Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein

Article abstract of DOI:10.1021/jm1002556

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC₅₀ of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC ₅₀ > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Full text of DOI:10.1021/jm1002556

6572 J. Med. Chem. 2010, 53, 6572–6583
DOI: 10.1021/jm1002556
Substituted 4-Carboxymethylpyroglutamic Acid Diamides as Potent and Selective Inhibitors
of Fibroblast Activation Protein
Ting-Yueh Tsai,† Teng-Kuang Yeh,† Xin Chen,† Tsu Hsu, Yu-Chen Jao, Chih-Hsiang Huang, Jen-Shin Song, Yu-Chen Huang,
Chia-Hui Chien, Jing-Huai Chiu, Shih-Chieh Yen, Hung-Kuan Tang, Yu-Sheng Chao, and Weir-Torn Jiaang*
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli
Country 350, Taiwan, R.O.C. †These authors contributed equally to this work.
Received February 26, 2010
Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected
to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage
products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II,
DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic
target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To
achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of
substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a
number of FAP inhibitors having IC50 of <100 nM with excellent selectivity over DPP-IV, DPP-II,
DPP8, and DPP9 (IC50 > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selec-
tive FAP inhibitors, which prompts us to further study the physiological role of FAP.
Introduction
in patients with type 2 diabetes.23,24 Therefore, inhibition of
DPP-IV prolongs the action of GLP-1, which offers a new
strategy for treating type 2 diabetes. Antidiabetic efficacy has
been demonstrated clinically with DPP-IV inhibitors; sita-
gliptin 1 (MK-0431) was approved by the U.S. Food and
Drug Administration for the treatment of type 2 diabetes,10
and vildagliptin 2 (LAF237) was approved for use in the
European market (Figure 1).9
Dipeptidyl peptidase II (DPP-II, EC 3.4.14.2) is believed to
be involved in the physiological breakdown of some proline-
containing neuropeptides and in the degradation of collagen
and substance P.25,26 Using 1-[(S)-2,4-diaminobutanoyl]pipe-
ridine as lead compound, Senten et al. developed a series of
potent and selective DPP-II inhibitors.12,13 The representative
DPP-II inhibitor 3 (Figure 1) showed IC50 = 0.23 nM and a
high selectivity toward DPP-IV (IC50 = 345 μM, Table 1).13
Our studies found thatcompound3 was inactive toward FAP,
DPP8, and DPP9 (Table 1). These selective DPP-II inhibi-
tors are outstanding tools to determine the physiological
function of DPP-II and the therapeutic potential of DPP-II
inhibitors.
Dipeptidyl peptidase 8/9 (DPP8/9) are cytoplasmic pro-
teases with a 51% homology in amino acid level with DPP-
IV.27 Previously, the administration of selective DPP8/9 inhi-
bitor 4 (Figure 1) may be associated with profound toxicities
in preclinical species, which included alopecia, thrombocyto-
penia, anemia, enlarged spleen, multiple histological patho-
logies, and animal mortality shown in rats.28 Highly specific
and potent DPP8/9 inhibitors were also developed by Jiaang
et al. The representative DPP8/9 inhibitor 5 (also called 1G-
244, Figure 1) had IC50 values of 14 and 53 nM against DPP8
and DPP9, respectively (Table 1).14 It did not inhibit DPP-IV,
FAP, or DPP-II, with IC50 values greater than 100 μM. Re-
cently, by using this potent and selective DPP8/9 inhibitor,
Fibroblast activation protein (FAPa) is a type II transmem-
brane serine protease belonging to the prolyl peptidase family,
which comprises serine proteases that cleave bioactive pep-
tides preferentially after proline residues. Members of this
family include dipeptidyl peptidase-IV (DPP-IV), DPP-II
(DPP7), DPP8, and DPP9, and this family has been impli-
cated in several diseases, including diabetes, cancer, and mood
disorder.1,2 FAPisexpressed on reactivestromalfibroblastsin
over 90% of common human epithelial cancers,3 in granula-
tion tissue of healing wounds, and in bone and soft tissue
sarcomas.4,5 It has been suggested thatFAP promotestumori-
genesis and that FAP inhibition may attenuate tumor
growth.6-8 The development of potent and specific inhibitors
for each of these DPP enzymes can be an important tool to
study the physiological functionand to validate their potential
as a therapeutic target. Specific inhibitors for DPP-IV,9-11
DPP-II,12,13 and DPP8/914 have been identified, and investi-
gations of selective inhibitors for FAP have only started
recently.15-17
Dipeptidyl peptidase IV (DPP-IV, also known as CD26)
(EC 3.4.14.5) is a drug target for type II diabetes. The active
form of glucagon-like peptide-1 (GLP-1) stimulates insulin
secretion, inhibits glucagons release,18,19 and slows gastric empty-
ing,20-22 each a benefit in the control of glucose homeostasis
*To whom correspondence should be addressed. Phone: 886-37-
246166, extension 35712. Fax: 886-37-586456. E-mail: wtjiaang@nhri.
org.tw.
a Abbreviations: FAP, fibroblast activation protein; DPP, dipeptidyl
peptidase; GLP-1, glucagon-like peptide-1; LiHMDS, lithium hexam-
ethyldisilazide; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EDC, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide; TFA, trifluoroacetic acid; LC-MS,
liquid chromatography coupled mass spectrometry; SAR, structure-
-activity relationship; HOBt, N-hydroxybenzotrizole.
r
pubs.acs.org/jmc
Published on Web 08/18/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6573
Wu et al. show that the inhibition is not associated with any
animal toxicity.29
Previously, N- and RC-substituted Gly-boro-Pro deriva-
tives have been developed, and these compounds strongly
inhibited DPP-IV, DPP7 (DPP-II), and FAP. In this class, a
representative compound is Val-boroPro 6 (Figure 1), having
IC50 of <40 nM against all the three DPPs (Table 1).15 On the
basis of FAP’s preference for Gly-Pro-based endopeptidase
substrates, Tran et al. developed a series of N-acyl-Gly-,
N-acyl-Sar- (sarcosine), and N-blocked-boroPro derivatives.
Representative compound in this class was inhibitor 7
(Figure 1), which preferentially inhibited FAP versus DPP-
IV, but the selectivity against DPP-II, DPP8, and DPP9 was
not reported (Table 1).16 In our studies, this compound had
weak inhibitory activity against DPP8 and DPP9 (11 and
6.5 μM, respectively) and was inactive toward DPP-II (Table 1).
In this paper, we report a systematic search for potent and
selective FAP inhibitors. A structure-activity relationship
was investigated starting from L-homoglutamic acid 8(Figure2),
a dual DPP-V and FAP inhibitor (IC50 = 10 and 54 nM, re-
spectively), as a lead compound (Table 1).30 To develop selec-
tive FAP inhibitors, introduction of ring constraint in the P2
portion of lead 8 and modification of the P2 site secondary
aminetoamidewere done asdepictedinFigure 2. Thereplace-
ment of secondary amine with amide is based on the fact that
FAP acts as both prolin-specific endopeptidase and dipeptidyl
peptidase, but DPP-IV prefers to display the latter activ-
ity.8,16,31 Further exploration of the 2-position pyrrolidine
derivatives (P1 site) and the 4-position amine substituents at
the carbonylmethyl group (P2 site) led to the discovery of
potent pyroglutamic acid-based FAP inhibitors with a high
selectivity for FAP over DPP-IV, DPP-II, DPP8, and DPP9.
Chemistry
Reference compounds N-acyl-Gly-boroPro 9 and N-acyl-
Gly-cyanoPro 10 were prepared according to the literature
Figure 1. DPP-IV inhibitors 1 and 2, DPP-II inhibitor 3, DPP8/9
inhibitors 4 and 5, nonselective inhibitor 6, and FAP inhibitor 7.
Table 1. Potency and Selectivity of Compounds 1-8
IC50 (μM)a
compd
name
FAP
DPP8
DPP9
DPP-II
DPP-IV
referenceb
1
2
3
sitagliptin
vildagliptin
DPP-II selective
>100
>100
ND
>100
14
>100
1.2
ND
>100
>50
0.023
0.056
345
this study
this study
13
ND
>100
0.15
0.014
ND
ND
11
0.00023
0.005
>100
>100
0.038
ND
>100
>100
>100
0.011
0.008
0.12
>100
0.24
0.053
ND
>100
>100
>100
0.0003
23
this study
this study
this study
15
4
5
6
7
DPP8/9 selective
1G-244
nonselective
FAP selective
ND
6.5
16
this study
this study
>100
2.6
8.0
0.010
8
1G-409
0.054
0.36
0.089
a Mean values of at least three experiments; standard deviations are (20%. ND, no data. b “This study” means in-house data.
Figure 2. Design of 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors.

6574 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Tsai et al.
Scheme 1. Synthesis of C(4)-Substituted Pyroglutamate Ester Urethanesa
a Reagents: (a) DIEPA, CH2Cl2; (b) (Boc)2O, 4-DMAP, CH2Cl2; (c) LHMDS, THF, -78 °C; (d) DBU, CH2Cl2, 0 °C to room temp.
Scheme 2. Synthesis of FAP Inhibitors 17-22, 24, 27, and 30a
a Reagents: (a) H2, Pd/C, MeOH; (b) EDC,HOBt, CH2Cl2/1,4-dioxane, various amines; (c) POCl3, imidazole, pyridine; (d) TFA, CH3CN, 0 °C to
room temp; (e) BCl3.
procedures with some modifications.9,16 (2S)-Cyanopyrroli-
dineanalogues 17-19, 24, 27, 30, and 31 were prepared as des-
cribed in Schemes 1 and 2 and are listed in Tables 2 and 3. The
synthesis (Scheme 1) proceeded in good yield following the
procedureof Young and co-workers to afford the fully protec-
ted pyroglutamate 13.32 The stereoselective alkylation using
lithium hexamethyldisilazide (LiHMDS) and tert-butyl bro-
moacetate 14 gave compound 15 in 70% yield with a cis/trans
ratio of 3:1. The cis isomer 15 was treated with 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) in methylene chloride for 24 h
to give inverted product in a 1:3 ratio and high yield. The
assignment of the cis/trans configuration was based on the results
reported in the literature.33,34 By use of the building block of C(4)-
substituted pyroglutamate ester urethanes cis- and trans-15,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6575
Table 2. Inhibition of FAP, DPP-IV, DPP8, DPP9, and DPP-II by Compounds 9, 10, 17, and 31
a Mean values of at least three experiments; standard deviations are (20%.

6576 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Tsai et al.
Table 3. Inhibition of FAP, DPP-IV, DPP8, DPP9, and DPP-II by Compounds 17-20, 24, 27, 30, and 32
a Mean values of at least three experiments; standard deviations are (20%.
pyroglutamic acid-based FAP inhibitors were synthesized. As
shown in Scheme 2, the fully protected pyroglutamate trans-
15 was deprotected by standard hydrogenation condition and
was 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-
coupledwithpyrrolidinederivatives togivetert-butylesters23
and 2517 or followed by dehydration of the amides to give
16.9,35 Removal of the tert-butyl protecting group of 16, 23,
and 25 with trifluoroacetic acid followed by EDC coupling
with primary or secondary amines provided the desired (2S)-
cyanopyrrolidine analogues17-19, thiazolidine 24, andboro-
nic ester 26, respectively. The free boronic acid 27 was ob-
tained through acid catalyzed transesterification of boronic
ester 26 with boron trichloride.17 The synthesis of cis-31 from
cis-15 was identical to that of trans-17 from trans-15. The
proposed mechanism for the formation of byproducts 20-22
is that the nitrile group of 17-19 trapped the tert-butyl car-
bocation followed by hydrolysis to produce amide com-
pounds 20-22, respectively. Therefore, the search for scaven-
gers of alkylating agents is required. When the thioanisole
was used as a scavenger for the intermediate carbocation, its
ability to suppress the formation of 20-22 was not effective.
The more promising result was observed when acetonitrile
(CH3CN) was applied as cosolvent with trifluoroacetic acid
(TFA), and thus, the nitrile group of CH3CN participated in
the reaction by acting as acceptor of the leaving tert-butyl
cation. As expected, this approach slashed the yield of 20-22
and produced another byproduct N-tert-butylacetamide
(Mw = 115, detected by LC-MS). Compound 30 was pre-
pared in five steps from the known building block 28, which
was synthesized according to literature report (cis or trans
configuration is not assigned).36
Results and Discussion
To establish an optimized P2 site for FAP inhibition, C(4)-
substituted pyroglutamic acid based inhibitors with various
amines were explored, including bicyclic ring system (17a-h),
piperazine ring system (17i,j), monocyclic system (17l,m), and
phenylamine (17n). These derivatives described above were tested
for inhibition of FAP, DPP-IV, DPP8, DPP9, and DPP-II, and
the data are summarized in Table 2. Patent search found that

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6577
N-acyl-Gly-boroPro 9 is a potent FAP inhibitor with an IC50
value of 1.8 nM.37 In our study, the reference compound 9 has
an IC50 of 19 nM for the inhibition of FAP and is inactive
toward DPP-IV and -II (IC50 greater than 20 and 100 μM,
respectively), but this compound shows moderate inhibition
against DPP8 and DPP9 (3.7 and 2.7 μM, respectively).
A structure-activity relationship (SAR) work was carried
out by replacing the boronic acid moiety of 9 with a cyano
group. This work has led to the identification of N-acyl-
Gly-cyanoPro 10 (FAP, IC50 = 0.61 μM), which is 30-fold
less potent than 9 and inactive toward the other related en-
zymes. In the pyroglutamic acid series of compounds, we star-
ted initially with the bicyclic system using the isoquinoline
ring. Compound 17a (trans configuration) inhibited FAPwith
an IC50 value of 79 nM and inactive toward DPP-IV, DPP-II,
DPP8, and DPP9 (IC50 > 100 μM). The cis epimer 31 was
around 3-fold less potent than trans-17a. Therefore, a focused
structure-activity profiling effort was initiated by modifying
the P2 site amine of trans isomer 17a. The introduction of
chloro (17b), fluoro (17c), or trifluoromethyl (17d) substituent
at the 5-position of the isoindoline gave similar potency (IC50
≈ 66-110 nM) compared to17a. When the isoindolinemoiety
of 17a was replaced with isoquinoline, compound 17e showed
a similar level of FAP inhibition as seen for 17a. Further
modification carried out with heterobicyclic building blocks,
such as 4,5,6,7-tetrahydrothieno[2,3-c]pyridine 17f, imidazo-
pyrazine 17g, and triazolopyrazine derivative 17h, led to at
least a 3-fold decrease in FAP inhibitory potency compared
to lead compound 17a. Compound 17h is the least potent
FAP inhibitor in the bicyclic ring system with an IC50 value
of 15 μM.
Replacement of the bicyclic ring system with piperazine
derivatives, such as 1-(4-fluorophenyl)piperazine 17i and
1-pyridin-4-ylpiperazine 17j, led to no improvement in FAP
potency. Among them, the potency of more polar 1-pyridin-4-
ylpiperazine 17j gave a relatively low IC50 of 3.2 μM. On the
basis of the results of 17h (bicyclic system) and 17j (piperazine
system), it seemed that polar substituents at the P2 site would
bedetrimental topotency; compound17h hadweakinhibition
(IC50 > 10μM), and compound 17j exhibited low micromolar
activity against FAP. Compound 17k with a 4-chlorophenyl
substituent at the 4-position of 1,2,3,6-tetrahydropyridine is a
close analogue of 17i. This compound showed 2.5-fold more
potency than 17i. As for the effects of monocyclic system,
pyrrolidine 17l (IC50 = 73 nM) is equipotent with bicyclic 17a
as FAP inhibitors. However, replacement of the pyrrolidine
ring of 17l with the piperidine (17m, IC50 = 730 nM) led to a
marked 10-fold decrease in potency. Next, when we brought
the nitrogen out of the ring system, the aniline 17n dramati-
cally decreased the inhibitory activity against FAP (IC50 > 20
μM). Therefore, the nitrogen out of the ring system was not
further modified. In general, these active FAP inhibitors
17a-n were inactive toward DPP-IV, DPP8, DPP9, and
DPP-II (IC50 > 20 μM).
Figure 3. Inactive 4-alkyl substituted pyroglutamic acid amides.
ring (18b) showed a decrease in activity (IC50 = 63 nM).
Compound 19 with a 4,4-difluoro substituent at the 2-cyano-
pyrrolidine ring exhibited very similar potency to monofluoro
substituted 18a. Changing the 2-position substituent of pyro-
glutamic acid from 2-cyanopyrrolidine 18a to thiazolidine
24a or pyrrolidine 24b lacking a nitrile moiety showed no
significant inhibition toward FAP (IC50 > 50 μM). All of the
active FAP inhibitors shown in Table 3 were inactive toward
the other related enzymes except boroproline 27. Even though
27 showed a potent FAP inhibition (IC50 = 31 nM), it also
exhibited strong inhibitory activity against the other DPP
enzymes (IC50 values ranged from 400 to 600 nM). So far,
from our studies and literature reports, we found that inhibi-
torsof DPPs containingprolineboronicacid (boro-Pro) inthe
P1site cause poor selectivity of the inhibitors. Compound20is
a byproduct in the synthesis of compound 17a. We also in-
vestigated the effect of carboxylic acid tert-butylamide at the
2-position of pyrrolidine. The bulky amide 20 was inactive
against FAP and the other DPP enzymes(IC50 > 100μM). As
for no substituent amide 32 was also inactive toward all the
DPP enzymes. It means that dipeptides containing prolina-
mide in the P1 site cannot be well tolerated by FAP. In com-
parison with 17a, 4-carboxypyroglutamic acid diamide 30,
shortened by one carbon at the 4-position of 17a, caused no
significant inhibition against FAP (IC50 > 20 μM) and the
other DPP enzymes (IC50 > 50 μM). In addition, we intro-
duced less polar alkyl groups, such as benzyl (33) and 3-methyl-
but-2-enyl (34) groups, to replace the 2-(1,3-dihydroisoindol-
2-yl)-2-oxoethyl moiety of the lead 17a at the 4-position of
pyroglutamic acid diamide, but none of them showed signifi-
cant inhibition against FAP (Figure 3, see Supporting Infor-
mation for details).
Next, we measured the inhibition constant Ki of potent
compound 19 against FAP. Assessment of reaction progress
curves in the presence of various concentrations of compound
19 revealed a clear, time-dependent approach toward steady
state, which is characteristic of slow-binding inhibition kine-
tics, and the Ki value of 19 is 1.3 nM. In addition, the kinetic
mechanism of the inhibition of FAP by the reference com-
pound N-acyl-Gly-boroPro 9 was also determined. It showed
a competitive inhibition pattern well-fitted to a Lineweaver-Burk
plot, and its Ki value was estimated to be 7.9 nM (Figure 4).29
Inhibitors that possessed excellent potency and selectivity
profiles were selected for plasma pharmacokinetic screening
in mice, and the data are summarized in Table 4. 4-Fluoropyr-
rolidine derivative 18a demonstrated short-to-moderate oral
half-life (1.8 h), low oral bioavailability (12%), and relatively
high clearance (94 (mL/min)/kg). Introducing a 3-chloro sub-
stituent at the isoindoline gave compound 18b, which showed
reduction of total body clearance (55 (mL/min)/kg) and pro-
longed oral half-life (t1/2 = 3.6 h) compared to 18a. On the
other hand, although compound 18b had a very low oral bio-
availability (F = 0.7%), it had a very long half-life (t1/2 (iv) =
13.3 h) and a favorable drug exposure (AUC) after iv dosing.
Therefore, compound 18b can be selected as an iv route in
After optimizing the P2 portion, we turned our attention to
the P1 portion of the molecule. The requirements of the S1
pocket for FAP inhibition were explored by keeping isoindo-
line as the 4-position substituent and varying the 2-position
substituent. Their inhibitory properties are shown in Table 3.
Incorporation of the (4S)-fluoro substituent at the (2S)-2-
cyanopyrrolidine ring (18a, IC50 = 20 nM) led to a 4-fold
increase in FAP inhibitory activity compared to that of un-
substituted analogue (17a, IC50 = 79 nM). However, intro-
duction of a chloro substituent at the 5-position of isoindoline

6578 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Tsai et al.
Figure 4. Determination of the Ki values for compounds 9 and 19 against human recombinant FAP (hFAP): (A) competitive inhibition of
hFAP by compound 9; (B) slow-binding kinetics for the inhibition of hFAP by compound 19. One representative experiment is shown in both
panels A and B.
Table 4. Pharmacokinetic Properties of Selected FAP Inhibitors in Mice
intravenous (dose, 2 mg/kg)
oral (dose, 20 mg/kg)
compd
CLP (mL/min/kg)
T
1/2 (h)
AUC (ng/(mL h))
3
Cmax (ng/mL)
T1/2 (h)
AUC ng/(mL h)
3
F (%)
18a
18b
19
94
58
87
2
460
593
495
210
21
1.8
3.6
3.9
408
43
12
0.7
10
13.3
0.8
108
336
animal models for therapeutic target validation of FAP. The
4,4-difluoropyrrolidine analogue 19 exhibited similar clear-
ance, po AUC, and oral bioavailability to 4-fluoropyrrolidine
18a. On the contrary, compound 19 showed 2-fold longer oral
half-life (3.9 h) and lower Cmax than 18a (1.8 h). An analysis of
the mouse pharmacokinetic data from the selected FAP
inhibitors indicated a strong trend where a more hydrophobic
chloro substituent at the isoindoline (18b) has a considerably
lower plasma clearance and longer half-life after iv dosing and
led toa compound withlow Cmax andoral bioavailabilityafter
oral dosing.
NMR spectra were obtained with a Varian Mercury-300 spectro-
meter operating at 300 or 400 MHz. Chemical shifts wererecorded
in parts per million (ppm, δ) and were reported relative to the
solvent peak or TMS. High-resolution mass spectra (HRMS)
were measured with a Finnigan (MAT-95XL) electron impact
(EI) mass spectrometer. LC-MS data were measured on an Agi-
lent MSD-1100 ESI-MS/MS System. Microanalyses were carried
out on a Heraeus VarioIII-NCH analyzer. Flash column chro-
matography was done using silica gel (Merck Kieselgel 60, no.
9385, 230-400 mesh ASTM). Reactions were monitored by TLC
using Merck 60 F254 silica gel glass backed plates (5 cm ꢀ 10 cm).
Zones were detected visually under ultraviolet irradiation (254
nm) or by spraying with phosphomolybdic acid reagent (Aldrich)
followed by heating at 80 °C. All starting materials and amines
were commercially available unless otherwise indicated. Purity of
target compounds were over 95% based on reversed-phase HPLC
analyses (Chromolith performance RP-18e 4.6 mm ꢀ100 mm
column) under the two elution conditions. Condition A was 40:60
MeOH-H2O, and condition B was 70:30 MeOH-H2O. The flow
rate was 0.2 mL/min, and the injection volume was 5 μL. The
system operated at 25 °C. Peaks were detected at λ = 220/254 nm.
Benzyl (2S,4S)-N-tert-Butoxylcarbonyl-4-tert-butoxycarbony-
lmethypyrglutamate (trans-15) and Benzyl (2S,4R)-N-tert-Butoxy-
lcarbonyl-4-tert-butoxycarbonylmethypyrglutamate (cis-15). Benzyl
(2S)-N-tert-butoxylcarbonylpyrglutamate 1332 (628 mg, 2 mmol)
was dissolved in tetrahydrofuran (THF, 10 mL) and cooled to
-78 °C, with stirring under nitrogen. LiHMDS (1.0 M in THF,
2.2 mL, 4.4 mmol) was added dropwise over 5 min, and stirring was
continued for 1 h. tert-Butyl bromoacetate 14 (0.32 mL, 4.0 mmol)
was added dropwise over 3 min, and stirring was continued at
-78 °C for 2 h. The reaction was quenched with saturated aqueous
ammonium chloride and extracted into ethyl acetate. The organic
layer was washed with water and saturated aqueous sodium
chloride and dried (MgSO4). The solvent was removed in vacuo
to give a brown oil, which was purified by column chromatography
on silica gel (eluted with hexane/CH2Cl2/EA = 4/5/1) to yield the
desired compounds trans-15 (130 mg, 15%) and cis-15 (390 mg,
45%) as white crystalline solids. trans-15:mp89-91 °C. [R]2D4 -24.0
(c 0.2, CH2Cl2). 1H NMR (300 MHz, CDCl3): δ 7.35 (m, 5H), 5.22
and 5.17 (AB quartet, J = 12.0 Hz, 2H), 4.62 (dd, J = 9.6, 1.2 Hz,
Conclusions
By introduction of ring-constraint in the lead compound 8
and carrying out SAR studies in a series of substituted 4-car-
boxymethylpyroglutamic acid diamides, some potent and
selective FAP inhibitors have been discovered. Notable among
these are compounds 18a and 19 with a 4-fluoro substituent at
the P1 site pyrrolidine ring, which are IC50 = 20 nM FAP
inhibitors with excellent selectivity profile over DPP-IV, DPP-
II, DPP8, and DPP9 (IC50 > 50 μM). The SAR suggests that
the polar substituents at the P2 site (17h and 17j) would be
detrimental to potency and that shortening by one carbon at
the 4-position of pyroglutamic acid diamide (30) results in a
loss in FAP inhibitory potency (IC50 > 20 μM). Our kinetic
analyses demonstrated that compound 19 is a slow-binding
inhibitor of FAP with Ki value of 1.3 nM. A survey of mouse
pharmacokinetic parameters showed that selected compounds
18a,b and 19 showed poor oral availability in mice. However,
18b has a longer half-life, lower total body clearance, and
favorable drug exposure (AUC) after iv treatment. Thus,
compound 18b can be dosed as iv route to evaluate the
potential therapeutic effects of FAP inhibitors in vivo.
Experimental Section
All commercial chemicals and solvents are reagent grade and
were used without further treatment unless otherwise noted. 1H

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6579
1H), 3.10-2.90 (m, 1H), 2.78 (dd, J = 17.0, 3.9 Hz, 1H), 2.39 (dd,
J = 17.0, 8.4 Hz, 1H), 2.32 (d, J = 9.0 Hz, 1H), 2.16-2.02 (m, 1H),
1.42 (s, 9H), 1.40 (s, 9H). MS (ESþ) m/z calcd for C23H31NO7:
433.49. Found: 456.1 (M þ Naþ). cis-15: mp 70-72 °C. [R]2D4 -15.3
(c 0.2, CH2Cl2). 1H NMR (400 MHz, CDCl3): δ 7.36 (m, 5H), 5.22
and 5.17 (AB quartet, J = 12.0 Hz, 2H), 4.55 (t, J = 7.8 Hz, 1H),
3.05-2.90 (m, 1H), 2.84 (dd, J = 17.0, 3.9 Hz, 1H), 2.60-2.70 (m,
1H), 2.34 (dd, J = 16.7, 10.0 Hz, 1H), 1.74-1.64 (m, 1H), 1.43 (m,
18H). MS (ESþ) m/z calcd for C23H31NO7: 433.49. Found: 456.2
(M þ Naþ).
J = 3.3 Hz, 0.5H), 5.38 (br s, 0.5H), 5.04 (d, J = 9.3 Hz, 1H),
4.64 (dd, J = 12.0, 1.5 Hz, 1H), 4.18-3.83 (m, 2H), 3.20 (m, 1H),
2.78-2.10 (m, 6H), 1.49 (s, 9H), 1.45 (s, 9H).
(3R)-3-tert-Butoxycarbonylmethyl-2-oxo-(5S)-5-[(2R)-2-((1S,-
2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.02,6]-
dec-4-yl)pyrrolidine-1-carbonyl]pyrrolidine-1-carboxylic Acid tert-
Butyl Ester (25). 1H NMR (300 MHz, CDCl3): δ 4.70 (d, J = 8.7
Hz, 1H), 4.27 (dd, J = 8.7, 1.8 Hz, 1H), 3.73-3.65 (m, 1H),
3.48-3.40 (m, 1H), 3.29 (dd, J = 9.0, 7.2 Hz, 1H), 3.17-3.06 (m,
1H), 2.77 (dd, J = 16.7, 4.2 Hz, 1H), 2.42-2.20 (m, 5H), 2.20-1.65
(m, 7H), 1.47 (s, 9H), 1.44 (s, 9H), 1.37 (s, 3H), 1.26 (m, 4H), 0.82 (s,
3H). MS (ESþ) m/z calcd for C30H47BN2O8: 574.51. Found: 597.2
(M þ Naþ), 419.1 (M - (Boc)-(tert-butyl) þ Hþ).
Isomerization from cis-15 to trans-15.34 The cis-15 (433 mg,
1 mmol) was dissolved in CH2Cl2 and cooled to 0 °C. DBU (0.44
mL, 3 mmol) was added dropwise and the mixture stirred at 0 °C
for 30 min and then at room temperature for 24 h. The mixture
was diluted with CH2Cl2 and washed once each with 1 N HCl
and water. The organic layer was dried over MgSO4, filtered,
and concentrated in vacuo to give the crude product, which was
purified by column chromatography as described above to yield
trans-15 (292 mg, 68%) and recovered cis-15 (100 mg, 23%).
General Procedure for the Synthesis of Compounds 16, 23, 25,
and 29. To a solution of compound 15 or 28 (1.0 mmol) in
MeOH (10 mL) was added 5% palladium on carbon under an
atmosphere of nitrogen. The mixture was stirred vigorously
under an atmosphere of hydrogen for 5 h at room temperature.
The mixture was filtered through Celite and concentrated in
vacuo to give (2S,4S)-N-tert-butoxylcarbonyl-4-tert-butoxycar-
bonylmethypyrglutamic acid or (2S,4S)-N-tert-butoxylcarbo-
nyl-4-tert-butoxycarbonylpyrglutamic acid as a pale yellow oil.
The crude acid was used for the next reaction without further
purification. To a solution containing this acid (343 mg, 1.0
mmol) and N-hydroxybenzotrizole (HOBt, 168 mg, 1.1 mmol)
in 1,4-dioxane (5 mL) was added a solution of EDC (211 mg, 1.1
mmol) in CH2Cl2 (5 mL). The mixture was stirred for 10 min at
room temperature. To the resulting solution was added the
appropriate proline derivatives (L-prolinamide, (2S,4S)-4-
fluoroprolinamide,35 (2S)-4,4-difluoroprolinamide,35 thiazolid-
ine or prolineboronic ester,17 1.2 equiv) in CH2Cl2 (4 mL), with
stirring. After 2 h, the reaction mixture was diluted with CH2Cl2
and washed with saturated aqueous NaHCO3 solution (10 mL),
1 N aqueous citric acid solution (10 mL), and brine (10 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated. The crude prolinamides 16 and 29 and thiazoli-
dine 23 were used for the next reaction without further purifica-
tion. The crude boronic ester 25 was purified over silica gel using
hexane/EA (7:3) as an eluant to yield the desired compound 25
(92%, two steps) as a colorless oil.
5-((2S)-2-Cyanopyrrolidine-1-carbonyl)-2-oxo-pyrrolidine-1,3-
dicarboxylic Acid di-tert-Butyl Ester (29). Compound 29 is a
single isomer (major). The absolute configuration of 29 at C-4 is
not assigned. 1H NMR (300 MHz, CDCl3): δ 4.77 (dd, J = 6.6,
2.1 Hz, 1H), 4.69 (dd, J = 9.2, 2.1 Hz, 1H), 3.73 (td, J = 9.8, 0.6
Hz, 1H), 3.72-3.54 (m, 2H), 2.64-2.52 (m, 1H), 2.30-2.03 (m,
5H), 1.43 (s, 15H). MS (ESþ) m/z calcd for C20H29N3O6: 407.46.
Found: 430.1 (M þ Naþ).
General Procedure for the Synthesis of Compounds 17-19,
20-22, 24, 26, and 30. To a solution of compound 16, crude 23,
25, or 29 (2.0 mmol) in CH3CN (5 mL) at 0 °C was added TFA
(5 mL) dropwise over 5 min. The reaction mixture was stirred for
1 h at room temperature. The reaction mixture was concentrated
under vacuum and crystallized with EA/ether to yield the
corresponding acids. To a stirred solution of these acids (0.5
mmol) and HOBt (0.55 mmol) in 1,4-dioxane (3 mL) was added
a solution of EDC (105 mg, 0.55 mmol) in CH2Cl2 (3 mL). The
mixture was stirred for 10 min at room temperature. The ap-
propriate 1° or 2° amine (0.55 mmol) in CH2Cl2 (2 mL) was
added with stirring. After 16 h, the reaction mixture was diluted
with CH2Cl2 (30 mL) and washed with saturated aqueous
NaHCO3 (30 mL), 1 N aqueous citric acid (30 mL), and brine
(30 mL). The organic layers were dried over MgSO4, filtered,
and concentrated. The residue was purified over silica gel
(eluting sequentially with CH2Cl2/MeOH = 98/2, 97/3, 96/4,
and 95/5) to yield compounds 17-19 (75-80%) and byproducts
20-22 (5-10%) from 16, compound 24 (85%) from 23, and
compound 30 (90%) from 29 as white solids or foam. The
elution condition for crude 26 was EA/CH2Cl2 = 1/1 followed
by EtOH/EA/CH2Cl2 = 5/45/50 to yield 26 (50%) from 25 as a
pale brown foam.
(2S)-1-{(2S, 4S)-4-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-
5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-carbonitrile (17a). Mp
170 °C (dec). 1H NMR (300 MHz, CDCl3): (6/1 mixture of trans/
cis amide rotomers) δ 7.30-7.26 (m, 4H), 6.56 (s, 0.86H), 6.06 (s,
0.14H), 4.82-4.77 (m, overlapped with two singlet at 4.82, 4.77,
4.86H), 4.60 (d, J = 6.6 Hz, 0.14H), 4.55 (d, J = 9.0 Hz, 0.14H),
4.40 (dd, J = 3.0, 9.0 Hz, 0.86H), 3.61-3.04 (m, 2H), 3.02-2.88
(m, 2H), 2.68-2.36 (m, 3H), 2.29-2.12 (m, 4H). MS (ESþ) m/z
calcd for C20H22N4O3: 366.17. Found: 367.1 (M þ Hþ). Anal.
To a mixture of the prolinamide 16 or 29 (1.6 mmol) and
imidazole (0.12 g, 1.8 mmol) in pyridine (5 mL) cooled to -20 °C
was added phosphoryl chloride (0.64 g, 4.2 mmol) under nitro-
gen. After being stirred for 30 min at -20 °C, the mixture was
evaporated to dryness in vacuo. The resulting browm solid was
dissolved in CH2Cl2 (40 mL) and washed with 1.0 N aqueous
citric acid (40 mL). The organic phase was dried over magne-
sium sulfate, filtered, and concentrated under reduced pressure
to yield the crude material as a viscous oil. The crude material
was purified by chromatography on silica gel (eluted with
hexane/EA = 2/1) to give the corresponding nitrile 16 or 29
(>90%) as a white powder. Only the data of representative
compounds are shown.
Calcd for C20H22N4O3 H2O: C, 62.49; H, 6.29; N, 14.57. Found:
3
C, 62.21; H, 6.14; N, 14.63.
(2S)-1-{(2S,4S)-4-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-
5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-carboxylic Acid tert-
Butylamide (20). 1H NMR (400 MHz, CD3OD): (2.6/1 mixture
of trans/cis amide rotomers) δ 8.05 (br s, 0.28H), 7.88 (br s,
0.28H), 7.56 (br s, 0.72H), 7.37-7.28 (m, 4H), 7.21 (br s, 0.72H),
4.90 (s, overlapped with H2O, 2H), 4.73 (s, 2H), 4.55 (dd, J =
9.6, 2.8 Hz, 0.72H), 4.39-4.32 (m, 1H), 4.10 (dd, J = 8.8, 2.4 Hz,
0.28H), 3.72-3.50 (m, 2H), 3.05-2.95 (m, 1H), 2.95-2.85 (m,
1H), 2.72-2.28 (m, 3H), 2.18-1.84 (m, 4H), 1.36 (s, 2.52H), 1.32
(s, 6.48H). MS (ESþ) m/z calcd for C24H32N4O4: 440.54. Found:
441.2 (M þ Hþ). HRMS (FAB) calcd for C22H33N4O4:
441.2502. Found: 441.2496.
(2S,4S)-3-tert-Butoxycarbonylmethyl-5-((2S)-2-cyanopyrroli-
dine-1-carbonyl)-2-oxopyrrolidine-1-carboxylic Acid tert-Butyl
1
Ester (16a). Mp 160-162 °C. H NMR (300 MHz, CDCl3): δ
4.83 (dd, J = 6.9, 2.1 Hz, 1H), 4.71 (dd, J = 9.2, 1.8 Hz, 1H),
3.79-3.58(m, 2H), 3.22-3.06 (m, 1H), 2.74 (dd, J =17.1, 3.9Hz,
1H), 2.50 (dd, J = 17.0, 8.1 Hz, 1H), 2.04-2.40 (m, 6H), 1.49 (s,
9H), 1.44 (s, 9H). MS (ESþ) m/z calcd for C21H31N3O6: 421.49.
Found: 444.1 (M þ Hþ), 266.0 (M - (Boc)-(tert-butyl) þ Hþ).
(2S,4S)-3-tert-Butoxycarbonylmethyl-5-((2S,4S)-2-cyano-4-
fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylic Acid
tert-Butyl Ester (16b). 1H NMR (300 MHz, CDCl3): δ 5.53 (br t,
(2S)-1-{(2S, 4R)-4-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-
5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-carbonitrile (31). 1H
NMR (400 MHz, CDCl3): (6/1 mixture of trans/cis amide

6580 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Tsai et al.
rotomers) δ 7.29-7.26 (m, 4H), 6.51 (s, 0.86H), 6.24 (s, 0.14H),
4.83-4.74 (m, 5H), 4.59-4.55 (m, 0.14H), 4.40-4.35 (m,
0.86H), 3.69-3.52 (m, 2H), 3.15-3.03 (m, 2H), 2.50-2.38 (m,
1H), 2.31-2.04 (m, 3H), 1.87-1.75 (m, 3H). MS (ESþ) m/z calcd
for C20H22N4O3: 366.17. Found: 367.1 (M þ Hþ). HRMS
(FAB) calcd for C20H22N4O3: 366.1692. Found: 366.1691.
(2S)-1-({(2S,4S)-4-[2-(5-Chloro-1,3-dihydro-2H-isoindol-2-yl)-
2-oxoethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-2-carbo-
nitrile (17b). 1H NMR (300 MHz, CDCl3): (7/1 mixture of trans/
cis amide rotomers) δ 7.37-7.18 (m, 3.88H), 6.56 (s, 0.12H),
5.31-4.69 (m, 5H), 4.53 (d, J = 9.3 Hz, 0.12H), 4.42 (dd, J =
2.1, 9.3 Hz, 0.88H), 3.61-3.57 (m, 2H), 3.01-2.87 (m, 2H),
2.57-2.18 (m, 7H). MS (ESþ) m/z calcd for C20H21ClN4O3:
400.13. Found: 400.9 (M þ Hþ), 402.9 (M þ 2 þ Hþ).
2.44-2.05 (m, 6H). MS (ESþ) m/z calcd for C18H20F3N7O3:
439.16. Found: 440.0 (M þ Hþ).
(2S)-1-({(2S,4S)-4-[2-[4-(4-Fluorophenyl)piperazin-1-yl]-2-oxo-
ethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-2-carbonitrile
(17i). H NMR (300 MHz, CDCl3): (7/1 mixture of trans/cis
1
amide rotomers) δ 7.27-6.85 (m, 4.88H), 6.27 (s, 0.12H), 4.83-
4.82 (m, 0.88H), 4.60 (d, J = 6.6 Hz, 0.12H), 4.48 (d, J = 8.4 Hz,
0.12H), 4.36 (d, J = 8.4 Hz, 0.88H), 3.80-3.55 (m, 6H), 3.10-
2.91 (m, 6H), 2.56-2.47 (m, 2H), 2.38-2.17 (m, 7H). MS (ESþ)
m/z calcd for C22H26FN5O3: 427.20. Found: 428.0 (M þ Hþ).
(2S)-1-({(2S,4S)-4-[2-[4-(Pyridine-4-yl)-piperazin-1-yl]-2-oxo-
ethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-2-carbonitrile
1
(17j). H NMR (400 MHz, CDCl3): (9/1 mixture of trans/cis
amide rotomers) δ 8.29 (d, J = 5.2 Hz, 2H), 6.95 (s. 0.9H), 6.65
(d, J = 5.2 Hz, 2H), 6.39 (s, 0.1H), 4.82-4.80 (m, 0.9H), 4.60 (d,
J = 8.0 Hz, 0.1H), 4.49 (d, J = 8.0 Hz, 0.1H), 4.36 (d, J = 8.0
Hz, 0.9H), 3.85-3.48 (m, 6H), 3.45-3.31 (m, 4H), 3.07-2.87
(m, 2H), 2.55-2.16 (m, 7H). MS (ESþ) m/z calcd for
C21H26N6O3: 410.21. Found: 411.3 (M þ Hþ), 433.3 (M þ
23þ). HRMS (FAB) calcd for C21H27N6O3: 411.2145. Found:
411.2144.
(2S)-1-{(2S,4S)-4-[2-(5-Fluoro-1,3-dihydroisoindol-2-yl)-2-oxo-
ethyl]-5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-carbonitrile (17c).
1H NMR (400 MHz, CDCl3): (7/1 mixture of trans/cis amide
rotomers) δ 7.24-7.19 (m, 1H), 7.02-6.95 (m, 2H), 6.70 (d, J =
4.0 Hz, 0.88H), 6.18 (d, J = 4.0 Hz, 0.12H), 4.83-4.72 (m,
4.88H), 4.61 (d, J = 7.2 Hz, 0.12H), 4.51 (d, J = 8.8 Hz, 0.12H),
4.39 (dd, J = 2.8, 8.8 Hz, 0.88H), 3.69-3.52 (m, 2H), 2.98-
2.87 (m, 2H), 2.61-2.39 (m, 3H), 2.30-2.17 (m, 4H). MS (ESþ)
m/z calcd for C20H21FN4O3: 384.16. Found: 385.3 (M þ Hþ).
HRMS (FAB) calcd for C20H22N4O3: 366.1692. Found:
366.1691.
(2S)-1-({(2S,4S)-4-[2-[4-(4-Chlorophenyl)-3,6-dihydropyridin-
1(2H)-yl]-2-oxoethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-
2-carbonitrile (17k). 1H NMR (300 MHz, CDCl3): (9/1 mixture
of trans/cis amide rotomers) δ 7.30 (s, 4H), 6.80 (d, J = 3.9 Hz,
0.9H), 6.19 (s, 0.1H), 6.06 (br s, 0.5H), 5.99 (brs, 0.5H), 4.83-
4.81 (m, 0.9H), 4.60 (d, J = 6.9 Hz, 0.1H), 4.58 (d, J = 9.3 Hz,
0.1H), 4.36(dd, J =2.4, 9.3Hz, 0.9H), 4.20(brs, 1H), 4.13 (d, J =
2.4 Hz, 1H), 3.82-3.52 (m, 4H), 3.02-2.87 (m, 2H), 2.59-2.46
(m, 4H), 2.39-2.14 (m, 5H). MS (ESþ) m/z calcd for C23H25-
ClN4O3: 440.16. Found: 440.9 (M þ Hþ), 442.0 (M þ 2 þ Hþ).
(2S)-1-({(2S,4S)-5-Oxo-4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]pyr-
(2S)-1-({(2S,4S)-4-[2-(5-Trifluoromethyl-1,3-dihydro-2H-iso-
indol-2-yl)-2-oxoethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-
2-carbonitrile (17d). 1H NMR (300 MHz, CDCl3): (7/1 mixture
of trans/cis amide rotomers) δ 7.60-7.55 (m, 2H), 7.42 (t, J =
7.5 Hz, 1H), 7.19 (d, J = 4.2 Hz, 0.88H), 6.55 (d, J = 4.2 Hz,
0.12H), 4.90-4.83 (m, overlapped two singlet at 4.90, 4.83, 4.88H),
4.69 (d, J = 7.8 Hz, 0.12H), 4.56 (d, J = 8.4 Hz, 0.12H), 4.65 (d,
J = 7.8 Hz, 0.88H), 3.64-3.60 (m, 2H), 3.09-2.91 (m, 2H),
2.63-2.20 (m, 7H). MS (ESþ) m/z calcd for C21H21F3N4O3:
434.16. Found: 435.0 (M þ Hþ).
1
rolidin-2-yl}carbonyl)pyrrolidine-2-carbonitrile (17l). H NMR
(300 MHz, CDCl3): (13/1 mixture of trans/cis amide rotomers)
δ 7.41 (s, 0.93H), 6.74 (s, 0.07H), 4.83-4.80 (m, 0.93H), 4.67 (d,
J = 4.5 Hz, 0.07H), 4.51 (d, J = 7.8 Hz, 0.07H), 4.42 (d, J = 7.8
Hz, 0.93H), 3.60-3.56 (m, 2H), 3.45-3.39 (q like, J = 6.3 Hz,
4H), 2.95-2.76 (m, 2H), 2.48-2.15 (m, 7H), 1.99-1.80 (m, 4H).
MS (ESþ) m/z calcd for C16H22N4O3: 318.17. Found: 319.1 (M þ
Hþ), 341.1 (M þ 23þ).
(2S)-1-({(2S,4S)-4-[2-(1,3-Dihydro-2H-isoquinol-2-yl)-2-oxo-
ethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-2-carbonitrile (17e).
1H NMR (300 MHz, CDCl3): (7/1 mixture of trans/cis amide
rotomers) δ 7.28-7.12 (m, 4.88H), 6.54 (s, 0.12H), 4.84 (t like, J =
5.1 Hz, 1H), 4.63 (s, 1H), 4.62 (s, 1H), 4.49 (d, J = 9.3 Hz, 0.12H),
4.38 (d, J = 9.3 Hz, 0.88H), 3.81-3.66 (m, 2H), 3.57-3.53 (m,
2H), 3.00-2.82 (m, 4H), 2.57-2.15 (m, 7H). MS (ESþ) m/z calcd
for C20H24N4O3: 380.18. Found: 381.0 (M þ Hþ).
(2S)-1-[{(2S,4S)-5-Oxo-4-(2-oxo-2-(piperidin-1-yl)ethyl)pyr-
rolidine-2-yl}carbonyl]pyrrolidine-2-carbonitrile (17m). 1H NMR
(300 MHz, CDCl3): (8/1 mixture of trans/cis amide rotomers)
δ 6.72 (s, 0.89H), 6.10 (s, 0.11H), 4.74-4.84 (m, 0.89H), 4.56 (br
d, J = 5.7 Hz, 0.11H), 4.45 (br d, J = 7.2 Hz, 0.11H), 4.36 (dd,
J = 9.6, 3.0 Hz, 0.89H), 3.76-3.30 (m, 6H), 3.10-2.80 (m, 2H),
2.60-2.00 (m, 7H), 1.74-1.54 (m, 6H). MS (ESþ) m/z calcd for
C17H24N4O3: 332.40. Found: 333.1 (M þ Hþ). HRMS (FAB)
calcd for C17H25N4O3: 333.1927. Found: 333.1920.
2S)-1-({(2S,4S)-4-[2-(6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-
yl)-2-oxoethyl]-5-oxopyrrolidin-2-yl}carbonyl)pyrrolidine-2-car-
bonitrile (17f). H NMR (300 MHz, CDCl3): (8/1 mixture of
1
trans/cis amide rotomers) δ 7.14 (t, J = 5.4 Hz, 1H), 6.88 (d, J =
5.4 Hz, 0.89H), 6.79 (dd, J = 3.3, 5.4 Hz, 1H), 6.43 (d, J = 5.4 Hz,
0.11H), 4.82-4.80 (m, 0.89H), 4.72-4.44 (m, 2.22H, overlapped
two singlet at 4.64, 4.56), 4.36 (d, J = 9.6 Hz, 0.89H), 3.96-3.48
(m, 4H), 3.04-2.83 (m, 4H), 2.62-2.11 (m, 5H). MS (ESþ) m/z
calcd for C19H22N4O3S: 386.14. Found: 386.9 (M þ Hþ). HRMS
(FAB) calcd for C19H22N4O3S: 386.1413. Found: 386.1411.
2S)-1-({(2S,4S)-5-oxo-4-[2-Oxo-[2-(trifluoromethyl)-5,6-dihy-
droimidazo[1,2-a]pyrazin-7(8H)-yl]ethyl]pyrrolidin-2-yl}carbonyl)-
2-[(3S,5S)-5-{[(2S)-2-Cyanopyrrolidin-1-yl]carbonyl}-2-oxo-
pyrrolidin-3-yl]-N-phenylacetamide (17n). 1H NMR (400 MHz,
CDCl3): (8/1 mixture of trans/cis amide rotomers) δ 8.96 (s, 1H),
7.54 (d, J= 7.6 Hz, 2H), 7.32-7.15 (m, 2.89H), 7.06 (t, J=7.6Hz,
1H), 6.47 (s, 0.11H), 4.75-4.73 (m, 0.89H), 4.61 (d, J = 7.6 Hz,
0.11H), 4.49 (d, J = 8.8 Hz, 0.11H), 4.33 (dd, J = 2.8, 8.8 Hz,
0.89H), 3.50-3.47 (m, 2H), 3.03-2.95 (m, 1H), 2.76 (dd, J =
6.4, 15.2 Hz, 1H), 2.52 (dd, J = 6.4, 15.2 Hz, 1H), 2.40-2.04 (m,
6H). MS (ESþ) m/z calcd for C18H20N4O3: 340.15. Found: 341.0
(M þ Hþ). HRMS (FAB) calcd for C18H20N4O3: 340.1535.
Found: 340.1531.
(2S,4S)-1-({(2S,4S)-4-[2-(1,3-Dihydro-2H-isoindol-2-yl)-2-oxo-
ethyl]-5-oxopyrrolidin-2-yl}carbonyl)-4-fluoropyrrolidine-2-car-
bonitrile (18a). Mp 130 °C (dec). 1H NMR (300 MHz, CDCl3):
(7/1 mixture of trans/cis amide rotomers) δ 7.28-7.25 (m, 4H),
7.23 (s, 0.88H), 6.28 (s, 0.12H), 5.54 (br s, 0.44H), 5.46 (br s,
0.06H), 5.36 (br s, 0.44H), 5.30 (br s, 0.06H), 5.09 (d, J = 8.4 Hz,
0.88H), 4.84 (d, J = 8.4 Hz, 0.12H), 4.80 (s, 2H), 4.75 (s, 2H),
4.57 (d, J = 8.4 Hz, 0.12H), 4.38 (d, J = 8.4 Hz, 0.88H), 3.39-
3.76 (m, 2H), 3.02-2.78 (m, 2H), 2.67-2.25 (m, 5H). MS (ESþ)
m/z calcd for C20H21FN4O3: 384.16. Found: 385.0 (M þ Hþ).
1
pyrrolidine-2-carbonitrile (17g). H NMR (400 MHz, CDCl3):
(6/1 mixture of trans/cis amide rotomers) δ 7.22 (d, J = 10.0 Hz,
0.86H), 7.08 (s, 1H), 6.58 (d, J = 10.0 Hz, 0.14H), 4.94-4.57(m,
3H), 4.47-4.46 (m, 0.14H), 4.32 (d, J = 9.6 Hz, 0.86H), 4.11-
3.92 (m, 4H, overlapped singlet at 4.01), 3.61-3.52 (m, 2H),
2.92-2.88 (m, 2H), 2.63-2.54 (m, 1H), 2.42-2.14 (m, 6H). MS
(ESþ) m/z calcd for C19H21F3N6O3: 438.16. Found: 439.1 (M þ
Hþ). HRMS (FAB) calcd for C19H21F3N6O3: 438.1627. Found:
438.1624.
(2S)-1-({(2S,4S)-5-Oxo-4-[2-oxo-2-[3-(trifluoromethyl)-5,6-
dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]ethyl]pyrrolidin-2-
yl}carbonyl)pyrrolidine-2-carbonitrile (17h). 1H NMR (300 MHz,
CD3OD): δ 5.04-4.98 (m, 2H), 4.78 (t, J = 5.4 Hz, 1H), 4.49 (dd,
J = 3.0, 8.7 Hz, 1H), 4.35-4.20 (m, 2H), 4.08-4.04 (m, 2H),
3.71-3.57 (m, 2H), 3.04-2.94 (m, 2H), 2.77-2.65 (m, 1H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6581
(2S)-1-[(2S)-4-(1,3-Dihydroisoindole-2-carbonyl)-5-oxopyrro-
lidine-2-carbonyl]pyrrolidine-2-carbonitrile (30). H NMR (300
Anal. Calcd for C20H21FN4O3 H2O: C, 59.69; H, 5.76; N, 13.92.
3
1
Found: C, 59.87; H, 5.51; N, 13.84.
(2S,4S)-1-{(2S,4S)-4-[2-(5-Chloro-1,3-dihydroisoindol-2-yl)-
2-oxoethyl]-5-oxopyrrolidine-2-carbonyl}-4-fluoropyrrolidine-2-
carbonitrile (18b). Mp 164-166 °C. 1H NMR (300 MHz, DMSO-
d6): (7/1 mixture of trans/cis amide rotomers) δ 8.02 (s, 1H), 7.46
(d, J = 8.4 Hz, 1H), 7.41-7.34 (m, 2H), 5.58 (s, 0.44H), 5.50 (s,
0.06H), 5.46 (s, 0.44H), 5.38 (s, 0.06H), 5.18 (d, J = 8.4 Hz, 0.12
H), 5.05 (d, J = 8.4 Hz, 0.88H), 4.82 (br s, 2H), 4.62 (d, J = 8.8
Hz, 2H), 4.32 (d, J = 8.8 Hz, 0.12H), 4.29 (d, J = 8.8 Hz, 0.88H),
3.89 (br s, 1H), 3.82 (br s, 1H), 2.84-2.62 (m, 2H), 2.52-2.10 (m,
5H). MS (ESþ) m/z calcd for C20H20ClFN4O3: 418.85. Found:
419.2 (M þ Hþ), 421.2 (M þ 2 þ Hþ). HRMS (FAB) calcd for
C20H20ClFN4O3: 418.1208. Found: 418.1215.
MHz, CDCl3): δ (major isomer) 7.30-7.21 (m, 4H), 6.86 (br s,
1H), 5.46 (d, J = 6.9 Hz, 1H), 4.94-4.76 (m, 4H), 4.58 (dd, J =
8.6, 5.1 Hz, 1H), 3.85 (dd, J = 9.0, 6.0 Hz, 1H), 3.70-3.56 (m,
2H), 3.16-3.04 (m, 1H), 2.34-2.08 (m, 5H). MS (ESþ) m/z calcd
for C19H20N4O3: 352.39. Found: 353.1 (M þ Hþ), 375.0 (M þ
Naþ). HRMS (FAB) calcd for C19H20N4O3: 352.1535. Found:
352.1536.
(2S)-1-({(2S,4S)-4-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-
5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-carboxylic Acid Amide
(32). The title compound was obtained as a white powder in a
manner similar to the preparation of 17 except for dehydration
step. Mp 150 °C (dec). 1H NMR (400 MHz, CD3OD): (6/1 mix-
ture of trans/cis amide rotomers) δ 7.34-7.28 (m, 4H), 4.91-
4.86 (m, overlapped with one singlet at 4.88, 1H), 4.81 (s, 2H),
4.55 (dd, J = 9.4, 2.4 Hz, 1H), 4.50 (dd, J = 8.6, 4.0 Hz, 1H),
3.71-3.60 (m, 2H), 3.04-2.96 (m, 1H), 2.91 (dd, J = 16.6, 4.0
Hz, 1H), 2.65 (dd, J = 16.2, 4.0 Hz, 1H), 2.58-2.50 (m, 1H),
2.40-1.80 (m, 6H). MS (ESþ) m/z calcd for C20H24N4O4:
384.43. Found: 385.1 (M þ Hþ). HRMS (FAB) calcd for
C20H25N4O4: 385.1876. Found: 385.1871.
Purification of Human Recombinant FAP, DPP-IV, DPP8,
DPP9, and DPP-II from Insect Cells. All the human DPP
enzymes used in this study and our previous publications are
recombinant proteins expressed individually in insect cells by
tagging with His-tag. DPP-IV and DPP8 were expressed and
purified as described in our previously published method.38-41
Specifically, the purification of DPP9 was carried out with
Strep-Tactin resin as we reported previously.42 FAP was pur-
ified and expressed as described previously.5 Specifically, the
medium of recombinant FAP from Hi5 insect cells was collected
and precipitated by adding 3.8 M ammonium sulfate up to 70%
saturation. The pellet was resuspended in the binding buffer
containing 20 mM Tris-HCl, pH 8.0, with 500 mM NaCl and
bound with the Ni-sepharose (Novagen). After the column was
washed with the binding buffer containing 10 and 20 mM
imidazole, FAP protein was eluted by 250 mM immidazole.
The purification of DPP-II was carried out with modifications
by ConA-sepharose and Q-sepharose HP chromatography.43
The culture medium of Hi5 cells expressing DPP-II was col-
lected and adjusted pH to 7.4. It was loaded into ConA-
sepharose column and washed with 20 mM Tris, 500 mM NaCl,
1 mM CaCl2, 1 mM MgCl2, pH 7.4. DPP-II was eluted by 250
mM methyl mannopyranoside in 20 mM Tris-HCl, 500 mM
NaCl, pH 7.4. The eluent of the ConA-sepharase column was
buffer with 20 mM Tris, pH 7.4, with Amicon YM 30 membrane
(Millipore). After binding to Q-sepharose HP column, DPP-II
was eluted with 0-200 mM NaCl gradient. All the enzymatic
preparations have purities of over 90% with a single predomi-
nant band on SDS-PAGE gel. Kinetic constants of each batch
of the enzymes were measured, and they were similar to the
reported values.
IC50 Determination. The enzyme activities were assayed in a
total volume of 100 μL for 30 min at 37 °C at an emission
wavelength of 405 nm with Power Wave X spectrometer (Bio-
Tek Instrument, Inc., Winooski, VT). The IC50 values for DPP8,
DPP9, and FAP were determined in phosphate-buffered saline
(PBS, pH 7.4) in the presence of 2.5 mM Gly-Pro-pNA, 1.5 mM
Gly-Pro-pNA, and 1.5 mM Ala-Pro-pNA, respectively. The
IC50 for DPP-IV was determined in 2 mM Tris-HCl (pH 8.0) in
the presence of 500 μM Gly-Pro-pNA. The IC50 for DPP2 was
determined in phosphate buffer (pH 5.5) in the presence of 1.5
mM Gly-Pro-pNA. The inhibitor concentrations ranged from
100 to 0.003 μM. IC50 values were computed with commercially
available curve-fitting programs such as SigmaPlot.
(2S)-1-({(2S,4S)-4-[2-(1,3-Dihydro-2H-isoindol-2-yl)-2-oxo-
ethyl]-5-oxopyrrolidin-2-yl}carbonyl)-4,4-difluoropyrrolidine-2-
carbonitrile (19). Mp 156 °C (dec). 1H NMR (300 MHz, DMSO-
d6): (6/1 mixture of trans/cis amide rotomers) δ 8.03 (s, 0.86H),
7.97 (s, 0.14H), 7.36-7.25 (m, 4H), 5.37 (d, J = 8.4 Hz, 0.14H),
5.09 (dd, J = 4.2, 8.4 Hz, 0.86H), 4.82 (s, 2H),4.62 (s, 2H), 4.32
(d, J = 8.7Hz, 1H), 4.25-4.04 (m, 2H), 2.93-2.66 (m, 4H), 2.49-
2.05 (m, 3H). MS (ESþ) m/z calcd for C20H20F2N4O3: 402.15.
Found: 403.1 (M þ Hþ). Anal. Calcd for C20H20F2N4O3 H2O:
3
C, 57.14; H, 5.27; N, 13.33. Found: C, 56.87; H, 5.17; N, 13.43.
(3S,5S)-3-[2-(1,3-Dihydro-2H-isoindol-2-yl)-2-oxoethyl]-5-(1,3-
thiazolidin-3-ylcarbonyl)pyrrolidin-2-one (24a). 1H NMR (300
MHz, CDCl3): δ 7.30-7.26 (m, 4H), 6.96 (d, J = 10.2 Hz, 1H),
4.82 (s, 2H), 4.77 (s, 2H), 4.66-4.40 (m, 3H), 3.94-3.67 (m, 2H),
3.11 (t, J = 6.3 Hz, 1H), 3.03-2.93 (m, 2H), 2.60-2.46 (m, 2H),
2.40-2.30 (m, 2H). MS (ESþ) m/z calcd for C18H21N3O3S:
359.13. Found: 360.1 (M þ Hþ).
(3S,5S)-3-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-5-(pyrroli-
dine-1-carbonyl)pyrrolidin-2-one (24b). 1H NMR (400 MHz,
CDCl3): δ 7.21-7.18 (m, 4H), 6.51 (s, 1H), 4.71 (d, J = 18.4
Hz, 4H), 4.26 (d, J = 10.0 Hz, 1H), 3.45-3.29 (m, 4H), 2.96-
2.85 (m, 2H), 2.50-2.19 (m, 3H), 1.93-1.73 (m, 4H). MS (ESþ)
m/z calcd for C19H23N3O3: 341.40. Found: 342.0 (M þ Hþ).
(3R)-3-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-(5S)-5-[(2R)-
2-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.-
1.02,6]dec-4-yl)pyrrolidine-1-carbonyl]pyrrolidin-2-one (26). 1H
NMR (300 MHz, CDCl3): δ 7.30-7.20 (m, 4H), 5.89 (s, 1H),
4.81 (s, 2H), 4.77 (s, 2H), 4.34 (dd, J = 9.0, 3.0 Hz, 1H), 4.25 (d,
J = 9.0 Hz, 1H), 3.55-3.35 (m, 2H), 3.18 (br t, J = 9.0 Hz, 1H),
3.00-2.85 (m, 2H), 2.60-2.45 (m, 2H), 2.45-2.20 (m, 3H),
2.20-1.50 (m, 7H), 1.37 (s, 3H), 1.23 (m 4H), 0.78 (s, 3H). MS
(ESþ) m/z calcd for C29H38BN3O5: 519.44. Found: 520.2 (M þ
Hþ), 542.2 (M þ Naþ).
(2R)-1-{(2S, 4S)-4-[2-(1,3-Dihydroisoindol-2-yl)-2-oxoethyl]-
5-oxopyrrolidine-2-carbonyl}pyrrolidine-2-boronic Acid (27).17
To a stirred solution of BCl3 (2.5 mL of a 1 M solution in
CH2Cl2) in CH2Cl2 (5 mL) at -78 °C was added (þ)-pinanediol
[(1R)-1-acetamido-2-(1-naphthyl)ethyl]boronate (26, 135 mg,
0.26 mmol). The mixture was stirred at -78 °C for 0.5 h and
the cooling bath then removed. The mixture was evaporated
under vacuum. Water (30 mL) and Et2O (70 mL) were added,
and the aqueous phase was separated and washed with Et2O
(3 ꢀ 10 mL). Lyophilization of the aqueous phase yielded crude
27, which was freed from the boric acid contaminant by suc-
cessive treatments with MeOH (100 mL) followed by distillation
until the distillate showed no green boron color in the flame
when a drop was ignited. The remaining MeOH was removed by
distillation under vacuum. The residue was dissolved in water
(10 mL), washed with Et2O (3 ꢀ 10 mL), and then lyophilized to
give 27 (39 mg, 40%) as a pale yellow solid. Mp 100-101 °C. 1H
NMR (400 MHz, D2O): δ (4/1 mixture of trans/cis amide
rotomers) 7.19-7.17 (m, 4H), 4.69-4.61 (m, 2H), 4.43 (d, J =
6.4 Hz, 0.8H), 4.24 (d, J = 6.4 Hz, 0.2H), 3.65-3.55 (m, 1H),
3.39-3.31 (m, 1H), 3.19 (s, 2H), 3.06-3.02 (m, 1H), 2.91-2.72
(m, 2H), 2.53-2.49 (m, 1H), 2.37-2.21 (m, 2H), 2.01-1.86 (m,
3H), 1.62-1.56 (m, 1H).
Inhibition Constant (Ki) Measurement. Ki was measured as
described previously,29 with a Beckman DU 800 spectrophot-
ometer (Beckman Coulter, Inc., Fullerton, CA). The assays were
performed in PBS in a total volume of 100 μL with 20 nM FAP.

6582 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Tsai et al.
For the competitive binding assays, the substrate concentrations
were 312.5-2500 μM and the concentrations of reference
compound 9 ranged from 0 to 40 nM. For the slow, tight
binding assay of FAP, the concentration of the substrate was
500 μM, and the concentrations of compound 19 ranged from
0 to 70 nM. The hydrolysis of the Ala-Pro-pNA substrate was
monitored continuously by measuring the light emission at 405
nm for 30 min. The data were fitted to slow-binding inhibition as
previously reported.29 The Ki values are represented as the
average of three independent experiments.
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Pharmacokinetic Evaluation of the Selected Compounds in
Mice. The Balb/c mice for the pharmacokinetic evaluation were
obtained from BioLASCO Taiwan Co., Ltd., Ilan, Taiwan, R.
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Research Institutes, Taiwan, R.O.C. The animal studies were
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recorded before the drug was administered. At 0 (immediately
before dosing), 2, 5 (intravenous only), 15, and 30 min and 1, 2,
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min to obtain plasma and then stored at -20 °C before analysis.
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chromatography-tandem mass spectrometry (LC-MS/MS).
The plasma concentration data were analyzed with a standard
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Acknowledgment. The study was financially supported by
National Health Research Institutes, National Research Pro-
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Science Council, Taiwan.
Supporting Information Available: Synthetic scheme and
experimental details for the synthesis of compounds 33 and 34
and a figure for recombinant human DPP proteins purified. This
material is available free of charge via the Internet at http://
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