Selective inhibitors of fibroblast activation protein (FAP) with a (4-quinolinoyl)-glycyl-2-cyanopyrrolidine scaffold

Article abstract of DOI:10.1021/ml300410d

Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp² hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10 ³) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.

Full text of DOI:10.1021/ml300410d

Letter
pubs.acs.org/acsmedchemlett
Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-
Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold
†
†
‡
†
†
§
Koen Jansen, Leen Heirbaut, Jonathan D. Cheng, Jurgen Joossens, Oxana Ryabtsova, Paul Cos,
§
∥
∥
†
,†
Louis Maes, Anne-Marie Lambeir, Ingrid De Meester, Koen Augustyns, and Pieter Van der Veken*
†Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-2610
Antwerp, Belgium
‡
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111-2497, United States
§
Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Departments of Pharmaceutical and Biomedical Sciences,
University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium
∥
Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp,
Belgium
*
S Supporting Information
ABSTRACT: Fibroblast activation protein (FAP) is a serine
protease that is generally accepted to play an important role in
tumor growth and other diseases involving tissue remodeling.
Currently there are no FAP inhibitors with reported selectivity
toward both the closely related dipeptidyl peptidases (DPPs) and
prolyl oligopeptidase (PREP). We present the discovery of a new
class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrro-
lidine) scaffold. We have explored the effects of substituting the
2
quinoline ring and varying the position of its sp hybridized nitrogen atom. The most promising inhibitors combined low
3
nanomolar FAP inhibition and high selectivity indices (>10 ) with respect to both the DPPs and PREP. Preliminary experiments
on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be
expected to be satisfactory.
KEYWORDS: Fibroblast activation protein (FAP), dipeptidyl peptidase IV (DPPIV), prolyl oligopeptidase (PREP), seprase
ibroblast activation protein (FAP, FAP-α, seprase) is a type
II transmembrane serine protease, belonging to the prolyl
Nonetheless, the relevance of these findings under in vivo
conditions has to be confirmed.
F
Currently no inhibitors with low nanomolar FAP affinity and
selectivity toward both PREP and the DPPs have been
reported. Most research effort in the domain of FAP-inhibitor
discovery to date has been centered around pyrrolidine-2-
oligopeptidase family. This family comprises serine proteases
that cleave peptides preferentially after proline residues. Other
important members of this family that are expressed in the
human proteome are prolyl oligopeptidase (PREP) and the
1
5,16
1
boronic acid derivatives.
These compounds in general also
dipeptidyl peptidases (DPPs): DPPIV, DPPII, and DPP8/9.
1
5,16
display significant affinity for one or several DPPs.
The
FAP expression is seen on activated stromal fibroblasts and
most representative of this class, Val-boroPro (Talabostat, PT-
100) 1 has reached phase II clinical trials (Table 1). It was
evaluated as a therapeutic drug for, among others, metastatic
kidney cancer, pancreatic adenocarcinoma, nonsmall cell lung
cancer, and chronic lymphocytary leukemia (Figure 1). While
talabostat in several of these trials was able to induce clinical
response, safety concerns potentially related to the drug’s lack
of selectivity led to its withdrawal from further develop-
pericytes of 90% of common human epithelial tumors
2
,3
examined. It has been established that FAP expression
promotes tumorigenesis in mouse models and that FAP
4
,5
inhibition can attenuate tumor growth. FAP is also highly
expressed in lesions characterized by activated stromal tissue
such as present in cirrhosis, fibrotic diseases, osteoarthritis and
6
−11
rheumatoid arthritis, keloidosis, and in healing wounds.
1
7,18
FAP has been demonstrated to possess both dipeptidyl
peptidase and endopeptidase activity, catalyzed by the same
active site. Several studies have identified in vitro substrates for
FAP. Peptides found to be substrates of FAP’s dipeptidyl
peptidase activity include Neuropeptide Y, B-type natriuretic
peptide, substance P, and peptide YY. Analogously, α2-
antiplasmin, type I collagen, and gelatin were found to behave
ment.
More recently, we and the group of Jiaang have focused on 2-
19,20
cyanopyrrolidine derivatives for FAP inhibitor discovery.
The 2-cyanopyrrolidine fragment has been successfully ex-
Received: November 20, 2012
Accepted: March 18, 2013
12−14
as in vitro substrates of the endopeptidase activity of FAP.
©
XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Table 1. IC s for Reference Compounds 1 and 2
50
IC50 (μM)
Nr
FAP
DPP IV
DPP9
DPP II
PREP
a
1
2
0.07 ± 0.01
0.37 ± 0.002
0.022 ± 0.001
0.0020 ± 0.0002
ND
0.086 ± 0.007
>100
0.98 ± 0.06
>100
>100
a
Not determined.
trifluoroacetic anhydride to yield the corresponding nitrile 5.
The nitrile was deprotected in acetonitrile to give hydrochloric
acid salt 6, which was then coupled with a carboxylic acid or
acyl chloride to give final compounds.
All compounds synthesized in this study were evaluated as
Figure 1. Structure of reference inhibitors used in this study: Val-
boroPro (1) and linagliptin (2).
inhibitors of FAP, DPP IV, DPP9, DPP II, and PREP using a
19
chromogenic substrate assay. It is worthwhile to stipulate that
DPP9 potencies reported can reasonably be expected to also be
indicative for inhibitor affinities toward the highly homologous
plored before for discovery of DPP inhibitors, exemplified, for
2
6,27
DPP8.
example, by the marketed DPP IV inhibitors vildagliptin and
2
1−24
The results summarized in Table 2 show that the N-(4-
quinolinoyl) substituted compound 7 has about 60 times more
FAP-affinity than the initial N-(1-naphthoyl) based ‘hit’ 3. Its
potency as an inhibitor of FAP also clearly stands out among
the other compounds in Table 2. The presence of other
azaheteroaromatic substituents, as, for example, in the N-
saxagliptin.
The work presented here deals with a group of
2
-cyanopyrrolidines that represent the first examples of
inhibitors combining low nanomolar FAP affinity and
significant selectivity with respect to both PREP and the
DPPs. Reference inhibitors used in this study are 1 and the
FDA-approved, clinically used DPP IV inhibitor linagliptin
25
(
indolyl-3-acyl) containing 8 and its regio-isomeric congener 9,
(
Tradjenta) 2 (Table 1). The latter compound is structurally
leads to a drastic drop in FAP-affinity. The same affinity trend
was observed for compounds 10−12: in these molecules, the
electron density in the naphthyl ring can reasonably be
expected to be higher than in compound 3. Directly inspired by
the docking study, these molecules were designed to gain
affinity from a stronger cation−π interaction with FAP’s
Arg123. Further deviation from ‘hit’ structure 3 was
investigated with compounds 13 and 14 in which the acylated
glycine amine function was changed for an oxygen atom. This,
however, was also found to be detrimental for FAP potency.
Likewise and in line with results obtained earlier, introduction
of a sulfonamide function replacing the P3 acyl group in 15
decreases potency very significantly. The ((5-cyanopyridin-2-
yl)amino)acetamide 16 and 3-hydroxyadamantane-1-carboxa-
mide 17 are nonbasic analogues of the N-substituted glycyl-(2-
cyanopyrroldine) DPPIV inhibitors NVP-DPP 728 and
distinct from 1 but has also been described to possess
significant FAP affinity.
Previous results pointed out that the N-acyl-glycyl-(2-
cyanopyrrolidine) scaffold has significant potential to deliver
FAP inhibitors with good selectivity toward PREP and the
19
dipeptidyl peptidases. The most potent compound identified
(
N-(1-naphthoyl)-Gly-(2-cyanopyrrolidine), 3) displayed high
19,20
nanomolar FAP-affinity.
For further optimization of this
compound’s potency, we proposed further exploration of the
P3 area, containing the naphtyl group. A docking study
furthermore indicated that the 1-naphthoyl residue could be
involved in a cation−π interaction with the guanidine side chain
of FAP’s Arg123. A set of analogues potentially capable of
corroborating this hypothesis and providing additional SAR
information in the parts of chemical space surrounding the 1-
naphthoyl group were prepared.
24
A general pathway for the synthesis of the target compounds
vildagliptin, respectively. By introducing an amide group
instead of the parent structures’ basic P2-amine function that is
critical for DPP IV interaction, we hoped to decrease DPP IV
potency, while retaining affinity for the phylogenetically very
closely related FAP. Only moderate inhibition of the latter was
observed however. Finally, an analogue of compound 18 with a
P1-pyrrolidine-2-boronate residue instead of a 2-cyanopyrroli-
dine group has been described as a low nanomolar FAP
3
−39 is displayed in Scheme 1. Commercially available
prolinamide was coupled to N-Boc-glycine giving N-Boc-
glycine-prolinamide 4 and subsequently dehydrated with
a
Scheme 1. Synthesis of Target Compounds 3-39
28
inhibitor. Nitrile 18, however, loses almost all FAP inhibition
when compared to its boronate analogue.
In terms of selectivity, all inhibitors in Table 2 display very
limited to no affinity for the DPPs. As mentioned earlier, this
can be rationalized by the absence of a basic P2 amine function
known to be engaged in salt bridging with two acidic glutamate
residues in the active center of the DPPs. The corresponding
Glu203 and Glu204 in FAP have a different orientation to
accommodate endopeptidase substrates, and thus also allow
acylated P2-amines to enter FAP’s active center. As reported
earlier, selectivity of N-acylated inhibitors with respect to the
endopeptidase PREP is far less evident. Nonetheless, a
comparison of initial ‘hit’ 3 (SI[FAP/PREP] = 5.3) and our new
a
Reagents and conditions: (a) Boc-glycine, HATU, DIPEA, DMF-
1
5
DCM, rt, 71%; (b) trifluoroacetic anhydride, pyridine, THF, 0 °C,
9
HATU, DIPEA, R-COOH or 1-chloro-N,N,2-trimethylprop-1-en-1-
amine, RCOOH, TEA or RCOCl, TEA, 20−76%.
2%; (c) (i) TFA, MeCN, 0 °C, 24h; (ii) HCl, diethylether, 84%; (d)
B
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ACS Medicinal Chemistry Letters
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Table 2. Enzymatic Evaluation Data for Compounds 3−18
Table 3. Effect of Substituents on the 4-Quinolinoyl Residue
a
b
FAP-affinity (K ) was determined to be 3.0 ± 0.4 nM. The mean
i
result of three separate measurements.
‘
lead’ 7 (SI[FAP/PREP] = 85) indicates that selectively optimizing
for FAP affinity is possible.
Next, we turned our attention to the effect of substituting the
N-(4-quinolinoyl) ring in 7 (Table 3). Overall, none of the
evaluated substituents was able to improve FAP potency
significantly, with substituents in the 2- and 3-position of the
quinoline ring as in compounds 19−24, having a clearly
negative effect on this parameter. Identifying the factors that
cause this effect is not evident from these six analogues, but
both steric (19, 22, and 23) and electronic (20, 21, and 24,
with an increased electron density in the pyridine ring
compared to 7) factors seem to be contributive. Remarkably,
PREP-affinity is affected in a more intricate manner by these
substitution types: the negative effect of 3-hydroxylation in 20
and 24 significantly contrasts with the influence of a 2-hydroxyl
functionality in 21. Introducing a chlorine or bromine
a
FAP-affinity (K ) was determined to be 4.1 ± 0.6 nM.
i
substituent at the 5-position of the quinoline ring such as in
analogues 25 and 26 has limited effect on FAP potency. Most
remarkably, the selectivity with respect to PREP of these
inhibitors sharply increases (SI[FAP/PREP] > 1000), rendering
C
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ACS Medicinal Chemistry Letters
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them the most favorable affinity/selectivity profile of all FAP-
inhibitors we had identified so far. Functionalizing the
quinoline ring’s 6-position as in compounds 27−30 does not
generally lead to marked differences in the affinity and
selectivity profiles of the corresponding inhibitors compared
to ‘lead’ 7. The most notable compound in this subseries of
molecules is the 6-methoxyquinoline 30, which displays 10-fold
less PREP affinity than its congeners. The 7-substituted
chloroquinoline 31 and 7-bromoquinoline 32 again do not
display relevantly changed FAP affinity when compared to 7,
but they do have moderately increased selectivity toward PREP.
Finally, 8-chloroquinoline 33 and 8-bromoquinoline 34 were
found to possess significantly reduced FAP potency, possibly
due to steric effects. These modifications, however, have little
effect on the PREP inhibitory potential of the corresponding
molecules.
derivatives (35 and 39, respectively) are characterized by very
low FAP-affinity, even when compared to ‘hit’ 3.
In conclusion, we have identified the N-(4-quinolinoyl)-
glycyl-(2-cyanopyrrolidine) scaffold as highly promising for
discovery of FAP-inhibitors. It is the first scaffold type
demonstrated to have potential for rendering compounds that
combine low nanomolar FAP-affinity and a selectivity index
3
>10 with respect to both the DPPs and PREP. Our SAR
investigations so far have mainly focused on the P3 part of the
scaffold but already allow to conclude that the N-(4-
quinolinoyl) residue is a critical element in determining FAP-
affinity. As such, it was found to hold a privileged position
within the series of different quinolinoyl and isoquinolinoyl
isomers evaluated. Furthermore, we have shown that
introduction of substituents at the 5-position of the quinolinoyl
ring system is a possible means of maximizing inhibitor
selectivity with respect to PREP. So far, however, we have not
been able to identify the eventual specific interactions between
FAP and the 4-quinolinoyl ring that could underlie the
observed affinities.
We subsequently studied the effect of varying the position of
2
the sp hybridized nitrogen atom of the quinoline ring (Table
4). We considered these investigations highly instrumental for
Table 4. Effect of Nitrogen Position in the (Iso-)Quinolinoyl
Ring
To this end, we are currently expanding our SAR-data set by
investigating additional modification types in the P3, P2, and
P1 region of these molecules. In addition, preclinical ADME
and in vivo pharmacokinetic parameters of the optimal
molecules will be determined. Inhibitor 7 has a kinetic
solubility of >200 μM and a log D of 0.51 at pH 7.4.
Furthermore, 7 was stable for >24 h both in PBS buffer at pH
7
.4 and in rat plasma. Furthermore, 26, which together with
inhibitor 25 shares low-nanomolar FAP-affinity and a FAP/
PREP selectivity index >10 , has a kinetic solubility of >200 μM
3
and a log D of 0.7 at pH 7.4. This molecule was stable for 24 h
in PBS and human plasma and 80% stable after 24 h in mouse
plasma. It displayed mouse microsomal stability of 70% over 24
h.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Analytical data and experimental procedures for synthetic
preparation, enzymatic evaluation, and physicochemical char-
acterization of compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*
E-mail: pieter.vanderveken@ua.ac.be. Tel: +32-3 265 27 08.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
understanding the striking difference in FAP affinity between
‘
hit’ 3 and ‘lead’ 7. If inhibitor affinity would depend
significantly on the ring nitrogen’s position, this could be
indicative for a specific interaction with the enzyme, other than
the cation−π complexation we originally hypothesized. On the
basis of our docking study, the favorable contribution to
binding of the latter would be less dependent on the nature of
the (iso-)quinolinoyl isomer selected.
Funding
This work was financially supported by the Fund for Scientific
Research Flanders/FWO-Vlaanderen (to I.D.M. and A-M.L.),
the research (BOF)-fund of the University of Antwerp (to K.J.,
I.D.M., K.A., and P.V.d.V.), and the Hercules foundation.
Notes
With FAP-affinities spanning almost 3 orders of magnitude,
evaluation results of compounds 35−39 nonetheless reveal a
pivotal importance of the nitrogen’s position. Of all the
positional isomers synthesized, the 4-quinolinoyl ring of ‘lead’ 7
clearly displays the best results and takes in a singular position
within this series. The 4-isoquinolinoyl and 8-quinolinoyl
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are indebted to Nicole Lamoen and Sophie Lyssens for
excellent technical assistance.
D
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(
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ABBREVIATIONS
■
the edge of vamidation-Cancer protease fibroblast activation protein.
FAP, fibroblast activation protein; PREP, prolyl oligopeptidase;
DPPII, dipeptidyl peptidase II; DPPIV, dipeptidyl peptidase IV;
DPP9, dipeptidyl peptidase 9; HATU, 2-(7-aza-1H-benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
DIPEA, N,N-diisopropylethylamine; SI, selectivity index
Mini-Rev. Med. Chem. 2008, 8, 719−727.
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Rozenkrants, N.; Minimo, L. C.; Ripka, W. C.; Szardenings, A. K.;
Kozarich, J. W.; Shreder, K. R. Synthesis and structure−activity
relationship of N-alkyl-Gly-boroPro inhibitors. Bioorg. Med. Chem. Lett.
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