyloxyca r bon yla m in o)-3-m eth yl-
bu tyl]p h osp h on a te (9). Compound 8 was converted into its
dimethyl ester by transesterification in methanol in the
presence of KF and 18-crown-6 ether.78 Yield: 91%; mp 43-
45 °C (lit. mp 34-35 °C,78 45-47 °C79); 31P NMR (CDCl3): δ
1
29.08; H NMR: δ 0.86 (d, J ) 6.6 Hz, 6H, CH(CH3)2), 1.50
(m, 2H, CHCH2CH), 1.66 (m, 1H, CH(CH3)2), 3.62 and 3.67 (d
each, J ) 10.8 Hz, 3H and 3H, P(OCH3)2), 4.12 (m, 1H, NCHP),
5.05 (AB system, J ) 12.2 Hz, 2H, CH2OC), 5.09 (m, 1H, C(O)-
NH), 7.27 (m, 5H, C6H5).
Meth yl Hyd r ogen [1-(N-Ben zyloxyca r bon yla m in o)-3-
m eth ylbu tyl]p h osp h on a te (10). The monomethyl ester was
obtained by alkaline hydrolysis of (9) in a 1 M NaOH/MeOH
solution. After overnight stirring methanol was removed under
reduced pressure. The crude product was precipitated by
acidification of the water phase to pH 1 and then extracted
with AcOEt. The organic phase was dried over Na2SO4,
filtered, and evaporated. Recrystallization from an AcOEt/
hexane mixture gave the pure product as a white crystalline
compound. Yield: 84%; mp 118-120 °C (lit. mp 117-119 °C79);
1
31P NMR (CDCl3): δ 29.56; H NMR: δ 0.85 (d, J ) 6.7 Hz,
6H, CH(CH3)2), 1.47 (m, 2H, CHCH2CH), 1.64 (m, 1H,
CH(CH3)2), 3.63 (d, J ) 10.8 Hz, 3H, POCH3), 4.11 (m, 1H,
NCHP), 5.05 (s, 2H, CH2OC), 7.26 (m, 5H, C6H5), 10.33 (s, 1H,
POH).
N-[[1-(N′-Ben zyloxyca r b on yla m in o)-3-m et h ylb u t yl]-
m eth oxyp h osp h in yl]glycin e Meth yl Ester (11). Th e Rep -
r esen ta tive P r oced u r e. The monomethyl ester (10) (0.63 g,
2 mmol) was dissolved in 10 mL of CHCl3, and thionyl chloride
was added (0.22 mL, 3 mmol). The solution was stirred for 2
h at room temperature and refluxed for additional 2 h. The
volatile components were evaporated under reduced pressure,
and the residue was treated with 10 mL of benzene and
evaporated again. The resulting phosphonochloridate was
dissolved in 10 mL of CHCl3 and added dropwise to the
mixture of glycine methyl ester hydrochloride (0.25 g, 2 mmol)
and triethylamine (0.70 mL, 5 mmol) in 15 mL of CHCl3 cooled
in an ice bath. The resulting solution was left at room
temperature overnight and then washed successively with 1
M NaOH, water, 5% HCl, water, 1 M Na2CO3, and brine (20
mL of each). The organic layer was dried over Na2SO4. After
removal of the solvent, the crude peptide was purified on
column chromatography using the AcOEt/hexane (3:1) solution
as the eluent. Yield: 55%; mp 62-79 °C; IR (KBr, cm-1): 3295
and 3225 (NH), 3065 and 2950 (CH), 1760 and 1700 (CdO),
1545 (δNH), 1270 (PdO), 1210, 1150, 1060, and 1035 (C-O,
P-O); 31P NMR (CDCl3): δ 31.39 and 32.72 (2:3 ratio); 1H
NMR: δ 0.85 (m, 6H, CH(CH3)2), 1.51 (m, 2H, CHCH2CH),
1.66 (m, 1H, CH(CH3)2), 3.05 and 3.15 (m each, 1H together,
PNH), 3.47-3.76 (m, 8H, POCH3, OCH3, and PNHCH2), 4.01
(m, 1H, NCHP) 4.95 and 5.24 (d each, J 1 ) 10.0 Hz, J 2 ) 10.1
Hz, 1H together, C(O)NH), 5.03 (AB system, J ) 12.2 Hz, 2H,
CH2OC), 7.27 (m, 5H, C6H5). Anal. (C17H27N2O6P) C, H, N.
Phosphonamidate dideptide analogues containing a
free R-amino group, designed as the most promising
LAP inhibitors, are stable only at strongly basic condi-
tions (pH > 12). Thus, this class of compounds is not
suitable for activity studies toward both studied ami-
nopeptidases. However, it is possible that a modified
phosphonamidate analogue, with the amino group at-
tached to the aromatic ring at P1, which would lower
its pKa and thus increase the stability, might be
considered as promising for inhibition of leucine ami-
nopeptidase. This hypothesis, however, requires elabo-
ration of synthetic procedures leading to this type of
compound and further experimental studies.
Exp er im en ta l Section
Ch em istr y. Gen er a l. Unless otherwise stated, materials
were obtained from commercial suppliers (Sigma-Aldrich,
Fluka, Merck) and used without purification. Isovaleryl alde-
hyde and thionyl chloride were freshly distilled. Triethylamine
was distilled and stored over potassium hydroxide. Ethanol-
and water-free chloroform was obtained by passing the solvent
through basic alumina followed by its distillation from over
P2O5. Anhydrous benzene was obtained by its distillation from
P2O5 and stored over sodium. Column chromatography was
performed on silica gel 60 (70-230 mesh).
N-[[1-(N′-Ben zyloxyca r b on yla m in o)-3-m et h ylb u t yl]-
m eth oxyp h osp h in yl]-(L)-leu cin e Meth yl Ester (12). The

2650 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
compound was prepared in the same manner as 11. Purifica-
tion on column chromatography using the AcOEt/hexane (3:
2) solution afforded the peptide as colorless oil. Yield: 52%;
IR (film, cm-1): 3295 and 3220 (NH), 2955 (CH), 1745 and
1720 (CdO), 1540 (δNH), 1265 (PdO), 1215, 1150, 1110, and
1050 (C-O, P-O); 31P NMR (CDCl3): δ 30.45, 30.96, 31.90,
N-[(1-Ben zyloxy-3-m et h ylb u t yl)et h oxyp h osp h in yl]-
glycin e Meth yl Ester (16). Starting from the phosphonate
15, the phosphonamidate peptide 16 was synthesized in the
same manner as described for the amino analogues (11, 12).
Purification on column chromatography using a AcOEt/
CH2Cl2 (2:1) solution as the eluent gave the product as colorless
oil. Yield: 44%; IR (film, cm-1): 3210 (NH), 2955 (CH), 1755
(CdO), 1260 (PdO), 1210, 1155 and 1045 (C-O, P-O); 31P
NMR (CDCl3): δ 28.78 and 30.75 (4:3 ratio); 1H NMR: δ 0.79,
0.81 and 0.93 (d each, J ) 6.6 Hz, 6H together, CH(CH3)2),
1.33 and 1.37 (t each, J ) 7.0 Hz, 3H together, POCH2CH3),
1.51 and 1.69 (m each, 2H together, CHCH2CH), 1.84 (m, 1H,
CH(CH3)2), 3.08 and 3.20 (m each, 1H together, PNH), 3.70
(s, 3H, OCH3), 3.72-3.93 (m, 3H, PNHCH2 and OCHP), 4.13
(m, 2H, POCH2CH3), 4.56, 4.64, 4.71 and 4.87 (d each, J )
11.1 Hz, 2H together, C6H5CH2O), 7.33 (m, 5H, C6H5). Anal.
(C17H28NO5P) C, H, N.
N -[(1-H y d r o x y -3-m e t h y lb u t y l)o x y p h o s p h in y l]g ly -
cin e Dilith iu m Sa lt (17). The removal of the protection
groups in peptide 16 was achieved by hydrogenolysis followed
by alkaline hydrolysis under the same conditions as described
for the amino analogues (11, 12). 31P NMR (D2O): δ 25.26; 1H
NMR: δ 0.81 and 0.86 (d each, J ) 6.6 Hz, 3H and 3H, CH-
(CH3)2), 1.36 and 1.49 (m each, 1H and 1H, CHCH2CH), 1.70
(m, 1H, CH(CH3)2), 3.41 (d, J ) 10.1 Hz, 2H, PNHCH2), 3.63
(m, 1H, OCHP).
1
and 32.23 (4:4:3:2 ratio); H NMR: δ 0.86 (m, 12H, 2 × CH-
(CH3)2), 1.47 (m, 4H, 2 × CHCH2CH), 1.64 (m, 2H, 2 ×
CH(CH3)2), 3.00 (m, 1H, PNH), 3.61 (m, 6H, POCH3 and
OCH3,), 3.99 (m, 2H, PNHCH and NCHP), 5.04 (m, 3H, C(O)-
NH and CH2OC), 7.26 (m, 5H, C6H5). Anal. (C21H35N2O6P) C,
H, N.
N-[(1-Am in o-3-m eth ylbu tyl)oxyph osph in yl]glycin e Dil-
ith iu m Sa lt (1). Th e Rep r esen ta tive P r oced u r e. To re-
move the benzyloxycarbonyl group, the peptide 11 (0.19 g, 0.5
mmol) was dissolved in 5 mL of MeOH and hydrogenated in
the presence of 0.1 g of 10% Pd/C catalyst. Hydrogen was
passed through the solution by gentle bubbling. The reaction
followed by TLC as well as by 31P NMR proceeded quantita-
tively, and it was completed within 2 h. The catalyst was
filtered off, and the solution was evaporated to dryness giving
a colorless oil. Removal of the methyl groups was achieved by
alkaline hydrolysis of N-[(1-amino-3-methylbutyl)methoxy-
phosphinyl]glycine methyl ester in the 1.5 M LiOH/MeOH
solution (2 mL, 1:1; 3 equiv of LiOH were used). After
evaporation to dryness at room temperature, the sample was
used directly for tests. Because of its instability at a pH lower
than 12, the deprotected peptide in form of the dilithium salt
was characterized only by NMR spectroscopy. 31P NMR
(D2O): δ 29.39; 1H NMR: δ 0.83 and 0.89 (d each, J ) 6.6 Hz,
3H and 3H, CH(CH3)2), 1.37 (m, 2H, CHCH2CH), 1.75 (m, 1H,
CH(CH3)2), 2.74 (m, 1H, NCHP), 3.42 (d, J ) 8.0 Hz, 2H,
PNHCH2).
Meth yl 3-[[1-(N-Ben zyloxyca r bon yla m in o)-3-m eth yl-
bu tyl]h yd r oxyp h osp h in yl]p r op ion a te (21). Th e Rep r e-
sen ta tive P r oced u r e. The phosphinic dipeptide was obtained
according to the procedures previously described in the
literature57-59 with only slight modification. [1-(N-Benzyloxy-
carbonylamino)-3-methylbutyl]phosphinic acid (18)62 (0.57 g,
2 mmol) was heated with hexamethyldisilazane (2.10 mL, 10
mmol) at 100-110 °C for 2 h under nitrogen. The reaction
mixture was cooled to 60 °C, and methyl acrylate was added
(0.27 mL, 3 mmol). Then the temperature was maintained at
85-90 °C for additional 3 h. After cooling to 60 °C, 5 mL of
methanol was added dropwise. The volatile products were
removed under reduced pressure, and the residue was dis-
solved in 25 mL of AcOEt and washed twice with 5% HCl (10
mL) and finally with brine (10 mL). The organic layer was
dried over Na2SO4, filtered, and evaporated to dryness. The
residue was worked up with 10 mL of Et2O and left overnight
for crystallization. Yield: 69%; mp 146-153 °C; IR (KBr, cm-1):
3285 (NH), 3035 and 2955 (CH), 1740 and 1710 (CdO), 1260
(PdO), 1230, 1115, 1065 and 1025 (C-O, P-O); 31P NMR
N-[(1-Am in o-3-m et h ylb u t yl)oxyp h osp h in yl]-(L)-leu -
cin e Dilith iu m Sa lt (2). 31P NMR (D2O): δ 26.58 and 26.99
1
(2:3 ratio); H NMR: δ 0.86 (m, 12H, 2 × CH(CH3)2), 1.29-
1.75 (m, 6H, 2 × CHCH2CH), 2.72 (m, 1H, NCHP), 3.72 (m,
1H, PNHCH).
Dieth yl (1-Hyd r oxy-3-m eth ylbu tyl)p h osp h on a te (13).
This compound was synthesized in the reaction of isovaleryl
aldehyde and diethyl phosphite in the presence of triethy-
lamine as described in the literature for the dimethyl ana-
1
logue.80 Yield: 46%; 31P NMR (CDCl3): δ 26.97; H NMR: δ
0.86 and 0.91 (d each, J ) 6.6 Hz, 3H and 3H, CH(CH3)2), 1.28
(t, J ) 7.0 Hz, 6H, P(OCH2CH3)2), 1.39 and 1.65 (m each, 1H
and 1H, CHCH2CH), 1.87 (m, 1H, CH(CH3)2), 3.89 (m, 1H,
OCHP), 4.09 (m, 4H, P(OCH2CH3)2).
1
(CDCl3): δ 55.38; H NMR: δ 0.83 and 0.85 (d each, J ) 6.6
Hz, 3H and 3H, CH(CH3)2), 1.47 (m, 2H, CHCH2CH), 1.62 (m,
1H, CH(CH3)2), 1.96 (m, 2H, PCH2), 2.53 (m, 2H, PCH2CH2),
3.58 (s, 3H, OCH3), 3.97 (m, 1H, NCHP), 5.03 (s, 2H, CH2OC),
5.08 (d, J ) 10.4 Hz, 1H, C(O)NH), 7.27 (m, 5H, C6H5), 7.92
(bs, 1H, POH). Anal. (C17H26NO6P) C, H, N.
Diet h yl
(1-Ben zyloxy-3-m et h ylb u t yl)p h osp h on a t e
(14). To protect the hydroxy function the phosphonate (13)
was stirred 48 h with 1.2 equiv of benzyl bromide and 1.5 equiv
of Ag2O in anhydrous DMF at room temperature. The result-
ing mixture was filtered through Celite, and DMF was
removed under reduced pressure. Purification by column
chromatography using AcOEt/hexane (2:1) gave the 1-benzyl-
oxyphosphonate as colorless liquid. Yield: 77%; 31P NMR
Meth yl 2-[[[1-(N-Ben zyloxyca r bon yla m in o)-3-m eth yl-
b u t y l ]h y d r o x y p h o s p h i n y l ]m e t h y l ]-4 -m e t h y l p e n -
ta n oa te (22). The phosphinic analogue: Z-Leu[CH2]Leu-OMe
was synthesized by Michael addition of methyl R-isobutylacry-
late81 to activated 18 as described above. Yield: 63%; mp 121-
137 °C; IR (KBr, cm-1): 3320 (NH), 2960 (CH), 1740 and 1685
(CdO), 1270 (PdO), 1230, 1170, 1130, and 1045 (C-O, P-O);
1
(CDCl3): δ 25.62; H NMR: δ 0.75 and 0.91 (d each, J ) 6.6
Hz, 3H and 3H, CH(CH3)2), 1.34 and 1.35 (t each, J ) 7.1 Hz,
3H and 3H, P(OCH2CH3)2), 1.47 and 1.78 (m each, 1H and
2H, CHCH2CH(CH3)2), 3.74 (m, 1H, OCHP), 4.15 (m, 4H,
P(OCH2CH3)2), 4.59 and 4.92 (d each, J ) 11.2 Hz, 1H and
1H, C6H5CH2O), 7.35 (m, 5H, C6H5).
1
31P NMR (CDCl3): δ 54.45 and 54.79 (1:1 ratio); H NMR: δ
0.79, 0.82, 0.85 and 0.88 (d each, J ) 6.5 Hz, 12H together, 2
× CH(CH3)2), 1.22, 1.48, 1.68 and 2.07 (m each, 1H, 4H, 2H
and 1H, 2 × CHCH2CH(CH3)2 and PCH2), 2.79 (m, 1H,
PCH2CH), 3.58 (s, 3H, OCH3), 3.97 (m, 1H, NCHP), 5.04 (AB
system, J ) 12.4 Hz, 2H, CH2OC), 5.25 and 5.26 (d each, J )
10.0 Hz, 1H together, C(O)NH), 7.25 (m, 5H, C6H5), 11.88 (bs,
1H, POH). Anal.(C21H34NO6P) C, H, N.
Eth yl Hyd r ogen (1-Ben zyloxy-3-m eth ylbu tyl)p h osp h o-
n a te (15). The monoethyl ester was obtained by alkaline
hydrolysis of 14 in a 1 M NaOH/EtOH solution. After 2 h of
gentle reflux, the solution was cooled and EtOH was removed
under reduced pressure. The water phase was acidified to pH
1 and extracted with three portions of AcOEt. Drying the
organic layer over Na2SO4 and evaporation afforded the
product as slightly yellow glass. 31P NMR (DMSO-d6): δ 19.41;
1H NMR: δ 0.71 and 0.84 (d each, J ) 6.4 Hz, 3H and 3H,
CH(CH3)2), 1.13 (t, J ) 6.9 Hz, 3H, POCH2CH3), 1.44 and 1.58
(m each, 1H and 1H, CHCH2CH), 1.73 (m, 1H, CH(CH3)2), 3.47
(m, 1H, OCHP), 3.96 (m, 2H, POCH2CH3), 4.47 and 4.86 (d
each, J ) 11.3 Hz, 1H and 1H, C6H5CH2O), 7.30 (m, 5H, C6H5).
Meth yl 2-[[[1-(R)-(N-Ben zyloxyca r bon yla m in o)-3-m e-
t h ylb u t yl]h yd r oxyp h osp h in yl]m e t h yl]-4-m e t h ylp e n -
ta n oa te (23). This compound was obtained using the same
procedure as for 21 and 22 using the (R) enantiomer of [1-(N-
benzyloxycarbonylamino)-3-methylbutyl]phosphinic acid (19).
The latter, optically active compound was separated by
crystallization of the diastereomeric salt of its racemic mixture

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2651
with (R)-(+)-1-phenylethylamine similarly as reported ear-
lier.62 The salt was crystallized several times from the EtOH/
acetone mixture (1:1) to the constant melting point and specific
rotation. Then it was treated with 1 M NaOH, the separated
amine was washed out with Et2O, and the water phase was
acidified to pH 1. The separated compound was extracted to
AcOEt. The organic phase was dried with Na2SO4, filtered,
and evaporated to dryness. The crude acid was recrystallized
from an AcOEt/hexane solution giving the enantiomer of
3.47 and 3.49 (s each, 3H together, OCH3), 3.83 (m, 1H,
NCHP), 5.05 (s, 2H, CH2OC), 5.86 (d, J ) 9.8 Hz, C(O)NH),
6.65 and 6.85 (d each, J ) 8.4 Hz, 2H and 2H, HOC6H4), 7.06-
7.30 (m each, 10H, 2 × C6H5). Anal. (C28H32NO7P) C, H, N.
3-[(1-Am in o -3-m e t h y lb u t y l)h y d r o x y p h o s p h in y l]-
p r op ion ic Acid (3). The benzyloxycarbonyl group was re-
moved by hydrogenolysis of the peptide 21 in the same manner
as described for phosphonamidates. Here, on the contrary to
the unstable P-N peptides, the same goal could be achieved
by the action of 33% HBr (1 mL for 0.1 g of the peptide) at
room temperature for 1 h. Removal of the methyl ester group
proceeded gently in alkaline conditions. However, because of
the formation of the phosphinic analogue of diketopiperazine
under these conditions, the acid hydrolysis was favorable. It
required 48 h in 40% HBr at 100 °C to cleave the ester
completely. After evaporation to dryness, the residue was
treated with MeOH and evaporated again to remove the traces
of HBr (three times). The resulting peptide was separated as
its hydrobromide salt upon treatment of the residue with Et2O,
which yielded an amorphous, slightly yellow solid of satisfac-
tory purity. Yield: 93%; IR (KBr, cm-1): 3415 (OH), 3300-
2600 (OH, NH, CH), 1735 (CdO), 1240 (PdO), 1195, 1170, and
1090 (C-O, P-O); 31P NMR (D2O, NaOD): δ 46.14; 1H NMR:
δ 0.79 and 0.85 (d each, J ) 6.6 Hz, 3H and 3H, CH(CH3)2),
1.27 (m, 2H, CHCH2CH), 1.66 (m, 3H, CH(CH3)2 and PCH2),
2.22 (m, 2H, PCH2CH2), 2.65 (dt, J H ) 6.5 Hz, J P ) 9.4 Hz,
1H, NCHP). Anal. (C8H18NO4P‚HBr) C, H, N.
20
specific rotation [R]D ) -56° (1% solution in EtOH).
[1-(N-Ben zyloxyca r bon yla m in o)-4-p h en ylbu tyl]p h os-
p h in ic Acid (20). The phosphinic analogue of homophenyl-
alanine was synthesized in the reaction of hydrocinnamalde-
hyde with the salt of diphenylmethylamine and hypophospho-
rous acid, followed by hydrolysis of the adduct and N-terminal
protection with benzyl chloroformate, similarly as described
in the literature.62,63 Total yield of the procedure: 26%; mp
136-140 °C (lit. mp 142-143 °C;63 31P NMR (DMSO-d6): δ
31.15; 1H NMR: δ 1.83 and 2.05 (m each, 1H and 1H,
PhCH2CH2), 2.60 and 2.74 (m each, 1H and 1H, PhCH2C), 3.85
(m, 1H, NCHP), 5.04 (s, 2H, CH2OC), 6.00 (bd, 1H, C(O)NH),
7.07-7.44 (m, 10H, 2 × C6H5), 7.83 (s, 1H, POH).
Meth yl 2-[[[1-(N-Ben zyloxyca r bon yla m in o)-4-p h en yl-
b u t y l ]h y d r o x y p h o s p h i n y l ]m e t h y l ]-4 -m e t h y l p e n -
ta n oa te (24). The phosphinic analogue: Z-hPhe[CH2]Leu-
OMe was obtained by addition of methyl R-isobutylacrylate
to activated 20 using the procedure described above for leucine
analogues 21-23. After workup the crude material was
purified on column chromatography using the CHCl3/MeOH/
AcOH (100:3:2) solution as the eluent and yielded the product
as a pale glass. Yield: 59%; IR (film, cm-1): 3290 (NH), 3030,
2955, and 2870 (CH), 1740 and 1715 (CdO), 1245 (PdO), 1160,
1130, and 1040 (C-O, P-O); 31P NMR (CDCl3): δ 49.85 and
2-[[(1-Am in o -3-m e t h y lb u t y l)h y d r o x y p h o s p h in y l]-
m eth yl]-4-m eth ylp en ta n oic Acid (4, 27). The peptide 4 was
obtained in the same manner as for 3. The final product was
separated upon treatment of the evaporated residue with Et2O/
hexane. Yield: 84%; IR (KBr, cm-1): 3405 (OH), 3300-2600
(OH, NH, CH), 1745 (CdO), 1240 (PdO), 1205, 1170, and 1100
(C-O, P-O); 31P NMR (D2O, NaOD): δ 45.04 and 45.79 (3:4
ratio); 1H NMR: δ 0.79 (m, 12H, 2 × CH(CH3)2), 1.24-1.91
(m, 8H, 2 × CHCH2CH(CH3)2 and PCH2), 2.49 (m, 1H,
PCH2CH), 2.63 (m, 1H, NCHP). Anal. (C12H26NO4P‚HBr) C,
H, N.
1
50.01 (2:1 ratio); H NMR: δ 0.81 (m, 6H, CH(CH3)2), 1.25-
2.10 (m, 7H, CH2CH(CH3)2, PhCH2CH2 and PCH2), 2.59-2.80
(m, 3H, PhCH2C and PCH2CH), 3.57 (s, 3H, OCH3), 3.85 (m,
1H, NCHP), 5.04 (s, 2H, CH2OC), 5.67 (m, 1H, C(O)NH), 7.07-
7.32 (m, 10H, 2 × C6H5). Anal. (C25H34NO6P) C, H, N.
Repetition of this procedure for compound 23 gave the
final peptide (R)-LeuP[CH2]-(R,S)-Leu (27) as mixture of two
diastereoisomers in a 1:1 ratio.
Meth yl 2-[[[1-(N-Ben zyloxyca r bon yla m in o)-4-p h en yl-
bu tyl]h yd r oxyp h osp h in yl]m eth yl]-3-p h en ylp r op ion a te
(25). The compound 25 was synthesized starting from 20 and
methyl R-benzylacrylate59 and purified the same manner as
24. Yield: 66%; IR (KBr, cm-1): 3320 (NH), 3060, 3030, 2950,
and 2860 (CH), 1730 and 1720 (CdO), 1245 (PdO), 1165, 1135,
and 1035 (C-O, P-O); 31P NMR (DMSO-d6): δ 43.75 (broad);
1H NMR: δ 1.66-1.98 (m, 4H, PhCH2CH2 and PCH2), 2.45,
2.78 and 2.96 (m each, 1H, 2H and 2H, 2 × PhCH2C and
PCH2CH), 3.41 and 3.44 (s each, 3H together, OCH3), 3.60 (m,
1H, NCHP), 5.06 (m, 3H, CH2OC and C(O)NH), 7.08-7.37 and
7.62 (m each, 15H together, 3 × C6H5). Anal. (C28H32NO6P) C,
H, N.
2-[[(1-Am in o -4-p h e n y lb u t y l)h y d r o x y p h o s p h in y l]-
m eth yl]-4-m eth ylp en ta n oic Acid (5). Because of solubility
limitations, pseudopeptides containing homophenylalanine
(24-26) were deprotected using a modified protocol. First, the
methyl ester was cleaved in a 1 M NaOH/MeOH solution. After
overnight stirring, methanol was removed under reduced
pressure. The acid was precipitated by acidification of the
water phase to pH 1 and then extracted with two portions of
AcOEt. The combined organic phases were dried over Na2SO4,
filtered, and evaporated. Then the residue was treated with
33% HBr (1 mL for 0.1 g of the peptide) at room temperature
for 1 h. After evaporation of the volatile components, the
residue was dissolved in methanol, and propylene oxide was
added to achieve neutral pH. The final product was precipi-
tated as a slightly yellow solid upon addition of Et2O. Yield:
85%; mp 141-149 °C; IR (KBr, cm-1): 3405 (OH), 3400-2600
(OH, NH, CH), 1705 (CdO), 1175, 1145, and 1040 (PdO, C-O,
P-O); 31P NMR (D2O, NaOD): δ 44.21 and 44.84 (2:3 ratio);
1H NMR: δ 0.77 (m, 6H, CH(CH3)2), 1.28-1.93 (m, 7H
together, CHCH2CH(CH3)2, C6H5CH2CH2 and PCH2), 2.42 and
2.55 (m each, 1H and 2H, C6H5CH2 and PCH2CH), 2.82 (m,
1H, NCHP), 7.25 (m, 5H, C6H5). Anal. (C16H26NO4P) C, H, N.
Meth yl 2-[[[1-(N-Ben zyloxyca r bon yla m in o)-4-p h en yl-
bu tyl]h yd r oxyp h osp h in yl]m eth yl]-3-(4-h yd r oxyp h en yl)-
p r op ion a te (26). The phosphinic pseudodipeptide: Z-hPhe-
[CH2]Tyr-OMe was synthesized by addition of methyl R-(4-
hydroxybenzyl)acrylate to activated 20. The acrylate was
obtained starting from the appropriate 4-hydroxybenzylma-
lonate in a usual manner.81 The latter compound was prepared
by reduction of 4-hydroxybenzylidenemalonate82 with NaBH4
(3 equiv) in absolute methanol. Purification on column chro-
matography using hexane/ethyl acetate (2:1) solution gave the
acrylate as brownish oil with 42% of total yield. 1H NMR
(CDCl3): δ 3.56 (s, 2H, HOC6H4CH2), 3.74 (s, 3H, OCH3), 5.40
(bs, 1H, OH), 5.47 and 6.21 (m each, 1H and 1H, H2CdC), 6.75
and 7.05 (d each, J ) 8.6 Hz, 2H and 2H, HOC6H4).
The crude phosphinic peptide was purified on column
chromatography using the CHCl3/MeOH/AcOH (100:5:2) solu-
tion yielding the final product 26 as a slightly yellow glass.
Yield: 45%; IR (KBr, cm-1): 3410 (OH), 3325 (NH), 3060, 3030,
2950, 2930, and 2855 (CH), 1720 and 1705 (CdO), 1245 (Pd
O), 1175, 1135, and 1035 (C-O, P-O); 31P NMR (CDCl3): δ
48.30 and 48.54 (2:1 ratio); 1H NMR: δ 1.76 (m, 2H,
PhCH2CH2), 2.05 (m, 2H, PCH2), 2.55 and 2.72 (m each, 1H,
and 3H, PhCH2C and HOC6H4CH2), 2.95 (m, 1H, PCH2CH),
2-[[(1-Am in o -4-p h e n y lb u t y l)h y d r o x y p h o s p h in y l]-
m eth yl]-3-p h en ylp r op ion ic Acid (6). Yield: 89%; mp 154-
161 °C; IR (KBr, cm-1): 3415 (OH), 3400-2600 (OH, NH, CH),
1700 (CdO), 1170 and 1040 (PdO, C-O, P-O); 31P NMR (D2O,
NaOD): δ 44.00 and 44.61 (1:2 ratio); 1H NMR: δ 1.56 (m,
2H, PhCH2CH2), 1.95 (m, 2H, PCH2), 2.60 (m, 2H, PhCH2CH2),
2.72 (m, 2H, PhCH2CH), 2.86 (m, 2H, PCH2CH and NCHP),
7.21 and 7.29 (m each, 4H and 6H, 2 × C6H5). Anal. (C19H24
-
NO4P) C, H, N.
2-[[(1-Am in o -4-p h e n y lb u t y l)h y d r o x y p h o s p h in y l]-
m eth yl]-3-(4-h yd r oxyp h en yl)p r op ion ic Acid (7). Yield:

2652 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Grembecka et al.
82%; mp 158-171 °C; IR (KBr, cm-1): 3400-2600 (OH, NH,
CH), 1700 (CdO), 1240, 1165, and 1040 (PdO, C-O, P-O);
31P NMR (D2O, NaOD): δ 44.52 and 45.01 (3:5 ratio); 1H
NMR: δ 1.53 (m, 2H, PhCH2CH2), 1.89 (m, 2H, PCH2), 2.59
(m, 5H, HOC6H4CH2, PhCH2, and PCH2CH), 2.81 (m, 1H,
NCHP), 6.65 and 6.85 (d each, J ) 8.2 Hz, 2H and 2H,
HOC6H4), 7.26 (m, 5H, C6H5). Anal. (C19H24NO5P) C, H, N.
2-Hyd r oxy-3,6-d iisobu tyl-2-oxo-2λ5[1,4,2]d ia za p h osp h i-
n a n -5-on e (28). The pseudodiketopiperazine analogue of
LeuP[CH2]Leu was obtained during alkaline hydrolysis of
methyl 2-[[(1-amino-3-methylbutyl)hydroxyphosphinyl]methyl]-
4-methylpentanoate. After removal of methanol, the residue
was acidified to pH 1 and extracted three times with CHCl3.
The organic layer was dried, filtered, and evaporated to
dryness giving the crude byproduct. 31P NMR (CDCl3): δ 54.14;
1H NMR: δ 0.98 (m, 12H, 2 × CH(CH3)2), 1.20 and 1.68 (m
each, 1H and 3H, 2 × CHCH2CH), 1.80 and 2.23 (m each, 3H
and 1H, 2 × CH(CH3)2 and PCH2CH2), 2.67 (m, 1H, PCH2CH),
3.66 (m, 1H, NCHP), 6.36 (bd, 1H, NH), 7.16 (bs, 1H, POH).
Kin etic Stu d ies. 1. En zym e P r ep a r a tion . Cytosolic leu-
cine aminopeptidase (LAP) from porcine kidney was obtained
from Sigma Chemical Co and prepared according to the
method described in the literature.66 After 2 h activation in
22 mM triethanolamine hydrochloride buffer, pH 8.5, contain-
ing MnCl2 (1mM), the enzyme solution was used directly in
the kinetic experiments.
APN from pig kidney was obtained as lyophilized powder
from Sigma Chemical Co. The enzyme was diluted with 50
mM potassium phosphate buffer, pH 7.2, and used directly in
kinetic experiments.
2. Assa y of LAP Activity. LAP was assayed at 25 °C in
7.5 mM triethanolamine hydrochloride buffer, pH 8.4, contain-
ing MgCl2 (5mM). The substrate L-leucine p-nitroanilide,
dissolved in DMSO, was added to the assay buffer followed
by the enzyme. The hydrolysis of the substrate was monitored
by following the change in the absorbance measured at 405
nm with a UV-Vis Spectrophotometer Pharmacia LKB, Bio-
chrom 4060. The measured Km value was 1.26 mM, which
remains in very good agreement with the data reported in the
literature.24 The determination of the inhibition constants was
performed as described in the literature.54 All the solutions of
the inhibitors were prepared in the assay buffer, and the pH
was adjusted to 8.4 by the addition of 0.1 M HCl or 0.1 M
NaOH solution, depending on inhibitor. Inhibitors were pre-
incubated with the enzyme for 60 min at room temperature,
which is required to obtain the linear change of absorbance
versus time progress. The assay mixture, which contained the
inhibitor solution (concentration dependent on the inhibitor),
the enzyme solution (20 µg/mL final concentration) and the
assay buffer, was adjusted to 1 mL final volume by addition
of 0.1 mL of substrate solution (0.2, 0.4, 0.6, 0.8, 1.0 mM final
concentrations), which initiated the reaction. Kinetic data were
collected over 10 min assay run at 25 °C. The concentration
of the enzyme was determined spectrophotometrically at 280
nm, assuming A2800.1% ) 0.83 cm-1 and LAP molecular weight
255.000 Da.54
3. Assa y of AP N Activity. APN activity was assayed at
25 °C in 50 mM potassium phosphate buffer, pH 7.2. The
substrate L-leucine p-nitroanilide, dissolved in DMSO, was
added to assay buffer followed by the enzyme. The hydrolysis
of the substrate was monitored by following the change in the
absorbance measured at 405 nm with the UV-Vis spectro-
photometer Pharmacia LKB, Biochrom 4060. The measured
Km value was 0.30 mM, which remains in a good agreement
with the literature data.54 The determination of inhibition
constants was performed as described in the literature.54 All
solutions of inhibitors were prepared in the assay buffer, and
pH was adjusted to 7.5 by the addition of 0.1 M HCl or 0.1 M
NaOH. All inhibitors were preincubated with APN for 60 min
at room temperature. The assay mixture, which contained the
inhibitor solution (concentration dependent on the inhibitor),
the enzyme solution (4 µg/mL final concentration), and the
assay buffer, was adjusted to 1 mL final volume by addition
of 0.1 mL of substrate solution (0.05, 0.1, 0.2, 0.4, 0.6 mM final
concentrations), which initiated the reaction. Kinetic data were
collected over 10 min assay run at 25 °C. The concentration
of the enzyme was determined spectrophotometrically at 280
nm, assuming A2800.1% ) 1.63 cm-1 and APN molecular weight
280.000 Da.54
The Ki values for the phosphinates toward LAP and APN
were determined from Lineweaver-Burk plots for the reaction
monitored in the presence and in the absence of the inhibitor,
using the velocities measured from the linear portion of the
absorbance versus time progress curves. Assays were per-
formed for three or four concentrations of each inhibitor, and
every measurement was repeated twice. The scattering of the
results (Ki values) did not exceed 20% for each tested inhibitor.
Com p u ta tion s. 1. Design of LAP In h ibitor s. The coor-
dinates of leucine aminopeptidase and LeuP were obtained
from the refined 1.65 Å X-ray structure of bovine lens leucine
aminopeptidase complexed with the inhibitor, the phosphonic
analogue of leucine25 (1lcp refcode in Brookhaven Protein Data
Bank). The hydrogen atoms were added using the Insight
97.0 (Accelrys/MSI) program.83 The protonation states of the
residues were selected for pH 7.0, excluding Lys250, which
is neutral, as the nitrogen atom electron lone pair in the
side chain of this residue is coordinated by Zn489.25 The
computer program Ludi84 was applied to design LAP inhibi-
tors. A systematic search of Ludi’s fragment library, containing
about 1000 structural fragments was performed. The LeuP
structure was fixed and Link mode of the program was used
to generate new LAP inhibitors. The structures of the designed
inhibitors were obtained by the replacement of the hydroxyl
group of LeuP with new structural moieties generated using
Ludi, assuming that they would be bound in the S1′ pocket of
the enzyme (Figure 1). New fragments at the P1 position of
inhibitors were obtained by substituting of LeuP side chain
by new ones found in Ludi_link library, which fit well to the
S1 pocket of LAP. The values of the most important Ludi
parameters used for design of new LAP inhibitors were as
follows: Min Separation
) 3; Link, Lipo and H-Bond
Weights were set to 1.0; Aliphatic_Aromatic and Reject
Bifurcated parameters were turned off; No•Unpaired•Polar,
Electrostatic•Check and Invert parameters were turned on;
Es Dist ) 2.5; Max RMS ) 0.5; Number of Rotatable Bonds -
two at a time, Radius of Search was changed from 5 Å to 10
Å.84 Visual inspection of the interactions of designed inhibitors
with LAP was performed within Insight•97.0 program.83
The structures of the designed inhibitors were optimized
within the enzyme active site using the AMBER and CFF97
force fields in Discover/Insight 97.0 program.85 The heavy
atoms of LAP were frozen during the optimization, because of
the lack of the structural changes of the enzyme upon ligand
binding, observed in the crystal structures of leucine ami-
nopeptidase complexed with the inhibitors of different size
(e.g., LeuP - amino acid analogue versus amastatin - tet-
rapeptide analogue).10,86 To maintain the appropriate positions
of the inhibitors with respect to zinc ions in the active site,
the distance constraints between these zinc ions and their
ligands with the force constants values 500 kcal/mol‚Å2 were
applied in the energy minimization procedure. The optimiza-
tion was performed using the conjugate gradient algorithm
until the maximum derivative was <0.1 kcal/mol‚Å. The
predicted binding affinities of the designed inhibitors were
estimated by use the empirical scoring function implemented
in the Ludi program.30,87
2. Ca lcu la t ion of AP N Act ive Sit e Mod el. The amino
acid sequence of APN from pig was obtained from GenBank,
available from the National Center for Biotechnology Infor-
mation (NCBI, www.ncbi.nlm.nih.gov, gi number: 1703286).
The search for homologues proteins in Protein Data Bank was
performed using BLAST program (www.ncbi.nlm.nih.gov/
blast)88,89 and resulted in finding one protein: human leuko-
triene A4 hydrolase/aminopeptidase (LTA4H, 1hs6 code in
PDB) with reasonable sequence similarity (27% identity for
204-526 amino acid fragment of APN, which is involved in
binding site formation). The pairwise sequence alignment of
APN with LTA4H was extracted from the multiple sequence

Organophosphorus Inhibitors of Leucine Aminopeptidase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2653
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alignment of peptidase M1 family members, performed using
CLUSTALW program (http://www.ebi.ac.uk/clustalw).90 This
alignment was then applied for calculation the model of part
of APN catalytic domain (residues 204-526), by application
of MODELLER program, with the default parameters (http://
guitar.rockefeller.edu/modeler/modeler.html).91 The structure
of the remaining part of APN was not modeled, as no templates
with the sequence identity above 20% to these regions were
found in PDB. However, the residues forming APN binding
site originate mostly from 204 to 526 fragment, thus neglecting
the remaining part of the protein in the model seems to be
admissible to analyze the interactions of inhibitors with APN.
The zinc ion was added to APN active site at the position,
which corresponds to zinc site in LTA4H,76 because of the
same residues surrounding zinc binding site in both enzymes
(Figure 3).
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stimulate post-proteasomal trimming of the N terminus of an
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Chem. 1998, 273, 18734-18742.
The hPheP[CH2]Phe and hPheP[CH2]Tyr were docked to the
model of APN based on the interactions of bestatin with
LTA4H (1hs6 in PDB). The model of APN was soaked in 5 Å
water layer and the structures of inhibitors were optimized
within the enzyme active site using the CVFF force field in
Discover/Insight 97.0 program.85 The backbone atoms of APN
were frozen during the optimization and the distance con-
straints between zinc ion and its ligands (His383, His387,
Glu406), with the force constants values 500 kcal/mol*Å2, were
applied in energy minimization procedure. Visual inspection
of interactions between the inhibitors and APN was performed
within Insight•97.0 program.83
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8478.
Ack n ow led gm en t. This work has been supported
by KBN grant 6 PO4B 02 18. Calculations were carried
out using hardware and software resources, including
Accelrys/MSI programs, of Interdisciplinary Center of
Mathematical and Computer Modeling at the University
of Warsaw (ICM), the Supercomputing and Networking
Center in Wrocław and in Poznan´ Supercomputing
Center.
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