Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26

Article abstract of DOI:10.1021/jm970640l

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is Characterized by its post-prolinecleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors

Full text of DOI:10.1021/jm970640l

2100
J . Med. Chem. 1998, 41, 2100-2110
Sp ecific a n d Ir r ever sible Cyclop ep tid e In h ibitor s of Dip ep tid yl P ep tid a se IV
Activity of th e T-Cell Activa tion An tigen CD26
Coralie Nguyen,† J ulia` Blanco,‡ J ean-Paul Mazaleyrat,† Bernard Krust,‡ Christian Callebaut,‡ Etienne J acotot,‡
Ara G. Hovanessian,‡ and Michel Wakselman*,†
SIRCOB, Universite´ de Versailles, Baˆtiment Lavoisier, 45 avenue des Etats Unis, F-78000 Versailles, France, and Unite´ de
Virologie et Immunologie Cellulaire, ERS 572 CNRS, Institut Pasteur, 28 rue du Dr. Roux, F-75724 Paris Cedex 15, France
Received September 26, 1997
The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-
cleaving capacity that plays an important but not yet understood role in biological processes.
Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking
into account the substrate specificity of DPP IV for P2-P1><-P1′ cleavage, we have designed
and synthesized cyclopeptides c[(RH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba ) 3-ami-
nobenzoic acid, R ) alkyl) possessing a proline at the P1 position and a lysine in the P2 position,
which allows the closing of the cycle on its side chain. These molecules show a free N-terminus,
necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide
electrophile, responsible for inactivation. Treatment of c[RZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn],
obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting
group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These
cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values
in the nanomolar range. Further studies were carried out to investigate the effect of the
modification of the ring size (n ) 2 or 4) and the nature of the sulfur substituents (R ) Me,
Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide,
whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other
aminopeptidases were not inhibited, and a much weaker activity was observed on a novel
isoform of DPP IV referred to as DPP IV-â. Thus, this new family of irreversible inhibitors of
DPP IV is highly specific to the peptidase activity of CD26.
In tr od u ction
phosphatase involved in signal transduction. Both
features of CD26, its signaling capacity and its pepti-
dase activity, contribute to the costimulatory function
of CD26 in T-cell activation events. However, the role
of DPP IV activity of CD26 in these events is unclear.
Some authors have described that small synthetic
inhibitors of DPP IV impair mitogen and antigen
stimulation of PBMC and other lymphocytic cell types.9
In contrast, others have found no effect of DPP IV
inhibitors on stimulated T-cells.10 Similarly, contradic-
tory results on the function of the DPP IV activity of
CD26 have been reported by using cell lines expressing
a mutated, catalytically inactive form of CD26.11,12 The
failure to understand the role of DPP IV activity is, in
part, due to the fact that no physiological substrates
have been identified. However, a broad spectrum of
bioactive peptides, including some interleukines, chemok-
ines, neuropeptides, and growth factors, can be poten-
tially cleaved by DPP IV.
The availability of stable specific irreversible inhibi-
tors or highly potent reversible inhibitors of DPP IV
should be useful in studies for the determination of the
physiological and pathological role(s) of this enzyme.
Several competitive, tight-binding, or irreversible in-
hibitors of the enzyme are already known: oligopeptides
with the N-terminal X-Pro sequence (X ) various amino
acid residues) such as the diprotins A and B (Ile-Pro-
Ile or Val-Pro-Leu),13,14 X-pyrrolidides and X-thiazo-
lidides,14-16 X-cyanoPro and X-cyanoThia,17-19 X-phospho-
noPro or Pip aryl esters,20,21 X-boroPro,22-25 X-ProCH2N+-
Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), a
membrane-bound exopeptidase, has been classically
associated to the T-cell activation antigen CD26, a
multifunctional sialoglycoprotein expressed on a variety
of different epithelia and also by different hematopoietic
cell types (for some recent reviews, see Yaron and
Naider.1 and Fleischer et al.2). Dipeptidyl peptidase IV
(CD26) is a serine protease which has the unique
specificity to cleave dipeptides from the N-terminus of
polypeptides provided that proline is the penultimate
residue.1 In HIV infection, CD26 has been implicated
in the viral entry process and its cytopathic effect.3
Furthermore, DPP IV activity inhibition by HIV-1 Tat
protein has been proposed as the mechanism of the lack
of response to recall antigens observed in early stages
of HIV infection;4 however, the relevance of this inhibi-
tion in physiological conditions is unclear.5 Whatever
is the case, the addition of soluble CD26 can restore
response to recall antigens of HIV-infected individuals
in vitro.6
Irrespective of its peptidase activity, CD26 is associ-
ated with other molecules on the cell surface. It has
been shown to be the main receptor of adenosine
deaminase,7 and on T-lymphocytes, CD26 is associated
with CD45,8 a cell-surface-expressed phosphotyrosine
* Corresponding author. Tel: 33 1 39254365. Fax: 33 1 39254452.
† Universite´ de Versailles.
‡ Institut Pasteur.
S0022-2623(97)00640-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/13/1998

Specific Irreversible Inhibitors of DPP IV
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2101
Ch a r t 1. General Structure of the Cyclopeptides A
Designed for Irreversible Inhibition of the DPP IV
Activity of CD26
Peptide synthesis in solution, using DCC/HOBt for the
formation of the amide bonds, was applied for the
synthesis of the linear precursors of these macrocycles.
The strategy involved the use of a nitro substituent as
a latent amino group.37 Coupling of the 2-(phenoxym-
ethyl)-5-nitrobenzoic acid (Nba-[CH2OPh], 538) with
either ethyl diglycinate or ethyl tetraglycinate gave the
substituted ethyl nitrobenzoyl polyglycinates 6a ,b. Se-
lective catalytic hydrogenation of the nitro substituent,
without hydrogenolysis of the benzyl ether function,
occurred in the presence of platinium oxide in MeOH/
DMF. The unstable aminobenzoyl polyglycine deriva-
tives were rapidly acylated with N-Boc-L-proline to
generate compounds Boc-Pro-Aba-[CH2OPh]-Glyn-OEt
7a ,b. Cleavage of the N-protecting group occurred in
trifluoroacetic acid. In the following coupling step, the
orthogonally diprotected Z-L-Lys(Boc)-OH derivative
was used. The linear peptides 8a ,b were obtained in
good yields. Cyclization of these precursors was achieved
by the azide method: hydrazinolysis of the ester func-
tion leading to protected hydrazides 9a ,b, selective
cleavage of the Nꢀ-Boc group of the lysine residue in the
presence of the NR-Z protecting group, treatment of the
resulting hydrazides with an alkyl nitrite, and dilution
in DMF in the presence of a tertiary amine. The
cyclization yields were 45% and 44% for 10a ,b, respec-
tively.
Me3,26 X-azaPro derivatives,27 X-Pro-N-(arylcarbonyloxy)-
amides,28,29 and one of its ψ(CFdC) fluoro olefin
isosteres.30,31 Owing to the presence of a free amino
N-terminus and the flexibility of the imino peptide bond,
several of these inhibitors easily cyclize and are not very
stable in solution.32 The diacylated hydroxylamines are
mechanism-based irreversible inhibitors of the pro-
tease: a demasked acylnitrene can react directly with
an active site nucleophile or can lead, through a Lossen
rearrangement, to an electrophilic isocyanate.33
We have previously designed and studied function-
alized cyclopeptides containing a latent quinoniminium
methide electrophile as suicide substrates for serine
proteases.34 Taking into account the substrate specific-
ity of the DPP IV enzyme for P2-Pro><-P′1 cleavages
(P1 ) Pro; Schechter and Berger notation35), we have
now designed, synthesized, and studied cyclopeptides
A (Chart 1): c[(RH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn]
2TFA- (Aba ) 3-aminobenzoic acid, R ) alkyl), pos-
sessing the same latent electrophile, as selective suicide
substrates for this exoprotease. These cyclopeptides
were able to induce the complete, rapid, and irreversible
inhibition of the DPP IV activity of CD26 with IC50 in
the nanomolar range. Their specificity was demon-
strated by the lack of its effect on the activity of other
peptidases, including the cell-surface-expressed DPP IV-
â, the recently described protein with typical DPP IV
activity.36
Organic sulfides, such as thioanisole, in trifluoroacetic
acid are known to deprotect O-benzyltyrosine without
the formation of O-to-C rearrangement products39 and
also to cleave the N-benzyloxycarbonyl protecting group.40
The reactions occur by a “push-pull mechanism”:
nucleophilic attack of the sulfide lone pair on the
benzylic carbon of a protonated ether or carbamate
function. The byproducts in these deprotection reac-
tions are sulfonium salts. We reasoned that treatment
of the c[RZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn] cyclopep-
tides with various dialkyl sulfides in trifluoroacetic acid
should cleave both the Z protecting group and the
phenyl ether function (Scheme 2), thus leading to
sulfonium salts having a protonated N-terminus. Ef-
fectively, such a treatment gave the bis trifluoroacetate
salts A: 11a (n ) 2, R ) Me, 62% yield), 11b (n ) 4, R
) Me, 60% yield), 12a (n ) 2, R ) Bu, 74.5% yield),
13a (n ) 2, R ) Oct, 71% yield).
For the preparation of the cyclopeptide having a
benzylic acetoxy substituent, the corresponding dim-
ethylsulfonium salt 11a was treated with potassium
acetate in dry DMF. The resulting acetate was not very
stable and decomposed during purification. The crude
product was therefore reacted with di-tert-butyl dicar-
bonate to give the stable N-protected derivative which
was easily purified by chromatography. Treatment with
trifluoroacetic acid cleaved the Boc protecting group and
led to the expected acetate salt 15a in 76% yield.
Resu lts
From the relative weak importance of the nature of
the P2 side chain on the rate of hydrolysis of the enzyme
substrates (vide infra), we hypothesized that the cy-
clization to the ꢀ-amino function of a P2 lysine residue
would result in new molecules able to bind the catalytic
site of the DPP IV activity in CD26. Moreover, this
cyclization would leave the N-terminal R-amino group
free and protonated, a necessary condition for the
recognition by the enzyme (Chart 1). In macrocycles
A, the substituted P′1 aminobenzoic acid residue Aba-
(6-CH2-S+R2) is a precursor of the latent electrophilic
quinoniminium methide cation. The presence of differ-
ent numbers of glycine residues (n) in the cyclopeptides
A allows the variation of the ring size, whereas the
nature of the sulfur substituents (R) will modify the bulk
and the lipophilicity in this part of the molecule. The
influence of the leaving group ability on the inhibition
efficiency and selectivity has been also examined by
replacing the benzylic sulfonium substituent by an
acetate group.
Finally, selective hydrogenolysis of the Z protecting
group of the cyclopeptide in the presence of the benzylic
phenyl ether function was achieved by using a pal-
ladium on carbon catalyst in aqueous methanol and
gave the cyclopeptide 16a possessing a phenoxy sub-
stituent (Scheme 2).
For comparison, simplified linear analogues of the
cyclopeptides A were studied. The trifluoroacetate salts
H2+-Ala-Pro-Aba(6-CH2-S+Me2)-OMe and H2+-Lys(H+)-
Ch em istr y. Cyclopeptides A were prepared accord-
ing to the reaction sequence shown in Scheme 1.

2102 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Nguyen et al.
Sch em e 1. Synthesis of the Cyclopeptides 10a ,b Having a Benzylic Phenoxy Substituent and a Terminal N-Z
Protecting Groupa
a
(i) GlynOEt, DCC/HOBt; (ii) 1. H2/PtO2, MeOH-DMF, 2. Boc-Pro-OH, DCC/HOBt; (iii) 1. CF3CO2H or HCl/CH2Cl2, 2. Z-Lys(Boc)-OH,
DCC/HOBt; (iv) NH2NH2/MeOH; (v) 1. CF3CO2H/CH2Cl2, 2. HCl/DMF-THF, 3. i-PrNEt2/DMF.
Sch em e 2. Last Steps of the Synthesis of Cyclopeptides 11a -16a a
a
(i) R2S/CF3CO2H; (ii) 1. KOAc/DMF, 2. Boc2O; (iii) CF3CO2H; (iv) H2/Pd/C, MeOH.
Pro-Aba(6-CH2-S+Me2)-OMe (18a ,b) were prepared as
above from the corresponding benzylic ethers H2+-X-Pro-
Aba(6-CH2-OC6H5)-OMe (17a ,b).
Biologica l Stu d ies. In all the studies described
here, the dipeptidyl peptidase activity of CD26 and DPP
IV-â was investigated using the enzymes in their
natural habitat, i.e., by using either crude cell extracts
or intact cells36 as described in the Experimental
Section. Indeed, CD26 and DPP IV-â are expressed on
the cell surface, and thus their enzymatic activity could
be assayed by using intact cells.36 As a source of CD26
and DPP IV-â, we used either human CEM cells
overexpressing CD26 by transfection49 or human C8166
cells which express only DPP IV-â.36 In some experi-
ments partially purified enzyme preparations were also
used.51 In the case of CD26, we also used immunoaf-
finity-purified preparations of CD26 as we have de-
scribed previously.36 For the measurement of the

Specific Irreversible Inhibitors of DPP IV
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2103
F igu r e 1. Specificity of molecule 11a . Effect of inhibitor 11a
on different types of peptidase activities. Crude MOLT4 cell
extracts36 were assayed for the effect of 10 µM compound 11a
on different aminopeptidase activities. The peptidases tested
were as follows: DPP IV by the cleavage of GP-pNA, RP-pNA,
and AP-pNA; Arg-peptidase by the cleavage of R-pNA; Ala-
peptidase by the cleavage of A-pNA; and Pro-peptidase by the
cleavage of P-pNA. At 10 µM 11a , the DPP IV activity against
different substrates was inhibited by more than 75%, while
no apparent effect on the other peptidases was observed.
Abbreviations of the amino acids: G ) glycine, P ) proline, A
) alanine, and R ) arginine.
F igu r e 2. Kinetics of molecule 11a -mediated inhibition of the
cell-surface-expressed DPP IV activity of CD26. Intact CEM
cells expressing high levels of CD26 (clone H01)49 were
incubated for 5, 10, 15, and 30 min in PBS with or without 5
µM compound 11a at 37 °C and then washed twice with PBS.
DPP IV activity of CD26 was then monitored by the cleavage
of GP-pNA as described in the Experimental Section. The
binding of the inhibitor 11a to DPP IV active site is very rapid
since 5 min is sufficient to obtain more than 75% inhibition.
activity of different peptidases, extracts from human
MOLT4 cells were used.36
Preliminary results pointed out that molecule 11a
was a potent inhibitor of DPP IV activity of CD26.
Indeed, the IC50 value for the inhibition of the DPP IV
activity of a purified preparation of CD2636 was found
to be 0.01 µM. For this reason, we first studied the
specificity of this inhibitor by determining its effect on
different aminopeptidases found in crude cell extracts.
The peptidases tested were as follows: arginine-pepti-
dase (EC 3.4.11.6) by the cleavage of Arg-pNA, alanine-
peptidase (EC 3.4.11.2) by the cleavage of Ala-pNA,
proline-peptidase (EC 3.4.11.5) by the cleavage of Pro-
pNA, and DPP IV by the cleavage of different substrates
(Gly-Pro-, Arg-Pro-, and Ala-Pro-pNA).36 At 10 µM
molecule 11a , which is 3 orders of magnitude higher
than its IC50, the DPP IV activity monitored with the
different substrates was inhibited by more than 80%
(Figure 1). The remaining residual 20% activity was
probably due to the background (i.e., a nonspecific and
CD26-independent cleavage of the substrates) since it
was observed even at 100 µM molecule 11a (not shown).
In contrast, molecule 11a exerted no inhibitory effect
on the activity of arginine-, alanine-, and proline-
aminopeptidase (Figure 1). These results therefore
demonstrated the specific nature of the DPP IV inhibi-
tion by molecule 11a .
To investigate the irreversible nature of the inhibitory
molecule 11a , its effect on the DPP IV activity of CD26
expressed on the cell surface was studied. For this
purpose, we established by transfection of CD26 cDNA,
human CEM cells which express high levels of CD26,
i.e., cells which express high levels of DPP IV activity
on the cell surface. Consequently, intact cells (clone
H01) could be assayed for DPP IV activity by incubation
with an appropriate substrate, such as Gly-Pro-pNA.36
Under these experimental conditions, molecule 11a was
found to be a potent inhibitor of cell-surface DPP IV
activity, with maximum inhibition occurring at 2 µM.
First, we investigated the kinetics of inhibition of cell-
surface DPP IV activity in these CEM cells preincubated
F igu r e 3. Irreversibility of DPP IV inhibition by molecule
11a demonstrated by the cell-surface-expressed CD26. Intact
CEM cells overexpressing CD26 (clone H01) were preincubated
for 15 min in PBS in the absence or presence of 5 µM 11a at
37 °C, then washed twice in PBS, and cultured in RPMI
supplemented medium. Aliquots of cells were taken at the
indicated times, washed twice with PBS, and then assayed for
DPP IV activity by incubating cells with the substrate GP-
pNA for 1 h at 37 °C. The samples at time 0 represent the
DPP IV activity just after the 15 min of preincubation.
with 5 µM molecule 11a . At times of 5, 10, 15, and 30
min, cells were washed extensively to remove unbound
molecule 11a before assay of the DPP IV activity. As
shown in Figure 2, inhibition was almost maximal after
5 min of incubation, since the degree of inhibition was
only slightly increased at 30 min. These results dem-
onstrated that molecule 11a has the capacity to bind
CD26 rapidly and thus inhibit irreversibly its DPP IV
activity. Second, to confirm the irreversible nature of
molecule 11a , cells were incubated with 5 µM inhibitor
for 15 min before extensive washing and further incuba-
tion in the culture medium for up to 3 days. At different
times during this period, cells were monitored for cell-
surface-expressed DPP IV activity (Figure 3). The
maximum inhibition observed following the 15-min
incubation of cells with molecule 11a (time 0 h) was
found to last for several hours. At 6 h, there was a very
slight difference on the maximum inhibition, whereas
at 24 h, there was about 50% inhibition. Interestingly,
this 50% reduction of the inhibition at 24 h coincided
with the doubling time of cells, which results in the

2104 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Nguyen et al.
Ta ble 1. Inhibition of DPP IV Activity by the Cyclopeptide
Ta ble 2. Characterization of the Inhibition Kinetics of CD26
and DPP IV-â by the Cyclopeptide 11a
Compoundsa
inhibition of
Gly-Pro-pNa
hydrolysis (IC50, µM)
inhibition constantsb
enzymea
KI (µM)
kinact (s-1
)
cyclopeptides A
CD26
DPP IV-â
0.085
0.470
11. 10-4
22. 10-5
ref
glycine (n)
leaving group
CD26
DPP IV-â
11a
11b
12a
13a
15a
16a
2
4
2
2
2
2
S+Me2
S+Me2
S+Bu2
S+Oct2
OAc
0.012
0.003
0.02
0.03
1.5
1
a
The DPP IV activities of CD26 and DPP IV-â were assayed
0.61
0.57
0.63
3.5
by the use of extracts from CEM H01 (cells expressing very high
levels of recombinant CD26) and C8166 cells (cells expressing only
DPP IV-â), respectively. The cleavage of the substrate Gly-Pro-
pNA (0.5 mM) was monitored in the presence of increasing
concentrations of inhibitor 11a by measuring absorbance at 405
nm. Preparation of extracts and assay conditions were as described
OPh
10
25
a
The DPP IV activity was monitored by the cleavage of Gly-
Pro-pNA in purified preparations of CD26 and DPP IV-â (Experi-
mental Section). CD26 was purified using extracts of CEM H01
cells expressing very high levels of CD26.49 DPP IV-â was purified
using extracts of CD26-negative C8166 cells.36 Both purification
procedures were as described elsewhere.51 Such purified prepara-
tions of CD26 and DPP IV-â were free of any significant contami-
nation by other peptidases. CD26 and DPP IV-â preparations were
preincubated for 15 min with different concentrations of each
inhibitor ranging from 1 nM to 10 µM before adding the substrate
Gly-Pro-pNA as indicated.
b
before36 and as in the Experimental Section. Calculations were
done as indicated in the Experimental Section. KI is the equilib-
rium constant of the inhibitor binding to the enzyme, whereas kinact
is the constant of the irreversible reaction that leads to the
inactivation of the enzyme.
essential to assess the action of the cyclopeptide inhibi-
tors on DPP IV-â. The results given in Table 1 show
that the inhibitory effect of the different irreversible
cyclopeptide inhibitors is much more pronounced for
CD26 compared to DPP IV-â. For example, it is
interesting to note that the inhibitory effect of molecules
11a ,b is 83- and 203-fold higher on the DPP IV activity
of CD26 compared to that of DPP IV-â, respectively
(Table 1). In contrast to such a significant selectivity,
molecules 15a and 16a exerted only about 2-fold dif-
ference between the two enzymes. These data might
suggest that the higher specificity of the inhibitors on
the DPP IV activity of CD26 could be related to the
reactivity of the cyclopeptide sulfonium salts.
To further investigate the significant differences in
the effect of the mostly studied inhibitorory molecule11a
on DPP IV activity of CD26 and DPP IV-â (Table 1), we
studied the kinetics of inhibition of both enzymes, by
using the approach previously described for irreversible
inhibitors of trypsin-like proteases.34 This approach
considers two steps in the inhibition process: first, the
reversible binding of the inhibitor to the enzyme and,
second, the cleavage and subsequent irreversible cova-
lent modification of the enzyme leading to the loss of
catalytic activity. The inhibition kinetics of the DPP
IV activities of CD26 and DPP IV-â fit well to this
model, and the equilibrium constant of the first step (KI)
as well as the kinetic constant of the second step, was
calculated. The results are summarized in Table 2 and
show that the higher potency of inhibitor 11a on the
DPP IV activity of CD26 is the consequence of a higher
affinity for this enzyme as demonstrated by the KI
values, along with a faster inactivation rate as pointed
out by the kinact values found for CD26.
production of cells that express newly synthesized CD26
molecules. At 3 days, no inhibition of cell-surface DPP
IV activity was observed. Taken together, these data
indicate that the inhibitory effect of molecule 11a is
irreversible, since the DPP IV activity could be resumed
only by the newly synthesized CD26 (Figure 3). CD26
being a cell-surface-expressed protein, its expression on
the cell surface could be modified with respect to the
different phases of the cell cycle. Consequently at 24
and 72 h, the DPP IV activity was variable in control
cells (without preincubation with the inhibitor) (Figure
3). It should also be noted that the cell-surface expres-
sion of CD26 was not affected by coupling with molecule
11a . Indeed, cells in the absence or presence of prein-
cubation with molecule 11a manifested similar levels
of cell-surface-expressed CD26, as monitored by FACS
analysis using anti-CD26-specific monoclonal antibodies
(data not shown).
The other cyclopeptides, possessing different leaving
groups (12a , 13a , 15a , 16a ) or a larger ring (11b) were
also assayed for their capacity to inhibit the DPP IV
activity of CD26 using crude extracts from CEM cells
(clone H01). The IC50 values for the inhibition of Gly-
Pro-pNA hydrolysis are given in Table 1. All of these
cyclopeptides showed different IC50 values in the nano-
molar or micromolar range. However, no correlation
was observed between these IC50 values and the length
or lipophilicity of the leaving group. Molecules 15a and
16a with leaving groups as acetate and phenoxy,
respectively, manifested significantly reduced IC50 val-
ues. In contrast, increasing the ring size in a given
molecule generated a compound with an increased
inhibitory activity (Table 1, compare molecules 11a ,b).
By use of the CD26-negative T-lymphoblastoid cell
line C8166, we have recently described a CD26-like cell-
surface protein with typical DPP IV activity.36 This
novel form of DPP IV, referred to as DPP IV-â, was
found to be distinct from CD26. However the pH
optimum and the profiles for substrate molecules were
found to be indistinguishable for both CD26 and DPP
IV-â. Similarly, several previously described inhibitors
of DPP IV exerted a very similar mode of action on both
CD26 and DPP IV-â.36 Consequently, it remained
The difference in the inhibitory effect of irreversible
inhibitors was further emphasized by testing the cell-
surface-expressed enzymes. For this purpose, we used
CEM cells expressing high levels of CD26 (clone H01)
as a source of CD26 and C8166 cells as a source of DPP
IV-â. Figure 4 shows the effect of different concentra-
tions of molecule 11a and a previously described revers-
ible inhibitor, Lys-[Z(NO2)]-pyrrolidide. As we had
reported previously,36 Lys-[Z(NO2)]-pyrrolidide inhibited
to a similar extent the DPP IV activity of both CD26
and DPP IV-â, with comparable IC50 values. On the
other hand, the molecule 11a completely inhibited the
DPP IV activity of cell-surface-expressed CD26 without

Specific Irreversible Inhibitors of DPP IV
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2105
F igu r e 5. Postulated mechanism of enzyme inactivation. Two
covalent bonds are formed between the inhibitor and the
enzyme (double-hit mechanism). First, an acyl-enzyme is
formed which should involve the catalytic serine 630 of CD26
and proline residue P1 of the inhibitory molecule (step 1).
Second, by means of the unmasked quinoniminium methide
(step 2), a second covalent bond is formed implicating a
nucleophile residue Nu in the vicinity of the catalytic serine
(step 3). Consequently, this second bond is responsible for the
irreversible blockade of the catalytic site of the enzyme.
F igu r e 4. Molecule 11a inhibits the DPP IV activity of the
cell-surface-expressed CD26 but not that of DPP IV-â. Intact
CEM cells overexpressing CD26 (clone H01) and C8166 cells
expressing only DPP IV-â were preincubated for 15 min in PBS
containing different concentrations of molecule 11a (upper
panel) or Lys-[Z(NO2)]-pyrrolidide (lower panel), before adding
GP-pNA to monitor residual DPP IV activity. Control activity
(100%) was determined in the absence of inhibitor.
IT1 by formation of an oxazolidine ring (IT2). Proton
transfer from the terminal ammonium function leads
to a third tetrahedral intermediate (IT3) and then
preferentially to a cis acyl-enzyme which isomerizes to
the trans isomer.
For the herein described cyclopeptides A, the postu-
lated mechanism of the observed enzyme inactivation
is presented in Figure 5. Provided that the protease
can accommodate the large molecule A, formation of an
acyl-enzyme, by selective nucleophilic attack of the
hydroxyl function of serine 630 on the P1 proline
carbonyl of the cyclopeptide (step 1), would unmask a
P′1-substituted aniline having a good benzylic leaving
group. Owing to the strong electron-releasing property
any apparent effect on cell-surface-expressed DPP
IV-â, even when used at high concentrations such as
10-20 µM (Figure 4). It should be noted that molecule
11a was active against the soluble form of DPP IV-â
with an IC50 value of 1 µM. Thus, the inability of
molecule 11a to affect cell-surface-expressed DPP IV-â
appears to be correlated with the structure of this
protein when expressed on the cell surface. Whatever
is the case, these results emphasize specific differences
in the overall structure of DPP IV-â compared to that
of CD26.
of the amino substituent (σ+
) -1.31, compared
p-NH2
+
to σ
) -0.6943 in the starting cyclopeptide),
p-NHCOR
a fast elimination of a neutral dialkyl sulfide (or an
anionic acetate) should give a substitued quinoniminium
methide ion34 (step 2) tethered in the active site during
the lifetime of the acyl-enzyme by means of the peptide
chain. A Michael-type addition of a second nucleophile
Nu, in the vicinity of the active serine, on this demasked
electrophile (step 3) would establish a second covalent
enzyme-inhibitor bond (elimination-addition pro-
cess).44 Such a bridge between the active site serine and
another nucleophilic residue active site leads to an
irreversible loss of the enzymatic activity. Moreover,
the inactivation is expected to be very selective as the
inhibitory activity of suicide substrates (mechanism-
based inhibitors) requires discrimination in the binding
steps, catalytic activation by the target enzyme, and
irreversible modification of the active center.45
Discu ssion
The DPP IV enzyme has an absolute requirement for
the L-configuration of the amino acid residues, both in
the penultimate and in the N-terminal positions. The
amide bond between the N-terminal P2 residue and the
P1 proline residue must be in a trans conformation. A
protonated amino end is necessary for enzymatic hy-
drolysis. Amino acids with aliphatic side chains at P2
are slightly favored over aromatic ones, but the effect
is not very large. On the basis of these literature data
and the results of directed mutagenesis studies, Brandt
et al. have recently proposed a model of the enzyme
active site41 and a new catalytic mechanism for this
glycoprotein,42 which has not yet been crystallized.
Compared to usual serine proteases, the main difference
is the stabilization of the first tetrahedral intermediate
It should be emphasized that the macrocyclic sulfo-
nium salts A are obtained in just one step by reacting
an alkyl sulfide in trifluoroacetic acid with the cyclo-
peptides having a benzylic phenoxy substituent and an

2106 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Nguyen et al.
NR-Z protecting group, themselves readily obtained by
peptide synthesis in solution and cyclized by the azide
method. These cyclopeptide sulfonium salts, as all
compounds 11-16, belonging to the A series of mol-
ecules, are stable and water-soluble, and their activity
does not decrease even after several months in solution.
Their cyclic structure prevents their decomposition by
cyclization due to the attack of the free amine on the
carbonyl of the proline. Moreover, the toxicity of these
compounds on CEM cells was observed only at concen-
trations higher than 250 µM.
Similar inhibitory activities were observed among the
compounds 11a , 12a , and 13a , suggesting that the
length of the alkyl chains on the sulfonium group has
little or no influence on the inhibition of DPP IV activity
of CD26. Interestingly, the size of the macrocycle
appeared to play an important role, since a strong
inhibition was observed with compound 11b which
contains four glycine residues in its cycle. The inhibi-
tory activity of this compound is 4 times more effective
than that of its homologue molecule 11a , which pos-
sesses only two glycine residues. The weak inhibition
observed with compounds 15a and 16a is not surprising,
since the acetate and the phenoxy groups are not good
leaving groups compared to the alkyl sulfide ones. For
the linear analogues of the cyclopeptides, no inhibition
was observed at 100 µM. In this case, a fast diffusion
of the quinoniminium methide cation out of the active
center may occur due to the lack of the peptide chain
which tethers the electrophile in the active site. Taken
together, our results show that large substituents on
the sulfur atom did not significantly modify the inhibi-
tion reaction. In contrast the ring size enlargement
improved it, probably by allowing more favorable in-
teractions with the enzyme active center and/or facili-
tating the approach to the nucleophilic group Nu in the
active site.
We have previously reported that the irreversible
inhibitor 11a affects HIV-1 Lai infection of CEM cells
in a dose-dependent manner, with an IC50
value of 10
µM.46 In contrast, another potent but reversible inhibi-
tor of the DPP IV activity, Lys-[Z(NO2)]-pyrrolidide, has
no apparent effect on HIV infection of CEM cells, even
at 100 µM.46 Interestingly, molecule 11a , which has no
inhibitory activity on the cell-surface-expressed DPP IV-
â, manifests no effect on the HIV-1 infection of CD26-
negative but DPP IV-â expressing C8166 cells.46 These
observations indicate that the irreversible inhibitors
behave as specific compounds affecting the DPP IV
activity of CD26, and consistent with previously pub-
lished reports,3,36,47-49 they demonstrate once again the
implication of CD26 in the mechanism of HIV infection.
Recently, Bekesi and collaborators have synthesized
reversible inhibitors of the type X-Pyrr-2-CN and have
shown that they inhibit HIV-1 infection with IC50 values
of 2.4-5.3 µM.17,50
Taken together, our results describe for the first time
potent irreversible inhibitors of DPP IV which could be
used specifically on CD26 and could be used in different
types of studies for better understanding the role of
CD26 in biological processes.
Exp er im en ta l Section
Syn th etic P r oced u r es. Melting points were recorded on
a Tottoli (Bu¨chi) or a Mettler FP61 and are uncorrected. The
1H NMR measurements were performed on a Bru¨ker AM 300
instrument. The chemical shifts are reported in ppm, the
deuterated solvents being used as internal standards: CD3-
OD (3.31 ppm) and CD3COOD (2.10 ppm). The coupling
constants are given in hertz. The optical rotations were
obtained on a Perkin-Elmer 241 polarimeter. Analyses were
performed by the Services of CNRS at ICSN (Gif sur Yvette).
The mass spectrometry measurements were performed by Mr.
Deroussent (Inst. G. Roussy, Villejuif) or Ms. Kargar (Ecole
Polytechnique) and for the FAB HRMS data, Mr. Fabre-Bonvin
(CNRS, Vernaison). Analytical thin-layer chromatography
(TLC) and preparative column chromatography were per-
formed on Kieselgel F 254 and Kieselgel 60, 0.063-0.5 and
0.040-00.063 mm (Merck), respectively, with the following
solvent systems (by vol): I (x/y), MeOH x%-CH2Cl2 y%; II,
AcOEt-n-BuOH-AcOH-H2O (1/1/1/1). UV light (254 nm)
allowed visualization of the spots after TLC runs for all
compounds, even at low concentration.
Nba -[CH2OP h ]-Gly2-OEt (6a ). 2-(Phenoxymethyl)-5-ni-
trobenzoic acid (5) (2.5 g, 9.15 mmol), prepared as previously
described38 was dissolved in a 50-mL solution of CH2Cl2/THF
(1/1). DCC (2.45 g, 11.89 mmol) and HOBt (1.36 g, 10.05
mmol) were added, and the solution was stirred at 0 °C for 15
min. Diglycine ethyl ester hydrochloride (1.98, 10.05 mmol)
and a solution of triethylamine (3.9 g, 27.45 mmol) in CH2-
Cl2/THF (1/1) (40 mL) were then added. The reaction mixture
was stirred for 1 h at 0 °C and for 24 h at room temperature.
Solvents were evaporated under reduced pressure at 50 °C,
the residue was dissolved in acetone, and the DCU was
filtered. The filtrate was then evaporated, dissolved in AcOEt,
and washed successively with H2O (100 mL), 0.5 N aqueous
HCl (100 mL), H2O (100 mL), 5% aqueous NaHCO3 (100 mL),
and H2O (100 mL). The organic phase was dried over Na2-
SO4, filtered, and concentrated. Chromatography on silica gel
with eluent I (2/98) led to 3.23 g (85.5%) of pure peptide 6a as
a white powder. Mp: 132.4 °C. Rf (I: 2/98) ) 0.5. 1H NMR
(CD3OD): 8.56, d (2.3), 1H (ArH); 8.39, dd (2.3, 8.5), 1H (ArH);
7.97, d (8.5), 1H (H3); 7.32, m, 2H and 7.01, m, 3H (ArH); 5.45,
s, 2H (CH2OPh); 4.21, q (7.1), 2H (OEt); 1.30, t (7.1), 3H (OEt).
Anal. Calcd for C20H21N3O7‚0.5H2O: C, 56.59; H, 5.18; N, 9.90.
Found: C, 56.61; H, 5.11; N, 10.21.
Comparative studies on the inhibition of the DPP IV
activity of CD26 and DPP IV-â revealed that the two
enzymes should be distinct from each other. In soluble
enzyme preparations, the irreversible cyclopeptide in-
hibitors were less active on DPP IV-â compared to
CD26. This latter is most probably due to both a
different affinity and a different inactivation rate, as
we have demonstrated to be the case with the cyclo-
peptide 11a (Table 2). Consequently, it is plausible to
suggest the presence of a less reactive active site
nucleophile Nu-DPP IV-â compared to the correspond-
ing Nu residue in CD26 and/or to a different localization
of this group with respect to the active catalytic serine
residue. These point out that there should be important
structural differences between DPP IV-â and CD26.
Such structural differences probably become accentu-
ated with the cell-surface-expressed proteins. Indeed,
cyclopeptide inhibitors active at nanomolar concentra-
tions on the cell-surface-expressed CD26 manifest no
apparent activity on the cell-surface-expressed DPP IV-
â. Accordingly, the catalytic domain necessary for the
DPP IV activity could be differentiated between CD26
and DPP IV-â. For the cell-surface-expressed DPP IV-
â, the particular structure of its catalytic domain
presumably cannot accommodate the inhibitor. On the
other hand, the solubilized DPP IV-â can bind the
functionalized cyclopeptide and therefore be inhibited.
Boc-P r o-Aba -[CH2OP h ]-Gly2-OEt (7a ). The peptide 6a
(2.5 g, 6.02 mmol) was dissolved in hot DMF/MeOH (25/75;

Specific Irreversible Inhibitors of DPP IV
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2107
60 mL), and platinium oxide (0.2 g) was added to the warm
solution, which was hydrogenated in a Parr apparatus at room
temperature for 2 h. The solution was filtered in order to
remove the catalyst. Analytical TLC of the filtrate with eluent
I (15/85) showed a new unique spot, highly fluorescent on UV
light at 254 and 366 nm, corresponding to the intermediate
aromatic amine. This compound was not isolated and was
used directly in the next step, because of its instability in
solution. Thus methanol and DMF were rapidly evaporated
in vacuo at 40 °C. The residue was dissolved in CH2Cl2/THF
(1/1; 30 mL). The clear solution was cooled at 0 °C, and a
solution of Boc-L-proline (1.295 g, 6.02 mmol) and HOBt (8.94
g, 6.62 mmol) in CH2Cl2/THF (1/1; 70 mL) and then DCC (1.613
g, 7.82 mmol) were added. The reaction mixture was stirred
from 0 °C during 2 h to room temperature overnight. The
solvents were evaporated, ethyl acetate (300 mL) was added,
and the dicyclohexylurea precipitate was removed by filtration.
The filtrate was extracted successively with 0.5 M aqueous
HCl (2 × 100 mL), H2O (100 mL), 5% aqueous NaHCO3 (2 ×
100 mL), and H2O (2 × 100 mL), dried over Na2SO4, filtered,
and evapored. The residue was chromatographed on a column
of silica gel. Elution with eluent system I (2/98) led to 2.05 g
(58%) of the pure peptide 7a . Rf (I: 10/90) ) 0.5. 1H NMR
(CD3OD): 7.84, d (1.8), 1H (ArH); 7.68, dd (1.8, 8.4), 1H (ArH);
7.54, d (8.4), 1H (ArH); 7.24, m, 2H and 6.94, m, 3H (ArH);
5.23, s, 2H (CH2OPh); 4.31, md, 1H (HRPro); 4.14, q (7.1), 2H
(OEt,); 3.89 and 3.68, 2s, 4H (HRGly); 3.50, m, 2H (HδPro);
2.29, m, 1H (HâPro); 1.97, m, 3H (HâPro, HγPro); 1.47 and
1.36, 9H (Boc); 1.24, t (7.1), 3H (OEt). Anal. Calcd for
in vacuo. The residue was dissolved in diethyl ether, tritu-
rated, and evaporated. After this operation was repeated three
times, the residue was dissolved in THF (20 mL) and N-
methylmorpholine (40 µL, 0.35 mmol) was added. This solu-
tion a was stirred at 0 °C for 30 min. To a solution of
Z-Lys(Boc)-OH (136 mg, 0.35 mmol) and N-methylmorpholine
(40 µL, 0.35 mmol) in THF (20 mL) cooled at - 10 °C was
added ethyl chloroformate (34 µL, 0.35 mmol). This mixture
b containing the mixed anhydride was stirred at -10 °C for
15 min and the former solution a was added. The reaction
mixture was stirred for 1 h at -10 °C and at room temperature
overnight. After evaporation of the solvents, the residue was
chromatographed on a column of silica gel. Elution with eluent
system I (10/90) led to the pure peptide 8b (0.14 g, 36%). Mp:
149.5 °C. Rf (I: 10/90) ) 0.4. 1H NMR (CD3OD): 7.75, s, 1H
(ArH); 7.56, m, 1H (ArH); 7.40, m, 1H (ArH); 7.28, m, 7H
(ArH); 6.88, m, 3H (ArH); 5.15, s, 2H (CH2OPh); 5.01, s, 2H
(CH2Z); 4.36, m, 1H (HRPro); 4.05, m, 5H (HRLys, OEt,
HRGly); 3.85, ∼s, 6H (HRGly); 3.54, m, 2H (HδPro); 2.99, m,
2H (HꢀLys); 2.21-1.58, m, 10H (HâPro, HγPro, HâLys, HγLys,
HδLys); 1.43, s, 9H (Boc); 1.16, t, 3H (OEt). Anal. Calcd for
C
48H62N8O13: C, 60.11; H, 6.52; N, 11.68. Found: C, 59.42;
H, 6.57; N, 10.51.
rZ-Lys(EBoc)-P r o-Aba-[CH2OP h ]-Gly2-NH-NH2 (9a). The
peptide 8a (1 g, 1.18 mmol) was dissolved in methanol (50 mL).
A large excess of hydrazine monohydrate (6 mL, 118 mmol)
was added, and the solution was stirred at room temperature
for 24 h. Methanol and the excess of hydrazine were evapo-
rated in vacuo. The residue was evacuated at 40 °C/0.1 mmHg
for 1 h, solubilized in methanol (20 mL), and precipitated by
addition of ether (200 mL). The precipitate was filtered,
washed with ether, and dried, leading to 0.69 g (70%) of
hydrazide 9a as a white powder. Rf (I: 10/90) ) 0.5. 1H NMR
(CD3OD): 7.83, s, 1H (ArH); 7.63, d (8.4), 1H (ArH); 7.51, d
(8.4), 1H (ArH); 7.28, m, 7H (ArH); 6.76, m, 3H (ArH); 5.22, s,
2H (CH2OPh); 5.07, s, 2H (CH2Z); 4.57, m, 1H (HRPro); 4.41,
m, 1H (HRLys); 4.23, 4.07, 4.04, and 3.94, 2sd, 4H (HRGly);
3.89 and 3.71, md, 2H (HδPro); 3.04, m, 2H (HꢀLys); 1.99-
1.46, m, 10H (HâPro, HγPro, HγLys, HγLys, HδLys); 1.41, s,
9H (Boc). Anal. Calcd for C42H54N8O10‚0.5H2O: C, 60.05; H,
6.60; N, 13.34. Found: C, 60.32; H, 6.75; N, 12.76.
C
30H38N4O8‚0.5H2O: C, 60.89; H, 6.64; H, 9.47. Found: C,
60.86; H, 6.57; N, 9.45.
Boc-P r o-Aba-[CH2OP h ]-Gly4-OEt (7b): obtained as above
from 6b32 (3.5 g, 6.6 mmol). Yield: 1.4 g (30%), white powder.
Rf (I: 10/90) ) 0.5. 1H NMR (CD3OD): 7.85, s, 1H (ArH); 7.65,
m, 1H (ArH); 7.52, m, 1H (ArH); 7.25, m, 2H and 6.95, m, 3H
(ArH); 5.16, s, 2H (CH2OPh); 4.26, md, 1H, (HRPro); 4.10, q,
2H (OEt); 4.05, s, 2H (HRGly); 3.90, 3.89 and 3.88, m, 6H
(HRGly); 3.53, m, 2H (HδPro); 2.29, m, 1H (HâPro); 2.03, m,
2H (HγPro); 1.89, m, 1H (HâPro); 1.47 and 1.36, 9H (Boc); 1.24,
t, 3H (OEt). Anal. Calcd for C34H44N6O10‚H2O: C, 57.12; H,
6.48; H, 11.75. Found: C, 57.11; H, 6.38; N, 11.44.
rZ-Lys(EBoc)-P r o-Aba-[CH2OP h ]-Gly2-OEt (8a). The pep-
tide 7a (1.5 g, 2.57 mmol) was dissolved in CH2Cl2 (15 mL) at
0 °C, and TFA (15 mL) was added. The reaction mixture was
stirred at 0 °C for 1.5 h. Excess of TFA was evaporated in
vacuo. The residue was dissolved in CH2Cl2, and the solution
was washed with 5% aqueous NaHCO3, dried over Na2SO4,
filtered, and evaporated. The residue was dissolved in CH2-
Cl2/THF (1/1; 15 mL) and added to a mixture of Z-Lys(Boc)-
OH (0.98 g, 2.57 mmol), DCC (0.689 g, 3.34 mmol), and HOBt
(0.382 g, 2.82 mmol) in CH2Cl2/THF (1/1; 30 mL) at 0 °C. The
reaction mixture was stirred from 0 °C during 2 h to room
temperature overnight. The solvents were evaporated, ethyl
acetate (300 mL) was added, and the dicyclohexylurea pre-
ciptate was removed by filtration. The filtrate was extracted
successively with 0.5 M aqueous HCl (2 × 100 mL), H2O (100
mL), 5% aqueous NaHCO3 (2 × 100 mL), and H2O (2 × 100
mL), dried over Na2SO4, filtered, and evapored. The residue
was chromatographed on a column of silica gel. Elution with
eluent system I (6/94) led to 1.4 g (65%) of the pure peptide
8a . Mp: 78.7 °C. Rf (I: 5/95) ) 0.5. 1H NMR (CD3OD): 7.74,
d (2.0), 1H (ArH); 7.60, dd (2.0, 8.4), 1H (ArH); 7.45, d (8.4),
1H (ArH); 7.26, m, 7H (ArH); 6.92, m, (ArH); 5.19, s, 2H (CH2-
OPh); 5.06, s, 2H (CH2Z); 4.57, m, 1H (HRPro); 4.41, m, 1H
(HRLys); 4.13, q (7.1), 2H (OEt); 4.03, s and 3.89, s, 4H
(HRGly); 3.86 and 3.69, m, 2H (HδPro); 3.03, m, 2H (HꢀLys);
2.19-1.48, m, 10H (HâPro, HγPro, HδLys, HâLys, HγLys);
1.41, s, 9H (Boc); 1.23, t (7.1), 3H (OEt). Anal. Calcd for
C44H56N6O11: C, 62.54; H, 6.68; N, 9.94. Found: C, 62.35; H,
6.63; N, 9.53.
rZ-Lys(EBoc)-P r o-Aba -[CH2OP h ]-Gly4-NH-NH2 (9b): ob-
tained as above from the peptide 8b (0.058 g, 0.6 mmol), white
powder (0.041 g, 72%). Rf (I: 8/92) ) 0.1. 1H NMR (CD3OD):
7.77, s, 1H (ArH); 7.61, d (8.4), 1H (ArH); 7.45, m, 1H (ArH);
7.32, m, 7H (ArH); 6.92, m, 3H (ArH); 5.18, dd∼s, 2H (CH2-
OPh); 5.05, dd∼s, 2H (CH2Z); 4.55, m, 1H (HRPro); 4.40, m,
1H (HRLys); 4.04, s, 2H (HRGly); 3.87, s, 3.86, s and 3.80, s,
6H (HRGly); 3.68, m, 2H (HδPro); 3.03, m, 2H (HꢀLys); 2.21-
1.62, m, 10H (HâPro, HγPro, HâLys, HγLys, HδLys); 1.40, s,
9H (Boc). Anal. Calcd for C46H60N10O12‚2H2O: C, 56.31; H,
6.57; N, 14.27. Found: C, 56.15; H, 6.55; N, 14.87.
Cyclo(-Lys(rZ)-P r o-Aba-[CH2OP h ]-Gly2-) (10a). The pep-
tide 9a (0.640 g, 0.77 mmol) was dissolved in CH2Cl2 (10 mL)
at 0 °C, and TFA (15 mL) was added. The solution was stirred
for 2 h. Then CH2Cl2 and excess of TFA were evaporated in
vacuo. The residue was evacuated at 40 °C/0.1 mmHg for 1 h
and triturated several times in diethyl ether to give a white
powder. This compound was dissolved in DMF (17 mL). After
cooling at -40 °C, a solution of 5.5 N HCl in THF (1.5 mL,
8.47 mmol) and isoamyl nitrite (0.145 mL, 1.08 mmol) were
successively added. The reaction mixture was stirred at -40
°C for 0.5 h. The resulting peptide azide was diluted with cold
DMF (200 mL). The solution was then brought to pH 9 by
addition of diisopropylethylamine (2 mL, 11.55 mmol) and kept
in a refrigerator for 24 h. DMF was evaporated, and distilled
water was added to the residue. The resulting solid was
filtered, dissolved in MeOH, and chromatographed on a column
of silica gel. Elution with eluent system I (10/90) led to 0.242
g (45%) of the pure cyclopeptide 10a . Mp: 144.6 °C. Rf (I:
10/90) ) 0.6. 1H NMR (DMSO-d6): 9.86, s, 1H (NHAr); 8.85,
∼t (5.5), 1H (NHGly1); 8.33, ∼t (5.5), 1H (NHGly2); 8.09, s,
1H (ArH); 7.62, ∼t (5.4), 1H (NHꢀLys); 7.50, m, 3H (2 ArH
and NHRLys); 7.32, m, 7H (ArH); 6.91, m, 3H (ArH); 5.23 and
rZ-Lys(EBoc)-P r o-Aba-[CH2OP h ]-Gly4-OEt (8b). The pep-
tide 7b (0.25 g, 0.35 mmol) was dissolved in ethanol (5 mL),
and a solution of HCl (3 N) in CH2Cl2 (20 mL) was added. The
reaction mixture was stirred at 0 °C for 1.5 h and evaporated

2108 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Nguyen et al.
5.16, 2d (12.3), 2H (CH2OPh); 5.01 and 5.00, 2d (12.7), 2H
(CH2Z); 4.35, m, 2H (HRPro, HRLys); 3.72, m, 5H (HRGly1,
HRGly2, HδPro); 3.56, ∼dd (15.9, 5), 1H (HRGly2); 3.09, m,
2H (HꢀLys); 2.19, m, 1H (HâPro); 3.79, m, 3H (HâPro, HγPro);
1.39, m, 5H (H2âLys, HδLys, HγLys); 0.85, m, 1H (HâLys).
1H NMR (CD3OD): 8.44, d, 1H (ArH); 7.62, d (8.3), 1H (ArH);
7.34, m, 5H (ArH); 7.32, m, 3H (ArH); 6.93, m, 3H (ArH);
5.28 and 5.23, 2d (12.7), 2H (CH2OPh); 5.07, s, 2H (CH2Z);
4.51, m, 2H (HRPro, HRLys); 4.11 and 3.97, 2d (16.6), 2H
(HRGly1); 3.93 and 3.86, 2d (16.8), 2H (HRGly2); 3.72 and
3.54, 2m, 2H (HδPro); 3.25, m, 2H (HꢀLys); 2.34 and 2.10,
2m, 2H (HâPro); 2.04, m, 2H (HγPro); 1.81, m, 2H (HâLys);
1.54, m, 2H (HδLys); 1.35, m, 2H (HγLys). Anal. Calcd for
C37H42N6O8‚H2O: C, 61.90; H, 6.18; N, 11.70. Found: C, 61.53;
H, 6.07; N, 11.31.
(FAB+): 617 (M+), 471 (M+ - Bu2S). HRMS (FAB+): calcd
for C31H49N6O5S+ (M+), 617.3485; found, 617.3498.
Cyclo(-Lys(rH2+)-P r o-Aba-[CH2S+Oct2]-Gly2-), 2CF3COO-
(13a ): obtained as above from the peptide 10a (0.035 g, 50
µmol) and dioctyl sulfide (0.5 mL, 50 mmol) in TFA (2 mL).
Yield: 0.034 g (71%), white powder. Mp: 174 °C dec. Rf (II)
) 0.6. 1H NMR (CD3OD): 8.29, s, 1H (ArH); 7.60, d (8.8), 1H
(ArH); 7.28, m, 1H (ArH); 4.58, m, 1H (HRPro); 4.22, m, 1H
(HRLys); 4.09, m, 2H (CH2S+); 3.98-3.80, m, 4H (HRGly); 3.67,
m, 2H (HδPro); 3.54, t (6.2), 4H (2CH2 S+); 3.32 and 3.20, 2m,
2H (HꢀLys); 2.38, m, 2H (HâPro); 2.04, m, 8H (HâLys, HγPro,
2CH2Oct); 1.80, m, 4H (HδLys, HδγLys); 1.50, m, 8H (CH2-
Oct); 1.29, s, 12H (6CH2Oct); 0.90, t, 6H (2CH3Oct). MS
(FAB+): 729 (M+), 471 (M+ - Oct2S). HRMS (FAB+): calcd
for C39H65N6O5S+ (M+), 729.4737; found, 729.4758.
Cyclo(-Lys(rZ)-P r o-Ab a -[CH 2OP h ]-Gly4-) (10b ): ob-
tained as above from the peptide 9b (0.05 g, 0.053 mmol).
Yield: 0.019 g (44%), white powder. Rf (I: 10/90) ) 0.4. 1H
(CD3OD): 7.66, s, 1H (ArH); 7.54, d, 1H (ArH); 7.34, m, 8H
(ArH); 6.92, m, 3H (ArH); 5.21, s, 2H (CH2OPh); 5.06, s, 2H
(CH2Z); 4.56, m, 1H (HRPro); 4.43, m, 1H (HRLys); 4.10-3.80,
m, 10H (HRGly1,HRGly2, HRGly3, HRGly4, HδPro); 3.13, m,
2H (HꢀLys); 2.20-1.50, m, 10H (HâPro, HγPro, HâLys, HγLys,
HδLys). MS (FAB+): 813 (MH+), 835 (MNa+), 851(MK+).
Cyclo(-Lys(rBoc)-P r o-Aba-[CH2OAc]-Gly2-) (14a). A mix-
ture of peptide 11a (0.02 g, 0.031 mmol) and dry potassium
acetate (0.05 g, 0.63 mmol) in 5 mL of DMF was stirred at
room temperature for 1.5 h. The solvent was evaporated in
vacuo. The residue was dissolved in dioxane (1 mL) and
aqueous NaOH (1.5 mg, 0.046 mmol) (1 mL). Boc2O (8.5 mg,
0.046 mmol) was added, and the reaction mixture was stirred
from 0 °C during 2 h to room temperature overnight. Solvents
were evaporated. Column chromatography of the residue
(silica gel, eluent I: 8/92), followed by evaporation of the
product containing pooled fractions, dissolution of the residue
in methanol, filtration, concentration to ca. 3 mL, precipitation
with ether, and treatment of the precipitate as before (cen-
trifugation, repeated washings with ether, and drying), yielded
0.011 g (70%) of acetate 14a as a white powder. Rf (I: 8/92)
) 0.4. 1H NMR (CD3OD): 8.41, d (2.5), 1H (ArH); 7.57, d (8.0),
1H (ArH); 7.31, dd (2.5, 8.0), 1H (ArH); 5.28, dd∼q, 2H (CH2-
OAc); 4.53, m, 1H (HRPro); 4.42, m, 1H (HRLys); 4.08, m, 2H
(HRGly1); 3.89, s, 2H (HRGly2); 3.71, m, 2H (HδPro); 3.22, m,
2H (HꢀLys); 2.34 and 2.10, 2m, 2H (HâPro); 2.06, s, 3H (CH3-
Ac); 1.94, m, 2H (HâLys); 1.62, m, 6H (HγPro, HδLys, HγLys);
1.43, s, 9H (Boc). MS (FAB+): 631 (MH+), 653 (MNa+), 669
(MK+). HRMS (FAB+): calcd for C30H43N6O9 (M+), 631.3091;
found, 631.3109.
Cyclo(-Lys(rH 2+)-P r o-Ab a -[CH2OAc]-Gly2-), CF 3COO-
(15a ). The peptide 14a (0.010 g, 0.016 mmol) was dissolved
in CH2Cl2 (10 mL) at 0 °C, and TFA (15 mL) was added. The
reaction mixture was stirred for 2 h. CH2Cl2 and excess of
TFA were evaporated in vacuo. The residue was evacuated
at 40 °C/0.1 mmHg for 1 h and triturated several times in
diethyl ether to give a white powder (7.7 mg, 76%). Rf (II) )
0.7. 1H NMR (CD3OD): 8.40, s, 1H (ArH); 7.60, d (8.0), 1H
(ArH); 7.30, d (8.0), 1H, (ArH); 5.30, dd∼q, 2H (CH2OAc); 4.55,
m, 1H (HRPro); 4.30, m, 1H (HRLys); 4.10, m, 2H (HRGly1);
3.90, s, 2H (HRGly2); 3.70, m, 2H (HδPro); 3.25, m, 2H (HꢀLys);
2.40 and 2.10, 2m, 2H (HâPro); 2.10, s, 3H (OAc); 1.95, m, 2H
(HâLys); 1.65, m, 6H (HγPro, HδLys, HγLys). MS (FAB+): 531
(MH+), 471 (M+ - OAc). HRMS (FAB+): calcd for C25H35N6O7
(MH+), 531.2567; found, 531.2591.
Cyclo(-Lys(rH2+)-P r o-Aba-[CH2SMe2+]-Gly2-), 2CF3COO-
(11a ). To a mixture of cyclopeptide 10a (0.05 g, 71 µmol) and
dimethyl sulfide (0.52 mL, 71 mmol) was added TFA (2.7 mL).
The solution was stirred at room temperature for 48 h.
Addition of a large excess of ether led to precipitation of a white
solid. The precipitate was collected by centrifugation, repeat-
edly washed with ether, and then centrifuged, dried, and
chromatographed on a column of silica gel. Elution with eluent
system II, followed by complete evaporation of the solvents in
vacuo at 25-30 °C, dissolution of the residue in ca. 2 mL of
methanol, precipitation with ether, repeated washings of the
precipitate with ether, centrifugation, and drying in vacuo at
25 °C, led to 0.025 g of the title product 11a (62%). Mp: 124.8
°C. Rf (II) ) 0.05. 1H (CD3COOD): 8.95, s, 1H (ArH); 7.64, d
(8.2), 1H (ArH); 7.45, m∼s (8.2), 1H (ArH); 4.83, s, 2H
(ArCH2S+); 4.76, t, 1H (HRPro); 4.61, t, 1H (HRLys); 4.33, 2d
(16.9), 2H (HRGly1); 4.09, s, 2H (HRGly2); 3.91 and 3.71, 2m,
2H (HδPro); 3.34, m, 2H (HꢀLys); 2.97, s, 6H (S+Me2); 2.44,
m, 2H (HâPro); 2.08, m, 4H (HâLys,HγPro); 1.52, m, 4H
(HδLys, HγLys). 1H NMR (CD3OD): 8.33, 1H (ArH); 7.57, d
(8.2), 1H (ArH); 7.31, m∼s (8.2), 1H (ArH); 4.72, s, 2H
(ArCH2S+); 4.59, m, 1H (HRPro); 4.50, m, 1H (HRLys); 4.26,
2d (16.9), 2H (HRGly1); 3.72, s, 2H (HRGly2); 3.74, m, 2H
(HδPro); 3.25, m, 2H (HꢀLys); 2.91, s, 6H (S+Me2); 2.41, m,
2H (HâPro); 1.98, m, 4H (HâLys,HγPro); 1.55, m, 4H (HδLys,
HδγLys). HRMS (FAB+): calcd for C25H37N6O5S+ (M+),
533.2546; found, 533.2567; calcd for C23H31N6O5 (M+ - Me2S),
471.2356; found, 471.2386.
Cyclo(-Lys(rH2+)-P r o-Aba-[CH2S+Me2]-Gly4-), 2CF3COO-
(11b): obtained as above from the peptide 10b (0.01 g, 0.012
1
mmol). Yield: 6.5 mg (60%), white powder. Rf (II) ) 0.5. H
Cyclo(H-Lys-P r o-Aba -[CH2OP h ]-Gly2-) (16a ). The pep-
tide 10a (0.055 g, 0.078 mmol) was dissolved in a mixture of
MeOH (5 mL) and H2O (1 mL). Palladium on charcoal (0.2 g)
was added, and the solution was hydrogenated in a Parr
apparatus at room temperature for 1 h. The solution was
filtered in order to remove the catalyst, and the solvent was
evaporated. The residue was chromatographed on a column
of silica gel. Elution with solvent system I (20/80), followed
by evaporation of the product containing pooled fractions,
dissolution of the residue in methanol, filtration, concentration
to ca. 3 mL, precipitation with ether, and treatment of the
precipitate as before (centrifugation, repeated washings with
ether, and drying), yielded 38.2 mg (80%) of pure peptide 16a .
Mp: 140.1 °C. Rf (I: 20/80) ) 0.1. 1H NMR (CD3OD): 8.43,
d, 1H (ArH); 7.53, d (8.1), 1H (ArH); 7.22, m, 3H (ArH); 6.89,
m, 3H (ArH); 5.22, dd∼q (12.5), 2H (CH2OPh); 4.57, m, 2H
(HRPro, HRLys); 4.10-3.71, m, 6H (2HRGly, HδPro); 3.25, m,
2H (HꢀLys); 2.36-1.99, m, 4H (HâPro, HγPro); 1.54-1.23, m,
NMR (CD3OD): 8.57, s, 1H (ArH); 7.57, m, 1H (ArH); 7.34,
m, 1H (ArH); 4.72, s, 2H (ArCH2S+); 4.21-3.92, m, 12H
(HRPro, HRLys, HRGly, HδPro); 3.19, m, 2H (HꢀLys); 2.91, s,
6H (S+Me2); 2.36 to 1.28, m, 10H (HâPro, HγPro, HâLys,
HγLys, HδLys). MS (FAB+): 647 (M+), 585 (M+ - Me2S).
HRMS (FAB+): calcd for C29H43N8O7S+ (M+), 647.2975; found,
647.2997.
Cyclo(-Lys(rH2+)-P r o-Aba-[CH2S+Bu 2]-Gly2-), 2CF3COO-
(12a ): obtained as above from the peptide 10a (0.05 g, 71 µmol)
and dibutyl sulfide (0.52 mL, 71 mmol) in TFA (2.7 mL).
Yield: 0.045 g (74,5%), white powder. Mp: 176 °C dec. Rf (II)
) 0.1. 1H NMR (CD3OD): 8.28, sd (2.2), 1H (ArH); 7.22, d
(8.4), 1H (ArH); 7.08, dd (2.2; 8.2), 1H (ArH); 4.57, m, 1H
(HRPro); 4.35, t∼m, 1H (HRLys); 4.09, s, 2H (ArCH2S+); 4.1-
3.8, m, 4H (HRGly); 3.79 and 3.67, 2m, 2H (HδPro); 3.54, t
(6.4), 4H (2CH2 S+); 3.20, m, 2H (HꢀLys); 2.36, m, 2H (HâPro);
2.05, m, 3H (HâLys, HγPro); 1.87, m, 1H (HγPro); 1.60-1.30,
m, 12H (HδLys, HδγLys, 4CH2Bu); 0.93, t, 6H (2CH3Bu). MS

Specific Irreversible Inhibitors of DPP IV
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2109
6H (HâLys, HδLys, HγLys). MS (FAB+): 565 (MH+), 471
(MH+ - OPh). HRMS (FAB+): calcd for C29H37N6O6 (MH+),
565.2775.
absorbance at 405 nM was determined every 15 min. Data
were plotted semilogarithmically as ln(absorbance increase/
time increase) against time, to obtain a straight line with slope
-π. Plotting this slope against 1/[I(S/Km + S)], where I )
absorbance increase/time increase and S ) substrate concen-
En zym a tic Stu d ies. Cells a n d P r ep a r a tion of Ex-
tr a cts: MOLT4 cells are lymphoblastoid CD4+ T-cells routinely
used in our laboratory for the assay of different peptidases.36
Cells (from Dr. G. Farrar) were generously provided by the
Medical Research Council AIDS Directed Programm Reagent
Project, U.K. CEM clones expressing enhanced levels of
human CD26 were obtained in the laboratory by transfection
of CEM cells as described previously;49 clones were selected
by their very high level of CD26 expression determined by
FACS analysis and by DPP IV activity measurement. All cells
were cultured in suspension medium RPMI-1640 (BioWhit-
taker, Verviers, Belgium) containing heat-inactivated (56 °C,
30 min) 10% (by vol) fetal calf serum. For preparation of cell
extracts, cells were first washed extensively with phosphate-
bufferd saline (PBS) before lysis in buffer E (20 mM Tris/HCl,
pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.2 mM phenylmethane-
sulfonyl fluoride (PhMeSO2F), 5 mM 2-mercaptoethanol, apro-
tinin (100 U/mL), and 0.5% Triton X-100) (75 µL/107 cells), and
the nuclei were pelleted by centrifugation (1000g for 8 min).
The supernatant was diluted with 1 volume of buffer I (20 mM
Tris/HCl, pH 7.6, 400 mM NaCl, 50 mM KCl, 1 mM EDTA,
0.2 mM PhMeSO2F, aprotinin (100 U/mL), 5 mM 2-mercap-
toethanol, 1% Triton X-100, and 20% glycerol) and centrifu-
gated at 12000g for 10 min. The supernatants (cell extracts)
were stored at -80 °C. Under these extraction conditions,
extracts from CEM cells (clone H01) showed more than 95%
of DPP IV activity associated to CD26; thus no further
purification was necessary. Extracts from C8166 cells showed
important levels of DPP II contamination; for this reason DPP
IV-â was purified by gel filtration and ion-exchange chroma-
tography using a FPLC system (Pharmacia, Uppsala, Sweden)
as described elsewhere.51
The DPP IV peptidase activity was also measured on the
surface of intact cells (2 × 106) in a total reaction volume of
0.5 mL of peptidase buffer PB (100 mM Hepes, pH 7.6, 120
mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, and 1%
BSA). Cells were incubated with 0.55 mM dipeptide-pNa at
37 °C for 90 min. The reactions were stopped by addition of
1 mM sodium acetate, pH 4.5 (1 mL). After centrifugation at
12000g, the production of p-nitroaniline in the supernatant
was assessed by measurement of the absorbance at 405 nm.
In this case, DPP II contamination of C8166 cells was not
detectable, due to the intracellular localization of this enzyme.
For determination of DPP IV peptidase activity in extracts
from CEM cells or partially purified preparations of DPP IV-
â, 50 µL of appropriately diluted samples was incubated in a
total volume of 200 µL of buffer PB with 1 mM Gly-Pro-pNA
at 37 °C for 120 min in flat-bottom 96-well microplates; activity
was determined by measuring absorption at 405 nm.
Inhibitory effect upon DPP IV in soluble preparations was
assayed in 96-well microplates, by preincubating CD26 and
DPP IV-â preparations with different concentrations of each
inhibitor ranging from 1 nM to 10 µM, for 15 min, before
adding the substrate as indicated. The inhibitory effect on
cell-surface-expressed cells was performed similarly; however,
cells were extensively washed after preincubation with the
inhibitor. The DPP IV activity-reversible inhibitor Lys-
[Z(NO2)]-pyrrolidide was kindly provided by Dr. A. Barth
(Halle, Germany). When its inhibitory effect was studied, no
washes were performed, due to the reversibility of this
inhibitor. IC50 values were calculated by nonlinear regression
as described.5 The values of KI and kinact were calculated using
the simplified approach described previously,34 which ignores
the inactivation of the inhibitor by the enzyme. Inhibition
kinetics of the DPP IV activities of both enzymes tested (CD26
and DPP IV-â) fit well to this model. Km values were
calculated as decribed.51 Briefly, in 96-well microtiter plates,
aliquots of each enzyme preparation were added to wells
containing 0.5 nM Gly-Pro-pNA and increasing concentrations
of inhibitor (ranging from 0.3- to 3-fold IC50 values) in a total
volume of 200 µL. Plates were incubated at 37 °C, and
tration, yields another straight line with an x-intercept of -KI
34
and a y-intercept of kinact
.
Ack n ow led gm en t. This work was supported by
grants from the Institut Pasteur. J .B. is a recipient of
a postdoctoral fellowship from the Spanish Ministerio
de Educacion y Ciencia. We thank Mrs. Nadine Robert
and J osette Svab for technical assistance.
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