Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP)

Article

pubs.acs.org/jmc

Extended Structure−Activity Relationship and Pharmacokinetic

Investigation of (4-Quinolinoyl)glycyl-2-cyanopyrrolidine Inhibitors

of Fibroblast Activation Protein (FAP)

Koen Jansen,^†Leen Heirbaut,^†Robert Verkerk,^∥Jonathan D. Cheng,^‡Jurgen Joossens,^†Paul Cos,^§

Louis Maes,^§Anne-Marie Lambeir,^∥Ingrid De Meester,^∥Koen Augustyns,^†and Pieter Van der Veken*^,†

^†Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, 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, Departments of Pharmaceutical Sciences and Biomedical Sciences,

University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium

^∥Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp,

Belgium

S

* Supporting Information

ABSTRACT: Fibroblast activation protein (FAP) is a serine

protease related to dipeptidyl peptidase IV (DPPIV). It has been

convincingly linked to multiple disease states involving remodeling

of the extracellular matrix. FAP inhibition is investigated as a

therapeutic option for several of these diseases, with most attention

so far devoted to oncology applications. We previously discovered the N-4-quinolinoyl-Gly-(2S)-cyanoPro scaﬀold as a possible

entry to highly potent and selective FAP inhibitors. In the present study, we explore in detail the structure−activity relationship

around this core scaﬀold. We report extensively optimized compounds that display low nanomolar inhibitory potency and high

selectivity against the related dipeptidyl peptidases (DPPs) DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The log D

values, plasma stabilities, and microsomal stabilities of selected compounds were found to be highly satisfactory. Pharmacokinetic

evaluation in mice of selected inhibitors demonstrated high oral bioavailability, plasma half-life, and the potential to selectively

and completely inhibit FAP in vivo.

PREP, which is an enzyme of strict endopeptidase capa-

bility.^14,15While designing out DPP aﬃnity in FAP inhibitors is

relatively straightforward, obtaining inhibitors possessing

selectivity for FAP over PREP is considered to be far more

challenging. This is, among others, illustrated by the signiﬁcant

overlap between in vitro processable substrate sequences for

FAP and PREP and the fact that numerous reported FAP

inhibitors have limited or no selectivity with respect to PREP.¹⁶

Given that recent ﬁndings indicate that PREP deﬁcient mice

have impaired spatial learning, memory, and neuronal develop-

ment, FAP over PREP selectivity could nonetheless be an

important feature for inhibitors.^17,18The availability of such

compounds can already have signiﬁcant impact when applied as

tool compounds to study the role of FAP in pathophysiology.

In some cancer types for example, FAP and PREP are

simultaneously overexpressed by cell types that are part of the

metastatic tumor microenvironments, and there are currently

no reports available that quantitatively discrimate between the

potential contributions of both proteolytic activities to disease

progression.¹⁷

INTRODUCTION

■

Fibroblast activation protein (FAP, FAP-α, seprase) belongs to

the prolyl oligopeptidase family S9, which consists of serine

proteases that cleave peptide substrates preferentially after

proline residues. Other members of this family include the

dipeptidyl peptidases (DPPs: DPPIV, DPP8, DPP9) and prolyl

oligopeptidase (PREP, POP).¹FAP has been linked to multiple

disease states involving remodeling of the extracellular matrix

such as hepatic and pulmonary ﬁbrosis, keloid formation,

rheumatoid arthritis, and osteoarthritis.²⁻⁷FAP is also highly

expressed on activated ﬁbroblasts in over 90% of common

human epithelial tumors.^8,9It has been demonstrated in

syngeneic mouse models that FAP activity promotes tumori-

genesis and that FAP inhibition attenuates tumor growth.¹⁰⁻¹²

The enzyme is furthermore expressed only transiently during

wound healing and is essentially absent in normal adult tissues

and in nonmalignant tumors.¹³These appealing characteristics

of FAP account for its ongoing evaluation as a drug target. Both

immunotherapy and small-molecule based approaches have so

far been reported, most of them focusing on applications in the

oncology domain (vide infra).

FAP possesses both dipeptidyl peptidase and endopeptidase

activity, catalyzed by the same active center. This is in contrast

with the DPPs, possessing only the former activity type, and

Received: January 8, 2014

Published: March 11, 2014

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Several of the relevant FAP inhibitors reported in the

literature were used as reference compounds in this study. Val-

boroPro 1 (talabostat, PT-100) is an nonselective boronic acid

inhibitor that reached phase II clinical trials for several cancer

types before it was withdrawn, apparently because of both

safety and eﬃcacy reasons (Figure 1 and Table 1).¹⁹⁻²¹

acetamide group as the N-substituent instead of a 4-pyridinoyl

group was found not to be selective toward PREP.²⁵

Nonetheless, no in vivo PK data have so far been published

for 6.²⁶

In our earlier report on FAP inhibitors related to 4, we have

investigated the quinolinoyl moiety’s substitution pattern and

the inﬂuence of the position of the heterocyclic N-atom on

FAP aﬃnity and selectivity. No heterocyclic scaﬀolds other than

quinoline and isoquinoline were so far built in at this position.

Evaluating the impact of other azaheteroaromatic rings was

therefore considered a ﬁrst goal of this study with which we

aimed to signiﬁcantly expand our current SAR knowledge for

this part of the molecule. Second, SAR for the P2 glycine

residue also remained underexplored, as no fragments other

than glycine have been introduced at this position. Several P2-

modiﬁed analogues were therefore made. Furthermore, these

molecules were deemed of additional interest to anticipate on

potential sensitivity of the P2 glycine amide bonds to unspeciﬁc

proteases in vivo. Third, we sought to further optimize the P1

cyanopyrrolidine residue of our inhibitors. This was done by

applying predictive SAR data that we derived earlier for

inhibitors containing a P1-(2-cyanopyrrolidine) moiety.^24,27In

the P1-modiﬁed series, we also investigated the importance of

the warhead on our molecules and the inﬂuence on inhibitory

potency of changing the carbonitrile function for other

electrophilic warhead types (boronic acid, chloromethyl

ketone). Fourth, in vitro PK and cellular toxicity data are

reported for ﬁve optimized inhibitors. On the basis of these

data, we selected four compounds for in vivo PK studies and in

vivo inhibition experiments in mice.

Figure 1. Relevant FAP inhibitors used as references in this study.

Linagliptin 2 has received clinical approval as a DPPIV inhibitor

but also displays substantial FAP aﬃnity.²²Compound 3 is a

representative of a series of pyroglutamyl(2-cyanopyrrolidine)

derivatives reported by Jiaang et al.²³Compounds 4 and 5 are

part of the quinolinoylglycyl(2-cyanopyrrolidine) class of FAP

inhibitors reported recently by our group.²⁴Although no

PREP-assay results were published by Jiaang, we report here

that compound 3 does have highly satisfactory FAP over PREP

selectivity. Therefore, Jiaang’s and our molecules represent the

only two reported inhibitor chemotypes with potential for full

FAP selectivity across the panel of related enzymes evaluated.

Jiaang’s compounds nonetheless were reported to have poor

pharmacokinetic (PK) behavior in mice. The corresponding PK

data for selected representatives of our own molecules are

reported in this manuscript. In addition, Bachovchin et al. have

also recently reported the ^D-Ala-boroPro based FAP inhibitor

6.²⁴This compound’s approximate 40-fold selectivity toward

PREP (determined under our assay conditions) is remarkable

for a boronic acid, certainly since a recently published pseudo

peptide boronic acid inhibitor with the same ^D-Ala-boroPro

scaﬀold but with an acetyl-Arg-2-(2-(2-aminoethoxy)ethoxy)-

CHEMISTRY

■

A total of around 60 novel inhibitors were synthesized for this

study. All compounds were prepared following the general

strategies in Schemes 1 and 2, in which target compounds are

clustered according to the modiﬁcation type they contain,

relative to reference compound 4. The preparation of inhibitors

containing an adapted P2 group (9−11 and 16−27) is

summarized in Scheme 1. Three types of modiﬁcations were

included in this series: (1) attachment of a side chain to the α-

methylene group of the P2 moiety, (2) introduction of N-

substituents, and (3) replacement of the P2−P3 amide bond by

a reduced analogue or a peptidomimetic triazole ring. For the

preparation of the α-methylene substituted series (Scheme 1,

entry 1), the corresponding, commercially available Boc-

protected P2-amino acids were coupled to 2-cyanopyrrolidine.

The Boc group of the coupled products was then removed

using tosylic acid in acetonitrile under conditions we identiﬁed

as optimal to avoid Ritter-type addition of the intermediate tert-

Table 1. IC₅₀of Reference FAP Inhibitors

a

IC₅₀(μM)

b

compd

FAP

PREP

DPPIV

DPP9

DPP2

SI (FAP/PREP)

c

1

2

3

4

5

6

0.066 0.011

0.37 0.002

0.98 0.06

>100

0.022 0.001

0.0020 0.0002

>100

ND

0.086 0.007

>100

14.8

>100

>50

>250

5882.4

83.5

0.017 0.001

0.0103 0.0004

0.011 0.0004

0.025 0.001

>100

0.86 0.07

>50

>100

>4500

39.6

0.99 0.04

>100

a

b

c

Determined under our own assay conditions. SI stands for “selectivity Index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]). ND stands for “not

determined”.

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a

Scheme 1. Synthesis of P2-Modiﬁed FAP Inhibitors 11−14 and 16−27

a

Reagents and conditions: (a) HATU, N-Boc-AA-OH, DIPEA, 75−80%; (b) TsOH 1.4 equiv, MeCN, 24 h, 84−86%; (c) 4-quinolinoyl chloride

hydrochloride, DIPEA, 25−86%; (d) (2S)-(2-cyanopyrrolidine) tosylate, HATU, DIPEA, 64%; (e) K₂CO₃, RNH₂, 13−52%; (f) K₂CO₃, quinolin-4-

ylmethanamine, 25−49%; (g) NaN₃, DMF; (h) CuI, THF, 17−50%.

butyl cation to the nitrile group of products 7−9. These

products were coupled with 4-quinolinoyl chloride hydro-

chloride to yield ﬁnal compounds 11−13. The 2-cyanopyrro-

lidine starting material was obtained as described earlier.²³For

molecules involving the introduction of N-substituents or P2−

P3 amide group modiﬁcations, (S)-1-(2-chloroacetyl)-

pyrrolidine-2-carbonitrile 15 was used as a central intermedi-

ate²⁸(Scheme 1, entry 2). Reaction of 15 with primary amines

and subsequent coupling with 4-quinolinoyl chloride hydro-

chloride yielded compounds 16−20. To obtain target

compounds 21 and 22 with a reduced P2−P3 amide group,

the chloroacetyl group of 15 was substituted with N-substituted

(4-quinolinyl)methylamines. Finally, the peptidomimetic tri-

azole containing compounds were made by nucleophilic

substitution of 15’s chloro group with sodium azide and

subsequent Cu(I)-catalyzed 1,3-dipolar addition with the

desired alkynes to give products 24−27.

prepared as reported earlier or as described in the Supporting

Information.²⁹Heteroarylcarboxylates were acquired commer-

cially or, in the case of substituted 4-quinolinoyl derivatives,

synthesized using the Sandmeyer isatin synthesis followed by

the Pﬁtzinger reaction and decarboxylation.^30,31Detailed

procedures for all intermediates and compounds in this report

are given in the Supporting Information. The boronate, the

chloromethyl ketone, and unsubstituted pyrrolidine derivatives

were synthesized in an analogous manner (Scheme 2, entry 2).

Again, N-(4-quinolinoyl)glycine 28 was coupled to the desired

pyrrolidine derivative using standard peptide coupling

techniques. Both the required 2-boronyl- and chloromethyla-

cylpyrrolidine derivatives were obtained as described in the

literature.^32,33To obtain boronic acid ﬁnal compound 66, its

pinanediol ester precursor 65 was deprotected using phenyl-

boronic acid.

Scheme 2 describes the preparation of P3- and P1-modiﬁed

analogues. Two strategies were followed for all reported

compounds bearing a 2-cyanopyrrolidine derivative in P1

(29−63) (Scheme 2, entry 1). For molecules designed to

mainly generate SAR data for the P3 position, diverse

heteroarylcarboxylates were coupled to a speciﬁc glycyl-(2-

cyanopyrrolidine) derivative using standard peptide coupling

reagents. Alternatively, for a number of compounds designed

mainly to render P1 SAR information, N-(4-quinolinoyl)glycine

28 was coupled to a desired 2-cyanopyrrolidine derivative. The

2-cyanopyrrolidine, its 4S-ﬂuoro, 4R-ﬂuoro, and 4,4-diﬂuoro

congeners and all glycine adducts of these molecules were

RESULTS AND DISCUSSION

■

All synthesized compounds were evaluated as inhibitors of FAP,

DPPIV, DPP9, DPPII, and PREP.²⁷No separate screening

experiments were carried out on DPP8. On the basis of the

enzyme’s close homology with DPP9 and the outcome of

earlier directed studies, potencies toward both enzymes can be

expected to be comparable with a high degree of conﬁdence.

Table 2 summarizes the assay results of the P2-modiﬁed

analogues. In a ﬁrst set of compounds (11−14), the P2-glycine

residue of 4 was replaced by ^D-Ser and related D-Ala, L-Ala, and

1-amino-1-carboxycyclopropane moieties. The choice for the

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a

Scheme 2. General Synthetic Strategies for the Synthesis of P1 and/or P3 Modiﬁed Target Compounds

a

Reagents and conditions: (a) YCOOH, HATU, DIPEA, 3 h, 43−67%, or YCOOH, HOBT, EDC, DIPEA, 16 h, 84−86%, or YCOOH, (CH₃)₂C

C(Cl)N(CH₃)₂, DIPEA, 25−70%, or quinoline-4-carbonyl chloride hydrochloride, DIPEA, 55−59%; (b) pyrrolidine derivative, HOBT, EDC,

DIPEA, 16 h, 65−86%; (c) phenylboronic acid, MTBE−H₂O, 79%.

ﬁrst two residues was based on an extensive screening of

ﬂuorogenic peptide substrates by Edosada et al., demonstrating

that FAP’s endopeptidase capability is strictly limited to

peptides with a P2-Gly, ^D-Ser, or D-Ala residue.³⁴As an

additional illustration, a P2-^D-Ala residue present in Bach-

ovchin’s boronic acid FAP-inhibitor 6 also demonstrated low

nanomolar FAP aﬃnity and good selectivity toward PREP.²⁶

When introduced as a replacement for compound 4’s glycine

residue however (11 and 12), the FAP aﬃnities observed

dropped 600- and 300-fold, respectively. FAP inhibition was

completely abolished by introducing the closely related amino

acids ^L-Ala and 1-amino-1-carboxycyclopropane at P2. While

these data underscore the importance of a P2-glycine in our

inhibitors, the lack of aﬃnity of 13 and 14 also serves as

conﬁrmation that Edosada’s original ﬁndings on substrates

translate similarly for inhibitors. Finally, ^L-Ala containing

analogue 13 has a PREP potency that stands out among the

four congeneric inhibitors discussed.

Moving further with our SAR exploration of the P2 position,

we decided to synthesize analogues with an N-alkylated and/or

a reduced P2−P3 amide bond. Taking into account that N-

alkylation of amide bonds in peptides is a well-known way of

reducing susceptibility to (non-FAP-related) proteolytic

activity, both modiﬁcation types could increase the metabolic

stability of this potentially labile part of the inhibitors’ basic

structure. In the N-alkylated amide series (compounds 16−20),

substituents of varying size and electronic properties were

tested. All these interventions were found to lead to a nearly

complete loss of FAP potency, even in the case of the smallest

methyl substituent. Remarkably, PREP aﬃnity was aﬀected to a

signiﬁcantly smaller extent, bringing on additional evidence that

the 4-quinolinoyl substituent of 4 is involved in a speciﬁc

interaction with FAP’s active center that signiﬁcantly adds to

target aﬃnity but is disrupted by small structural changes.

Similar conclusions were drawn from the assay results of the

reduced analogues: tertiary amines 21−23 also had strongly

reduced FAP inhibition. It is, however, impossible to state to

what extent the increased conformational freedom of the

quinolinylmethyl substituent or the lack of a conjugated,

electron withdrawing carbonyl function contributes to these

ﬁndings. Furthermore, a basic amine functionality is present in

these molecules. Apparently this feature does not allow picking

up additional potency from salt-bridge formation with FAP’s

Glu203 and Glu204 residues, the interaction responsible for

FAP’s recognition of dipeptide substrates. Neither does it seem

to be involved in a signiﬁcant interaction with the other DPPs’

homologous GluGlu motifs, as reﬂected by the assay results for

these enzymes. On the basis of the obtained results with the set

of inhibitors 16−23, we did not prepare any additional

analogues in this series. Fitting in the same framework of

metabolic P2−P3 amide stabilization, molecules (24−27) were

then prepared in which this amide group is replaced by an

isosteric, 4-substituted 1,2,3-triazole ring. The 4-alkynyl

derivatized quinolines required to obtain triazole analogues of

4 were, however, found not to be readily accessible. This led us

to prepare analogues of older FAP inhibitors that we reported:

N-acylated glycyl(2-cyanopyrrolidines) with a P3-benzoyl or

naphthoyl substituent. The compounds obtained were again

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Table 2. IC₅₀Data of Inhibitors with a Modiﬁed P2 Residue

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Table 2. continued

a

SI stands for “selectivity index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]).

found to have reduced inhibitory aﬃnity, with phenyl analogue

24 possessing around 8-fold less potency compared to the

benzoyl amide. Naphthyl-containing 27 is characterized by an

even higher aﬃnity loss compared to its 1-naphthoyl

substituted parent compound that possesses FAP submicro-

molar potency. Not satisﬁed by these results, we concluded that

the 4-substituted-1,2,3-triazole is not a good amide isostere at

the glycine P2 position.

Identiﬁcation of these other potentially interesting parts of

chemical space was then attempted with compounds 38−44.

These contain heteroaromatic P3 substituents that are more

distant from the pyridine- or quinoline-based systems that we

so far discovered to be optimal. Pyridazine (in inhibitor 38) was

the only azine with more than one nitrogen heteroatom that we

evaluated. Already, this compound had a 3-fold decreased FAP

potency relative to 29. This ﬁnding withheld us from investing

eﬀort in preparing target molecules with a triazine isomer at P3.

Conversely, we shifted attention to more electron-rich, ﬁve-

membered azaheterocycles. Pyrrole was not selected for this

subset because of stability reasons, but a suitable (vide supra)

imidazole derivative and two triazole isomers were included

(compounds 39−41). These molecules, together with the

homologated 42 and the thia- and oxazole derivatives 43 and

44, all performed signiﬁcantly less than 29 in the FAP

inhibition assay. Finally, mutation of the pyridine ring into an

aniline was found not to be beneﬁcial for FAP aﬃnity either:

the aniline 45 and its triﬂuoroacetylated precursor 46 have IC₅₀

values in the low micromolar range, more than 100-fold less

than pyridine 29. Taken altogether, the data in Table 3 were

not compelling enough to immediately continue further

structural exploration of monocyclic heteroaromatic P3 rings:

FAP potencies observed in this series were not able to compete

with reference 4. In the case of the highly eﬃciently binding

inhibitor 29, further optimization did not seem obvious. In

addition, the FAP/PREP selectivity indices observed, so far not

mentioned in the framework of this series, were generally very

satisfactory for the pyridine subset but again not signiﬁcantly

better than the optimal quinolines described earlier. It deserves

highlighting however that as reported by Poplawski et al.,

introduction of a P3−P4-pyridinoyl substituent has been used

to obtain a series of highly potent, boronate-based FAP

inhibitors represented by reference compound 6.²⁶

Investigations of the P3 region of inhibitors related to 4 was

aimed at replacing its quinoline ring with various other

azaheterocycles. Our earlier studies, during which we had

already investigated quinoline and isoquinoline isomers, clearly

demonstrated the position of the azaheteroaromatic nitrogen to

be crucial, with appreciable FAP aﬃnity only present in case of

the 4-quinolinoyl isomer of compound 4. In all compounds

reported here, we therefore limited the selection of heterocycles

to systems that have a azaheteroatom topology for at least one

nitrogen that is comparable to that of the 4-quinolinoyl ring

nitrogen. Table 3 summarizes the data obtained for a number of

(substituted) ﬁve- and six-membered heteroaromatic contain-

ing analogues of 4. The closely related pyridine 29 was found to

have a 6-fold reduced FAP potency compared to 4. In spite of

its lower absolute aﬃnity, 29 has the highest ligand eﬃciency

(0.38) of all compounds we hitherto prepared. Introduction of

substituents in 2-position of the pyridine ring as in compounds

30−35 resulted in a further decrease of inhibitory potency, but

no additional steric or electronic determinants of FAP potency

could be discerned here: all compounds from this subset seem

to have inhibitory potential within the same order of

magnitude. Additionally, an analogue containing an extra

methylene linker (36) and a reduced P3-heteroaromatic ring

system (piperidine 37) were prepared. Similarly, these

molecules did not demonstrate better FAP aﬃnities than 29

and also convey the hypothesis that high FAP-aﬃnity-

conferring P3 substituents are mainly present in limited parts

of heteroaromatic chemical space.

For the reasons cited above, we then turned attention to

bicyclic P3 heteroaromatics with at least one nitrogen atom that

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Table 3. IC₅₀Data of Five- and Six-Membered P3 Heterocycles

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Table 3. continued

a

SI stands for “selectivity index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]).

is equivalent to the azaheteroatom in quinoline 4 (Table 4).

With the set of inhibitors 47−51, 2,3-annelated pyridines were

evaluated. Pyrrolopyridine 47 was found to be a nanomolar

FAP inhibitor but still almost 5-fold less potent than 4.

Selectivity indices with respect to PREP were largely

comparable for both (SI_[FAP/Prep]values were calculated to be

∼50 and 80 for 47 and 4, respectively). Substituted

imidazopyridines 48 and 49 and certainly triazolopyridine 50

were signiﬁcantly less potent as FAP inhibitors than 4,

suggesting that introduction of additional nitrogen atoms in

the annelated ﬁve-membered heterocycle leads to lower target

aﬃnities. A similar eﬀect seems to be in place when a six-

membered heterocycle is annelated with pyridine (1,7-

naphthyridine 51). The IC₅₀value of this azaquinoline

derivative is roughly 3 times higher than that of quinoline 4;

nonetheless, selectivity toward PREP is slightly better than that

of the reference.

Since a P3-quinoline residue out of all the heterocycles

investigated still proved to be optimal, we decided to return to

this scaﬀold and investigate the eﬀect of hitherto unexplored

substitution patterns at its 5 and 7 positions. Our preliminary

data had already indicated that introduction of 5- or 7-chloro

and bromo substituents resulted in low nanomolar FAP

inhibitors with optimal selectivity toward PREP (SI_[FAP/Prep]

of up to 10³). Similar FAP potencies were obtained here with

the methyl-substituted analogues 52 and 53, of which the 5-

substituted congener clearly had the best FAP/PREP selectivity

seen so far (SI_[FAP/Prep]= 2 × 10³). Introduction of bulkier

phenyl-based groups (compounds 54−56) seems less desirable

at both the 5 and 7 positions: FAP potencies drop close to 1

order of magnitude. Because of the relatively small size of the

methoxy substituent, one would furthermore expect the 5-

methoxylated inhibitor 57 to have potencies in the same range

as 5 or 53. Surprisingly however, potency was reduced more

than 1000-fold, again indicating that the supposed speciﬁc

interaction of the quinolinoyl system with FAP’s active center

can be critically disturbed by small structural changes.

Additional investigation of this ﬁnding was undertaken during

the optimization of the P1 position (vide infra, Table 5).

As part of an earlier study, we had shown that in the glycyl(2-

cyanopyrrolidine) series, FAP’s S1 pocket preferentially

accommodates 2-cyanopyrrolidine and a limited number of

derivatives with 4-substituents of minimal steric bulk, e.g., a 4S-

ﬂuoride. Similarly, (2S,4S)-2-cyano-4-ﬂuoropyrrolidine and its

4,4-diﬂuorinated analogue were also used earlier as P1 residues

by Jiaang in FAP inhibitors related to 6. Depending on the

target, these residues have also been investigated with

considerable success in compounds targeting DPPIV and

DPP8/9. The same strategy was applied to the inhibitor series

studied here.²⁷Complementarily, we also prepared the

(2S,4R)-2-cyano-4-ﬂuoropyridine diastereomer and introduced

it as a P1 residue. Supposedly relating to its more elaborate

synthesis involving a chirality inversion step on the 4-position

of starting material trans-4-hydroxyproline, this isomer so far

had not been investigated in FAP or related DPP inhibitors. To

allow eﬃcient assessment of the eﬀect of introducing each of

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Table 4. IC₅₀Data of Bicyclic P3 Heterocycles

a

b

SI stands for “selectivity index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]). Solubility problems upon dilution of compounds in aqueous buﬀer,

resulting in poor reducibility of inhibition curves.

the three mono- and diﬂuorinated P1 isomers, direct analogues

of reference compound 4 were prepared (58−60). Grossly in

line with expectations, the 4S-ﬂuoropyrrolidine 58 displayed

around 3-fold higher FAP aﬃnity than the parent compound.

PREP-aﬃnity however had also increased slightly more than

proportionally with FAP-potency. This is consistent with our

earlier mentioned report that included 4S-ﬂuorinated (2-

cyano)pyrrolidine inhibitors.²⁷Most remarkably, the 4R-

ﬂuoropyrrolidine isomer 59 has lost FAP aﬃnity compared to

4 and 58, amounting to up to 3 orders of magnitude. Making

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Table 5. IC₅₀Data of Inhibitors with a Modiﬁed P1 Residue

a

SI stands for “selectivity index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]).

Table 6. Inﬂuence of the Warhead on IC₅₀

a

SI stands for “selectivity index” (calculated as [IC₅₀(PREP)/IC₅₀(FAP)]).

steric factors less likely to be accountable for this eﬀect, the

corresponding 4,4-diﬂuorinated analogue of 4 (UAMC-1110,

vide infra) was one of the most potent FAP inhibitors identiﬁed

to date. Seminal studies by Raines et al. have shown that 4S-

ﬂuoroproline derivatives possess a hyperconjugatively stabilized

endo-puckered ring conformation, while in 4R-ﬂuoroprolines

an exo-puckered ring conformer is more favored. This

diﬀerence, which should also be present with 2-cyanopyrroli-

dine derivatives, could be contributing to the large potency

diﬀerences observed between 58 and 59.³⁵Analogously, 4,4-

diﬂuoroproline derivatives have been demonstrated to have

endo- and exo-puckered conformations of comparable energy,

similar to unsubstituted prolines.³⁶Although this would imply

that the slightly higher FAP aﬃnity of 60 compared to 4 cannot

be rationalized using conformational arguments, increased

hydrophobicity resulting from diﬂuorination might be in play

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a

Table 7. In Vitro Pharmacokinetic Properties of Selected FAP Inhibitors

parameter

4

5

68

>200

60

>200

61

kinetic solubility (μM)

log D

>200

nd

0.5

0.7

0.8

1

plasma stability (% unchanged at 6 h) (mouse/rat/human)

microsomal stability (% unchanged at 6 h) (mouse/rat)

cytotoxicity (MRC-5 cells) (μM)

100/90/nd

nd

90/nd/100

75/nd

>64

nd/100/80

90/nd

>64

85/95/nd

90/94/nd

>64

nd

>64

a

nd stands for “not determined”.

Table 8. In Vivo PK Properties of Selected FAP Inhibitors in Rats

T_max(h)

C_max(μg/mL)

T_1/2(h)

AUC (μg·h/mL)

Cl (mL/min)

V_d(L)

relative bioavailability (%)

52

a

5 (iv)

6.5

5.6

1.73

1.7

6.1

13.4

23.4

76.7

11.1

39.39

11.7

23.0

1.75

3.5

b

5 (po)

0.33

0.75

a

60 (iv)

11.8

14.6

8.5

1.74

3.4

2.83

0.43

0.34

0.77

0.86

74

79

b

60 (po)

1.55

6.5

a

61 (iv)

1.40

1.22

b

61 (po)

14.7

8.2

a

b

Compound was formulated in PEG₂₀₀and administered via single intravenous injection at 5 mg/kg. Compound was formulated in PEG₂₀₀and

administered per os (gavage) at 20 mg/kg.

here. Noteworthy, 60 was also found to have better FAP/PREP

selectivity and a very proﬁcient ligand eﬃciency of 0.34, which

is signiﬁcantly higher than the corresponding value calculated

for 4 (0.27).

for FAP inhibitors without a warhead group. Next, the boronic

acid warhead, present in many of the older series of (mostly

nonselective) FAP inhibitors, was evaluated in 66. This

molecule was found to be highly potent but also considerably

less selective toward PREP when compared to its nitrile-based

counterpart 4 ((SI_[FAP/Prep]of 3 vs ∼85, respectively).

Evaluation data of Bachovchin et al.’s most promising boronate

6 (that under our conditions had SI_[FAP/Prep]= 40) together

with the PREP aﬃnities of our own inhibitors 12 and 13

nonetheless suggest that this selectivity could be improved by

selecting a ^D-Ala P2 group. Finally, the enzymatic assay data for

chloromethyl ketone 67show this compound to be 17 times

less potent than nitrile 4. Detailed kinetic analysis also revealed

this molecule not to bind irreversibly to the target enzyme.

Taking into account the number of low nanomolar FAP

inhibitors with high FAP/PREP selectivity that we had

discovered as part of this and the foregoing study, four of the

most promising compounds were then selected for which in

vitro pharmacokinetic parameters were determined (log D_7.4,

kinetic solubility, stabilities in mouse, rat, and/or human

plasma) (Table 7). Compound 4, for which we reported these

parameters earlier, was used as a reference. In general, no large

diﬀerences between individual inhibitors were observed for the

evaluated parameters. Compounds with a P1-(2-cyano-4,4-

diﬂuoropyrrolidine) residue were found to behave similarly to

the nonﬂuorinated molecules in the series (4, 5, and 46 vs 60

and 61). The log D_7.4values determined varied between 0.5 and

1. Kinetic solubility in all cases was >200 μM, and plasma

stabilities were satisfactory throughout the assay results with

half-lives exceeding 6 h for all inhibitors and in all media tested.

For compounds 5, 60, and 61, stability in the presence of

mouse and/or rat hepatic microsomes was also tested.

Gratifyingly, these molecules were found not to be subject of

fast oxidative metabolization and, again, displayed half-lives of

more than 6 h. Finally, cytotoxicity was evaluated on MRC-5

cells. At the highest concentration measured (64 μM), no signs

of cellular toxicity could be observed for the compounds in

Table 7. Additional cytotoxicity data for other compounds in

this report can be retrieved from the Supporting Information.

These are comparable with the data shown here, although

To further expand the P1-4,4-diﬂuorinated subset, com-

pounds 61−63 were synthesized. These diﬀer by their P3-

quinoline residues’ substitution patterns. The 6-methoxyquino-

line 61 is the diﬂuorinated analogue of an inhibitor that we

reported in our foregoing study.²³Both the low nanomolar

FAP potency and thousandfold FAP/PREP selectivity of these

two molecules are highly comparable. Compounds 62 and 63

were speciﬁcally included to extract additional information on

the SAR of the P3-quinoline’s 5-position (vide supra).

Surprisingly, 62 and 63 were found to be micromolar FAP

inhibitors. Although the 5-methoxylated compound 57 (Table

5) had a similar proﬁle, drawing the general conclusion that

introduction of substituents at the 5-position reduces FAP

aﬃnity, would oversee reference molecule 5. The latter all but

compares to its diﬂuorinated counterpart 62, but together with

its 5-chlorinated analogue that we also published earlier, it

belongs to the set of most potent and selective molecules

discovered.²⁹More subtle hypotheses related to the com-

pound’s binding kinetics might therefore be required to

tentatively explain the observed behavior. It is clear that

invoking only the steric or electronic parameters of the

quinolone substituents is not suﬃcient to rationalize the assay

data of all 5-substituted molecules we prepared.

Finally, we investigated how either omitting or changing the

warhead function to a boronic acid would impact FAP potency

and selectivity of our molecules. We also selected the well-

known chloromethyl ketone warhead to verify whether

modiﬁcation to irreversible inhibitors could be possible. All

molecules prepared were analogues of reference 4 (Table 6).

First, building in an unsubstituted pyrrolidine residue at P1 in

64 was found to give a 3 log reduction in potency compared to

reference compound 4. Qualitatively, this ﬁnding is in line with

earlier reports by our own group and by Jiaang et al. and

underscores the very considerable contribution to inhibitor

aﬃnity from the warhead function. Compared to the

mentioned examples though, the residual micromolar aﬃnity

of 64 still is signiﬁcant and one of the best potencies reported

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compound 57 did show slight toxicity at concentrations greater

than 24 μM.

CONCLUSION

■

In this study we have built a thorough structure−activity

relationship around the 4-quinolinoyl-Gly-cyanoPro scaﬀold.

First, replacing the P2 glycine residue with a limited set of other

amino acids was found to decrease FAP potency signiﬁcantly

for each alternative residue evaluated. Adding substituents to

the P2−P3 amide bond or changing it with a triazole peptide

bond isostere abolished almost all FAP aﬃnity. Second, an

extended series of azaheterocycles was evaluated at the P3

position in order to identify alternatives for the quinoline

group. Of all moieties evaluated, only pyridines resulted in

inhibitors with appreciable FAP aﬃnity, albeit in absolute terms

still less potent than their quinoline-bearing analogues.

Furthermore, we extended our investigation of the quinoline

ring’s substitution pattern. We conﬁrmed with more potent

inhibitors our earlier preliminar observation that substituents at

the 5-position of the quinoline moiety can improve selectivity

toward PREP while maintaining FAP aﬃnity. Nonetheless,

notable outliers of this hypothesis were identiﬁed as well (vide

infra). Third, the P1 position was investigated. We found that

either 4S-ﬂuoro or 4,4-diﬂuoro substitution of the 2-

cyanopyrrolidine ring can be used to optimize FAP aﬃnity

and selectivity with respect to PREP. Introduction of a 4R-

ﬂuoro substituent on the other hand was found to give rise to

far less potent compounds, a feature that we tentatively

rationalized using stereoelectronic arguments. Furthermore, we

found that the combined presence of a 5-substituted

quinolinoyl residue in P3 and a 2-cyano-4,4-diﬂuoropyrrolidine

in P1 unexpectedly leads to drastic FAP aﬃnity loss. Although

we do not have a clear explanation, this ﬁnding suggests that

FAP imposes strict structural requirements on its inhibitors that

seem related to the possibility of covalent bond formation

between the enzyme and the nitrile warhead present in these

molecules. Investigating this feature in depth, we prepared

compounds in which the nitrile warhead was either omitted or

replaced by a boronate or chloromethyl ketone. The presence

of a warhead in the studied series of molecules was shown to be

indispensable for high aﬃnity, and a nitrile group was found to

be preferable above the boronate function, mainly in terms of

inhibitor selectivity. The chloromethyl ketone function,

although of potential interest for the synthesis of activity-

based probes for FAP, did not lead to irreversible inhibition or

to high target aﬃnity, again suggesting defected interaction with

the enzyme’s catalytic serine.

The in vivo PK parameters were then determined for

inhibitors 4, 5, 60, and 61 in rats. Six male rats were treated for

each inhibitor tested, three of which received the compound via

a single intravenous (iv) administration at 5 mg/kg. The other

three animals were dosed per os (po) at 20 mg/kg. Blood

samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 h

after administration. Inhibitor concentrations were determined

using UPLC−MS/MS, and pharmacokinetic parameters were

calculated using standard algorithms. Notably, all rats subjected

to 4 were found to die within 6 h. No signs of toxicity were

observed during the observation period or upon autopsy in any

of the other animals treated with compounds 5, 60, and 61, and

parameters for these molecules are summarized in Table 8.

Taking into account the high degree of structural similarity

between the four compounds evaluated, we do not have a clear

view on possible factors that could explain the singular toxicity

to rats of 4.

All inhibitors evaluated displayed signiﬁcant, roughly

comparable oral bioavailabilities (50−79%) and reasonable

elimination half-lives (1.5−3 h). These data were comple-

mented with experiments in which we followed up the extent of

plasma FAP inhibition in the inhibitor-treated rats. A soluble

form of the enzyme is present in plasma, where it was originally

characterized as α₂-antiplasmin cleaving enzyme (APCE).²⁵In

brief, residual FAP activity was determined in the same plasma

samples that were collected for the determination of inhibitor

concentrations after iv and po administration. FAP activity was

measured using the commercially available substrate Z-Gly-Pro-

AMC under conditions that excluded interference from soluble

PREP activity.³⁷It deserves mentioning that for the ex vivo

assay plasma samples are diluted 100-fold, potentially leading to

underestimation of the amount of FAP that is blocked in vivo.

At each of the investigated time points (5 min to 24 h) after iv

or oral administration of compound 60, the residual FAP

activity measured ex vivo was less than 15% when compared

with vehicle controls. Also after oral administration of

compounds 5 and 61, more than 85% inhibition of FAP

activity was observed for all time points. Whereas at the 4 h

time point after iv administration of the latter two compounds,

the measured % inhibition still reached more than 75%. The

FAP activity measured at the 24 h time point was around 50%

compared to vehicle controls. All ex vivo FAP measurements

point to a complete or near complete and prolonged inhibition

of plasma FAP activity in vivo after single doses of compounds

5, 60, and 61. The in vivo observations corroborate the in vivo

pK parameters C_maxand T_1/2and the in vitro stability testing.

To check whether slow, tight binding might be contributing to

maintaining long-term FAP inhibition, a kinetic and mecha-

nistic investigation was performed for compound 60, as this

compound resulted in the most extensive and prolonged

inhibition of FAP in the PK studies. No tight binding behavior

was observed, and the inhibitor proved to bind reversibly to

FAP. We therefore conclude that the longer in vivo half-life of

60 (compared to 5 and 61) is the main contributor to the

prolonged ex vivo inhibition of FAP activity observed for this

compound. On the basis of the high degree of structural

similarity between 5, 60, and 61, we assume comparable kinetic

behavior for these molecules. A more extended in vivo study

using several, especially lower doses of these compounds is

needed to conﬁrm this hypothesis.

A selection was made among the most promising FAP

inhibitors discovered so far in the studied series. In vitro PK

and toxicity parameters were determined for these, generally

predicting potential for satisfactory in vivo behavior. Four of

these compounds were then submitted to in vivo PK analysis in

rats. The corresponding data show that all four compounds

tested had good oral bioavailability and half-lives. However,

some questions remain with respect to compound 4. We were

surprised to see that all rats treated with this compound died

within 6 h after compound administration, while the rats

treated with the other, structurally highly similar inhibitors did

not show signs of toxicity under an identical dosing regimen.

Related to this, we do not have indications of a likely oﬀ-target

of 4. It is also highly speculative to suggest that the toxicity

mechanism of 4 could be related to that of allo-Ile-isoindoline, a

DPP8/9 inhibitor for which a putative oﬀ-target eﬀect has been

invoked to rationalize in vivo toxicity in rats and dogs.³⁸

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for PREP assays. DPPIV assays used Gly-Pro-p-nitroanilide (100 μM)

at pH 8.3. DPPII assays were carried out using Lys-Ala-p-nitroanilide

(1 mM) at pH 5.5, and DPP9 assays used Ala-Pro-p-nitroanilide (300

μM) at pH 7.4 . The substrate concentrations were chosen around the

K_mvalue obtained under the assay conditions used. Mouse and rat

plasma came from Innovative Research. The turbidity in the kinetic

solubility experiments was measured using the UV/vis spectropho-

tometer Synergy MX, Biotek with Gen5. MRC-5SV2 cells (human

embryonic diploid ﬁbroblasts) were obtained from Sigma and cultured

in MEM + Earl’s salts medium, supplemented with ^L-glutamine,

NaHCO₃, and 5% inactivated fetal calf serum. All cultures and assays

were conducted at 37 °C under an atmosphere of 5% CO₂. Male

Wistar rats (body weight of ∼250 g) were acquired from Janvier

(France).

The following section comprises the synthetic procedures and

analytical data for all ﬁnal compounds reported in this manuscript. The

corresponding data for synthesis intermediates en route to ﬁnal

compounds can be found in the Supporting Information. Synthesis

procedures that were used in the preparation of several ﬁnal products

are summarized here as “General Procedures”.

General Procedure A (Formation of the P2−P3 Amide

Bond). The carboxylic acid (0.065 g, 0.377 mmol) was dissolved in 3

mL of DMF, and N-ethyl-N-isopropylpropan-2-amine (0.210 mL,

1.168 mmol) and HATU (0.121 g, 0.377 mmol) were added. After 15

min a solution of (S)-1-(2-aminoacetyl)-pyrrolidine-2-carbonitrile

triﬂuoroacetate or tosylate (0.085 g, 0.377 mmol) in DMF was

added. The mixture was stirred for 3−4 h at room temperature. The

volatiles were evaporated. The residue was dissolved in ethyl acetate

and washed with 1 N citric acid, saturated sodium bicarbonate, and

brine. The solution was dried over sodium sulfate, ﬁltered, and

evaporated. It was puriﬁed using column chromatography using

DCM−methanol.

General Procedure B (Deprotection of Boc Groups). Tosylic

acid (1.4 equiv) was added to a cold (0 °C) solution of the Boc-

protected product (1 equiv) in acetonitrile (0.4 M) at 0 °C. After 30

min the mixture was allowed to warm to room temperature and stirred

for 24 h. The volatiles were evaporated and the mixture was washed

with cold ethyl acetate to remove N-tert-butylacetamide. The white

precipitate formed is the product.

General Procedure C (Formation of the P2−P3 Amide Bond

of Compounds with a P3-(4-Quinolinoyl) Residue). Quinoline-4-

carbonyl chloride hydrochloride (0.102 g, 0.448 mmol) was added to a

solution of the amine (0.448 mmol) and DIPEA (0.217 mL, 1.242

mmol) in DCM and stirred until completion as followed with LC−MS

(usually 2−3 h). On completion of the reaction, it was diluted with

DCM−isopropanol (4:1), washed with saturated aqueous NaHCO₃

solution (10 mL), 0.1 N aqueous citric acid solution (10 mL), and

brine (10 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated and puriﬁed with ﬂash chromatography.

General Procedure D (Preparation of Final Products with an

N-Alkylated P2−P3 Amide Bond). (S)-1-(2-Chloroacetyl)-

pyrrolidine-2-carbonitrile 15 (0.400 g, 2.317 mmol, 1 equiv) in THF

(0.5M) was added dropwise to a 0.3 M ice−water cooled mixture of

primary amine and K₂CO₃(0.961 g, 6.95 mmol, 3 equiv) in THF. The

mixture was stirred for 2 h at 0 °C, then another 3 days at room

temperature. After ﬁltration of K₂CO₃and evaporation of volatiles, the

concentrate was dissolved in 30 mL of DCM and washed with

saturated sodium bicarbonate (3×), brine, dried over Na₂SO₄, ﬁltered,

and concentrated to give the secondary amine as an oily substance. If

necessary the product was puriﬁed using silica gel column

chromatography with 0.1 N ammonia as an additive. Quinoline-4-

carbonyl chloride hydrochloride (0.102 g, 0.448 mmol) was added to a

solution of the secondary amine and DIPEA (0.217 mL, 1.242 mmol)

in DCM and stirred until completion as followed with LC−MS

(usually 2−3 h). On completion of the reaction, it was diluted with

DCM−isopropanol (4:1) and washed with saturated aqueous

NaHCO₃solution (10 mL), 0.1 N aqueous citric acid solution (10

mL), and brine (10 mL). The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated and puriﬁed with ﬂash chromatography

using a DCM−methanol gradient and/or preparative HPLC.

Summarizing, we have identiﬁed a series of highly potent

FAP inhibitors with promising pharmacokinetic behavior. We

believe that our selective, in vivo active inhibitors are currently

best placed among all published compounds to help elucidate

the function of FAP in diﬀerent animal models of disease and

to allow its continuing validation as a drug target.

EXPERIMENTAL SECTION

■

Unless otherwise stated, laboratory reagent grade solvents were used.

Reagents were obtained from Sigma-Aldrich, Acros Organics, Apollo

Scientiﬁc, Manchester Organics, or Fluorochem and were used

without further puriﬁcation unless otherwise indicated. Character-

ization of all compounds was done with ¹H NMR and mass

1

spectrometry. H NMR spectra were recorded on a 400 MHz Bruker

Avance III Nanobay spectrometer with Ultrashield. Chemical shifts are

in ppm, and coupling constants are in hertz (Hz). Minor rotamers of

the amide bond, which were less than 10% of the major rotamer, are

not reported in the NMR data. ES mass spectra were obtained from an

Esquire 3000plus ion trap mass spectrometer from Bruker Daltonics.

Purity was veriﬁed using two diﬀerent LC/MS systems, and purities of

all ﬁnal products were found to be >95%. Water (A) and MeCN (B)

were used as eluents. LC−MS spectra were recorded on an Agilent

1100 series HPLC system using a Alltech Prevail C18 column (2.1 mm

× 50 mm, 3 μm) coupled with an Esquire 3000plus as MS detector,

and a “method A” 5−100% B, 20 min gradient was used with a ﬂow

rate of 0.2 mL/min. Formic acid, 0.1%, was added to solvents A and B.

UPLC involved the following: Waters Acquity H-class UPLC system

coupled to a Waters TQD ESI mass spectrometer and Waters TUV

detector. A Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50

mm column was used. Solvent A consisted of water with 0.1% formic

acid. Solvent B consisted of acetonitrile with 0.1% formic acid. Method

I involved the following: 0.15 min 95% A, 5% B, then in 1.85 min from

95% A, 5% B to 95% B, 5% A, then 0.25 min (0.350 mL/min), 95% B,

5% A. The wavelength for UV detection was 254 nm. Method II

involved the following: ﬂow 0.4 mL/min, 0.25 min 95% A, 5% B, then

in 4.75 min to 95% B, 5% A, then 0.25 min 95% B, 5% A, followed by

0.75 min 95% A, 5% B. The wavelength for UV detection was 214 nm.

Where necessary, ﬂash puriﬁcation was performed on a Biotage

ISOLERA One ﬂash system equipped with an internal variable dual-

wavelength diode array detector (200−400 nm). For normal phase

puriﬁcations SNAP cartridges (10−340 g, ﬂow rate of 10−100 mL/

min) were used, and reversed phase puriﬁcations were done making

use of KP-C18 containing cartridges. Dry sample loading was done by

self-packing samplet cartridges using silica and Celite 545, respectively,

for normal and reversed phase puriﬁcations. Gradients used varied for

each puriﬁcation. However, typical gradients used for normal phase

were 30 min gradient of 0−50% EtOAc in hexane to 100% hexane or

0−5% methanol in DCM to 20% methanol in DCM. For reversed

phase a gradient of 5% MeCN in water to 50% MeCN in water was

used. HRMS involved the following: The dry samples were dissolved

1

in 1 mL of methanol and diluted /₁₀₀in MeCN/H₂O, 0.1% formic

acid. Then 10 μL of each sample was injected using the CapLC system

(Waters, Manchester, U.K.) and electrosprayed through the Nanomate

(Advion, Ithaca, NY) nanoelectrospray source. The Nanomate was

operated in positive ion mode at an electrospray potential of 1.5 kV.

Samples were injected with an interval of 3 min. Positive ion mode

accurate mass spectra were acquired using a Q-TOF II instrument

(Waters, Manchester, U.K.). The mass spectrometer was calibrated

prior to use with a 0.2% H₃PO₄solution. The spectra were lock-mass-

corrected using the known mass of the nearest H₃PO₄cluster or the

phthalate background ions. The Waters Acquity UPLC system

coupled to a Waters TQD ESI mass spectrometer was also used for

LC/MS/MS measurements. Enzymes used in the IC₅₀measurements

were puriﬁed from human or animal sources (human seminal plasma

for DPPIV and DPPII, bovine testes for DPP9) or were isolated from

recombinant sources (recombinant murine FAP from human HEK293

kidney cells; recombinant human PREP from E. coli). Substrates used

in the IC₅₀measurements were Ala-Pro-p-nitroanilide (2 mM) at pH

7.4 for FAP assays and Z-Gly-Pro-p-nitroanilide (0.25 mM) at pH 7.5

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General Procedure E (Preparation of Final Products with a

P2−P3 Triazole Isostere). Sodium azide (0.863 g, 13.27 mmol) was

added to a solution of (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile

15 (1.27 g, 7.4 mmol) in DMF (25 mL) and stirred for 24 h. After

evaporation of the volatiles, the residue was dissolved in ethyl acetate

and washed with water and brine. Puriﬁcation was done using column

chromatography with a hexane−ethyl acetate gradient (20−35%) to

give (1.1 g, 83%) white crystals of (S)-1-(2-azidoacetyl)pyrrolidine-2-

to DCM−MeOH (95:5), yielding white crystals. UPLC I (ESI) t_R1.44

min, m/z 268.5 [M + H]⁺(96%); 0.47g, 83%. Then the Boc

deprotection was done using general procedure B. ¹H NMR (400

MHz, D₂O) δ 7.73−7.65 (d, J = 8.0 Hz, 3H), 7.40−7.34 (d, J = 7.9 Hz,

3H), 4.79−4.75 (m, 1H), 4.39−4.29 (q, J = 7.1 Hz, 1H), 3.83−3.73

(dt, J = 10.6, 5.7 Hz, 1H), 3.60−3.49 (q, J = 8.8 Hz, 1H), 2.45−2.36

(s, 3H), 2.37−2.12 (m, 4H), 1.54−1.44 (dd, J = 7.1 Hz, 3H); UPLC I

(ESI) t_R0.25 min, m/z 168.4 [M + H]⁺(95%); 0.5 g, 84%.

1

carbonitrile. H NMR (400 MHz, CDCl₃) (4:1 mixture of trans−cis

(S)-1-((S)-2-Aminopropanoyl)pyrrolidine-2-carbonitrile 4-

Methylbenzenesulfonate (9). tert-Butyl (S)-1-((S)-2-cyanopyrroli-

din-1-yl)-1-oxopropan-2-ylcarbamate was made using general proce-

dure A and puriﬁed by column chromatography with a gradient DCM

to DCM−MeOH (95:5). ¹H NMR (400 MHz, CDCl₃) δ 5.26 (d, J =

7.9 Hz, 1H), 4.78 (dd, J = 6.8, 3.7 Hz, 1H), 4.47−4.37 (m, 1H), 3.77−

3.58 (m, 2H), 2.33−2.12 (m, 4H), 1.42 (s, J = 6.3 Hz, 9H), 1.35 (d, J

= 6.9 Hz, 3H); UPLC I (ESI) t_R1.45 min, m/z 268.5 [M + H]⁺

(96%); 0.46 g, 81%. Then Boc deprotection was done using general

rotamers) δ 4.80 (d, J = 5.8 Hz, 0.8H), 4.63 (dd, J = 12.8, 6.4 Hz,

0.2H), 3.93 (d, J = 14.5 Hz, 1H), 3.86 (d, J = 16.0 Hz, 1H), 3.64−3.54

(m, 1H), 3.47−3.38 (m, 1H), 2.38−2.02 (m, 4H); MS (ESI) m/z

180.2 [M + H]⁺. The alkyne (0.096 mL, 0.837 mmol) was added to a

mixture of (S)-1-(2-azidoacetyl)pyrrolidine-2-carbonitrile (0.075 g,

0.419 mmol) and copper(I) iodide (7.97 mg, 0.042 mmol) in THF

(volume, 4 mL) with one drop of triethylamine. The reaction mixture

was stirred at room temperature for 24 h. The reaction mixture was

then ﬁltered, the ﬁltrate evaporated and the residue chromatographed,

yielding the title compound as a slightly yellowish transparent colorless

oil.

1

procedure B, yielding a white powder. H NMR (400 MHz, D₂O) δ

7.73−7.66 (d, J = 7.9, 2H), 7.41−7.34 (d, J = 7.9 Hz, 2H), 4.87−4.80

(dd, J = 7.5, 5.2 Hz, 1H), 4.38−4.29 (q, J = 7.1 Hz, 1H), 3.75−3.59

(m, 2H), 2.43−2.38 (s, 3H), 2.37−2.09 (m, 4H), 1.58−1.51 (d, J = 7.1

Hz, 3H); UPLC I (ESI) t_R0.19 min, m/z 168.4 [M + H]⁺(97%); 0.43

g, 77%. Optical purity was conﬁrmed with Marfey’s reagent.

General Procedure F (Formation of the P2−P3 Amide Bond).

The carboxylic acid (0.092 g, 0.814 mmol) was dispersed in dry

dioxane (3 mL) in a round-bottom ﬂask with nitrogen. To this 1-

chloro-N,N,2-trimethylprop-1-en-1-amine (0.151 mL, 1.139 mmol)

was added, and the mixture was stirred for 30 min at room

temperature. The starting material dissolved over time. Then (S)-1-(2-

aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride (0.247 g, 1.302

mmol) with N-ethyl-N-isopropylpropan-2-amine (0.307 mL, 1.709

mmol) was added, and the mixture was stirred for 2 h, evaporated to

dryness, and redissolved in ethyl acetate. This was followed by washing

with 0.1 N citric acid and saturated sodium bicarbonate and brine.

After drying over sodium sulfate, ﬁltration, and evaporation, the

product was puriﬁed using column chromatography.

General Procedure G (Formation of the P2−P3 Amide

Bond). To a solution containing the carboxylic acid (0.058 g, 0.333

mmol) and hydroxybenzotriazole (0.056 g, 0.366 mmol) in 1,4-

dioxane (3 mL) was added a solution of EDC (0.070 g, 0.366 mmol)

in DCM (2 mL). The mixture was stirred for 10 min at room

temperature. To the resulting solution were added the appropriate

amine (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile 4-methylbenze-

nesulfonate (0.130 g, 0.400 mmol) and DIPEA (0.122 mL, 0.699

mmol) in DCM (4 mL), with stirring. After 2 h or when the reaction

was complete according to HPLC, the reaction mixture was diluted

with DCM and washed with saturated aqueous NaHCO₃solution (10

mL), 0.1 N aqueous citric acid solution (10 mL), and brine (10 mL).

The organic layer was dried over Na₂SO₄, ﬁltered, and concentrated

and puriﬁed with ﬂash chromatography.

Methyl 1-(Quinoline-4-carboxamido)cyclopropane-

carboxylate (10). The title compound was prepared using general

procedure C starting from 1-aminocyclopropanecarboxylic acid,

1

producing a colorless oil. H NMR (400 MHz, CDCl₃) δ 8.92 (d, J

= 4.3 Hz, 1H), 8.33 (dd, J = 8.5, 0.8 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H),

7.77 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.63 (ddd, J = 8.3, 6.9, 1.3 Hz,

1H), 7.44 (d, J = 4.3 Hz, 1H), 6.66 (s, 1H), 3.81−3.76 (m, 3H), 1.76−

1.72 (m, 2H), 1.38 (dd, J = 8.1, 4.9 Hz, 2H); UPLC I (ESI) t_R1.14

min, m/z 271.6 [M + H]⁺(93%).

N-((R)-1-((S)-2-Cyanopyrrolidin-1-yl)-3-hydroxy-1-oxopro-

pan-2-yl)quinoline-4-carboxamide (11). The title product was

prepared using general procedure C, yielding a white powder. ¹H

NMR (400 MHz, CDCl₃) cis/trans amide conformers (3/10) δ 8.91

(d, J = 4.35 Hz, 0.3H), 8.89 (d, J = 4.33 Hz, 1H), 8.19 (dd, J = 1.29,

8.69 Hz, 1H), 8.19−8.16 (m, 0.3H), 8.13 (d, J = 8.75 Hz, 0.3H), 8.11

(dd, J = 1.09, 8.75 Hz, 1H), 7.77−7.74 (m, 0.3H), 7.73 (ddd, J = 1.39,

6.87, 8.40 Hz, 1H), 7.63−7.59 (d, J = 1.35 Hz, 0.3H), 7.58 (ddd, J =

1.30, 6.82, 8.36 Hz, 1H), 7.51 (d, J = 7.87 Hz, 1H), 7.46 (d, J = 4.31

Hz, 1.3H), 7.43 (d, J = 7.26 Hz, 0.3H), 5.62 (dd, J = 2.08, 7.73 Hz,

0.3H), 5.11 (dt, J = 4.58, 7.87 Hz, 1H), 4.94 (dt, J = 3.82, 7.23 Hz,

0.3H), 4.60−4.55 (m, 1H), 4.15 (dd, J = 3.43, 11.74 Hz, 0.3H), 4.02

(dd, J = 4.18, 11.36 Hz, 1,3H), 3.97−3.89 (m, 2.3H), 3.73−3.62 (m,

2H), 3.59−3.50 (m, 0.6H), 2.46−2.10 (m, 5.2H); major conformer,

¹³C NMR (101 MHz, CDCl₃) δ 169.42, 167.55, 149.86, 148.70,

General procedures A−G were used for the preparation of all ﬁnal

140.63, 130.31, 129.98, 128.14, 125.17, 124.37, 119.00, 117.90, 77.36,

63.36, 53.19, 47.01, 30.08, 25.24; minor conformer, ¹³C NMR (101

MHz, CDCl₃) δ 170.61, 168.09, 149.86, 148.76, 140.21, 130.34,

130.10, 128.18, 125.02, 124.37, 119.07, 117.90, 77.36, 62.35, 52.11,

46.65, 32.30, 23.27; UPLC I (ESI) t_R1.10 min, m/z 339.6 [M + H]⁺

(95%); LC−MS (A) (ESI) t_R10.5 min, m/z 339.0 [M + H]⁺(95%);

42 mg, 40%.

compounds present in the publication.

(S)-1-((R)-2-Amino-3-hydroxypropanoyl)pyrrolidine-2-car-

bonitrile 4-Methylbenzenesulfonate (7). tert-Butyl (R)-3-(tert-

butyldimethylsilyloxy)-1-((S)-2-cyanopyrrolidin-1-yl)-1-oxopropan-2-

ylcarbamate was prepared according to general procedure A using (S)-

pyrrolidine-2-carbonitrile 4-methylbenzenesulfonate and (R)-2-(tert-

butoxycarbonylamino)-3-(tert-butyldimethylsilyloxy)propanoic acid.

¹H NMR (400 MHz, CDCl₃) δ 5.31−5.22 (d, J = 8.4 Hz, 1H),

4.72−4.66 (m, 1H), 4.66−4.56 (td, J = 8.9, 5.3 Hz, 1H), 3.86−3.70

(m, 3H), 3.69−3.61 (t, J = 9.1 Hz, 1H), 2.36−2.07 (m, 4H), 1.50−

1.37 (s, 9H), 0.88−0.78 (s, 9H), 0.03−0.02 (s, 3H), 0.015−0.00 (s,

3H); UPLC I (ESI) t_R2.25 min, m/z 398.67 [M + H]⁺(96%); 0.260g,

65%). Then Boc deprotection was done using general procedure B. ¹H

NMR (400 MHz, D₂O) δ 7.73−7.66 (m, 2H), 7.40−7.35 (m, 2H),

4.84−4.81 (m, 1H), 4.43 (dd, J = 5.5, 4.3 Hz, 1H), 4.02−3.91 (m,

1H), 3.99 (dd, J = 12.6, 4.3 Hz, 1H), 3.94 (dd, J = 12.6, 5.6 Hz, 1H),

3.62 (dt, J = 9.8, 7.9 Hz, 1H), 2.41 (s, 3H), 2.39−2.16 (m, 4H); white

powder, 0.220 g, 95%.

N-((R)-1-((S)-2-Cyanopyrrolidin-1-yl)-1-oxopropan-2-yl)-

quinoline-4-carboxamide (12). The title product was prepared

1

using general procedure C. Major conformer, H NMR (400 MHz,

CDCl₃) δ 8.94 (d, J = 4.31 Hz, 1H), 8.24 (dd, J = 1.16, 8.99 Hz, 1H),

8.16−8.11 (m, 1H), 7.76 (ddd, J = 1.41, 6.93, 8.38 Hz, 1H), 7.62 (ddd,

J = 1.36, 6.84, 8.32 Hz, 1H), 7.49 (d, J = 4.29 Hz, 1H), 7.19 (d, J =

7.55 Hz, 1H), 5.04 (qd, J = 2.10, 6.79 Hz, 1H), 4.68 (dd, J = 2.00, 8.01

Hz, 1H), 4.00−3.91 (m, 1H), 3.59−3.51 (m, 1H), 2.49−2.10 (m, 4H),

1

1.52 (d, J = 6.83 Hz, 3H); minor conformer, H NMR (400 MHz,

CDCl₃) δ 8.93 (d, J = 4.30 Hz, 1H), 8.20 (dd, J = 1.15, 8.41 Hz, 1H),

8.16−8.11 (m, 1H), 7.76 (ddd, J = 1.41, 6.93, 8.38 Hz, 1H), 7.62 (ddd,

J = 1.36, 6.84, 8.32 Hz, 1H), 7.46 (d, J = 4.29 Hz, 1H), 6.93 (d, J =

7.24 Hz, 1H), 5.38 (dd, J = 2.16, 7.68 Hz, 1H), 5.01−4.95 (m, 1H),

4.00−3.91 (m, 1H), 3.68−3.61 (m, 1H), 2.49−2.10 (m, 4H), 1.62 (d, J

= 6.87 Hz, 3H); major conformer, ¹³C NMR (101 MHz, CDCl₃) δ

171.10, 166.71, 149.92, 148.79, 141.13, 130.17, 130.02, 127.96, 125.29,

(S)-1-((R)-2-Aminopropanoyl)pyrrolidine-2-carbonitrile 4-

Methylbenzenesulfonate (8). tert-Butyl (R)-1-((S)-2-cyanopyrroli-

din-1-yl)-1-oxopropan-2-ylcarbamate was made using general proce-

dure A and puriﬁed by column chromatography with a gradient DCM

3066

dx.doi.org/10.1021/jm500031w | J. Med. Chem. 2014, 57, 3053−3074

Journal of Medicinal Chemistry

Article

124.50, 118.86, 117.97, 47.67, 47.06, 46.59, 30.15, 25.23, 18.31; minor

conformer, ¹³C NMR (101 MHz, CDCl₃) δ 171.07, 166.63, 149.88,

148.76, 141.09, 130.23, 130.07, 128.03, 125.12, 124.43, 118.91, 117.97,

47.56, 47.06, 46.59, 29.82, 23.23, 17.93; UPLC I (ESI) t_R1.18 min, m/

z 323.6 [M + H]⁺(96%); LC−MS (A) (ESI) t_R10.2 min, m/z 323.0

[M + H]⁺(97%); 48 mg, 50%.

4H), 0.62−0.37 (m, 3H), 0.31−0.20 (m, 1H); UPLC (ESI) I t_R1.26,

m/z 349.40 [M + H]⁺; LC−MS (A) (ESI) t_R11.7 min, m/z 349.1 [M

+ H]⁺(98%); yellowish oil, 0.050 g, 18%.

(S)-N-(4-Chlorobenzyl)-N-(2-(2-cyanopyrrolidin-1-yl)-2-

oxoethyl)quinoline-4-carboxamide (18). The title product was

1

prepared using general procedure D. H NMR (CDCl₃, 400 MHz)

(cis/trans amide rotamer, 1/4) δ 8.98−8.94 (m, 0.2H), 8.93 (d, J =

4.35, 0.8H), 8.35 (br s, 1H), 8.18 (dd, J = 16.44, 3.43 Hz, 0.2H), 8.13

(d, J = 8.36 Hz, 0.8H), 7.91−7.83 (m, 0.2H), 7.78 (ddd, J = 8.30, 6.69,

1.44 Hz, 0.8H), 7.69 (ddd, J = 8.23, 6.92, 1.29 Hz, 0.8H), 7.64−7.58

(m, 0.2H), 7.43 (d, J = 3.70, 0.8H), 7.41−7.36 (m, 0.2H), 7.34 (q, J =

6.69, 5.04 Hz, 1.6H), 7.33−7.27 (m, J = 6.69, 5.04 Hz, 0.4H), 7.13−

7.03 (m, 2H), 4.94−4.72 (m, 2H), 5.37−5.33 (m, 0.8H), 5.47−5.43

(m, 0.2H), 4.43 (d, J = 3.14 Hz, 2H), 3.75 (dt, J = 11.03, 5.33 Hz,

0.4H), 3.67−3.43 (d, J = 26.63 Hz, 1.6H), 2.41−2.06 (m, 3.6H),

2.40−2.05 (m, 0.4H); UPLC(ESI) I t_R1.69, m/z 433.30 [M + H]⁺;

LC−MS (A) (ESI) t_R15.6 min, m/z 433.3 [M + H]⁺(98%); yellowish

oil, 0.030 g, 18%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-N-(2-

methoxyethyl)quinoline-4-carboxamide (19). The title product

was prepared using general procedure D. ¹H NMR (CDCl₃, 400

MHz) (cis/trans mixture of amide rotamers) δ 8.96 (d, J = 4.4, 1H),

8.34−8.29 (m, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.78 (td, J = 7.9, 7.4, 1.6

Hz, 1H), 7.68 (s, 1H), 7.44−7.38 (m, 1H), 4.97−4.85 (m, 1H), 4.07

(m, 1H), 3.70 (m, 2H), 3.47−3.30 (m, 5H), 3.27 (s, 3H), 2.41−2.11

(m, 4H); UPLC (ESI) I t_R1.21, m/z 367.30 [M + H]⁺; LC−MS (A)

(ESI) t_R11.2 min, m/z 367.1 [M + H]⁺(99%); yellowish oil, 0.049 g,

28%.

N-((S)-1-((S)-2-Cyanopyrrolidin-1-yl)-1-oxopropan-2-yl)-

quinoline-4-carboxamide (13). The title product was prepared

1

using general procedure C to give a white powder. H NMR (400

MHz, CDCl₃) δ 8.91−8.88 (d, J = 4.3 Hz, 1H), 8.26−8.20 (dd, J = 8.7,

1.5 Hz, 1H), 8.15−8.09 (dt, J = 8.5, 1.0 Hz, 1H), 7.78−7.71 (ddd, J =

8.4, 6.9, 1.4 Hz, 1H), 7.63−7.56 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.46−

7.43 (d, J = 4.2 Hz, 1H), 7.43−7.41 (s, 1H), 5.23−5.16 (p, J = 7.1 Hz,

0.05H), 5.05−4.93 (p, J = 7.1 Hz, 0.95H), 4.86−4.84 (d, J = 7.5 Hz,

0.05H), 4.74−4.67 (m, 0.95H), 3.88−3.78 (qd, J = 7.0, 3.1 Hz, 1H),

3.76−3.68 (m, 1H), 2.36−2.16 (m, 4H), 1.56 (d, J = 6.96 Hz, 3H);

¹³C NMR (101 MHz, CDCl₃) δ 171.35, 166.89, 149.79, 148.57,

141.08, 130.03, 129.84, 127.76, 125.21, 124.39, 118.85, 118.09, 47.39,

46.65, 46.52, 29.78, 25.34, 17.89; UPLC I (ESI) t_R1.20 min, m/z

323.6 [M + H]⁺(96%); LC−MS (A) (ESI) t_R10.5 min, m/z 323.0 [M

+ H]⁺(97%); 53 mg, 59%.

(S)-N-(1-(2-Cyanopyrrolidine-1-carbonyl)cyclopropyl)-

quinoline-4-carboxamide (14). Methyl 1-(quinoline-4-

carboxamido)cyclopropanecarboxylate was prepared using general

procedure C starting from methyl 1-aminocyclopropanecarboxylate.

¹H NMR (400 MHz, CDCl₃) δ 8.92 (d, J = 4.3 Hz, 1H), 8.33 (dd, J =

8.5, 0.8 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.77 (ddd, J = 8.4, 6.9, 1.4

Hz, 1H), 7.63 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.44 (d, J = 4.3 Hz, 1H),

6.66 (s, 1H), 3.81−3.76 (m, 3H), 1.76−1.72 (m, 2H), 1.38 (dd, J =

8.1, 4.9 Hz, 2H); UPLC I (ESI) t_R1.14 min, m/z 271.6 [M + H]⁺

(93%). Then methyl 1-(quinoline-4-carboxamido)cyclopropane-

carboxylate (0,130 g, 0,481 mmol) was added to a solution of KOH

(0.033 g, 0.495 mmol) in methanol (1 mL). The mixture was stirred

for 2 h and evaporated till dryness. The resulting potassium 1-

(quinoline-4-carboxamido)cyclopropanecarboxylate (0.140 g, 0.476

mmol, 99% yield) was used without further puriﬁcation in the next

step. Compound 14 was made according to general procedure A with

cyanoproline tosylate and methyl 1-(quinoline-4-carboxamido)-

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-N-isopentyl-

quinoline-4-carboxamide (20). The title product was prepared

1

using general procedure D. H NMR (400 MHz, CDCl₃) (cis/trans

amide rotamer mixture) δ 8.94 (dd, J = 4.2, 2.5 Hz, 1H), 8.13 (dd, J =

8.6, 1.3 Hz, 1H), 8.25 (br s, 1H), 7.76 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H),

7.66 (t, J = 7.6, 1H), 7.33 (br s, 1H), 4.92−4.68 (m, 1.5H), 4.50−4.10

(m, 0.5H), 3.84−3.54 (m, 3H), 3.21−3.06 (m, 2H), 2.42−2.05 (m,

4H), 1.45−1.15 (m, 3H), 0.55 (d, J = 6.3 Hz, 6H); LC−MS (A) (ESI)

t_R14.5 min, m/z 379.1 [M + H]⁺(99%); yellowish oil, 0.056 g, 33%.

(S)-1-(2-((2-Methoxyethyl)(quinolin-4-ylmethyl)amino)-

acetyl)pyrrolidine-2-carbonitril (21). A solution of (S)-1-(2-

chloroacetyl)pyrrolidine-2-carbonitrile 11 (0.272 g, 1.576 mmol) in

2.5 mL of THF was added dropwise over 0.5 h into an ice−water

cooled mixture of the appropriate 2-methoxy-N-(quinolin-4-ylmethyl)-

ethanamine S10 (0.150 g, 0.694 mmol) and potassium carbonate

(0.392 g, 2.84 mmol) in 5 mL of THF. The resulting mixture was

stirred at ice−water temperature for 2 h and then at room temperature

for 1−3 days. The resulting mixture was then ﬁltered to remove

K₂CO₃, concentrated, and then partitioned between DCM and

saturated aqueous NaHCO₃. The aqueous layer was then washed

with DCM (2×), and the combined organic layers were washed with

water, dried over Na₂SO₄, and concentrated in vacuo. Puriﬁcation was

done using column chromatography with a DCM−MeOH gradient to

1

cyclopropanecarboxylate, giving a colorless oil. H NMR (400 MHz,

MeOD) δ 8.92 (d, J = 4.41 Hz, 1H), 8.17 (d, J = 7.27 Hz, 1H), 8.09

(d, J = 8.42 Hz, 1H), 7.83 (ddd, J = 1.42, 6.78, 8.37 Hz, 1H), 7.68

(ddd, J = 1.21, 6.92, 8.30 Hz, 1H), 7.59 (d, J = 4.51 Hz, 1H), 3.74 (dq,

J = 6.88, 19.58 Hz, 2H), 3.35 (s, 1H), 2.38−2.15 (m, 2H), 2.11 (ddt, J

= 6.08, 14.42, 17.60 Hz, 2H), 1.57 (s, 2H), 1.24 (d, J = 2.75 Hz, 2H);

¹³C NMR (101 MHz, MeOD) δ 171.74, 170.51, 151.01, 149.19,

142.80, 131.61, 129.88, 129.18, 126.26, 125.85, 120.22, 119.89, 55.81,

43.79, 37.07, 30.80, 26.56, 15.48, 15.38; LC−MS (A) (ESI) t_R10.4

min, m/z 335.0 [M + H]⁺(96%); UPLC I (ESI) t_R1.15 min, m/z

335.6 [M + H]⁺(96%); 76 mg, 47%.

(S)-1-(2-Chloroacetyl)pyrrolidine-2-carbonitrile (15). The title

compound was made as described by Singh et al.²⁸

1

yield a yellowish oil. H NMR (CDCl₃, 400 MHz) (cis/trans amide

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-N-methylqui-

noline-4-carboxamide (16). The title product was prepared using

rotamer, 1/4) δ 8.84 (d, J = 4.4 Hz, 0.2H), 8.83 (d, J = 4.3 Hz, 0.8H),

8.26 (dd, J = 8.4 Hz, J′ = 0.7 Hz, 0.8H), 8.09 (dd, J = 8.5 Hz, J′ = 0.7

Hz, 0.8H), 8.1 (dd, J = 8.5 Hz, J′ = 0.7 Hz, 0.2H) 8.18 (dd, J = 8.7 Hz,

J′ = 1.0 Hz, 0.2H), 7.68 (ddd, J = 8.4 Hz, J′ = 6.8 Hz, J″ = 1.4 Hz, 1H),

7.54 (ddd, J = 8.3 Hz, J″ = 6.8 Hz, J′ = 1.3 Hz, 1H), 7.45 (dd, J = 4.4

Hz, J′ = 0.9 Hz, 0.8H), 7.41 (d, J = 4.3 Hz, 0.2H), 5.20 (dd, J = 8.0 Hz,

J′ = 1.6 Hz, 0.2H), 4.70−4.57 (m, 0.8H), 4.38 (d, J = 14.0 Hz, 0.8H),

4.30 (d, J = 14.2 Hz, 0.8H), 4.24 (d, J = 14.1 Hz, 0.2H), 4.16 (d, J =

14.2 Hz, 0.2H), 3.71 (d, J = 14.4 Hz, 0.4H), 3.66 (td, J = 7.7 Hz, J′ =

3.9 Hz, 0.4H), 3.53 (t, J = 5.2 Hz, 1.6H), 3.50−3.44 (m, 0.2H), 3.39

(d, J = 15.0 Hz, 1.6H), 3.32 (s, 3H), 3.38−3.31 (m, 0.8H), 3.27−3.11

(m, 1H), 2.99 (dt, J = 5.3 Hz, J′ = 2.4 Hz, 1.6H), 2.81 (ddd, J = 13.9

Hz, J′ = 5.5 Hz, J″ = 3.2 Hz, 0.4H), 2.25−1.89 (m, 4H); UPLC (ESI) I

t_R1.18, m/z 353.50 [M + H]⁺; LC−MS (A) (ESI) t_R10.3 min, m/z

353.1 [M + H]⁺(97%); 0.055 g, 25%.

1

general procedure D. H NMR (CDCl₃, 400 MHz) (cis/trans amide

rotamer, 1/3) δ 8.98 (dd, J = 4.2, 2.5 Hz, 1H), 8.20−8.09 (m, 1.75H),

7.88 (dd, J = 8.2, 1.1 Hz, 0.25H), 7.78 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H),

7.67 (ddd, J = 8.3, 6.9, 1.3 Hz, 0.75H), 7.60 (ddd, J = 8.3, 7.0, 1.3 Hz,

0.25H), 7.38 (d, J = 4.4 Hz, 0.75H), 7.33 (d, J = 4.3, 0.25H), 4.94−

4.86 (m, 1H), 3.84−3.69 (m, 1H), 3.70−3.59 (m, 1H), 2.98 (d, J =

11.5 Hz, 1H), 2.95 (s, 3H), 2.94 (d, J = 11.4 Hz, 1H), 2.44−2.09 (m,

4H); UPLC (ESI) I t_R1.05, m/z 323.50 [M + H]⁺; LC−MS (A) (ESI)

t_R10.4 min, m/z 323.1 [M + H]⁺(97%); yellowish oil, 0.022 g, 13%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-N-cyclopropyl-

quinoline-4-carboxamide (17). The title product was prepared

1

using general procedure D. H NMR (CDCl₃, 400 MHz) (cis/trans

amide rotamer, 1/9) δ 8.95 (d, J = 4.4 Hz, 1H), 8.25−8.09 (m, 2H),

7.74 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.62 (ddd, J = 8.2, 6.9, 1.3 Hz,

1H), 7.44 (d, J = 4.7, 1H), 4.91−4.87 (m, 0.1H), 4.85 (dd, J = 7.6, 2.5

Hz, 0.9H), 4.66 (d, J = 16.1 Hz, 1H), 4.05 (d, J = 16.1 Hz, 1H), 3.74−

3.65(m, 1H), 3.64−3.55 (m, 1H), 2.89−2.78 (m, 1H), 2.42−2.06 (m,

(S)-1-(2-(Methyl(quinolin-4-ylmethyl)amino)acetyl)-

pyrrolidine-2-carbonitrile (22). The title compound was prepared

in a similar manner as compound 21 starting from N-methyl-1-

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dx.doi.org/10.1021/jm500031w | J. Med. Chem. 2014, 57, 3053−3074

Journal of Medicinal Chemistry

Article

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(quinolin-4-yl)methanamine (S9). Puriﬁcation was done using column

chromatography with a DCM−MeOH gradient with an ammonia

4g, 82%. H NMR (sodium salt) (400 MHz, D₂O) δ 8.94 (d, J = 4.5

Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.90 (t, J =

7.1 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.71 (d, J = 4.5 Hz, 1H), 4.07 (s,

2H).

1

additive to yield a yellowish oil. H NMR (CDCl₃, 400 MHz) (cis/

trans amide rotamer, 1/3) δ 8.79 (s, 0.25H), 8.77 (d, J = 4.3 Hz,

0.75H), 8.17 (dd, 0.75H), 8.13−8.09 (m, 0.25H), 8.06 (d, J = 8.5 Hz,

0.25H), 8.03 (d, J = 8,5 Hz, 0.75H), 7.62 (ddd, J = 8.4, 6.8, 1.4 Hz,

1H), 7.49 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.32 (d, J = 4.4, 0.75H), 7.29

(d, J = 4.3, 0.25H), 4.78 (dd, J = 7.8, 1.8 Hz, 0.25H), 4.63−4.52 (m,

0.75H), 4.07 (d, J = 13.4 Hz, 0.75H), 3.98 (d, J = 11.4 Hz, 0.75H),

3.96 (d, J = 13.6 Hz, 0.25H), 3.92 (d, J = 13.6 Hz, 0.25H), 3.50−3.37

(m, 1H), 3.20 (d, J = 1.5 Hz, 2H), 3.24−3.09 (m, 1H), 2.38 (s,

0.75H), 2.35 (s, 2.25H), 2.22−1.59 (m, 4H); UPLC (ESI) I t_R0.57,

m/z 309.50 [M + H]⁺; LC−MS (A) (ESI) t_R8.8 min, m/z 309.1 [M +

H]⁺(97%); 0.131 g, 48.8%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-

isonicotinamide (29). The title product was prepared using general

procedure A. ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, J = 6 Hz, 2H),

7.99 (br s, 1H), 7.61 (d, J = 6 Hz, 2H), 4.76−4.72 (m, 1H), 4.53−4.46

(m, 1H), 4.12−4.05 (m, 1H), 3.74−3.66 (m, 1H), 3.54−3.45 (m, 1H),

2.36−2.13 (m, 4H); MS (ESI) m/z 259.1 [M + H]⁺; LC−MS (A) t_R

3.7 min, m/z 259.1 [M + H]⁺(96%); yellowish oil, 24 mg, 16%.

(S)-2-Chloro-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-

isonicotinamide (30). The title product was prepared using general

procedure F. ¹H NMR (400 MHz, CDCl₃) (mixture of cis/trans

amide rotamers, 1/9) δ 8.51 (dd, J = 5.1, 0.6 Hz, 1H), 7.72 (dd, J =

1.5, 0.7 Hz, 1H), 7.57 (dd, J = 5.1, 1.5 Hz, 1H), 7.44 (s, 1H), 4.81−

4.75 (m, 0.9H), 4.68 (d, J = 5.7 Hz, 0.1H), 4.44 (dd, J = 17.6, 5.0 Hz,

0.1H), 4.35 (dd, J = 17.9, 5.0 Hz, 0.9H), 4.30−4.22 (m, 0.1H), 4.16

(dd, J = 17.9, 3.7 Hz, 0.9H), 3.74−3.66 (m, 1H), 3.55−3.46 (m, 1H),

2.42−2.14 (m, 4H); UPLC I (ESI) t_R1.20 min, 293.3 m/z [M + H]⁺

(96%); white powder, 53 mg, 57%)

(S)-2-Bromo-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-

isonicotinamide (31). The title product was prepared using general

procedure A. ¹H NMR (400 MHz, CDCl₃) (mixture of cis/trans

amide rotamers, 1/9) δ 8.50 (dd, J = 5.1, 0.6 Hz, 1H), 7.88 (dd, J =

1.4, 0.7 Hz, 1H), 7.62 (dd, J = 5.1, 1.5 Hz, 1H), 7.36 (s, 1H), 4.80−

4.76 (m, 0.9H), 4.68 (d, J = 6.2 Hz, 0.1H), 4.43 (dd, J = 17.6, 5.2 Hz,

0.1H), 4.33 (dd, J = 17.9, 4.8 Hz, 0.9H), 4.29−4.23 (m, 0.1H), 4.17

(dd, J = 17.9, 3.7 Hz, 0.9H), 3.73−3.65 (m, 1H), 3.58−3.45 (m, 1H),

2.42−2.17 (m, 4H); UPLC I (ESI) t_R1.23 min, m/z 337.5, 339.6 [M +

H]⁺(96%); colorless oil, 36 mg, 41%.

(2S,2′S)-1,1′-(2,2′-(Methylazanediyl)bis(acetyl))bis-

(pyrrolidine-2-carbonitrile) (23). The title product was prepared

using general procedure D, but only 0.5 equiv of 33% methylamine in

1

EtOH was used as the primary amine. H NMR (400 MHz, CDCl₃)

(mixture of trans/cis rotamers) δ 5.69 (d, J = 7.3 Hz, 0.4H), 4.80−4.62

(m, 1.6H), 3.72−3.66 (m, 1H), 3.62−3.56 (m, 1H), 3.53−3.44 (m, J =

13.7, 8.1 Hz, 3H), 3.43−3.36 (m, J = 14.6, 3.3 Hz, 3H), 2.54 (s, 1.5H),

2.42 (s, J = 7.7 Hz, 1.5H), 2.35−1.99 (m, 8H); UPLC (ESI) I t_R0.29,

m/z 304.50 [M + H]⁺; LC−MS (A) (ESI) t_R2.2 min, m/z 304.1 [M +

H]⁺(98%); yellowish oil, 0.028 g, 13%.

(S)-1-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)acetyl)pyrrolidine-2-

carbonitrile (24). The title product was prepared using general

1

procedure E. H NMR (400 MHz, CDCl₃) δ 8.1 (s, 1H), 7.85−7.74

(m, 2H), 7.53−7.40 (m, 3H), 5.35 (d, J = 16.5 Hz, 1H), 5.12 (d, J =

16.5 Hz, 1H), 4.79−4.73 (m, 1H), 3.81−3.72 (m, 1H), 3.66−3.58 (m,

1H), 2.40−2.13 (m, 4H); LC−MS (A) (ESI) t_R12.8 min, m/z 282.3

[M + H]⁺(96%); white powder, 54 mg, 62%.

(S)-2-Amino-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-

isonicotinamide (32). (S)-tert-Butyl 4-(2-(2-cyanopyrrolidin-1-yl)-2-

oxoethylcarbamoyl)pyridin-2-ylcarbamate was prepared using general

procedure A using potassium 2-(tert-butoxycarbonylamino)-

isonicotinate to give a Boc-protected intermediate. Then TFA

(0.827 mL, 10.74 mmol) was slowly added to a solution of (S)-tert-

butyl 4-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethylcarbamoyl)pyridin-2-yl-

carbamate (0.537 mmol) in acetonitrile (2 mL) at 0 °C. After 1 h, the

mixture was allowed to warm to room temperature and was stirred for

24 h. The volatiles were evaporated. The products were puriﬁed by

reversed phase ﬂash column using a acetonitrile−water gradient or

converted into the HCl salt by stirring for 15 min in 1 N HCl in ether,

followed by decantation of the ether and washing with cold ethyl

(S)-1-(2-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)acetyl)-

pyrrolidine-2-carbonitrile (25). The title product was prepared

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using general procedure E. H NMR (400 MHz, CDCl₃) δ 8.04 (s,

1H), 7.84−7.75 (m, 2H), 7.09 (t, J = 8.84 Hz, 2H), 5.35 (d, J = 16.38

Hz, 1H), 5.14 (d, J = 16.40 Hz, 1H), 4.79−4.73 (m, 1H), 3.81−3.72

(m, 1H), 3.66−3.58 (m, 1H), 2.40−2.13 (m, 4H); LC−MS (A) (ESI)

t_R13.5 min, m/z 300.0 [M + H]⁺(97%); white powder, 39 mg, 37%.

(S)-1-(2-(4-(3-(Isoindolin-2-yl)-3-oxopropyl)-1H-1,2,3-triazol-

1-yl)acetyl)pyrrolidine-2-carbonitrile (26). The title product was

1

prepared using general procedure E. H NMR (400 MHz, CDCl₃) δ

7.65 (s, 1H), 7.32−7.22 (m, 4H), 5.22 (d, J = 16.33 Hz, 1H), 5.08 (d, J

= 16.33 Hz, 1H), 4.79 (d, J = 5.59 Hz, 4H), 4.74−4.70 (m, 1H), 3.76−

3.68 (m, 1H), 3.62−3.53 (m, 1H), 3.16 (t, J = 7.19 Hz, 2H), 2.84 (t, J

= 7.19 Hz, 2H), 2.36−2.16 (m, 4H); LC−MS (A) (ESI) t_R12.9 min,

m/z 379.1 [M + H]⁺(97%); white powder, 36 mg, 23%.

(S)-1-(2-(4-(Naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)acetyl)-

pyrrolidine-2-carbonitrile (27). The title product was prepared

using general procedure E. ¹H NMR (400 MHz, CDCl₃) δ 8.43−8.34

(m, 1H), 8.10 (s, 1H), 7.94−7.87 (m, 2H), 7.76 (dd, J = 1.23, 7.14 Hz,

1H), 7.57−7.49 (m, 3H), 5.43 (d, J = 16.32 Hz, 1H), 5.23 (d, J = 16.30

Hz, 1H), 4.79 (dd, J = 2.60, 7.46 Hz, 1H), 3.84−3.74 (m, 1H), 3.72−

3.61 (m, 1H), 2.40−2.14 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ

164.05, 147.28, 133.89, 131.10, 129.05, 128.46, 127.76, 127.40, 126.77,

126.05,125.37, 125.35, 124.41, 117.74, 51.55, 46.97, 46.28, 29.87,

25.24; LC−MS (A) (ESI) t_R15.0 min, m/z 332.1 [M + H]⁺(95%);

white powder, 140 mg, 63.1%.

2-(Quinoline-4-carboxamido)acetic Acid (28). Ethyl 2-(Quino-

line-4-carboxamido)acetate. To a solution of quinoline-4-carbonyl

chloride hydrochloride (5 g, 21.9 mmol) in DCM were added a

solution of ethyl 2-aminoacetate hydrochloride (3.06 g, 21.92 mmol)

and triethylamine (9.72 mL, 70.2 mmol) in DCM. The mixture was

stirred for 4 h at room temperature. It was washed with 1 N citric acid.

The precipitate that formed was the product (5.66 g, 90%). UPLC I

(ESI) t_R1.20 min, m/z 259.6 [M + H]⁺(96%). Then sodium

hydroxide (21.30 mL, 21.30 mmol) was added to a mixture of ethyl 2-

(quinoline-4-carboxamido)acetate (5.5 g, 21.30 mmol) in MeOH (10

mL) and water (10.00 mL). The mixture was stirred for 16 h,

evaporated, redissolved in water, washed with DCM, acidiﬁed, and the

precipitate was 2-(quinoline-4-carboxamido)acetic acid: white powder,

1

acetate. H NMR (400 MHz, DMSO-d₆) (9/1 mixture of trans/cis

amide rotamers) δ 9.13 (t, J = 5.87 Hz, 1H), 8.06 (d, J = 6.40 Hz, 1H),

7.95 (s, 2H), 7.26 (s, 1H), 7.10 (dd, J = 1.56, 6.44 Hz, 1H), 5.24 (dd, J

= 2.26, 7.39 Hz, 0.1H) 4.76 (dd, J = 3.74, 7.38 Hz, 0.9H), 4.30 (dd, J =

5.74, 16.59 Hz, 0.2H), 4.20−4.02 (m, 1.8H), 3.70 (ddd, J = 3.88, 7.60,

9.51 Hz, 1H), 3.55−3.48 (m, 1H), 2.23−1.96 (m, 4H); UPLC I (ESI)

t_R0.45 min, m/z 274.6 [M + H]⁺(98%); LC−MS (A) t_R2.2 min, m/z

274.0 [M + H]⁺(97%); yellowish oil, 40 mg, 60%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-phenyl-

isonicotinamide (33). The title product was prepared using general

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procedure A using 2-phenylisonicotinic acid S11. H NMR (400 M

Hz, CDCl₃) (9/1 mixture of trans/cis amide rotamers) δ 8.83−8.79

(dd, J = 5.0, 0.9 Hz, 1H), 8.13−8.10 (dd, J = 1.6, 0.9 Hz, 1H), 8.07−

8.02 (m, 2H), 7.60−7.56 (dd, J = 5.0, 1.6 Hz, 1H), 7.53−7.42 (m,

3H), 7.41−7.36 (s, 1H), 4.83−4.77 (dd, J = 8.0, 2.4 Hz, 0.9H), 4.75−

4.71 (d, J = 8.3 Hz, 0.1H), 4.53−4.46 (dd, J = 17.6, 5.2 Hz, 0.1H),

4.40−4.31 (dd, J = 18.0, 4.7 Hz, 0.9H), 4.26−4.17 (dd, J = 18.0, 3.7

Hz, 1H), 3.75−3.67 (ddd, J = 14.9, 8.4, 4.0 Hz, 1H), 3.58−3.49 (td, J

= 8.6, 7.6, 5.9 Hz, 1H), 2.43−2.15 (m, 4H); UPLC I (ESI) t_R1.45

min, m/z 335.6 [M + H]⁺(98%); LC−MS (A) t_R1.32min, 335.6 m/z

[M + H]⁺(99%); white powder, 0.037 g, 52%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-(3,4-

dimethoxyphenyl)isonicotinamide (34). The title product was

prepared using general procedure A using 2-(3,4-dimethoxyphenyl)-

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isonicotinic acid S12. H NMR (400 MHz, CDCl₃) (9/1 mixture of

trans/cis amide rotamers) δ 8.82−8.69 (dd, J = 5.0, 0.9 Hz, 1H),

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dx.doi.org/10.1021/jm500031w | J. Med. Chem. 2014, 57, 3053−3074

Journal of Medicinal Chemistry

Article

8.11−8.00 (s, 1H), 7.73−7.66 (d, J = 2.1 Hz, 1H), 7.61−7.57 (dd, J =

8.4, 2.1 Hz, 1H), 7.52−7.48 (dd, J = 5.1, 1.6 Hz, 1H), 7.40−7.33 (t, J =

3.9 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.83−4.76 (dd, J = 8.1, 2.8 Hz,

0.9H), 4.74−4.72 (d, J = 8.1 Hz, 0.1H), 4.52−4.46 (dd, J = 17.3, 5.1

Hz, 0.1H), 4.36 (dd, J = 18.0, 4.7 Hz, 0.9H), 4.21 (dd, J = 18.0, 3.7 Hz,

1H), 4.03−3.99 (s, 3H), 3.97−3.92 (s, 3H), 3.78−3.68 (m, 1H),

3.60−3.50 (m, 1H), 2.42−2.16 (m, 4H); UPLC I (ESI) t_R1.34 min,

m/z 395.5 [M + H]⁺(100%); LC−MS (A) t_R12.6 min, m/z 395.1 [M

+ H]⁺(99%); white powder, 36 mg, 43%.

(m, 4H); minor conformer, ¹H NMR (400 MHz, CDCl₃) δ 9.56 (dd, J

= 1.26, 2.38 Hz, 1H), 9.34 (dd, J = 1.25, 5.28 Hz, 1H), 8.05 (t, J = 4.70

Hz, 1H), 7.87 (dd, J = 2.36, 5.28 Hz, 1H), 4.85 (dd, J = 2.12, 7.69 Hz,

1H), 4.47 (dd, J = 5.30, 17.11 Hz, 1H), 4.29 (dd, J = 4.40, 17.09 Hz,

1H), 3.71 (ddd, J = 3.41, 7.40, 10.39 Hz, 1H), 3.58−3.47 (m, 1H),

2.48−2.05 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 168.07, 163.44,

151.93, 148.47, 130.64, 123.93, 118.16, 46.98, 45.97, 42.34, 29.82,

25.22; LC−MS (A) (ESI) t_R5.0 min, m/z 259.9 [M + H]⁺(96%);

white crystals, 134 mg, 65%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-1-methyl-1H-

imidazole-5-carboxamide (39). The title product was prepared

using general procedure A. ¹H NMR (400 MHz, CDCl₃) (9/1 mixture

of trans/cis amide rotamers) δ 7.57−7.51 (s, 1H), 7.51−7.44 (s, 1H),

7.08−7.02 (d, J = 5.6 Hz, 1H), 4.81−4.75 (m, 0.9H), 4.72−4.68 (dd, J

= 7.8, 1.9 Hz, 0.1H), 4.43−4.34 (dd, J = 17.3, 5.2 Hz, 0.1H), 4.33−

4.24 (dd, J = 17.7, 5.2 Hz, 0.9H), 4.23−4.15 (m, 0.1H), 4.13−4.05

(dd, J = 17.7, 3.9 Hz, 0.9H), 3.97−3.84 (s, 3H), 3.71−3.64 (ddd, J =

9.4, 7.0, 3.0 Hz, 1H), 3.54−3.44 (td, J = 9.0, 8.4, 6.3 Hz, 1H), 2.40−

2.12 (m, 4H); UPLC I (ESI) t_R0.29 min, m/z 262.6 [M + H]⁺(98%);

LC−MS (A) t_R1.5 min, m/z 262.0 [M + H]⁺(97%); colorless oil, 46

mg, 38%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-1H-1,2,3-tria-

zole-5-carboxamide (40). The title product was prepared using

general procedure A. ¹H NMR (400 MHz, DMSO-d₆) (9/1 mixture of

trans/cis amide rotamers) δ 15.53 (s, 1H), 8.58−8.44 (s, 1H), 8.39 (s,

1H), 5.28−5.22 (m, 0.1H), 4.77 (dd, J = 3.76, 7.32 Hz, 1H), 4.30 (dd,

J = 5.61, 16.77 Hz, 0.2H), 4.10 (d, J = 5.76 Hz, 2H), 3.68 (ddd, J =

4.04, 7.68, 9.39 Hz, 1H), 3.50 (td, J = 6.84, 9.09 Hz, 1H), 2.31−1.88

(m, 4H); UPLC I (ESI) t_R0.72 min, m/z 249.6 [M + H]⁺(96%);

LC−MS (A) t_R4.0 min, m/z 249.0 [M + H]⁺(96%); colorless oil, 34

mg, 40%.

(S)-2-(4-Cyanophenyl)-N-(2-(2-cyanopyrrolidin-1-yl)-2-

oxoethyl)isonicotinamide (35). The title product was prepared

using general procedure A using 2-(4-cyanophenyl)isonicotinic acid

1

S13. H NMR (400 MHz, CDCl₃): (9/1 mixture of trans/cis amide

rotamers) δ 8.76 (d, J = 5.0 Hz, 1H), 8.16−8.05 (m, 3H), 7.81−7.72

(m, 3H), 7.61 (dd, J = 5.0, 1.5 Hz, 1H), 4.84−4.74 (m, 0.9H), 4.73−

4.71 (dd, J = 9.6, 1.9 Hz, 0.1H), 4.53−4.43 (dd, J = 17.8, 5.7 Hz, 1H),

4.34−4.28 (dd, J = 17.4, 3.6 Hz, 0.1H), 4.23−4.13 (dd, J = 17.8, 3.5

Hz, 0.9H), 3.81−3.70 (m, 1H), 3.58−3.51 (m, 1H), 2.42−2.18 (m,

4H); UPLC I (ESI) t_R1.44 min, m/z 360.6 [M + H]⁺(100%); LC−

MS (A) t_R13.5 min, m/z 360.0 [M + H]⁺(99%); white powder, 45

mg, 52%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-(pyridin-4-

yl)acetamide (36). The title product was prepared using general

1

procedure A. H NMR (400 MHz, MeOD) δ 8.49−8.44 (d, J = 4.5

Hz, J′ = 1.6 Hz, 2H), 7.46−7.41 (d, J = 4.5 Hz, J′ = 1.6 Hz, 2H), 5.05

−5.02 (m, 0.14H), 4.79−4.74 (t, J = 5.4 Hz, 0.86H), 4.26−4.16 (m,

0.2H), 4.12−4.00 (m, 1.8H), 3.73−3.66 (m, 3H), 3.57−3.49 (dt, J =

9.6, 7.6 Hz, 1H), 2.28−2.12 (m, 4H); ¹³C NMR (101 MHz, MeOD) δ

172.54, 169.57, 150.0, 147.3, 126.35, 119.51, 47.99, 46.90, 42.90,

42.50, 30.97, 26.12; UPLC I (ESI) t_R0.28 min, m/z 273.6 [M + H]⁺

(97%); LC−MS (A) t_R1.5 min, m/z 273.0 [M + H]⁺(97%); colorless

oil, 72 mg, 66%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-1H-1,2,4-tria-

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)piperidine-4-

carboxamide (37). Step 1. 1-(tert-Butoxycarbonyl)piperidine-4-carbox-

ylic Acid. The piperidine-4-carboxylic acid (1 g, 7.74 mmol) was added

to a stirred solution of guanidine hydrochloride (0.111 g, 1.161 mmol)

and di-tert-butyl dicarbonate (4.22 g, 19.36 mmol) in EtOH (7 mL) at

35−40 °C. The mixture was stirred overnight. Then the ethanol was

evaporated and the residue was dissolved in DCM and ﬁltered to

separate the catalyst. The ﬁltrate was evaporated and washed with

zole-3-carboxamide (41). The title product was prepared using

1

general procedure A. H NMR (400 MHz, D₂O) (9/1 mixture of

trans/cis amide rotamers) δ 8.56 (s, 1H), 5.12 (dd, J = 2.05, 7.76 Hz,

0.1H), 4.85−4.81 (m, 0.9H), 4.43 (d, J = 2.63 Hz, 0.2H), 4.32 (s, 2H),

3.79 (ddd, J = 4.53, 7.02, 9.82 Hz, 1H), 3.63 (dt, J = 7.65, 9.63 Hz,

1H), 2.48−2.10 (m, 4H); UPLC I (ESI) t_R0.77 min, m/z 271.5 [M +

Na]⁺(97%); LC−MS (A) t_R2.8 min, m/z 249.0 [M + H]⁺(97%);

yellowish oil, 90 mg, 55%.

1

hexane to yield a white powder (1.5g, 84%). H NMR (400 MHz,

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-(1H-imida-

MeOD) δ 4.02−3.94 (td, J = 4.0, 1.3 Hz, 2H), 2.96−2.83 (br s, 2H),

2.55−2.43 (tt, J = 11.0, 4.0 Hz, 1H), 1.94−1.83 (dq, J = 13.9, 3.6 Hz,

2H), 1.61−1.47 (m, 2H), 1.52−1.38 (s, 9H); MS (ESI) m/z 230.4 [M

+ H]⁺

zol-4-yl)acetamide (42). The title product was prepared using

1

general procedure A. H NMR (400 MHz, MeOD) δ 7.68−7.63 (s,

1H), 7.05−6.99 (s, 1H), 4.78−4.73 (t, J = 5.3 Hz, 1H), 4.10−3.98 (m,

2H), 3.71−3.64 (ddd, J = 9.7, 6.4, 4.7 Hz, 1H), 3.63−3.58 (s, 2H),

3.56−3.47 (dt, J = 9.5, 7.5 Hz, 1H), 2.36−2.06 (m, 4H); UPLC I

(ESI) t_R0.29 min, m/z 262.6 [M + H]⁺(99%); LC−MS (A) t_R1.2

min, m/z 262.0 [M + H]⁺(96%); yellowish oil, 72 mg, 66%.

Step 2: (S)-tert-Butyl 4-(2-(2-Cyanopyrrolidin-1-yl)-2-

oxoethylcarbamoyl)piperidine-1-carboxylate. The title product was

prepared using general procedure A using 1-(tert-butoxycarbonyl)-

piperidine-4-carboxylic acid to give the product (0.1 g, 63%). UPLC I

(ESI) t_R1.47 min, m/z 365.6 [M + H]⁺(90%). Then (S)-N-(2-(2-

cyanopyrrolidin-1-yl)-2-oxoethyl)piperidine-4-carboxamide (40) was

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-4-methylthia-

zole-5-carboxamide (43). The title product was prepared using

1

general procedure A. H NMR (400 MHz, CDCl₃) (9/1 mixture of

made using general procedure B. H NMR (400 MHz, MeOD) (9/1

trans/cis amide rotamers) δ 8.71−8.67 (s, 1H), 7.13−7.04 (d, J = 7.3

Hz, 1H), 4.79−4.74 (d, J = 7.7 Hz, 0.9H), 4.74−4.71 (d, J = 7.7 Hz,

0.1H), 4.47−4.40 (dd, J = 17.4, 5.1 Hz, 0.1H), 4.38−4.27 (dd, J = 17.8,

5.1 Hz, 0.9H), 4.26−4.18 (dd, J = 17.4, 3.5 Hz, 0.1H), 4.16−4.05 (dd,

J = 18.0, 3.4 Hz, 0.9H), 3.71−3.64 (ddd, J = 9.3, 7.2, 3.3 Hz, 1H),

3.54−3.44 (m, 1H), 2.76−2.73 (s, 3H), 2.38−2.14 (m, 4H); UPLC I

(ESI) t_R1.08 min, m/z 279.6 [M + H]⁺(100%); LC−MS (A) t_R9.3

min, m/z 279.0 [M + H]⁺(99%); whitish powder, 57 mg, 39%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-4-methyloxa-

zole-5-carboxamide (44). The title product was prepared using

mixture of trans/cis amide rotamers) δ 5.07−5.03 (m, 0.1H), 4.80−

4.72 (dd, J = 6.1, 4.6 Hz, 0.1H), 4.12−3.99 (d, J = 17.1 Hz, 1H), 4.04−

3.92 (d, J = 17.1 Hz, 1H), 3.77−3.63 (m, 1H), 3.61−3.48 (dt, J = 9.5,

7.5 Hz, 1H), 3.15−3.09 (t, J = 3.4 Hz, 1H), 3.12−3.06 (t, J = 3.3 Hz,

1H), 2.73−2.58 (td, J = 12.5, 2.9 Hz, 2H), 2.54−2.39 (tt, J = 11.6, 3.8

Hz, 1H), 2.31−2.19 (m, 2H), 2.22−2.11 (m, 2H), 1.89−1.77 (m, 2H),

1.76−1.58 (m, 2H; ¹³C NMR (101 MHz, MeOD) δ 178.10, 169.80,

119.55, 47.9, 46.90, 46.11, 43.66, 42.70, 42.48, 30.97, 29.85, 26.13,

23.76; UPLC I (ESI) t_R0.26 min, m/z 265.5 [M + H]⁺(98%);

yellowish oil, 40 mg, 70%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)pyridazine-4-

carboxamide (38). The title product was prepared using general

procedure F. Major conformer (4:1 mixture of trans/cis amide

rotamers), ¹H NMR (400 MHz, CDCl₃) δ 9.51 (dd, J = 1.24, 2.31 Hz,

1H), 9.24 (dd, J = 1.24, 5.31 Hz, 1H), 8.50 (dd, J = 3.60, 6.74 Hz, 1H),

7.81 (dd, J = 2.33, 5.30 Hz, 1H), 4.72 (dd, J = 3.04, 7.21 Hz, 1H), 4.54

(dd, J = 6.78, 17.41 Hz, 1H), 4.10 (dd, J = 3.62, 17.41 Hz, 1H), 3.71

(ddd, J = 3.41, 7.40, 10.39 Hz, 1H), 3.58−3.47 (m, 1H), 2.48−2.05

1

general procedure A. H NMR (400 MHz, CDCl₃) (9/1 mixture of

trans/cis amide rotamers) δ 7.83−7.75 (s, 1H), 7.19−7.12 (s, 1H),

4.83−4.76 (dd, J = 8.0, 2.6 Hz, 0.9H), 4.83−4.74 (d, 8.0 Hz, 0.1H),

4.46−4.39 (dd, J = 17.3, 5.6 Hz, 0.1H), 4.31−4.22 (dd, J = 17.9, 4.9

Hz, 0.9H), 4.22−4.17 (m, 0.1H), 4.16−4.09 (dd, J = 17.9, 4.0 Hz,

0.9H), 3.72−3.64 (ddd, J = 9.5, 7.1, 2.8 Hz, 1H), 3.56−3.43 (m, 1H),

2.58−2.45 (s, 3H), 2.40−2.18 (m, 4H); UPLC I (ESI) t_R1.04 min, m/

z 263.4 [M + H]⁺(96%); whitish powder, 34 mg, 36%.

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Article

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-4-(2,2,2-

triﬂuoroacetamido)benzamide (46) and (S)-4-Amino-N-(2-(2-

cyanopyrrolidin-1-yl)-2-oxoethyl)benzamide 2,2,2-Triﬂuoroa-

cetate (45). TFA (0.228 mL, 2.95 mmol) was added to a solution

of (S)-tert-butyl 4-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethylcarbamoyl)-

phenylcarbamate S22 (0.055 g, 0.148 mmol) in acetonitrile (2 mL) at

0 °C. The mixture was allowed to warm to room temperature slowly

and was stirred for 24 h. The volatiles were evaporated and the

products were puriﬁed by reversed phase ﬂash column using a

acetonitrile−water gradient to yield (S)-N-(2-(2-cyanopyrrolidin-1-yl)-

2-oxoethyl)-4-(2,2,2-triﬂuoroacetamido)benzamide (whitish crystals,

0.02 g, 0.054 mmol, 36% yield) and (S)-4-amino-N-(2-(2-

cyanopyrrolidin-1-yl)-2-oxoethyl)benzamide 2,2,2-triﬂuoroacetate

(colorless oil, 0.03 g, 0.078 mmol, 52% yield).

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-4-(2,2,2-

triﬂuoroacetamido)benzamide (45). ¹H NMR (400 MHz,

DMSO-d₆) δ 8.21 (t, J = 5.9 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H),

6.55 (d, J = 8.6 Hz, 2H), 5.64 (s, 2H), 4.75 (dd, J = 7.3, 3.7 Hz, 1H),

4.07 (dd, J = 16.8, 5.6 Hz, 1H), 3.98 (dd, J = 16.8, 5.5 Hz, 1H), 3.72−

3.64 (m, 1H), 3.54−3.44 (m, 1H), 2.22−1.95 (m, 4H); LC−MS (A)

(ESI) t_R6.5 min, m/z 273.0 [M + H]⁺(97%).

405.6 [M + H]⁺(96%); LC−MS (A) (ESI) t_R14.6 min, m/z 405.0 [M

+ H]⁺(97%); white powder, 67 mg, 75%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)[1,2,4]triazolo-

[1,5-a]pyrimidine-7-carboxamide (50). The title product was

prepared using general procedure A. ¹H NMR (400 MHz, DMSO-d₆)

δ 9.59 (d, J = 6.96 Hz, 1H), 9.28 (s, 1H), 8.86 (s, 1H), 7.87 (d, J =

6.95 Hz, 1H), 4.79 (dd, J = 3.72, 7.35 Hz, 1H), 4.18 (d, J = 5.11 Hz,

2H), 3.76−3.67 (m, 1H), 3.52 (q, J = 8.33 Hz, 1H), 2.36−1.97 (m,

4H); UPLC I (ESI) t_R1.02 min, m/z 300.6 [M + H]⁺(96%); LC−MS

(A) (ESI) t_R9.0 min, m/z 300.0 [M + H]⁺(97%); white powder, 54

mg, 57%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-1,7-naphthyri-

dine-4-carboxamide (51). The title product was prepared using

general procedure G, using 1,7-naphthyridine-4-carboxylic acid S23. A

1

5:1 mixture of trans/cis amide rotamers, major rotamer, H NMR

(400 MHz, CDCl₃) δ 9.44 (d, J = 1.02 Hz, 1H), 8.97 (d, J = 4.29 Hz,

1H), 8.59 (d, J = 5.90 Hz, 1H), 8.12 (dd, J = 0.98, 5.86 Hz, 1H), 7.72

(d, J = 4.34 Hz, 1H), 7.64−7.57 (m, 1H), 4.76−4.69 (m, 1H), 4.41

(dd, J = 5.58, 17.60 Hz, 1H), 4.19 (dd, J = 4.00, 17.59 Hz, 1H), 3.69

(ddd, J = 3.37, 6.58, 11.84 Hz, 1H), 3.58−3.46 (m, 1H), 2.39−2.07

1

(m, 4H); minor rotamer, H NMR (400 MHz, CDCl₃) δ 9.48 (d, J =

(S)-4-Amino-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-

benzamide 2,2,2-Triﬂuoroacetate (46). ¹H NMR (400 MHz,

CDCl₃) (mixture of cis/trans amide rotamers, 1/5) δ 8.09 (s, 1H),

7.87 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.27 (s, 1H), 4.81−

4.75 (m, 0.8H), 4.76−4.72 (m, 0.2H), 4.52−4.43 (m, 0.2H), 4.35 (dd,

J = 17.9, 5.0 Hz, 0.8H), 4.27−4.21 (m, 0.2H), 4.16 (dd, J = 17.9, 3.7

Hz, 0.8H), 3.76−3.66 (m, 1H), 3.55−3.47 (m, 1H), 2.41−2.18 (m,

4H); LC−MS (A) (ESI) t_R12.4 min, m/z 369.0 [M + H]⁺(97%).

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-1H-pyrrolo-

[2,3-b]pyridine-4-carboxamide (47). The title product was

0.94 Hz, 1H), 9.02 (d, J = 4.29 Hz, 1H), 8.61 (d, J = 4.26 Hz, 1H),

8.10 (d, 5.86 Hz, 1H), 7.70 (d, J = 4.12 Hz, 1H), 7.44 (t, J = 4.78 Hz,

1H), 4.79 (dd, J = 2.04, 7.77 Hz, 1H), 4.54 (dd, J = 5.44, 17.23 Hz,

1H), 4.29 (dd, J = 3.98, 17.27 Hz, 1H), 3.69 (ddd, J = 3.37, 6.58, 11.84

Hz, 1H), 3.58−3.46 (m, 1H), 2.39−2.07 (m, 4H); ¹³C NMR (101

MHz, CDCl₃) δ 167.24, 166.00, 154.45, 151.46, 144.79, 143.45,

139.40, 127.89, 122.74, 118.00, 117.76, 46.82, 45.79, 42.45, 29.91,

25.16; UPLC I (ESI) t_R1.03 min, m/z 310.7 [M + H]⁺(96%); LC−

MS (A) t_R9.0 min, m/z 309.9 [M + H]⁺(96%); white powder, 58 mg,

57%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-7-methylqui-

noline-4-carboxamide (52). The title product was prepared using

general procedure A, using 7-methylquinoline-4-carboxylic acid S31.

¹H NMR (400 MHz, CDCl₃) δ 8.89 (d, J = 4.36 Hz, 1H), 8.17 (d, J =

8.64 Hz, 1H), 7.92−7.88 (m, 1H), 7.47−7.41 (m, 2H), 7.16 (t, J =

4.64 Hz, 1H), 4.79−4.73 (m, 1H), 4.58 (dd, J = 5.36, 17.37 Hz, 0.1H),

4.40 (dd, J = 5.05, 17.76 Hz, 0.9H), 4.31 (dd, J = 3.70, 17.23 Hz,

0.1H), 4.24 (dd, J = 3.98, 17.77 Hz, 0.9H), 3.70 (ddd, J = 2.85, 6.84,

10.97 Hz, 1H), 3.58−3.49 (m, 1H), 2.57 (s, 3H), 2.39−2.17 (m, 4H);

¹³C NMR (101 MHz, CDCl₃) δ 167.66, 167.14, 149.91, 149.10,

140.66, 140.60, 130.21, 128.98, 124.96, 122.52, 118.18, 117.96, 46.81,

45.75, 42.56, 30.03, 25.20, 21.93; UPLC I (ESI) t_R1.14 min, m/z

323.5 [M + H]⁺(98%); LC−MS (A) t_R10.1 min, m/z 323.0 [M +

H]⁺(97%); white powder, 55 mg, 56%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-5-methylqui-

noline-4-carboxamide (53). The title product was prepared using

general procedure G, using 5-methylquinoline-4-carboxylic acid S30.

¹H NMR (400 MHz, CDCl₃) δ 8.93−8.84 (m, 1H), 8.04−7.96 (m,

1H), 7.62 (dd, J = 7.06, 8.49 Hz, 1H), 7.38 (dt, J = 1.20, 7.04 Hz, 1H),

7.35 (d, J = 4.24 Hz, 1H), 7.06 (t, J = 4.50 Hz, 1H), 4.75 (dd, J = 1.99,

7.79 Hz, 0.1H), 4.72−4.67 (m, 0.9H), 4.56 (dd, J = 5.53, 17.38 Hz,

0.1H), 4.37 (dd, J = 4.95, 17.82 Hz, 0.9H), 4.32 (dd, J = 5.53, 17.38

Hz, 0.1H), 4.23 (dd, J = 3.95, 17.81 Hz, 0.9H), 3.70 (ddd, J = 2.77,

7.09, 11.25 Hz, 1H), 3.52 (dt, J = 7.63, 10.69 Hz, 1H), 2.65 (s, 3H),

2.42−2.09 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.38, 166.83,

149.68, 149.13, 141.90, 134.49, 130.19, 129.73, 128.76, 123.43, 119.96,

117.89, 46.78, 45.74, 42.61, 30.05, 25.19, 21.31; UPLC I (ESI) t_R1.07

min, m/z 323.5 [M + H]⁺(99%); LC−MS (A) t_R10.0 min, m/z 323.1

[M + H]⁺(98%); white powder, 72 mg, 47%.

1

prepared using general procedure A. H NMR (400 MHz, DMSO-

d₆) (9/1 mixture of trans/cis amide rotamers) δ 11.88 (s, 1H), 8.68 (t,

J = 5.69 Hz, 1H), 8.33 (d, J = 4.91 Hz, 1H), 7.61 (dd, J = 2.51, 3.44

Hz, 1H), 7.42 (d, J = 4.92 Hz, 1H), 6.86 (dd, J = 1.86, 3.43 Hz, 1H),

5.29 (d, J = 7.22 Hz, 0.1H), 4.80 (dd, J = 3.62, 7.39 Hz, 0.9H), 4.38

(dd, J = 5.71, 16.63 Hz, 0.1H), 4.22 (dd, J = 6.09, 16.95 Hz, 0.9H),

4.14 (dd, J = 5.47, 16.93 Hz, 1H), 3.73 (ddd, J = 3.72, 7.57, 9.60 Hz,

1H), 3.54 (td, J = 6.79, 8.95 Hz, 1H), 2.34−2.01 (m, 4H); UPLC I

(ESI) t_R1.08 min, m/z 298.6 [M + H]⁺(96%); LC−MS (A) (ESI) t_R

9.2 min, m/z 298.0 [M + H]⁺(97%); colorless oil, 50 mg, 35%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-(4-ﬂuoro-

phenyl)-3H-imidazo[4,5-b]pyridine-7-carboxamide (48). The

title product was prepared using general procedure A using 2-(4-

ﬂuorophenyl)-3H-imidazo[4,5-b]pyridine-7-carboxylic acid S26. ¹H

NMR (400 MHz, DMSO-d₆) (9/1 mixture of trans/cis amide

rotamers) δ 14.02 (s, 1H), 10.09 (s, 1H), 8.55−8.41 (m, 3H), 7.75 (d,

J = 5.13 Hz, 1H), 7.49 (t, J = 8.60 Hz, 2H), 5.29 (dd, J = 2.78, 7.47 Hz,

0.1H), 4.88 (dd, J = 3.96, 6.56 Hz, 0.9H), 4.56 (d, J = 4.53 Hz, 0.1H),

4.45 (dd, J = 5.16, 17.77 Hz, 0.9H), 4.36 (dd, J = 4.92, 17.84 Hz, 1H),

3.79−3.69 (m, 1H), 3.55 (q, J = 8.30 Hz, 1H), 2.36−1.94 (m, 4H);

¹³C NMR (101 MHz, DMSO-d₆) δ 167.34, 166.85, 163.21, 162.70,

152.85, 144.21, 129.72, 129.63, 127.99, 125.40, 119.28, 116.58, 116.42,

116.21, 46.36, 45.35, 42.40, 29.57, 24.80; UPLC I (ESI) t_R1.52 min,

m/z 393.5 [M + H]⁺(96%); LC−MS (A) (ESI) t_R14.7 min, m/z

393.0 [M + H]⁺(97%); white powder, 53 mg, 58%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-2-(4-methoxy-

phenyl)-3H-imidazo[4,5-b]pyridine-7-carboxamide (49). The

title product was prepared using general procedure A using 2-(4-

ﬂuorophenyl)-3H-imidazo[4,5-b]pyridine-7-carboxylic acid S28. ¹H

NMR (400 MHz, DMSO-d₆) (9/1 mixture of trans/cis amide

rotamers) δ 10.15 (s, 1H), 8.44 (d, J = 5.02 Hz, 1H), 8.37 (d, J = 8.74

Hz, 2H), 7.72 (d, J = 4.99 Hz, 1H), 7.17 (d, J = 8.78 Hz, 2H), 5.32−

5.26 (m, 0.1H), 4.89 (dd, J = 4.13, 6.70 Hz, 0.9H), 4.58 (dd, J = 4.45,

17.77 Hz, 0.1H), 4.45 (dd, J = 4.92, 17.95 Hz, 0.9H), 4.36 (dd, J =

4.78, 17.81 Hz, 1H), 3.88 (s, 3H), 3.74 (ddd, J = 3.71, 7.64, 11.08 Hz,

1H), 3.55 (td, J = 6.92, 9.11 Hz, 1H), 2.23−1.96 (m, 4H); ¹³C NMR

(101 MHz, DMSO-d₆) δ 170.68, 166.32, 162.77, 161.25, 153.30,

149.94, 142.98, 132.23, 128.45, 120.49, 118.63, 115.75, 114.05, 54.98,

45.82, 44.80, 41.88, 29.03, 24.26; UPLC I (ESI) t_R1.50 min, m/z

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-7-phenylqui-

noline-4-carboxamide (54). The title product was prepared using

general procedure G, using 7-phenylquinoline-4-carboxylic acid S24.

¹H NMR (400 MHz, CDCl₃) δ 8.96 (d, J = 4.35 Hz, 1H), 8.38−8.33

(m, 2H), 7.89 (dd, J = 2.01, 8.66 Hz, 1H), 7.79−7.71 (m, 2H), 7.55−

7.46 (m, 3H), 7.47−7.37 (m, 1H), 7.22 (t, J = 4.58 Hz, 1H), 7.12 (t, J

= 4.28 Hz, 0.1H), 4.77 (dd, J = 2.41, 7.67 Hz, 1H), 4.60 (dd, J = 5.54,

17.33 Hz, 0.1H), 4.43 (dd, J = 5.07, 17.75 Hz, 0.9H), 4.34 (dd, J =

3.51, 17.28 Hz, 0.1H), 4.26 (dd, J = 4.02, 17.77 Hz, 0.9H), 3.72 (ddd, J

= 2.95, 7.09, 9.42 Hz, 1H), 3.59−3.50 (m, 1H), 2.51−2.12 (m, 4H);

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Article

¹³C NMR (101 MHz, CDCl₃) δ 167.35, 166.97, 150.32, 149.12,

(t, J = 14.69 Hz, 1H), 2.35 (dddd, J = 3.51, 9.44, 14.83, 40.62 Hz, 1H);

¹³C NMR (101 MHz, CDCl₃) δ 167.53, 167.30, 149.58, 148.15,

141.17, 130.28, 129.49, 127.94, 125.24, 124.35, 118.90, 117.24, 92.83,

91.03, 52.46, 52.22, 45.09, 42.34, 36.09, 35.88; UPLC I (ESI) t_R

1.11min, m/z 327.6 [M + H]⁺(98%); LC−MS (A) t_R9.5 min, m/z

327.0 [M + H]⁺(98%); white crystals, 95 mg, 60%.

142.68, 140.57, 139.78, 129.07, 128.16, 127.50, 127.47, 127.39, 125.73,

123.49, 118.69, 117.83, 46.72, 45.65, 42.48, 29.94, 25.10; LC−MS (A)

(ESI) t_R14.6 min, m/z 385.1 [M + H]⁺(95%); white powder, 86 mg,

74%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-7-

(phenylamino)quinoline-4-carboxamide (55). The title product

was prepared using general procedure G, using 7-(phenylamino)-

N-(2-((2S,4R)-2-Cyano-4-ﬂuoropyrrolidin-1-yl)-2-oxoethyl)-

quinoline-4-carboxamide (59). The title product was prepared

using general procedure G with 2-(quinoline-4-carboxamido)acetic

1

quinoline-4-carboxylic acid S25. H NMR (400 MHz, CDCl₃) δ 8.67

1

(d, J = 4.43 Hz, 1H), 8.07 (d, J = 9.10 Hz, 1H), 7.57 (d, J = 2.39 Hz,

1H), 7.42 (t, J = 4.78 Hz, 1H), 7.33−7.27 (m, 2H), 7.20 (ddd, J =

1.60, 2.58, 8.15 Hz, 4H), 7.02 (tt, J = 1.21, 7.35 Hz, 1H), 6.53 (s, 1H),

4.75 (dd, J = 1.94, 7.81 Hz, 0.1H), 4.72−4.66 (m, 0.9H), 4.50 (dd, J =

5.62, 17.21 Hz, 0.1H), 4.33 (dd, J = 5.36, 17.59 Hz, 0.9H), 4.24 (dd, J

= 3.96, 17.21 Hz, 0.1H), 4.13 (dd, J = 4.18, 17.59 Hz, 0.9H), 3.62

(ddd, J = 3.23, 7.04, 9.02 Hz, 1H), 3.44 (td, J = 4.49, 8.52, 9.05 Hz,

1H), 2.32−2.08 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 167.88,

167.29, 150.41, 150.23, 145.40, 141.28, 140.57, 129.56, 126.41, 122.81,

120.71, 119.91, 119.07, 118.12, 115.97, 110.03, 46.77, 45.72, 42.42,

29.93, 25.16; LC−MS (A) (ESI) t_R11.8 min, m/z 400.0 [M + H]⁺

(98%); white powder, 78 mg, 73%.

acid and S15. H NMR (400 MHz, CDCl₃) (1:9 mixture of cis/trans

amide rotamers) δ 8.94 (d, J = 4.39 Hz, 1H), 8.31−8.24 (m, 1H), 8.17

(dd, J = 1.16, 8.63 Hz, 1H), 7.77 (ddd, J = 1.41, 6.87, 8.45 Hz, 1H),

7.62 (ddd, J = 1.24, 6.92, 8.32 Hz, 1H), 7.56 (d, J = 4.33 Hz, 1H), 7.35

(s, 0.9H), 7.12 (s, 0.1H), 5.41−5.25 (m, 1H), 4.93 (t, J = 8.15 Hz,

0.1H), 4.80 (dd, J = 7.86, 9.23 Hz, 0.9H), 4.63 (dd, J = 5.23, 17.31 Hz,

0.1H), 4.43 (dd, J = 4.62, 17.54 Hz, 0.9H), 4.28 (dd, J = 4.24, 17.50

Hz, 1H), 3.96 (dd, J = 12.24, 20.03 Hz, 1H), 3.82 (ddd, J = 3.13,

12.30, 34.74 Hz, 0.9H), 3.60 (dd, J = 12.96, 33.60 Hz, 0.1H), 2.91−

2.76 (m, 1H), 2.49 (dddd, J = 3.91, 9.27, 14.76, 37.98 Hz, 1H); ¹³C

NMR (101 MHz, CDCl₃) δ 167.44, 167.24, 149.32, 147.91, 141.58,

130.68, 129.38, 128.29, 125.45, 124.60, 119.03, 117.18, 91.70, 89.88,

52.52, 52.29, 45.29, 42.74, 36.63, 36.41. 3; UPLC I (ESI) t_R1.09min,

m/z 327.6 [M + H]⁺(99%); LC−MS (A) t_R9.1 min, m/z 327.0 [M +

H]⁺(98%); white crystals, 86 mg, 63%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-5-phenylqui-

noline-4-carboxamide (56). The title product was prepared using

general procedure G, using 5-phenylquinoline-4-carboxylic acid S32.

¹H NMR (400 MHz, CDCl₃) (mixture of 1:9 cis/trans amide

(S)-N-(2-(2-Cyano-4,4-diﬂuoropyrrolidin-1-yl)-2-oxoethyl)-

quinoline-4-carboxamide (60). The title product was prepared

rotamer) major rotamer, δ 8.98 (d, J = 4.21 Hz, 1H), 8.20 (dd, J =

1.30, 8.46 Hz, 1H), 7.80 (dd, J = 7.14, 8.47 Hz, 1H), 7.52 (dd, J = 1.35,

7.16 Hz, 1H), 7.49−7.46 (m, 1H), 7.43 (s, 1H), 7.33 (dd, J = 8.72,

11.73 Hz, 2H), 7.05 (s, 1H), 6.49−6.43 (m, 2H), 4.77−4.71 (m, 1H),

4.43 (dd, J = 4.80, 17.69 Hz, 1H), 4.29 (dd, J = 4.01, 17.76 Hz, 1H),

3.44 (ddd, J = 3.19, 7.76, 10.27 Hz, 1H), 3.32−3.10 (m, 1H), 2.44−

1

using general procedure A. H NMR (400 MHz, CDCl₃) (8.5/1.5

mixture of trans/cis amide rotamers) δ 8.96−8.86 (m, 1H), 8.23 (t, J =

9.99 Hz, 1H), 8.12 (d, J = 8.42 Hz, 1H), 7.74 (t, J = 7.69 Hz, 1H), 7.60

(dd, J = 11.22, 4.11 Hz, 1H), 7.49 (dd, J = 10.11, 4.30 Hz, 1H), 7.30

(s, 1H), 5.15 (d, J = 9 Hz, 0.15H), 4.99−4.92 (m, 0.85H), 4.70 (dd, J

= 17.4, 5.7 Hz, 0.15H), 4.39 (dd, J = 17.4, 5.6 Hz, 0.85H), 4.33 (dd, J

= 17.4, 4.3 Hz, 0.15H), 4.21 (dd, J = 17.4, 4.2 Hz, 0.85H), 4.07−3.91

(m, 2H), 2.83−2.72 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 167.68,

167.40, 149.93, 148.82, 140.67, 130.30, 130.11, 128.08, 125.24, 124.41,

118.93, 116.09, 52.54, 52.22, 51.90, 44.44, 42.42, 37.78, 37.53, 37.28,

31.07; MS (ESI) m/z 345.0 [M + 1]⁺; LC−MS (A) t_R10.8 min, m/z

345.0 [M + H]⁺(98%); HRMS calcd for C₁₇H₁₅N₄O₂F₂[M + H]⁺,

345.1163; found, 345.1172; oﬀ-white powder, 350 mg, 67%.

(S)-N-(2-(2-Cyano-4,4-diﬂuoropyrrolidin-1-yl)-2-oxoethyl)-6-

methoxyquinoline-4-carboxamide (61). The title product was

prepared using general procedure A. ¹H NMR (400 MHz, CDCl₃) (9/

1 mixture of trans/cis amide rotamers) δ 8.63 (d, J = 4.43 Hz, 1H),

7.90 (d, J = 9.19 Hz, 1H), 7.54 (d, J = 2.75 Hz, 1H), 7.47−7.40 (br s,

1H), 7.35 (d, J = 4.40 Hz, 1H), 7.31 (dd, J = 2.78, 9.24 Hz, 1H), 5.19−

5.12 (m, 0.1H), 4.91 (dd, J = 4.58, 8.37 Hz, 0.9H), 4.52 (d, J = 5.77

Hz, 0.1H), 4.30 (dd, J = 5.71, 17.35 Hz, 0.9H), 4.12 (dd, J = 4.73,

17.33 Hz, 1H), 4.08−3.86 (m, 2H), 3.86 (s, 3H), 2.83−2.65 (m, 4H);

¹³C NMR (101 MHz, CDCl₃) δ 168.18, 167.71, 158.77, 147.05,

1

2.10 (m, 4H); mionor rotamer, H NMR (400 MHz, CDCl₃) δ 8.95

(d, J = 4.26 Hz, 1H), 8.16 (dd, J = 1.24, 8.46 Hz, 1H), 7.60 (dd, J =

7.55, 8.47 Hz, 1H), 7.55 (d, J = 4.33 Hz, 1H), 7.49 (d, J = 4.27 Hz,

1H), 7.43 (s, 1H), 7.33 (dd, J = 8.72, 11.73 Hz, 2H), 6.94 (s, 1H),

6.49−6.43 (m, 2H), 4.79 (d, J = 5.81 Hz, 1H), 4.43 (dd, J = 4.80,

17.69 Hz, 1H), 4.29 (dd, J = 4.01, 17.76 Hz, 1H), 3.76−3.66 (m, 1H),

3.62−3.50 (m, 1H), 2.44−2.10 (m, 4H); ¹³C NMR (101 MHz,

CDCl₃) δ 167.91, 166.54, 149.70, 141.36, 139.43, 133.70, 131.05,

129.81, 129.32, 128.11, 127.02, 122.45, 121.11, 118.86, 117.70, 100.00,

46.55, 45.37, 42.20, 29.98, 24.97; LC−MS (A) (ESI) t_R13.3 min, m/z

385.1 [M + H]⁺(96%); colorless oil, 52 mg, 84%.

(S)-N-(2-(2-Cyanopyrrolidin-1-yl)-2-oxoethyl)-5-methoxyqui-

noline-4-carboxamide (57). The title product was prepared using

general procedure G, using 5-methoxyquinoline-4-carboxylic acid S33.

¹H NMR (400 MHz, CDCl₃) (9:1 mixture of trans/cis amide

rotamers) δ 9.01 (d, J = 5.51 Hz, 1H), 8.70 (dd, J = 0.83, 8.64 Hz,

1H), 8.21 (d, J = 8.64 Hz, 1H), 7.74 (dt, J = 0.92, 8.66 Hz, 1H), 7.66

(dd, J = 7.62, 8.60 Hz, 1H), 6.92 (dd, J = 1.01, 7.73 Hz, 1H), 4.88 (d, J

= 6.04 Hz, 0.1H), 4.85−4.81 (m, 0.9H), 4.58 (dd, J = 6.21, 17.03 Hz,

0.1H), 4.42 (dd, J = 5.53, 17.72 Hz, 0.9H), 4.34−4.27 (m, 0.1H), 4.25

(dd, J = 4.34, 17.75 Hz, 0.9H), 4.02 (s, 3H), 3.78−3.68 (m, 1H),

3.63−3.49 (m, 1H), 2.41−2.17 (m, 4H); ¹³C NMR (101 MHz,

CDCl₃) δ 167.50, 164.82, 155.19, 149.17, 147.28, 132.81, 130.53,

122.20, 121.83, 118.15, 118.00, 105.71, 56.01, 46.80, 45.81, 42.34,

30.08, 25.29; UPLC I (ESI) t_R1.61 min, m/z 339.6 [M + H]⁺(97%);

LC−MS (A) t_R14.0 min, m/z 339.1 [M + H]⁺(97%); white crystals,

63 mg, 64%.

144.85, 139.04, 131.13, 127.88, 125.54, 123.14, 119.15, 116.40, 102.85,

55.83,52.05 (t, J = 32.25 Hz), 44.41, 42.20, 37.26 (t, J = 25.29 Hz);

UPLC I (ESI) t_R1.28 min, m/z 375.6 [M + H]⁺(99%); LC−MS (A)

(ESI) t_R11.6 min, m/z 374.9 [M + H]⁺(98%); white powder, 61 mg,

33%.

(S)-5-Bromo-N-(2-(2-cyano-4,4-diﬂuoropyrrolidin-1-yl)-2-

oxoethyl)quinoline-4-carboxamide (62). The title product was

1

prepared using general procedure A. H NMR (400 MHz, CDCl₃)

(cis/trans mixture of amide rotamers, 1:9) δ 9.10 (dd, J = 4.46, 6.43

Hz, 0.9H), 8.88 (s, 0.1H), 8.41 (d, J = 8.76 Hz, 1H), 8.05 (d, J = 8.73

Hz, 1H), 7.96 (d, J = 8.55 Hz, 1H), 7.84 (dd, J = 1.06, 7.58 Hz, 1H),

7.55 (dd, J = 7.47, 8.49 Hz, 1H), 5.34 (d, J = 8.83 Hz, 0.1H), 5.03 (dd,

J = 5.05, 8.22 Hz, 0.9H), 4.64 (dd, J = 6.13, 17.08 Hz, 0.1H), 4.56 (dd,

J = 6.74, 17.53 Hz, 0.9H), 4.30 (dd, J = 4.51, 17.10 Hz, 0.1H), 4.17

(dd, J = 4.17, 17.48 Hz, 0.9H), 4.14−4.05 (m, 1H), 4.04−3.95 (m,

1H), 2.84−2.73 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 168.40,

164.31, 149.25, 146.80, 136.27, 131.65, 130.18, 129.81, 128.24, 121.69,

119.40, 116.80, 53.55,51.98 (t, 32.1 Hz, 1C), 44.42, 41.96, 37.19 (t,

25.4 Hz, 1C); UPLC I (ESI) t_R1.83 min, m/z 423.5, 425.3 [M + H]⁺

(96%); LC−MS (A) t_R15.8 min, m/z 265.9, 267.9 (95%); white

powder, 89 mg, 54%.

N-(2-((2S,4S)-2-Cyano-4-ﬂuoropyrrolidin-1-yl)-2-oxoethyl)-

quinoline-4-carboxamide (58). The title product was prepared

using general procedure G with 2-(quinoline-4-carboxamido)acetic

1

acid and S16. H NMR (400 MHz, CDCl₃) (1:9 mixture of cis/trans

amide rotamers) δ 8.87 (d, J = 4.45 Hz, 1H), 8.23 (ddd, J = 0.66, 1.36,

8.60 Hz, 1H), 8.13−8.09 (m, 1H), 7.76−7.69 (m, 1H), 7.60−7.54 (m,

1H), 7.48 (d, J = 4.33 Hz, 1H), 7.41−7.35 (m, 1H), 5.43 (dt, J = 3.29,

51.00 Hz, 0.9H), 5.34 (dt, J = 3.38, 51.11 Hz, 0.1H), 5.04 (d, J = 8.96

Hz, 0.1H), 4.92 (d, J = 9.29 Hz, 0.9H), 4.62 (dd, J = 5.79, 17.21 Hz,

0.1H), 4.37 (dd, J = 5.64, 17.48 Hz, 0.9H), 4.29 (dd, J = 4.02, 17.22

Hz, 0.1H), 4.17 (dd, J = 4.19, 17.49 Hz, 0.9H), 3.96 (ddd, J = 1.21,

11.79, 22.38 Hz, 1H), 3.76 (ddd, J = 3.62, 12.05, 36.05 Hz, 1H), 2.66

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(S)-5-Cyano-N-(2-(2-cyano-4,4-diﬂuoropyrrolidin-1-yl)-2-

oxoethyl)quinoline-4-carboxamide (63). To a round-bottom ﬂask

were added (S)-5-bromo-N-((2-cyano-4,4-diﬂuoropyrrolidin-1-yl)-

methyl)quinoline-4-carboxamide 65 (0.085 g, 0.215 mmol), zinc

cyanide (0.015 g, 0.129 mmol) (caution, highly toxic), 10% palladium

on carbon (0.023 g, 0.022 mmol), 1,1′-bis(diphenylphosphino)-

ferrocene (4.82 mg, 8.60 μmol), and DMAC (volume, 2 mL). The

resulting slurry was sparged with subsurface nitrogen for 10 min, and

zinc(II) formate dihydrate (6.2 mg, 0.032 mmol) was added to the

reaction mixture. The reaction mixture was again sparged with

subsurface nitrogen for 10 min and was heated under nitrogen to 110

°C for 2 h. The reaction mixture was diluted with 10 mL of EtOAc.

The resulting slurry was ﬁltered, and the cake was rinsed with EtOAc

(2 mL). The product was isolated by washing the ﬁltrate with water (2

× 10 mL) and 5% NH₄OH (1 × 10 mL). The organic layer was dried

with Na₂SO₄. The volatile was removed in vacuo to give a residue,

which was further puriﬁed by silica gel chromatography (heptanes/

EtOAc) to provide the product (white powder, 40 mg, 50%). ¹H NMR

(400 MHz, DMSO-d₆) δ 9.20 (t, J = 5.73 Hz, 1H), 8.73 (d, J = 8.75

Hz, 1H), 8.49 (d, J = 8.53 Hz, 1H), 8.44−8.37 (m, 2H), 8.05 (dd, J =

7.08, 8.62 Hz, 1H), 5.13 (dd, J = 2.65, 9.28 Hz, 1H), 4.37−4.27 (m,

1H), 4.27 (d, J = 5.92 Hz, 2H), 4.14 (dt, J = 10.32, 21.10 Hz, 1H),

3.00−2.73 (m, 2H); UPLC II (ESI) t_R3.24 min, m/z 370.6 [M + H]⁺

(95%).

was extracted with a mixture 4:1 of DCM−isopropanol. The DCM

layer was dried over sodium sulfate, ﬁltered, evaporated, and further

puriﬁed using column chromatography (DCM−MeOH, 0−10%

MeOH) to yield a clear oil of 0.119 g (79%) of (R)-1-(2-

(quinoline-4-carboxamido)acetyl)pyrrolidin-2-ylboronic acid. ¹H

NMR (400 MHz, D₂O) δ 8.92−8.84 (m, 1H), 8.15 (ddd, J = 0.64,

1.43, 8.58 Hz, 1H), 8.09−8.00 (m, 1H), 7.84 (ddd, J = 1.41, 6.92, 8.47

Hz, 1H), 7.70 (ddd, J = 1.18, 6.94, 8.29 Hz, 1H), 7.64 (d, J = 4.46 Hz,

1H), 4.32−4.27 (m, 2H), 3.67 (ddd, J = 3.33, 8.37, 10.14 Hz, 1H),

3.55 (ddd, J = 6.54, 8.44, 10.90 Hz, 1H), 3.12 (dd, J = 7.01, 10.34 Hz,

1H), 2.13 (tdd, J = 3.42, 7.02, 9.03 Hz, 2H), 2.08−1.87 (m, 1H),

1.82−1.68 (m, 1H); ¹³C NMR (101 MHz, D₂O) δ 170.63, 168.11,

150.41, 147.54, 142.15, 131.50, 128.92, 128.64, 125.66, 124.61, 119.99,

48.94 (¹³C-B splitting), 47.22, 42.32, 27.48, 27.43; UPLC I (ESI) t_R

1.01 min, m/z 328.6 [M + H]⁺(99%); LC−MS (A) t_R9.3min, m/z

328.0 [M + H]⁺(98%); HRMS calcd for C₁₆H₁₉BN₃O₄[M + H]⁺,

328.1469; found, 328.1469.

(S)-N-(2-(2-(2-Chloroacetyl)pyrrolidin-1-yl)-2-oxoethyl)-

quinoline-4-carboxamide (67). 4 M HCl (10.90 mL, 43.6 mmol)

in dioxane was added to (S)-tert-butyl 2-(2-chloroacetyl)pyrrolidine-1-

carboxylate (1.2 g, 4.84 mmol) S35 and stirred for 1 h. Then the

volatiles were evaporated and the residue was washed with ether to

give 0.9 g of (S)-2-chloro-1-(pyrrolidin-2-yl)ethanone hydrochloride.

The product was used in the next reaction without further puriﬁcation.

To a solution containing 2-(quinoline-4-carboxamido)acetic acid

(0.180 g, 0.782 mmol) and HOBT (0.132 g, 0.860 mmol) in 1,4-

dioxane (5 mL) was added a solution of EDC (0.165 g, 0.860 mmol)

in DCM (5 mL). The mixture was stirred for 30 min at 0 °C. To the

resulting solution was added the appropriate amine (S)-2-chloro-1-

(pyrrolidin-2-yl)ethanone hydrochloride (0.144 g, 0.782 mmol) and

DIPEA (0.150 mL, 0.860 mmol) in DCM (4 mL), with stirring. After

2 h (on completion of the reaction), the reaction mixture was diluted

with DCM and washed with saturated aqueous NaHCO₃solution (10

mL), 0.1 N aqueous citric acid solution (10 mL), and brine (10 mL).

The combined organic layers were dried over MgSO₄, ﬁltered,

concentrated, and puriﬁed with ﬂash chromatography using DCM−

MeOH with 0−5% methanol (slightly yellowish oil, 0.120 g, 43%). ¹H

NMR (400 MHz, CDCl₃) δ 8.96 (d, J = 4.38 Hz, 1H), 8.29 (dd, J =

1.36, 8.17 Hz, 1H), 8.19 (d, J = 8.17 Hz, 1H), 7.78 (ddd, J = 1.41, 6.99,

8.50 Hz, 1H), 7.64 (ddd, J = 1.22, 6.91, 8.30 Hz, 1H), 7.54 (d, J = 4.32

Hz, 1H), 7.02 (s, 1H), 4.83 (dd, J = 5.10, 8.35 Hz, 1H), 4.43 (dd, J =

4.78, 17.74 Hz, 1H), 4.33 (d, J = 15.29 Hz, 1H), 4.35−4.25 (m, 1H),

4.23 (d, J = 15.33 Hz, 1H), 3.71 (ddd, J = 5.79, 7.27, 9.78 Hz, 1H),

3.63 (dt, J = 6.68, 9.75 Hz, 1H), 2.35−1.97 (m, 4H); ¹³C NMR (101

MHz, CDCl₃) δ 200.81, 167.32, 166.61, 149.78, 148.58, 141.31,

130.25, 129.78, 127.98, 125.42, 124.54, 118.95, 62.52, 47.19, 46.54,

42.41, 28.81, 25.16; UPLC I (ESI) t_R1.25min, m/z 360.6 [M + H]⁺

(95%); LC−MS (A) t_R11.0 min, m/z 360.1 [M + H]⁺(98%).

N-(2-Oxo-2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide

(64). The title product was prepared using general procedure A using

2-amino-1-(pyrrolidin-1-yl)ethanone hydrochloride S34. The product

was puriﬁed using column chromatography with a DCM-MeOH

1

gradient, followed by recrystallization from methanol. H NMR (400

MHz, DMSO-d₆) δ 8.99 (d, J = 4.30 Hz, 1H), 8.92 (t, J = 5.90 Hz,

1H), 8.41 (d, 8.16 Hz, 1H), 8.09 (d, J = 8.32 Hz, 1H), 7.82 (ddd, J =

1.45, 6.88, 8.44 Hz, 1H), 7.68 (ddd, J = 1.31, 6.84, 8.25 Hz, 1H), 7.55

(d, J = 4.27 Hz, 1H), 4.17−4.07 (m, 2H), 3.51 (t, J = 6.75 Hz, 2H),

3.37 (t, J = 6.89 Hz, 2H), 1.94 (h, J = 6.12, 6.65 Hz, 2H), 1.87−1.75

(m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 167.55, 166.67, 150.69,

148.38, 142.83, 130.28, 129.66, 127.63, 126.45, 124.74, 119.44, 46.16,

45.46, 42.13, 26.16, 24.22; UPLC I (ESI) t_R1.11min, m/z 284.7 [M +

H]⁺(98%); LC−MS (A) t_R7.8 min, m/z 284.1 [M + H]⁺(98%);

white powder, 0.187 g, 73%.

N-(2-Oxo-2-((2R)-2-((3aS,7aR)-3a,5,5-trimethylhexahydro-

4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)-

ethyl)quinoline-4-carboxamide (65). (2R)-2-((3aS,7aR)-3a,5,5-

Trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)-

pyrrolidine hydrochloride³³(0.190 g, 0.665 mmol) was added to a

mixture of 2-(quinoline-4-carboxamido)acetic acid (0.153 g, 0.665

mmol), HATU (0,253 g, 0,665 mmol), and DIPEA (0.360 mL, 2.062

mmol) in dichloromethane (5 mL). The mixture was stirred for 3 h

and washed with 0.5 N citric acid, saturated sodium bicarbonate, and

brine, followed by ﬁltration and evaporation. Puriﬁcation was done

using column chromatography DCM−MeOH, 0−6% MeOH. ¹H

NMR (400 MHz, CDCl₃) δ 8.97 (d, J = 4.36 Hz, 1H), 8.30 (ddd, J =

0.72, 1.48, 8.50 Hz, 1H), 8.17 (dd, J = 1.25, 8.28 Hz, 1H), 7.77 (ddd, J

= 1.44, 6.92, 8.47 Hz, 1H), 7.62 (ddd, J = 1.29, 6.94, 8.30 Hz, 1H),

7.53 (d, J = 4.34 Hz, 1H), 7.19 (t, J = 4.10 Hz, 1H), 4.32−4.27 (m,

3H), 3.61−3.39 (m, 2H), 3.21 (dd, J = 6.93, 10.00 Hz, 1H), 2.50−

2.25(m, 1H), 2.25−1.95 (m, 5H), 1.94−1.78 (m, 3H), 1.41 (s, 3H),

1.28−1.24 (m, 4H), 0.82 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ

167.01, 165.44, 149.56, 130.42, 129.57, 128.03, 125.63, 124.73, 119.02,

86.32, 78.05, 77.36, 53.57, 51.35, 45.86, 42.35, 39.61, 38.75, 38.35,

35.62, 28.59, 27.53, 27.34, 27.20, 26.41, 24.15; colorless oil, 0.270 g,

88%.

ASSOCIATED CONTENT

* Supporting Information

■

S

Detailed synthetic procedures and analytical data for inter-

mediates, protocols of the enzymatic assays used, and

procedures and extended data series for in vitro and in vivo

pharmacokinetic analyses. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: pieter.vanderveken@uantwerpen.be. Phone: +32-3

265 27 08.

■

(R)-1-(2-(Quinoline-4-carboxamido)acetyl)pyrrolidin-2-yl-

boronic Acid (66). To a stirred solution of the N-(2-oxo-2-((2R)-2-

((3aS,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]-

dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide 69

(0.27 g, 0.585 mmol) in water (8 mL) at pH 3 (adjusting as

necessary with 2 N aqueous HCl) were added phenylboronic acid

(0.143 g, 1.17 mmol) and methyl tert-butyl ether (7 mL). The mixture

was stirred for 2 days (pH 4). Water and the organic layer were

separated. The water layer was adjusted to pH 3, and the water layer

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank Nicole Lamoen, Sophie Lyssens, and Ann

Mattheussen for excellent technical assistance with the

■

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dx.doi.org/10.1021/jm500031w | J. Med. Chem. 2014, 57, 3053−3074

Journal of Medicinal Chemistry

Article

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ABBREVIATIONS USED

■

FAP, ﬁbroblast activation protein; PREP, prolyl oligopeptidase;

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Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP)

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