Bioorganic & Medicinal Chemistry Letters 22 (2012) 3412–3417
`
`Contents lists available at SciVerse ScienceDirect
`
`Bioorganic & Medicinal Chemistry Letters
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c l
`
`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:
`Received 19 February 2012
`Revised 27 March 2012
`Accepted 29 March 2012
`Available online 4 April 2012
`
`Keywords:
`Fibroblast activation protein-a
`FAP
`Seprase
`DPP IV, DPP4
`PREP
`Prolyl oligopeptidase
`Val-boroPro
`PT-100
`Talabostat
`
`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.
`
`Ó 2012 Elsevier Ltd. All rights reserved.
`
`Fibroblast activation protein (FAP, FAP-a, seprase) is a Clan SC
`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
`
`⇑ 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
`
`reports that identify for example, a2-antiplasmin, type I collagen
`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
`
`Petitioner GE Healthcare – Ex. 1029, p. 3412
`
`

`

`O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
`
`3413
`
`N
`
`N
`
`O
`
`HN
`
`HO
`
`3
`vildagliptin
`
`O
`
`N
`
`N
`
`N
`
`O
`
`2
`KYP-2047
`
`
`
`H2N
`
`B
`
`OH
`
`OH
`
`N
`
`O
`
`1
`Val-boroPro
`PT-100
`
`F
`
`F
`
`F
`
`R2
`
`N
`
`N
`
`5
`O
`target compounds
`
`HN
`
`R1
`
`CF3
`
`N
`
`N
`
`N
`
`N
`
`NH2
`
`O
`
`4
`sitagliptin
`
`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
`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
`
`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-
`
`Table 1
`Affinity data for reference compounds 1–4
`
`Compd
`
`1
`2
`3
`4
`
`a n.d. = not determined.
`
`FAP
`
`0.066 ± 0.011
`>100
`52 ± 18
`>100
`
`PREP
`
`0.98 ± 0.06
`0.006 ± 0.004
`>100
`>100.
`
`IC50 (lM)
`
`DPP II
`
`0.086 ± 0.007
`>100
`>1000
`>100
`
`DPP IV
`
`0.022 ± 0.001
`>100
`0.12 ± 0.001
`0.04 ± 0.001
`
`DPP9
`
`n.d.a
`>100
`0.68 ± 0.02
`>100
`
`Petitioner GE Healthcare – Ex. 1029, p. 3413
`
`

`

`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)
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`FAP
`
`PREP
`
`DPPII
`
`DPPIV
`
`DPP9
`
`3.5 ± 0.1a
`
`10.7 ± 0.5
`
`>100a
`
`>100
`
`>100
`
`2.4 ± 0.1
`
`15.9 ± 0.9
`
`>100
`
`>100
`
`>100
`
`14 ± 0.4
`
`9.4 ± 1.1
`
`>100
`
`>100
`
`>100
`
`9.4 ± 0.4
`
`1.6 ± 0.1
`
`>100
`
`>100
`
`>100
`
`6.8 ± 0.2
`
`2.6 ± 0.2
`
`>100
`
`>100
`
`>100
`
`3.9 ± 0.2
`
`0.60 ± 0.03
`
`>100
`
`>100
`
`>100
`
`>100
`
`>100
`
`>100
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`20
`
`1.9 ± 0.1
`
`2.9 ± 0.1
`
`4.7 ± 0.2
`
`5.0 ± 0.2
`
`>100
`
`>100
`
`>100
`
`3.7 ± 0.2
`
`>10
`
`>100
`
`>100
`
`>100
`
`14.6 ± 0.5
`
`>100
`
`>100
`
`>100
`
`>100
`
`8.1 ± 0.2
`
`1.7 ± 0.2
`
`>100
`
`13.1 ± 0.7
`
`20.2 ± 1.3
`
`1.4 ± 0.1
`
`5.8 ± 0.6
`
`>100
`
`>100
`
`>100
`
`7.5 ± 0.6
`
`5.4 ± 0.4
`
`>100
`
`>100
`
`>10
`
`0.67 ± 0.04
`
`3.3 ± 0.2
`
`>100
`
`>100
`
`>10
`
`5.1 ± 0.5
`
`>10
`
`>100
`
`>100
`
`>100
`
`Petitioner GE Healthcare – Ex. 1029, p. 3414
`
`

`

`Table 2 (continued)
`
`Compd
`
`R1
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
`
`3415
`
`FAP
`
`PREP
`
`DPPII
`
`DPPIV
`
`DPP9
`
`IC50 (lM)
`
`2.7 ± 0.1
`
`>10
`
`>100
`
`>100
`
`>100
`
`12 ± 1
`
`>50
`
`>100
`
`>100
`
`>100
`
`20 ± 1
`
`>50
`
`>100
`
`>100
`
`>>100
`
`10.3 ± .5
`
`>50
`
`>100
`
`>100
`
`>100
`
`19.9 ± 1.3
`
`>50
`
`>100
`
`>100
`
`>100
`
`1.3 ± 0.1
`
`>50
`
`>100
`
`>100
`
`69 ± 2
`
`2.7 ± 0.1
`
`>100
`
`>100
`
`>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.
`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-
`
`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
`
`Petitioner GE Healthcare – Ex. 1029, p. 3415
`
`

`

`3416
`
`O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
`
`Table 3
`P1-modifications studied
`
`Compd
`
`P1
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`R1
`
`Benzoyl–
`
`FAP
`
`>100a
`
`IC50 (lM)
`
`PREP
`
`DPPII
`
`DPPIV
`
`DPP9
`
`—
`
`>>100
`
`>100
`
`>10b
`
`Benzoyl–
`
`>25
`
`>100
`
`>100
`
`>100
`
`>10
`
`Benzoyl–
`
`17.5 ± 0.7
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`4.1 ± 0.4
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`>100
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`0.126 ± 0.007
`
`1.1 ± 0.2
`
`>100
`
`>100
`
`>10
`
`>100
`
`>10
`
`34
`
`35
`
`36
`
`37
`
`38
`
`39
`
`40
`
`41
`
`Benzoyl–
`
`0.85 ± 0.07
`
`>10
`
`>100
`
`1-Naphthoyl–
`
`0.110 ± 0.007
`
`4.84 ± 0.4
`
`>100
`
`>100
`
`>10
`
`Benzoyl–
`
`>100
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`6.7 ± 0.4
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`42 ± 3
`
`>100
`
`>100
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`>100
`
`>100
`
`>100
`
`>100
`
`>10
`
`Benzoyl–
`
`>100
`
`>100
`
`23.3 ± 0.4
`
`>100
`
`>10
`
`1-Naphthoyl–
`
`>50
`
`>100
`
`>100
`
`>100
`
`>10
`
`a ‘>’ means that residual enzymatic activity is higher than 50% at the indicated concentration.
`b DPP9 assays: highest conc tested was 10 lM.
`
`Petitioner GE Healthcare – Ex. 1029, p. 3416
`
`

`

`O. Ryabtsova et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3412–3417
`
`3417
`
`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.
`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.
`
`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.
`
`Supplementary data
`
`Supplementary data (detailed experimental conditions and en-
`zyme sources used in the FAP and PREP assays) associated with this
`article can be found, in the online version, at http://dx.doi.org/
`10.1016/j.bmcl.2012.03.107.
`
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`Petitioner GE Healthcare – Ex. 1029, p. 3417
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