Transesterification of monophenyl phosphonamidates - Chemical modelling of serine protease inhibition

Article abstract of DOI:10.1016/S0040-4020(02)00561-6

O-Phenyl phosphonamidates have been designed to bind covalently by nucleophilic substitution to the serine residue in the active site of serine proteases, similarly to the diphenyl phosphonates used as standard. The synthesis of these compounds as well as their phosphonylating reactivity towards methanol, which served as mimetic of the serine nucleophile, is described. The stereochemistry of the substitution in basic solutions was studied in some detail. The stability of the phosphonamidates in aqueous solutions and their selectivity in the reaction against alcohols versus thiols proved that they constitute a class of potential inhibitors of serine proteases, as well as valuable tools to investigate the mechanism of inhibition.

Full text of DOI:10.1016/S0040-4020(02)00561-6

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 5855±5863
Transesteri®cation of monophenyl phosphonamidatesÐchemical
modelling of serine protease inhibition
Artur Muchap and Paweø Kafarski
Â
Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocøaw University of Technology, WybrzezÇe Wyspianskiego 27,
50-370 Wroclaw, Poland
Received 11 February 2002; revised 10 May 2002; accepted 30 May 2002
AbstractÐO-Phenyl phosphonamidates have been designed to bind covalently by nucleophilic substitution to the serine residue in the active
site of serine proteases, similarly to the diphenyl phosphonates used as standard. The synthesis of these compounds as well as their
phosphonylating reactivity towards methanol, which served as mimetic of the serine nucleophile, is described. The stereochemistry of
the substitution in basic solutions was studied in some detail. The stability of the phosphonamidates in aqueous solutions and their selectivity
in the reaction against alcohols versus thiols proved that they constitute a class of potential inhibitors of serine proteases, as well as valuable
tools to investigate the mechanism of inhibition. q 2002 Published by Elsevier Science Ltd.
1. Introduction
intermediate, which is subsequently hydrolysed by a water
molecule to regenerate the active enzyme and release the
product of reaction. The hydrolysis step also proceeds via a
tetrahedral transition state. The whole process is facilitated
by basic±acidic catalysis coming from the presence of the
imidazole ring of His57 and carboxylate residue of Asp102.
More than one hundred members form the serine protease
familyÐone of the four main superfamilies of enzymes that
hydrolyse the amide bond of peptide substrates. Under
normal conditions serine proteases are involved in various
physiological processes, namely blood coagulation,
immune defence, digestion and reproduction. Moreover,
their activity is also associated with many pathological
disorders and consequently with various human diseases
including pulmonary emphysema, rheumatoid arthritis,
in¯ammatory diseases and others. This important role
makes them potential targets for the development of inhibi-
tors in the hope that they might serve as new therapeutic
agents against many diseases.
The synthesis of compounds able to bind covalently to one
of the amino acid residues crucial for activity, and thus
blocking active-site function, was proven to be one of the
most effective strategies in the construction of inhibitors for
serine proteases.1±3 Among phosphorylating agents, the
most popular is diisopropyl phosphoro¯uoridate4 (1),
which nonselectively inactivates enzymes of this class,
and is considered as a diagnostic tool for them. The
structural features of substrate±enzyme selectivity can
be assured by the use a-aminoalkylphosphono¯uoridates
(2)Ðphosphorus containing analogues of amino acids.5,6
However, their application is strongly limited by poor
stability in water solutions. Although less reactive, diphenyl
esters of a-aminoalkylphosphonic acids and their peptide
derivatives (3) are resistant to hydrolysis and since the
beginning of the last decade they have been intensively
exploited as effective and selective inhibitors of various
serine proteases.7±19 They are considered as stable ana-
logues of high-energy tetrahedral transition states of peptide
hydrolysis able to phosphonylate the serine residue in the
active site of enzymes (Fig. 1).
The rational design of new inhibitors and drugs demands
knowledge of chemical mechanisms of enzymatic catalysis.
Generally the proteases belonging to the same family cleave
peptide bonds using the same basic mechanism and the
same or similar catalytic residues. In the case of the serine
family, the catalytic mechanism is based on the presence of
three amino acid residues in the catalytic centre, namely
Ser195, His57 and Asp102 (chymotrypsin numbering
system). The reactive hydroxyl group of Ser195 attacks
the carbonyl group of the scissile amide bond of the
substrate to form a tetrahedral intermediate stabilised by a
speci®c network of hydrogen bonding. In the next step
the substrate±enzyme complex collapses to the acyl
The molecular mechanism of the action of diphenyl phos-
phonates has not yet been fully elucidated. After noncova-
lent binding of the inhibitor, the nucleophilic substitution
proceeding on the phosphorus atom leads to the formation
of an enzyme-Ser-O-phosphonate mixed ester which can be
Keywords: phenyl phosphonates; phosphonamidates; transesteri®cation;
serine proteases; inhibitors.
Corresponding author. Tel.: 148-71-3203354; fax: 148-71-3284064;
e-mail: amucha@kchf.ch.pwr.wroc.pl
p
0040±4020/02/$ -see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020(02)00561-6

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A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
Inhibitors with designed stereochemistry at phosphorus
atom and possessing two different leaving groups are very
interesting tools to facilitate the elucidation of the mecha-
nism of inhibition. In this study we report the synthesis and
the chemical behaviour of a series of such, the O-mono-
phenyl phosphonamidates and mixed esters considered as
potential inhibitors of serine proteases. We compare their
relative phosphonylating reactivity in a model system, using
methanol and a base to mimic the activity of serine protease
active-sites. We have proven that in these conditions
the substitution proceeds much more readily for P±N
compounds than for commonly applied diphenyl phospho-
nates. We also describe a quantitative procedure of their
transesteri®cation to methyl derivatives with the use of
potassium ¯uoride. By using diastereoisomers with a chiral
phosphorus atom we have demonstrated the stereospeci-
®city of the transesteri®cation and showed the selectivity
of the reaction against alcohols versus thiols. The results
of our study on the chemical behaviour of O-phenyl
phosphonamidates indicate that they may represent a new
promising class of compounds useful for the design and
construction of effective and speci®c inhibitors for serine
protease family members.
Figure 1. Organophosphorous inhibitors of serine proteases.
observed in 31P NMR.9 The transesteri®cation process is
accompanied by release of a phenoxy group. However the
crystal structures of enzymes complexed with inhibitors
show that both phenoxy groups were removed,20,21 presu-
mably as a result of another nucleophilic attack of water
molecule. It is not clear how fast this second `ageing'
process is. Both displacements proceed most probably
via pentacoordinate transition states. Such a trigonal
bipyramidal intermediate was recently determined in the
crystal structure of human a-thrombin complexed with the
phosphonate tripeptide in crystals of varying age.22 Interest-
ingly, in this case the nucleophilic attack of water and the
formation of the monoester is postulated to precede
phosphonylation of the enzyme. This assumption is in
good agreement with the study of Pratt and Rahil on the
inhibition of serine-dependent b-lactamases by monophenyl
phosphonate monoanions.23±26 The inhibitors ®rst bind
noncovalently to the enzyme and then, similar to above,
nucleophilic displacement leads to O-phosphonylation
via dianionic transition state containing a pentacoordinate
phosphorus atom.27±29
2. Results and discussion
The synthetic strategy to obtain O-phenyl phosphonami-
dates and mixed esters is illustrated in Scheme 1. Initially,
it involved the synthesis of monophenyl [(N-benzyloxy-
carbonyl)aminobenzyl]phosphonate (5) by two-step pro-
cedure: condensation of benzyl carbamate, benzaldehyde
and triphenyl phosphite30 followed by alkaline hydrolysis
of the resulting diphenyl ester (4).31 The obtained monoester
(5) was converted into the ®nal products using the phospho-
nochloridate strategy, namely the activation of a monoester
with thionyl chloride, which usually ensures medium to
Scheme 1.

A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
5857
Scheme 2.
Scheme 3.
good yields of the appropriate chloridate. The latter can
easily react with an amine or an alcohol nucleophile afford-
ing the ®nal products.32±36 The progress of the reaction
leading to phosphonochloridates was controlled by means
of 31P NMR. The phosphonochloridate (6) was obtained as a
diastereoisomeric mixture in 1:1 ratio, which was seen as
two signals at d 32.98 and 33.36 ppm. The complex signal
appearing at d 10.9 ppm belongs presumably to the pyro-
phosphonate byproduct.35 After aminolysis or alcoholysis
and standard work-up, the compounds (7a±e) were ®nally
puri®ed by column chromatography on silicagel. The yields
of products obtained as mixtures of diastereoisomers varied
between 50±70%.
carried out in these conditions allowed the recovery of
50±75% of the starting material and produced a complex
mixture of byproducts with cleaved amidate, carbamate and
ester moieties. The complete phosphonamidate decompo-
sition, however, also accompanied by signi®cant Cbz loss,
can be achieved by the action of gaseous HCl generated in
methanolic solution in situ by addition of acetyl chloride
(Scheme 2). The yield of mixed phosphonate (8), represent-
ing the main product of this reaction, is 50%.
The satisfactory stability of phosphonamidates encouraged
us to compare their phosphonylating reactivity versus
diphenyl phosphonates in nucleophilic substitutions.
Similarly to phosphonate inhibitors in the active sites of
serine proteases, the studied compounds (4, 7a±e) undergo
transesteri®cation in the presence of a nucleophile (here
methanol) releasing phenol. Basic catalysis is assured by
the addition of triethylamine. The substitution process can
be easily followed by means of 31P NMR and leads to
O-monomethyl derivatives (8, 9a±e, Scheme 3) with the
formation of traces of byproducts being observed (,5%).
The phosphonamidate moiety is frequently considered to
be unstable37±39 even though pseudopeptides containing
this structural fragment are reported as very potent enzyme
inhibitors.40±43 From our recent study44 we know that P±N
lability strongly depends on the state of the N-terminal
amino group and the character of the phosphonamidate
moiety alone. The fully blocked phosphonamidates (7a±d)
obtained in this work appeared to be stable across the whole
pH range. For example, stirring the compound (7b) for three
days in methanol containing hydrochloric acid (pH 1) did
not result in the P±N bond cleavage. In more drastic condi-
tions (methanolic HCl or HBr, pH ,1, elevated temperature
or elongated time) it is partially susceptible to the acidoly-
sis, to approximately the same extent as the carbamate bond
of benzyloxycarbonyl (Cbz) group. Reactions of (7b)
The P±N bond in the resulting phosphonamidates (9a±d)
remains intact. Generally, the displacement proceeds much
more readily for phosphonamidates than for phosphonates
(4, 7e). The yields of the transesteri®cation with presence of
Et3N established on the basis of 31P NMR spectroscopy are
listed in Table 1.
The possibility of reaching quantitative conversion (with
yield exceeding 95%) of O-phenyl phosphonamidates or
phosphonates into their methyl derivatives requires the
application of potassium ¯uoride in the excess of alcohol31
(for the details see Section 4). This reaction, proceeding via
¯uorophosphonates, gave products in roughly 1:1 diastereo-
meric ratio not depending on the diastereomeric compo-
sition of the substrate. On the contrary, transesteri®cation
in basic conditions proceeded stereospeci®cally and the
resulting product has the same stereoisomer ratio, however
most probably of the opposite con®guration as is seen from
the 31P NMR spectra. To check this hypothesis the attempt
to synthesise a single diastereoisomer bearing a chiral
phosphorus atom was undertaken (Scheme 4).
Table 1. Transesteri®cation of phosphonamidates 7 with methanol in the
presence of triethylamine
Starting compound
The yield of the
transesteri®cation product
(%)a
7a, X±RˆNHPh
25 (9a)
90 (9b)
20 (9c)
75 (9d)
5 (9e)
7b, X±RˆNHCH2Ph
7c, X±RˆNHCH2CH2CH(CH3)2
7d, X±RˆL-ValOMe
7e, X±RˆOCH2Ph
4, X±RˆOph
20 (8)
a
The yield established by 31P NMR after 3 days of reaction.

5858
A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
Scheme 4.
The enantiomers of monophenyl [(N-benzyloxycarbonyl)-
aminobenzyl]phosphonate (5) were resolved via their
diastereoisomeric 1-phenylethylamine salts. The obtained
salts were recrystallised several times from acetone until
constant speci®c rotations were achieved. After work-up
and ®nal recrystallisation white, crystalline, optically active
compounds (10a, b) were obtained. As known from the
literature for their analogues,45,46 the absolute con®guration
(R) can be matched to the (1) enantiomer separated using
(R)-1-phenylethylamine (10a) and (S) to the (2) enantiomer
(10b).
recrystallised several times from ethyl acetate to achieve
constant physical data, as well as a single 31P NMR signal.
The two preferentially crystallising compounds obtained in
each of two parallel separations (11a, b) were enantiomeric
to each other.
The obtained diastereoisomers were transesteri®ed in basic
conditions. The reaction proceded with high stereospeci-
®city giving the diastereoisomers of the O-methyl phos-
phonamidates with de exceeding 99% when started from
(R)-(2) O-phenyl derivative and 95% de from (S)-(1) one.
The optically active monophenyl esters were converted into
their N-benzyl phosphonamidates and puri®ed as described
above for the racemic mixtures. The product obtained in
each synthesis was a mixture of two diastereoisomers
in roughly 1:3 ratio (determined from 31P NMR spectra).
To obtain a single diastereoisomer, the compounds were
O-Phenyl phosphonamidates appeared to be resistant to the
action of thiols, what was demonstrated by the lack of any
reaction and in basic conditions with HSCH2COOEt. The
same stability was observed in the presence of KF even
though the substrates are partially converted into phos-
phono¯uoridates, which did not undergo further reaction.

A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
5859
appropriate stereoisomer of phosphonopeptide might exhibit
inhibitory potency. However, the phosphonamidate
compounds reported here, given their potential usefulness
as enzyme inhibitors, may serve as valuable tools to under-
stand the molecular mechanisms of inhibitory action of
phosphorus containing compounds towards serine proteases.
4. Experimental
4.1. General
Figure 2. Organophosphorous pseudopeptide analogues of transition state
of peptide hydrolysis.
3. Conclusions
Unless otherwise stated, starting materials were obtained
from commercial suppliers (Sigma±Aldrich, Merck) and
used without further puri®cation. Thionyl chloride was
freshly distilled. Triethylamine was distilled and stored
over potassium hydroxide. Ethanol-and water-free chloro-
form were obtained by passing the solvent through basic
alumina followed by its distillation from over phosphorus
pentoxide. Anhydrous benzene was obtained by its distil-
lation from over P2O5 and was stored over sodium. Column
chromatography was performed on silica gel 60 (60±230
mesh).
Despite the fact that they appear to be close analogues of
peptides, phosphonamidates were predicted to be poor
inhibitors of serine proteases, due to the fact that the
respective transition state differs from that of tetrahedral
intermediate.9 Additionally, the electrophilicity of the phos-
phorus atom and consequently their phosphonylating
reactivity was supposed to be signi®cantly decreased in
comparison to diphenyl derivatives.9 The results of their
chemical behaviour towards methanol in basic conditions
obtained in this work are quite contradictory and suggest
that phosphonamidates could be successfully applied for
construction of potent and selective inhibitors of serine
proteases. Including the additional amino acid moiety into
the structure of the inhibitor also offers a possibility of
extension the interactions for the S0 subsite of enzymes.
These types of phosphonamidate,6 phosphonate47,48 and
phosphinate49 pseudopeptide analogues (13) have only
been marginally studied until now (Fig. 2).
Melting points were taken on Boetius apparatus and were
not corrected. IR spectra were recorded in KBr pellets on
Perkin±Elmer System 2000 FT IR spectrometer. Proton
and phosphorus NMR spectra were recorded on Bruker
1
DRX spectrometer operating at 300.13 MHz for H and
121.50 MHz for 31P. Measurements were made in CDCl3
or DMSO-d6 solutions. Proton chemical shifts are reported
in relation to tetramethylsilane used as internal standard. 31P
1
Furthermore the mechanism of their action remains unclear.
The con®guration of the phosphorus atom was shown to be
of little consequence regarding their activity.6,47 Final
enzyme±inhibitor complexes exhibit the same geometry at
phosphorus, independent of the starting diasteroisomer
used.48 This indicates the operation of various mechanisms
of substitution, which may proceed either with overall
inversion or with retention of con®guration. In the case of
mixed phosphonate esters, the lack of binding with the S0
subsite even in the case of compounds with conserved
C-terminal fragment is quite surprising (partial cleavage
of both O-phosphonate ester groups is observed instead).48
NMR spectra were obtained with use of broad-band H
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,
Wrocøaw University of Technology.
4.1.1. Diphenyl [(N-benzyloxycarbonyl)aminobenzyl]-
phosphonate (4). Diphenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonate was synthesised by condensation of
benzyl carbamate, benzaldehyde and triphenyl phosphite
as described previously.30 Yield 52%; mp: 137±1408C (lit.
mp: 138±1408C30); 31P NMR (CDCl3): d 15.02.
The mechanism of transesteri®cation described in this
study is obviously much simpler, with presumably a typical
SN2(P) process taking place as indicated by observed
inversion of con®guration (Fig. 3).
4.1.2.
Monophenyl
[(N-benzyloxycarbonyl)amino-
benzyl]phosphonate (5). The monophenyl ester was
obtained by alkaline hydrolysis of the diphenyl ester in
1 M NaOH/dioxane solution in the presence of potassium
¯uoride and 18-crown-6 as described in the literature.31
Yield (after recrystallisation from ethyl acetate) 86%; mp:
163±1648C (lit. mp: 161±1628C31); 31P NMR (DMSO-d6):
This cannot be simply compared to displacement in restricted
geometry of the active site and suggests that only one,
1
d 16.58; H NMR: d 5.04 (AB system, Jˆ12.6 Hz, 2H,
CH2OC(O)), 5.14 (dd, JPHˆ22.3 Hz, JHHˆ10.1 Hz, 1H,
NCHP), 7.03±7.51 (m, 15H, 3£C6H5), 8.39 (bd, Jˆ
10.1 Hz, 1H, C(O)NH).
4.2. The general procedure for the preparation of mixed
O-phenyl esters and phosphonamidates
Monophenyl [(N-benzyloxycarbonyl)aminobenzyl]phos-
phonate (5) (0.80 g, 2 mmol) was suspended in 20 ml of
Figure 3. Intermediate of SN2(P) process of transesteri®cation of phenyl
phosphonamidates.

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A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
dry benzene and thionyl chloride was added (0.22 ml,
3 mmol). The mixture was stirred for 2 h at room tempera-
ture and re¯uxed gently for additional 2 h. Then the volatile
components were evaporated under reduced pressure.
Another portion of 20 ml of benzene was added and the
evaporation was repeated. The resulting phosphonochlori-
date was dissolved in 10 ml of chloroform and added
dropwise to the cooled (08C) mixture of a nucleophile
(2 mmol) and triethylamine (0.42 ml, 3 mmol or 0.70 ml,
5 mmol in the case of aminoester hydrochloride) in 15 ml
of chloroform or benzene. The resulting solution was stirred
at room temperature overnight and then evaporated to
dryness. The mixture was redissolved in ethyl acetate and
washed successively with: 1 M sodium hydroxide, water,
5% hydrochloric acid, water, 1 M sodium carbonate and
brine (20 ml of each). The organic layer was dried over
anhydrous sodium sulphate. After removal of the solvent,
the crude compounds were puri®ed by recrystallisation from
an ethyl acetate/hexane solution or by silica gel column
chromatography using an ethyl acetate/hexane solution as
the eluent to yield white crystalline products.
4.2.4. Methyl {phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonyl}-(L)-valinate (7d). Yield 48%; mp:
155±1728C; IR (KBr, cm21): 3330 and 3230 (NH), 3065
and 2965 (CH), 1735 and 1710 (CvO), 1550 (dNH),
1265 (PvO), 1230, 1205, 1140 and 1025 (C±O, P±O);
31P NMR (CDCl3): d 23.47, 24.00 and 24.32 (2:3:2 ratio);
1H NMR: d 0.60, 0.69, 0.70, 0.79 and 0.85 (d each,
Jˆ6.8 Hz, 6H together, CH(CH3)2), 1.75 and 1.91 (m
each, 1H together, CH(CH3)2), 3.12, 3.37 and 3.50 (dd
each, J1<J2<10.5 Hz, 1H together, PNH), 3.52, 3.54 and
3.59 (s each, 3H together, OCH3), 3.67, 3.80 and 3.91 (m
each, 1H together, NCHC(O)), 5.01±5.14 (AB systems,
Jˆ12.3 Hz, 2H together, CH2OC(O)), 5.25 and 5.34 (dd
each, JHPˆ18.9 Hz, JHHˆ9.6 Hz, 1H together, NCHP),
5.90, 6.05 and 6.30 (m each, 1H together, C(O)NH),
7.08±7.48 (m, 15H, 3£C6H5). Calcd for C27H31N2O6P: C,
63.52; N, 5.49; P, 6.07; found: C, 63.63; N, 5.38; P, 6.20.
4.2.5. Benzyl phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonate (7e). Yield 56%; mp: 133±1368C;
IR (KBr, cm21): 3235 (NH), 3060, 3030 and 2960 (CH),
1715 (CvO), 1550 (dNH), 1255 (PvO), 1200, 1055 and
1020 (C±O, P±O); 31P NMR (CDCl3): d 19.42 and 19.52
(1:2 ratio); 1H NMR: d 4.80 (ABX system, JHaHbˆ11.6 Hz,
JPxHaˆ8.4 Hz, JPxHbˆ7.3 Hz, 2H, POCH2), 5.057 and 5.064
(AB system each, Jˆ12.2 Hz, 2H together, CH2OC(O)),
5.39 and 5.40 (dd each, JHPˆ22.1 Hz, JHHˆ9.6 Hz, 1H
together, NCHP), 5.86 and 5.95 (m each, 1H together,
C(O)NH), 7.05±7.45 (m, 20H, 4£C6H5). Calcd for
C28H26NO5P: C, 68.98; N, 2.87; P, 6.36; found: C, 69.23;
N, 2.73; P, 6.26.
4.2.1. N0-Phenyl O-phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (7a). Yield 64%; mp: 177±
1808C; IR (KBr, cm21): 3345 and 3190 (NH), 3065 and
3030 (CH), 1690 (CvO), 1535 (dNH), 1240 (PvO),
1225, 1205 and 1030 (C±O, P±O); 31P NMR (CDCl3): d
1
20.16 and 20.43 (1:3 ratio); H NMR: d 4.99 and 5.81 (bd
each, 1H together, PNH), 5.10 (AB system, Jˆ12.2 Hz, 2H,
CH2OC(O)), 5.41 and 5.61 (dd each, JHPˆ18.4 Hz, JHH
ˆ
9.7 Hz, 1H together, NCHP), 6.29 and 6.42 (m each, 1H
together, C(O)NH), 6.90±7.50 (m, 20H, 4£C6H5). Calcd
for C27H25N2O4P: C, 68.63; N, 5.93; P, 6.56; found: C,
68.82; N, 5.98; P, 6.46.
4.3. The general procedure for transesteri®cation of
mixed O-phenyl esters or phosphonamidates in
methanol
4.2.2. N0-Benzyl O-phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (7b). Yield 61%; mp: 144±
1468C; IR (KBr, cm21): 3300 and 3230 (NH), 3060 and
3030 (CH), 1720 (CvO), 1550 (dNH), 1255 (PvO),
1205 and 1020 (C±O, P±O); 31P NMR (CDCl3): d 24.55
and 24.78 (3:2 ratio); 1H NMR: d 3.04 and 3.42 (m each, 1H
together, PNH), 3.90 and 4.10 (m each, 2H together,
PNHCH2), 5.04 and 5.06 (AB system each, J1ˆ12.2 Hz,
J2ˆ12.3 Hz, 2H together, CH2OC(O)), 5.33 (dd, JHPˆ
18.7 Hz, JHHˆ9.2 Hz, 1H, NCHP), 6.12 and 6.32 (m each,
1H together, C(O)NH), 6.90±7.47 (m, 20H, 4£C6H5). Calcd
for C28H27N2O4P: C, 69.12; N, 5.76; P, 6.37; found: C,
68.98; N, 5.83; P, 6.35.
The phenyl ester or phosphonamidate (4, 7a±e) (0.2 mmol)
was dissolved in 2 ml of methanol (in certain cases the
addition of 1 ml chloroform was necessary to achieve the
solubility). The solution was stirred overnight after addition
of potassium ¯uoride (60 mg, 1.0 mmol) or for three days
when triethylamine (0.5 ml) was used as catalyst. The yield
of the transesteri®cation was established on the basis of 31P
NMR spectra of the reaction mixture and exceeded 95% for
the reactions carried out in the presence of KF. To obtain the
pure products methanol was evaporated and the resulting
residue dissolved in 10 ml of ethyl acetate, washed with
1 M sodium hydroxide to remove phenol and then washed
with brine (10 ml of each). The organic layer was dried over
anhydrous sodium sulphate. After removing the solvent,
crude products were puri®ed by column chromatography
using an ethyl acetate/hexane solution as the eluent to yield
white crystalline compounds. The yields of the transesteri®-
cation carried out with presence of Et3N are listed in Table 1.
4.2.3. N0-Isoamyl O-phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (7c). Yield 63%; mp: 164±
1688C; IR (KBr, cm21): 3295 (NH), 3065, 3035 and 2960
(CH), 1720 (CvO), 1550 (dNH), 1250 (PvO), 1215, 1200
and 1020 (C±O, P±O); 31P NMR (CDCl3): d 25.16
1
and 25.28 (1:5 ratio); H NMR: d 0.72 and 0.78 (d each,
Jˆ6.6 Hz, 6H together, CH(CH3)2), 1.20 (m, 2H,
CH2CH2CH), 1.45 (m, 1H, CH2CH2CH), 2.74 (m, 1H,
PNH), 2.96 (m, 2H, PNHCH2), 5.06 and 5.11 (AB
system each, Jˆ12.2 Hz, 2H, CH2OC(O)), 5.26 (dd,
JHPˆ18.2 Hz, JHHˆ9.6 Hz, 1H, NCHP), 6.19 (m, 1H,
C(O)NH), 6.94±7.51 (m, 15H, 3£C6H5). Calcd for
C26H31N2O4P: C, 66.94; N, 6.00; P, 6.64; found: C, 66.93;
N, 6.16; P, 6.43.
4.3.1. Dimethyl [(N-benzyloxycarbonyl)aminobenzyl]-
phosphonate. This was obtained by transesteri®cation of
(4) in the presence of KF.31 Mp: 115±1178C (lit. mp:
1
117±118.58C31); 31P NMR (CDCl3): d 5.12; H NMR: d
3.48 and 3.72 (d each, Jˆ10.8 Hz, 3H together, POCH3),
5.10 (AB system, Jˆ12.3 Hz, 2H, CH2OC(O)), 5.19 (dd,
JPHˆ22.0 Hz, JHHˆ9.9 Hz, 1H, NCHP), 5.90 (m, 1H,
C(O)NH), 7.20±7.44 (m, 10H, 2£C6H5).

A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
5861
1
4.3.2. O-Methyl N0-phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (9a). Mp: 164±1688C; IR (KBr,
cm21): 3340 and 3205 (NH), 3060, 3030 and 2950 (CH),
1690 (CvO), 1535 (dNH), 1250 (PvO), 1215 and 1045
(C±O, P±O); 31P NMR (CDCl3): d 24.58 and 24.71 (3:2
NMR (CDCl3): d 24.19 and 24.35 (1:1 ratio); H NMR: d
3.36 and 3.58 (d each, J1ˆ10.8 Hz, J2ˆ10.9 Hz, 3H
together, POCH3), 4.68 (ABX system, JHaHbˆ11.7 Hz,
JPxHaˆ8.5 Hz, JPxHbˆ7.3 Hz, 2H, POCH2), 4.97 and 4.99
(AB system each, J1ˆ12.4 Hz, J2ˆ12.2 Hz, 2H together,
CH2OC(O)), 5.15 (dd, JHPˆ21.9 Hz, JHHˆ9.7 Hz, 1H,
NCHP), 5.90 (m, 1H, C(O)NH), 7.07±7.34 (m, 15H,
3£C6H5). Calcd for C23H24NO5P: C, 64.93; N, 3.29; P,
7.29; found: C, 64.82; N, 3.23; P, 7.21.
1
ratio); H NMR: d 3.48 and 3.66 (d each, J1ˆ10.6 Hz,
J2ˆ11.1 Hz, 3H together, POCH3), 4.91 and 5.05 (AB
system each, Jˆ12.3 Hz, 2H together, CH2OC(O)), 5.19
(dd, JHPˆ17.7 Hz, JHHˆ9.6 Hz, 1H, NCHP), 5.44 and
5.92 (m each, 1H together, PNH) 6.23 and 6.32 (m each,
1H together, C(O)NH), 6.77±7.37 (m, 15H, 3£C6H5). Calcd
for C22H23N2O4P: C, 64.38; N, 6.83; P, 7.55; found: C,
64.28; N, 6.79; P, 7.50.
4.3.7. Methyl phenyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonate (8). This compound was obtained by
transesteri®cation of (4) in the presence of NEt3 or by
cleavage of the P±N bond during the addition of acetyl
chloride to the methanol solution of the N0-benzyl O-phenyl
phosphonamidate (9b) and puri®ed in the same manner as
described above. Mp: 114±1168C; IR (KBr, cm21): 3245
(NH), 3060 and 3030 (CH), 1710 (CvO), 1545 (dNH),
1250 (PvO), 1045 and 1025 (C±O, P±O); 31P NMR
4.3.3. N0-Benzyl O-methyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (9b). Mp: 125±1288C; IR (KBr,
cm21): 3340 and 3235 (NH), 3060, 3035 and 2955 (CH),
1710 (CvO), 1555 (dNH), 1255 (PvO), 1220, 1145 and
1030 (C±O, P±O); 31P NMR (CDCl3): d 29.11 and 29.35
(1:1 ratio); 1H NMR: d 2.98 and 3.34 (m each, 1H together,
PNH), 3.35 and 3.56 (d each, J1ˆ10.8 Hz, J2ˆ10.9 Hz, 3H
together, POCH3), 3.61±4.08 (m, 2H, PNHCH2), 4.94±5.13
(m, 3H, CH2OC(O) and NCHP), 6.25 and 6.44 (m each, 1H
together, C(O)NH), 7.04±7.33 (m, 15H, 3£C6H5). Calcd for
C23H25N2O4P: C, 65.08; N, 6.60; P, 7.30; found: C, 64.99;
N, 6.61; P, 7.37.
1
(CDCl3): d 20.37 and 20.52 (2:3 ratio); H NMR: d 3.54
and 3.79 (d each, Jˆ10.8 Hz, 3H together, POCH3), 5.07
and 5.09 (AB system each, J1ˆ12.6 Hz, J2ˆ12.1 Hz,
2H together, CH2OC(O)), 5.37 (dd, JPHˆ21.9 Hz, JHH
ˆ
9.3 Hz, 1H, NCHP), 5.80 (m, 1H, C(O)NH), 6.91±7.47
(m, 15H, 3£C6H5). Calcd for C22H22NO5P: C, 64.23; N,
3.40; P, 7.54; found: C, 64.26; N, 3.33; P, 7.55.
4.3.4. N0-Isoamyl O-methyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonamidate (9c). Mp: 121±1238C; IR (KBr,
cm21): 3335 and 3230 (NH), 3065, 3035 and 2955 (CH),
1715 (CvO), 1550 (dNH), 1245 (PvO), 1210 and 1025
(C±O, P±O); 31P NMR (CDCl3): d 9.27 and 29.44 (1:5
4.4. Synthesis and transesteri®cation of the diastereo-
isomers of N0-benzyl O-phenyl [(N-benzyloxycarbonyl)-
aminobenzyl]phosphonamidate
The enantiomers of monophenyl [(N-benzyloxycarbonyl)-
aminobenzyl]phosphonate (5) were resolved via their dia-
stereomeric 1-phenylethylamine salts. These salts were
obtained by addition of (R)-(1) or (S)-(2) amine (2.44 g,
0.02 mol) dissolved in 10 ml of ethyl acetate to the suspen-
sion of the monoester (7.95 g, 0.02 mol) in 25 ml of ethyl
acetate. After 10 min of stirring and another 10 min of re¯ux
the solution was left for crystallisation. The separated salts
were recrystallised several times from acetone as far as
1
ratio); H NMR: d 0.72, 0.78 and 0.86 (d each, Jˆ6.6 Hz,
6H together, CH(CH3)2), 1.15 (m, 2H, CH2CH2CH), 1.46
(m, 1H, CH(CH3)2), 2.70 (m, 2H, PNHCH2), 2.90 (m, 1H,
PNH), 3.47 and 3.68 (d each, J1ˆ10.3 Hz, J2ˆ10.7 Hz, 3H
together, POCH3), 5.05 (m, 1H, NCHP), 5.08 (AB system,
Jˆ12.3 Hz, 2H, CH2OC(O)), 5.87 (m, 1H, C(O)NH), 7.29±
7.42 (m, 10H, 2£C6H5). Calcd for C21H29N2O4P: C, 62.36;
N, 6.93; P, 7.66; found: C, 62.55; N, 6.88; P, 7.54.
20
constant speci®c rotations were achieved: [a]D ˆ19.8
20
4.3.5. Methyl {methyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonyl}-L-valinate (9d). Mp: 115±1308C; IR
(KBr, cm21): 3325 and 3235 (NH), 3065, 3035 and 2960
(CH), 1730 and 1695 (CvO), 1535 (dNH), 1255 (PvO),
1215 and 1040 (C±O, P±O); 31P NMR (CDCl3): d 27.81,
for the case of (R)-(1) amine and [a]D ˆ210.2 for
(S)-(2) amine (1% solutions in ethanol). Then the salts
were treated with 10 ml of 1 M NaOH and washed with
ether to remove the separated amine. The enantiomers
were precipitated by acidi®cation of the basic solution to
pH 1. The ®nal recrystallisation from ethyl acetate gave
white, crystalline compounds showing the speci®c rotation
1
28.04, 28.56 and 28.66 (1:1:1:3 ratio); H NMR: d 0.66,
0.70, 0.74, 0.83 and 0.87 (d each, Jˆ6.8 Hz, 6H together,
CH(CH3)2), 1.80 and 1.95 (m each, 1H together, CH(CH3)2),
3.00, 3.10, 3.24 and 3.39 (dd each, J1<J2<10.8 Hz, 1H
together, PNH), 3.42, 3.60 and 3.62 (d each, J1ˆ10.9 Hz,
J2ˆ10.8 Hz, J3ˆ10.9 Hz, 3H together, POCH3), 3.53, 3.56,
3.59 and 3.62 (s each, 3H together, OCH3), 3.69±3.84 (m,
1H, NCHC(O)), 4.95±5.15 (m including two AB systems,
Jˆ12.2 Hz, 3H, CH2OC(O) and NCHP), 5.86 and 6.09 (m
each, 1H together, C(O)NH), 6.74±7.34 (m, 10H, 2£C6H5).
Calcd for C22H29N2O6P: C, 58.92; N, 6.25; P, 6.91; found:
C, 58.64; N, 6.13; P, 6.65.
20
values as follows: [a]D ˆ118.0 for the enantiomer
20
crystallised with the (R)-(1) amine and [a]D ˆ217.9 for
the enantiomer crystallised with the (S)-(2) amine (1%
solutions in 1 M NaOH). The absolute con®guration
(R) can be matched to the (1) enantiomer and (S) to the
(2) enantiomer.45,46 The total yield of the resolution was
25% for each case.
The optically active monophenyl esters (10a, b) were
converted into their N-benzyl phosphonamidates and
puri®ed as described above for the racemic mixture. Each
product was obtained as a mixture of two diastereoisomers
in roughly 1:3 ratio by 31P NMR. To obtain the single
diastereoisomer each mixture was recrystallised several
times from ethyl acetate to achieve constant physical data.
4.3.6. Benzyl methyl [(N-benzyloxycarbonyl)amino-
benzyl]phosphonate (9e). Mp: 87±918C; IR (KBr, cm21):
3265 (NH), 3060 and 3030 (CH), 1725 (CvO), 1545
(dNH), 1255 (PvO), 1060 and 1010 (C±O, P±O); 31P

5862
A. Mucha, P. Kafarski / Tetrahedron 58 (2002) 5855±5863
3. Walker, B.; Lynas, J. F. Cell. Mol. Life Sci. 2001, 58, 596±
624.
The values of speci®c rotations and melting points were
monitored as well as 31P NMR spectra The data for the
preferentially crystallising phosphonamidate diastereo-
isomer (11a) obtained from (R)-(1) phenyl monoester:
4. Jansen, E. F.; Nutting, M. D. F.; Balls, A. K. Adv. Enzymol.
1952, 13, 321±343.
20
5. Lamden, L. A.; Bartlett, P. A. Biochem. Biophys. Res.
Commun. 1983, 112, 1085±1090.
mp: 188±1898C and [a]D ˆ234.7 and for the (11b)
20
obtained from (S)-(2) monoester: mp: 187±1888C,
6. Bartlett, P. A.; Lamden, L. A. Bioorg. Chem. 1986, 14, 356±
377.
[a]D ˆ133.4 (1% solutions in MeOH). The obtained two
compounds are enantiomeric to each other. The yield of the
resolution was 20% for each case.
7. Oleksyszyn, J.; Powers, J. C. Biochem. Biophys. Res.
Commun. 1989, 161, 143±149.
4.4.1. (R)-(2)-N0-Benzyl O-phenyl [(N-benzyloxycarbo-
nyl)aminobenzyl]phosphon-amidate (11a). Mp: 188±
8. Fastrez, J.; Jespers, L.; Lison, D.; Renard, M.; Sonveaux, E.
Tetrahedron Lett. 1989, 30, 6861±6864.
1
1898C; 31P NMR (CDCl3): d 24.78; H NMR: d 3.34 (m,
9. Oleksyszyn, J.; Powers, J. C. Biochemistry 1991, 30, 485±
493.
1H, PNH), 4.10 (m, 2H, PNHCH2), 5.06 (AB system,
Jˆ12.3 Hz, 2H, CH2OC(O)), 5.33 (dd, JHPˆ18.1 Hz,
JHHˆ9.8 Hz, 1H, NCHP), 6.29 (d, Jˆ7.8 Hz, 1H,
C(O)NH), 6.90±7.49 (m, 20H, 4£C6H5). Calcd for
C28H27N2O4P: C, 69.12; N, 5.76; P, 6.37; found: C, 69.07;
N, 5.73; P, 6.45.
10. Cheng, L.; Goodwin, C. A.; Scully, M. F.; Kakkar, V. V.;
Claeson, G. Tetrahedron Lett. 1991, 32, 7333±7336.
11. Wang, C.-L. J.; Taylor, T. L.; Mical, A. J.; Spitz, S.; Reilly,
T. M. Tetrahedron Lett. 1992, 33, 7667±7670.
12. Oleksyszyn, J.; Boduszek, B.; Kam, C.-M.; Powers, J. C.
J. Med. Chem. 1994, 37, 226±231.
13. Boduszek, B.; Oleksyszyn, J.; Kam, C.-M.; Selzler, J.; Smith,
R. E.; Powers, J. C. J. Med. Chem. 1994, 37, 3969±3976.
14. Oleksyszyn, J.; Powers, J. C. Meth. Enzymol. 1994, 244, 423±
441.
The obtained single diastereoisomers (11a, b) (25 mg of
each) were transesteri®ed in basic conditions (in the
presence of triethylamine) as described above. The reaction
followed by 31P NMR proceeded with high stereospeci®city
giving the diastereoisomers of the N0-benzyl O-methyl
phosphonamidate (12a, b) with de exceeding 99% starting
from (R)-(2)-N0-benzyl O-phenyl phosphonamidate (11a)
(the puri®cation and the data described below) and 95% de
starting from (S)-(1)-N0-benzyl O-phenyl phosphonamidate
15. Bergin, C.; Hamilton, R.; Walker, B.; Walker, B. J. Chem.
Commun. 1996, 1156±1156.
16. Hamilton, R.; Walker, B.; Walker, B. J. Bioorg. Med. Chem.
Lett. 1998, 8, 1655±1660.
17. Jackson, D. S.; Fraser, S. A.; Ni, L.-M.; Kam, C.-M.; Winkler,
U.; Johnson, D. A.; Froelich, C. J.; Hudig, D.; Powers, J. C.
J. Med. Chem. 1998, 41, 2289±2301.
1
(11b) (established on the basis of H and 31P NMR).
To collect the spectroscopic data of the pure product (11a)
the reaction mixture was evaporated to dryness and the
residue was redissolved in 5 ml of ethyl acetate. The organic
layer was successively washed with: 1 M sodium hydroxide,
5% hydrochloric acid, 1 M sodium carbonate and brine
(5 ml of each) and then dried over anhydrous sodium
sulphate. Crude product obtained after evaporation of
solvents was puri®ed by column chromatography using
ethyl acetate/hexane (3:1) solution as the eluent.
18. Morty, R. E.; Troeberg, L.; Powers, J. C.; Ono, S.; Lonsdale-
Eccles, J. D.; Coetzer, T. H. T. Biochem. Pharm. 2000, 60,
1497±1504.
19. Oleksyszyn, J. Aminophosphonic and Aminophosphinic Acid
Derivatives in the Design of Transition-state Analogue
Inhibitors: Biomedical Opportunities and Limitations. In
Aminophosphonic and Aminophosphinic Acids. Chemistry
and Biological Activity; Kukhar, V. P., Hudson, H. R., Eds.;
Wiley: Chichester, 2000; pp 537±558.
20. Bertrand, J. A.; Oleksyszyn, J.; Kam, C.-M.; Boduszek, B.;
Presnell, S.; Plaskon, R. R.; Suddath, F. L.; Powers, J. C.;
Williams, L. D. Biochemistry 1996, 35, 3147±3155.
21. Hof, P.; Mayr, I.; Huber, R.; Korzus, E.; Travis, J.; Powers,
J. C.; Bode, W. EMBO J. 1996, 15, 5481±5491.
22. Skordalakes, E.; Dodson, G. G.; Green, D. S. C.; Goodwin,
C. A.; Scully, M. F.; Hudson, H. R.; Kakkar, V. V.; Deadman,
J. J. J. Mol. Biol. 2001, 311, 549±555.
4.4.2. (R)-(1)-N0-Benzyl O-methyl [(N-benzyloxycarbo-
nyl)aminobenzyl]phosphon-amidate (12a). Mp: 164±
20
1888C; [a]D ˆ15.0 (1% solutions in MeOH); 31P NMR
1
(CDCl3): d 29.04; H NMR: d 2.60 (m, 1H, PNH), 3.61
(d, Jˆ10.8 Hz, POCH3), 3.61±4.08 (m, 2H, PNHCH2),
5.02 (d, Jˆ12.2 Hz, 2H, CH2OC(O)), 5.07 (m, 1H,
NCHP), 5.75 (m, 1H, C(O)NH), 7.06±7.28 (m, 15H,
3£C6H5). Calcd for C23H25N2O4P: C, 65.08; N, 6.60; P,
7.30; found: C, 65.33; N, 6.68; P, 7.11.
23. Pratt, R. F. Science 1989, 246, 917±919.
24. Rahil, J.; Pratt, R. F. Biochem. J. 1991, 275, 793±795.
25. Rahil, J.; Pratt, R. F. Biochemistry 1993, 32, 10763±10772.
26. Rahil, J.; Pratt, R. F. Biochem. J. 1993, 296, 389±393.
27. Rahil, J.; Pratt, R. F. Biochemistry 1992, 31, 5869±5878.
28. Chen, C. C. H.; Rahil, J.; Pratt, R. F.; Herzberg, O. J. Mol.
Biol. 1993, 234, 165±178.
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