Structure-activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase IV inhibitors.

Article abstract of DOI:10.1021/jm981033g

The previously reported diphenyl 1-(S)-prolylpyrrolidine-2(R, S)-phosphonate (5) was used as a lead compound for the development of potent and irreversible inhibitors of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). The synthesis of a series of diaryl 1-

Full text of DOI:10.1021/jm981033g

J . Med. Chem. 1999, 42, 1041-1052
1041
Str u ctu r e-Activity Rela tion sh ip of Dia r yl P h osp h on a te Ester s a s P oten t
Ir r ever sible Dip ep tid yl P ep tid a se IV In h ibitor s
Alexander Belyaev,† Xuemei Zhang,† Koen Augustyns,† Anne-Marie Lambeir,‡ Ingrid De Meester,‡
Irina Vedernikova,† Simon Scharpe´,‡ and Achiel Haemers*,†
Departments of Pharmaceutical Chemistry and Medical Biochemistry, University of Antwerp (UIA), Universiteitsplein 1,
B-2610 Antwerp, Belgium
Received October 9, 1998
The previously reported diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate (5) was used as
a lead compound for the development of potent and irreversible inhibitors of dipeptidyl peptidase
IV (DPP IV, EC 3.4.14.5). The synthesis of a series of diaryl 1-(S)-prolylpyrrolidine-2(R,S)-
phosphonates with different substituents on the aryl rings (hydroxyl, methoxy, acylamino,
sulfonylamino, ureyl, methoxycarbonyl, and alkylaminocarbonyl) started from the corresponding
phosphites. A good correlation was found between the electronic properties of the substituent
and the inhibitory activity and stability. The most striking divergence of this correlation was
the high potency combined with a high stability of the 4-acetylamino-substituted derivative
11e. This compound shows low cytotoxicity in human peripheral blood mononuclear cells and
also has favorable properties in vivo. Therefore bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-
2(R,S)-phosphonate (11e) is considered as a major improvement and will be a highly valuable
DPP IV inhibitor for further studies on the biological function of the enzyme and the therapeutic
value of its inhibition.
Ch a r t 1
In tr od u ction
Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is a
highly specific exopeptidase with serine-type mechanism
protease activity, cleaving off dipeptides from the ami-
noterminus of peptides with proline at the penultimate
position. It is constitutively expressed on epithelial or
endothelial cells of a variety of different tissues and is
also found in body fluids. In the hematopoietic system
DPP IV was identified as the leukocyte antigen CD26,1
where its expression depends strongly on the dif-
ferentiation and activation status. T cells with a high
surface density of CD26 have been shown to be respon-
sible for proliferation in response to recall antigen in
vitro.
Several inhibitors of DPP IV are described belonging
to different chemical classes, most of them dipeptide
analogues with a proline mimic in P-1 (Chart 1).
Pyrrolidides and thiazolidides2,3 1 are reversible, com-
petitive inhibitors with inhibition constants in the
micromolar range. Pyrrolidine-2-nitriles4,5 2 are revers-
ible inhibitors in the submicromolar range. Diacyl-
hydroxylamines6 3 are irreversible inhibitors, and bo-
ronic acids7 (e.g., Pro-boroPro 4) are very potent and
specific transition-state analogues with a reversible,
slow-binding inhibition. Unfortunately they are un-
stable in aqueous solution at neutral pH, due to a
cyclization reaction between the free amino group of the
P-2 amino acid and the boronic acid.8 The pinanediol
esters of these boronic acids are highly potent inhibitors
not only because of hydrolysis to the free boronic acid
but also because they are active on themselves.8 Dipep-
tide diphenyl phosphonates9,10 (e.g., diphenyl 1-(S)-
prolylpyrrolidine-2(R,S)-phosphonate hydrochloride, (S)-
Pro-(S,R)-ProP(OPh)2, 5) are potent, specific, and irrevers-
ible inhibitors leading to a phosphonylated serine at the
active site of the enzyme. In this case, the recovery of
activity of DPP IV was very slow, i.e., only around 10%
was regained after 4 weeks.11 Recently it was shown
that peptides containing a N-terminal X-X-Pro sequence
also present DPP IV inhibitory activity, like the HIV-1
Tat protein and the shorter Tat(1-9) fragment as well
as other synthetic X-X-Pro peptides with a minimum
length of 6 amino acids.12
Several biological functions for DPP IV are reported.
The unique proteolytic specificity points to participation
in peptide metabolism. Dipeptide cleavage has been
proven for several biologically active peptides,13,14 e.g.,
the CC-chemokine RANTES.15 Moreover, DPP IV is
essential for intestinal and renal transport of proline-
containing peptides.16,17 The ability to interact with
proteins of the extracellular matrix18 might be impor-
tant during cell migration. The function of DPP IV in
the immune system is thoroughly investigated.19 Trig-
gering of CD26 provides a costimulatory signal for T
cell receptor (TcR/CD3)-mediated T cell activation,
leading to IL-2 production, IL-2 receptor upregulation,
and T cell proliferation. The use of specific DPP IV
† Department of Pharmaceutical Chemistry.
‡ Department of Medical Biochemistry.
10.1021/jm981033g CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/25/1999

1042 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
Sch em e 1a
a
Reagents: (i) PCl3; (ii) HCl; (iii) HOAc, 90 °C, 2 h; (iv) HCl/EtOAc (1 M); (v) H2, Pd/C. For 8-11: R1 ) (a ) H, (b) 4-OMe, (c) 4-OAc
(4-OH for 11), (d ) 3-NHAc, (e) 4-NHAc, (f) 4-NHSO2Me, (g) 3-NHCONH2, (h ) 4-(N-Bz-Gly-NH), (i) 4-(N-Z-Gly-NH) [4-(H-Gly-NH) for 11],
(j) 4-(N-Z-(S)-Ala-NH) [4-(H-(S)-Ala-NH) for 11], (k) 4-((S)-Pyr-NH), (l) 4-[(2S)-MeO2CCH(NHAc)CH2], (m ) 4-COOMe, (n ) 4-(CONHCH2COOEt),
(o) 4-[CONH(CH2)2COOMe], (p ) 4-(CONH(CH2)2CH3).
inhibitors (Pro-boroPro 4) suppresses T cell proliferation
in vitro7 and decreases antibody production in mice
immunized with BSA.20 Several DPP IV inhibitors also
suppressed alkyldiamine-induced arthritis in a dose-
dependent manner.21 The disadvantage of the boronic
acid 4 is that frequent administration was required to
maintain serum DPP IV activity at a low level, due to
rapid inactivation of the inhibitor. In this respect, the
dipeptide diphenyl phosphonate 5 is advantageous
because of profound and long-lasting irreversible inhibi-
tion and higher stability (t1/2 in human citrated plasma
) 5 h).11 Intravenous injection of a single dose of 5 (1,
5, or 10 mg) in rabbits causes a decrease in plasma DPP
IV activity with more than 80%, and it takes more than
20 days for complete recovery. Not only plasma DPP
IV was inhibited, but also DPP IV in circulating
lymphocytes and peripheral tissues.22 This prompted us
to investigate the role of DPP IV/CD26 in alloantigen-
driven T cell activation. Heart transplantation in rats
gives an early (24-h) increase in cellular CD26 expres-
sion followed by a rise in DPP IV serum activity, which
peaked at day 6, i.e., before the time of actual graft loss.
Diphenyl phosphonate 5 abrogates acute rejection and
prolongs cardiac allograft survival from 7 to 14 days.
This was associated with severely impaired host cyto-
toxic T lymphocyte responses in vitro.23
Pyrrolidine-2-nitrile inhibitors of DPP IV have shown
to inhibit HIV-1 infection in a T cell line, suggesting
that DPP IV/CD26 enzymatic acitivity may play a role
in facilitating HIV-1 infection of human CD4+ T cells
at the entry process.24
The recent results showing that DPP IV/CD26 plays
an important role in the mechanism of allograft rejection
in vivo prompted us to investigate in more detail the
structure-activity relationship of dipeptide diphenyl
phosphonates as inhibitors. We have shown previously11
in a study on the role of the P-2 amino acid that proline
at this position gave one of the most active, and certainly
the most stable, inhibitors in human citrated plasma.
It was described9 that bis(4-chlorophenyl) phosphonates
are more active than their nonsubstituted congeners.
These data prompted us to investigate the influence of
different functional groups on the inhibitory activity of
these phosphonates. We synthesized prolylpyrrolidine
diphenyl phosphonates, substituted on the phenyls with
hydroxyl, methoxy, acylamino, sulfonylamino, ureyl,
methoxycarbonyl, and alkylaminocarbonyl groups. The
phenyl esters were also replaced by other groups with
good leaving group capacities such as trichloroethyl and
trifluoroethyl. The DPP IV inhibitory activity in vitro
and in vivo, the stability, and the specificity of these
compounds will be described.
Resu lts a n d Discu ssion
Syn th esis. The synthesis of a series of prolylpyrrol-
idine diaryl phosphonates started with the coupling of
4-aminobutyraldehyde diethyl acetal to N-protected
proline, activated as mixed anhydride with isobutyl
chloroformate, as published previously.10 The acetal 6
was hydrolyzed with HCl, and the crude aldehyde 7 was
treated with the corresponding triaryl phosphite 9 in
acetic acid to give diastereoisomeric mixtures of the
protected prolylpyrrolidine diaryl phosphonate 1010
(Scheme 1). Deprotection using standard methods af-
forded the final compounds 11.
For the preparation of substituted phenyl phosphon-
ates, it was necessary to prepare some commercially
unavailable phenols. The 4-(methylsulfonylamino)-
phenol (8f) was prepared from the corresponding sulf-
onyl chloride and 4-aminophenol. Likewise, the 4-(acyl-
amino)phenols 8h -k were prepared by condensation of
the corresponding carboxylic acid with 4-aminophenol
using the mixed anhydride method. N-Acetyl-L-tyrosine
methyl ester (8l) was prepared as described.25 The
glycine derivative 8n was obtained after condensation
of glycine ethyl ester with 4-hydroxybenzoic acid using
diphenyl phosphorylazide (DPPA). The synthesis of the
4-hydroxybenzoic acid amides 8o and 8p was ac-
complished by coupling of 4-acetoxybenzoic acid with the
corresponding amine using the mixed anhydride method

SAR of Phosphonate Esters as DPP IV Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 1043
Sch em e 2a
acid in dipeptide diphenyl phosphonates, we showed
that proline in this position gives one of the most potent
inhibitors. A major advantage of 5 was its greater
stability in human citrated plasma compared to the
other dipeptide derivatives. The stability of 5 in plasma
equals the stability in buffer, whereas for the other
compounds the stability in plasma was reduced com-
pared to buffer. This reflects the relative stability of a
Pro-Pro amino acid sequence to proteolytic breakdown
and indicates that the decrease in activity of 5 is mainly
caused by hydrolysis of the phosphonate ester. To
increase the inhibitory activity while retaining the
stability, we introduced several substituents on the
phenyl rings that act as leaving groups. Powers et al.
showed that a 4-chlorophenoxy group improved the
inhibition considerably in the case of DPP IV9, but not
for other serine proteases.28 The influence of electron-
donating or -withdrawing substituents on enzyme in-
hibition and stability was investigated as shown in
Table 1. All compounds were irreversible inhibitors of
DPP IV, probably due to the formation of a phosphon-
ylated serine at the active site of the enzyme. The
inactivation rate constants were calculated from experi-
mental IC50 values and were in reasonable agreement
with the measured inactivation rate constants, where
available.
a
We observed a good correlation between the electron-
withdrawing properties of the substituent and its activ-
ity (Figure 1). Introduction of an electron-donating
substituent (4-OH, 11c) decreases potency, whereas an
electron-withdrawing substituent (4-methoxycarbonyl,
11m ) increases potency. The most striking divergence
in the correlation between the Hammett constant and
the inhibitory activity is the strong inhibition caused
by the 4-(acetylamino)phenyl (11e) and the 4-(methyl-
sulfonylamino)phenyl (11f) phosphonate esters. These
compounds have similar Hammett constants compared
to the diastereoisomeric mixture of 5 but are neverthe-
less about 100 times more potent. This indicates that
other features might also be important for binding. The
equal potency of the 3-acetylamino (11d ) and the
4-acetylamino (11e) also points to an extra interaction
of 11e with the active site of the enzyme, considering
the fact that 11d should be much more active because
of a higher electron-withdrawing potential. The pos-
sibility of an extra interaction was further investigated
by introduction of other acylamino substituents. The
4-glycylamino (11i) and the 4-alanylamino (11j) have a
comparable activity but are considerably less stable in
plasma than the 4-acetylamino (11e)-substituted di-
phenyl phosphonate. We suppose that this is due to
proteolytic cleavage of 11i and 11j to the 4-amino
derivative, which will be considerably less active be-
cause of its strong electron-donating properties (Ham-
mett constant of -0.57 gives a calculated IC50 of 975
µM). This conversion of a very active to an almost
inactive molecule gives a rapid decrease of the apparent
IC50 value, reflected in a short half-life. To prove this
assumption, we measured the stability of 11i and 11j
in buffer (50 mM Tris‚HCl, pH ) 8). The half-lives of
11i (96 ( 7 min) and 11j (99 ( 4 min) in buffer are
higher than in plasma and equal the half-life of the
N-benzoyl-protected 4-glycylamino derivative 11h in
plasma. This indicates that the breakdown of 11i and
Reagents: (i) PCl3; (ii) AcOH, Et3N; (iii) 7, HOAc, 90 °C, 2 h;
(iv) H2, Pd/C; (v) HCl/EtOAc (1 M).
followed by mild alkaline hydrolysis of the phenyl esters
12 and 13.26 The triaryl phosphites 9 were then syn-
thesized from the corresponding substituted phenols 8
and phosphorus trichloride.
Cyclic N-protected 2,2′-biphenylyl derivatives 17a and
17b were prepared by reacting 2,2′-biphenylylacetyl
phosphite (16) with aldehyde 7a or 7b in acetic acid
(Scheme 2). Attempted removal of benzyloxycarbonyl (Z)
protection from phosphonate 17a by hydrogenolysis
with a Pd/C catalyst in methanol resulted in the opening
of the dioxaphosphepan ring to give the mixed methyl
aryl ester 18. Deprotection of the Boc derivative 17b
with HCl/EtOAc yielded free 2,2′-biphenylyl phos-
phonate 19.
Phosphonate analogues with 2,2,2-trichloroethyl and
2,2,2-trifluoroethyl as leaving groups27 were prepared
similarly to the aryl esters by reaction of aldehyde 7a
with the corresponding phosphites, followed by depro-
tection of the resulting phosphonates 20 and 21 to the
target compounds 22 and 23.
Generally, with this method we obtained a mixture
of diastereoisomers of the phosphonates 10. To obtain
the pure diastereoisomers of 5, it was necessary to
introduce a trityl protection after removal of the Z-
protection, which resulted in easily separable isomers
(10a (S,R) and 10a (S,S)). We suppose that the most
active isomer has the R configuration at the carbon atom
next to phosphorus, and this was confirmed by com-
parison of the relative mobility on TLC, optical activity,
1H NMR spectrum, and biological activity with that of
(S)-Ile-(R)-ProP(OPh)2. The configuration of this refer-
ence compound was unambiguously determined with
X-ray crystallography.10
DP P IV In h ibition a n d Sta bility of th e In h ibi-
tor s. In a previous study11 on the role of the P-2 amino

1044 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
Ta ble 1. Potency and Stability of DPP IV Inhibitorsa
config at
kcalc (M-1
s-1
t1/2 (min)
in plasma
compd
R1
ProP C(2)
IC50 (µM)
)
5
H
H
H
R,S
R
S
32 (n ) 1)
2.4 × 101
250 ( 10
300 ( 30
11a
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
18
15 ( 3 (n ) 3)
5.1 × 101
>104
4-MeO
4-HO
3-AcNH
4-AcNH
4-MeSO2NH
3-H2NCONH
4-(N-Bz-Gly-NH)
4-(H-Gly-NH)
4-(H-(S)-Ala-NH)
4-((S)-Pyr-NH)
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
R,S
22 ( 11 (n ) 4)
190 ( 80 (n ) 3)
0.8 ( 0.1 (n ) 2)
0.4 ( 0.2 (n ) 5)
0.40 ( 0.02 (n ) 2)
2.3 ( 0.3 (n ) 2)
0.7 ( 0.3 (n ) 2)
0.5 ( 0.1 (n ) 2)
0.6 ( 0.2 (n ) 2)
5.0 ( 0.8 (n ) 2)
1.4 ( 0.5 (n ) 4)
0.016 ( 0.004 (n ) 2)
0.023 ( 0.007 (n ) 2)
0.036 ( 0.006 (n ) 4)
0.03 ( 0.01 (n ) 2)
47 ( 18 (n ) 2)
31 ( 6 (n ) 2)
3.5 × 101
4.1
470 ( 90
.200
9.6 × 102
1.9 × 103
1.9 × 103
3.3 × 102
1.1 × 103
1.5 × 103
1.3 × 103
1.5 × 102
5.5 × 102
4.8 × 104
3.3 × 104
2.1 × 104
2.6 × 104
1.6 × 101
2.5 × 101
4.3 × 10-1
8.5 × 10-2
220 ( 30
320 ( 140
150 ( 30
210 ( 80
93 ( 3
28 ( 3
8 ( 1
170 ( 30
190 ( 150
19 ( 1
4-[(2S)-MeO2CCH(NHAc)CH2]
4-MeO2C
4-(EtO2CCH2NHCO)
4-[MeO2C(CH2)2NHCO]
4-[CH3(CH2)2NHCO]
P(OMe)(OC6H4(2-OH-C6H4))
P-2,2′-biphenylyl
P(OCH2CCl3)2
35 ( 2
26 ( 1
12 ( 1
140 ( 80
6 ( 1
19
22
23
1800 (n ) 1)
9100 (n ) 1)
P(OCH2CF3)2
a
IC50 values were determined after 15-min preincubation with DPP IV at 37 °C. The listed values are the average of n independent
measurements ( the standard deviation. Functional stability in plasma was estimated by fitting the inverse of the apparent IC50 values
versus time with a single-exponential decay. The half-life is listed ( the standard error of fit.
F igu r e 1. Correlation between Hammett constant and log
calc. A good correlation (log k ) 4.6504σ + 2.5472, r2 ) 0.82)
was observed between the Hammett constant and log kcalc, the
most striking divergence being the difference between the
unsubstituted 5 and the 4-acetylamino 11e.
F igu r e 2. Correlation between Hammett constant and log
half-life. A good correlation (log t1/2 ) -2.2138σ + 2.3043, r2
) 0.77) was observed between the Hammett constant and log
k
t1/2
.
by hydrolysis of the phosphonate esters. The more
electron-withdrawing derivatives (methoxycarbonyl and
alkylaminocarbonyl, 11m -p ) are very potent inhibitors
(IC50 ) 16 nM) but are also unstable (t1/2 ) 19 min).
The only exceptions to the correlation between electronic
properties and half-life are the aminoacyl derivatives
11i and 11j (vide supra) and the dioxaphosphepan ring
19. The chemical instability of this ring is reflected in
its short half-life in plasma. This compound has the
same potency as 5, but its hydrolyzed derivative shows
11j in plasma is caused by hydrolysis of the phosphonate
ester and also by cleavage of the amino acid substituent
and that this cleavage can be overcome with a N-benzoyl
protection.
The half-life of the inhibitors seems to correlate also
with the Hammett constant (Figure 2). Compounds with
similar electronic properties (e.g., 5 and 11e) have a
similar stability in plasma. This is in agreement with
the fact that the decrease in activity is mainly caused

SAR of Phosphonate Esters as DPP IV Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 1045
cultures. The compounds 11e and 11n are therefore
promising tools for further studies on a cellular level.
The irreversible mechanism of inhibition overcomes the
rather limited stability of the compounds in biological
media.
DP P IV In h ib it ion in Vivo. We reported previ-
ously22 that a single intravenous injection of 5 (0.3-5
mg/kg) in rabbits caused a decrease in plasma DPP IV
activity with more than 80%, and it took more than 20
days for complete recovery.
In rats, a comparable dose per weight intravenously
did not result in a sufficient inhibition of circulating
DPP IV, because adequate inhibition could not be
reached without severe systemic toxicity. However, a
combination of subcutaneous and intraperitoneal injec-
tions of 11a allowed us to bring plasma DPP IV to less
than 15% of pretreatment values. The observation that
monotherapy with the diastereoisomeric mixture of 11a
(5) not only significantly prolonged graft acceptance
upon alloantigen challenge23 but occasionally also caused
systemic toxicity and more often local ulcerations stimu-
lated the in vivo testing of 11e and 11n in rabbits, rats,
and mice.
In rabbits, the higher in vitro potency of 11n com-
pared to 11e was also observed. The IC50 values for
inhibition of plasma DPP IV in rabbits upon single
intravenous injection was below 20 µg/kg for 11n and
was around 0.2 mg/kg for 11e. In this species single
intravenous injection of 2 mg/kg of 11n inhibited plasma
DPP IV activity for more than 95% during at least 24
h, without side effects.
In rats, 11e as well as 11n could keep plasma DPP
IV activity below 10% of pretreatment values by daily
subcutaneous injection of 50 mg/kg (initial dose 100 mg/
kg), without any sign of acute systemic or local toxicity.
In mice, a pharmacologically useful inhibition was
only obtained with 11n and not with 11a and 11e. The
molecular basis for the large interspecies differences in
efficiency remains to be elucidated. Due to the higher
intrinsic stability, 11e was selected for further inves-
tigations in rat disease models.
Sp ecificity for DP P IV. The specificity for DPP IV/
CD26 was examined by comparison with inhibition rates
for other proteases (Table 2). Prolyl endopeptidase
(PEP), dipeptidyl peptidase II (DPP II), membrane
alanyl aminopeptidase (MAAP) and elastase were used
for compounds 11a , 11e, and 11n , also studied in vitro
and in vivo. At concentrations equal to the IC50 value
for DPP IV, these compounds were specific, because only
a small inhibition was noticed for 11n with PEP (15%)
and DPP II (22%). This is remarkably specific knowing
that DPP II is quite similar to DPP IV, being also a
serine-type exopeptidase, cleaving off dipeptides from
the amino terminus, with some specificity for proline
at the penultimate position. At a higher concentration
(1 mM, 40000-fold higher than the IC50 value for DPP
IV) 11n gives complete inhibition of PEP and elastase,
some inhibition of DPP II, and almost no inhibition of
MAAP. Some other compounds were only used to
measure their inhibitory potency for PEP. Here also, a
concentration equal to the IC50 value for DPP IV gives
a very small inhibition of PEP. In general, 50% inhibi-
tion of PEP was obtained at concentrations 1250-1700-
fold higher and complete inhibition at concentrations
F igu r e 3. Correlation between log kcalc and log half-life. A
good correlation (log t1/2 ) -0.4051(log k) + 3.2899, r2 ) 0.75)
was observed between log half-life and log k. Compounds 11i,
11j, 18, and 19 were left out. Compounds 11i and 11j have a
considerably shorter half-life than could be expected, presum-
ably because they suffer from another metabolism, extra to
the phosphonate ester hydrolysis (see Discussion). Compounds
18 and 19 cannot be compared directly to the other diaryl
phosphonate esters.
no inhibitory activity. On the contrary, the O-methyl
derivative 18 is almost as potent and stable as 5. The
phosphonate esters of trichloroethanol (22) and trifluoro-
ethanol (23) have rather disappointing inhibitory activi-
ties.
Unfortunately, the correlation between electronic
properties and inhibitory activity and between electronic
properties and stability results also in an inverse
correlation between inhibitory activity and stability
(Figure 3). This means that more active compounds are
also more unstable. Therefore, the higher potency (IC50
) 0.4 µM) and equal stability (t1/2 ) 320 min) of the
paracetamol-substituted phenyl phosphonate 11e is a
major improvement to the unsubstituted 5.
In Vitr o Cytotoxicity a n d Effica cy in Hu m a n
P er ip h er a l Blood Mon on u clea r Cells (P BMC). On
the basis of inhibition potency, stability in plasma, and
synthesis efficiency, compounds 11e and 11n were
selected for further in vitro and in vivo studies. Both
compounds were evaluated in human peripheral blood
mononuclear cells (PBMC) and did not show cyto-
toxicity in freshly isolated mononuclear cells or phyto-
hemagglutinin-stimulated blasts when concentrations
up to 100 µM were used. Under these circumstances,
more than 90% of the DPP IV activity in cell lysates as
well as in supernatants was inhibited. This is in
contrast with the results obtained for the active dia-
stereoisomer of the unsubstituted diphenyl phosphonate
11a , where no satisfactory inhibition of DPP IV activity
could be reached without cytotoxic effects on PBMC

1046 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
Ta ble 2. Specificity of the Inhibitors 11 for DPP IV/CD26a
PEP
DPP II
1 mM
MAAP
1 mM
elastase
compd
R1
IC50
1 mM
IC50
102
IC50
100
IC50
1 mM
82
11a
11d
11e
11f
11h
11j
H (R at carbon next to phosphorus)
3-AcNH
4-AcNH
107
89
96
73
92
88
102
73
85
83
97
71
50
60
4
73
83
83
96
100
106
100
99
105
4-MeSO2NH
4-(N-Bz-Gly-NH)
4-(H-(S)-Ala-NH)
4-[(2S)-MeO2CCH(NHAc)CH2]
4-MeO2C
4-(EtO2CCH2NHCO)
4-[MeO2C(CH2)2NHCO]
4-[CH3(CH2)2NHCO]
32
47
35
4
1
0
11l
11m
11n
11o
11p
78
66
104
94
102
6
5
a
The effect of the different inhibitors 11 on the activity of prolyl endopeptidase (PEP), dipeptidyl peptidase II (DPP II), membrane
alanyl aminopeptidase (MAAP), and elastase was evaluated at the IC50 concentration for DPP IV (see Table 1) and at 1 mM. The residual
enzyme activity is given as a percentage toward a control experiment without inhibitor.
2500-62500-fold higher than the IC50 value for DPP IV.
So, we can say that the selectivity index is at least 1250
compared to PEP and much higher for the other
enzymes.
The advantage of this compound compared to the
pyrrolidine-2-nitrile reversible inhibitors is its long-
lasting irreversible inhibition. Moreover, it is more
stable than the frequently used boronic acid inhibitors.
Con clu sion
Exp er im en ta l Section
The recent result that a dipeptide diphenyl phosphon-
ate inhibitor of DPP IV/CD26 abrogated acute rejection
and prolonged cardiac allograft survival in rats prompted
us to investigate in more detail the structure-activity
relationship of these compounds using diphenyl 1-(S)-
prolylpyrrolidine-2(R,S)-phosphonate as a lead com-
pound. We synthesized a series of diaryl 1-(S)-prolylpyr-
rolidine-2(R,S)-phosphonates with different substituents
on the aryl rings (hydroxyl, methoxy, acylamino, sulfon-
ylamino, ureyl, methoxycarbonyl, and alkylaminocar-
bonyl) and measured their DPP IV inhibitory potency
and stability in plasma. All compounds were irreversible
inhibitors. A good correlation was observed between the
electron-withdrawing properties of the substituents and
the inhibition of DPP IV. The methoxycarbonyl- and
alkylaminocarbonyl-substituted derivatives were the
most potent inhibitors with IC50 values around 20 nM
Gen er a l. 4-Methoxyphenol (8b), 1-(3-hydroxyphenyl)urea
(8g), methyl 4-hydroxybenzoate (8m ), triphenyl phosphite (9a ),
2,2′-biphenol (14), L-proline, L-tyrosine, 4-aminophenol, glycine,
L-pyroglutamic acid, 4-hydroxybenzoic acid, glycine ethyl ester
hydrochloride, â-alanine, and all common chemicals were
purchased from Acros Chimica N.V., Belgium. 3-Acetamido-
phenol (8d ), 4-aminobutyraldehyde diethyl acetal, PCl3, di-
phenyl phosphorylazide (DPPA), and 4-acetoxybenzoic acid
were obtained from Sigma-Aldrich Chemie BV, Belgium.
4-Acetamidophenol (paracetamol) (8e) was obtained from
Sterling Organics Ltd., England. Purity of all new synthesized
1
compounds was checked by TLC, H NMR, 13C NMR, or MS.
1
The final products were checked by TLC, H NMR, 13C NMR,
FAB-MS, and(or) elemental analysis. Thin-layer chromatog-
raphy was performed with POLYGRAM SIL G/UV254 plates
precoated with silica gel (Machinery-Nagel GmbH, Germany)
using EtOAc-petroleum ether, EtOAc-MeOH, or n-BuOH-
AcOH-H2O (4:1:1) mixtures as eluent. Silica gel H, 5-40 µm
(Fluka, Switzerland), was used for preparative vacuum column
chromatography. The NMR spectra were recorded on a Varian
EM360L or a Brucker Avance DRX 400 spectrometer. Mass
spectra were recorded on a VG 70-SEQ spectrometer. Optical
rotation was measured on a Perkin-Elmer 241 polarimeter.
Melting points were determined on a digital melting points
apparatus (Electrothermal) and are uncorrected. Elemental
analyses were performed at The Institute of Organic Chem-
istry, University of Tu¨bingen, Germany.
Z- and Boc-protected amino acids were prepared according
to standard procedures29 using Z-Cl and (Boc)2O, respectively.
â-Alanine methyl ester was synthesized by treatment of the
amino acid in methanolic HCl similarly to described proce-
dure.29 The preparation of 4-(Z-(S)-prolyl)aminobutyraldehyde
diethyl acetal (6a ) was described earlier.10 Tris(2,2,2-trichlo-
roethyl) phosphite and tris(2,2,2-trifluoroethyl) phosphite were
prepared as described.30 2-Chlorodibenzo[d,f]-1,3,2-dioxaphos-
phepan (15) was prepared as described.31
and inactivation rate constants around 3000 M-1 s-1
.
The same correlation was also observed between the
electron-donating properties of the substituents and the
stability in plasma. The most potent inhibitors are also
the most unstable compounds. A notable exception is
the good stability of the 4-(acetylamino)phenyl phos-
phonate ester 11e (t1/2 ) 320 min), together with a
higher potency than could be expected (IC50 ) 0.4 µM,
k ) 1900 M-1 s-1). Therefore, this compound together
with the very potent 11n were further investigated in
vitro and in vivo. These inhibitors showed no cytotox-
icity in human peripheral blood mononuclear cells in
concentrations up to 100 µM. The IC50 values of 11e and
11n for inhibition of plasma DPP IV in rabbits upon
single intravenous injection were around 0.2 mg/kg and
below 20 µg/kg, respectively. The compounds also showed
no acute systemic or local toxicity, as was observed with
the unsubstituted lead compound 5.
4-(Boc-(S)-p r olyl)a m in obu tyr a ld eh yd e Dieth yl Aceta l
(6b). To a solution of Boc-(S)-proline (10 mmol, 2.14 g) in dry
CHCl3 (20 mL) was added Et3N (10 mmol, 1.4 mL) with
stirring at -10 °C followed by isobutyl chloroformate (10 mmol,
1.31 mL). After 20 min, 4-aminobutyraldehyde diethyl acetal
(10 mmol, 1.73 mL) was added, and the mixture was stirred
overnight (-10 °C to room temperature). Chloroform was
removed, the residue was distributed between EtOAc (100 mL)
and water (100 mL), and the organic layer was dried (MgSO4)
and purified by column chromatography: yield 2.96 g (83%),
Due to the higher stability of 11e compared to 11n ,
we believe that bis(4-acetamidophenyl) 1-(S)-prolylpyr-
rolidine-2(R,S)-phosphonate (11e) is considered as a
major improvement and will be a highly valuable
inhibitor for further studies on the biological function
of the enzyme and the therapeutic value of its inhibition.
1
oil; H NMR (CDCl3) δ 1.0-2.4 (m, 23H, 3- and 4-CH2 (Pro),

SAR of Phosphonate Esters as DPP IV Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 1047
N-(4-Acetoxyben zoyl)-â-a la n in e m eth yl ester (12): yield
CH2CH2, C(CH3)3, CH3), 2.9-3.9 (m, 9H, 5-CH2, NCH2, OCH2
and (EtO)2CH), 4.25 (m, 1H, 2-CH), 4.5 (t, 1H, NH).
1
69%; mp 84-88 °C; H NMR (CDCl3) δ 2.35 (s, 3H, COCH3),
2.6 (t, 2H, CO2CH2), 3.7 (m, 5H, NCH2 and CO2CH3), 7.15 (m,
3H, 2Harom and NH), 7.75 (d, 2Harom).
N-n -P r op yl-4-a cetoxyben zoyla m id e (13): yield 73%; mp
94-98 °C; 1H NMR (CDCl3) δ 0.9 (t, 3H, CH3), 1.6 (m, 2H,
CH2), 2.25 (s, 3H, COCH3), 3.4 (m, 2H, NCH2), 7.1 (m, 3H,
2Harom and NH), 7.8 (d, 2Harom).
N-(4-Hydr oxyben zoyl)-â-alan in e m eth yl ester (8o): yield
65%; mp 124-126 °C; 1H NMR (DMSO) δ 2.55 (t, 2H, CO2CH2),
3.7 (m, 5H, NCH2 and CO2CH3), 6.9 (m, 3H, 2Harom and NH),
7.55 (d, 2Harom), 8.7 (br s, 1H, OH).
N-n -P r op yl-4-h yd r oxyben zoyla m id e (8p ): yield 78%; oil;
1H NMR (DMSO) δ 1.1 (t, 3H, CH3), 1.55 (m, 2H, CH2), 3.3
(m, 2H, NCH2), 6.9 (m, 3H, 2Harom and NH), 7.65 (d, 2Harom),
8.5 (br s, 1H, OH); MS (FAB+) m/z 180 (M + H)+.
Syn th esis of Tr ia r yl P h osp h ites 9. Gen er a l P r oced u r e.
To a stirred solution of the corresponding phenol (90 mmol)
and Et3N (90 mmol, 12.6 mL) in dry DMF (30 mL) was added
a solution of PCl3 (30 mmol, 2.62 mL) in dry CHCl3 (10 mL)
dropwise at 0 °C. The mixture was stirred overnight (0 °C to
room temperature) and diluted with CHCl3 (500 mL), and the
resulting solution was washed with water (2 × 500 mL). The
organic layer was dried (MgSO4) and evaporated and the
residue, eventually together with the product insoluble in both
layers, was purified by column chromatography with an
EtOAc-methanol or an EtOAc-petroleum ether mixture as
eluents.
4-(Meth ylsu lfon yla m in o)p h en ol (8f). To a suspension of
4-aminophenol (200 mmol, 21.8 g) in MeOH (250 mL) was
added CH3SO2Cl (100 mmol, 7.75 mL) at 10-15 °C with
stirring. The resulting solution was stirred for 1 h at room
temperature and evaporated. The residue was suspended in
1 N HCl (250 mL); the solid was filtered, washed with water,
and dried in a vacuum over NaOH: yield 13.6 g (81%); mp
1
165-166 °C; H NMR (DMSO) δ 2.85 (s, 3H, CH3S), 6.70 (d,
2Harom), 7.10 (d, 2Harom), 8.70 (s, 1H, OH), 8.85 (s, 1H, NH).
Syn th esis of 4-(Acyla m in o)p h en ols 8h -k . Gen er a l
P r oced u r e. To a solution of carboxylic acid (100 mmol) in
DMF (100 mL), was added Et3N (100 mmol, 14 mL) at -10
°C, followed by isobutyl chloroformate (100 mmol, 13 mL).
After 0.5 h at this temperature, 4-aminophenol (115 mmol,
12.5 g) was added and the mixture was stirred overnight (-10
°C to room temperature). After dilution with 1 N HCl (500
mL), the precipitate was collected by filtration and washed
on the filter with water, NaHCO3 solution, water, and, finally,
ether. The product was dried and, if necessary, recrystallized
from an appropriate solvent.
N2-Ben zoyl-N-(4-h yd r oxyp h en yl)glycin a m id e (8h ): re-
crystallized from EtOH-acetone; yield 35%; mp 244-245 °C;
1H NMR (DMSO) δ 4.05 (d, 2H, CH2CO), 6.5-8.0 (m, 9Harom),
8.7 (m, 1H, NH), 9.15 (s, 1H, OH), 9.75 (s, 1H, NH).
N2-(Ben zyloxyca r b on yl)-N-(4-h yd r oxyp h en yl)glycin -
1
a m id e (8i): yield 44%; mp 184-185 °C; H NMR (DMSO) δ
Tr is(4-m eth oxyp h en yl) p h osp h ite (9b): yield 85%, oil;
1H NMR (CDCl3) δ 3.51 (s, 9H, OCH3), 6.51-7.15 (m, 12Harom).
Tr is(4-a cetoxyp h en yl) p h osp h ite (9c): yield 55%, oil; 1H
NMR (CDCl3) δ 2.21(s, 9H, CH3), 7.05 (m, 12H, Harom).
Tr is(3-a ceta m id op h en yl) p h osp h ite (9d ): yield 42%,
3.80 (d, 2H, CH2CO), 6.5-7.5 (m, 10H, 9Harom and NH), 8.85
(s, 1H, OH), 9.40 (s, 1H, NH).
N2-(Ben zyloxycar bon yl)-N-(4-h ydr oxyph en yl)-(S)-alan -
1
in a m id e (8j): yield 73%; mp 97-100 °C; H NMR (DMSO) δ
1.36 (d, 3H, CH3), 4.29 (m, 1H, CH of L-Ala), 5.00 (s, 2H, CH2),
6.00 (m, 1H, NH), 6.35-7.38 (m, 9H, Harom), 8.69 (br s, 1H,
OH), 9.24 (br s, 1H, NHCO)
1
solid foam; H NMR (DMSO) δ 2.1 (s, 9H, COCH3), 6.7-7.7
(m, 12Harom), 9.7 (s, 3H, NH).
Tr is(4-a ceta m id op h en yl) p h osp h ite (9e): yield 37%,
N-(4-Hyd r oxyp h en yl)-(S)-p yr oglu ta m a m id e (8k ): yield
64%; mp 303-305 °C dec; 1H NMR (DMSO) δ 1.7-2.7 (m, 4H,
CH2CH2), 4.2 (m, 1H, CHCO), 6.65 (d, 2Harom), 7.40 (d, 2Harom),
7.8 (s, 1H, NH), 9.1 (br s, 1H, OH), 9.7 (s, 1H, NH).
1
solid foam; H NMR (DMSO) δ 2.1 (s, 9H, COCH3), 6.9-7.7
(dd, 12Harom), 9.2 (s, 3H, NH).
Tr is[4-(m eth ylsu lfon yla m in o)p h en yl] p h osp h ite (9f):
yield 60%, solid foam; 1H NMR (DMSO) δ 2.95 (s, 9H, SO2CH3),
7.2 (m, 12Harom), 9.15 (s, 3H, NH).
N-(4-Hyd r oxyben zoyl)glycin e Eth yl Ester (8n ). To a
solution of 4-hydroxybenzoic acid (100 mmol, 13.8 g) in DMF
(70 mL) was added H-Gly-OEt‚HCl (100 mmol, 14 g) followed
by DPPA (100 mmol, 21.6 mL) and Et3N (200 mmol, 28 mL)
with stirring at 0 °C. The mixture was stirred overnight (0 °C
to room temperature), Et3N‚HCl was removed by filtration,
and the filtrate was evaporated to one-half of the initial
volume. The residue was mixed with 5% NaHCO3 (200 mL)
and extracted with EtOAc (500 mL). The organic layer was
washed with brine, dried (MgSO4), and evaporated until
crystallization. The mixture was diluted with ether (100 mL),
and the crystals were collected: yield 9 g (40%); mp 205-209
°C; 1H NMR (DMSO) δ 1.25 (t, 3H, CH3), 4.05 (m, 4H, CO2CH2
and COCH2N), 6.8 (d, 2Harom), 7.75 (d, 2Harom), 8.35 (tr, 1H,
NH), 9.7 (s, 1H, OH).
Syn th esis of 4-Hyd r oxyben zoic Acid Am id es 8o a n d
8p . Gen er a l P r oced u r e. To a solution of 4-acetoxybenzoic
acid (100 mmol, 18 g) in THF (250 mL) was added Et3N (100
mmol, 14 mL) followed by isobutyl chloroformate (100 mmol,
13 mL) with stirring at -10 °C. After 15 min the corresponding
amine (free base or HCl salt) was added followed by Et3N (100
mmol, 14 mL) in case of a hydrochloride. The mixture was
stirred overnight (-10 °C to room temperature), the solid was
removed by filtration, the filtrate was evaporated, and the
residue was crystallized from an ether-hexane mixture.
4-Acetoxybenzoic acid amide 12 or 13 thus obtained (70 mmol)
was dissolved in MeOH (240 mL); H2O (90 mL); was added
followed by saturated NaHCO3 solution (120 mL). An excess
of NaHCO3 precipitated. The heterogeneous mixture was
stirred for 4 h at room temperature, methanol was removed
in a vacuum, the residue was extracted with EtOAc (250 mL),
and the organic layer was washed with 1 N HCl and brine,
then dried (MgSO4), and evaporated. The residue crystallized
from ether or was purified on a silica gel column.
Tr is(3-u r eylp h en yl) p h osp h ite (9g): yield 30%, solid
1
foam; H NMR (DMSO) δ 5.85 (m, 6H, CONH2), 6.5-7.5 (m,
12Harom), 8.65 (s, 3H, NH).
Tr is[4-(N-ben zoylglycyla m in o)p h en yl] p h osp h ite (9h ):
yield 90%; mp 210 °C dec; 1H NMR (DMSO) δ 4.1 (d, 6H,
COCH2N), 6.9-8.0 (m, 27Harom), 8.5 (m, 3H, NH), 9.9 (s, 3H,
NH).
Tr is[4-(N-ben zyloxycar bon ylglycylam in o)ph en yl] ph os-
p h ite (9i): yield 76%; mp 173 °C dec; 1H NMR (DMSO) δ 3.85
(d, 6H, COCH2N), 5.05 (s, 6H, CH2Ph), 6.5-8.0 (m, 30H, NH
and Harom), 9.8 (br s, 3H, NH).
Tr is[4-(N-b en zyloxyca r b on yl-(S)-a la n yla m in o)p h en -
yl] p h osp h ite (9j): yield 85%, solid foam; 1H NMR (DMSO) δ
1.42 (d, 9H, CH3), 4.29 (m, 3H, CH), 5.01(s, 6H, CH2Ph), 6.45-
7.15 (d, 3H, NH), 7.25 (m, 27H, Harom), 9.76 (br s, 3H, NH).
Tr is[4-((S)-pyr oglu tam ylam in o)ph en yl] ph osph ite (9k):
1
yield 31%, solid foam; H NMR (DMSO) δ 1.8-2.8 (m, 12H,
CH2CH2), 4.25 (m, 3H, COCHN), 7.1 (d, 6Harom), 7.65 (d,
6Harom), 8.25 (s, 3H, NH), 10.3 (s, 3H, NH).
Tr is[4-(S)-(2-m eth oxycar bon yl-2-acetam idoeth yl)ph en -
yl] p h osp h ite (9l): yield 73%, solid foam; 1H NMR (DMSO) δ
2.0 (s, 9H, COCH3), 3.1 (d, 6H, ArCH2), 3.7 (s, 9H, CO2CH3),
4.8 (m, 3H, COCHN), 6.6 (d, 3H, NH), 7.1 (br s, 12Harom).
Tr is[4-(m eth oxycar bon yl)ph en yl] ph osph ite (9m ): yield
1
57%, oil; H NMR (CDCl3) δ 3.95 (s, 9H, CO2CH3), 6.65-8.05
(m, 12Harom).
T r is {4-[(e t h o x y c a r b o n y l)m e t h y la m in o c a r b o n y l]-
p h en yl} p h osp h ite (9n ): yield 70%, solid foam; 1H NMR
(CDCl3) δ 1.3 (t, 9H, CH3), 3.9-4.3 (m, 12H, COCH2N and
OCH2), 6.7-8.0 (m, 15H, NH and Harom).
Tr is{4-[2-(m e t h oxyca r b on yl)e t h yla m in oca r b on yl]-
p h en yl} p h osp h ite (9o): yield 71%, solid foam; 1H NMR

1048 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
(CDCl3) δ 2.65 (t, 6H, CO2CH2), 3.7 (m, 15H, NCH2 and
CO2CH3), 7.1 (d, 6Harom), 7.9 (m, 9H, Harom and NH).
2-CH), 5.1 (br s, 2H, CH2Ph), 6.8-7.5 (m, 13Harom), 8.8 (s, 2H,
NH); MS (FAB+) m/z 649 (M + H)+.
Bis[4-(m eth ylsu lfon yla m in o)p h en yl] 1-(ben zyloxyca r -
bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-p h osp h on a te (10f):
yield 66%, solid foam; 1H NMR (CDCl3) δ 1.2-2.7 (m, 8H,
3-CH2 and 4-CH2), 2.8 (s, 6H, CH3SO2), 3.3-3.8 (m, 4H, 5-CH2),
4.3-5.2 (m, 4H, 2-CH and CH2Ph), 6.8-7.4 (m, 13Harom), 8.0
(s, 2H, NH); MS (FAB+) m/z 721 (M + H)+.
Bis(3-u r eylp h en yl) 1-(ben zyloxyca r bon yl-(S)-p r olyl)-
pyr r olidin e-2(R,S)-ph osph on ate (10g): yield 8%, solid foam;
1H NMR (CDCl3) δ 1.3-2.4 (m, 8H, 3-CH2 and 4-CH2), 2.8-
3.8 (m, 4H, 5-CH2), 4.3-5.2 (m, 4H, 2-CH and CH2Ph), 5.6 (m,
4H, NH2), 6.8-7.5 (m, 13Harom), 8.65 (br s, 2H, NH); MS (FAB+)
m/z 651 (M + H)+.
Bis[4-(N-b en zoylglycyla m in o)p h en yl]-1-(b en zyloxy-
car bon yl-(S)-pr olyl)pyr r olidin e-2(R,S)-ph osph on ate (10h ):
yield 47%, solid foam; 1H NMR (CDCl3) δ 1.2-2.7 (m, 8H,
3-CH2 and 4-CH2), 3.2-3.8 (m, 4H, 5-CH2), 4.0 (m, 4H, NCH2-
CO), 4.4-5.0 (m, 2H, 2-CH), 5.1 (br s, 2H, CH2Ph), 6.6-8.3
(m, 25H, 23Harom and NH), 9.5 (br s, 2H, NH); MS (FAB+) m/z
887 (M + H)+.
Bis[4-(N-b en zyloxyca r b on ylglycyla m in o)p h en yl]-1-
(ben zyloxyca r bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-p h os-
p h on a te (10i): yield 19%, solid foam; 1H NMR (CDCl3) δ 1.2-
2.6 (m, 8H, 3-CH2 and 4-CH2), 3.2-4.1 (m, 8H, 5-CH2 and
COCH2N), 4.3-5.2 (m, 8H, 2-CH and CH2Ph), 6.1 (m, 2H, NH),
6.7-7.6 (m, 23Harom), 8.95 (br s, 2H, NH); MS (FAB+) m/z 947
(M + H)+.
Tr is[4-(n -pr opylam in ocar bon yl)ph en yl] ph osph ite (9p):
yield 54%; mp 207 °C; 1H NMR (DMSO) δ 1.1 (t, 9H, CH3), 1.6
(m, 6H, CH2), 3.25 (m, 6H, CH2N), 7.15 (d, 6Harom), 8.1 (m,
9H, NH and Harom).
Sep a r a tion of th e Dia ster eoisom er s of 10a : Dip h en yl
1-(Trityl-(S)-prolyl)pyrrolidine-2(R)-phosphonate (10a(S,R))
a n d Dip h en yl 1-(Tr ityl-(S)-p r olyl)p yr r olid in e-2(S)-p h os-
p h on a te (10a (S,S)). A solution of diphenyl 1-(Z-(S)-prolyl)-
pyrrolidine-2(R,S)-phosphonate10 (13.1 mmol, 7 g) in MeOH
(130 mL) was hydrogenated over 10% Pd/C for 6 h at room
temperature. The catalyst was removed on Celite, the filtrate
was evaporated, and the residue was distributed between
EtOAc (100 mL) and saturated NaHCO3 solution (50 mL). The
organic layer was dried (MgSO4) and evaporated. The resulting
oil (2 g) was dissolved in dry CHCl3 (50 mL); Et3N (5 mmol,
0.7 mL) was added with stirring at room temperature followed
by trityl chloride (5 mmol, 1.39 g). The solution was stirred
overnight and washed with water (100 mL); the organic layer
was dried, evaporated, and purified by column chromatography
(petroleum ether/EtOAc, 4/1 to 2/1). The faster running isomer
was 10a (S,R).
Isom er 10a (S,R): yield 0.78 g (9%), solid foam; 1H NMR
(CDCl3) δ 0.7-2.8 (m, 10H, 3-CH2 and 4-CH2, 5-CH2 Pro), 3.3-
3.5 (m, 2H, 5-CH2 ProP), 4.0 (dd, 1H, J ) 8.95, 2.8 Hz, 2-CH
Pro), 5.15 (m, 1H, 2-CH ProP), 6.9-7.6 (m 25Harom); [R]D
)
20
-79.5° (c 1, CHCl3); MS (FAB+) m/z 643 (M + H)+.
Bis[4-(N-ben zyloxyca r bon yl-(S)-a la n yla m in o)p h en yl]-
1-(ben zyloxycar bon yl-(S)-pr olyl)pyr r olidin e-2(R,S)-ph os-
1
Isom er 10a (S,S): yield 0.82 g (11%), solid foam; H NMR
(CDCl3) δ 0.7-2.6 (m, 8H, 3-CH2 and 4-CH2), 2.7-3.7 (m, 4H,
5-CH2), 3.9 (m, 0.5H, 2-CH ProP), 4.2 (dd, 1H, J ) 13.5, 7.4
Hz, 2-CH Pro), 5.05 (br t, 0.5H, 2-CH ProP), 6.9-7.8 (m,
1
p h on a te (10j): yield 40%; mp 200-203 °C; H NMR (CDCl3)
δ 1.35 (d, 6H, CH3), 1.65-2.35 (m, 8H, 3-CH2 and 4-CH2),
2.95-3.95 (m, 4H, 5-CH2), 4.0-4.65 (m, 4H, 2-CH, CH), 5.00
(s, 6H, CH2Ph), 6.20 (br, 2H, NH), 7.04 (s, 23H, Harom), 9.25
(br, 2H, NH); MS (FAB+) m/z 975 (M + H)+.
25Harom); [R]D ) -27.2° (c 1, CHCl3); MS (FAB+) m/z 643 (M
20
+ H)+.
Syn th esis of N-Boc- or N-Z-P r otected Dia r yl 1-((S)-
P r olyl)pyr r olidin e-2(R,S)-ph osph on ates 10. Gen er al P r o-
ced u r e. Diethyl acetal 6a or 6b (10 mmol) was dissolved in a
mixture of THF (60 mL) and 0.5 N HCl (30 mL, 15 mmol) with
stirring, at room temperature. After 2 h ether (90 mL) was
added with stirring, and the mixture was neutralized with an
excess of solid NaHCO3 (ca. 5 g). The organic layer was
separated, dried (MgSO4), and evaporated. The residue (crude
aldehyde 7a or 7b) was dissolved in acetic acid (30 mL)
together with the corresponding triaryl phosphite 9 (10 mmol),
and the resulting solution was stirred at 85-90 °C for 1.5-2
h. After cooling to room temperature, acetic acid was evapo-
rated; the residue was dissolved in chloroform (250 mL) and
washed with water (200 mL), saturated NaHCO3 solution (100
mL), and water (100 mL). The organic layer was dried (MgSO4)
and evaporated, and the residue was purified by column
chromatography with an ethyl acetate-petroleum ether or
ethyl acetate-methanol mixture as eluents.
Bis(4-m et h oxyp h en yl) 1-(ter t-b u t yloxyca r b on yl-(S)-
p r olyl)p yr r olid in e-2(R,S)-p h osp h on a te (10b): yield 33%,
oil; 1H NMR (CDCl3) δ 1.15-2.65 (m, 17H, C(CH3)3, 3-CH2 and
4-CH2), 3.67 (s, 6H, OCH3), 3.15-4.3 (m, 4H, 5-CH2), 4.35-
5.4 (m, 2H, 2-CH), 6.45-7.35 (m, 8Harom).
Bis(4-a cet oxyp h en yl) 1-(b en zyloxyca r b on yl-(S)-p r o-
lyl)p yr r olid in e-2(R,S)-p h osp h on a te (10c): yield 50%, solid
foam; 1H NMR (CDCl3) δ 1.65-2.85 (m, 8H, 3-CH2 and 4-CH2),
2.29 (s, 6H, COCH3), 3.00-3.85 (m, 4H, 5-CH2), 4.1-4.85 (m,
2H, 2-CH), 5.15 (s, 2H, CH2), 6.92 (m, 8Harom), 7.33 (s, 5H,
Bis[4-((S)-p yr oglu t a m yla m in o)p h en yl]-1-(b en zyloxy-
car bon yl-(S)-pr olyl)pyr r olidin e-2(R,S)-ph osph on ate (10k):
yield 25%; mp 170 °C dec; 1H NMR (CDCl3) δ 1.2-2.9 (m, 16H,
3-CH2 and 4-CH2), 3.55 (m, 4H, 5-CH2), 4.1 (m, 2H, 5-CH Pyr),
4.3-5.2 (m, 4H, 2-CH and CH2Ph), 6.7-8.1 (m, 15H, NH and
13Harom), 9.55 (br s, 2H, NH); MS (FAB+) m/z 787 (M + H)+.
Bis[4-(S)-(2-m eth oxyca r bon yl-2-a ceta m id oeth yl)p h en -
yl] 1-(ben zyloxyca r bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-
1
p h osp h on a te (10l): yield 57%, solid foam; H NMR (CDCl3)
δ 1.3-2.7 (m, 8H, 3-CH2 and 4-CH2), 1.9 (s, 6H, COCH3), 3.1
(d, 4H, CH2Ar), 3.7 (s, 6H, COOCH3), 3.2-3.9 (m, 4H, 5-CH2),
4.5-5.0 (m, 4H, 2-CH), 5.1 (br s, 2H, CH2Ph), 6.1 (d, 2H, NH),
7.0 (s, 8Harom), 7.3 (br s, 5H, C6H5); MS (FAB+) m/z 821 (M +
H)+.
Bis[4-(m eth oxyca r bon yl)p h en yl] 1-(ter t-bu tyloxyca r -
bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-p h osp h on a te (10m ):
1
yield 5.5%, solid foam; H NMR (CDCl3) δ 1.05-2.55 (m, 8H,
3-CH2 and 4-CH2), 1.42 (s, 9H, C(CH3)3), 3.33-4.06 (m, 4H,
5-CH2), 3.83 (s, 6H, OCH3), 4.45 (m, 1H, 2-CH Pro), 5.04 (m,
1H, 2-CH ProP), 7.25 (m, 4H, Harom), 7.96 (m, 4H, Harom).
Bis{4-[(eth oxyca r bon yl)m eth yla m in oca r bon yl]p h en -
yl} 1-(ben zyloxyca r bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-
p h osp h on a te (10n ): yield 32%, solid foam; 1H NMR (CDCl3)
δ 1.2-2.7 (m, 8H, 3-CH2 and 4-CH2), 1.3 (t, 6H, CH3), 3.2-3.9
(m, 4H, 5-CH2), 4.0 (m, 4H, NCH2CO), 4.2 (q, 4H, OCH2), 4.3-
5.1 (m, 2H, 2-CH), 5.1 (br s, 2H, CH2Ph), 6.9-7.8 (m, 15H,
13Harom and NH); MS (FAB+) m/z 793 (M + H)+.
Bis {4-[2-(m e t h oxyc a r b on yl)e t h yla m in oc a r b on yl]-
p h en yl} 1-(b en zyloxyca r b on yl-(S)-p r olyl)p yr r olid in e-
H
arom).
1
Bis(3-a ceta m idop h en yl) 1-(ben zyloxyca r bon yl-(S)-p r o-
2(R,S)-p h osp h on a te (10o): yield 22%, solid foam; H NMR
lyl)p yr r olid in e-2(R,S)-p h osp h on a te (10d ): yield 31%, solid
(CDCl3) δ 1.2-2.8 (m, 8H, 3-CH2 and 4-CH2), 2.65 (t, 4H,
CH2CO2), 3.2-3.8 (m, 8H, 5-CH2 and CH2N), 3.75 (s, 6H,
CO2CH3), 4.3-5.2 (m, 4H, 2-CH and CH2Ph), 6.7-7.9 (m, 15H,
13Harom and NH); MS (FAB+) m/z 793 (M + H)+.
1
foam; H NMR (CDCl3) δ 1.0-2.7 (m, 8H, 3-CH2 and 4-CH2),
2.1 (s, 6H, CH3CO), 4.0-3.2 (m, 4H, 5-CH2), 4.3-5.1 (m, 2H,
2-CH), 5.1 (br s, 2H, CH2Ph), 6.6-7.7 (m, 13Harom), 9.1 (s, 2H,
NH); MS (FAB+) m/z 649 (M + H)+.
Bis[4-(n -p r op yla m in oca r bon yl)p h en yl] 1-(ben zyloxy-
car bon yl-(S)-pr olyl)pyr r olidin e-2(R,S)-ph osph on ate (10p):
Bis(4-a ceta m idoph en yl) 1-(ben zyloxyca r bon yl-(S)-p r o-
lyl)p yr r olid in e-2(R,S)-p h osp h on a te (10e): yield 60%, solid
1
yield 4.3%, solid foam; H NMR (CDCl3) δ 0.85 (t, 6H, CH3),
1
foam; H NMR (CDCl3) δ 1.6-2.7 (m, 8H, 3-CH2 and 4-CH2),
1.3-2.8 (m, 12H, 3-CH2, 4-CH2 and CH2), 3.1-3.9 (m, 8H,
5-CH2 and CH2N), 4.5 (m, 1H, 2-CH Pro), 4.7-5.2 (m, 3H,
2.1 (s, 6H, CH3CO), 3.3-3.8 (m, 4H, 5-CH2), 4.3-5.1 (m, 2H,

SAR of Phosphonate Esters as DPP IV Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 1049
CH2Ph and 2-CH ProP), 6.9-8.1 (m, 15H, 13Harom and NH);
MS (FAB+) m/z 705 (M + H)+.
(m, 1H, 2-CH), 4.85 (m, 1H, 2-CH), 7.15 (m, 8Harom), 8.6 (br s,
1H, N+H), 9.79 (s, 2H, SO2NHPh), 10.3 (br s, 1H, N+H). Anal.
(C23H31N4O8PS2‚HCl) C, H, N.
Bis(3-u r eylp h en yl) 1-((S)-p r olyl)p yr r olid in e-2(R,S)-
p h osp h on a te h yd r och lor id e (11g): B; yield 90%, solid foam;
1H NMR (CD3OD) δ 1.4-2.5 (m, 8H, 3-CH2 and 4-CH2), 3.3-
3.8 (m, 4H, 5-CH2), 4.5 (m, 1H, 2-CH), 4.8 (m, 1H, 2-CH), 6.4-
7.2 (m, 6Harom), 7.4 (m, 2Harom); MS (FAB+) m/z 517 (M + H)+.
Anal. (C23H29N6O6P‚HCl‚2.3H2O) C, H, N.
Bis[4-(N-b en zoylglycyla m in o)p h en yl] 1-((S)-p r olyl)-
p yr r olid in e-2(R,S)-p h osp h on a te h yd r och lor id e (11h ): B;
yield 87%, solid foam; 1H NMR (DMSO) δ 1.6-2.6 (m, 8H,
3-CH2 and 4-CH2), 3.2-3.6 (m, 4H, 5-CH2), 4.2 (d, 4H, NCH2-
CO), 4.5-4.9 (m, 2H, 2-CH), 6.9-7.9 (m, 20H, 18Harom and
NH), 8.5 (m, 1H, N+H), 10.0 (s, 2H, NH), 10.2 (m, 1H, N+H);
MS (FAB+) m/z 753 (M + H)+. Anal. (C39H41N6O8P‚HCl‚2H2O)
C, H, N.
Dia r yl 1-((S)-P r olyl)p yr r olid in e-2-p h osp h on a t e Hy-
d r och lor id es 11. P r oced u r e A. The diaryl 1-(tert-butyloxy-
carbonyl-(S)-prolyl)pyrrolidine-2-phosphonate 10 or diaryl 1-(tri-
tyl-(S)-prolyl)pyrrolidine-2-phosphonate 10 (5 mmol) was
dissolved in a 1 M HCl solution in EtOAc (25 mL), and the
solution was stirred for 2 h at room temperature. Dry ether
(30 mL) was added, and the mixture was left in the fridge
overnight. If the product crystallized, the crystals were col-
lected by filtration; otherwise the oil was triturated in dry
ether. The material obtained was dried in a vacuum over
NaOH pellets.
P r oced u r e B. The diaryl 1-((S)-benzyloxycarbonylprolyl)-
pyrrolidine-2-phosphonate 10 (2 mmol) was hydrogenated over
Pd/C in methanol (50 mL) for 5-6 h. The catalyst was removed
by filtration through Celite, the filtrate was acidified with 1
M HCl/EtOAc (2.2 mL) and evaporated, and the residue was
precipitated with dry ether from methanol (5 mL). The
resulting product was dried in a vacuum over NaOH pellets.
Dip h en yl 1-((S)-p r olyl)p yr r olid in e-2(R)-p h osp h on a te
h yd r och lor id e (11a (S,R)): A; yield 81%; mp 175-177 °C; 1H
NMR (CDCl3) δ 1.6-2.8 (m, 8H, 3-CH2 and 4-CH2), 3.3-3.9
(m, 4H, 5-CH2), 4.8 (m, 1H, 2-CH), 5.0 (m, 1H, 2-CH), 7.2 (m,
5Harom), 7.3 (m, 5Harom), 8.1 (br s, 1H, N+H), 10.2 (br s, 1H,
N+H); 13C NMR (CDCl3) δ 24.1(4-CH2), 24.6 (4-CH2), 26.1 (3-
CH2), 28.8 (3-CH2), 46.2 (5-CH2), 47.1 (5-CH2), 54.5 (d, 2-CH-
P, 1J (C-P) ) 162 Hz), 59.1 (2-CH), 120.3, 125.2, 129.7, 150.3
Bis[4-(N-ben zyloxycar bon ylglycylam in o)ph en yl] 1-((S)-
p r olyl)p yr r olid in e-2(R,S)-p h osp h on a te tr ih yd r och lor id e
1
(11i): B; yield 81%, solid foam; H NMR (DMSO) δ 1.55-2.5
(m, 8H, 3-CH2 and 4-CH2), 3.3-3.8 (m, 4H, 5-CH2), 3.8 (s, 4H,
COCH2N), 4.55 (m, 1H, 2-CH), 4.85 (m, 1H, 2-CH), 7.15 (m,
4Harom), 7.65 (m, 4Harom), 8.35 (br s, 6H, N+H), 8.5 (br s, 1H,
N+H), 10.35 (br s, 1H, N+H), 11.0 (s, 2H, NH). Anal.
(C25H33N6O6P‚3HCl‚2.5H2O) C, H, N.
Bis[4-((S)-a la n yla m in o)p h en yl] 1-((S)-p r olyl)p yr r oli-
d in e-2(R,S)-p h osp h on a te tr ih yd r och lor id e (11j): B; yield
70%, solid foam; 1H NMR (D2O) δ 1.49 (d, 6H, CH3), 1.65-
2.60 (m, 8H, 3-CH2and 4-CH2), 3.23-3.75 (m, 4H, 5-CH2), 4.12
(m, 2H, CH of Ala), 7.05 (m, 4H, Harom), 7.35 (m, 4H, Harom).
Anal. (C27H37N6O6P‚3HCl‚0.5H2O) C, H; N: calcd, 12.16; found,
11.73.
20
(Carom), 168.2 (CO); [R]D ) -100.4° (c 1, CHCl3). Anal.
(C21H25N2O4P‚1.25HCl) C, H, N.
Dip h en yl 1-((S)-p r olyl)p yr r olid in e-2(S)-p h osp h on a te
h yd r och lor id e (11a (S,S)): A; yield 88%, solid foam; 1H NMR
(CDCl3) δ 1.7-2.6 (m, 8H, 3-CH2 and 4-CH2), 3.2-3.9 (m, 4H,
5-CH2), 4.75 (m, 1H, 2-CH), 4.95 (m, 0.7H, 2-CH), 5.4 (m, 0.3H,
2-CH), 6.9-7.4 (m, 10Harom), 8.6 (br s, 1H, N+H), 10.3 (br s,
0.3H, N+H), 10.6 (br s, 0.7H, N+H); 13C NMR (CDCl3) δ 22.1,
24.0 (4-CH2), 23.5, 23.9 (4-CH2), 26.4, 27.7 (3-CH2), 28.5, 29.1
(3-CH2), 45.6, 46.2 (5-CH2), 46.6, 47.1 (5-CH2), 54.4, 55.3 (d,
Bis[4-((S)-p yr oglu ta m yla m in o)p h en yl] 1-((S)-p r olyl)-
p yr r olid in e-2(R,S)-p h osp h on a te h yd r och lor id e (11k ): B;
1
yield 95%, solid foam; H NMR (DMSO) δ 1.5-2.4 (m, 16H,
3-CH2 and 4-CH2), 3.1-4.2 (m, 6H, 5-CH2 and 5-CH), 4.5 (m,
1H, 2-CH Pro), 4.85 (m, 1H, 2-CH ProP), 7.1 (m, 4Harom), 7.65
(m, 4Harom), 7.9 (s, 2H, NH), 8.6 (m, 1H, N+H), 10.1 (m, 1H,
N+H), 10.3 (s, 2H, NH). Anal. (C31H37N6O8P‚1.5HCl‚3H2O) C,
H, N.
1
2-CH-P, J (C-P) ) 160 Hz), 58.3, 58.7 (2-CH), 120.1, 125.0,
20
129.4, 149.5 (Carom), 167.3, 168.4 (CO); [R]D ) 25.9° (c 1,
CHCl3); MS (FAB+) m/z 401 (M + H)+.
Bis[4-(S)-(2-m eth oxyca r bon yl-2-a ceta m id oeth yl)p h en -
yl] 1-((S)-p r olyl)p yr r olid in e-2-p h osp h on a te h yd r och lo-
r id e (11l): B; yield 85%, solid foam; 1H NMR (DMSO) δ 1.6-
2.6 (m, 8H, 3-CH2 and 4-CH2), 1.9 (s, 6H, COCH3), 3.0 (d, 4H,
CH2Ar), 3.2-3.8 (m, 4H, 5-CH2), 3.7 (s, 6H, COOCH3), 4.4-
4.9 (m, 4H, 2-CH), 7.1 (m, 8Harom), 8.2 (d, 2H, AcNH), 8.7 (m,
1H, N+H), 10.4 (m, 1H, N+H); MS (FAB+) m/z 687 (M + H)+.
Anal. (C33H43N4O10P‚1.5HCl‚2H2O) C, N; H: calcd, 6.29; found,
5.81.
Bis(4-m eth oxyph en yl) 1-((S)-pr olyl)pyr r olidin e-2(R,S)-
p h osp h on a te h yd r och lor id e (11b): A; yield 70%; 1H NMR
(CDCl3) δ 1.75-2.60 (m, 8H, 3-CH2 and 4-CH2), 3.28-3.65 (m,
4H, 5-CH2), 3.75 (s, 6H, OCH3), 5.35-5.50 (m, 2H, 2-CH),
6.70-7.60 (m, 8H, Harom), 8.45 (br s, 1H, N+H), 10.55 (br s,
1H, N+H); MS (FAB+) m/z 461 (M + H)+; 31P NMR (CDCl3) δ
16.22, 16.72. Anal. (C23H29N2O6P‚2HCl‚2H2O) C, H, N.
Bis(4-h ydr oxylph en yl) 1-((S)-pr olyl)pyr r olidin e-2(R,S)-
1
p h osp h on a te h yd r och lor id e (11c): B; yield 80%; H NMR
Bis[4-(m eth oxyca r bon yl)p h en yl] 1-((S)-p r olyl)p yr r ol-
idin e-2(R,S)-ph osph on ate h ydr och lor ide (11m ): yield 82%,
(CDCl3) δ 1.70-2.68 (m, 8H, 3-CH2 and 4-CH2), 3.27-3.75 (m,
4H, 5-CH2), 4.45-4.95 (m, 2H, 2-CH), 6.55-7.16 (m, 8H, Harom),
7.84 (br s, 2H, OH), 8.65 (br s, 1H, N+H), 10.18 (br s, 1H, N+H);
MS (FAB+) m/z 433 (M + H)+; 31P NMR (DMSO) δ 17.31, 17.40.
Anal. (C21H25N2O6P‚HCl‚1.5H2O) C, H, N.
1
solid foam; H NMR (CDCl3) δ 1.62-2.78 (m, 8H, 3-CH2 and
4-CH2), 3.21-3.64 (m, 4H, 5-CH2), 3.82 (s, 6H, OCH3), 4.71,
4.90 (m, 2H, 2-CH), 7.21 (m, 4H, Harom), 7.91 (m, 4H, Harom),
8.15 (br, 1H, N+H), 10.65 (br, 1H, N+H); MS (FAB+) m/z 517
(M + H)+. Anal. (C25H29N2O8P‚2HCl‚2H2O) C, H; N: calcd,
4.48; found, 5.21.
Bis(3-a cet a m id op h en yl)
1-((S)-p r olyl)p yr r olid in e-
2(R,S)-p h osp h on a t e h yd r och lor id e (11d ): B; yield 83%,
solid foam; 1H NMR (DMSO) δ 1.6-2.6 (m, 8H, 3-CH2 and
4-CH2), 2.1 (s, 6H, COCH3), 3.3-3.8 (m, 4H, 5-CH2), 4.7 (m,
1H, 2-CH), 4.9 (m, 1H, 2-CH), 6.7-7.6 (m, 8Harom), 8.5 (m, 1H,
N+H), 9.7 (m, 1H, N+H), 9.9 (m, 2H, CONHPh). Anal.
(C25H31N4O6P‚1.5HCl‚0.5H2O) C, H, N.
B i s {4-[(e t h o x y c a r b o n y l)m e t h y la m i n o c a r b o n y l]-
p h en yl} 1-((S)-p r olyl)p yr r olid in e-2(R,S)-p h osp h on a t e
(11n ): B; yield 86%, solid foam; 1H NMR (DMSO) δ 1.3 (t, 6H,
CH3), 1.4-2.6 (m, 8H, 3-CH2 and 4-CH2), 3.2-3.8 (m, 4H,
5-CH2), 4.0 (m, 4H, NCH2CO), 4.2 (q, 4H, OCH2), 4.6 (m, 1H,
2-CH), 4.9 (m, 1H, 2-CH), 7.3 (m, 4Harom), 7.9 (m, 4Harom), 8.7
(m, 4H, N+H and NH); MS (FAB+) m/z 659 (M + H)+. Anal.
(C31H39N4O10P‚HCl‚1.5H2O) C, H, N.
Bis {4-[2-(m e t h oxyc a r b on yl)e t h yla m in oc a r b on yl]-
ph en yl} 1-((S)-pr olyl)pyr r olidin e-2(R,S)-ph osph on ate h y-
d r och lor id e (11o): B; yield 79%, solid foam; 1H NMR (DMSO)
δ 1.2-2.8 (m, 8H, 3-CH2 and 4-CH2), 2.65 (t, 4H, CH2COO),
3.2-3.9 (m, 8H, 5-CH2 and CH2N), 3.8 (s, 6H, COOCH3), 4.7
(m, 1H, 2-CH), 4.9 (m, 1H, 2-CH), 7.55 (m, 4Harom), 8.15 (m,
4Harom), 8.55 (m, 4H, N+H and NH).
Bis(4-a cet a m id op h en yl)
1-((S)-p r olyl)p yr r olid in e-
2(R,S)-p h osp h on a te h yd r och lor id e (11e): B; yield 90%,
solid foam; 1H NMR (DMSO) δ 1.6-2.6 (m, 8H, 3-CH2 and
4-CH2), 2.1 (s, 6H, COCH3), 3.3-3.8 (m, 4H, 5-CH2), 4.6 (m,
1H, 2-CH), 4.9 (m, 1H, 2-CH), 7.1 (m, 4Harom), 7.6 (m, 4Harom),
8.7 (m, 1H, N+H), 10.0 (m, 2H, CONHPh), 10.2 (m, 1H, N+H);
31P NMR (DMSO) δ 17.48, 17.62. Anal. (C25H31N4O6P‚HCl‚
2H2O) C, H, N.
Bis[4-(m eth ylsu lfon yla m in o)p h en yl] 1-((S)-p r olyl)p yr -
r olid in e-2(R,S)-p h osp h on a te h yd r och lor id e (11f): B; yield
1
80%, solid foam; H NMR (DMSO) δ 1.55-2.5 (m, 8H, 3-CH2
Bis[4-(n -p r op yla m in oca r bon yl)p h en yl] 1-((S)-p r olyl)-
p yr r olid in e-2(R,S)-p h osp h on a te h yd r och lor id e (11p ): B;
and 4-CH2), 2.95 (s, 6H, SO2CH3), 3.3-3.8 (m, 4H, 5-CH2), 4.55

1050 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
1
yield 80%, solid foam; H NMR (CD3OD) δ 0.95 (t, 6H, CH3),
4.1 (m, 4H, 5-CH2), 4.2-5.0 (m, 6H, CH2CF3 and 2-CH), 5.2
(br s, 2H, CH2Ph), 7.35 (s, 5H, Harom); MS (FAB+) m/z 546 (M
+ H)+.
Bis(2,2,2-tr ich lor oeth yl) 1-((S)-pr olyl)pyr r olidin e-2(R,S)-
p h osp h on a te h yd r och lor id e (22): prepared from the Z-
protected derivative 20 using procedure B for diaryl phosphon-
ates; yield 26%, solid foam; 1H NMR (CDCl3) δ 1.5-3.0 (m,
8H, 3-CH2 and 4-CH2), 3.2-4.1 (m, 4H, 5-CH2), 4.4-5.1 (m,
6H, CH2CCl3 and 2-CH), 6.55 (m, 1H, N+H), 8.4 (m, 1H, N+H);
MS (FAB+) m/z 511 (M + H)+.
1.6 (m, 4H, CH2), 1.7-2.6 (m, 8H, 3-CH2 and 4-CH2), 3.2-3.9
(m, 8H, 5-CH2 and CH2N), 4.65 (m, 1H, 2-CH Pro), 5.1 (m,
1H, 2-CH ProP), 7.3 (m, 4Harom), 7.85 (m, 4Harom); 13C NMR
(CD3OD) δ 11.7 (CH3), 23.5 (CH2), 25.0 (4-CH2), 25.8 (4-CH2),
27.3 (3-CH2), 29.7 (3-CH2), 42.8 (NCH2), 47.4 (5-CH2), 48.3 (5-
CH2), 55.9 (d, 2-CH-P, 1J (C-P) ) 161 Hz), 60.6 (2-CH), 121.4,
130.4, 133.1, 153.7 (Carom), 168.8 (CO), 169.2 (CO).
2,2′-Bip h en ylyl Acetyl P h osp h ite (16). A solution of
chloroanydride 15 (0.152 mol, 38 g) in dry THF (50 mL) was
added dropwise to a solution of AcOH (0.152 mol, 9.1 mL) and
Et3N (0.152 mol, 21.3 mL) in dry THF (100 mL) with stirring
at 0 °C. The cooling was removed, and the mixture was stirred
at room temperature for 3 h. The solid was filtered off, and
the filtrate was evaporated. The crude product was used in
the next step without further purification: yield 40 g (96%),
viscous oil; 1H NMR (CDCl3) δ 2.1 (s, 3H, CH3CO), 6.9-7.5
(m, 8H, Harom).
Bis(2,2,2-tr iflu or oeth yl) 1-((S)-pr olyl)pyr r olidin e-2(R,S)-
p h osp h on a te h yd r och lor id e (23): prepared from the Z-
protected derivative 21 using procedure B for diaryl phos-
1
phonates; yield 44%, solid foam; H NMR (CDCl3) δ 1.3-2.8
(m, 8H, 3-CH2 and 4-CH2), 3.2-4.0 (m, 4H, 5-CH2), 4.4-5.1
(m, 6H, CH2CF3 and 2-CH), 6.9 (m, 1H, N+H), 8.4 (m, 1H,
N+H); MS (FAB+) m/z 413 (M + H)+. Anal. (C13H19F6N2O4P‚
HCl‚0.8H2O) C; H: calcd, 4.70; found, 5.51. N: calcd, 6.05;
found, 6.72.
2,2′-Bip h en ylyl 1-(ben zyloxyca r bon yl-(S)-p r olyl)p yr -
r olid in e-2(R,S)-p h osp h on a te (17a ): prepared according to
the general procedure for diaryl phosphonates with acetal 6a
DP P IV In h ibition in Vitr o a n d Sta bility Mea su r e-
m en ts. DPP IV was purified from human seminal plasma as
described previously.32 Enzymatic activity was measured at
37 °C in a Spectramax 340 (Molecular Devices) microtiter plate
reader using Gly-Pro-p-nitroanilide (Sigma) as a chromogenic
substrate. The reaction was monitored at 405 nm, and the
initial rate was determined between 0 and 0.25 absorbance
units. The reaction mixture contained 2 mM substrate, ap-
proximately 1 mU of DPP IV, 40 mM TRIS-HCl buffer, pH
8.3, and a suitable amount of inhibitor (ranging between 0 and
10 mM) in a total volume of 0.2 mL. Activity measurements
were routinely performed in duplicate. The IC50 value is
defined as the concentration of inhibitor required to reduce
the DPP IV activity to 50% after a 15-min preincubation with
the enzyme at 37 °C before addition of the substrate. Inhibitor
stock solutions (100 mM) were prepared in DMSO or phos-
phate buffer, pH 7.4, depending on the solubility of the
compound and stored at -20 °C. Stock solutions were diluted
with 50 mM TRIS-HCl buffer, pH 8.3, as required immediately
before the experiment. Since the compounds described in this
paper completely inactivate DPP IV following pseudo-first-
order kinetics, the IC50 value is inversely correlated with the
second-order rate constant of inactivation.11 For a simple
pseudo-first-order inactivation process, the activity after in-
cubation with inhibitor (vi) varies with the inhibitor concentra-
1
and phosphite 16; yield 27%, solid foam; H NMR (CDCl3) δ
1.2-2.9 (m, 8H, 3-CH2 and 4-CH2), 3.1-4.0 (m, 4H, 5-CH2),
4.55 (m, 1H, 2-CH Pro), 4.85 (m, 1H, 2-CH ProP), 5.2 (br s,
2H, CH2Ph), 7.1-7.85 (m, 15Harom); MS (EI) m/z (relative
intensity) 532 (M, 40), 397 (20), 300 (26), 232 (29), 215 (41),
204 (45), 168 (42), 160 (74), 91 (100), 70 (48).
2,2′-Bip h en ylyl 1-(ter t-b u t yloxyca r b on yl-(S)-p r olyl)-
p yr r olid in e-2(R,S)-p h osp h on a te (17b): prepared according
to the general procedure for diaryl phosphonates with acetal
6b and phosphite 16; yield 24%, solid foam; 1H NMR (CDCl3)
δ 1.3-2.55 (m, 8H, 3-CH2 and 4-CH2), 1.5 (s, 9H, CH3), 3.35-
3.9 (m, 4H, 5-CH2), 4.55 (m, 1H, 2-CH), 4.9 (m, 1H, 2-CH),
6.95-7.75 (m, 8H, Harom); MS (FAB+) m/z 499 (M + H)+.
2-(2′-Hyd r oxyp h en yl)p h en yl m eth yl 1-(ben zyloxyca r -
bon yl-(S)-p r olyl)p yr r olid in e-2(R,S)-p h osp h on a te h yd r o-
ch lor id e (18): prepared from the Z-protected derivative 17a
using procedure B for diaryl phosphonates; yield 92%, solid
1
foam; H NMR (CDCl3) δ 1.5-2.6 (m, 8H, 3-CH2 and 4-CH2),
3.1-3.9 (m, 7H, 5-CH2 and OCH3), 4.4-4.8 (m, 2H, 2-CH Pro
and 2-CH ProP), 6.85 (br s, 1H, PhOH), 7.1-7.6 (m, 8Harom),
8.4 (m, 1H, N+H), 9.6 (m, 1H, N+H); 13C NMR (CDCl3) δ 25.7
(4-CH2), 26.1 (4-CH2), 28.2 (3-CH2), 30.5 (3-CH2), 48.1 (5-CH2),
48.6 (5-CH2), 55.6 (OCH3), 55.6 (2-CH-P, 1J (C-P) ) 142 Hz),
60.8 (2-CH), 117.8, 121.0, 126.3, 130.1, 132.5, 133.8 149.7,
156.2 (Carom), 168.9 (CO); MS (FAB+) m/z 431 (M + H)+. Anal.
(C22H27N2O5P‚HCl‚H2O) C, H; N: calcd, 5.78; found, 6.26.
tion (i) as described in the following equation: vi ) vo × e-kit
,
where vo is the activity in absence of inhibitor, k is the second-
order rate constant of inactivation, and t is the time. Since at
1
i ) IC50 by definition vi ) /2vo, it follows from the equation
that k ) ln 2/(ti × IC50), where ti ) 15 min. These calculated
k values are listed in Table 1. For some inhibitors (5, 11a , 11b,
11d , 11e, 11h , 11n ) the inactivation rate constant was
determined from the time course of inhibition as described
before.11
The functional stability of the inhibitors was estimated by
measuring the inhibitory potency (apparent IC50) of a 1 mM
dilution of the compounds in citrated human plasma at three
or four time points between 0 and 300 min at 37 °C. Fitting
the inverse of the apparent IC50 values versus time with a
single-exponential decay gives the half-lives reported in Table
1.
2,2′-Bip h en ylyl 1-((S)-p r olyl)p yr r olid in e-2(R,S)-p h os-
p h on a t e h yd r och lor id e (19): prepared from the Boc-
protected derivative 17b using procedure A for diaryl phos-
phonates; yield 81%, solid foam; 1H NMR (CDCl3) δ 1.3-2.55
(m, 8H, 3-CH2 and 4-CH2), 3.35-3.75 (m, 4H, 5-CH2), 4.75 (m,
2H, 2-CH), 7.0-7.5 (m, 8H, Harom), 10.9 (m, 2H, N+H); 13C NMR
(CDCl3) δ 24.4 (4-CH2), 24.9 (4-CH2), 26.7 (3-CH2), 28.7 (3-
CH2), 46.6 (5-CH2), 51.9 (5-CH2), 53.4 (2-CH-P, 1J (C-P) )
151 Hz), 59.2 (2-CH), 116.9, 120.9, 121.7, 121.9, 125.8, 126.6,
128.4, 129.0, 130.3, 131.5, 147.9 (Carom), 167.8 (CO); MS (FAB+)
m/z 399 (M + H)+. Anal. (C21H23N2O4P‚1.4HCl‚1.1H2O) C, H;
N: calcd, 5.97; found, 5.43.
In Vitr o Cytotoxicity a n d Effica cy in Hu m a n P er ip h -
er a l Blood Mon on u clea r Cells (P BMC). Peripheral blood
mononuclear cells were isolated from buffy coats (obtained
from the blood transfusion center of Antwerp). After dilution
(1/4) in phosphate buffer saline (PBS), cells were layered onto
Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Swe-
den) and centrifuged at room temperature at 550g during 20
min. The interfaces were collected and washed three times in
RPMI-1640. Finally the cells were resuspended at 1 × 106 cells/
mL in RPMI-1640 containing 10% heat-inactivated fetal calf
serum and antibiotics (penicillin/streptomycin) (Gibco). These
freshly isolated cells were used immediately for inhibitor
studies or first stimulated with phytohemagglutinin (Murex
diagnostics) at 1 µg/mL during 3 days at 37 °C in a 5% CO2
Bis(2,2,2-tr ich lor oeth yl) 1-(ben zyloxyca r bon yl-(S)-p r o-
lyl)p yr r olid in e-2(R,S)-p h osp h on a te (20): Prepared accord-
ing to the general procedure for diaryl phosphonates with
acetal 6a and tris(2,2,2-trichloroethyl) phosphite; yield 31%,
oil; 1H NMR (CDCl3) δ 1.2-2.8 (m, 8H, 3-CH2 and 4-CH2), 3.3-
4.1 (m, 4H, 5-CH2), 4.3-5.1 (m, 6H, CH2CCl3 and 2-CH), 5.2
(br s, 2H, CH2Ph), 7.35 (s, 5H, Harom); MS (FAB+) m/z 645 (M
+ H)+.
Bis(2,2,2-tr iflu or oeth yl) 1-(ben zyloxyca r bon yl-(S)-p r o-
lyl)p yr r olid in e-2(R,S)-p h osp h on a te (21): prepared accord-
ing to the general procedure for diaryl phosphonates with
acetal 6a and tris(2,2,2-trifluoroethyl) phosphite; yield 48%,
oil; 1H NMR (CDCl3) δ 1.2-2.7 (m, 8H, 3-CH2 and 4-CH2), 3.2-

SAR of Phosphonate Esters as DPP IV Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 1051
(9) Boduszek, B.; Oleksyszyn, J .; Kam, C. M.; Selzler, J .; Smith, R.
E.; Powers, J . C. Dipeptide Phosphonates as Inhibitors of
Dipeptidyl Peptidase IV. J . Med. Chem. 1994, 37, 3969-
3976.
(10) Belyaev, A.; Borloo, M.; Augustyns, K.; Lambeir, A. M.; De
Meester, I.; Scharpe´, S.; Blaton, N.; Peeters, O. M.; De Ranter,
C.; Haemers, A. A New Synthetic Method for Proline Diphenyl
Phosphonates. Tetrahedron Lett. 1995, 36, 3755-3758.
(11) Lambeir, A. M.; Borloo, M.; De Meester, I.; Belyaev, A.; Au-
gustyns, K.; Hendriks, D.; Scharpe´, S.; Haemers, A. Dipeptide-
derived Diphenyl Phosphonate Esters: Mechanism-based In-
hibitors of Dipeptidyl Peptidase IV. Biochim. Biophys. Acta 1996,
1290, 76-82.
(12) Wrenger, S.; Reinhold, D.; Hoffmann, T.; Kraft, M.; Frank, R.;
Faust, J .; Neubert, K.; Ansorge, S. The N-terminal X-X-Pro
sequence of the HIV-1 Tat protein is important for the inhibition
of dipeptidyl peptidase IV (DP IV/CD26) and the suppression of
mitogen-induced proliferation of human T cells. FEBS Lett.
1996, 383, 145-149.
(13) Yaron, A.; Naider, F. Proline-dependent Structural and Biologi-
cal Properties of Peptides and Proteins. Crit. Rev. Biochem. Mol.
Biol. 1993, 28, 31-81.
humidified incubator. Inhibitor (stock solution at -80 °C in
phosphate buffer, diluted ex tempore in RPMI-1640) or vehicle
alone was added to the cells (5 × 106/test) at different
concentrations. After overnight incubation at 37 °C, an aliquot
was taken for cytotoxicity evaluation by 0.4% trypan blue
exclusion. The remaining cells were washed three times in
PBS, and the final cell pellet was solubilized in 200 µL of PBS
containing 1% v/v Triton X-100 and 100 KIU/mL aprotinin
(Bayer) and centrifuged during 10 min at 20000g. Super-
natants were used immediately for enzyme assay and protein
determination by the Bradford microassay. Specific activities
(U/g protein) are compared, and the percent inhibition is given
toward control samples without inhibitor.
DDP IV In h ibit ion in Vivo. Male New Zealand white
rabbits (2.5-3.5 kg), Wistar rats (250-350 g), and Swiss mice
(23-36 g) were allowed to adjust to their environment for at
least 7 days. They received standard diet and water ad lib.
Test compounds were dissolved in 50 mM phosphate buffer,
pH 7.4, at concentrations ranging from 10 to 100 mg/mL,
stored in aliquots at -80 °C, and thawed ex tempore. Rabbits
received a single slow intravenous bolus injection of test
compound or vehicle alone in the marginal ear vein. Blood was
sampled from the central ear artery. Rats and mice were
injected subcutaneously or intraperitoneally. Rat blood samples
were obtained under anesthesia (Forene) from the vena
femoralis by puncture after incision of the skin. The mice were
bled by orbita puncture after induction of anesthesia with
pentobarbital. After clotting, blood samples were centrifuged
(3000g, 10 min), and the resulting sera were stored at -80 °C
until assayed for DPP IV activity.
(14) Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.; Scharpe´,
S. Proline Motifs in Peptides and their Biological Processing.
FASEB J . 1995, 9, 736-744.
(15) (a) Oravecz, T.; Pall, M.; Roderiquez, G.; Gorrell, M. D.; Ditto,
M.; Nguyen, N. Y.; Boykins, R.; Unsworth, E.; Norcross, M. A.
Regulation of the Receptor Specificity and Function of the
Chemokine RANTES (Regulated on Activation, Normal T Cell
Expressed and Secreted) by Dipeptidyl Peptidase IV (CD26)-
mediated Cleavage. J . Exp. Med. 1997, 186, 1865-1872. (b)
Proost, P.; De Meester, I.; Schols, D.; Struyf, S.; Lambeir, A. M.;
Wuyts, A.; Opdenakker, G.; De Clercq, E.; Scharpe´, S.; Van
Damme, J . Amino-terminal truncation of chemokines by CD26/
dipeptidyl peptidase IV: Conversion of RANTES into a potent
inhibitor of monocyte chemotaxis and HIV-1 infection. J . Biol.
Chem. 1998, 273, 7222-7227.
(16) Morita, A.; Chung, Y.; Freeman, H.; Erickson, R.; Sleisenger,
M.; Kim, Y. Intestinal Assimilation of a Proline-containing
Tetrapeptide. J . Clin. Invest. 1983, 72, 610-616.
(17) Tiruppathi, C.; Miyamoto, Y.; Ganapathy, V.; Roesel, R. A.;
Whitford, G. M.; Leibach, F. H. Hydrolysis and Transport of
Proline-containing Peptides in Renal Brush-border Membrane
Vesicles from Dipeptidyl Peptidase IV-positive and Dipeptidyl
Peptidase IV-negative Rat Strains. J . Biol. Chem. 1990, 265,
1476-1483.
(18) Piazza, G.; Callanan, H.; Mowery, J .; Hixson, D. Evidence for a
Role of Dipeptidyl Peptidase IV in Fibronectin-mediated Interac-
tions of Hepatocytes with Extracellular Matrix. Biochem. J .
1989, 262, 327-334.
(19) Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune
Response; Fleisher, B., Ed.; Springer-Verlag: Heidelberg, Ger-
many, 1995.
(20) Kubota, T.; Flentke, G. R.; Bachovchin, W. W.; Stollar, B. D.
Involvement of Dipeptidyl Peptidase IV in an in vivo Immune
Response. Clin. Exp. Immunol. 1992, 89, 192-197.
(21) Tanaka, S.; Murakami, T.; Horikawa, H.; Sugiura, M.; Ka-
washima, K. Sugita, T. Suppression of arthritis by the inhibitors
of dipeptidyl peptidase IV. Int. J . Immunopharmacol. 1997, 19,
15-24.
(22) De Meester, I.; Belyaev, A.; Lambeir, A. M.; De Meyer, G. R. Y.;
Van Osselaer, N.; Haemers, A.; Scharpe´, S. In vivo Inhibition of
Dipeptidyl Peptidase IV Activity by Pro-Pro-diphenyl-phos-
phonate (Prodipine). Biochem. Pharmacol. 1997, 54, 173-179.
(23) Korom, S.; De Meester, I.; Stadlbauer, T. H. W.; Chandraker,
A.; Schaub, M.; Sayegh, M. H.; Belyaev, A.; Haemers, A.;
Scharpe´, S.; Kupiec-Weglinski, J . W. Inhibition of CD26/Dipep-
tidyl Peptidase IV Activity in vivo prolongs Cardiac Allograft
Survival in Rat Recipients. Transplantation 1997, 63, 1495-
1500.
Deter m in a tion of Sp ecificity for DP P IV. Prolyl endo-
peptidase and elastase activity were determined as previously
described.33,34 Dipeptidyl peptidase II activity was determined
by the hydrolysis of Lys-Ala-4-MeO-2-NA (1.4 mM) (Sigma,
L-2270) in 100 mM acetate buffer, pH 5.5, containing 2 mM
EDTA. Hydrolysis of L-Ala-4-MeO-2-NA (5 mM) (Sigma,
A-5414) in 60 mM phosphate buffer (pH 7.4) was chosen for
the determination of membrane alanyl aminopeptidase activ-
ity.
Refer en ces
(1) Reviewed in Fleischer, B. CD26: a Surface Protease involved in
T-cell Activation. Immunol. Today 1994, 15, 180-184.
(2) Scho¨n, E.; Born, I.; Demuth, H. U.; Faust, J .; Neubert, K.;
Steinmetzer, T.; Barth, A.; Ansorge, S. Dipeptidyl Peptidase IV
in the Immune System: Effects of Specific Enzyme Inhibitors
on Activity of Dipeptidyl Peptidase IV and Proliferation of
Human Lymphocytes. Biol. Chem. Hoppe-Seyler 1991, 372, 305-
311.
(3) Augustyns, K.; Lambeir, A. M.; Borloo, M.; De Meester, I.;
Vedernikova, I.; Vanhoof, G.; Hendriks, D.; Scharpe´, S.; Haem-
ers, A. Pyrrolidides: Synthesis and Structure-activity Relation-
ship as Inhibitors of Dipeptidyl Peptidase IV. Eur. J . Med. Chem.
1997, 32, 301-309.
(4) Li, J .; Wilk, E.; Wilk, S. Aminoacylpyrrolidine-2-nitriles: Potent
and Stable Inhibitors of Dipeptidyl-Peptidase IV (CD26). Arch.
Biochem. Biophys. 1995, 323, 148-154.
(5) Ashworth, D. M.; Atrash, B.; Baker, G. R.; Baxter, A. J .; J enkins,
P. D.; J ones, D. M.; Szelke, M. 2-Cyanopyrrolidides as Potent,
Stable Inhibitors of Dipeptidyl Peptidase IV. BioMed. Chem.
Lett. 1996, 6, 1163-1166.
(6) Demuth, H. U.; Neumann, U.; Barth, A. Reactions between
dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic
investigations. J . Enzyme Inhib. 1989, 2, 239-248.
(7) Flentke, G. R.; Munoz, E.; Huber, B. T.; Plaut, A. G.; Kettner,
C. A.; Bachovchin, W. W. Inhibition of Dipeptidyl Aminopepti-
dase IV (DP-IV) by Xaa-boroPro Dipeptides and use of these
Inhibitors to examine the Role of DP-IV in T-cell Function. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 1556-1559.
(8) Snow, R. J .; Bachovchin, W. W.; Barton, R. W.; Campbell, S. J .;
Coutts, S. J .; Freeman, D. M.; Gutheil, W. G.; Kelly, T. A.;
Kennedy, C. A.; Krolikowski, D. A.; Leonard, S. F.; Pargellis, C.
A.; Tong, L.; Adams, J . Studies on Proline Boronic Acid Dipeptide
Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic
Species Containing a B-N Bond. J . Am. Chem. Soc. 1994, 116,
10860-10869.
(24) J iang, J . D.; Wilk, S.; Li, J .; Zhang, H.; Bekesi, J . G. Inhibition
of human immunodeficiency virus type 1 infection in a T-cell
line (CEM) by new dipeptidyl-peptidase IV (CD26) inhibitors.
Res. Virol. 1997, 148, 255-266.
(25) J ackson, E. L. O-p-Toluenesulfonyl-L-tyrosine, its N-acetyl and
N-benzoyl Derivatives. J . Am. Chem. Soc. 1952, 74, 837-838.
(26) Bu¨chi, G.; Weinreb, S. M. Total Syntheses of Aflatoxins M1 and
G1 and an Improved Synthesis of Aflatoxin B1. J . Am. Chem.
Soc. 1971, 93, 746-752.
(27) Walsh, E. N. Conversion of Tertiary Phosphites to Secondary
Phosphonates. Diphenyl Phosphonate. J . Am. Chem. Soc. 1959,
81, 3023-3026.
(28) Boduszek, B.; Brown, A. D.; Powers, J . C. R-Aminoalkylphos-
phonate di(chlorophenyl) esters as inhibitors of serine proteases.
J . Enzyme Inhib. 1994, 8, 147-158.
(29) Bodanszky, M.; Bodanszky, A. In The Practice of Peptide
Synthesis, 2nd ed.; Springer-Verlag: Berlin Heidelberg, 1994;
pp 11-18 and 28-29.

1052 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Belyaev et al.
(30) Sasse, K. Phosphorigsa¨ure-derivate. In Methoden der Organis-
chen Chemie (Houben-Weyl), 4th ed.; Mu¨ller, E., Bayer, O.,
Meerwein, H., Ziegler, K., Eds.; Georg Thieme Verlag: Stuttgart,
1964; pp 54-56.
(31) Veriznikov, L. V.; Kirpichnikov, P. A. Synthesis of the o,o′-
Biphenylphosphinic Acid Esters. Zh. Obshch. Khim. 1967, 37,
1355-1358.
purification of native human CD26/dipeptidyl peptidase IV (EC
3.4.14.5). J . Immunol. Methods 1996, 189, 99-105.
(33) Goossens, F.; De Meester, I.; Vanhoof, G.; Scharpe´, S. A sensitive
method for the assay of serum prolyl endopeptidase. Eur. J . Clin.
Chem. Clin. Biochem. 1992, 30, 235-238.
(34) Bergmeyer, H. U. Methods of enzymatic analysis, Volume V:
Enzymes 3, Peptidase, Proteinases and their inhibitors; Verlag
Chemie: Germany; pp 176-184.
(32) De Meester, I.; Vanhoof, G.; Lambeir, A.-M.; Scharpe´, S. Use of
immobilized adenosine deaminase (EC 3.5.4.4) for the rapid
J M981033G