Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity

Article abstract of DOI:10.1016/j.bmcl.2012.03.107

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery.

Full text of DOI:10.1016/j.bmcl.2012.03.107

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3412–3417
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation
protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity
Oxana Ryabtsova a, Koen Jansen a, Sebastiaan Van Goethem a, Jurgen Joossens a, Jonathan D. Cheng b,
Anne-Marie Lambeir c, Ingrid De Meester c, Koen Augustyns a, Pieter Van der Veken a,
⇑
a Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium
b Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA
c Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating struc-
ture–activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation pro-
tein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification
of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the
micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline
selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with
respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant
overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the
importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these
two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic struc-
tures for future FAP-selective-inhibitor discovery.
Received 19 February 2012
Revised 27 March 2012
Accepted 29 March 2012
Available online 4 April 2012
Keywords:
Fibroblast activation protein-
FAP
Seprase
DPP IV, DPP4
PREP
a
Prolyl oligopeptidase
Val-boroPro
PT-100
Ó 2012 Elsevier Ltd. All rights reserved.
Talabostat
Fibroblast activation protein (FAP, FAP-
a, seprase) is a Clan SC
reports that identify for example, a2-antiplasmin, type I collagen
protease of the prolyl oligopeptidase subfamily, occurring as a cell
surface homodimer. FAP has been demonstrated to possess both
dipeptidyl peptidase and endopeptidase activity, catalyzed by the
same active center. Its expression is associated with activated stro-
mal fibroblasts and pericytes in over 90% of human epithelial tu-
mors examined and with normal or excessive wound healing, for
example, tissue remodeling sites or during chronic inflammation.
The enzyme is generally not expressed in normal adult tissues
and in nonmalignant tumors.1
During the last decade, numerous reports have been published
that claim an important role for FAP in tumor growth and prolifer-
ation and several other pathologic processes that involve degrada-
tion of the extracellular matrix.2 The exact mechanism by which
FAP takes part in these processes is unknown, but direct modula-
tion of tumor growth or disease progression by proteolytic pro-
cessing of growth factors, cytokines, collagenase activity
regulating proteins and even collagen derived proteins, is currently
the subject of intense research. Several studies have tried to map
the physiological substrate spectrum of FAP, including very recent
and gelatin as in vitro substrates of the endopeptidase activity of
FAP.3 Analogously, Neuropeptide Y, B-type natriuretic peptide,
substance P and peptide YY have been identified as in vitro sub-
strates of the dipeptidyl peptidase activity of FAP.4 Nonetheless,
the relevance of these findings under in vivo conditions remains
debatable and the unambiguous definition of FAP’s physiological
substrate spectrum remains a largely untouched matter so far.
While awaiting the detailed functional characterization of the
enzyme, several groups currently focus on FAP’s status as a poten-
tial cancer biomarker whose presence or activity in tumors could
also be used for site-directed delivery of oncology drugs.5 Equally
important, FAP or its activity are being targeted by several groups
as a direct way to reduce tumor growth and proliferation by means
of immunotherapeutic and small molecule inhibitor approaches.6,7
For the latter, a number of in vivo proof-of-concept studies have
been published. These all involve the dipeptide derived boronic
acid talabostat (PT-100, Val-boroPro) or close analogues, and report
significant activity on tumor stromagenesis and growth.8 In addi-
tion, talabostat has been evaluated as a therapeutic drug in various
clinical trials through phase II, for the treatment of, for example,
metastatic kidney cancer, chronic lymphocytary leukemia, pancre-
atic adenocarcinoma and non-small cell lung cancer (Fig. 1). While
⇑
Corresponding author.
E-mail address: pieter.vanderveken@ua.ac.be (P. Van der Veken).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.107

O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
3413
OH
O
B
N
N
N
N
N
OH
H
N
HO
H2N
N
O
O
O
1
2
3
Val-boroPro
KYP-2047
vildagliptin
CF3
PT-100
R2
O
F
N
N
N
N
N
N
H
N
R1
NH2
O
4
5
F
sitagliptin
target compounds
F
Figure 1. Reference compounds used in this study (1–4) and generic structure of products reported in this publication (5).
talabostat in several of these trials was able to induce clinical re-
sponses, questions were raised with regards to the safety profile
of the compound, potentially related to its well-known lack of
selectivity with respect to other Subfamily S9B proteases.9
With the number of reported FAP-inhibitors being small and
most of them belonging to the class of boronic acids, we focused
on compounds that contain a carbonitrile warhead in place of
the boronic acid, but retain an overall dipeptide derived architec-
ture (Fig. 1, generic structure 5). The latter is a hallmark of most
chemotypes of published Subfamily S9B inhibitors. The carboni-
trile function itself is also a popular affinity-enhancing moiety in
reported series of inhibitors of DPP IV, DPP8, DPP9 and PREP.10
Compared to other warheads that are used in serine protease
inhibitor design (e.g., –B(OH)2, –CHO, chloromethylketones, keto-
amides,. . .) the relatively mildly electrophilic carbonitrile could ac-
count for making the inhibitor more selective in vivo, a hypothesis
that has been raised in literature earlier.11
The common N-acyl glycyl-(2-cyano)pyrrolidine scaffold of our
compounds was inspired by earlier work from Edosada et al. in
which library screening of acetyl-P2-Pro-AMC fluorogenic peptides
was used to identify FAP as a protease with particular endopepti-
dase activity toward acetyl-Gly-Pro sequences.12 In addition, we
anticipated the absence of a basic amino terminus to render com-
pounds with far less affinity for S9B dipeptidyl peptidases, com-
pared to for example, ValboroPro and related inhibitors.13 As part
of this study, two types of modifications were investigated: (1) var-
iation of the N-acyl substituent (R1) and (2) modifications of the (2-
cyanopyrrolidine) moiety (R2) (Fig. 1, generic structure 5). At the
outset of our activities, only isolated cases of carbonitrile inhibitory
activities against FAP were reported, mostly in the framework of
selectivity assessment of DPP IV inhibitors. Recently, Tsai et al.
published a paper that also reports directed investigations aiming
at the identification of dipeptide derived carbonitriles as inhibitors
of FAP.14
Furthermore, as was anticipated by taking into account the ab-
sence of a basic P2-amine function in the target molecules, these
molecules in general do not display measurable affinity for any
of the dipeptidyl peptidases tested (vide infra, Tables 2 and 3).
Additionally, PREP assay data were considered relevant for this
study taking into account the related proline selective endopepti-
dase activity of the enzyme and the directly related risk of poten-
tially overlapping inhibitor pharmacophores.17 This is illustrated
i.a. by a publication by Tran et al. in which N-blocked Gly-boroPro’s
are presented as dual leads for FAP and PREP inhibitor
development.18
For a set of representative literature inhibitors of Clan SC en-
zymes, activities were determined for use as reference standards
in this study (Fig. 1, Table 1). This set consists of Val-boroPro, the
aforementioned, non-selective boronate that has been extensively
applied for i.a. in vitro and in vivo blocking of FAP activity.8,9 Inhib-
itor KYP-2047 (2) can be regarded as a selective PREP-inhibitor
with respect to the set of target enzymes tested, notwithstanding
the fact that a prolylpyrrolidine skeleton is present in several com-
pounds that were described to possess FAP- and DPP-affinity.11,19
The clinically used DPP IV inhibitors sitagliptin (3) and vildagliptin
(4) were also found to lack FAP affinity.
The first set of inhibitors synthesized in the framework of this
study, differ by variation in the N-acyl residue (R1 in generic struc-
ture 5, R2 = H) (Table 2, 26 and 27). All compounds in this series
were prepared by acylation of the amino terminus of Gly-(2-
cyano)pyrrolidine, either by reaction with commercially available
acyl chlorides (or sulfonyl chloride in 20), or by TBTU-mediated
coupling using the appropriate carboxylic acid. A considerable
number of compounds in Table 2 on the other hand, display dual
FAP and PREP affinity with often roughly comparable IC50-values.
Only compounds 21, 22, 26 and 27 possess inhibitory profiles in
which substantial FAP potency (IC50 <5 lM) is decoupled from
PREP binding potential. The common structural feature that poten-
tially accounts for this profile, is an (azaheterocyclyl)acetyl group
as the N-acyl scaffold substituent. Certainly, both the scope of this
claim and the possibility to improve FAP affinity by further explo-
ration of this structural characteristic, should be the subject of fur-
All inhibitors were assayed for potency toward FAP, PREP and
the dipeptidyl peptidases DPP IV, DPP II and DPP9.15 Additionally,
DPP9 potencies reported can reasonably be expected to be indica-
tive for inhibitor affinities toward the highly homologous DPP8.16
Table 1
Affinity data for reference compounds 1–4
Compd
IC50
(lM)
FAP
PREP
DPP II
DPP IV
DPP9
1
2
3
4
0.066 0.011
>100
52 18
>100
0.98 0.06
0.006 0.004
>100
0.086 0.007
>100
>1000
0.022 0.001
>100
0.12 0.001
0.04 0.001
n.d.a
>100
0.68 0.02
>100
>100.
>100
a
n.d. = not determined.

3414
O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
Table 2
Acylglycyl-(2-cyanopyrrolidine) based inhibitors: variation of the acyl moiety (R1 in generic structure 5, R2 = H)
Compd
R1
IC50 (lM)
FAP
PREP
DPPII
DPPIV
>100
DPP9
>100
6
7
3.5 0.1a
10.7 0.5
>100a
2.4 0.1
15.9 0.9
>100
>100
>100
8
14 0.4
9.4 0.4
6.8 0.2
3.9 0.2
9.4 1.1
1.6 0.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
10
11
2.6 0.2
0.60 0.03
12
13
1.9 0.1
4.7 0.2
2.9 0.1
5.0 0.2
>100
>100
>100
>100
>100
>100
14
15
3.7 0.2
>10
>100
>100
>100
>100
>100
>100
14.6 0.5
>100
16
17
8.1 0.2
1.4 0.1
1.7 0.2
5.8 0.6
>100
>100
13.1 0.7
>100
20.2 1.3
>100
18
19
20
7.5 0.6
0.67 0.04
5.1 0.5
5.4 0.4
3.3 0.2
>10
>100
>100
>100
>100
>100
>100
>10
>10
>100

O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
3415
Table 2 (continued)
Compd
R1
IC50 (lM)
FAP
PREP
>10
DPPII
DPPIV
>100
DPP9
>100
21
22
23
2.7 0.1
>100
12
20
1
1
>50
>50
>100
>100
>100
>100
>100
>>100
24
25
10.3 .5
>50
>50
>100
>100
>100
>100
>100
>100
19.9 1.3
26
1.3 0.1
2.7 0.1
>50
>100
>100
>100
>100
69
2
27
>100
>100
a
‘>’ means that residual enzymatic activity is higher than 50% at the indicated concentration.
ther investigation. Finally, the presence of a sulfonyl instead of an
acyl linkage (7 vs 20) does not seem to have significant implica-
tions for either FAP affinity of FAP/PREP selectivity.
sponding pyrrolidine carboxamide (or pyrrolidine in the case of
28). Dehydration of the carboxamide group using trifluoroacetic
acid/pyridine was used to install the carbonitrile group. The prep-
aration of the different pyrrolidine carboxamides used in this study
was achieved based on literature procedures.21 From the evalua-
tion results of inhibitors 28 and 29 (compared to 6), it is clear that
the carbonitrile group significantly contributes to FAP affinity. This
observation is indicative for the interaction of the enzyme’s cata-
lytically active serine-OH with the carbonitrile–carbon, potentially
involving the formation of an enzyme-bound imidate, as has been
demonstrated for example, DPP IV by X-ray crystallography.22
In addition, subsitution of the (2-cyano)pyrrolidine group at the
4-position, was explored. Earlier reports from our group have indi-
cated that introduction of substituents at this position can lead to
significant increase of enzyme affinity in P1-pyrrolidine containing
inhibitors of DPP IV and DPP8/9.23 In the case of FAP however,
available space at this position seems very limited. First, com-
pounds 30–32 that contain either a 4R or 4S-azido substituent
clearly display lower inhibitory activity compared to their non-
substituted congeners 6 and 19. The 4R-azido substituent in 32
even seems not to be accepted by the enzyme. Likewise, results
measured for 4-methyl-, methylene-, ethyl- and 4R-trifluoro-
methyl- substituted analogues consistently are indicative of the
same conclusion. Notably, this effect is least pronounced for the
4-methylene substituted analogue 37, which can be expected to
also impose substantial conformational constraints on the pyrroli-
dine ring system. Further, fluorinated inhibitors 33–35, are the
only compounds from Table 3 that outperform FAP-potency of
their non-substitued analogues with no significant difference ob-
served between the mono- and di-fluorinated compounds. With
regards to the FAP/PREP selectivity issue, available space in PREP’s
Regardless of the selectivity issue, extracting structure–activity
relationship (SAR)-data from Table 2 with specific regards to affinity
for FAP was another primary goal of this study. The P3-region of FAP,
in which the N-acyl susbtituents can be expected to be accommo-
dated, clearly does not impose strict requirements with respect to
steric bulk: even large substituents still allow binding of the inhibi-
tors, as demonstrated by compounds 12 and 14–27. More detailed
information on the available space in the P3 region can be derived
by comparing the rigid and bulky regio-isomeric inhibitor pairs 16
and 17 (the former containing a thiophene ring as an isosteric ben-
zene replacement) and 18 and 19. Both indicate FAP’s preference
of almost an order of magnitude for the compounds in which the dis-
tal part of the ring system is in a skewed position relative to the acyl
group. In addition, compound 19, containing a 1-naphthoyl substi-
tuent, was found to be the most potent inhibitor in this series. Our
selection of the 1-naphthoyl residue was based on a patent by Bac-
hovchin and Lai in which the activity of N-(1-naphthoyl)-substi-
tuted Gly-boroPro was claimed to possess superior FAP-affinity
relative to the N-benzoyl substituted congener, an observation we
found to also hold for the corresponding nitriles.20 In addition, com-
pound 19 was also reported in the aforementioned publication by
Tsai et al. with comparable FAP potency, but not including PREP
assay data.14
In a second compound series, the influence of modifications at
the pyrrolidine ring was investigated (R2 in generic structure 5,
R1 = benzoyl or 1-naphthoyl, results summarized in Table 3). All
compounds reported were prepared by TBTU-mediated coupling
of N-benzoylglycine or N-(1-naphthoyl)glycine with the corre-

3416
O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
Table 3
P1-modifications studied
Compd
P1
R1
IC50 (lM)
FAP
PREP
—
DPPII
DPPIV
>100
DPP9
>10b
28
29
Benzoyl–
Benzoyl–
>100a
>>100
>25
>100
>100
>100
>100
>100
>100
>10
>10
30
31
Benzoyl–
17.5 0.7
1-Naphthoyl–
4.1 0.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
>10
>10
>10
32
33
34
1-Naphthoyl–
1-Naphthoyl–
Benzoyl–
>100
>100
0.126 0.007
0.85 0.07
1.1 0.2
>10
35
1-Naphthoyl–
0.110 0.007
4.84 0.4
>100
>100
>10
36
37
38
Benzoyl–
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
>10
>10
1-Naphthoyl–
1-Naphthoyl–
6.7 0.4
42
3
39
1-Naphthoyl–
>100
>100
>100
>100
>10
40
41
Benzoyl–
>100
>50
>100
>100
23.3 0.4
>100
>100
>100
>10
>10
1-Naphthoyl–
a
‘>’ means that residual enzymatic activity is higher than 50% at the indicated concentration.
DPP9 assays: highest conc tested was 10 lM.
b

O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
3417
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S1 pocket seems even more limited than for FAP: only in the case of
the fluorinated compounds, introduction of a 4-substituent does
not completely delete enzyme affinity.
Taking into account its positive effect on FAP-inhibitory activity,
(di-)fluorination of the 4-position of the pyrrolidine ring could be
regarded upon as a viable strategy to improve FAP-selectivity of
promising inhibitors. Finally, as an additional illustration of the al-
leged limited dimensions of FAP and PREP’s S1 pockets, (2-
cyano)piperidines 40 and 41, were found to possess no significant
affinity toward the two enzymes.
7. (a) Cheng, J. D.; Valianou, M.; Canutescu, A. A.; Jaffe, E. K.; Lee, H. O.; Wang, H.;
Lai, J. H.; Bachovchin, W. W.; Weiner, L. M. Mol. Cancer Ther. 2005, 4, 351; (b)
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In conclusion, we have shown that the class of N-acylated Gly-
(2-cyano)pyrrolidines holds significant potential for identification
of promising FAP-inhibitors. In general, compounds of this type
possess a high degree of selectivity with respect to the phylogenet-
ically related dipeptidyl peptidases. Selectivity toward the endo-
peptidase PREP was found to be less evident.
A similar
observation has been published before by Tran et al. with a series
of acylated boronic acid inhibitors, but most other authors how-
ever have hitherto neglected to simultanously report PREP and
FAP-affinity for their inhibitors.18 Nonetheless, systematic deter-
mination of a FAP/PREP selectivity index might be advisable for
all compound classes intended as inhibitors of either of these
two enzymes. During this study, we have identified several struc-
tural features that can serve to increase both FAP-activity and
selectivity in the reported class of N-acylated Gly-(2-cyano)pyrrol-
idines. These include an (azaheterocyclyl)acetyl group as the N-
acyl scaffold substituent and mono- or di-fluorosubstitution at
the 4-position of the P1 pyrrolidine ring. Further effort to obtain
inhibitors of this type with maximal FAP affinity and selectivity
is currently underway.
15. For experimental conditions and enzyme sources used in the DPP IV, DPP9 and
DPP II assays, vide: Van Goethem, S.; Matheeussen, V.; Joossens, J.; Lambeir, A.
M.; Chen, X.; De Meester, I.; Haemers, A.; Augustyns, K.; Van der Veken, P. J.
Med. Chem. 2011, 54, 5737. Assay conditions and enzyme sources used for the
FAP and PREP-assays are reported in the Supplementary data of this
publication.
Acknowledgments
This work was financially supported by the Fund for Scientific
Research Flanders/FWO-Vlaanderen (A.M.L., I.D.M.) the BOF-fund
of the University of Antwerp (O.R., K.J., I.D.M., K.A., P.V.d.V.) and
the Hercules Foundation. We are indebted to Nicole Lamoen and
Willy Bollaert for excellent technical assistance.
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