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
`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 (>103) 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
`
`F ibroblast activation protein (FAP, FAP-α, seprase) is a type
`
`II transmembrane serine protease, belonging to the prolyl
`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
`dipeptidyl peptidases (DPPs): DPPIV, DPPII, and DPP8/9.1
`FAP expression is seen on activated stromal fibroblasts and
`pericytes of 90% of common human epithelial
`tumors
`examined.2,3 It has been established that FAP expression
`promotes tumorigenesis in mouse models and that FAP
`inhibition can attenuate tumor growth.4,5 FAP is also highly
`expressed in lesions characterized by activated stromal tissue
`such as present in cirrhosis, fibrotic diseases, osteoarthritis and
`rheumatoid arthritis, keloidosis, and in healing wounds.6−11
`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
`as in vitro substrates of the endopeptidase activity of FAP.12−14
`
`Nonetheless, the relevance of these findings under in vivo
`conditions has to be confirmed.
`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-
`boronic acid derivatives.15,16 These compounds in general also
`display significant affinity for one or several DPPs.15,16 The
`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-
`ment.17,18
`More recently, we and the group of Jiaang have focused on 2-
`cyanopyrrolidine derivatives for FAP inhibitor discovery.19,20
`The 2-cyanopyrrolidine fragment has been successfully ex-
`
`Received: November 20, 2012
`Accepted: March 18, 2013
`Published: March 18, 2013
`
`© 2013 American Chemical Society
`
`491
`
`dx.doi.org/10.1021/ml300410d | ACS Med. Chem. Lett. 2013, 4, 491−496
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`

`ACS Medicinal Chemistry Letters
`
`Table 1. IC50s for Reference Compounds 1 and 2
`
`Letter
`
`Nr
`
`1
`2
`aNot determined.
`
`FAP
`
`0.07 ± 0.01
`0.37 ± 0.002
`
`DPP IV
`
`0.022 ± 0.001
`0.0020 ± 0.0002
`
`IC50 (μM)
`
`DPP9
`NDa
`>100
`
`DPP II
`
`0.086 ± 0.007
`>100
`
`PREP
`
`0.98 ± 0.06
`>100
`
`Figure 1. Structure of reference inhibitors used in this study: Val-
`boroPro (1) and linagliptin (2).
`
`plored before for discovery of DPP inhibitors, exemplified, for
`example, by the marketed DPP IV inhibitors vildagliptin and
`saxagliptin.21−24 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
`(Tradjenta) 2 (Table 1).25 The latter compound is structurally
`distinct
`from 1 but has also been described to possess
`significant FAP affinity.
`the N-acyl-glycyl-(2-
`that
`Previous results pointed out
`cyanopyrrolidine) scaffold has significant potential to deliver
`FAP inhibitors with good selectivity toward PREP and the
`dipeptidyl peptidases.19 The most potent compound identified
`(N-(1-naphthoyl)-Gly-(2-cyanopyrrolidine), 3) displayed high
`nanomolar FAP-affinity.19,20 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.
`A general pathway for the synthesis of the target compounds
`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
`
`Scheme 1. Synthesis of Target Compounds 3-39a
`
`aReagents and conditions: (a) Boc-glycine, HATU, DIPEA, DMF-
`DCM, rt, 71%; (b) trifluoroacetic anhydride, pyridine, THF, 0 °C,
`92%; (c) (i) TFA, MeCN, 0 °C, 24h; (ii) HCl, diethylether, 84%; (d)
`HATU, DIPEA, R-COOH or 1-chloro-N,N,2-trimethylprop-1-en-1-
`amine, RCOOH, TEA or RCOCl, TEA, 20−76%.
`
`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
`inhibitors of FAP, DPP IV, DPP9, DPP II, and PREP using a
`chromogenic substrate assay.19 It is worthwhile to stipulate that
`DPP9 potencies reported can reasonably be expected to also be
`indicative for inhibitor affinities toward the highly homologous
`DPP8.26,27
`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
`in the N-
`azaheteroaromatic substituents, as,
`for example,
`(indolyl-3-acyl) containing 8 and its regio-isomeric congener 9,
`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
`vildagliptin, respectively.24 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
`inhibitor.28 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.15 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
`
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`

`ACS Medicinal Chemistry Letters
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`Letter
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`Table 2. Enzymatic Evaluation Data for Compounds 3−18
`
`Table 3. Effect of Substituents on the 4-Quinolinoyl Residue
`
`aFAP-affinity (Ki) was determined to be 3.0 ± 0.4 nM. bThe mean
`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
`
`aFAP-affinity (Ki) was determined to be 4.1 ± 0.6 nM.
`
`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
`
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`ACS Medicinal Chemistry Letters
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`Letter
`
`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.
`We subsequently studied the effect of varying the position of
`the sp2 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
`
`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.
`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
`
`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
`>103 with respect to both the DPPs and PREP. Our SAR
`investigations so far have mainly focused on the P3 part of the
`the N-(4-
`scaffold but already allow to conclude that
`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.
`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 >103, has a kinetic solubility of >200 μM
`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.
`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
`The authors declare no competing financial interest.
`
`■ ACKNOWLEDGMENTS
`We are indebted to Nicole Lamoen and Sophie Lyssens for
`excellent technical assistance.
`
`494
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`ACS Medicinal Chemistry Letters
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`Letter
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`■ ABBREVIATIONS
`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
`
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