Design, Synthesis, and SAR of Potent and Selective Dipeptide-Derived Inhibitors for Dipeptidyl Peptidases

Article abstract of DOI:10.1021/jm0308803

In this paper we report the systematic search for new, potent, and selective DPP II inhibitors. A study of the structure-activity relationship was conducted starting from aminoacyl pyrrolidides as lead compounds. Rational exploration of the P₁

Full text of DOI:10.1021/jm0308803

J . Med. Chem. 2003, 46, 5005-5014
5005
Design , Syn th esis, a n d SAR of P oten t a n d Selective Dip ep tid e-Der ived
In h ibitor s for Dip ep tid yl P ep tid a ses
Kristel Senten,† Pieter Van der Veken,† Ingrid De Meester,‡ Anne-Marie Lambeir,‡ Simon Scharpe´,‡
Achiel Haemers,† and Koen Augustyns*,†
Department of Medicinal Chemistry and Department of Medical Biochemistry, University of Antwerp (UIA),
Universiteitsplein 1, B-2610 Antwerp, Belgium
Received April 24, 2003
In this paper we report the systematic search for new, potent, and selective DPP II inhibitors.
A study of the structure-activity relationship was conducted starting from aminoacyl
pyrrolidides as lead compounds. Rational exploration of the P1 and P2 building blocks led to
the discovery of some very potent DPP II inhibitors which can be characterized by their high
selectivity for DPP II with regard to DPP IV. Dab-Pip and Dab-Pip-2-CN were selected as the
most promising inhibitors (IC50 nM range) and will enable us to study the physiological role of
DPP II and to differentiate between DPP II and DPP IV in biological systems.
In tr od u ction
tissues and body fluids. Recently it is suggested to be
identical to human quiescent cell proline dipeptidase
(QPP), based on the significant sequence homology
(79.4%) found between human QPP and rat DPP II.5
Human QPP6,7 is a 58-kDa glycoprotein functionally
active as a homodimer formed with a leucine zipper
motif.8 It has been shown that QPP inhibitors cause
apoptosis in quiescent lymphocytes, but not in activated
or transformed lymphocytes. This process is believed to
be independent of DPP IV, because both DPP IV+ and
DPP IV- T cells undergo apoptosis.6 No sequence
homology has been found between DPP II/QPP and DPP
IV.
DPP IV has been studied extensively over the last
three decades and a broad array of diverse functional
properties in the immune, nerve, and endocrine system
is suggested.9-13 DPP IV is bound to the cell membrane
and expressed quite ubiquitously in mammalian tissues.
In the hematopoietic system it was identified as the
leukocyte antigen CD26. Inhibition of DPP IV prolongs
the lifetime of incretin hormones and is therefore
potentially valuable in the treatment of type 2 diabetes.
Some DPP IV inhibitors are currently under clinical
evaluation in this field.10,14-16
Development of highly specific and potent inhibitors
of both enzymes will contribute to the unraveling of
their physiological functions and will be helpful to
differentiate between DPP II and DPP IV in biological
systems. More importantly, to assess the therapeutic
potential of DPP IV and DPP II inhibitors, selectivity
will be an important issue. The resembling substrate
specificity and catalytic mechanism make this a chal-
lenging task. In contrast to DPP IV,9,10 only a few DPP
II inhibitors are reported. They will be summarized
here.
DPP IV and DPP II inhibitors often resemble the
dipeptide cleavage product with a proline mimic at the
P1-site. Aminoacyl pyrrolidides (Pyrr) (1) and thiazoli-
dides (Thia) (2) are known as potent, competitive
inhibitors of DPP IV17 and DPP II (Figure 1). Thiazo-
lidides (2) are more effective inhibitors than the corre-
sponding pyrrolidides (1) for both enzymes (Table 1).
Dipeptidyl peptidases (DPPs, EC 3.4.14.x) have been
identified in various mammalian tissues and catalyze
the sequential release of dipeptides from peptides.
Among these enzymes, DPP II (EC 3.4.14.2) and DPP
IV (EC 3.4.14.5) preferentially release N-terminal dipep-
tide moieties (Xaa-Pro) at acidic (DPP II) or weakly basic
(DPP IV) pH from some oligopeptides or polypeptides.
Both enzymes are members of the recently described
‘DPP IV activity- and/or structure-homologues’ (DASH)
proteins, comprising enzymes with a common post-
proline-cleaving serine dipeptidase mechanism.1 A large
number of proteins and peptides contain proline at the
penultimate position, and due to the unique structure
of proline relatively few peptidases are able to cleave
the peptide bond after a proline residue. Dipeptidyl
peptidases from this family of DASH proteins play an
important role in the regulation of the function of these
molecules, as is clearly established for DPP IV-mediated
degradation of incretin hormones. Although DPP IV and
DPP II share proline specificity, they can be functionally
and biochemically distinguished. Using a positional
scanning synthetic combinatorial dipeptide substrate
library, it was shown that both enzymes strongly prefer
proline at P1, but that the next most preferred residue
for DPP II is norleucine (Nle), whereas for DPP IV this
is alanine. For the P2 subsite, DPP II has a preference
for Lys, Nle, Met, and Ala; whereas DPP IV can tolerate
a variety of residues at this position.2
DPP II, first identified by McDonald at al.,3 is believed
to be involved in the physiological breakdown of some
proline-containing neuropeptides and in the degradation
of collagen4 together with tripeptidyl peptidase I (EC
3.4.14.9). However, its physiological functions remain
elusive and the potential therapeutic value of DPP II
inhibitors remains unclear. DPP II is generally localized
in lysosomes and is found in a number of mammalian
* To whom correspondence should be addressed. Tel. +32-(0)3-
8202703; fax: +32-(0)3-8202739; e-mail: Koen.Augustyns@ua.ac.be.
† Department of Medicinal Chemistry.
‡ Department of Medical Biochemistry.
10.1021/jm0308803 CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

5006 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Senten et al.
phosphonates in which various P1 diphenyl phosphonate
building blocks were combined with commercially avail-
able or easily accessible amino acids. These compounds
were used for the rapid profiling of DPP II. N-(Cyclo-
pentyl)glycyl-NHCH(C6H5)PO(OPh)2 (7) was the most
selective and potent DPP II inhibitor in these series.24
In this paper we report the systematic search for new,
potent, and selective DPP II inhibitors: the structrure-
activity relationship of several classes of typical serine
protease inhibitors is investigated. Preliminary results
were reported earlier.25 Pyrrolidide 1 was used as lead
compound. After selection of an optimal P2 substitution,
the pyrrolidine ring was optimized by substituting for
different ring sizes and open amines and by introduction
of substituents in position 3. The most interesting
compound was further modified (thioamide; 2-carboni-
trile), and an optimal compound considering potency
and selectivity was selected.
Resu lts a n d Discu ssion
F igu r e 1. Examples of reported dipeptidyl peptidase inhibi-
tors.
Ch em istr y. Most compounds were prepared by par-
allel synthesis. Commercially available amino acids with
tert-butyloxycarbonyl (Boc) as R-amino protection and
acid-labile side-chain protecting groups were coupled
with the appropriate amine using a polymer-assisted
solution-phase procedure as reported earlier.26 In this
two-step synthesis we were able to produce a large
number of compounds without difficult purification
steps. Some compounds, however, were purified by
preparative TLC before final deprotection in order to
ensure the 95% purity needed for biological evaluation.
Compounds 10.3-10.5 were prepared by thioxylation
using Lawesson’s reagent after the parallel synthesis
of the corresponding protected amides.18 Inhibitory
activities for DPP IV of pyrrolidide analogues with
different ring sizes, open ring structures, and other
homologues have been reported earlier.27 These com-
pounds (8) were now also evaluated for their DPP II
inhibitory capacity. The 3-substituted pyrrolidides were
obtained by coupling of Boc protected amino acid (Ile
or Lys) to 3-hydroxypyrrolidine. Various reactions at the
hydroxyl function and cleavage of the protecting group
resulted in the 3-substituted pyrrolidides.27 An azide
(8.13, 8.14) was obtained from a tosylate intermediate.
Treatment with benzoyl chloride afforded the benzoate
(8.15, 8.16). Fluorine (8.17) was introduced with di-
ethylaminosulfur trifluoride (DAST). The dipeptide ni-
triles (11, 12) were obtained by coupling of the required
Boc protected amino acid with respectively 2-pyrrolidi-
necarboxamide or 2-piperidinecarboxamide, followed by
dehydration of the primary amide function to the nitrile
using phosphorus oxychloride19 and subsequent acid-
catalyzed deprotection.
Ta ble 1. Inhibitory Activity of DPP II Inhibitors Reported in
Literature
compounds DPP II/ QPP inhibition DPP IV inhibition
SIa
1,b P2 ) Ile
2,b P2 ) Ile
3,b P2 ) Ala
4,b P2 ) Ala
5c
Ki ) 24.7 µM
Ki ) 8.17 µM
Ki ) 1.43 µM
Ki ) 0.277 µM
Ki ) 125 nM
IC50 ) 110 µM
IC50 ) 3.8 µM
Ki ) 0.218 µM
Ki ) 0.126 µM
Ki ) 47.6 µM
Ki ) 7.88 µM
Ki ) 2 nM
Ki ) 0.2 µM
IC50 > 125 µM
0.0088
0.015
33
28
0.016
6d
7e
>33
a
SI ) selectivity index ) value for DPP IV divided by value for
DPP II. Values taken from ref 18. c Values taken from ref 6.
b
Values taken from ref 20. e Values taken from ref 24.
d
From a series with Ala, Phe, Val, Ile at P2, Ile-Thia (2,
P2 ) Ile) is the most potent DPP IV inhibitor, while Ile-
Pyrr (1, P2 ) Ile) is the most selective DPP IV inhibitor
with respect to DPP II.18 Introduction of a thioamide
bond results in thioxo amino acid pyrrolidides (3) and
thiazolidides (4).18 Thioxylation increased DPP II inhi-
bition up to 10 times, whereas thioamides are 20 times
less efficient at inhibition of DPP IV than the corre-
sponding amides.18 Thioamide analogues such as Alaψ-
[CS-N]-Pyrr (3, P2 ) Ala) and Alaψ[CS-N]-Thia (4, P2
) Ala) are the only DPP II-DPP IV inhibitors described
in this series to have some selectivity toward DPP II.18
The boronic acid dipeptide analogue Val-boroPro (5),
used to inhibit QPP in the apoptosis studies,6 is in fact
a more effective inhibitor for DPP IV (Table 1).
Substitution with a nitrile group in 1 or 2 at positions
2 and 4 affords competitive DPP IV inhibitors with
approximately a 1000-fold increase in potency compared
to the parent compounds.19-21 Ala-Pyrr-2-CN (6) was
reported20 as a weak inhibitor for DPP II, but with
higher potency toward DPP IV. Depending on the
substituent, several nitrogen-substituted Gly-Pyrr-2-CN
are even more potent and more selective DPP IV
inhibitors.14
Bioch em ica l Eva lu a tion . To establish an optimized
N-terminal amino acid (P2) for DPP II inhibition, we
prepared a series of pyrrolidides (1). IC50 values for DPP
II and DPP IV inhibition of compounds 1 are sum-
marized in Table 2. A selectivity index is given as a
means to evaluate the selectivity for DPP II with respect
to DPP IV. Lys-Pyrr (1.8 IC50 ) 9.9 µM) and His-Pyrr
(1.6 IC50 ) 1.16 µM) are the most active DPP II
inhibitors in this series and are also the most selective
for DPP II with respect to DPP IV. From this set of
pyrrolidides (1) we conclude that basic (His (1.6), Lys,
Incorporation of an electrophilic phosphonate group
on the proline mimic at P1 affords dipeptide proline
diphenyl phosphonates, that are well-known irreversible
inhibitors of DPP IV, with good selectivity over DPP II
and other enzymes.22,23 Recently, our laboratory re-
ported a series of dipeptide R-aminoalkyl diphenyl

Inhibitors for Dipeptidyl Peptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5007
Ta ble 2. Inhibitory Activities and Selectivity Index of
Pyrrolidides (1)
Ta ble 3. P1 Optimization: Inhibitory Activities and Selectivity
Index
DPP II
inhibition
IC50 (µM)
DPP IV
inhibition
IC50 (µM)
1.x
P2
Ala
Asn
Asp
Chab
Gly
His
Ile
Lys
Ser
SIa
0.2
1.2
<0.2
0.4
1
20
0.04
3.9
2.9
0.3
<0.03
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
179 ( 15
152 ( 50
>500
41 ( 6
188 ( 6
122 ( 2
17 ( 2
42 ( 3
>1000
>1000
1.2 ( 0.1
110 ( 7
9.9 ( 1.3
65 ( 30
79 ( 29
>500
23.1 ( 1.0
4 ( 1
39 ( 2
1.9
190 ( 120
21 ( 4
1.10
1.11
1.12
1.13
1.14
Phe
Pro
Thiapro
Tyr
15 ( 2
>500
14 ( 2.4
4 ( 0.4
>500
150 ( 24
223 ( 13
0.09
0.02
Val
a
SI ) selectivity index ) IC50 value for DPP IV divided by IC50
b
value for DPP II. Cha ) cyclohexylalanine.
(1.8)) amino acids are preferred above neutral amino
acids at P2. Acidic amino acids (Asp (1.3)), imino acids
(Pro (1.11), Thiapro (1.12)) and nonsubstituted amino
acids (Gly (1.5)) at this position are not favorable. This
is in agreement with the reported substrate specificity28
and more or less also compares with the results of a
positional scanning synthetic combinatorial dipeptide
substrate library (Xaa-Xaa-AMC)2. The results for DPP
IV illustrate that more residues are tolerated at the S2
subsite2, confirming the preference for lipophilic, â-
branched-R-amino acids (Ile (1.7), Val (1.14)).19
In a search for an optimized C-terminal residue (P1),
we replaced the pyrrolidine ring by several four- to
seven-membered ring structures, acyclic amines, and
3-substituted pyrrolidines. Results are summarized in
Table 3. Ile, previously reported and here confirmed
(Table 2) to afford potent DPP IV inhibition, was used
as a standard P2 amino acid. Lys, identified as a good
P2 amino acid for DPP II inhibition in the pyrrolidide
series, was used in combination with the most promising
P1 building blocks. The results of compounds 8 were
compared with Ile-Pyrr (1.7) and Lys-Pyrr (1.8).
Compared to pyrrolidine, cyclopentylamine (8.1), pyr-
roline (8.2), azetidine (8.9, 8.10), and acyclic amines
(8.18-8.21) reduce potency considerably on both en-
zymes. As already pointed out in the Introduction,
thiazolidides have been reported to give an increase in
both DPP II and DPP IV inhibition compared to the
corresponding pyrrolidides. Ile-Thia (2.1) is more active
on both enzymes, keeping selectivity index to about the
same value. In our hands, thiazolidide 2.2 gave unex-
pected results: Lys-Thia (2.2) is 3 times more active as
DPP II inhibitor, but less active against DPP IV,
increasing the selectivity for DPP II compared to Lys-
Pyrr (1.8). Therefore the increase in potency of thiazo-
lidides may not be generalized.
a
SI ) selectivity index ) IC50 value for DPP IV divided by IC50
value for DPP II.
IV. Introducing a tetrahydropyridine in the P1-position
decreases the DPP IV inhibition as reported.27 Com-
pared to pyrrolidine as P1, tetrahydropyridine improves
DPP II inhibition with Ile (8.5) in P2; no influence is
seen with Lys (8.6). Increasing the ring size further to
a seven-membered ring decreased both DPP II and DPP
IV inhibition.
Replacing the pyrrolidine ring by piperidine in 8.3 and
8.4, we observe a 2- to 6-fold improvement in the DPP
II inhibition. As reported earlier,25,27 this six-membered
ring gives a substantial decrease in potency for DPP IV
inhibitors. Introduction of piperidine at P1 is therefore
a good way of enhancing selectivity for DPP II over DPP
For the 3-substituted pyrrolidides, a 2-fold increase
of DPP II inhibition is seen for the azide substituent
(8.13 and 8.14). Lys-Pyrr-3-N3 (8.14) has an IC50 of 4.9
µM and shows an enhanced selectivity for DPP II with
regard to DPP IV compared to Lys-Pyrr (1.8). With
hydroxy and benzoyloxy the DPP II inhibition decreases,

5008 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Senten et al.
Ta ble 4. Inhibitory Activities and Selectivity Index of
Piperidides (9)
His (9.3), and Lys (8.4)), high DPP II inhibitory activi-
ties are observed.
The side chain length ((CH2)nNH2) in Lys-Pip was
investigated by replacing the P2 amino acid lysine (n )
4) (8.4) with respectively ornithine (n ) 3) (9.6), 2,4-
diaminobutyric acid (n ) 2) (9.7), and 2,3-diaminopro-
pionic acid (n ) 1) (9.11). Decreasing the side chain
length to n ) 2 enhanced the DPP II inhibitory potency.
Further decrease of the side chain revealed a reduction
in potency (9.11, n ) 1). Also selectivity was signifi-
cantly improved since inhibition of DPP IV declined
tremendously with decreasing side chain length. Dab-
Pip (9.7) with an IC50 ) 0.13 µM and a selectivity index
of more than 7000 is the most active and most selective
DPP II inhibitor in this series.
DPP II
inhibition
IC50 (µM)
DPP IV
inhibition
IC50 (µM)
9.x
P2
SIa
29
22
704
1.1
46
154
64
9.1
9.2
9.3
8.3
9.4
8.4
9.5
9.6
9.7
9.8
9.9
9.10
9.11
9.12
9.13
9.14
Arg
0.63 ( 0.11
9.9 ( 0.9
0.33 ( 0.06
62 ( 27
21.7 ( 0.8
1.6 ( 0.3
2.1 ( 0.2
0.45 ( 0.08
0.13 ( 0.01
130 ( 5
1.15 ( 0.08
191 +
1.84 ( 0.13
88.7 ( 7.3
57.1 ( 3.9
32.4 ( 2.7
18.1 ( 0.5
217 ( 13
213 ( 42
67 ( 11
>1000
Chab
His
Ile
Ser
Lys
Lys(Z)
Orn
247 ( 20
134.9 ( 1.2
>500
>1111
>7592
.8
Dabc
D-Dab
Dab(Z)
Z-Dab
Dapd
Abue
Nvaf
Nleg
>1000
.1000
Blocking the side chain amino function in Lys-Pip and
Dab-Pip with benzyloxycarbonyl (9.5, 9.9, respectively)
afforded for Dab(Z)-Pip (9.9) a decrease in potency up
to 9 times, whereas selectivity was reduced by a factor
17. The significance of this unprotected side chain amino
function is also revealed in compounds 9.12 to 9.14
where the amino function is omitted: the DPP II
inhibition is decreased tremendously, and compounds
show no selectivity at all toward DPP IV. The highest
potency here is observed with norleucine (9.14, n ) 3,
IC50 ) 32.4 µM) and decreases slightly with decreasing
chain length ((CH2)nCH3). This does not compare with
Nle-Pro-AMC being a substrate with a lower Km value
than Lys-Pro-AMC.2 From these results we learn that
basic amino acids with a certain side chain length at
P2 are preferable for potent and selective DPP II
inhibition. A 1000-fold decrease in potency of D-Dab-
Pip (9.8) confirms the importance of the L-configuration
of the P2-amino acid for DPP II inhibition. A similar
result is published for DPP IV inhibitors.9 Like for DPP
IV, the low potency of Z-Dab-Pip (9.10) shows the
importance of a free R-aminofunction and proves that
DPP II is an exopeptidase.
500 ( 25
>1000
435
>1000
>544
2.8
4
250 ( 25
229 ( 44
250 ( 25
8
a
SI ) selectivity index ) IC50 value for DPP IV divided by IC50
value for DPP II. Cha ) cyclohexylalanine. c Dab ) 2,4-diami-
b
nobutyric acid. Dap ) 2,3-diaminopropionic acid. e Abu ) 2-ami-
d
nobutyric acid. f Nva ) norvaline. Nle ) norleucine.
g
but the DPP IV inhibition is even more decreased. With
a small substituent such as fluorine (8.17), the inhibi-
tion of both enzymes is not affected.
Lys-Piperidide (8.4) is the most potent DPP II-
inhibitor in this series exhibiting an IC50 of 1.6 µM and
a selectivity index of 154 for DPP II versus DPP IV.
Conclusively, series 8 show that S1 specificity of DPP
II is less stringent than in the case of DPP IV. This is
in agreement with recent studies of the substrate
specificity, in which substrates with norleucine in P1
instead of proline are also recognized.2 A combination
of Lys at P2 with thiazolidine, 3-azidopyrrolidine and
especially piperidine considerably enhances potency and
selectivity compared to pyrrolidine. These results are
clearly in disagreement with the results of Leiting et
al. where Lys-Thia and Lys-Pip were equipotent against
both enzymes.2 Tetrahydropyridine, hexamethylene-
imine, and 3-hydroxy- and 3-benzoyloxypyrrolidine have
lower affinity for the S1 site of both DPP II and DPP
IV, but they are better accepted by DPP II compared to
DPP IV. Our diphenyl phosphonate inhibitor 7 seems
to indicate that phenyl can also be accommodated in the
S1 site of DPP II. Considering these results, we assume
that the S1 site of DPP II is somewhat larger than the
one of DPP IV. Indeed, the crystal structure of DPP IV
shows a hydrophobic pocket that is optimally suited to
fit five-membered rings.29
Recognizing the importance of the piperidine ring, a
broad series of aminoacyl piperidides (Pip) (9) with basic
and neutral amino acids at P2 were synthesized. Results
are summarized in Table 4. The significance of this
piperidine ring is again confirmed: compared to the
pyrrolidide series (1), an increase in DPP II inhibition
up to 6 times is observed, whereas inhibition of DPP
IV decreased simultaneously with a factor between 6
and 17. Therefore, changing pyrrolidine to piperidine
results in a considerable increase in potency and
selectivity for DPP II. With basic amino acids (Arg (9.1),
Having identified Dab-Pip (9.7) as the most potent
and selective inhibitor so far, some analogues were
prepared by replacing the piperidine ring (Table 5). The
DPP II inhibitory activity is declined tremendously for
Dab-piperazide (10.2), whereas Dab-morpholide (10.1)
can still be considered as a potent DPP II inhibitor.
As reported by Sto¨ckel et al,18 thioxylation of pyrro-
lidides improved DPP II inhibition, whereas inhibition
of DPP IV was negatively affected. Using this knowl-
edge, we investigated the effect of thioxylating some
aminoacyl piperidides. Previous findings are true for
compounds 10.3 and 10.4 with respectively lysine and
ornithine as P2 amino acid. Unfortunately, this increase
in DPP II inhibition and selectivity is not seen with
thioxylation of the more potent Dab-Pip (10.7). Dab Ψ-
[CS-N]-piperidide (10.5) exhibits an IC50 ) 0.22 µM and
is therefore a less active and less selective DPP II
inhibitor compared to Dab-Pip (10.7).
The thiazolidides (2.2-2.4) (Table 6) show that these
compounds are slightly less active as DPP II inhibitor
then the corresponding piperidides (respectively 8.4, 9.6,
and 9.7). More importantly, selectivity is significantly
decreased due to a more potent DPP IV inhibition.
Thiazolidides (2) are therefore less selective DPP II
inhibitors compared to the corresponding piperidides (9).

Inhibitors for Dipeptidyl Peptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5009
Ta ble 5. Inhibitory Activities and Selectivity Index of Dab-Pip
(9.7) Analogues
F igu r e 2. Structures of the most potent and selective DPP
II inhibitors.
selectivity index declines as well and is therefore less
selective with respect to DPP IV compared to Dab-Pip
(9.7).
Conclusively, based on these compounds, introduction
of a nitrile in aminoacylpiperidides moderately increases
potency for DPP II, but at the same time improves DPP
IV inhibition. As a result, aminoacylpiperidide-2-nitriles
are very potent DPP II inhibitors. Although their
selectivity generally is lower than the corresponding
piperidides, they still can be considered to be highly
selective for DPP II with respect to DPP IV.
Con clu sion s
a
SI ) selectivity index ) IC50 value for DPP IV divided by IC50
In this structure-activity investigation we were able
to identify an optimized N-terminal and C-terminal
residue for potent and selective DPP II inhibition. 2,4-
Diaminobutyric acid (Dab) was selected as the most
promising N-terminal amino acid for the development
of potent and selective DPP II inhibitors. A basic side
chain amino function proved to be favorable. Omitting
this function or neutralizing it by substitution with
carbamate decreased DPP II inhibitory potency consid-
erably and more importantly, negatively affects selectiv-
ity. Also histidine (His) shows excellent properties at
this position. Since His is expected to be almost com-
pletely protonated under the assay conditions (pH )
5.5), we assume that a positively charged nitrogen
separated by two carbons from the R-amino group
represents an optimized situation. Furthermore, the
amino acid at this position needs to be in the L-
configuration with a free R-amino function.
b
value for DPP II. Dab ) 2,4-diaminobutyric acid.
Ta ble 6. Inhibitory Activities and Selectivity Index of
Dipeptide Nitriles (11, 12)
a
SI ) selectivity index ) IC50 value for DPP IV divided by IC50
b
value for DPP II. The compound tested was L-Lys-Pip-2(R,S)-
CN.
In search for an optimized P1 building block piperidine
was recognized as being superior over pyrrolidine and
thiazolidine. For aminoacylthiazolidides (2), the DPP II
inhibition is slightly decreased compared to the corre-
sponding piperidides (9), but they are regarded as less
selective with respect to DPP IV. We showed that S1
specificity of DPP II is less stringent than in the case
of DPP IV. Considering our results, we assume that the
S1 site of DPP II is somewhat larger than the one of
DPP IV. The crystal structure of DPP IV shows a
hydrophobic pocket that is optimally suited to fit five-
membered rings.29
On the basis of our compounds, introduction of a
nitrile at P1 only slightly improves DPP II inhibition
but results in a decreased selectivity. Unexpectedly,
thioxylation of the amide bond does not always result
in an increased DPP II inhibition and a decreased DPP
IV inhibition.18
Aminoacylpyrrolidide-2-nitriles are slow-binding, re-
versible inhibitors of DPP IV with approximately a
1000-fold increase in potency compared to the parent
aminoacylpyrrolidides (low nM Ki) and a more than 500-
fold selectivity against DPP II.20 However, we investi-
gated some dipeptide nitriles (11,12) for their DPP II
inhibitory activity (Table 6). Introduction of a nitrile on
Lys-Pyrr (1.8), results in a 10-fold increase in DPP II
inhibition and a 100-fold increase in DPP IV inhibition.
Hence, Lys-Pyrr-2-CN (11) is a nonselective inhibitor
of both enzymes. The selectivity could be enhanced by
replacing the pyrrolidine ring with piperidine. Com-
pared to 11, Lys-Pip-2-CN (12.1, mixture of diastereo-
mers) only slightly affects DPP II inhibition while
inhibitory activity of DPP IV declines with a factor 160.
However compared to Lys-Pip (8.4), introduction of a
nitrile does not improve DPP II inhibitory potency and
decreases selectivity 4-fold.
Dab-Pip (10.7) has an IC50 of 130 nM and a selectivity
index of more than 7000. Incorporation of a nitrile,
leading to Dab-Pip-2-CN (12.2), increased potency with
a factor 3 (Figure 2). Although the selectivity index of
this nitrile compound (12.2) declined to approximately
4000, it still can be regarded as highly selective for DPP
Incorporation of a nitrile in Dab-Pip (9.7) enhances
inhibition of DPP II with a factor 3. Dab-Pip-2-CN (12.2)
exhibits an IC50 ) 38 nM and is the most active DPP II
inhibitor in our investigation. However, for 12.2 the

5010 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Senten et al.
was < 0.5, an IC50 value was determined experimentally using
at least 6 different concentrations of inhibitor. For those
compounds with IC50 values below 5 µM for one of the enzymes,
the analysis was repeated using a new stock of compound.
Generally, independent measures of IC50 differ less than 20%
from each other. IC50 value was defined as the inhibitor
concentration, which caused a 50% decrease of the activity
under assay conditions. The errors given in the tables repre-
sent errors of the fit.
II. Both compounds are far more active and selective
DPP II inhibitors than earlier reported compounds.
Especially, the high selectivity of these two compounds
(9.7 and 12.2) must be emphasized.
Val-Pyrr (1.14) and Ile-Thia (2.1), identified as the
most selective DPP IV inhibitor in our pyrrolidide and
thiazolidide series respectively, are extensively used in
animal studies to evaluate the effects of DPP IV
inhibitors in the treatment of type II diabetes.10 How-
ever, selectivity for DPP IV in comparison with DPP II
remains rather limited. One can question the in vivo
selectivity for DPP IV and results should be interpreted
with caution. In this respect some of the Pro-Pro diaryl
phosphonates reported30 by our group and the N-
substituted glycylpyrrolidide-2-nitriles,14 withsdepending
on the N-substituentsvery low DPP II inhibitory activ-
ity, seem a better choice to selectively inhibit DPP IV.
Our two compounds (9.7 and 12.2) should offer the
opportunity to study the physiological role of DPP II and
possible therapeutic benefits of DPP II inhibition. Their
high selectivity will enable to differentiate between DPP
II and DPP IV in biological systems. Both compounds
will also serve as lead compounds for further develop-
ment of DPP II inhibitors in our laboratory.
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 1, 2,
8.3, 8.4, 8.6, 8.8, 8.10, 9, 10.1, a n d 10.2. These series were
prepared by parallel synthesis using a PASP-protocol26: R-ami-
no Boc-protected amino acids (0.375 mmol) (side chain protec-
tion was as followed: -Lys(Boc), -Dab(Boc), -Orn(Boc), -Dap-
(Boc), -His(Trt), -Asn(Trt), -Ser(tBu), -Asp(OtBu), -Tyr(tBu),
-Arg(diZ)), HOBt (0.425 mmol), and PS-Carbodiimide (0.75
mmol) were added to a dry reaction vessel. Dichloromethane
(4 mL) was added, and the mixture was stirred for 10 min
prior to the addition of the appropriate amine (respectively
pyrrolidine (1), thiazolidine (2), 1,2,5,6-tetrahydropyridine (8.5,
8.6), hexamethyleneimine (8.7, 8.8), azetidine (8.9, 8.10),
piperidine (8.3, 8.4, 9, 10.3-10.5), morpholine (10.1), and Boc-
piperazine (10.2)). After the mixture was stirred at room-
temperature overnight the polymer-bound polyamine (1.5
mmol) was added, and stirring was continued for 5 h. The
reaction mixture was filtered, and the amide product was
collected in the filtrate. The resins are washed two times with
4 mL of dichloromethane, and the combined fractions were
evaporated under reduced pressure. The purity of the com-
pounds was checked by TLC and reversed phase HPLC.
Compounds were purified by preparative TLC using a mixture
of EtOAc and hexane (usually 40/60) as eluent. Isolation from
the silica gel of the preparative TLC was done by suspending
the silica gel in EtOAc/MeOH (90:10), followed by filtration.
Gen er a l P r oced u r e for ter t-Bu t yloxyca r b on yl (Boc)
Dep r otection . Deprotection was done by dissolving in 4 mL
of a TFA/dichloromethane (1:1) mixture. The solution was
stirred for 3 h, and the volatile part was removed under
reduced pressure. After coevaporation several times with
ether, the residues were lyophilized from tert-butyl alcohol/
water (4:1).
1-(L-Ala n yl)p yr r olid in e tr iflu or oa ceta te (1.1): 1H NMR
(D2O, 400 MHz) δ 1.44 (d, 3H, CH3), 1.84-1.97 (m, 4H, CH2),
3.34-3.56 (m, 4H, CH2), 4.26 (m, 1H, R-CH); MS (ES+) m/z
143 (M + H)+; LC-MS: rt 1.3 min, 100%, m/z 143; HPLC (214
nm): rt 3.54 min, 98%.
1-(L-Asp a r a gin yl)p yr r olid in e tr iflu or oa ceta te (1.2): 1H
NMR (D2O, 400 MHz) δ 1.78-1.95 (m, 4H, CH2), 2.71 (dd, 1H,
CH2), 2.84 (dd, 1H, CH2), 3.28-3.56 (m, 4H, CH2), 4.47 (t, 1H,
R-CH); MS (FAB+) m/z 186 (M + H)+; LC-MS: rt 1.0 min,
100%, m/z 186; HPLC (214 nm): rt 3.60 min, 98%.
1-(L-Aspar tyl)pyr r olidin e tr iflu or oacetate (1.3): 1H NMR
(D2O, 400 MHz) δ 1.84-1.97 (m, 4H, CH2), 2.86 (dd, 1H, CH2),
3.02 (dd, 1H, CH2), 3.36-3.59 (m, 4H, CH2), 4.54 (t, 1H, R-CH);
MS (FAB+) m/z 187 (M + H)+; LC-MS: rt 1.1 min, 100%, m/z
187; HPLC (214 nm): rt 2.54 min, 100%.
Exp er im en ta l Section
Ma ter ia ls. Parallel synthesis was performed using the
Quest 210 Organic Synthesizer (Argonaut Technologies). Boc-
protected amino acids, N-cyclohexylcarbodiimide, N′-methyl-
polystyrene resin (PS-carbodiimide), and tris(2-aminoethyl)-
amine polystyrene resin were purchased from Novabiochem.
Other reagents were obtained from Sigma-Aldrich or Acros.
Compounds 8.1, 8.2, 8.5, 8.7, 8.9, 8.11, 8.13, 8.15, 8.17-8.21
were synthesized as reported earlier.27
An a lysis. Characterization of all compounds was done with
1H NMR and mass spectrometry. 1H NMR were recorded on a
Bruker Avance DRX-400 spectrometer (400 MHz). Fast atom
bombardment (FAB+) mass spectra were obtained on a VG 70-
SEQ hybrid mass spectrometer (Micromass, Manchester, UK),
equipped with a cesium ion gun. Electrospray (ES+) mass
spectra were acquired on a Autospec-ao-TOF mass spectrom-
eter (Micromass, Manchester, UK) or a triple quadrupole mass
spectrometer (Quattro II, Micromass, Manchester, UK) or an
ion trapp mass spectrometer (Esquire 3000, Bruker Daltonics).
Purity was verified using two diverse HPLC systems using
respectively mass and UV-detector. LC-MS were recorded on
an Agilent 1100 Series HPLC system using a Discovery Cyano
column (2.1 × 50 mm, 5 µm, Supelco, Sigma-Aldrich) coupled
with a Bruker Esquire 3000 plus mass spectrometer (0-80%
ACN, 22 min, 0.2 mL/min). Reversed phase HPLC was run
on a Gilson instrument (Viliers-le-bel, France) equipped with
an Ultrasphere ODS column (4.6 × 250 mm, 5 µm, Beckman,
Fullerton, CA) and a UV-detection (10-100% ACN, 35 min,
214 nm, 1 mL/min). Preparative TLC was performed on silica
gel 60PF254 containing gypsum.
Bioch em ica l Eva lu a tion . DPP IV was purified from
human seminal plasma as described previously.31 DPP II was
isolated from the same source using techniques described
previously for purification of the enzyme from porcine seminal
plasma,32 supplemented with adenosine deaminase affinity
chromatography to eliminate contaminating DPP IV.31 Enzyme
activity was measured kinetically with the chromogenic sub-
strates Gly-Pro-p-nitroanilide at pH 8.3 and Lys-Ala-p-nitro-
anilide at pH 5.5 for DPP IV and DPP II, respectively. Test
compounds were dissolved and diluted in DMSO (final con-
centration DMSO during assay 5% v/v). All measurements
were carried out in duplicate. The initial evaluation of
compounds was carried out at 1 mM, or in case of solubility
limits, the highest concentration possible. If vi/vo (velocity in
the presence of inhibitor/velocity in the presence of control)
1-(S-Cycloh exylalan yl)pyr r olidin e tr iflu or oacetate (1.4):
1H NMR (DMSO-d6), 400 MHz) δ 0.70-2.00 (m, 17H, CH2,
CH), 3.20-3.70 (m, 4H, CH2), 3.95-4.10 (m, 1H, R-CH), 8.30
(brs, 3H, NH3+); MS (FAB+) m/z 225 (M + H)+; LC-MS: rt
16.0 min, 100%, m/z 225; HPLC (214 nm): rt 14.34 min, 100%.
1-(L-Glycyl)p yr r olid in e tr iflu or oa ceta te (1.5): 1H NMR
(D2O, 400 MHz) δ 1.82-1.98 (m, 4H, CH2), 3.36-3.42 (m, 4H,
CH2), 3.86 (s, 2H, CH2); MS (ES+) m/z 129 (M + H)+; LC-MS:
rt 1.1 min, 100%, m/z 129; HPLC (214 nm): rt 3.39 min, 95%.
1-(L-Histid yl)p yr r olid in e bis tr iflu or oa ceta te (1.6): 1H
NMR (D2O, 400 MHz) δ 1.90-2.01 (m, 4H, CH2), 3.22-3.28
(m, 1H, â-CH2), 3.44-3.57 (m, 4H, CH2), 3.64-3.69 (m, 1H,
â-CH2), 4.65 (t, 1H, R-CH), 7.52 (s, 1H, 4H-His), 8.78 (s, 1H,
2H-His); MS (ES+) m/z 209 (M + H)+; LC-MS: rt 0.7 min,
100%, m/z 209; HPLC (214 nm): rt 2.57 min, 100%.
1-(L-Isoleu cyl)p yr r olid in e tr iflu or oa ceta te (1.7): 1H
NMR (DMSO-d6, 250 MHz) δ 0.85 (t, 3H, δ-CH3), 0.93 (d, 3H,
γ-CH3), 1.05-1.20 (m, 1H, γ-CH2), 1.40-1.60 (m, 1H, γ-CH2),

Inhibitors for Dipeptidyl Peptidases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 5011
1.70-2.00 (m, 5H, â-CH, CH2), 3.25-3.70 (m, 4H, CH2), 3.93
(m, 1H, R-CH), 8.14 (s, 3H, NH3+); MS (FAB+) m/z 185 (M +
H)+; LC-MS: rt 9.8 min, 100%, m/z 185; HPLC (214 nm): rt
9.68 min, 100%.
(t, 0.5H, R-CH), 5.75-5.82 (m, 1H, 4-CH), 5.96-6.06 (m, 1H,
3-CH); MS (ES+) m/z 212 (M + H)+; LC-MS: rt 0.9 min, 100%,
m/z 212; HPLC (214 nm): rt 3.87 min, 100%.
1-(L-Lysyl)h exa m et h ylen eim in e b is t r iflu or oa cet a t e
(8.8): 1H NMR (D2O, 400 MHz) δ 1.51-1.66 (m, 6H, CH2),
1.72-1.86 (m, 6H, CH2), 1.94-2.00 (m, 2H, CH2), 3.06 (t, 2H,
ꢀ-CH2), 3.39-3.56 (m, 2H, CH2), 3.62-3.74 (m, 2H, CH2), 4.53
(t, 1H, R-CH); MS (ES+) m/z 228 (M + H)+; LC-MS: rt 1.2
min, 100%, m/z 228; HPLC (214 nm): rt 5.27 min, 100%.
1-(L-Lysyl)a zetid in e bis tr iflu or oa ceta te (8.10): 1H NMR
(D2O, 400 MHz) δ 1.43-1.57 (m, 2H, CH2), 1.70-1.80 (m, 2H,
CH2), 1.85-1.96 (m, 2H, CH2), 2.35-2.48 (m, 2H, 3-CH2), 3.05
(br s, 2H, ꢀ-CH2), 4.06-4.29 (m, 3H, CH2, R-CH), 4.39-4.44
(m, 1H, 3-CH); MS (ES+) m/z 186 (M + H)+; LC-MS: rt 0.6
min, 97%, m/z 186; HPLC (214 nm): rt 3.10 min, 98%.
1-(L-Ar gin yl)p ip er id in ebistr iflu or oa ceta te (9.1). Depro-
tection was done by hydrogenolysis, followed by Boc-depro-
tection.
1-(L-Lysyl)p yr r olid in e bis tr iflu or oa ceta te (1.8): 1H
NMR (D2O, 400 MHz) δ 1.42-1.48 (m, 2H, CH2), 1.63-1.71
(m, 2H, CH2), 1.83-2.00 (m, 6H, â-CH2, CH2), 2.96 (t, 2H,
ꢀ-CH2), 3.35-3.60 (m, 4H, CH2), 4.26 (t, 1H, R-CH); MS (ES+)
m/z 200 (M + H)+; LC-MS: rt 0.7 min, 95%, m/z 200; HPLC
(214 nm): rt 4.42 min, 100%.
1
1-(L-Ser yl)p yr r olid in e tr iflu or oa ceta te (1.9): H NMR
(D2O, 400 MHz) δ 1.78-1.95 (m, 4H, CH2), 3.32-3.56 (m, 4H,
CH2), 3.81 (dd, 1H, CH2), 3.90 (dd, 1H, CH2), 4.29 (t, 1H, R-CH);
MS (FAB+) m/z 159 (M + H)+; LC-MS: rt 6.3 min, 90%, m/z
159; HPLC (214 nm): rt 7.96 min, 90%.
1-(L-P h en yla la n yl)p yr r olid in e tr iflu or oa ceta te (1.10):
1H NMR (D2O, 400 MHz) δ 1.50-1.78 (m, 4H, CH2), 2.56-
2.62 (m, 1H, CH2), 3.06-3.19 (m, 2H, CH2), 3.24-3.39 (m, 3H,
CH2), 4.42 (t, 1H, R-CH), 7.21 (m, 2H, o-Harom), 7.31-7.38 (m,
3H, m-, p-Harom); MS (ES+) m/z 219 (M + H)+; LC-MS: rt 13.0
min, 100%, m/z 219; HPLC (214 nm): rt 12.43 min, 98%.
1-(L-P r olyl)p yr r olid in e tr iflu or oa ceta te (1.11): 1H NMR
(D2O, 400 MHz) δ 1.86-2.07 (m, 7H, â-CH2-, γ-CH2, CH2),
2.45-2.54 (m, 1H, â-CH2), 3.31-3.56 (m, 6H, δ-CH2, CH2), 4.51
(t, 1H, R-CH); MS (ES+) m/z 169 (M + H)+; LC-MS: rt 2.1
min, 100%, m/z 169; HPLC (214 nm): rt 6.44 min, 99%.
1-(S-Th ia p r olyl)p yr r olid in e tr iflu or oa ceta te (1.12): 1H
NMR (D2O, 400 MHz) δ 1.86-2.01 (m, 4H, CH2), 3.17 (dd, 1H,
â-CH2), 3.37-3.59 (m, 4H, CH2), 3.63 (m, 1H, â-CH2), 4.39 (d,
1H, δ-CH2), 4.48 (d, 1H, δ-CH2), 4.75-4.84 (m, 1H, R-CH); MS
(ES+) m/z 187 (M + H)+; LC-MS: rt 2.3 min, 95%, m/z 187;
HPLC (214 nm): rt 6.83 min, 96%.
1-(L-Tyr osyl)pyr r olidin e tr iflu or oacetate (1.13): 1H NMR
(D2O, 400 MHz) δ 1.51-1.80 (m, 4H, CH2), 2.56-2.62 (m, 1H,
CH2), 2.98-3.14 (m, 2H, CH2), 3.24-3.40 (m, 3H, CH2), 4.37
(t, 1H, R-CH), 6.84 (d, 2H, 3-,5-Harom), 7.12 (d, 2H, 2-,6-Harom);
MS (ES+) m/z 235 (M + H)+; LC-MS: rt 9.4 min, 100%, m/z
235; HPLC (214 nm): rt 6.09 min, 100%.
1-(L-Va lyl)p yr r olid in e tr iflu or oa ceta te (1.14): 1H NMR
(D2O, 400 MHz) δ 0.97 (d, 3H, CH3), 1.02 (d, 3H, CH3), 1.82-
1.98 (m, 4H, CH2), 2.18-2.26 (m, 1H, â-CH2), 3.38-3.60 (m,
4H, CH2), 4.06 (d, 1H, R-CH); MS (ES+) m/z 171 (M + H)+;
LC-MS: rt 0.6 min, 100%, m/z 171; HPLC (214 nm): rt 6.14
min, 100%.
1-(L-Isoleu cyl)th ia zolid in e tr iflu or oa ceta te (2.1): 1H
NMR (CDCl3, 400 MHz) δ 0.9-1.38 (m, 7H, CH3, CH2), 1.52-
1.67 (m, 1H, CH2), 1.90-2.02 (m, 1H, CH), 2.98-3.15 (m, 2H,
5-CH2), 3.69-3.80 (m, 1H, 4-CH2), 3.88-4.02 (m, 1H, 4-CH2),
4.12-4.23 (m, 1H, R-CH), 4.41-4.68 (m, 2H, 2-CH2); MS (ES+)
m/z 203 (M + H)+; LC-MS: rt 9.8 min, 100%, m/z 203; HPLC
(214 nm): rt 9.31 min, 100%.
1-(L-Lysyl)th ia zolid in e bis tr iflu or oa ceta te (2.2): 1H
NMR (D2O, 400 MHz) δ 1.81-2.11 (m, 6H, CH2), 3.15-3.29
(m, 4H, ꢀ-CH2, 5-CH2), 3.82-4.04 (m, 2H, 4-CH2), 4.60-4.82
(m, 3H, 2-CH2, R-CH); MS (ES+) m/z 218 (M + H)+; LC-MS:
rt 0.8 min, 98%, m/z 218; HPLC (214 nm): rt 5.84 min, 98%.
1-(S-Or n ith yl)th ia zolid in e bis tr iflu or oa ceta te (2.3): 1H
NMR (D2O, 400 MHz) δ 1.83-1.91 (m, 2H, γ-CH2), 2.05-2.10
(m, 2H, â-CH2), 3.12 (t, 2H, 5-CH2), 3.20 (t, 1H, δ-CH2), 3.27
(t, 1H, δ-CH2), 3.83-4.08 (m, 2H, 4-CH2), 4.52 (t, 0.5H, R-CH),
4.57 (t, 0.5H, R-CH), 4.59-4.84 (m, 2H, 2-CH2); MS (ES+) m/z
204 (M + H)+; LC-MS: rt 0.6 min, 98%, m/z 204; HPLC (214
nm): rt 4.09 min, 95%.
1H NMR (D2O, 400 MHz) δ 1.50-1.80 (m, 8H, CH2), 1.92-
2.00 (m, 2H, CH2), 3.25-3.38 (m, 2H, δ-CH2), 3.44-3.77 (m,
4H, CH2), 4.60-4.70 (m, 1H, R-CH); MS (ES+) m/z 242 (M +
H)+; LC-MS: rt 0.4-0.5 min, m/z 242 (M + H)+; HPLC (214
nm): rt 4.58 min, 91%.
1-(S-Cycloh exylalan yl)piper idin e tr iflu or oacetate (9.2):
1H NMR (D2O, 400 MHz) δ 0.91-1.79 (m, 19H, CH2), 3.40-
3.53 (m, 4H, 2-CH2, 6-CH2), 4.49 (t, 1H, R-CH); MS (ES+) m/z
239 (M + H)+; LC-MS: rt 1.0-1.4 min, m/z 239 (M + H)+;
HPLC (214 nm): rt 23.49 min, 100%.
1
1-(L-Histid yl)p ip er id in e bis tr iflu or oa ceta te (9.3): H
NMR (D2O, 400 MHz) δ 1.52-1.79 (m, 6H, 3-CH2, 4-CH2,
5-CH2), 3.31-3.72 (m, 6H, â-CH2, 2-CH2, 6-CH2), 4.81-4.93
(m, 1H, R-CH), 7.54 (s, 1H, 4-CH-His), 8.81 (s, 1H, 2-CH-
His); MS (ES+) m/z 223 (M + H)+; LC-MS: rt 0.3-0.5 min,
m/z 223 (M + H)+; HPLC (214 nm): rt 4.04 min, 88%.
1-(L-Isoleu cyl)piper idin e tr iflu or oacetate (8.3). 1H NMR
(DMSO-d6, 400 MHz) δ 0.85 (t, 3H, δ-CH3), 0.94 (d, 3H, γ-CH3),
1.00-1.25 (m, 1H, γ-CH), 1.35-1.70 (m, 7H, γ-CH, 3-CH2,
4-CH2, 5-CH2), 1.70-1.85 (m, 1H, â-CH), 3.20-3.65 (m, 4H,
2-CH2, 6-CH2), 4.25 (d, 1H, R-CH), 8.07 (br s, 3 H, NH3+); MS
(ES+) m/z 199 (M + H)+; LC-MS: rt 0.6-0.7 min, m/z 199 (M
+ H)+; HPLC (214 nm): rt 11.30 min, 100%.
1-(L-Ser yl)p ip er id in e tr iflu or oa ceta te (9.4): 1H NMR
(D2O, 400 MHz) δ 1.59-1.73 (m, 6H, CH2), 3.50-3.64 (m, 4H,
CH2), 3.90-3.95 (m, 1H, â-CH2), 4.02-4.06 (m, 1H, â-CH2),
4.62-4.68 (m, 1H, R-CH); MS (ES+) m/z 173 (M + H)+; LC-
MS: rt 0.5-0.6 min, m/z 173 (M + H)+; HPLC (214 nm): rt
4.91 min, 93%.
1-(L-Lysyl)piper idin e bis tr iflu or oacetate (8.4): 1H NMR
(D2O, 400 MHz) δ 1.34-1.87 (m, 12H, CH2), 2.95 (t, 2H, ꢀ-CH2),
3.43-3.53 (m, 4H, CH2), 4.50 (t, 1H, R-CH), MS (ES+) m/z 214
(M + H)+; LC-MS: rt 0.3-0.5 min, m/z 214 (M + H)+; HPLC
(214 nm): rt 2.59 min, 86%.
1-[N-ꢀ-(Ben zyloxyca r bon yl)-L-Lysyl]p ip er id in e tr iflu o-
r oa ceta te (9.5): 1H NMR (D2O, 400 MHz) δ 1.37-1.76 (m,
10H, CH2), 1.84-1.98 (m, 2H, CH2), 3.14-3.28 (m, 2H, ꢀ-CH2),
3.41-3.68 (m, 4H, CH2), 4.47-4.57 (m, 1H, R-CH), 5.11-5.26
(m, 2H, CH2-Z), 7.49 (s, 5H, Harom); MS (ES+) m/z 348 (M +
H)+; LC-MS: rt 1.6-1.9 min, m/z 348 (M + H)+; HPLC (214
nm): rt 14.59 min, 100%.
1-(S-Or n ith yl)p ip er id in e bis tr iflu or oa ceta te (9.6): 1H
NMR (D2O, 400 MHz) δ 1.59-1.91 (m, 8H, CH2), 1.97-2.03
(m, 2H, CH2), 3.11 (t, 2H, δ-CH2), 3.53-3.66 (m, 4H, CH2),
4.66 (t, 1H, R-CH); MS (ES+) m/z 200 (M + H)+; LC-MS: rt
0.4-0.6 min, m/z 200 (M + H)+; HPLC (214 nm): rt 3.74 min,
100%.
1-[(S)-2,4-Dia m in obu ta n oyl]th ia zolid in e bis tr iflu or o-
1
a ceta te (2.4): H NMR (D2O, 400 MHz) δ 2.34-2.40 (m, 2H,
â-CH2), 3.18-3.28 (m, 4H, γ-CH2, 5-CH2), 3.84-4.07 (m, 2H,
4-CH2), 4.55-4.84 (m, 3H, 2-CH2, R-CH); MS (ES+) m/z 190
(M + H)+; LC-MS: rt 0.6 min, 98%, m/z 190; HPLC (214 nm):
rt 3.99 min, 99%.
1-[(S)-2,4-Dia m in obu ta n oyl]p ip er id in e bis tr iflu or o-
1
a ceta te (9.7): H NMR (D2O, 400 MHz) δ 1.59-1.73 (m, 6H,
CH2), 2.27-2.34 (m, 2H, â-CH2), 3.12-3.23 (m, 2H, γ-CH2),
3.50-3.71 (m, 4H, CH2), 4.72 (t, 1H, R-CH); MS (ES+) m/z 186
(M + H)+; LC-MS: rt 0.5-0.6 min, m/z 186 (M + H)+; UV-
HPLC rt 3.62 min, 100%.
1-(L-Lysyl)-1,2,5,6-tetr a h yd r op yr id in e bis tr iflu or oa c-
et a t e (8.6): 1H NMR (D2O, 400 MHz) δ 1.45-1.55 (m, 2H,
CH2), 1.69-1.81 (m, 2H, CH2), 1.91-2.00 (m, 2H, CH2), 2.23-
2.36 (m, 2H, 5-CH2), 3.05 (b, 2H, ꢀ-CH2), 3.62-3.81 (m, 2H,
6-CH2), 4.03-4.17 (m, 2H, 2-CH2), 4.55 (t, 0.5H, R-CH), 4.62
1-[(R)-2,4-Dia m in obu ta n oyl]p ip er id in e bis tr iflu or o-
1
a ceta te (9.8): H NMR (D2O, 400 MHz) δ 1.50-1.62 (m, 6H,
CH2), 2.15-2.21 (m, 2H, â-CH2), 3.02-3.11 (m, 2H, γ-CH2),

5012 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Senten et al.
3.41-3.59 (m, 4H, CH2), 4.61 (t, 1H, R-CH); MS (ES+) m/z 186
(M + H)+; LC-MS: rt 0.4-0.5 min, m/z 186 (M + H)+; HPLC
(214 nm): rt 4.66 min, 100%.
1-[(S)-2-Am in o,4-b en zyloxyca r b on yla m in ob u t a n oyl]-
p ip er id in e tr iflu or oa ceta te (9.9): 1H NMR (D2O, 400 MHz)
δ 1.20-1.71 (m, 6H, CH2), 1.85-2.05 (m, 2H, â-CH2), 3.29-
3.55 (m, 6H, CH2, γ-CH2), 4.44 (m, 1H, R-CH), 5.13 (s, 2H,
CH2), 7.43 (s, 5H, Harom); MS (ES+) m/z 320 (M + H)+; LC-MS:
rt 1.1-1.2 min, m/z 320 (M + H)+; HPLC (214 nm): rt 14.85
min, 97%.
4.90 (t, 1H, R-CH); MS (ES+) m/z 216 (M + H)+; LC-MS: rt
1.1 min, 100%, m/z 216; HPLC (214 nm): rt 7.07 min, 100%.
1-[(S)-2,4- Dia m in obu ta n oyl]th ioxop ip er id in e bis tr i-
1
flu or oa ceta te (10.5): H NMR (D2O, 400 MHz) δ 1.61-1.63
(m, 6H, 3-CH2, 4-CH2, 5-CH2), 2.30-2.35 (m, 2H, â-CH2), 3.10-
3.26 (m, 2H, γ-CH2), 3.82-3.98 (m, 2H) and 4.12-4.20 (m, 1H)
and 4.36-4.44 (m, 1H) (2-CH2, 6-CH2), 4.91 (t, 1H, R-CH); MS
(ES+) m/z 202 (M + H)+; LC-MS: rt 1.1 min, 95%, m/z 202;
HPLC (214 nm): rt 5.97 min, 99%.
3-Su bstitu ted P yr r olid id e An a logu es w er e Syn th e-
sized fr om 1-[N-(ter t-Bu tyloxyca r bon yl)-L-lysyl]-3(R,S)-
h yd r oxyp yr r olid in e. 1-(L-Lysyl)-3(R,S)-h yd r oxyp yr r oli-
d in e Bis Tr iflu or oa ceta te (8.12). To a mixture of N-R,γ-
di(tert-butyloxycarbonyl)-L-lysine (1.1 equiv, 2.28 g), triethyl-
amine (3 equiv, 2.53 mL), and TBTU (1.1 equiv, 2.12 g) in DMF
(40 mL) was added (R,S)-3-hydroxypyrrolidine (1 equiv, 522
mg). After the mixture was stirred at room-temperature
overnight, water was added and the mixture was extracted
with EtOAc (3 × 50 mL). The combined organic layers were
washed with 1 N HCl (2 × 25 mL), 5% NaHCO3 (2 × 25 mL),
and brine (25 mL). The organic layer was dried over NaSO4,
evaporated, and purified by column chromatography yielding
1-[N-R,γ-di(tert-butyloxycarbonyl)-L-lysyl]-3(R,S)-hydroxypyr-
rolidine (87%). This Boc-protected compound was treated with
a mixture of TFA/DCM (1/1, 10 mL) at room temperature. The
title compound was obtained after evaporation and coevapo-
ration with diethyl ether: 1H NMR (D2O, 400 MHz) δ 1.48-
1.68 (m, 2H, CH2), 1.71-1.80 (m, 2H, CH2), 1.93-2.25 (m, 4H,
CH2), 3.06 (br s, 2H, ꢀ-CH2), 3.54-3.88 (m, 4H, CH2), 4.27-
4.42 (m, 1H, R-CH), 4.57-4.66 (m, 1H, 3-CH); MS (ES+) m/z
216 (M + H)+; LC-MS: rt 0.5 min, 95%, m/z 216; HPLC (214
nm): rt 2.75 min, 95%.
1-(L-Lysyl)-3(R,S)-a zid op yr r olid in e Bis Tr iflu or oa c-
eta te (8.14). To a solution of 1-[N-R,γ-di(tert.-butyloxycarbo-
nyl)-L-lysyl]-3(R,S)-hydroxypyrrolidine (1.5 mmol, 620 mg) in
dry 1.2-dichloroethane (20 mL) were added triethylamine (4.5
mmol, 574 µL) and p-toluenesulfonyl chloride (2.5 mmol, 457
mg) at 0 °C. The mixture was stirred at room temperature for
48 h (after 24 h an second addition of p-toluenesulfonyl
chloride (1.5 mmol) occurred). Water (30 mL) was added, and
the mixture was extracted with CH2Cl2 (2 × 80 mL). The
organic layer was washed with 5% NaHCO3 (2 × 50 mL), dried,
evaporated, and purified by column chromatography (CHCl3)
yielding 1-[N-R,γ-di(tert.-butyloxycarbonyl)-L-lysyl]-3(R,S)-azi-
doyrrolidine (70%). A solution of this compound (0.95 mmol,
540 mg) in DMF (15 mL) was treated with NaN3 (4.75 mmol,
390 mg) and stirred at 80 °C for 5 h. EtOAc (50 mL) was added,
and the resulting mixture was washed with 5% NaCO3 (2 ×
30 mL). The organic layer was dried, evaporated, and purified
by column chromatography yielding a pale yellow oil. Depro-
tection was done according to the general procedure to yield
the title compound. 1H NMR (D2O, 400 MHz) δ 1.49-1.60 (m,
2H, CH2), 1.72-1.82 (m, 2H, CH2), 1.94-2.03 (m, 2H, CH2),
2.17-2.38 (m, 2H, 4-CH2), 3.07 (br s, 2H, ꢀ-CH2), 3.56-3.99
(m, 4H, CH2), 4.30-4.42 (m, 1H, R-CH), 4.46-4.55 (m, 1H,
3-CH); MS (ES+) m/z 241 (M + H)+; LC-MS: rt 0.7 min, 100%,
m/z 241; HPLC (214 nm): rt 3.79 min, 100%.
1-[(S)-2-Ben zyloxyca r b on yla m in o,4-a m in ob u t a n oyl]-
p ip er id in e tr iflu or oa ceta te (9.10). 1H NMR (D2O, 400
MHz) δ 1.41-1.71 (m, 6H, CH2), 1.98-2.17 (m, 2H, â-CH2),
3.02-3.76 (m, 6H, γ-CH2, CH2), 4.70-4.85 (m, 1H, R-CH), 5.17
(s, 2H, CH2), 7.46 (s, 5H, Harom); MS (ES+) m/z 320 (M + H)+;
LC-MS: rt 0.4-0.6 min, m/z 320 (M + H)+; HPLC (214 nm):
rt 14.48 min, 96%.
1-[(S)-2,3-Dia m in op r op a n oyl]p ip er id in e bis tr iflu or o-
a ceta te (9.11): 1H NMR (D2O, 400 MHz) δ 1.57-1.81 (m, 6H,
CH2), 3.42-3.85 (m, 6H, CH2, â-CH2), 4.97 (m, 1H, R-CH); MS
(ES+) m/z 172 (M + H)+; LC-MS: rt 0.4-0.5 min, m/z 172 (M
+ H)+; HPLC (214 nm): rt 3.60 min, 100%.
1-[(S)-2-Am in ob u t a n oyl]p ip er id in e t r iflu or oa cet a t e
(9.12): 1H NMR (D2O, 400 MHz) δ 1.05 (t, 3H, CH3), 1.63-
1.77 (m, 6H, CH2), 1.89-2.00 (m, 2H, â-CH2), 3.50-3.68 (m,
4H, CH2), 4.52 (t, 1H, R-CH); MS (ES+) m/z 171 (M + H)+;
LC-MS: rt 0.5-0.6 min, m/z 171 (M + H)+; HPLC (214 nm):
rt 7.92 min, 96%.
1-(S-Nor va lyl)p ip er id in e tr iflu or oa ceta te (9.13): 1H
NMR (D2O, 400 MHz) δ 1.01 (t, 3H, CH3), 1.41-1.51 (m, 2H,
γ-CH2), 1.59-1.78 (m, 6H, CH2), 1.85-1.89 (m, 2H, â-CH2),
3.50-3.68 (m, 4H, CH2), 4.55 (t, 1H, R-CH); MS (ES+) m/z 185
(M + H)+; LC-MS: rt 0.7-0.8 min, m/z 185 (M + H)+; HPLC
(214 nm): rt 9.91 min, 100%.
1-(S-Nor leu cyl)p ip er id in e t r iflu or oa cet a t e (9.14): 1H
NMR (D2O, 400 MHz) δ 0.95 (t, 3H, CH3), 1.30-1.42 (m, 4H,
CH2), 1.59-1.76 (m, 6H, CH2), 1.87-1.90 (m, 2H, â-CH2),
3.49-3.69 (m, 4H, CH2), 4.54 (t, 1H, R-CH); MS (ES+) m/z 199
(M + H)+; LC-MS: rt 0.5-0.7 min, m/z 199 (M + H)+; HPLC
(214 nm): rt 11.96 min, 96%.
4-[(S)-2,4-Dia m in obu ta n oyl]m or folin e bis tr iflu or oa c-
1
eta te (10.1): H NMR (D2O, 400 MHz) δ 2.27-2.38 (m, 2H,
â-CH2), 3.12-3.28 (m, 2H, γ-CH2), 3.61-3.85 (m, 8H, CH2),
4.71 (t, 1H, R-CH); MS (ES+) m/z 188 (M + H)+; LC-MS: rt
0.6 min, 100%, m/z 188; HPLC (214 nm): rt 3.41 min, 100%.
1-[(S)-2,4-Dia m in obu ta n oyl]p ip er a zin e tr is tr iflu or o-
a ceta te (10.2): 1H NMR (D2O, 400 MHz) δ 2.25-2.40 (m, 2H,
â-CH2), 3.12-3.28 (m, 2H, γ-CH2), 3.33-3.50 (m, 4H, CH2),),
3.75-4.15 (m, 4H, CH2) 4.76 (t, 1H, R-CH); MS (ES+) m/z 187
(M + H)+; LC-MS: rt 0.5 min, 95%, m/z 187; HPLC (214 nm):
rt 3.40 min, 99%.
Gen er al P r ocedu r e for Syn th esis of Th ioam ides (10.3-
10.5). The protected amino acids amides (9) were prepared by
paralel synthesis using the PASP-protocol. Thioxylation of
these compounds was performed according to the following
procedure: To a solution of the Boc-protected amino acid
amides (2 equiv) in 5 mL of toluene was added 2,4-bis(p-
methoxyphenyl)-1,3-dithiadiphosphatane 2,4-disulfide (Lawes-
son’s reagent) (1 equiv). The reaction mixture was stirred for
2 h at 80 °C. The solvent was removed by evaporation and
the crude compound was purified by preparative TLC (EtOAc/
hexane, 40:60). Pure compounds were deprotected according
to the general procedure.
1-(L-Lysyl)-3(R,S)-ben zoyloxyp yr r olid in e Bis Tr iflu o-
r oa ceta te (8.16). A solution of 1-[N-R,γ-di(tert-butyloxycar-
bonyl)-L-lysyl]-3(R,S)-hydroxypyrrolidine (1.16 mmol, 480 mg)
in dry pyridine (15 mL) was treated with benzoyl chloride (1.28
mol, 148 µL) at 0 °C and stirred for 3 h at room temperature.
After cooling the reaction mixture to 0 °C, water was added
to the residue and solvents were evaporated. Dichloromethane
was added to the redidue and the mixture was washed with
5% NaHCO3. The organic layer was dried, evaporated and
purified by preparative TLC using EtOAc as eluent to yield
the pure 1-[N-R,γ-di(tert-butyloxycarbonyl)-L-lysyl]-3(R,S)-ben-
zoyloxypyrrolidine as an oil (53%). Deprotection was done
according to the general procedure to yield the title com-
pound: 1H NMR (D2O, 400 MHz) δ 1.44-1.60 (m, 2H, CH2,
4H, CH2), 2.31-2.52 (m, 2H, CH2), 2.55-2.70 (m, 0.5H, ꢀ-CH2),
2.90 (br s, 0.5H, ꢀ-CH2), 3.08 (br s, 1H, ꢀ-CH2), 3.72-4.05 (m,
4H, CH2), 4.34-4.46 (m, 1H, R-CH), 5.62-5.73 (m, 1H, 3-CH),
1-(L-Lysyl)th ioxop ip er id in e bis tr iflu or oa ceta te (10.3):
1H NMR (D2O, 400 MHz) δ 1.43-1.62 (m, 2H) and 1.71-1.83
(m, 8H) (γ-CH2, δ-CH2, 3-CH2, 4-CH2, 5-CH2), 1.93-2.00 (m,
2H, â-CH2), 3.04 (t, 2H, ꢀ-CH2), 3.80-3.96 (m, 2H) and 4.07-
4.22 (m, 1H), 4.34-4.41 (m, 1H) (2-CH2, 6-CH2), 4.74-4.79 (m,
1H, R-CH); MS (ES+) m/z 230 (M + H)+; LC-MS: rt 1.4 min,
100%, m/z 230; HPLC (214 nm): rt 5.94 min, 100%.
1-(S-Or n ith in e)th ioxop ip er id in e bis tr iflu or oa ceta te
1
(10.4): H NMR (D2O, 400 MHz) δ 1.82-1.99 (m, 8H, CH2),
2.03-2.09 (m, 2H, â-CH2), 3.12 (t, 2H, δ-CH2), 3.89-4.03 (m,
2H, CH2), 4.20-4.27 (m, 1H, CH2), 4.42-4.48 (m, 1H, CH2),

Inhibitors for Dipeptidyl Peptidases
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7.56-7.64 (m, 2H, m-Harom), 7.72-7.81 (m, 1H, p-Harom), 8.05-
8.12 (m, 2H, o-Harom); MS (ES+) m/z 320 (M + H)+; LC-MS: rt
9.9 min, 100%, m/z 320; HPLC (214 nm): rt 11.76 min, 100%.
Gen er a l P r oced u r e for th e Syn th esis of Dip ep tid e
Nitr iles (11, 12). To a mixture of Boc-Xaa-OH (1.1 equiv),
triethylamine (3 equiv) and TBTU (1.1 equiv) in DMF was
added YaaNH2 (1 equiv) [2(S)-pyrrolidinecarboxamide was
commercially available; 2(S)-piperidinecarboxamide was pre-
pared from L-pipecolinic acid (1 equiv) by reaction with
N-hydroxysuccinimide (1.05 equiv) and dicyclohexylcarbodi-
imide (DCC, 1.05 equiv) in DCM (yield: 90%), followed by
treatment of a solution in dioxane with ammonium gas
(yield: 99%).] After the mixture was stirred overnight at room
temperature, water was added and the mixture was extracted
with EtOAc (3 × 50 mL). The combined organic layers were
washed with 1 N HCl (2 × 25 mL), 5% NaHCO3 (2 × 25 mL)
and brine (25 mL). The organic layer was dried over NaSO4,
evaporated, and purified by column chromatography, yielding
Boc-Xaa-YaaNH2 (86%). Dehydratation of the amide function
to the nitrile was done according to the following procedure:19
To a solution of Boc-Xaa-YaaNH2 (1 equiv) and imidazol (2
equiv) in pyridine at -30 °C was slowly added phosphoru-
soxychloride (4 equiv). The solution was allowed to attain room
temperature, and the reaction was monitored by TLC. After
completion of the reaction, the solvent was evaporated and the
residue was extracted with 1 N HCl and diethyl ether. The
organic layer was dried and evaporated, and the residue was
purified by prepartive TLC to yield the Boc protected dipeptide
nitrile (60%). Boc deprotection was done according to the
general procedure.
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1-(L-Lysyl)-2(S)-cya n op yr r olid in e bis tr iflu or oa ceta te
(11): 1H NMR (D2O, 400 MHz) δ 1.50-1.53 (m, 2H, CH2),
1.72-1.77 (m, 2H, CH2), 1.98-2.12 (m, 2H, CH2), 2.14-2.26
(m, 2H, CH2), 2.32-2.43 (m, 2H, CH2), 3.02-3.06 (m, 2H,
ꢀ-CH2), 3.70-3.74 (m, 2H, 5-CH2), 4.37 (t, 1H, R-CH), 4.83-
4.88 (m, 1H, R-CH); MS (FAB+) m/z 225 (M + H)+; HPLC (214
nm): rt 3.20 min, 100%.
1-(L-Lysyl)-2(R,S)-cya n op ip er id in e bis tr iflu or oa ceta te
1
(12.1): H NMR (D2O, 400 MHz) δ 1.42-1.63 (m, 3H, CH2),
1.71-2.02 (m, 8H, CH2), 2.18-2.29 (m, 1H, CH2), 3.00-3.41
(m, 2H, 6-CH2), 3.46-3.50 (m, 1H, ꢀ-CH2), 3.88-4.00 (m, 1H,
ꢀ-CH2), 4.53-4.67 (m, 1H, R-CH), 5.69-5.88 (m, 1H, 2-CH);
MS (ES+) m/z 239 (M + H)+; LC-MS: rt 1.0 min, 100%, m/z
239; HPLC (214 nm): rt 5.78 min, 99%.
1-[(S)-2,4-Dia m in obu ta n oyl]-2(S)-cya n op ip er id in e bis
tr iflu or oa ceta te (12.2): 1H NMR (D2O, 400 MHz) δ 1.50-
1.68 (m, 1H, CH2), 1.72-2.00 (m, 4H, CH2), 2.08-2.19 (m, 1H,
CH2), 2.35-2.49 (m, 2H, CH2), 3.10-3.29 (m, 2H, 6-CH2),
3.48-3.53 (m, 1H, γ-CH2), 3.85-3.98 (m, 1H, γ-CH2), 4.70-
4.82 (m, 1H, R-CH), 5.69-5.89 (m, 1H, 2-CH2); MS (ES+) m/z
211 (M + H)+; LC-MS: rt 0.8 min, 100%, m/z 211; HPLC (214
nm): rt 6.56 min, 100%.
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Ack n ow led gm en t. This work received support from
The Fund for Scientific ResearchsFlanders (Belgium)
(F.W.O.) and The Special Fund for ResearchsUniversity
of Antwerp (BOF UA). P. Van der Veken is a fellow of
the Institute for the Promotion of Innovation by Science
and Technology in Flanders (IWT). The excellent tech-
nical assistance of N. Lamoen and W. Bollaert is greatly
appreciated.
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