Structure-activity relationship studies on isoindoline inhibitors of dipeptidyl peptidases 8 and 9 (DPP8, DPP9): Is DPP8-selectivity an attainable goal?

Article abstract of DOI:10.1021/jm200383j

This work represents the first directed study to identify modification points in the topology of a representative DPP8/9-inhibitor, capable of rendering selectivity for DPP8 over DPP9. The availability of a DPP8-selective compound would be highly instrumental for studying and untwining the biological roles of DPP8 and DPP9 and for the disambiguation of biological effects of nonselective DPP-inhibitors that have mainly been ascribed to blocking of DPPIV's action. The cell-permeable DPP8/9-inhibitor 7 was selected as a lead and dissected into several substructures that were modified separately for evaluating their potential to contribute to selectivity. The obtained results, together with earlier work from our group, clearly narrow down the most probable DPP8-selectivity imparting modification points in DPP8/9 inhibitors to parts of space that are topologically equivalent to the piperazine ring system in 7. This information can be considered of high value for future design of compounds with maximal DPP8 selectivity.

Full text of DOI:10.1021/jm200383j

ARTICLE
pubs.acs.org/jmc
StructureꢀActivity Relationship Studies on Isoindoline Inhibitors of
Dipeptidyl Peptidases 8 and 9 (DPP8, DPP9): Is DPP8-Selectivity an
Attainable Goal?
Sebastiaan Van Goethem,† Veerle Matheeussen,‡ Jurgen Joossens,† Anne-Marie Lambeir,‡ Xin Chen,§
Ingrid De Meester,‡ Achiel Haemers,† Koen Augustyns,† and Pieter Van der Veken*,†
†Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1,
B-2610 Wilrijk, Antwerp, Belgium
‡Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1,
B-2610 Antwerp, Belgium
§Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhu Nan, Miaoli 350, Taiwan, ROC
S Supporting Information
b
ABSTRACT: This work represents the first directed study to
identify modification points in the topology of a representative
DPP8/9-inhibitor, capable of rendering selectivity for DPP8
over DPP9. The availability of a DPP8-selective compound
would be highly instrumental for studying and untwining the
biological roles of DPP8 and DPP9 and for the disambiguation
of biological effects of nonselective DPP-inhibitors that have
mainly been ascribed to blocking of DPPIV’s action. The cell-
permeable DPP8/9-inhibitor 7 was selected as a lead and dissected into several substructures that were modified separately for
evaluating their potential to contribute to selectivity. The obtained results, together with earlier work from our group, clearly narrow
down the most probable DPP8-selectivity imparting modification points in DPP8/9 inhibitors to parts of space that are
topologically equivalent to the piperazine ring system in 7. This information can be considered of high value for future design of
compounds with maximal DPP8 selectivity.
’ INTRODUCTION
Since the discovery of DPPIV in the late 1960s, more than 30
pharmaceutical and biotechnology companies have started up
development projects for DPPIV inhibitors as therapeutics for
the treatment of type 2 diabetes.8 By prolonging the lifetime of
the incretin hormones glucagon-like-peptide 1 (GLP-1) and
glucose dependent insulinotropic peptide (GIP), DPPIV inhibi-
tors are able to stimulate postprandial insulin production and
blood glucose clearance.9 Up until now sitagliptin 1, vildagliptin
2, and saxagliptin 3 have all gained regulatory approval, while
others have successfully advanced into late stage clinical trials and
several other “gliptins” are still under investigation.10ꢀ14
(Figure 1, Table 1) Besides these incretin enhancers, other
potential therapies comprise GLP-1 receptor agonists or incretin
mimetics, i.e. long acting GLP-1 analogues.15ꢀ17 Besides the
important function as incretin regulating protein, the ubiqui-
tously expressed DPPIV is under investigation for its role in cell
biological processes and immune function. This is extensively
reviewed in recent literature reports.2,3,18ꢀ20
In recent years, several serine proteases belonging to clan SC
have gained pharmaceutical interest in the areas of immunol-
ogy, metabolic disorders, and cancer.1,2 The most widely
investigated protein in this class of enzymes is dipeptidyl
peptidase IV (DPPIV/CD26). Other members of the so-called
DPPIV activity and/or structure-homologues (DASH) family
are DPPII, DPP8, DPP9, fibroblast activating protein (FAP),
and prolyl oligopeptidase (POP/PREP).3ꢀ6 These prolyl spe-
cific peptidases are unique among the proteases because they
are able to cleave a peptide bond flanked by a proline residue.
Because of the specific recognition of the pyrrolidine ring, these
serine proteases are responsible for the cleavage of what is
accepted to be a relatively resistant peptide bond, giving unique
biochemical properties. The biological importance of the prolyl
peptidases is underscored by the fact that many bioactive
peptides, i.e. hormones, chemokines, and neuropeptides, con-
tain one or more proline residues and the processing and
degradation of such biologically important regulatory peptides
requires prolyl specific peptidases.7 Despite their enzymatic and
structural similarity, the DASH proteins are most likely to have
distinct physiological functions, as indicated by their difference
in expression and localization.
Fibroblast activation protein (FAP), a 95 kD membrane-
bound glycoprotein, has both endopeptidase and exopeptidase
Received: March 31, 2011
Published: June 29, 2011
r
2011 American Chemical Society
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Table 1. Potency and Selectivity of Reference Compounds
IC50a (μM)
compd
name
DPP8
DPP9
>100
DPPIV
DPPII
reference
1
2
3
4
5
6
7
sitagliptin
>50
0.04 ( 0.001
0.12 ( 0.01
0.60 ( 0.06 nM
165 ( 9
>100
>1000
b
vildagliptin
9.0 ( 0.1
0.68 ( 0.07
b
saxagliptin
c
12
25
45
43
46
UAMC-00039
UAMC-00141
UAMC-00491
142 ( 27
0.53 ( 0.11
0.160 ( 0.016
0.014
78.6 ( 9.1
1.57 ( 0.07
0.07 ( 0.04
0.053
0.48 ( 0.04 nM
>1000
>500
>100
21.5 ( 3.2
>100
>100
a Values expressed in μM unless stated otherwise. b In house data. c 3 did not show any significant inhibition of DPPII at concentrations up to 30 μM.
activity and is a member of the S9B family most closely related to
DPPIV. In general, FAP is absent in the adult tissues. It is
expressed in pathologies comprising tissue remodelling, such as
tumors and liver disease.20 Research on small molecule inhibitors
of FAP is currently ongoing in our group as well as in other
laboratories.21
Dipeptidyl peptidase II (DPPII, QPP) is assigned to family S28 of
class SC. Despite the numerous reports with suggestions on possible
functions of the enzyme, the physiological role of this serine protease
still has to be elucidated.22 To unravel the physiological role of the
latter, our group developed the most potent and specific hitherto
described, orally bioavailable DPPII inhibitor 4.23ꢀ25
During the past decade, DPP8 and DPP9 have been dis-
covered as two members of the prolyl oligopeptidase S9b
subfamily, which also contains DPPIV and FAP. Compared
to the two latter extensively studied enzymes, relatively little is
known about DPP8 and DPP9.26 Neither the tertiary structures
nor in vivo substrates or the physiological role of these two
enzymes have been firmly established. The open reading frame
(ORF) of DPP8 codes for two splice variants of 882 or 898
amino acids. DPP8 shares 27% amino acid (aa) identity and
51% aa similarity with the protein sequences of DPPIV, and this
increases to 35% aa identity and 57% aa similarity in the
hydrolase domain.27 Unlike DPPIV and FAP, DPP8 is a soluble
protein localized in the cytoplasm.28 DPP9 was identified in
silico by Abbott et al. and was first cloned and expressed
recombinantly by Olsen and Wagtmann.26,28 Recently, natural
DPP9 was purified and identified from bovine testes.29,30
Similar to DPP8, DPP9 is also ubiquitously expressed as two
variants comprising the short 863 aa version and the longer 892
aa version. Whether both forms are biologically active remains
controversial.30ꢀ32 DPP8 shares 57% aa identity and 77% aa
similarity with DPP9, and this increases to more than 90% and
100% respectively within the active site, following an in-house
homology modeling study. This study is based on the X-ray
structures of DPPIV (pdb code 3KWJ), FAP (pdb code 1Z68),
and PREP (pdb code 1H2W), and on the homology models of
DPP8 and DPP9, constructed by Rummey et al.33 The depicted
sequence alignment clearly shows that in DPP8 and DPP9
almost identical amino acid residues make up the active sites:
only at positions 174DPP8 and 433DPP8 a difference is seen.
However, this alteration is moderate with possibly little overall
effect, as the basic aa Lys at position 174 in DPP8 is replaced
by the analogous amino acid residue Arg in DPP9. Similarly, the
hydrophobic residue Ile at position 433 in DPP8 is replaced
by ValDPP9, also a hydrophobic amino acid.
Figure 1. DPPIV inhibitors 1, 2, and 3, DPPII inhibitor 4, irreversible
DPP8/9 inhibitor 5, and reversible DPP8/9 inhibitor 6.
The distribution of the cytosolic enzymes, DPP8 and DPP9,
and their expression in different types of endothelial cells is
summarized by Yu et al.34 Because of the recent insight that
DPP8 and DPP9 have a similar enzymatic activity profile as
DPPIV, it is very likely that some physiological functions
attributed to the latter could in fact be credited to DPP8 and/
or DPP9.19,35 Furthermore, because studies on the involvement
of DPPIV in various pathologies such as inflammatory bowel
disease, rheumatoid arthritis, and multiple sclerosis were per-
formed with nonselective DPPIV inhibitors, it is suspected that
DASH proteins other than DPPIV could be responsible for
this effect.19 Recent literature reports show DPP8/9 activity in
human leukocytes as well as in B and T cell lines and in endothelial
cells.35,36 Moreover, several cytokines, e.g. SDF1, ITAC, and IP-
10, are demonstrated to be in vitro substrates for DPP8/9.32 In
addition, DPP9 appears to be a rate-limiting enzyme for degrada-
tion of RU134ꢀ42, a proline containing antigenic peptide.37 These
findings suggest a potential role for DPP8/9 in the immune
system. Furthermore, these enzymes may be involved in other
physiological processes, such as spermatogenesis, because bovine
testes appear to be a source of DPP9 and DPP8 could be detected
in spermatogonia and spermatids.28ꢀ30,38 Previously, attention
was drawn to DPP8 and DPP9, following research results reported
by Lankas et al., suggesting that the use of the DPP8/9 inhibitor
allo-Ile-isoindoline is associated with severe toxicity in animal
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Figure 2. Protein sequence comparison of the active sites of DPPIV, FAP, PREP, DPP8, and DPP9. After alignment and superposition of the proteins
amino acid sequences, residues were selected within 4.5 Å from a docked inhibitor. Differences in the secondary structure between DPP8 and DPP9 are
highlighted in italic and bold.
Figure 3. Lead compound 7 and structural modifications investigated in this study.
models.39 Whether the observed toxicity was DPP8/DPP9 related,
or solely due to compound mediated off-target effects, remains to
be unambiguously established.40 Nevertheless, several groups
provide sufficient evidence to question the alleged toxicity caused
by DPP8/9 inhibition.41,42
From this series, we selected isoindoline and 5-fluoroisoindo-
line as fragments that offer an optimal balance of potency and
selectivity for DPP8/9 with respect to DPP IV and DPP II (e.g., 6).43
Additionally, this study indicated the limited potential of modifying
the P1 position for obtaining compounds able to discriminate
between DPP8 and DPP9. Furthermore, our previously described
(2S,4S)-4-azido-2-cyanopyrrolidines yielded nanomolar DPP8/9
inhibitors with modest overall selectivity toward DPPIV and
DPPII.44 Thus far, neither our strategies, nor the attempts of other
groups, resulted in compounds with either only DPP8 or only DPP9
affinity. To establish the biological functions and identify the in vivo
substrates of the enzymes, the availability of at least a highly selective
inhibitor of DPP8 (or a highly selective inhibitor of DPP9) is very
desirable.
As depicted in Figure 2, the high degree of sequence
homology between DPP8 and DPP9 on one side and DPPIV
and FAP on the other makes even dual DPP8/9 inhibitor
design a challenging task that hitherto has been the subject of
only a limited number of publications, several of which have
come from our groups.29,43ꢀ47 Strong inhibition of DPP8 was
reported by us using several irreversible, in-house synthesized,
isoindolin-1-yl phosphonate analogues of 5.45 However, after
our reported successful investigations to force access to highly
purified bovine DPP9, the compounds turned out to show
concomitant affinity for this enzyme.29 Subsequently, we
investigated an extended group of pyrrolidine analogues
and a focused series of substituted isoindolines and azaheter-
oaromatic analogues as P1 fragments in DPP8/9 inhibitors.
The principal objective of this work was to establish topolo-
gical insight in a representative DPP8/9 inhibitor structure for
identifying positions in its molecular framework that, via chemi-
cal modification, allow improvement of DPP8 selectivity. As the
model inhibitor in this study, compound 7 (1G244) was selected
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ARTICLE
and synthesized by Jiaang et al.46 (Figure 3) This lead compound,
already displaying some selectivity for DPP8, possesses the
overall dipeptide-derived structure of all known DPP8/9 inhibi-
tors, is the most active DPP8/9 inhibitor described to date and
readily penetrates plasma membranes.42 In addition, compared
to the dimensions of several other reported DPP8/9 inhibitors, 7
is a large molecule that can be expected to probe a proportionally
larger part of DPP8’s and DPP9’s active site, hence offering
higher probability for detecting differences between both. As the
starting point of our study, compound 7 was dissected in four
substructures that conceptually can each be envisaged as modifi-
able modules: (1) the P1-isoindoline, (2) the P2 Asp-residue, (3)
the piperazine ring system, and (4) the bis(4-fluorophenyl)-
methyl substituent. In all molecules prepared under this study,
however, isoindoline or 5-fluoroisoindoline were incorporated at
the P1 position. As explained earlier, these two residues were
selected from a large number of P1 candidates based on earlier
work by our group that among other things also indicated that
the P1 position is not an obvious modification point for impart-
ing DPP8 (or DPP9) selectivity. Structural modifications of the
P2 position investigated included homologation and deletion of
the free amino function (Figure 3). As for the piperazine module,
this was altered into (i) acyclic aminoalkyl chains, (ii) substituted
piperazines, and (iii) piperazine analogues like homopiperazine
and 3-aminopyrrolidine. Finally, a series of both alkyl- and acyl-
replacements of comparable bulk size or smaller were selected for
the bis(4-fluorophenyl)methyl group in 7. In the following parts,
we will describe the synthesis, the obtained biochemical evaluat-
ing data, and the derived SAR for the prepared analogues of 7.
For clarity’s sake, the different compounds prepared are grouped
into six classes (AꢀF) as summarized in Figure 4.
’ CHEMISTRY
Target compounds from class AꢀF were prepared using the
following synthetic strategy (Scheme 1) and are listed in Table 2.
Starting from isoindoline (X = H) or 5-fluoroisoindoline (X = F),
which was synthesized as reported previously,43 these favorable
P1 L-proline surrogates were coupled with succinyl acid 4-methyl
ester, N-Boc-aspartic acid 4-methyl ester, or N-Boc-glutamic acid
4-methyl ester, yielding succinyl (R1 = H and n1 = 1), N-Boc
protected glutamylisoindoline (R1 = NH-Boc and n1 = 2), or N-
Boc protected aspartylisoindoline (R1 = NH-Boc and n1 = 1)
derivatives, respectively. Standard peptide coupling procedures
were employed. Subsequent hydrolysis with potassium hydro-
xide in methanol proceeded smoothly to give the aminoacyli-
soindoline derivatives ready for peptide coupling with a diverse
set of primary and secondary amines. Final deprotection with
trifluoroacetic acid (TFA) or hydrochloric acid (HCl) yielded
the final compounds.
For the preparation of the piperazine analogues, mainly five
synthetic strategies were followed. First, the piperazine moieties
from target compounds 7 and 12(aꢀo) were bought as such or
were synthesized starting from commercially available piperazine
analogues, which were first N-Boc protected using Boc-anhy-
dride and Et3N. These intermediates were employed as nucleo-
philes in an SN2 reaction on different alkyl halides using Et3N as
base in dimethylformamide at 70 °C. Subsequently, this alkyla-
tion was followed by acidolytic cleavage of the Boc functionality
with TFA in dichloromethane (DCM), furnishing the corre-
sponding alkylated piperazines.
Figure 4. Overview of compounds divided in six classes. (A) Alkylated
piperazine analogues 7 and 12(aꢀo); (B) acylated piperazine analogues
12(qꢀs); (C) L-proline derivatives 12(tꢀu); (D) derivatized aspartic
acid compounds 12(vꢀy); (E) 3-aminopyrrolidine derivative 12z; (F)
4-amino-L-proline derivatives 16(aꢀb).
In a second strategy, two different approaches to form the
acylated piperazine analogues 12(pꢀs) were selected. The first
approach started with mono-Boc-piperazine, which was coupled
to different commercially available carboxylic acids, for example,
Scheme 1. General Synthesis of DPP8/9 Inhibitorsa
a Reagents: (a) TBTU, Et3N, aa derivative, CH2Cl2; (b) KOH, MeOH; (c) TBTU, Et3N, various amines, CH2Cl2 ; (d) TFA, CH2Cl2 or HCl in dioxane.
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Table 2. Structural Characteristics of Isoindoline Compounds
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Scheme 2. General Synthesis of 4-Amino-L-proline Derivatives 16a and 16ba
a Reagents: (a) TBTU, Et3N, (2S,4S)-4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid, CH2Cl2; (b) Pd/C, H2,
methanol; (c) 15a: quinoline-4-carboxaldehyde, acetic acid, NaCNBH3, DCM; 15b: 2-methoxy-1-carboxaldehyde, acetic acid, NaCNBH3, CH2Cl2; (d)
TFA, CH2Cl2 followed by HCl in isopropyl alcohol, CH2Cl2.
Figure 5. Determination of the Ki values for compound 12f (A) and 12n (B) against both DPP8 and DPP9, assuming competitive inhibition.
9H-fluorene-9-carboxylic acid, under normal peptide coupling
conditions as described above. In a second approach, mono-Boc-
piperazine was reacted with different acid chlorides and Et3N in
DCM. Finally, the treatment of Boc-protected acylated inter-
mediates with TFA gave four acylated piperazine analogues,
using conditions as described above.
Third, preparation of the L-proline derivatives began with the
coupling of the free carboxylic acid from N-Boc-proline to com-
mercially available amines: bis(4-fluorophenyl)methanamine and
1-(bis(4-fluorophenyl)methyl)piperazine. A Boc-deprotecting step
yielded a piperazine-nitrogen ready for coupling with the P1 moiety
with formation of compounds 12(tꢀu).
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Table 3. Inhibitory Properties of Isoindoline Compounds
block (2S,4S)-4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy-
carbonyl)pyrrolidine-2-carboxylic acid, the preparation of which
is described in the Supporting Information section of this paper.
Prior to reductive alkylation of the 4-amino functionality with the
commercially available aldehydes quinoline-4-carbaldehyde and
2-methoxy-1-naphthaldehyde, the benzyloxycarbonyl (Cbz)
protecting group was removed with catalytic hydrogenation.
Final deprotection yielded compounds 16a and 16b (Scheme 2).
IC50 (μM)a
compd class
DPP8
DPP9
DPPIV
DPPII
7
A
A
A
A
A
A
A
A
A
A
A
0.012 ( 0.001 0.084 ( 0.002 >50
>50
12a
12b
12c
12d
12e
12f
0.96 ( 0.04
4.0 ( 0.1
>10
4.0 ( 0.17
5.0 ( 0.4
>10
>100
>100
>100
19.3 ( 0.7
14.0 ( 0.6
>100
0.0094 ( 0.0004 0.0254 ( 0.0025 >50
0.0088 ( 0.0007 0.0269 ( 0.0026 >100
0.0099 ( 0.0004 0.050 ( 0.003 >50
0.063 ( 0.002 0.224 ( 0.023 >100
>50
’ RESULTS AND DISCUSSION
>25
For all compounds synthesized, inhibitory potency toward
DPP8, DPP9, DPPIV, and DPPII was assessed using IC50
measurements. Experimentally determined Ki values were found
to be in line with the IC50 results as depicted in Figure 5.29
Table 3 summarizes the inhibitory profiles of these inhibitors: 7,
12aꢀz, and 16aꢀb. In general, the compounds tested showed no
affinity for DPPIV, comparable to what was observed for lead 7.
Because of the presumed larger S2-pockets of DPP8 and DPP9,
selectivity over DPP IV is most likely achieved by the combined
presence of a P1 isoindoline and the sterically demanding
substituents of the P2 aspartyl residue.33 In general, all synthe-
sized compounds consistently display at least some selectivity for
DPP8 over DPP9, a feature already present in the lead. During
our initial attempts to increase this DPP8 selectivity, we homo-
logated the P2 aspartyl fragment to a glutamyl residue, yielding
compounds 12a and 12b, which proved to be less favorable
inhibitors of both DPP8 and DPP9 and displayed limited but
measurable affinity for DPP II. Therefore, no further analogues
containing a P2-glutamyl residue were prepared. In addition,
removing the amine functionality from the P2 fragment in 12c
resulted in a dramatic decrease in inhibitory potency that was not
compensated for by affinity contribution from the P2 substituting
(bis(4-fluorophenyl)methyl)piperazine fragment. This finding
indicates the pivotal importance of the free amine group,
reflecting the substrate selectivity of the dipeptidyl peptidases,
and was convincing enough not to further explore this type of
derivatization. Subsequently, modification of the piperazine ring
was investigated. First, piperazine analogues or substituted
acyclic secondary amines were found to induce a decrease in
potency toward both DPP8 and DPP9 or, again, to diminish the
selectivity with regards to DPPII. Inhibitors 12j, 12(tꢀy), and
12z might serve as illustrative examples thereof. Surprisingly,
compound 12z roughly lost all DPP8/9 potency and turned out
to be a potent DPPII inhibitor, albeit less pronounced than
reference DPP II inhibitor 4. Compound 12z is characterized by
the presence of a less conformationally constrained 3-aminopyr-
rolidine ring. As a rationale for its DPPII activity, one might
hypothesize this molecule, if bound to the enzyme, to be able to
align its basic amine functions to the same positions as the
corresponding amines of the dibasic 1,4-diaminobutyryl phar-
macophore in 4. From the foregoing data, a 1,4-diazacyclohexane
ring was deduced to be preeminently tolerated by both DPP8 and
DPP9. Next, C-substitution of the piperazine ring with a methyl
group, was investigated for its potential to uncouple DPP9
activity in the diastereomeric pair 12m and 12n. Both were
found to possess a roughly 10-fold selectivity for DPP8 over
DPP9 and approximately thousand-fold selectivity over DPPIV
and DPPII, with the S-methyl congener displaying slightly higher
affinity. However, converting the piperazine ring into a piperazin-
2-one in compound 12o was shown not to be beneficial, as it
reduced potency toward both DPP8 and DPP9. Additionally, the
>50
12g
12h
12i
>50
0.141 ( 0.003 0.53 ( 0.06
34.8 ( 1.4 54 ( 2
0.080 ( 0.004 0.26 ( 0.02
>100
>100
>100
>100
>100
>100
>50
>25
>100
12j
3.3 ( 0.17
>25
12k
12l
Ac 0.23 ( 0.03
1.13 ( 0.12
0.24 ( 0.01
A
A
A
A
B
B
B
0.08 ( 0.01
>25
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
12y
12z
16a
16b
0.032 ( 0.002 0.26 ( 0.02
>50
0.05 ( 0.005
0.52 ( 0.03
0.54 ( 0.04
2.6 ( 0.2
>25
>100
>100
>100
>50
0.166 ( 0.006 0.63 ( 0.08
0.278 ( 0.006 0.68 ( 0.03
1.9 ( 0.1
17.3 ( 1.1
>25
0.029 ( 0.001 0.065 ( 0.006 >100
Bb 0.31 ( 0.01
0.54 ( 0.05
22.6 ( 2.5
1.7 ( 0.1
1.4 ( 0.1
29.1 ( 2.2
35.0 ( 1.8
>10
>100
>100
>100
>100
>100
>100
>100
>100
>25
C
C
D
D
D
D
E
6.8 ( 0.4
0.91 ( 0.07
0.73 ( 0.02
9.4 ( 0.3
4.5 ( 0.2
>10
23 ( 3
2.10 ( 0.07
>100
0.17 ( 0.01
0.65 ( 0.03
15.2 ( 0.8
0.033 ( 0.001
3.91 ( 0.18
1.1 ( 0.1
15.9 ( 0.18
16.4 ( 1.9
2.1 ( 0.1
>50
F
17.7 ( 0.7 3.6 ( 0.1
>100 0.29 ( 0.01
F
a IC50 values expressed in μM, except stated otherwise. b Sulfonylated
R2. c Bridged piperazine ((1R,4R)-2,5-diazabicyclo[2.2.1]heptane).
Alternatively, converting commercially available amines, e.g.
tert-butyl (2-aminoethyl)carbamate, 2,2-diphenylethanamine,
and bis(4-fluorophenyl)methanamine, into primary and second-
ary amides with N-Boc aspartylisoindoline yielded target com-
pounds 12(vꢀy). The same procedure was used for 4-hydroxy-
amantadine, which was synthesized in excellent yield following a
literature procedure.48 Finally, the amino-pyrrolidine moiety of
12z was synthesized as follows. The starting product 4S-hydroxy-
L-proline was decarboxylated to the corresponding 3-hydroxy-
pyrrolidine, which was N-Boc protected. Successive mesylation
of the alcohol was accomplished using methanesulfonyl chloride
and Et3N in DCM. Nucleophilic substitution of the mesylated
alcohol with sodium azide gave compound X in quantitative
yield. The Boc protecting group was converted to the base-labile
trifluoroacetic acid (TFA) protecting group. Catalytic hydroge-
nation of the azide functionality yielded the primary amine,
which was immediately Boc protected. Removal of the TFA
protecting group, subsequent alkylation of 4,40-(chloromethy-
lene)bis(fluorobenzene), Boc deprotection, and final coupling
afforded compound 12z.
Compounds 16a and 16b were synthesized, starting from
isoindoline, which was coupled to the 4-amino-L-proline building
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bridged piperazine ((1R,4R)-2,5-diazabicyclo[2.2.1]heptane) of
compound 12k did not result in increased potency nor selec-
tivity. Finally, modification of the bis(4-fluorophenyl)methyl
group with piperazine N-substituents of comparable or
slighty decreased bulk size, was investigated. This effort
involved the preparation of compounds 12fꢀi and 12l,
grouping inhibitors with N-alkyl type substituents and
12pꢀs in which susbtituents were grafted by acylation or
sulfonylation. Of all substituents introduced by alkylation,
only the 1,1-phenyl-(4-chloro)phenylmethyl substituent of
12f led to a comparable inhibitory profile compared to the
lead compound. Mono N-substitution of the piperazine ring
with a 4-fluorobenzyl moiety in compound 12l did not result
in increased DPP8/9 potency. For the acylated piperazine
compounds 12pꢀs, no general trend could be observed.
While compounds 12pꢀq and 12s are just reasonable
DPP8/9 inhibitors, compound 12r is a potent inhibitor of
DPP8 and DPP9 with a favorable selectivity profile toward
the other assessed DPPs. In summary, these results indicate
that both acylated and alkylated piperazine N-substitution
types allow for preparation of potent DPP8/9 inhibitors, but
neither is significantly better at inducing selectivity for DPP8.
Finally, combining the P1-(5-fluoroisoindoline), optimized
by us earlier, in conjunction with optimized P2 fragments,
gave compounds 12d and 12e, with potencies equivalent to
that of the reference compound 7.
the only modification type to further improve uncoupling of
DPP9 activity, compared to the lead molecule. Further structural
modification is currently being investigated as a means to obtain
compounds with maximal affinity and selectivity for DPP8.
Finally, by narrowing down the most probable DPP8-selectivity
imparting modification points in DPP8/9 inhibitors to parts of
space that are topologically equivalent to the piperazine ring
system, this study can be considered valuable to future DPP8-
selective inhibitor discovery programs, eventually involving
novel compound chemotypes.
’ EXPERIMENTAL SECTION
Amino acids and TBTU were purchased from Novabiochem. Other
reagents were obtained from Sigma-Aldrich or Acros and used as such,
unless otherwise specified. Characterization of intermediates and final
compounds was done using NMR spectroscopy and mass spectrometry,
and final purity was controlled using HPLC analysis. 1H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded on a
Bruker Advance III ultrashield 400 spectrometer. CDCl3, CD3OD, or
DMSO-d6 were used as the solvents. Chemical shifts in the spectra are
given in ppm and coupling constants (J) in Hz. ES mass spectra were
obtained with an Esquire 3000plus ion trap mass spectrometer from
Bruker Daltonics, using the direct infusion mode.
Purity of final products was controlled using HPLC analysis. (1)
LC/MS chromatograms were recorded on an AGILENT 1100 series
HPLC system equipped with an Alltech Prevail C18 column (2.1 mm ꢁ
50 mm ꢁ 3 μm) connected to an Esquire 3000plus ion trap mass
spectrometer from Bruker Daltonics. A 5ꢀ100% acetonitrile/water,
20 min gradient was used with a flow rate of 0.2 mL/min. Formic acid
(0.1%) was added to both solvents. (2) In addition, reversed phase
HPLC chromatograms were recorded on a Gilson instrument equipped
with an Ultra sphere ODS column (4.6 mm ꢁ 250 mm ꢁ 5 μm) and a
UV detector. A 10ꢀ100% acetonitrile, 35 min gradient was used with a
flow rate of 1 mL/min. Then 0.1% trifluoroacetic acid was added to both
solvents. An indicated purity of 100% indicates that no other peaks in the
chromatogram occur. All compounds reported in this publication were
determined to have purities of g95%, except for compounds 12i (93%)
and 12p (92%), which could not be purified further.
’ CONCLUSIONS
This work represents the first directed study to identify
modification points in the topology of a representative DPP8/
9-inhibitor, capable of rendering selectivity for DPP8 over DPP9.
The highly challenging character of this task was discussed in
view of the remarkably similar amino acid sequences within these
enzymes’ active sites. As the starting point of our investigations,
the cell-permeable DPP8/9-inhibitor 7 was selected as a lead and
dissected into several substructures that were modified separately
for evaluating their potential to contribute to selectivity. The
analogues of 7 generated in this way were assessed for DPP8 and
DPP9 activity and, in addition, for activity toward DPPIV and
DPPII. SAR data obtained from this study reflect the anticipated
difficulties in designing DPP8-selective compounds, with the
most promising structures possessing a selectivity index of
approximately 1 order of magnitude in favor of DPP8. In
summary, the series of inhibitors presented here all contain an
isoindoline and 5-fluoroisoindoline as the optimal P1 residues, a
feature investigated earlier in extenso by our group. The SAR
generated in this publication demonstrates that an aspartyl
residue at the P2 position is preferable over succinyl and glutamyl
moieties for inhibitory potency. Further, a piperazin-1-yl moiety,
with a bulky alkyl or acyl substituent at its 4-position, seems to be
the most evident choice to supplement the optimal P2 aspartyl
residue. Practically all synthesized compounds, compared to lead
structure 7, turned out to conserve excellent selectivity with
respect to DPPIV. Compounds, however, in which the P2-
supplementing piperazine was either deleted or replaced by a
3-aminopyrrolidine analogue, turned out to regain binding
potential toward DPPII. Maximal selectivity for DPP8 over
DPP9, roughly in the range of 1 order of magnitude, was
observed with compounds 12m and 12n, methylpiperazine
analogues of 7. While this degree of selectivity toward DPP8 is
still modest, C-alkylation of the piperazine ring was found to be
General Experimental and Analytical Data for Key Products
12e, 12f, 12m, and 12n. Detailed experimental and analytical data for
all intermediates and final products in the manuscript can be found in the
Supporting Information part of this publication.
(2S)-2-Amino-4-(4-((4-chlorophenyl)(phenyl)methyl)pipe-
razin-1-yl)-1-(5-fluoroisoindo-lin-2-yl)butane-1,4-dione bis-
(2,2,2-trifluoroacetate) (12e). Compound 11e (0.1 mmol) was
dissolved in dichloromethane (2 mL). Next, trifluoroacetic acid (2 mL)
was added and the reaction was stirred for 30 min. The volatiles were
evaporated under reduced pressure. The crude product was purified by
precipitation with cold, dry diethyl ether and obtained as a white,
amorphous powder; yield 94%. 1H NMR (400 MHz, CDCl3) δ
7.59ꢀ7.54 (m, 4H), 7.41ꢀ7.28 (br m, 5H), 7.19ꢀ7.13 (m, 1H),
7.01ꢀ6.89 (m, 2H), 4.98ꢀ4.83 (m, 2H), 4.79ꢀ4.73 (m, 1H),
4.68ꢀ4.57 (m, 3H), 4.12ꢀ4.07 (m, 1H), 3.83ꢀ3.71 (m, 3H),
3.14ꢀ3.07 (m, 4H), 2.97ꢀ2.93 (m, 2H). ESI+-MS: m/z = 521 (M +
H). Purity determination: (1) LC/MS tR = 14.8 min, 99%; ESI+-MS m/z =
521.3 (M + H), 1063.2 (2M + Na); (2) HPLC tR = 17.3 min, 100%
(2S)-2-Amino-4-(4-((4-chlorophenyl)(phenyl)methyl)pipe-
razin-1-yl)-1-(isoindolin-2-yl)butane-1,4-dione bis(2,2,2-tri-
fluoroacetate) (12f). This product was obtained as a white, amor-
phous solid byacidolyticremoval of theBoc-protecting groupin11f, using
1
an identical procedure as described for 12e; yield 87%. H NMR (400
MHz, CDCl3) δ 7.57ꢀ7.56 (m, 4H), 7.44ꢀ7.38 (m, 5H), 7.28ꢀ7.26
(m,2H), 7.21ꢀ7.19 (m, 2H), 4.99ꢀ4.93 (m, 2H), 4.84ꢀ4.81 (m, 1H),
5744
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Journal of Medicinal Chemistry
ARTICLE
4.74ꢀ4.70 (m, 2H), 4.63ꢀ4.60 (m, 1H), 3.88ꢀ3.77 (m, 2H), 3.27ꢀ3.10
(m, 5H), 2.88 (s, 3H). ESI+-MS: m/z = 503 (M + H). Purity determina-
tion: (1) LC/MS tR = 14.5 min, purity: 99% ESI+-MS m/z = 503 (M +
H), 1027 (2M + Na); (2) HPLC tR = 16.5 min, 98%.
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(S)-2-Amino-4-((S)-4-(bis(4-fluorophenyl)methyl)-3-methyl-
piperazin-1-yl)-1-(isoindolin-2-yl)butane-1,4-dione Bis(2,2,2-tri-
fluoroacetate) (12m). This product was obtained as a white, amorphous
solid by acidolytic removal of the Boc-protecting group in 11m, using an
identical procedure as described for 12e; yield 91%. 1H NMR (400 MHz,
CDCl3) δ7.71ꢀ7.59 (m, 4H), 7.21ꢀ6.08 (m, 8H), 4.98 (s, 1H), 4.82ꢀ4.68
(m, 5H), 4.43 (m, 1H), 3.69 (m, 2H), 3.31 (m, 1H), 3.16ꢀ2.76 (br m, 5H),
1.30ꢀ1.15 (m, 3H). ESI+-MS: m/z = 519 (M + H). Purity determination:
(1) LC/MS tR = 14.1 min, purity 97%; (2) HPLC tR = 15.9 min, 98%.
(2S)-2-Amino-4-((3R)-4-((3-fluorophenyl)(4-fluorophenyl)-
methyl)-3-methylpiperazin-1-yl)-1-(isoindolin-2-yl)butane-
1,4-dione Bis(2,2,2-trifluoroacetate) (12n). This product was
obtained as a white, amorphous solid by acidolytic removal of the Boc-
protecting group in 11n, using an identical procedure as described for
1
12e; yield 84%. H NMR (400 MHz, CDCl3) δ 7.50ꢀ7.44 (m, 4H),
7.26ꢀ6.99 (m, 8H), 5.00 (s, 1H), 4.82ꢀ4.68 (m, 5H), 4.20 (m, 1H),
3.65 (m, 2H), 3.30 (m, 1H), 3.14 (m, 2H), 2.91ꢀ2.73 (m, 2H), 2.64 (m,
1H), 1.11ꢀ1.05 (m, 3H). ESI+-MS: m/z = 519 (M + H). Purity
determination: (1) LC/MS tR = 14.2 min, purity 96%; (2) HPLC tR =
15.9 min, 97%.
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’ ASSOCIATED CONTENT
S
Supporting Information. General synthetic procedures,
b
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He, H.; Hickey, G. J.; Kowalchick, J. E.; Leiting, B.; Lyons, K.; Marsilio, F.;
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Wu, J. K.; Wyvratt, M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N. A.; Weber,
A. E. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-
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orally active dipeptidyl peptidase IV inhibitor for the treatment of type
2 diabetes. J. Med. Chem. 2005, 48, 141–151.
(11) Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.;
Dunning, B. E.; Prasad, K.; Mangold, B. L.; Russell, M. E.; Hughes, T. E.
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Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.;
Huang, Q.; Han, S. P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo,
E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller,
S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and
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compound characterization data, and enzymatic assay condi-
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +32-(0)3 265 27 08. E-mail: pieter.vanderveken@ua.ac.be.
’ ACKNOWLEDGMENT
This work was supported by the Fund for Scientific Research
Flanders (Belgium, FWO-Vlaanderen), by BOF GOA (Research
Council of University of Antwerp), and by the Hercules Founda-
tion. Pieter Van der Veken and Veerle Matheeussen are indebted
to the Flemish Fund for Scientific research (FWO-Vlaanderen)
for financial support. We are grateful to Nicole Lamoen and Willy
Bollaert for their excellent technical assistance. The laboratory of
Medicinal Chemistry and the Laboratory of Medical Biochem-
istry, belonging to the department of Pharmaceutical Sciences,
are partners of the Antwerp Drug Discovery Network (ADDN,
www.addn.be).
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dipeptidyl peptidase-4 inhibitors: a brief overview. Tetrahedron 2010,
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’ ABBREVIATIONS USED
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DPP, dipeptidyl peptidase; DASH, DPPIV activity and/or struc-
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glucagon-like peptide-1; GIP, glucose dependent insulinotropic
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oligopeptidase; PREP, prolyl endopeptidase; QPP, quiescent cell
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