Discovery of the Potent, Selective, Orally Available CXCR7 Antagonist ACT-1004-1239

pubs.acs.org/jmc

Article

Discovery of the Potent, Selective, Orally Available CXCR7

Antagonist ACT-1004-1239

̈

Sylvia Richard-Bildstein,* Hamed Aissaoui, Julien Pothier, Gabriel Schafer, Carmela Gnerre,

Eleanor Lindenberg, Francois Lehembre, Laetitia Pouzol, and Philippe Guerry *

̧

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01588

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: The chemokine receptor CXCR7, also known as ACKR3, is a seven-transmembrane G-protein-coupled receptor

(GPCR) involved in various pathologies such as neurological diseases, autoimmune diseases, and cancers. By binding and scavenging

the chemokines CXCL11 and CXCL12, CXCR7 regulates their extracellular levels. From an original high-throughput screening

campaign emerged hit 3 among others. The hit-to-lead optimization led to the discovery of a novel chemotype series exempliﬁed by

the trans racemic compound 11i. This series provided CXCR7 antagonists that block CXCL11- and CXCL12-induced ß-arrestin

recruitment. Further structural modiﬁcations on the trisubstituted piperidine scaﬀold of 11i yielded compounds with high CXCR7

antagonistic activities and balanced ADMET properties. The eﬀort described herein culminated in the discovery of ACT-1004-1239

(28f). Biological characterization of ACT-1004-1239 demonstrated that it is a potent, insurmountable antagonist. Oral

administration of ACT-1004-1239 in mice up to 100 mg/kg led to a dose-dependent increase of plasma CXCL12 concentration.

migration, adhesion, proliferation, diﬀerentiation, and cell

survival.⁹Dysregulation of the CXCR7 axis has been associated

with various disease states such as, neurological disorders,¹⁰⁻¹³

autoimmune diseases,¹⁴⁻¹⁷inﬂammation,¹⁸⁻²⁰cancers,²¹⁻²⁶

and cardiac disorders.^21,27,28

Antibodies and/or small interfering RNA (siRNA) have

demonstrated the utility of targeting CXCR7 in some of these

diseases.²⁹⁻³³Although a number of small molecules were

reported as CXCR7 antagonists,¹⁷⁻¹⁹they were later on

described as bona ﬁde agonists, recruiting β-arrestin to CXCR7

upon binding to the receptor (“functional antagonists”).³⁴⁻³⁶

Very recently, a moderately potent CXCR7 antagonist

inhibiting the recruitment of ß-arrestin to CXCR7 following

CXCL12 stimulation was reported (“true antagonist”).³⁷We

describe here the hit-to-lead process followed by the lead

INTRODUCTION

■

C-X-C chemokine receptor type 7 (CXCR7) is a seven-

transmembrane G-protein-coupled receptor (GPCR), also

known as ACKR3 (atypical chemokine receptor 3), expressed

mainly by endothelial cells and other nonlymphoid cells,

including mesenchymal cells, cardiomyocytes, neurons,

astrocytes, and oligodendrocyte precursor cells (OPCs).^1,2

CXCR7 binds, internalizes, and degrades the chemokines

CXCL11 (C-X-C chemokine ligand 11) and CXCL12 (C-X-C

chemokine ligand 12). Importantly, CXCL11 and CXCL12

also bind to the chemokine receptors CXCR3 and CXCR4,

respectively. Unlike classical GPCRs, CXCR7 does not couple

with G proteins and consequently does not mediate signaling

cascades but rather functions primarily as a scavenger receptor.

Binding of its ligands leads to the recruitment of β-arrestin,

internalization of the CXCR7-ligand complex,^1,3and degrada-

tion of the ligand in lysosomes.⁴Through its scavenging

function, CXCR7 regulates the extracellular levels of CXCL11

and CXCL12 and, therefore, modulates CXCR3 and CXCR4

activities.⁵CXCL11-CXCR3 signaling regulates migration,

diﬀerentiation, and activation of immune cells including T

helper type 1 cells and macrophages.⁶⁻⁸Activation of CXCR4

by CXCL12 mediates a wealth of cellular functions such as

Received: September 10, 2020

American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

A

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Structures of the CXCR7 agonist 1 and primary screening hits 2−6.

optimization of a novel chemotype series providing potent,

selective, insurmountable, and orally available “true” CXCR7

antagonists.

more than 30% eﬀect (inhibition) in the primary screen were

selected, and their IC₅₀values were determined.

Following our screening cascade, another β-arrestin recruit-

ment assay (CXCR7 Tango assay from Invitrogen, “CXCR7-

CXCL12 assay” thereafter) was selected to further proﬁle the

active compounds using CXCL12 as a ligand. This assay was

used as a biological in vitro assay to establish the SAR of the

compounds identiﬁed in the primary screen. The CXCR7-β-

lactamase U2OS cell line contains human CXCR7 fused at its

intracellular C-terminus to the Gal4-VP16 transcription factor

(CXCR7-Gal4-VP16). Interposed between CXCR7 and Gal4-

VP16 is the tobacco etch virus (TEV) protease site that can be

recognized and cleaved by the TEV protease. The parental cell

line stably expresses a β-arrestin/TEV protease fusion protein

and the β-lactamase reporter gene under the control of a UAS

response element. Upon ligand binding and receptor

activation, the protease-tagged β-arrestin molecule is recruited

to CXCR7-Gal4-VP16. The protease cleaves the transcription

factor Gal4-VP16 from CXCR7, which translocates to the

nucleus and activates the expression of β-lactamase. A FRET-

enabled substrate allows detecting β-lactamase activity. The

results of this assay are reported as IC₅₀values.

RESULTS AND DISCUSSION

■

Biological Assays. Binding of agonists to CXCR7 has been

reported to mediate β-arrestin recruitment to the receptor.

This recruitment can be monitored using the PathHunter

CHO-K1 CXCR7 β-arrestin assay from DiscoverX (“CXCR7-

agonist 1 assay” thereafter). The system is based on the

enzyme fragment complementation technology. Two com-

plementing fragments of the β-galactosidase (β-gal) enzyme

are expressed within stably transfected cells. The larger portion

of β-gal, termed EA for enzyme acceptor, is fused to the C-

terminus of β-arrestin 2. The smaller fragment, termed ProLink

tag, is fused to CXCR7 at the C-terminus. Upon activation, β-

arrestin 2 is recruited to CXCR7, which forces the interaction

of ProLink and EA, allowing complementation of the two

fragments of β-gal, and the formation of a functional enzyme,

which is able to hydrolyze the substrate and generating a

chemiluminescent signal.

This assay was used to screen the Idorsia, formerly Actelion,

screening compound collection. For practical and cost reasons,

we decided to screen our library against the low-molecular-

weight agonist 1³⁸(Figure 1) instead of the natural ligands

CXCL11 and CXCL12. It was also postulated that this

innovative approach could lead to the identiﬁcation of weak

antagonists that we might not have identiﬁed by screening

against the natural ligands. The CHO-K1 CXCR7 β-arrestin

cell line was preincubated with the compounds to be screened

for 15 min before the addition of 5 nM of the small molecule

agonist 1. Compounds from our screening compound

collection were screened at 10 μM. Compounds displaying

Structure−Activity and Structure−Property Relation-

ships. The 300,000 compounds of the HTS deck of Idorsia

were composed of vendor screening compounds and

proprietary compounds. The high-throughput screening of

this compound collection against CXCR7 activated with the

agonist 1³⁸(CXCR7-agonist 1 assay) identiﬁed a small cluster

of ﬁve basic molecules 2, 3, 4, 5, and 6, all of them devoid of

antagonistic activity against the natural ligand in the CXCR7-

CXCL12 assay. Herein, we describe the conversion of this

unique cluster of antagonists active exclusively against the

agonist 1 to potent CXCR7 antagonists active against the

natural ligands CXCL11 and CXCL12.

B

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. First SAR Iteration on Amides A and B

a

The CHO-K1 CXCR7 β-arrestin cell line was preincubated with the compounds to be screened for 15 min before the addition of 5 nM of the

small molecule agonist 1, used as surrogate of endogenous CXCR7 ligands; data are the geometric mean (+/−2-fold) of at least three independent

experiments. For synthesis and assay details, see the Supporting Information.

C

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Having no crystal structure of the target protein available to

guide chemistry and in view of the structural features of the

hits, a classical high-throughput chemistry approach was

followed to explore the chemical space around these

compounds using the CXCR7-agonist 1 assay. In the hit-to-

lead phase of the project, several libraries were produced in an

iterative manner starting mainly from commercially available

building blocks. The ﬁve compounds depicted in Figure 1

shared some common features. To conﬁrm the emerging SAR,

two amide libraries have been prepared (Table 1). The

truncation of the thiophene moiety in hits 2 and 3

(compounds 7a and 7b) led to similar or improved ligand

eﬃciency (LE) showing that this moiety was not a key part of

the pharmacophore. Combination of amine and carboxylic acid

parts of the hits was either inactive (7c, 7d) or equipotent (7e)

to the original hits. As shown by the inactive compound 7f, the

basic amine seemed to be important for potency. Bridging of

the tertiary amine to a pyrrolidine (7g) decreased the LE. The

extension of the amide linker from two to three carbons led to

similar (7h, 7j) or improved LE (7k). Introduction of

additional ring constraints in the linker was investigated. The

4-amino-piperidine cores of compounds 7i and 7l gave only a

moderate LE, but the substitution of the piperidine N-atom

with larger alkyl groups as analogs of 7l provided a signiﬁcant

increase in potency with improved or at least similar LE as

shown by compounds 7m to 7p, 7o exhibiting an IC₅₀value of

78 nM. Considering the chemical structure of the compounds,

a cardiac potassium channel hERG liability was anticipated and

Table 2. Modulation of the Carboxamide Part

conﬁrmed by the activity of 7o in the hKV11.1 assay (IC₅₀

=

4.7 μM). To minimize the risk of inhibition of this ion channel,

the compounds were designed by ideally keeping the cLog D_7.4

in the range of 0.5−1.5.³⁹

For the second synthetic iteration, 1-cyclohexylpiperidin-4-

amine was kept constant and coupled with a series of

carboxylic acids to map the right-hand side amide part

(Table 2). An unsubstituted phenyl triazole 8a and

unsubstituted phenyl-4-oxobutanamide 8c surprisingly led to

a complete loss in activity, highlighting the importance of the

substituents of the phenyl ring. It appeared that ﬂuorine atoms

had a synergistic eﬀect and important role for reaching low

nanomolar potency as shown by the p-ﬂuorophenyl compound

8d (IC₅₀= 374 nM, 5-fold less potent than 7o). A p-bromo

halogen atom was substantially less active (8b) and compound

8f bearing a p-chloro substituent led to a 4-fold reduction in

potency. A pyridine moiety as a bioisostere⁴⁰of the 2,4-

diﬂuorophenyl group led to the inactive compound 8j. The

combination of an isoxazole and the key ﬂuorine substituents

as illustrated by compounds 8g, 8h, and 8i gave an activity cliﬀ

to IC₅₀= 5 nM for 8g and conﬁrmed the important role of the

two ﬂuorine atoms. Based on the low nanomolar potency

reached, compound 8g was further characterized. Tested in the

CXCR7-CXCL12 assay with CXCL12 as a ligand, 8g showed

an IC₅₀of 463 nM (Table 3). Despite an acceptable measured

log D_7.4of 2.8, 8g showed an hERG inhibition of 1.5 μM in the

hKV11.1 assay. But the compound exhibited encouraging

solubilities in fasted (FaSSIF) and fed state simulated intestinal

ﬂuid (FeSSIF) with 144 and 196 mg/L, respectively, and an

aqueous solubility of 15 mg/mL at pH 7.

a

The CHO-K1 CXCR7 β-arrestin cell line was preincubated with the

compounds to be screened for 15 min before the addition of 5 nM of

the small molecule agonist 1, used as surrogate of endogenous

CXCR7 ligands; data are the geometric mean (+/−2-fold) of at least

three independent experiments. For synthesis and assay details, see

the Supporting Information.

channel liability (Table 3). Diverse alkyl chains were tolerated

and led to nanomolar activities in the CXCR7-agonist 1 assay.

Unfortunately, the shift in IC₅₀between the CXCR7-agonist 1

assay and CXCR7-CXCL12 assay was still signiﬁcant and the

potency slightly decreased when the ring size was reduced (9c

and 9d). The amide analog 9a conﬁrmed that an amine group

was mandatory for potency. In addition, attempts to reduce the

basicity of the tertiary amine led also to a decrease in activity as

depicted by the ﬂuorinated example 9e and ether analogue 9f.

A benzyl substituent 9g led to the low IC₅₀shift of 12 between

the assays but with the price of an increased lipophilicity and

potentially an added metabolic hotspot. The shift observed

with the secondary amine 9b was the lowest (5.8), but the

potency was too low for further investigation.

For the next generation of compounds, the most potent ones

were screened in the CXCR7-CXCL12 assay in addition to the

CXCR7-agonist 1 assay. The left-hand part N-R¹amine

substituent was modiﬁed with the double aim of further

improving the activity and tackling the identiﬁed hERG

The replacement of the isoxazole ring with other 5-

membered or 6-membered heterocycles was then explored.

The modiﬁcation had a marked impact on the lipophilicity of

the compounds as shown by the clog D_7.4values. Table 4

D

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3. Exploration of the Tertiary Amine Moiety

a

The CHO-K1 CXCR7 β-arrestin cell line was preincubated with the compounds to be screened for 15 min before the addition of 5 nM of the

b

c

small molecule agonist 1, used as surrogate of endogenous CXCR7 ligands. CXCR7-CXCL12 assay using CXCL12 as a ligand. nd: not

determined. Data are the geometric mean (+/−2-fold) of at least three independent experiments. For synthesis and assay details, see the

Supporting Information.

depicts a selection of active molecules prepared. A decrease in

aﬃnity was observed for the pyrazole and triazole derivatives

(10a, 10b) in the CXCR7-agonist 1 assay. The inverted 3-(2,4-

diﬂuorophenyl)isoxazole analogue 10c of 8g was 5 times less

potent in the CXCR7-agonist 1 assay but only 2 times less

potent in the CXCR7-CXCL12 assay. Six-membered rings as

exempliﬁed by the pyrimidine 10f led to a decrease in potency.

Interestingly, the IC₅₀shift between the assays was in the same

range for all these analogues and lower than observed

previously. The oxadiazoles 10d and 10e showed potencies

in the same range than 8g, with IC₅₀values of 554 and 203 nM

in the CXCR7-CXCL12 assay, respectively. Despite a

measured log D_7.4of 1.6, the latest compound did not exhibit

an improvement on the hERG inhibitory activity with an IC₅₀

of 2.7 μM.

As the SAR observed so far remained steep in the CXCR7-

CXCL12 assay, it was decided to investigate the potential of a

new exit vector on the 4-amino-piperidine core with the aim of

further increasing activity in this assay and improving the

selectivity over the hERG channel. All the compounds

described in Table 5 are racemates unless otherwise stated,

and their synthesis is described in the Supporting Information

part.

Alkylation of the secondary amide function was rapidly

abandoned due to a dramatic decrease in potency of the

examples produced. Among them, only the benzyl analogue

11a is shown. The focus was then set on the introduction of

substituents in position 3 of the piperidine core. The decrease

of the basicity of the tertiary amine by the addition of two

ﬂuorine atoms (11b) led to an important decrease in potency

as observed previously (compound 9e). The introduction of a

methoxy group provided a signiﬁcant improvement in potency

as shown by compound 11c with IC₅₀values of 2 nM in the

CXCR7-agonist 1 assay and 61 nM in the CXCR7-CXCL12

assay. Larger alkoxy groups like ethoxy (11d) caused a loss in

potency by 2-fold in both assays. The same exercise with a

hydroxy group (11e) gave potencies in a similar low

nanomolar range in the CXCR7-agonist 1 assay but a 2-fold

potency decrease in the CXCR7-CXCL12 assay as compared

with the methoxy analogue. Extending the alcohol function by

one carbon (11f) did not improve the potency in the CXCR7-

CXCL12 assay. The introduction of a methyl ester in position

3 of the piperidine core (11g) delivered a very potent

compound in the CXCR7-agonist 1 assay that unfortunately

exhibited a 100-fold IC₅₀shift in the CXCR7-CXCL12 assay.

Saponiﬁcation of the ester to the acid 11h, which could have

helped to modulate the hERG liability, caused a loss in

E

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 4. Exploration of the Core Heteroaromatic Ring

Table 5. Exploration of Exit Vectors and Substituents on the

4-Amino-piperidine Core

a

The CHO-K1 CXCR7 β-arrestin cell line was preincubated with the

compounds to be screened for 15 min before the addition of 5 nM of

the small molecule agonist 1, used as surrogate of endogenous

b

CXCR7 ligands. CXCR7-CXCL12 assay using CXCL12 as a ligand.

Data are the geometric mean (+/−2-fold) of at least three

independent experiments. For synthesis and assay details, see the

Supporting Information.

potency. Interestingly, by the introduction of a dimethyl amide,

the potency was regained as shown by compound 11i that

exhibited an IC₅₀of 66 nM in the CXCR7-CXCL12 assay. The

shift of the amide group to position 4 of the piperidine core

exempliﬁed by the achiral compound 11j led to a complete loss

in activity.

Compound 11i was selected for further characterization.

Aqueous solubility (192 mg/L at pH 7) and solubilities in

FaSSIF and FeSSIF (569 and 652 mg/L) were improved

compared to 8g. Some ADMET parameters were promising,

especially with an intrinsic clearance of 13 μL/min/mg protein

in human microsomes and 274 μL/min/mg protein in rat

microsomes. Finally, compound 11i demonstrated moderate

inhibitory potency (IC₅₀= 5.5 μM) in the hKV11.1 assay.

Separation of the racemate 11i by chiral prep. HPLC delivered

both enantiomers (R,R)-11i and (S,S)-11i, which, respectively,

display IC₅₀CXCR7-agonist 1 assay/IC₅₀CXCR7-CXCL12

assay of 1.5 nM/37.6 nM and 7.4 nM/388 nM. A 5-fold shift

in the CXCR7-agonist 1 assay and a 10-fold shift in the

CXCR7-CXCL12 assay were observed between both enan-

tiomers, highlighting that the inﬂuence of the absolute

stereochemistry had to be assessed. The absolute stereo-

chemistry of enantiomers (R,R)-11i and (S,S)-11i was

a

The CHO-K1 CXCR7 β-arrestin cell line was preincubated with the

compounds to be screened for 15 min before the addition of 5 nM of

the small molecule agonist 1, used as surrogate of endogenous

b

CXCR7 ligands. CXCR7-CXCL12 assay using CXCL12 as a ligand.

c

nd: not determined. Data are the geometric mean (+/−2-fold) of at

least three independent experiments. For synthesis and assay details,

see the Supporting Information.

conﬁrmed by an enantioselective synthesis described in the

chemistry part.

F

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 6. SAR of 3-Carboxamide Moiety of the trans Racemic Series

a

b

c

CXCR7-CXCL12 assay using CXCL12 as a ligand. Separated (3R,4R) enantiomer. Separated (3S,4S) enantiomer. Data are the geometric mean

(+/−2-fold) of at least three independent experiments.

To ensure a sustainable antagonistic eﬀect, the ideal

compound should be insurmountable, meaning that its

potency should be constant even in the presence of potentially

increasing concentrations of the natural ligands. A selection of

compounds was tested in an insurmountability assay

(Supporting Information). Whereas compounds 11c and 11e

were found surmountable, the (R,R)-enantiomer of the

dimethyl analog 11i was the ﬁrst compound showing

insurmountability. This exciting ﬁnding encouraged us to

investigate further this exit vector and to produce large amide

libraries from the racemic trans carboxylic acid intermediate

11h. Some of the most interesting analogs are shown in Table

6.

pyrrolidine (20e), respectively. More hydrophilic groups did

not improve potency as shown by compounds 20f and 20g.

Interestingly, the coupling with amines bearing an aromatic

group gave a signiﬁcant increase in potency as depicted by the

benzylamide 20h with an IC₅₀of 36 nM and the pyridin-2-yl-

methanamide 20j with an IC₅₀of 51 nM. The phenylamide 20i

was less potent. A further surprising breakthrough was

achieved with the phenethyl amides 20k and 20l with low

nanomolar potencies for the ﬁrst time of IC₅₀= 10 nM and

IC₅₀= 2 nM, respectively. Another major improvement was

realized when the pyridin-2-yl-methanamide 20j was shielded

with a methyl group at the benzylic position. Indeed,

compound 20m, a mixture of four trans-diastereomers showed

a 10-fold increase in potency with an IC₅₀of 6.7 nM.

The coupling with diverse small alkyl amines (20a−20d)

gave compounds with potencies in a similar range than the

dimethylamide 11i in the CXCR7-CXCL12 assay. The

introduction of cyclic secondary amines led to a 3- and 10-

fold decrease in potency for the azetidine (20d) and the

Having established the absolute stereochemistry of (3S,4S)-

11i and (3R,4R)-11i, the same exercise was repeated with both

enantiomeric (R)-1-pyridinyl-2-yl-ethylamine and (S)-1-pyr-

idinyl-2-yl-ethylamine that were coupled with both enan-

G

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 7. Structural Modiﬁcation and Properties: 3-Carboxamide Moiety

a

b

CXCR7-CXCL12 assay using CXCL12 as a ligand. nd: not determined. Data are the geometric mean (+/−2-fold) of at least three independent

experiments.

tiomers (S,S)-19 and (R,R)-19 intermediates to deliver the

four possible trans-enantiomers. The diastereomer 20o was the

most potent antagonist of this series, followed by 20n, then

20p, and ﬁnally 20q. Compound 20o was selected for further

characterization. Its aqueous solubility was low (4 mg/L at pH

7), whereas solubility values in FaSSIF and FeSSIF were 38

and > 910 mg/L, respectively. Several ADMET parameters

were not yet optimal, especially CL_intin HLM (100 μL/min/

mg) and in RLM (823 μL/min/mg). On the other hand,

did not improve the potency, the focus was to further improve

potency and properties for oral administration by exploring

(S,S)-3-carboxamides containing an aromatic or heteroaro-

matic ring.

The in vitro characteristics of this panel of 3-carboxamides

are detailed in Table 7. The (S,S)-benzylic analogue 21a

displayed the same potency as the racemic 20h. Methylation of

the benzylic position improved the potency further but

increased the lipophilicity, which translated in a decreased

metabolic stability. Further structural modiﬁcations were

attempted to reduce lipophilicity and thereby increase

metabolic stability. Hydroxylation on the benzylic methyl

group (21c) reduced the potency. Spacer extension was

tolerated but 21d was rather metabolically unstable. The

dimethylated compound 21e was very potent. The 1-(pyridin-

2-yl)cyclopropyl amide 21f was slightly less potent but not

inhibiting the hERG channel up to 10 μM in comparison to

many analogues of this series, and it exhibited a metabolic

stability of 47 μL/min/mg. Finally, the 1-(pyrimidin-2-

compound 20o still demonstrated inhibitory potency (IC₅₀

=

3.4 μM) in the hERG assay. 20o was also found

insurmountable. Stereochemistry of the central scaﬀold

appeared to play an important role in combination with the

substitution on the 3-carboxamide, a large amide group being

more potent when combined with the (S,S) scaﬀold. On the

other hand, a small dimethyl amide combined with the (R,R)

scaﬀold was more potent than the corresponding (S,S)

analogue as shown before (compound (R,R)-11i). As

replacement of the dimethyl amide with other small amides

H

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 8. Structural Modiﬁcation and Properties: N-1-Piperidine Substituents

a

b

CXCR7-CXCL12 assay using CXCL12 as a ligand. nd: not determined. Data are the geometric mean (+/−2-fold) of at least three independent

experiments.

yl)cyclopropyl amide 21g was a very potent antagonist,

displaying a metabolic stability of 22 μL/min/mg but

inhibiting hERG with an IC₅₀of 2.6 μM. Protein binding

was measured in human plasma for the most promising

antagonists and found to be in a range of 84.1−98.4%. This

exploration of the amide substitution of the 3-carboxamide led

to the identiﬁcation of very potent antagonists, however with

moderate Cl_intvalues in HLM and still showing hERG

inhibitory activities.

Table 8 summarizes the impact of N-substitution on the

potency, the Cl_intin HLM, the IC₅₀against the hERG channel,

and on some additional physico-chemical properties. These

modiﬁcations showed that the lipophilicity of substituents was

an important feature for low nanomolar potency on the

receptor, small substituents like H (27), Me (28a), or ethyl

(28b) giving less potent compounds. On the other hand, larger

substituents gave compounds that displayed higher Cl_intvalues

in HLM and had a stronger aﬃnity toward hERG. Basicity of

I

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 1. Preparation of (3S,4S)-1-Cyclohexyl-4-{[carboxyamino-3-carbonyl]-amino}-piperidine-3-carboxylic Acid-amide

a

Derivatives (3S,4S)-11i

a

Reagents and conditions: (a) p-toluensulfonic acid, toluene, reﬂux, 18 h, quantitative; (b) NaBH₄, isobutyric acid, toluene, 0 °C, 6 h; (c) NaOEt,

EtOH, 50 °C, 18 h, then HCl diethylether/dioxane, 16%; (d) 10% Pd/C, HCO₂NH₄, MeOH, reﬂux, 2 h, 82%; (e) 5-(2,4-diﬂuoro-phenyl)-

isoxazole-3-carboxylic acid, HATU, DIPEA, DMF, rt., 18 h, 51%; (f) HCl 4 N in dioxane, DCM, rt., 30 min, 99%; (g) cyclohexanone,

NaBH(OAc)₃, AcOH, DCM, rt., 24 h, 94%; (h) NaOH aq., THF, rt., 6 h, quantitative; and (i) dimethylamine, (40% in water), HATU, DIPEA,

DMF, rt., 6 h, 36%.

the piperidine nitrogen was measured through the pK_aof the

conjugated acid. The nitrogen needed to be basic enough for

highly potent CXCR7 antagonism. The corresponding neutral

N-acetamide 28o was indeed only weakly active. Introduction

of ﬂuorine in compounds 28k, 28l, 28m, or of a methoxy

group as in derivative 28n on the N-alkyl substituent did

reduce the basicity and the hERG blockage but at the same

time reduced the aﬃnity to the receptor. Protein binding was

measured in human plasma for the most promising antagonists

and was in a range of 73.1−91.5%. Among all the compounds

of Table 8, the N-cyclopropylmethyl compound 28f displayed

the most balanced proﬁle.

The interesting proﬁle of 28f prompted us to conﬁrm the

inﬂuence of stereochemistry on the antagonist potency. The

stereochemistry had a strong impact as the corresponding

trans-(3R,4R)-enantiomer 29, obtained by the same synthetic

approach and is described in Scheme 2 but starting with the

chiral auxiliary (R)-(+)-α-methylbenzylamine, was 150 time

less potent with an IC₅₀of 490 nM. This stereochemical eﬀect

was even more pronounced in the case of the cis substitution as

both cis-(3R,4S)-30 and cis-(3S,4R)-31 enantiomers were

found to be rather weak antagonists with IC₅₀of 2250 nM

and above 5500 nM, respectively. The cis enantiomers 30 and

31 were obtained from the same synthetic pathway and are

described in Scheme 2, starting either from (S)-(−)-α-

methylbenzylamine or (R)-(+)-α-methylbenzylamine, without

conducting the equilibration step. The structural assignment

based on the synthetic access,⁴¹in particular the absolute

(3S,4S) stereochemistry, was conﬁrmed by X-ray analysis of

crystals of compound 28f (Figure 2).

To summarize the important features in vitro, potent

antagonists required some basic character on N-1 of the

piperidine and suﬃcient lipophilicity to achieve a low

nanomolar potency on the target receptor. Ideal basicity

values were found to be close to pKa 7.4 to keep potency on

the receptor and should not be higher than pKa 7.8 to avoid

stronger hERG inhibition. Lipophilicity (log D_7.4) was kept

below 2.7 for the same reasons. Despite the surprisingly narrow

navigation space between these molecular properties, com-

pound 28f, named with the corporate code ACT-1004-1239,

ﬁtted most requirements and was therefore selected as the

ideal candidate for in vivo evaluation. Compound 28f is a weak

base (pK_a= 7.7) and displays an aqueous solubility of 374 mg/

L at pH 7, whereas solubility values in FaSSIF and FeSSIF

J

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 2. Preparation of 1-Substituted (3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-piperidine-3-

a

carboxylic Acid (1-Pyrimidin-2-yl-cyclopropyl)-amide Derivatives

a

Reagents and conditions: (a) p-toluensulfonic acid, toluene, reﬂux, 18 h, quantitative; (b) NaBH₄, triﬂuoracetic acid, THF, −18 °C, 1 h; (c) 20%

Pd(OH)₂/C, H₂, MeOH, rt., 18 h, quantitative; (d) 5-(2,4-diﬂuoro-phenyl)-isoxazole-3-carboxylic acid, T₃P, DCM, TEA, rt., 18 h, 79%; (e)

NaOEt, EtOH, EtOAc, rt., 2 h, chiral chromatography, 63%; (f) NaOH 1 N aq., THF, rt., 8 h, HCl 2 N, rt., 30 min, 98%; (g) 1-pyrimidin-2-yl-

cyclopropylamine, HATU, DIPEA, DMF, rt., 18 h, 87%; (h) 4 N HCl in 1,4-dioxane, MeOH, rt., 1 h, NaHCO₃aq. DCM, 98%; (i) aldehyde or

ketone, NaBH(OAc)₃, AcOH, DCM, rt., 24 h, 94%; and (j) alkyl bromide or iodide, base, acetone.

were 513 and > 880 mg/L. The capacity of 28f to inhibit the

cytochrome P450 2C9, 2D6, and 3A4 was assessed in vitro in

HLM. All IC₅₀values were above 50 μM. ACT-1004-1239 was

very potent on CXCR7 from other species and inactive on

human CXCR4 (Table 9). ACT-1004-1239 was tested in a

panel of 87 enzymatic and radioligand binding assays and was

found to be selective for CXCR7 (data not shown).

Insurmountability Assessment and Binding Kinetic of

ACT-1004-1239. We then wished to conﬁrm that ACT-1004-

1239 displayed insurmountable antagonism against both

CXCR7 ligands, CXCL11 and CXCL12. Thus, the IC₅₀of

ACT-1004-1239 was determined in the presence of increasing

concentrations of CXCL11 or CXCL12 (up to 1 μM). As

shown in Figure 3, the IC₅₀values of ACT-1004-1239 fell into

a narrow range at diﬀerent ligand concentrations. The data

show that ACT-1004-1239 is a potent insurmountable CXCR7

antagonist against both CXCL11 and CXCL12.

tag-hCXCR7 fusion protein were labelled with SNAP-Lumi4-

Tb. A ﬂuorescent derivative of CXCL12, hereafter named “red-

CXCL12”, was used. The dissociation constant (K_d) of red-

CXCL12 was determined to be 1.33 nM at room temperature

(data not shown). Competitive binding assays were carried out

using ACT-1004-1239 as a competitor. As expected, Figure 4

shows a decrease of the HTRF signal upon the addition of

increasing concentrations of ACT-1004-1239. In this experi-

ment, ACT-1004-1239 had a K_iof 0.53 nM, a K_offof 0.06945

min⁻¹, and a K_onof 1.309 10⁸M⁻¹min⁻¹at room temperature.

The dissociation rate constant of ACT-1004-1239 (resi-

dence time, t = 1/K_off) was 14.4 min, and its half-life (t_1/2

ln2/K_off) was 9.98 min.

=

In Vivo Pharmacokinetic Studies. Pharmacokinetic

studies were performed in rat and dog after oral (10 and 1

mg/kg) and intravenous (1 mg/kg) dosing. The pharmacoki-

netic parameters of ACT-1004-1239 are described in Table 10,

which also displays the plasma protein binding and intrinsic

clearance in the liver microsomal preparation of each species.

After intravenous dosing in the rat, ACT-1004-1239 displayed

a systemic plasma clearance of 70 mL/min per kilogram. After

To determine the association (K_on) and dissociation (K_off)

rate constants of ACT-1004-1239, a HEK293 cell line

expressing a SNAP-tag-fused hCXCR7 receptor was estab-

lished for the binding assay. Cells stably expressing the SNAP-

K

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Molecular structure of compound 28f as determined by X-

ray structural analysis. Anisotropic thermal ellipsoids are drawn at a

50% probability level. The cyclopropyl rest was not reﬁned

anisotropically (due to a high disorder present, for details see the

Experimental Section and Supporting Information). For clarity, the

disordered part and all hydrogen atoms have been omitted. The

solvate molecule present (MeOH) was squeezed using software tool

Platon SQUEEZE.⁴⁴

a

Table 9. In Vitro Pharmacology of ACT-1004-1239 (28f)

human CXCR7^aIC₅₀(nM)

dog CXCR7^bIC₅₀(nM)

rat CXCR7^cIC₅₀(nM)

3.2

2.3

3.1

mouse CXCR7^dIC₅₀(nM)

guinea pig CXCR7^eIC₅₀(nM)

macaque CXCR7^fIC₅₀(nM)

human CXCR4^gIC₅₀(nM)

2.3

0.6

1.5

>10000

a

Note: CXCR7-CXCL12 assay. ^a−fAssays performed with CXCL12

as a ligand. ^b−fData are the average of at least ﬁve independent

experiments and were determined using the DiscoverX assay. ^gHuman

CXCR4 FlipR assay. For assay details, see the Supporting

Information.

correction for blood/plasma partitioning, blood clearance was

approximatively 70% of the liver blood ﬂow. Plasma and blood

clearances in the dog were 2.8 and 3.8 mL/min per kilogram,

the latter representing 9% of the liver blood ﬂow. These data

show that ACT-1004-1239 is a high-clearance drug in the rat

and a low-clearance drug in the dog. Also, these in vivo

clearance data are in line with the in vitro intrinsic clearance

values determined in rat and dog liver microsomes, i.e., of 140

versus 5 μ/mL per milligram protein, respectively. The volume

of distribution at the steady state (V_ss) in rat and dog was in

excess of total body water with 3.6 and 1.6 L per kilogram,

respectively. Oral absorption was rapid in the rat with peak

plasma concentrations being reached within 0.5 h. In the dog,

T_maxwas around 1.5 h. Bioavailability reached 35% in the rat

and was 61% in the dog.

Figure 3. Determination of ACT-1004-1239 insurmountability

against CXCL11 (upper panel) and CXCL12 (lower panel) in the

CXCR7-agonist 1 assay.

Evaluation of in Vivo Target Engagement. The potency

and rodent pharmacokinetic properties of compound ACT-

1004-1239 administrated orally were suitable for evaluating

this compound in vivo. The eﬀect of ACT-1004-1239 on

plasma CXCL12 was evaluated in naive male DBA/1 mice at a

range of 1−100 mg/kg. Single oral dosing resulted in a rapid,

signiﬁcant, dose-dependent increase in CXCL12 plasma

Figure 4. Determination of the K_i, K_on, and K_offconstants (speciﬁc

binding) of ACT-1004-1239.

concentrations over 24 h in comparison to vehicle-treated

mice. These data demonstrate the inhibition of CXCR7-

mediated degradation of circulating CXCL12 with ACT-1004-

1239. At the highest dose tested (100 mg/kg), a single oral

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 10. Pharmacokinetic Parameters of ACT-1004-1239 in Rat and Dog

AUC_0‑last(ng/h/mL) C_max(ng/h/mL) T_max(h) F (%) AUC_0‑Inf(ng/h/mL) V_ss(L/kg) Cl (mL/min/kg) T_1/2(h) Cl_int(mL/min/mg) PPB (%)

rat, 10 mg/kg

827

dog, 1 mg/kg

3620

rat, 1 mg/kg

238

dog, 1 mg/kg

5940

RLM

140

DLM

5

rat

600

314

0.5

1.5

35

61

3.6

1.6

70

1.3

6.6

91.8

dog

83.9

2.8

a

Observed pharmacokinetic parameters are average values for rat, p.o. (n = 3), iv (n = 2), dog p.o./iv (n = 2). T_maxis given as median. Blood/

plasma ratios for rat and dog are 1.1 and 0.7, respectively. Mean conc. 8 h post oral dose was 18.3 ng/mL in the rat and 185 ng/mL in the dog.

Figure 5. Eﬀect of a single oral administration (p.o.) of ascending doses of ACT-1004-1239 on CXCL12 plasma concentration (upper row) and

compound plasma concentration (lower row) in naive male DBA/1 mice. CXCL12 plasma concentration was measured at 0.5 (A), 6 (B), or 24 h

(C) after a single oral administration of ACT-1004-1239. Results are expressed as fold change to vehicle-treated mice. ACT-1004-1239 plasma

concentration measured 0.5 (D), 6 (E), or 24 h (F) after a single oral administration. Results are expressed as mean + standard error of the mean

(SEM) (n = 3−5/dose group). **p < 0.01, ***p < 0.001, ****p < 0.0001 vs vehicle, using one-way analysis of variance (ANOVA) followed by the

Dunnett’s multiple comparisons test.

dose of ACT-1004-1239 induced a 4.1- to 4.9-fold mean

CXCL12 plasma elevation relative to control mice, 0.5 and 6 h

after dosing, respectively (Figure 5, panels A and B). The

duration of this increase was also dose dependent. At doses up

to 30 mg/kg, CXCL12 plasma concentrations returned to

vehicle levels within 24 h, whereas with 100 mg/kg of ACT-

1004-1239, 24 h after administration, CXCL12 plasma levels

were still substantially increased (2.6-fold; p < 0.01 vs vehicle-

treated mice) (Figure 5, panel C). Nevertheless, even at this

dose, CXCL12 plasma concentrations returned to levels similar

to vehicle-treated mice within 96 h post dosing (data not

shown). The second ligand of CXCR7, CXCL11, an inducible

chemokine was also measured in plasma but did not

6

2 ng/mL (1 mg/kg single oral dose) to 4285 579 ng/

mL (100 mg/kg single oral dose) (Figure 5, panel D). At doses

of 30 mg/kg and below, no compound was quantiﬁable 24 h

after oral administration (below the limit of quantiﬁcation:

1.52 ng/mL) whereas at 100 mg/kg, 14

5 ng/mL of

compound was still measured in the plasma (Figure 5, panel

F). Based on these results, twice daily oral administration of

ACT-1004-1239 might be needed in murine disease models to

reach high enough drug plasma concentration at trough, to

maintain sustained CXCL12 elevation at all times.

CHEMISTRY

■

An enantioselective synthesis⁴¹of N-boc-trans-4-aminopiper-

idine-3-carboxylic acid ethyl ester 15 employing α-methyl-

benzylamine as a chiral auxiliary and nitrogen source was used

to generate both trans enantiomers (Scheme 1) and allowed

the establishment of the absolute stereochemistry of both

́

signiﬁcantly increase in naive mice after a single oral dose

(data not shown). The plasma concentrations of ACT-1004-

1239 increased in a dose-dependent manner and reached a

maximum after 0.5 h, with plasma concentrations ranging from

M

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

enantiomers, (3R,4R)-11i and (3S,4S)-11i. This synthetic

route further oﬀers the advantage that it can be used to explore

stepwise substitution at the three exit vectors of the piperidine

scaﬀold.

27, which was alkylated under reductive amination condition

with sodium triacetoxyborohydride in the presence of acetic

acid or by reaction with the corresponding alkyl halides in the

presence of a base to furnish ﬁnal compounds 28a−28o.

4-Oxo-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester 3-

ethyl ester was reacted with the chiral auxiliary (S)-(−)-α-

methylbenzylamine in reﬂuxing toluene containing a catalytic

amount of p-toluenesulfonic acid to form enamine 12. The

enamine was reduced with a mixture of NaBH₄and isobutyric

acid at 0 °C. The resulting 4:1 mixture of the two cis-

diastereomers of the β-amino ester 13 was treated with sodium

ethanolate in ethanol causing epimerization to the trans-β-

aminoester 14. The pure (S,S)-β-aminoester 14 was isolated

from the mixture as the hydrochloride salt in 16% yield. A

cleavage of the α-methylbenzyl group was achieved with

ammonium formiate in the presence of 10% Pd/C in reﬂuxing

MeOH. During this reaction, a transesteriﬁcation to the methyl

ester occurred. The trans primary amine 15 was acylated with

5-(2,4-diﬂuoro-phenyl)-isoxazole-3-carboxylic acid to deliver

the trans-(3S,4S)-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-3-car-

bonyl]-amino}-piperidine-1,3-dicarboxylic acid 1-tert-butyl

ester 3-methyl ester 16. N-Boc deprotection with HCl in

1,4-dioxane to the secondary amine 17 followed by N-

alkylation with cyclohexanone under reductive amination

conditions with sodium triacetoxy borohydride in the presence

of acetic acid furnished the amine 18. The ester moiety of 18

was hydrolyzed with NaOH in a THF/water mixture to 19.

HATU-mediated amide bond formation with aq. dimethyl

amine was executed in a moderate yield to furnish the dimethyl

amide, which after comparison of the chromatogram on chiral

stationary phase was found to correspond to the less potent

enantiomer (3S,4S)-11i. Repetition of the same sequence of

reactions with (R)-(+)-α-methylbenzylamine as chiral auxiliary

delivered the more potent (R,R)-enantiomer (3R,4R)-11i.

Because of the poor isolated yield of the pure diastereomer

hydrochloride 14, a modiﬁed procedure was needed to allow

for further structural modiﬁcations of the 1-(pyrimidin-2-

yl)cyclopropyl amide at 3-position through modiﬁcation of

substituents on N-1 by alkylation of the versatile secondary

amine (Scheme 2). Our modiﬁed synthetic approach toward

the (S,S)-4-aminopiperidine-3-carboxylic ethyl ester followed

the strategy we have previously employed in Scheme 1, but

equilibration toward the more stable trans isomer was done

after introduction of the 4-amide. 4-Oxo-piperidine-1,3-

dicarboxylic acid 1-tert-butyl ester 3-ethyl ester was reacted

with the chiral auxiliary (S)-(−)-α-methylbenzylamine in

reﬂuxing toluene containing catalytic amount of p-toluene-

sulfonic acid to form enamine 12. The enamine was reduced

with a mixture of NaBH₄and 3 eq. triﬂuoracetic acid in THF

at −20 °C to give 13. The 4-α-methylbenzylamine was cleaved

by hydrogenation with H₂in the presence of 20% Pd(OH)₂/C

in EtOH and the cis primary amine 22 was acylated with 5-

(2,4-diﬂuoro-phenyl)-isoxazole-3-carboxylic acid to deliver the

6:1 mixture of cis-(3R,4S)-and cis-(3S,4R)-4-{[5-(2,4-diﬂuoro-

phenyl)-isoxazole-3-carbonyl]-amino}-piperidine-1,3-dicarbox-

ylic acid 1-tert-butyl ester 3-ethyl ester 23. Treatment with

sodium ethanolate in ethanol and ethyl acetate causing

epimerization to the trans-β-aminoesters, followed by chiral

chromatography, delivered the pure (3S,4S)-diastereomer 24.

The ester was hydrolyzed with NaOH in a THF/water

mixture. HATU-mediated amide bond formation on 25 with 1-

pyrimidin-2-yl-cyclopropylamine delivered the di-amide 26. N-

Boc-deprotection with HCl in 1,4-dioxane gave the piperidine

CONCLUSIONS

■

In summary, ACT-1004-1239 (compound 28f) was identiﬁed

and broadly characterized as a potent, insurmountable,

selective, and orally bioavailable antagonist of CXCR7, starting

from hits obtained in an innovative HTS assay using an in-

house agonist of CXCR7 to allow for a diﬀerent assay window.

The introduction of an additional exit vector in position 3 of

the 4-amino-piperidine core led to a breakthrough in terms of

potency, binding kinetics, and overall physicochemical and

ADMET properties. Further studies demonstrated that

modulation of lipophilicity and basicity by combination of

substituents on this trisubstituted 4-amino-piperidine scaﬀold

yielded compounds with good CXCR7 antagonism. Com-

pound ACT-1004-1239 demonstrated target engagement in

vivo. ACT-1004-1239 showed a dose-dependent increase of

CXCL12 plasma concentration in mice. Overall, ACT-1004-

1239 has favorable ADMET properties. The Cl_intmeasured in

HLM is low and with the understanding generated in PK

studies in rat and dog; the compound has the potential to be

well absorbed and to display a low systemic clearance in

humans.

EXPERIMENTAL SECTION

■

Commercially available starting materials were used as received

without further puriﬁcation. All reactions were carried out in oven-

dried glassware under an atmosphere of nitrogen or argon. Flash

column chromatography (FC) was performed using RediSEp Rf

normal-phase silica ﬂash columns on silica gel (average particle size

0.035−0070 mm) from Teledyne Isco.; elution was performed with

EtOAc, heptane, DCM, MeOH, or mixture thereof. Analytical LC−

MS data were obtained on a binary gradient pump Agilent G4220A

with mass spectrometry detection (single-quadrupole mass analyzer,

Thermo Finnigan MSQPlus or equivalent); acidic conditions

(Method A): column Zorbax SB-aq (3.5 μm, 4.6 × 50 mm); eluents:

A, H₂O + 0.04% TFA. B: MeCN; gradient: 5−95% B over 1.5 min

(ﬂow: 4.5 mL/min). Detection: UV/vis + MS or basic conditions

(Method B): column Waters BEC C18 (2.5 μm, 3.0 × 50 mm);

eluents: A, H₂O + 0.5% NH₄OH (25% aq.); B, MeCN gradient: 5% B

to 95% B, over 1.5 min (ﬂow: 1.6 mL/min). Detection: UV/vis + MS.

Prep-HPLCs were performed on binary gradient pump Gilson 333/

334 or equivalent with mass spectrometry detection (single-

quadrupole mass analyzer, Thermo Finnigan MSQPlus or equiv-

alent); XBridge Prep C18 columns from Waters. Eluents: A, H₂O +

0.5% acidic or basic additive (HCO₂H or NH₄OH 25% in H₂O); B,

MeCN; gradient 5% B to 95% B over 5 min. Detection: UV/vis and/

or MS. Retention time t_Ris given in min; molecular weight obtained

from the mass spectrum is given in g/mol. Purity of all ﬁnal

compounds was checked by an additional LC−MS analysis (QC LC−

MS) on a Waters Acquity UPLC-SQD system equipped with a Waters

Acquity binary pump, a Waters SQ Detector, an Acquity UPLC PDA

detector; columns: Acquity UPLC CSH C18 1.7 μm, 2.1 mm × 50

mm from Waters, gradient: 2−98% MeCN containing 0.045% formic

acid in H₂O containing 0.05% formic acid over 2 min; ﬂow: 1.0 mL/

min; 60 °C. According to these LC−MS analyses, ﬁnal compounds

showed a purity of greater than 95% (UV at 214 and at 230 nm).

Most compounds were obtained as lyophilizates or dry ﬁlms. Chiral

integrity was proven by HPLC (chiral stationary phase): Hardware

from UltiMate instrument series (Dionex): HPG-3200SD binary

pump, WPS-3000 autosampler, TCC-3200 thermostated column

compartment, DAD-3000 detector, SRD-3400 degasser; ValveMate 2

(Gilson) solvent valves. Chiral integrity was also proven by SFC: CO₂

N

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

supply: Aurora Fusion A5 Evolution; pump: Agilent G4302A; UV

detector: Agilent G1315C. Column, solvent, and retention time (t_R)

as indicated, ﬂow 1 mL/min. No racemisation/epimerization was

observed during the synthesis of the target compounds. Chiral

preparative HPLC chromatography were performed with a Varian

SD1 pump equipped with a Dionex DAD-3000 UV detector with

columns from ChiralPak IA, IB, IC, IE, or IF, 5 μm, 20 × 250 mm, or

Regis (R,R) Whelk-O1, 21.1 × 250mm, 5 μm; eluent: appropriate

mixture of A (0−90% Hept) and B (10−100% EtOH, 0.1% DEA),

ﬂow: appropriate ﬂow of 16, 23, or 34 mL/min. Chiral SFC

chromatography was performed with: CO₂supply: Maximator

DLE15-GG-C; pumps: 2 SSI HF CP 300; UV detector: Dionex

DAD-3000 with columns Regis (R,R) Whelk-O1, 30 × 250 mm, 5 μm

or ChiralPak IC, 30 × 250 mm, 5 μm; eluent: appropriate mixture of

A (60−80% CO₂), and B (30−40% of DCM/EtOH/DEA 50:50:0.1),

ﬂow 160 mL/min. LC-HRMS analysis was performed on a Waters

Acquity UPLC-SYNAPTG2MS system equipped with a Waters

Acquity Binary pump, a SYNAPT G2MS MS Solvent Manager;

source temperature: 150 °C, desolvation temperature: 500 °C;

desolvation gas ﬂow: 800 L/h; cone gas ﬂow: 20 L/h; extraction cone:

5, RF lens: 0.1 V; sampling cone: 30; capillary: 2.5 kV; high-resolution

mode; gain: 1.0; MS function: 0.15 s per scan, 50−1200 amu in full

scan, centroid mode. Lock spray: leucine enkephalin 2 ng/mL

(556.2771 Da) scan time 0.2 s with interval of 10 s and average of 10

scans; DAD: Acquity UPLC PDA Detector. Column: Acquity UPLC

CSH C18 1.7 μm, 2.1 mm × 50 mm from Waters, thermostatted in

the Acquity UPLC Column manager at 60 °C. Eluents: H₂O and

0.05% formic acid; B:CH₃CN and 0.05% formic acid. Gradient: 2−

98% B over 2.0 min; ﬂow: 1.0 mL/min. Detection: UV 214 nm and

and ammonium formate (3.35 g, 53.28 mmol) in MeOH (60 mL)

under N₂. The mixture was reﬂuxed for 6 h. After the reaction was

complete (disappearance of the starting material but also exchange of

the ethyl− for the methyl ester), the cooled solution was ﬁltered

through Celite and the ﬁltrate was concentrated to obtain the title

product (1.48 g, 86%) as a slightly yellow oil. LC−MS method A: t_R=

0.53 min; [M + H]⁺= 259.23. ¹H NMR (400 MHz, CDCl₃) δ: 4.32−

4.58 (m, 1H), 4.16−4.25 (m, 1H), 3.77 (s, 3H), 3.41 (td, J₁= 11.2

Hz, J₂= 4.1 Hz, 1H), 2.67−2.89 (m, 3H), 2.10 (dd, J₁= 13.1 Hz, J₂=

3.5 Hz, 1H), 1.64 (m, 1H), 1.48 (m, 11H).

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-1,3-dicarboxylic Acid 1-tert-Butyl Ester 3-

Methyl Ester (16). To a solution of (3S,4S)-4-amino-piperidine-1,3-

dicarboxylic acid 1-tert-butyl ester 3-methyl ester 15 (1.463 g, 5.37

mmol) in DMF (17 mL) at rt. was added 5-(2,4-diﬂuorophenyl)-

isoxazole-3-carboxylic acid (1.21 g, 5.37 mmol). DIPEA (2.3 mL,

13.34 mmol) was then added followed by HATU (2.451 g, 6.44

mmol). The reaction mixture was stirred overnight at rt. The reaction

mixture was concentrated, dissolved in DCM (100 mL), and washed

twice with aq. sat. NaHCO₃(2 × 100 mL). The organic layer was

dried over MgSO₄and evaporated. The crude residue was puriﬁed by

ﬂash chromatography over 40 g of silica gel with heptane/EtOAc (1:0

to 3:1) as eluent to yield 16 (2.38 g, 95%) as a white powder; LC−

1

MS method A: t_R= 0.94 min; [M + H]⁺= 466.04. H NMR (400

MHz, D₆-DMSO) δ: 8.90 (d, J = 8.8 Hz, 1H), 8.06 (td, J₁= 6.5 Hz, J₂

= 8.7 Hz, 1H), 7.58 (m, 1H), 7.33 (td, J₁= 2.3 Hz, J₂= 8.4 Hz, 1H),

7.13 (d, J = 2.9 Hz, 1H), 4.26 (m, 1H), 4.04−4.10 (m, 1H), 3.94 (d, J

= 11.8 Hz, 1H), 3.56 (s, 3H), 2.86−3.04 (m, 2H), 2.66 (td, J₁= 11.0

Hz, J₂= 4.1 Hz, 1H), 1.80 (dd, J₁= 13.6 Hz, J₂= 4.5 Hz, 1H), 1.53

(m, 1H), 1.42 (m, 9H).

1

MS. H and ¹³C NMR spectra were recorded at rt. with a Bruker

1

Avance II 400 (400 MHz for H, 100 MHz for ¹³C) or a Bruker

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-3-carboxylic Acid Methyl Ester Hydro-

chloride (17). (3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-car-

bonyl]-amino}-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester 3-

methyl ester 16 (1.13 g, 2.42 mmol) was dissolved in MeOH (12

mL). HCl in dioxane 4 M (12 mL, 48 mmol) was added dropwise.

The mixture was stirred at rt. for 1 h. The solvents were evaporated,

and the residue was dried on HV to deliver the title crude compound

17, (0.97 g, quantitative) as a white powder. LC−MS method A: t_R=

Avance HD (500 MHz for ¹H; 125 MHz for ¹³C). Chemical shifts (δ)

are reported in parts per million (ppm) relative to the deuterated

solvent as the internal standard (δ H: CDCl₃7.26 ppm, d₆-DMSO

2.50 ppm); multiplicities, s = singlet, d = doublet, t = triplet, q =

quadruplet, m = multiplet, br = broad signal; coupling constants are

given in Hz. X-ray structure analysis: crystals are sensitive to the

solvate loss and therefore to the loss of crystallinity. To avoid solvent

loss, the crystals were treated, picked, and mounted at low

temperature (approx. 243 K) using an inert gas (N₂) device to

protect and cool down the crystals. A single crystal (0.112 × 0.036 ×

0.020 mm³) was mounted on a Rigaku-Oxford Diﬀraction XtaLAB

Synergy-S dual source diﬀractometer equipped with a kappa-axis four-

circle goniometer and a Dectris Pilatus3 R 200 K HPC (Hybrid

Photon Counting) detector and Cu and Mo PhotonJet microfocus X-

ray sources. Reﬂections were collected at 253 K using monochromatic

Cu Kα radiation. For the structure solution, the program SHELXT⁴²

implemented in the APEX-III Bruker AXS, V.2018.7-2 software was

used. The structure reﬁnement was done with SHELXL⁴³as

implemented in APEX-III software. Full matrix least-squares reﬁne-

ment was performed with anisotropic temperature factors for all the

nondisordered atoms. The disordered part (see Supporting

Information for details) was reﬁned with isotropic temperature

factors to achieve stability of the reﬁnement cycles and avoid the

introduction of constrains in the molecule. The solvate molecule

present (MeOH) is disordered over at least 3 positions. Due to the

“not so good” data quality, the solvate positions cannot be fully

reﬁned, nor found completely. Therefore, to avoid such problems and

instability of the reﬁnement, the MeOH solvate was SQUEEZED out

using the program Platon⁴⁴Squeeze. Most of the hydrogen atoms

present were ﬁxed in the reﬁnement, the two hydrogen atoms

involved in donor-acceptor hydrogen bonds were reﬁned freely.

Coordinates, anisotropic temperature factors, bond lengths, angles,

and special reﬁnement issues are deposited with the Cambridge

Crystallographic Data Centre, CCDC 1993933.

1

0.61 min; [M + H]⁺= 366.18. H NMR (400 MHz, D₆-DMSO) δ:

9.21 (d, J = 8.6 Hz, 1H), 9.09−9.10 (m, 1H), 8.08 (m, 1H), 7.60 (m,

1H), 7.35 (td, J₁= 2.2 Hz, J₂= 8.6 Hz, 1H), 7.19 (d, J = 2.9 Hz, 1H),

4.34 (m, 1H), 3.58 (s, 5H), 3.47−3.50 (m, 1H), 3.14 (dd, J₁= 0.9 Hz,

J₂= 2.4 Hz, 2H), 3.03 (m, 1H), 1.97 (d, J = 2.9 Hz, 2H).

(3S,4S)-1-Cyclohexyl-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-

3-carbonyl]-amino}-piperidine-3-carboxylic Acid Methyl Ester

(18). To a suspension of (3S,4S)-4-{[5-(2,4-diﬂuoro-phenyl)-

isoxazole-3-carbonyl]-amino}-piperidine-3-carboxylic acid methyl

ester hydrochloride 17 (0.95 g, 2.36 mmol) in DCM (20 mL) at rt.

was added cyclohexanone (0.57 mL, 5.47 mmol) followed by acetic

acid (0.35 mL, 6.13 mmol) and sodium triacetoxyborohydride (1.23

g, 5.93 mmol). The reaction mixture was stirred overnight at rt. The

reaction mixture was diluted with DCM (30 mL) and washed twice

with aq. sat. NaHCO₃(2 × 50 mL). The organic phase was dried over

MgSO₄and evaporated. Puriﬁcation by ﬂash chromatography over 40

g of silica gel with heptane/EtOAc (1:0 to 0:1) as eluent yielded 18,

(1.00 g, 95%) as an oﬀ-white solid. LC−MS method A: t_R= 0.71 min;

1

[M + H]⁺= 448.17. H NMR (400 MHz, D₆-DMSO) δ: 8.87−8.92

(m, 1H), 8.06 (q, J = 7.7 Hz, 1H), 7.59 (t, J = 10.2 Hz, 1H), 7.34 (t, J

= 8.5 Hz, 1H), 7.13 (s, 1H), 3.96−4.10 (m, 1H), 3.54 (s, 3H), 2.96−

3.07 (m, 1H), 2.68−2.89 (m, 2H), 2.34−2.42 (m, 3H), 1.73−1.81

(m, 5H), 1.56−1.64 (m, 2H), 1.14−1.20 (m, 4H), 1.04−1.10 (m,

1H).

(3S,4S)-1-Cyclohexyl-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-

3-carbonyl]-amino}-piperidine-3-carboxylic Acid Hydrochlor-

ide (19). (3S,4S)-1-Cyclohexyl-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-

3-carbonyl]-amino}-piperidine-3-carboxylic acid methyl ester 18

(0.95 g, 2.11 mmol) was dissolved in THF (14 mL), and 1 M aq.

LiOH solution (5.27 mL, 5.27 mmol) was added. The mixture was

stirred overnight at rt., 1 M aq. HCl solution (5.27 mL, 5.27 mmol)

was added, and the reaction was stirred for 5 min. THF was

(3S,4S)-4-Amino-piperidine-1,3-dicarboxylic Acid 1-tert-

Butyl Ester 3-Methyl Ester (15). A solution of (3S,4S)-4-((S)-1-

phenyl-ethylamino)-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester

3-ethyl ester 14⁴¹(2.41 g, 6.66 mmol) in MeOH (10 mL) was added

to a suspension of palladium on activated charcoal (10%) (250 mg)

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

evaporated and the product 19 precipitated. The resulting white solid

was ﬁltered oﬀ and dried in vacuo to yield 19, (0.788 g, 86%) as a

white powder. LC−MS method A: t_R= 0.66 min; [M + H]⁺= 434.06.

¹H NMR (400 MHz, D₆-DMSO) δ: 12.86 (d, 1H), 10.36−10.38 (m,

1H), 9.24 (d, J = 8.2 Hz, 1H), 8.07 (q, J = 7.8 Hz, 1H), 7.60 (t, J =

10.4 Hz, 1H), 7.35 (t, J = 8.4 Hz, 1H), 7.19 (s, 1H), 4.36 (d, J = 7.5

Hz, 1H), 3.60 (d, J = 8.0 Hz, 1H), 3.42 (m, 1H), 3.18−3.26 (m, 4H),

2.07−2.12 (m, 3H), 2.02 (m, 1H), 1.82 (d, J = 11.8 Hz, 2H), 1.61 (m,

1H), 1.43 (q, J = 11.3 Hz, 2H), 1.26 (q, J = 12.2 Hz, 2H), 1.12 (td, J

= 12.8 Hz, 1H).

Ethyl Ester (23). To a solution of (3R,4S)-4-amino-piperidine-1,3-

dicarboxylic acid 1-tert-butyl ester 3-ethyl ester 22 (7.44 g, 27.2

mmol) in DCM (200 mL) at rt. was added 5-(2,4-diﬂuorophenyl)-

isoxazole-3-carboxylic acid (6.12 g, 27.3 mmol). TEA (15.2 mL, 109

mmol) was then added followed by T₃P 50% in DCM (32.4 mL, 54.4

mmol). The reaction mixture was stirred for 24 h at rt. The reaction

mixture was washed twice with sat. aq. NaHCO₃(2 × 100 mL). The

organic layer was dried over MgSO₄and evaporated. The crude

residue was puriﬁed by ﬂash chromatography over 100 g of silica gel

with heptane/EtOAc (1:0 to 85:15) as eluent to yield the title

compound 23, as a white powder (10.25 g, 79%). The title compound

contains ∼10% of the corresponding (3S,4R)-isomer; LC−MS

(3S,4S)-1-Cyclohexyl-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-

3-carbonyl]-amino}-piperidine-3-carboxylic Acid Dimethyla-

mide ((3S,4S)-11i). To a solution of (3S,4S)-1-cyclohexyl-4-{[5-

(2,4-diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-piperidine-3-car-

boxylic acid hydrochloride 19 (0.102 g, 0.23 mmol) in DMF (5 mL)

was added dimethylamine 2 M in THF (0.177 mL, 0.35 mmol).

DIPEA (0.13 mL, 0.75 mmol) was then added followed by HATU

(0.144 g, 0.38 mmol). The reaction mixture was stirred overnight at

rt. The crude mixture was directly puriﬁed by prep. HPLC to yield

(3S,4S)-11i (0.040 g, 37%) as a white powder. LC−MS method A: t_R

= 0.71 min; [M + H]⁺= 461.22. Chiral HPLC: t_R= 6.8 min; 99% ee;

column: Regis (R,R) Whelk-O1, 4.6 × 250 mm, 5 μm; Detector

wavelength: 254 nm; Eluent: 30% heptane 0.05% DEA; 70% ethanol

0.05% DEA; ﬂow: 0.8 mL/min; BPR: 150 bar; temperature: 25 °C.

Injection volume: 2.5 μL. LC-HRMS: t_R= 0.59 min; m/z = 460.2285,

1

method A: t_R= 1.15 min; [M + H]⁺= 480.1. H NMR (400 MHz,

CDCl₃) δ: 8.92 (d, J = 8.9 Hz, 1H), 8.06 (m, 1H), 7.60 (ddd, J₁=

11.5 Hz, J₂= 9.3 Hz, J₃= 2.5 Hz, 1H), 7.33 (td, J₁= 8.4 Hz, J₂= 2.2

Hz, 1H), 7.13 (d, J = 2.9 Hz, 1H), 4.35−4.47 (m, 1H), 3.96−4.11 (m,

3H), 3.63−3.82 (m, 1H), 3.38−3.44 (m, 1H), 3.06−3.16 (m, 1H),

2.97−3.01 (m, 1H), 1.90−1.99 (m, 1H), 1.65−1.72 (m, 1H), 1.40 (s,

9H), 1.16 (t, J = 6.9 Hz, 3H).

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-1,3-dicarboxylic Acid 1-tert-Butyl Ester 3-

Ethyl Ester (24). Sodium ethoxide 95% (4.086 g, 57 mmol) was

added portion wise to a solution of (3R,4S)-4-{[5-(2,4-diﬂuoro-

phenyl)-isoxazole-3-carbonyl]-amino}-piperidine-1,3-dicarboxylic

acid 1-tert-butyl ester 3-ethyl ester 23 (6.837 g, 14.3 mmol) in a

mixture of EtOH (80 mL) and EtOAc (40 mL). The mixture was

stirred at rt. for 1 day. Sat. aq. NH₄Cl (50 mL) was added to the

reaction mixture. EtOH and EtOAc were evaporated at reduced

pressure. DCM (150 mL) was added. The organic phase was

separated, and the aq. layer extracted thrice with DCM (3 × 100 mL).

The combined organic layers were dried over MgSO₄, ﬁltered, and

concentrated. The crude residue was puriﬁed by prep-HPLC with

basic conditions. The title compound was obtained as a colorless

powder (5.04 g, 74%), containing ∼10% of the corresponding

(3S,4R)-isomer. The enantiomerically pure title compound was

obtained by chiral preparative SFC of the mixture of (3S,4S)-4-{[5-

(2,4-diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-piperidine-1,3-di-

carboxylic acid 1-tert-butyl ester 3-ethyl ester containing ∼10% of

(3R,4R)-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-pi-

peridine-1,3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester using a

column ChiralPak IC, 5 μm, 30 × 250 mm; with a mixture of A (80%

CO₂) and B (20%: DCM (50%), MeOH (50%), 0.1% DEA) as eluent

and a ﬂow of 160 mL/min.; BPR: 100 bar; temperature = 40 °C to

deliver 24 (4.32 g, 63%) as a light yellow powder. Chiral SFC HPLC:

t_R= 3.24 min; 99% ee; column: ChiralPak IC 4.6 × 250 mm, 5 μm;

detector wavelength: 254 nm; eluent A (80% CO₂) and eluent B

(20%: DCM (50%), MeOH (50%), 0.1% DEA) and a ﬂow of 4.0

mL/min.; BPR: 150 bar; temperature = 40 °C; Flow: 0.8 mL/min;

BPR: 150 bar; temperature: 25 °C. Injection volume: 4 μL. LC−MS

method A: t_R= 1.06 min; [M + H]⁺= 480.08. ¹H NMR (D₆-DMSO)

δ: 8.93 (d, J = 8.9 Hz, 1H), 8.05−8.10 (m, 1H), 7.59 (ddd, J₁= 11.5

Hz, J₂= 9.5 Hz, J₃= 2.3 Hz, 1H), 7.34 (td, J₁= 8.4 Hz, J₂= 2.2 Hz,

1H), 7.14 (d, J = 2.9 Hz, 1H), 4.23−4.32 (m, 1H), 4.03 (q, J = 7.1

Hz, 3H), 3.90−3.98 (m, 1H), 2.82−3.03 (m, 2H), 2.59−2.61 (m,

1H), 1.77−1.81 (m, 1H), 1.48−1.59 (m, 1H), 1.43 (s, 9H), 1.08 (t, J

= 7.1 Hz, 3H).

1

found = 461.2373 [M + H]⁺. H NMR (500 MHz, D₆-DMSO) δ:

8.69 (d, J = 8.6 Hz, 1H), 8.05 (td, J₁= 8.7 Hz, J₂= 6.4 Hz, 1H), 7.58

(m, 1H), 7.33 (m, J₁= 8.1 Hz, J₂= 2.0 Hz, 1H), 7.09 (d, J = 2.9 Hz,

1H), 4.08 (m, 1H), 3.13 (td, J₁= 10.7 Hz, J₂= 3.6 Hz, 1H), 3.06 (s,

3H), 2.82−2.88 (m, 2H), 2.77 (s, 3H), 2.24−2.32 (m, 2H), 2.19 (t, J

= 11.3 Hz, 1H), 1.81−1.85 (m, 1H), 1.73 (d, J = 8.0 Hz, 4H), 1.58

(m, 2H), 1.19 (m, 4H), 1.05−1.09 (m, 1H).

(3R,4S)-4-((S)-1-Phenyl-ethylamino)-piperidine-1,3-dicar-

boxylic Acid 1-tert-Butyl Ester 3-Ethyl Ester (13). Sodium

borohydride (1.43 g, 37.85 mmol) was dissolved in THF (100 mL) at

−15 °C under N₂. TFA (10.7 mL, 0.14 mmol) was added dropwise

over 20 min. 4-((S)-1-Phenyl-ethylamino)-5,6-dihydro-2H-pyridine-

1,3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester⁴¹12 (10.5 g, 28

mmol) was added over 10 min at −14 to −18 °C. The resulting

mixture was stirred for 60 min at 0 °C. Iced water (100 mL) was

added carefully, and the reaction mixture was stirred for 10 min at rt.

A 3 M aq. NaOH solution was added to bring the mixture to pH 11.

The reaction mixture was extracted with DCM (2 × 100 mL), the

combined organic layers were washed with brine (2 × 100 mL) and

dried over MgSO₄, and the solvent was evaporated under reduced

pressure. The resulting oil was puriﬁed by ﬂash chromatography over

120 g of silica gel with heptane/EtOAc (1:0 to 4:1) as eluent to give

the title product 13 as a yellowish oil, (9.6 g, 91%). The title

compound was contaminated by ∼10% of the corresponding (3S,4R)-

1

isomer. LC−MS method A: t_R= 0.71 min; [M + H]⁺= 377.33. H

NMR (400 MHz, CDCl₃) δ: 7.32−7.35 (m, 4H), 7.29−7.30 (m, 1H),

7.24 (m, 1H), 4.19 (q, J = 7.3 Hz, 2H), 4.00 (d, J = 9.2 Hz, 1H), 3.88

(q, J = 6.8 Hz, 1H), 3.69−3.76 (m, 1H), 3.19 (dd, J₁= 13.8 Hz, J₂=

3.9 Hz, 1H), 3.01 (m, 1H), 2.87 (m, 1H), 2.79 (s, 1H), 1.81 (s, 1H),

1.76 (m, 1H), 1.51 (m, 1H), 1.44 (m, 9H), 1.28−1.33 (m, 5H).

(3R,4S)-4-Amino-piperidine-1,3-dicarboxylic Acid 1-tert-

Butyl Ester 3-Ethyl Ester (22). A solution of (3R,4S)-4-((S)-1-

phenyl-ethylamino)-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester

3-ethyl ester 13 (9.6 g, 25.5 mmol) in MeOH (250 mL) was added to

a suspension of Pd(OH)₂/C 20% (1 g) under H₂. The mixture was

stirred for 18 h at rt. The suspension was ﬁltered through Celite, and

the ﬁltrate is concentrated under vacuo to obtain the title product as a

slightly yellow oil 22, (6.94 g. quantitative). The title compound

contained ∼10% of the corresponding (3S,4R)-isomer. LC−MS

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-1,3-dicarboxylic Acid 1-tert-Butyl Ester

(25). (3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-1,3-dicarboxylic acid 1-tert-butyl ester 3-ethyl

ester 24 (4.98 g, 10.7 mmol) was dissolved in THF (80 mL). Aq. 1

M NaOH solution (20 mL, 20 mmol) was added, and the mixture

stirred at rt. for 3 h. The reaction mixture was acidiﬁed to around pH

= 3 with 2 M aq. HCl solution (10 mL) and extracted thrice with

DCM (3 × 50 mL). The combined organic phases were dried over

MgSO₄, ﬁltered, and concentrated. The title compound 25 was

obtained as a white powder (4.73 g, 98%); LC−MS method A: t_R=

1

method A: t_R= 0.54 min; [M + H]⁺= 273.26. H NMR (400 MHz,

D₆-DMSO) δ: 4.07 (q, J = 7.0 Hz, 2H), 3.28−3.36 (m, 5H), 2.57−

2.61 (m, 1H), 1.54−1.68 (m, 2H), 1.34−1.41 (m, 11 H), 1.20 (t, J =

7.3 Hz, 3H).

(3R,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-1,3-dicarboxylic Acid 1-tert-Butyl Ester 3-

1

0.99 min; [M + H]⁺= 452.33. H NMR (500 MHz, D₆-DMSO) δ:

12.51 (s, 1H), 8.06 (td, J₁= 8.7 Hz, J₂= 6.4 Hz, 1H), 7.59 (m, 1H),

7.34 (td, J₁= 8.4 Hz, J₂= 2.3 Hz, 1H), 7.14 (d, J = 2.9 Hz, 1H), 4.25

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(m, 1H), 4.05−4.16 (m, 1H), 3.92−3.94 (m, 1H), 2.79−3.00 (m,

2H), 2.59 (td, J₁= 11.0 Hz, J₂= 4.1 Hz, 1H), 1.78−1.82 (m, 1H),

1.49 (m, 2H), 1.42 (m, 9H).

Hz), 63.0 (s), 55.7 (s), 52.5 (s), 49.2 (s), 47.8 (s), 36.5 (s), 31.5 (s),

19.5 (d, J = 17 Hz), 9.0 (s), 4.3 (d, J = 12 Hz).

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-3-(1-pyrimidin-2-yl-cyclopropylcarbamoyl)-piperi-

dine-1-carboxylic Acid tert-Butyl Ester (26). To a solution of

(3S,4S)-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-pi-

peridine-1,3-dicarboxylic acid 1-tert-butyl ester 25 (2.5 g, 5.54 mmol)

in DMF (25 mL) were added 1-(pyrimidin-2-yl)cyclopropan-1-amine

hydrochloride (0.97 g, 5.54 mmol), DIPEA (2.37 mL, 13.8 mmol),

and HATU (2.527 g, 6.65 mmol). The reaction mixture was stirred at

rt. for 4 h. The volatiles were evaporated, and the crude mixture

puriﬁed by ﬂash chromatography over 40 g of silica gel with heptane/

EtOAc (1:1 to 0:1) as eluent to yield 26 as a white powder (2.74 g,

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01588.

Detailed experimental procedures and analytical data of

HTS hits 2−6; synthesized compounds 7a−31 and

respective precursors; X-ray crystallographic structure

parameters of compound 28f; method for the determi-

nation of the melting point by diﬀerential scanning

calorimetry (DSC); method for the determination of log

D_7.4and pK_avalues; method describing the CXCR7-

agonist 1 assay, including insurmountability determi-

nation; method describing the CXCR7-CXCL12 assay;

experimental details for the biological assays to assess

the potency of 28f on CXCR7 from various species, on

human CXCR4 and to determine its K_i, K_on, and K_off

constants; and experimental details for the cytochrome

P450 enzyme inhibition assays, for the metabolic

stability studies, the detailed description of the

pharmacokinetic studies in rat and dog, and the target

engagement in vivo (PDF)

1

87%); LC−MS method A: t_R= 0.95 min; [M + H]⁺= 568.97. H

NMR (400 MHz, D₆-DMSO) δ: 8.63 (d, J = 8.6 Hz, 1H), 8.53 (d, J =

4.9 Hz, 3H), 8.08 (td, J₁= 8.7 Hz, J₂= 6.4 Hz, 1H), 7.57 (ddd, J₁=

11.5 Hz, J₂= 9.3 Hz, J₃= 2.5 Hz, 1H), 7.34 (td, J₁= 8.2 Hz, J₂= 2.1

Hz, 1H), 7.19 (t, J = 4.9 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 4.12−4.25

(m, 2H), 3.96−3.99 (m, 1H), 2.77−2.99 (m, 2H), 2.58 (td, J₁= 11.2

Hz, J₂= 4.0 Hz, 1H) 1.82−1.86 (m, 1H), 1.44−1.53 (m, 11 H),

1.35−1.39 (m, 1H), 1.03−1.13 (m, 2H).

(3S,4S)-4-{[5-(2,4-Diﬂuoro-phenyl)-isoxazole-3-carbonyl]-

amino}-piperidine-3-carboxylic Acid (1-Pyrimidin-2-yl-cyclo-

propyl)-amide Hydrochloride (27). (3S,4S)-4-{[5-(2,4-Diﬂuoro-

phenyl)-isoxazole-3-carbonyl]-amino}-3-(1-pyrimidin-2-yl-cyclopro-

pylcarbamoyl)-piperidine-1-carboxylic acid tert-butyl ester 26 (1.81 g,

3.18 mmol) was dissolved in dioxane (10 mL). HCl in dioxane 4 M

(4 mL, 16 mmol) was added dropwise. The mixture was stirred at rt.

for 1 h. The solvents were evaporated, and the residue dried in HV to

deliver crude 27 as a white powder (1.57 g, 98%). LC−MS method A:

t_R= 0.64 min; [M + H]⁺= 469.17. ¹H NMR (400 MHz, D₆-DMSO)

δ: 8.54 (d, J = 8.2 Hz, 1H), 8.50 (d, J = 4.8 Hz, 2H), 8.41 (s, 1H),

8.07 (q, J = 8.8 Hz, 1H), 7.59 (m, 1H), 7.33 (td, J₁= 8.5 Hz, J₂= 1.6

Hz, 1H), 7.18 (m, 2H), 4.03−4.11 (m, 1H), 3.11 (d, J = 9.5 Hz, 1H),

2.96 (d, J = 11.9 Hz, 1H), 2.59 (m, 2H), 1.81 (d, J = 9.5 Hz, 1H),

1.35−1.50 (m, 4H), 1.24 (m, 1H), 1.04−1.14 (m, 3H).

Molecular formula strings (CSV)

Accession Codes

The corresponding data set has been deposited at the

Cambridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, United Kingdom, http://www.ccdc.cam.

c.uk/, under the following deposition number: CCDC

1993933 (compound 28f, see the Supporting Information).

(3S,4S)-1-Cyclopropylmethyl-4-{[5-(2,4-diﬂuoro-phenyl)-

isoxazole-3-carbonyl]-amino}-piperidine-3-carboxylic Acid (1-

Pyrimidin-2-yl-cyclopropyl)-amide (28f). To a suspension of

(3S,4S)-4-{[5-(2,4-diﬂuoro-phenyl)-isoxazole-3-carbonyl]-amino}-pi-

peridine-3-carboxylic acid methyl ester hydrochloride 27 (0.200 g,

0.396 mmol) in DCM (20 mL) at rt. was added cyclopropanecarbox-

aldehyde (0.03 mL, 0.396 mmol) followed by DIPEA (0.2 mL, 1.2

mmol) and sodium triacetoxyborohydride (0.221 g, 1 mmol). The

reaction mixture was stirred for 2 h at rt. The reaction mixture was

washed twice with aq. sat. NaHCO₃(2 × 50 mL). The organic phase

was dried over MgSO₄and evaporated. The crude residue was

puriﬁed by prep. HPLC under basic conditions to yield 28f (0.148 g,

72%) as a colorless solid, which can be crystallized from MeCN or

MeCN/H₂O. LC−MS method A: t_R= 0.69 min; [M + H]⁺= 523.04.

Chiral HPLC: t_R= 7.0 min; >99% ee; column: ChiralPak IC 4.6 × 250

mm, 5 μm; Detector wavelength: 254 nm; eluent: 10% heptane 0.05%

DEA; 90% ethanol 0.05% DEA; Flow: 1.2 mL/min; BPR: 150 bar;

temperature: 25 °C. Injection volume: 2 μL. LC-HRMS: t_R= 0.57

AUTHOR INFORMATION

■

Corresponding Authors

Sylvia Richard-Bildstein − Drug Discovery, Idorsia

Pharmaceuticals Ltd., Allschwil CH-4123, Switzerland;

orcid.org/0000-0001-6588-1599; Phone: +41-58-844-

06-87; Email: sylvia.richard@idorsia.com

Philippe Guerry − Drug Discovery, Idorsia Pharmaceuticals

Ltd., Allschwil CH-4123, Switzerland; orcid.org/0000-

0001-5631-1279; Phone: +41-58-844-02-88;

Email: philippe.guerry@idorsia.com

Authors

Hamed Aissaoui − Drug Discovery, Idorsia Pharmaceuticals

Ltd., Allschwil CH-4123, Switzerland

Julien Pothier − Drug Discovery, Idorsia Pharmaceuticals

Ltd., Allschwil CH-4123, Switzerland

̈

1

Gabriel Schafer − Drug Discovery, Idorsia Pharmaceuticals

min; m/z = 522.2191, found = 523.2265 [M + H]⁺. H NMR (500

Ltd., Allschwil CH-4123, Switzerland; orcid.org/0000-

0002-7792-9542

MHz, D₆-DMSO) δ: 8.54 (d, J = 8.5 Hz, 1H), 8.51 (d, J = 4.8 Hz,

2H), 8.45 (s, 1H), 8.08 (td, 1H), 7.59 (ddd, J₁= 11.4 Hz, J₂= 9.4 Hz,

J₃= 2.4 Hz, 1H), 7.34 (td, J₁= 2.1 Hz, J₂= 8.4 Hz, 1H), 7.18 (t, J =

4.8 Hz, 1H), 7.16 (d, J = 2.9 Hz, 1H), 3.99 (m, 1H), 3.14 (d, J = 9.5

Hz, 1H), 2.99 (d, J = 11.3 Hz, 1H), 2.72 (td, J₁= 3.6 Hz, J₂= 11.0 Hz,

1H), 2.23 (m, 2H), 2.12 (t, J = 11.4 Hz, 1H), 2.01 (t, J = 10.5 Hz

1H), 1.84−1.87 (d, J = 8.7 Hz 1H), 1.62 (m, 1H), 1.48−1.51 (m,

1H), 1.35−1.39 (m, 1H), 1.05−1.12 (m, 2H), 0.83−0.88 (m, 1H),

0.47−0.50 (m, 2H), 0.10 (m, 2H). ¹³C NMR (125 MHz, D₆-DMSO)

δ: 172.5 (s), 170.2 (s), 164.2 (dd, J₁= 252 Hz, J₂= 13 Hz), 160.2 (s),

159.4 (dd, J₁= 256 Hz, J₂= 13 Hz), 157.8 (s), 157.3 (s), 130.2 (dd, J₁

= 3 Hz, J₂= 10 Hz), 118.7 (s), 113.5 (dd, J₁= 3 Hz, J₂= 22 Hz),

111.9 (dd, J₁= 4 Hz, J₂= 12 Hz), 106.0 (t, J = 26 Hz), 103.0 (d, J = 8

Carmela Gnerre − Drug Discovery, Idorsia Pharmaceuticals

Ltd., Allschwil CH-4123, Switzerland

Eleanor Lindenberg − Drug Discovery, Idorsia

Pharmaceuticals Ltd., Allschwil CH-4123, Switzerland

Franco̧ is Lehembre − Drug Discovery, Idorsia

Pharmaceuticals Ltd., Allschwil CH-4123, Switzerland

Laetitia Pouzol − Drug Discovery, Idorsia Pharmaceuticals

Ltd., Allschwil CH-4123, Switzerland

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01588

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Inflammatory Conditions. J. Neuroimmune Pharmacol. 2016, 11, 26−

35.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Funding

No funding was provided.

(3) Balabanian, K.; Lagane, B.; Infantino, S.; Chow, K. Y. C.;

Harriague, J.; Moepps, B.; Arenzana-Seisdedos, F.; Thelen, M.;

Bachelerie, F. The Chemokine SDF-1/CXCL12 Binds to and Signals

Through the Orphan Receptor RDC1 in T lymphocytes. J. Biol. Chem.

2005, 280, 35760−35766.

(4) Naumann, U.; Cameroni, E.; Pruenster, M.; Mahabaleshwar, H.;

Raz, E.; Zerwes, H. G.; Rot, A.; Thelen, M. CXCR7 Functions as a

Scavenger for CXCL12 and CXCL11. PLoS One 2010, 5, No. e9175.

Notes

The authors declare no competing ﬁnancial interest.

́

(5) Sanchez-Alcaniz, J. A.; Haege, S.; Mueller, W.; Pla, R.; Mackay,

̃

ACKNOWLEDGMENTS

We thank Dr. Eric A. Ertel and Dr. Martin Holdener for helpful

discussions, Viktor Ribic, Siefke Siefken, Clement Popineau,

Pascal Rebmann, Audrey Izard, Aurelien Merot, Philippe

■

́

F.; Schulz, S.; Lopez-Bendito, G.; Stumm, R.; Marín, O. CXCR7

Controls Neuronal Migration by Regulating Chemokine Responsive-

ness. Neuron 2011, 69, 77−90.

́

(6) Zohar, Y.; Wildbaum, G.; Novak, R.; Salzman, A. L.; Thelen, M.;

Alon, R.; Barsheshet, Y.; Karp, C. L.; Karin, N. CXCL11-Dependent

Induction of FOXP3-Negative Regulatory T Cells Suppresses

Autoimmune Encephalomyelitis. J. Clin. Invest. 2014, 124, 2009−

2022.

́

Risch, Melanie Garnier, Ann Natalie de Leon for the synthesis

and characterization of the compounds, Julien Grimont,

Franco̧ is Le Goﬀ and Marco Caldarone for the analysis of

the compounds, Benedicte Haenig, Panja Strickner and Manon

Kiry for performing the biological assays, Romain Sube,

(7) Tokunaga, R.; Zhang, W.; Naseem, M.; Puccini, A.; Berger, M.

D.; Soni, S.; McSkane, M.; Baba, H.; Lenz, H. J. CXCL9, CXCL10,

CXCL11/CXCR3 Axis for Immune Activation - a Target for Novel

Cancer Therapy. Cancer Treat. Rev. 2018, 63, 40−47.

(8) Torraca, V.; Cui, C.; Boland, R.; Bebelman, J. P.; van der Sar, A.

M.; Smit, M. J.; Siderius, M.; Spaink, H. P.; Meijer, A. H. The

CXCR3-CXCL11 Signaling Axis Mediates Macrophage Recruitment

and Dissemination of Mycobacterial Infection. Dis. Model Mech. 2015,

8, 253−269.

́

Camille Forni and Cedric Etien for hERG blockage measure-

ments, the DMPK as well as the preformulation and preclinical

galenics groups for their continuous support with routine in

vitro and in vivo experiments, Anna Sassi, Nadege Baumlin and

Melanie Tunis for performing in vivo experiments and

biomarker analysis, Dr. G. Santiso-Quinones from Crystallise

AG for crystallization experiments and preparation of ﬁles for

the CSD, and Dr. Gianvito Vile for the synthesis of some key

intermediates by continuous ﬂow chemistry.

̀

́

(9) Quinn, K. E.; Mackie, D. I.; Caron, K. M. Emerging Roles of

Atypical Chemokine Receptor 3 (ACKR3) in Normal Development

and Physiology. Cytokine+ 2018, 109, 17−23.

(10) Wang, Y.; Huang, J.; Li, Y.; Yang, G. Y. Roles of Chemokine

CXCL12 and its Receptors in Ischemic Stroke. Curr. Drug Targets

2012, 13, 166−172.

ABBREVIATIONS USED

■

AcOH, acetic acid; BPR, back pressure regulator; CHO,

Chinese hamster ovary; Cl_int, intrinsic clearance; clog D_7.4,

calculated log D at pH 7.4; C_max, maximal (peak) plasma

concentration; Compd, compound; DEA, diethylamine;

DIPEA, diisopropylethylamine; DLM, dog liver microsomes;

EtOAc, ethyl acetate; EtOH, ethanol; HATU, 2-(7-aza-1H-

benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuoro-

phosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-

(11) Puchert, M.; Pelkner, F.; Stein, G.; Angelov, D. N.; Boltze, J.;

Wagner, D. C.; Odoardi, F.; Flugel, A.; Streit, W. J.; Engele, J.

̈

Astrocytic Expression of the CXCL12 Receptor, CXCR7/ACKR3 is a

Hallmark of the Diseased, but not Developing CNS. Mol. Cell.

Neurosci. 2017, 85, 105−118.

(12) Xu, T.; Yu, X.; Deng, J.; Ou, S.; Liu, X.; Wang, T.; Liu, Y.; Yang,

J.; Tan, C.; Yuan, J.; Chen, Y. CXCR7 Regulates Epileptic Seizures by

Controlling the Synaptic Activity of Hippocampal Granule Cells. Cell

Death Dis. 2019, 10, 825.

̀

fonic acid; hERG, human ether-a-go-go related gene (also

called hKV11.1); HLM, human liver microsomes; HTRF,

homogeneous time resolved technology; hPPB, human plasma

protein binding; HV, high vacuum; KO^tBu, potassium tert-

butoxide; log D_7.4, measured log D at pH 7.4; MeCN,

acetonitrile; MeOH, methanol; NaBH(OAc)₃, sodium triace-

toxyborohydride; NaOAc, sodium acetate; NaOEt, sodium

ethanolate; nM, nanomolar; PDA, photo diode array; Pd-

(dppf)Cl₂·DCM, (1,1′-bis(diphenylphosphino)ferrocene) di-

chloropalladium (II) dichloromethane; p.o., per os; prep.,

preparative; QC, quality control; rac, racemic; RLM, rat liver

microsomes; TBTU, 2-(1H-benzotriazole-1-yl)-1,2,3,3-tetra-

methyluronium tetraﬂuoroborate; T_1/2, terminal half-life;

TEA, triethylamine; T_max, time of maximal plasma concen-

tration; T₃P, propylphosphonic anhydride; UAS, Upstream

Activation Sequence

́

(13) Trojan, E.; Slusarczyk, J.; Chamera, K.; Kotarska, K.; Glombik,

K.; Kubera, M.; Basta-Kaim, A. The Modulatory Properties of

Chronic Antidepressant Drugs Treatment on the Brain Chemokine -

Chemokine Receptor Network: A Molecular Study in an Animal

Model of Depression. Front. Pharmacol. 2017, 8, 779.

(14) McCandless, E. E.; Piccio, L.; Woerner, B. M.; Schmidt, R. E.;

Rubin, J. B.; Cross, A. H.; Klein, R. S. Pathological Expression of

CXCL12 at the Blood-Brain Barrier Correlates with Severity of

Multiple Sclerosis. Am. J. Pathol. 2008, 172, 799−808.

(15) Moll, N. M.; Cossoy, M. B.; Fisher, E.; Staugaitis, S. M.; Tucky,

B. H.; Rietsch, A. M.; Chang, A.; Fox, R. J.; Trapp, B. D.; Ransohoff,

R. M. Imaging Correlates of Leukocyte Accumulation and CXCR4/

CXCL12 in Multiple Sclerosis. Arch. Neurol. 2009, 66, 44−53.

(16) Cruz-Orengo, L.; Holman, D. W.; Dorsey, D.; Zhou, L.; Zhang,

P.; Wright, M.; McCandless, E. E.; Patel, J. R.; Luker, G. D.; Littman,

D. R.; Russell, J. H.; Klein, R. S. CXCR7 Influences Leukocyte Entry

into the CNS Parenchyma by Controlling Abluminal CXCL12

Abundance During Autoimmunity. J. Exp. Med. 2011, 208, 327−339.

(17) Watanabe, K.; Penfold, M. E. T.; Matsuda, A.; Ohyanagi, N.;

Kaneko, K.; Miyabe, Y.; Matsumoto, K.; Schall, T. J.; Miyasaka, N.;

Nanki, T. Pathogenic Role of CXCR7 in Rheumatoid Arthritis.

Arthritis Rheum. 2010, 62, 3211−3220.

REFERENCES

■

(1) Burns, J. M.; Summers, B. C.; Wang, Y.; Melikian, A.;

Berahovich, R.; Miao, Z.; Penfold, M. E. T.; Sunshine, M. J.;

Littman, D. R.; Kuo, C. J.; Wei, K.; McMaster, B. E.; Wright, K.;

Howard, M. C.; Schall, T. J. A Novel Chemokine Receptor for SDF-1

and I-TAC Involved in Cell Survival, Cell Adhesion, and Tumor

Development. J. Exp. Med. 2006, 203, 2201−2213.

(18) Konrad, F. M.; Meichssner, N.; Bury, A.; Ngamsri, K. C.;

Reutershan, J. Inhibition of SDF-1 Receptors CXCR4 and CXCR7

Attenuates Acute Pulmonary Inflammation via the Adenosine A2B-

Receptor on Blood Cells. Cell Death Dis. 2017, 8, No. e2832.

(2) Banisadr, G.; Podojil, J. R.; Miller, S. D.; Miller, R. J. Pattern of

CXCR7 Gene Expression in Mouse Brain Under Normal and

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

̈

(19) Ngamsri, K.-C.; Mu

̈

ller, A.; Bosmuller, H.; Gamper-Tsigaras, J.;

with TMZ Activates Immune Responses and Extends Survival in

Reutershan, J.; Konrad, F. M. The Pivotal Role of CXCR7 in

Stabilization of the Pulmonary Epithelial Barrier in Acute Pulmonary

Inflammation. J. Immunol. 2017, 198, 2403−2413.

Mouse GBM Models. Mol. Ther. 2018, 26, 1354−1365.

́

(33) Maussang, D.; Mujic-Delic, A.; Descamps, F. J.; Stortelers, C.;

Vanlandschoot, P.; Walsum, M. S.-v.; Vischer, H. F.; van Roy, M.;

Vosjan, M.; Gonzalez-Pajuelo, M.; van Dongen, G. A.; Merchiers, P.;

van Rompaey, P.; Smit, M. J. Llama-Derived Single Variable Domains

(Nanobodies) Directed Against Chemokine Receptor CXCR7

Reduce Head and Neck Cancer Cell Growth in vivo. J. Biol. Chem.

2013, 288, 29562−29572.

(20) Chang, H.-C.; Huang, P.-H.; Syu, F.-S.; Hsieh, C.-H.; Chang, S.

L.-Y.; Lu, J.; Chen, H.-C. Critical Involvement of Atypical Chemokine

Receptor CXCR7 in Allergic Airway Inflammation. Immunology 2018,

154, 274−284.

(21) Wang, C.; Chen, W.; Shen, J. CXCR7 Targeting and Its Major

Disease Relevance. Front. Pharmacol. 2018, 9, 641.

́

(34) Zabel, B. A.; Wang, Y.; Lewen, S.; Berahovich, R. D.; Penfold,

M. E. T.; Zhang, P.; Powers, J.; Summers, B. C.; Miao, Z.; Zhao, B.;

Jalili, A.; Janowska-Wieczorek, A.; Jaen, J. C.; Schall, T. J. Elucidation

of CXCR7-Mediated Signaling Events and Inhibition of CXCR4-

Mediated Tumor Cell Transendothelial Migration by CXCR7

Ligands. J. Immunol. 2009, 183, 3204−3211.

(22) Walters, M. J.; Ebsworth, K.; Berahovich, R. D.; Penfold, M. E.

T.; Liu, S.-C.; Al Omran, R.; Kioi, M.; Chernikova, S. B.; Tseng, D.;

Mulkearns-Hubert, E. E.; Sinyuk, M.; Ransohoff, R. M.; Lathia, J. D.;

Karamchandani, J.; Kohrt, H. E. K.; Zhang, P.; Powers, J. P.; Jaen, J.

C.; Schall, T. J.; Merchant, M.; Recht, L.; Brown, J. M. Inhibition of

CXCR7 Extends Survival Following Irradiation of Brain Tumours in

Mice and Rats. Br. J. Cancer 2014, 110, 1179−1188.

(23) Guillemot, E.; Karimdjee-Soilihi, B.; Pradelli, E.; Benchetrit,

M.; Goguet-Surmenian, E.; Millet, M. A.; Larbret, F.; Michiels, J. F.;

Birnbaum, D.; Alemanno, P.; Schmid-Antomarchi, H.; Schmid-

Alliana, A. CXCR7 Receptors Facilitate the Progression of Colon

Carcinoma within Lung not within Liver. Br. J. Cancer 2012, 107,

1944−1949.

(24) Luker, K. E.; Lewin, S. A.; Mihalko, L. A.; Schmidt, B. T.;

Winkler, J. S.; Coggins, N. L.; Thomas, D. G.; Luker, G. D.

Scavenging of CXCL12 by CXCR7 Promotes Tumor Growth and

Metastasis of CXCR4-Positive Breast Cancer Cells. Oncogene 2012,

31, 4750−4758.

(25) Goguet-Surmenian, E.; Richard-Fiardo, P.; Guillemot, E.;

Benchetrit, M.; Gomez-Brouchet, A.; Buzzo, P.; Karimdjee-Soilihi, B.;

Alemanno, P.; Michiels, J. F.; Schmid-Alliana, A.; Schmid-

Antomarchi, H. CXCR7-Mediated Progression of Osteosarcoma in

the Lungs. Br. J. Cancer 2013, 109, 1579−1585.

(26) Wani, N. A.; Nasser, M. W.; Ahirwar, D. K.; Zhao, H.; Miao, Z.;

Shilo, K.; Ganju, R. K. C-X-C Motif Chemokine 12/C-X-C

Chemokine Receptor Type 7 Signaling Regulates Breast Cancer

Growth and Metastasis by Modulating the Tumor Microenvironment.

Breast Cancer Res. 2014, 16, R54.

(27) Sierro, F.; Biben, C.; Martinez-Munoz, L.; Mellado, M.;

Ransohoff, R. M.; Li, M.; Woehl, B.; Leung, H.; Groom, J.; Batten,

M.; Harvey, R. P.; Martinez-A, C.; Mackay, C. R.; Mackay, F.

Disrupted Cardiac Development But Normal Hematopoiesis in Mice

Deficient in the Second CXCL12/SDF-1 Receptor, CXCR7. Proc.

Natl. Acad. Sci. U. S. A. 2007, 104, 14759−14764.

(28) Menhaji-Klotz, E.; Hesp, K. D.; Londregan, A. T.; Kalgutkar, A.

S.; Piotrowski, D. W.; Boehm, M.; Song, K.; Ryder, T.; Beaumont, K.;

Jones, R. M.; Atkinson, K.; Brown, J. A.; Litchfield, J.; Xiao, J.;

Canterbury, D. P.; Burford, K.; Thuma, B. A.; Limberakis, C.; Jiao,

W.; Bagley, S. W.; Agarwal, S.; Crowell, D.; Pazdziorko, S.; Ward, J.;

Price, D. A.; Clerin, V. Discovery of a Novel Small-Molecule

Modulator of C-X-C Chemokine Receptor Type 7 as a Treatment for

Cardiac Fibrosis. J. Med. Chem. 2018, 61, 3685−3696.

(35) Luker, K. E.; Gupta, M.; Luker, G. D. Imaging Chemokine

Receptor Dimerization with Firefly Luciferase Complementation.

FASEB J. 2008, 23, 823−834.

(36) Gustavsson, M.; Wang, L.; van Gils, N.; Stephens, B. S.; Zhang,

P.; Schall, T. J.; Yang, S.; Abagyan, R.; Chance, M. R.; Kufareva, I.;

Handel, T. M. Structural Basis of Ligand Interaction with Atypical

Chemokine Receptor 3. Nat. Commun. 2017, 8, 14135.

(37) Menhaji-Klotz, E.; Ward, J.; Brown, J. A.; Loria, P. M.; Tan, C.;

Hesp, K. D.; Riccardi, K. A.; Litchfield, J.; Boehm, M. Discovery of

Diphenylacetamides as CXCR7 Inhibitors with Novel β -Arrestin

Antagonist Activity. ACS Med. Chem. Lett. 2020, 11, 1330−1334.

(38) Fretz, H. G.; Guerry, P.; Kimmerlin, T.; Lehembre, F.; Pfeifer,

T.; Valdenaire, A. 1 -[m-Carboxamido(hetero)aryl-methyl]-piper-

idine-4-carboxamide Derivatives as CXCR7 Modulators and their

Preparation. US20130345199A1, 2013.

(39) Waring, M. J. Lipophilicity in Drug Discovery. Expert Opin.

Drug Discov. 2010, 5, 235−248.

(40) Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design

and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018,

61, 5822−5880.

(41) Schinnerl, M.; Murray, J. K.; Langenhan, J. M.; Gellman, S. H.

Asymmetric Synthesis of a New Helix-Forming β -Amino Acid: trans-

4-Aminopiperidine-3-carboxylic Acid. Eur. J. Org. Chem. 2003, 721−

726.

(42) Sheldrick, G. M. SHELXT - Integrated Space-Group and

Crystal-Structure Determination. Acta Crystallogr. A Found Adv. 2015,

71, 3−8.

(43) Sheldrick, G. M. Crystal Structure Refinement with SHELXL.

Acta Crystallogr. C Struct. Chem. 2015, 71, 3−8.

(44) Spek, A. L. PLATONSQUEEZE: A Tool for the Calculation of

the Disordered Solvent Contribution to the Calculated Structure

Factors. Acta Crystallogr., Sect. C 2015, 71, 9−18.

(29) Dong, B. C.; Li, M. X.; Wang, X. Y.; Cheng, X.; Wang, Y.; Xiao,

T.; Jolkkonen, J.; Zhao, C. S.; Zhao, S. S. Effects of CXCR7-

Neutralizing Antibody on Neurogenesis in the Hippocampal Dentate

Gyrus and Cognitive Function in the Chronic Phase of Cerebral

Ischemia. Neural Regener. Res. 2020, 15, 1079−1085.

(30) Bao, J.; Zhu, J.; Luo, S.; Cheng, Y.; Zhou, S. CXCR7

Suppression Modulates Microglial Chemotaxis to Ameliorate

Experimentally-Induced Autoimmune Encephalomyelitis. Biochem.

Biophys. Res. Commun. 2016, 469, 1−7.

(31) Peng, H.; Zhang, H.; Zhu, H. Blocking CXCR7-Mediated

Adipose Tissue Macrophages Chemotaxis Attenuates Insulin

Resistance and Inflammation in Obesity. Biochem. Biophys. Res.

Commun. 2016, 479, 649−655.

(32) Salazar, N.; Carlson, J. C.; Huang, K.; Zheng, Y.; Oderup, C.;

́

Gross, J.; Jang, A. D.; Burke, T. M.; Lewen, S.; Scholz, A.; Huang, S.;

Nease, L.; Kosek, J.; Mittelbronn, M.; Butcher, E. C.; Tu, H.; Zabel, B.

A. A Chimeric Antibody Against ACKR3/CXCR7 in Combination

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01588

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of the Potent, Selective, Orally Available CXCR7 Antagonist ACT-1004-1239

DOI: 10.1021/acs.jmedchem.0c01588

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01588

Full text of DOI:10.1021/acs.jmedchem.0c01588

Products guided by the article

R&D Labs maybe for 859855-38-2

Relevant to this article

Hot Product