Discovery of a Novel Small-Molecule Modulator of C-X-C Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis

Article abstract of DOI:10.1021/acs.jmedchem.8b00190

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 K_i = 13 nM, adrenergic α 1a K_b > 10-?000 nM, and adrenergic β 2 K_b > 10-?000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

Full text of DOI:10.1021/acs.jmedchem.8b00190

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Discovery of a Novel Small Molecule Modulator of C-X-C
Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis
Elnaz Menhaji-Klotz, Kevin D. Hesp, Allyn Timothy Londregan, Amit S. Kalgutkar, David W.
Piotrowski, Markus Boehm, Kun Song, Tim Ryder, Kevin Beaumont, Rhys M Jones, Karen
Atkinson, Janice A. Brown, John Litchfield, Jun Xiao, Daniel P Canterbury, Kristen Burford,
Benjamin A. Thuma, Chris Limberakis, Wenhua Jiao, Scott W. Bagley, Saket Agarwal,
Danielle Crowell, Stephen Pazdziorko, Jessica Ward, David A. Price, and Valerie Clerin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00190 • Publication Date (Web): 08 Apr 2018
Downloaded from http://pubs.acs.org on April 9, 2018
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Discovery of a Novel Small Molecule Modulator of
CꢀXꢀC Chemokine Receptor Type 7 as a Treatment
for Cardiac Fibrosis
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†
Elnaz Menhaji-Klotz,*, Kevin D. Hesp,‡ Allyn T. Londregan,‡ Amit S. Kalgutkar,† David W.
Piotrowski,‡ Markus Boehm,† Kun Song,† Tim Ryder,‡ Kevin Beaumont,† Rhys M. Jones,† Karen
Atkinson,‡ Janice A. Brown,‡ John Litchfield,† Jun Xiao,‡ Daniel P. Canterbury,‡ Kristen
Burford,‡ Benjamin A. Thuma,‡ Chris Limberakis,‡ Wenhua Jiao,‡ Scott W. Bagley,‡ Saket
Agarwal,† Danielle Crowell,† Stephen Pazdziorko,† Jessica Ward,† David A. Price† and Valerie
Clerin†
†Pfizer Worldwide Research & Development, Cambridge, Massachusetts 02139, United States
‡Pfizer Worldwide Research & Development, Groton, Connecticut 06340, United States
KEYWORDS: CꢀXꢀC chemokine receptor type 7, CXCR7 modulator, chemokine receptor,
ACKR3, CXCL12, SDFꢀ1α.
ABSTRACT: CꢀXꢀC chemokine receptor type 7 (CXCR7) is involved in cardiac and immune
pathophysiology. We report the discovery of a novel 1,4ꢀdiazepine CXCR7 modulator,
demonstrating for the first time, the role of pharmacological CXCR7 intervention in cardiac
repair. Structureꢀactivity relationship (SAR) studies demonstrated that a net reduction in
lipophilicity (logD) and incorporation of saturated ring systems yielded compounds with good
CXCR7 potency and improvements in oxidative metabolic stability in human liver microsomes
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(HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., compound 18
CXCR7 Ki = 13 nM, adrenergic α 1a Kb >10,000 nM, adrenergic β 2 Kb >10,000 nM). Subꢀ
cutaneous administration of 18 in mice led to a statistically significant increase in circulating
concentrations of plasma stromal cellꢀderived factor 1 alpha (SDFꢀ1α) by approximately twoꢀ
fold. Chronic dosing of compound 18 in a mouse model of isoproterenolꢀinduced cardiac injury
further resulted in a statistically significant reduction of cardiac fibrosis.
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INTRODUCTION
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CXCR7, also known as atypical chemokine receptor 3 (ACKR3), is a seven transꢀmembrane
domain Gꢀprotein coupled receptor (GPCR) expressed in monocytes, endothelial cells, B cells
and cardiomyocytes. Two ligands have been identified: stromal cellꢀderived factor 1 alpha (SDFꢀ
1α) also known as CXC chemokine ligand 12 (CXCL12) as well as interferonꢀinducible Tꢀcell
alpha chemoattractant (IꢀTAC), also known as CXC chemokine ligand 11 (CXCL11). Unlike
most chemokine receptors, CXCR7 does not participate in any Gꢀprotein signaling cascades.
Rather, ligand binding induces recruitment of βꢀarrestin and receptor internalization.1
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CXCR7 is reported to be a ‘scavenger’ receptor for SDFꢀ1α which is an 8 kDa, 89ꢀamino acid
peptide that also binds to CꢀXꢀC chemokine receptor type 4 (CXCR4).2ꢀ3 The scavenger activity
of CXCR7 is thought to control the local levels of SDFꢀ1α thereby modulating the activation
state of CXCR4. Activation of CXCR4 by SDFꢀ1α leads to Gꢀprotein signaling cascades
involved in cardiomyocyte survival and angiogenesis. As such, SDFꢀ1α has been reported to
play a protective role after cardiac injury to promote repair processes, reduce fibrosis and
improve cardiac function.4
While SDFꢀ1α gene therapy is being explored in the clinic as a treatment for patients with a
prior myocardial infarction (MI),5 we hereby propose a small molecule modulator of CXCR7 as
a novel approach to block its SDFꢀ1α scavenging activity, increase local SDFꢀ1α concentration
and promote activation of CXCR4 to improve cardiac function in the setting of ischemic heart
disease.
Peptide GPCRs have been noted to be challenging drug discovery targets. Optimized ligands
for peptide GPCRs often suffer from poor physiochemical properties including low drugꢀlike
properties (i.e., median MW = 518 and cLogP = 5).6 In addition, small molecule chemical
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matter targeting chemokine receptors is often basic and lipophilic7 with undesired polyꢀ
pharmacology making it challenging to identify suitable in vivo tools. A number of CXCR7
small molecule8ꢀ22 and peptidic23ꢀ25 modulators have been reported in the literature, but none
have demonstrated efficacy in animal models of cardiac injury. In this report, we describe our
efforts in the identification of novel small molecules from the 1,4ꢀdiazepine series with
nanomolar affinity for CXCR7 with improved physicochemical properties and offꢀtarget
selectivity for use in proofꢀofꢀmechanism studies in a rodent model of heart failure.
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RESULTS AND DISCUSSION
Synthesis and in vitro pharmacology
Estimates of the binding affinity (Ki) for CXCR7 were determined by a radioligand
competition binding assay utilizing 125IꢀSDFꢀ1α and membrane preparations from cells
overexpressing the human25 or mouse CXCR7 receptor. As CXCR7 is not known to signal via
Gꢀproteins, we evaluated the functional effects of our compounds on the receptor using a βꢀ
arrestin recruitment assay from DiscoverX.25 The potency value reported for this assay is an
EC50 or the concentration of compound required to reach 50% of the maximum effect.
Interestingly, all compounds that bound to the receptor in a competitive manner with the native
SDFꢀ1α ligand also promoted recruitment of βꢀarrestin. To ensure selectivity for CXCR7 over
CXCR4, which shares the SDFꢀ1α ligand, binding to the CXCR4 receptor was also monitored
using a whole cell CXCR4 125IꢀSDFꢀ1α competition binding assay with Jurkat cells, an
immortalized line of human T lymphocyte cells, which endogenously express the CXCR4
receptor.
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Figure 1. Representative CXCR7 Modulator
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Under the present experimental conditions, the previously reported19 1,4ꢀdiazepaneꢀbased
CXCR7 ligand 1 revealed high binding affinity for CXCR7 (Ki = 9 nM). However, 1 is
lipophilic (shake flask (sF)logD26 = 3.7) and demonstrates inhibitory potency (Ki < 13 nM) in the
hERG membrane competitive binding assay, which is comparable to its CXCR7 binding affinity.
Our work, along with an analysis of related compounds from the literature suggested that the
core 1,4ꢀdiazepane motif was optimal for affinity. Therefore, we elected to retain this core as a
key element of future analogues and explore the pendant quinoline and phenylthiazole regions of
the molecule using parallel enabled methods.
Our immediate design goal centered on the minimization of the oxidative metabolic instability
and the idiosyncratic toxicity risk posed from the potential oxidative bioactivation of the 1,4ꢀ
diazepaneꢀ7ꢀmethoxyquinoline framework to a potentially electrophilic quinoneꢀimine species.27
A number of analogues (5a–5h) of CXCR7 modulator 1 were synthesized in a parallel medicinal
chemistry fashion through a robust CꢀN bond formation, reductive amination synthetic sequence
(Scheme 1). We focused our efforts on head groups (quinoline replacements) that either
removed the methoxy substituent (i.e., 2a, 2b) and/or replaced the pyridine motif with different
heteroꢀbicyclic systems (i.e., 2e). Heteroaryl bromides or chlorides (2a–2h) were treated with Nꢀ
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Boc homopiperazine (3), followed by NꢀBOC deprotection to form the desired Nꢀaryl bond (4a–
4h). Intermediates 4a-4h were used without further purification wherein reductive amination
with 2ꢀphenylthiazoleꢀ4ꢀcarbaldehyde provided the substituted 1,4ꢀdiazepanes 5a–5h.
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Scheme 1. Parallel Synthesis of 1,4ꢀDiazepanes 5a–5h. Reaction conditions: (a) For heteroaryl
bromides: Pd2dba3, RuPhos, NaOtꢀBu, 1,4ꢀdioxane, 95 oC; (b) For heteroaryl chlorides: iꢀPr2NEt,
DMF, 100 oC; (c) TFA, DCM, rt; (d) 2ꢀphenylthiazoleꢀ4ꢀcarbaldehyde, NaBH(OAc)3, DCE, 50
oC.
Analogues 5a–5h were evaluated in the CXCR7 binding assay to understand the effect of
various heterocyclic systems on binding affinity (Table 1). Replacement of the methoxy group
with a trifluoromethoxy functionality yielded 5a, which maintained CXCR7 binding affinity and
showed only a small loss in lipophilic efficiency (LipE).28 We found quinoline head group
replacements to pose a bigger challenge. For example, naphthyridine analogues 5b (Ki = 730
nM) and 5c (Ki = 1,900 nM) were considerably less potent, and indazole 5d (Ki = 18,000 nM)
virtually lost binding affinity for CXCR7. The imidazopyridines (5e–5h), on the other hand,
were found to be suitable replacements for the quinoline. Thus, 7ꢀmethoxyꢀimidazopyridine 5e
demonstrated good CXCR7 binding affinity (Ki = 52 nM) with high LipE. Based on the LipE
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value of 5e, we also made the 7ꢀtrifluoromethylꢀ (5f) and 7ꢀmethylꢀimidazopyridine (5g)
analogues both of which showed subꢀmicromolar potency for CXCR7. In particular, 5g
exhibited good binding affinity (Ki = 29 nM) and LipE = 4.8. In contrast, the corresponding 6ꢀ
methylꢀimidazopyridine 5h isomer exhibited approximately 30ꢀfold loss of binding affinity (Ki =
900 nM) in comparison to 5g.
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Table 1. CXCR7 In Vitro Binding Affinity, Physicochemical Properties and Metabolic Intrinsic
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Clearance (CLint) of 1,4ꢀdiazepines.
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29, c
HLM CLint
CXCR7 Ki
[nM]25, a
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Compd.
R
sFlogD26
3.4
LipE28, b
[ꢀL/min/mg]
4.7
98
1
(8.1±0.6)
42
3.0
4.4
143
5a
(7.4±0.1)
730
2.5
3.6
218
5b
(6.1±0.2)
1,900
(5.7±0.5)
18,000
(4.7±0.3)
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3.4
3.8
1.7
5.1
2.8
2.3
0.9
5.5
1.0
4.8
171
150
67
5c
5d
5e
5f
(7.3±0.2)
760
145
>320
(6.1±0.2)
29
5g
(7.5±0.3)
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900
3.7
2.3
290
5h
(6.0±0.7)
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Assays were run using established protocols with references provided where appropriate.
aValues were determined by a radioligand binding assay and are reported as geometric mean of
at least three independent experiments with pKi (negative logarithm) ± SD in parentheses.
bLipE, a measure of lipophilic efficiency, is the difference between pKi and sFLogD. cHLM
CLint values were generated following incubation of the test compound (1 ꢀM) in NADPH (1.3
mM)ꢀsupplemented HLM (protein concentration = 0.25 mg/mL) at 37 °C for 30 min.
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In addition to CXCR7 binding affinity measurements, we evaluated the HLM stability of these
compounds and found that the majority of analogues demonstrated high intrinsic clearance
(CLint). Imidazopyridine 5e demonstrated the lowest metabolic clearance (CLint = 67
ꢀL/min/mg) and was also the least lipophilic compound (sFlogD = 1.7) of the analogues in
Table 1. We hypothesized that lowering the lipophilicity (sFlogD <2) within this series would
improve the overall metabolic clearance30 and also address potential offꢀtarget pharmacology and
affinity against the hERG channel, often associated with lipophilic and basic compounds.31
In order to lower lipophilicity across the 1,4ꢀdiazepine series, we sought to replace the 2ꢀ
phenylthiazole substituent with a saturated ring system, specifically a piperidine ring.32 Besides
lowering the lipophilicity of analogues, the piperidine template would allow for more facile SAR
studies through amide coupling chemistry and provide access to a wide array of commercially
available carboxylic acid reagents. We selected 7ꢀmethyl and 7ꢀethyl imidazopyridine groups
for further SAR evaluation because we found that homologation of the methyl group by one
carbon provided improvements in affinity (see Table 2). A representative synthesis of an
advanced piperidine template is illustrated in Scheme 2 wherein CꢀN bond formation is followed
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by reductive amination with tꢀbutyl 4ꢀformylpiperidineꢀ1ꢀcarboxylate. Scheme 3 depicts the
general parallel medicinal chemistry strategy where piperidines 7 and 8 were converted to the
corresponding amides. The experimental data for a subset of amides synthesized in parallel
medicinal fashion using commercially available carboxylic acids is shown in Table 2.
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Scheme 2. Representative synthesis of piperidine template in support of parallel synthesis
efforts. Reaction conditions: (a) Pd2dba3, RuPhos, NaOtꢀBu, PhCH3, 95 oC, 6 h, 73%; (b) TFA,
DCM, rt, 5 h; (c) tꢀbutyl 4ꢀformylpiperidineꢀ1ꢀcarboxylate, DCE, rt, 1 h, then NaBH(OAc)3, rt,
16 h, 59%.
Scheme 3. Parallel Synthesis of Piperidine Amides
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Cyclopentyl piperidine amide 10a had modest CXCR7 binding affinity (Ki = 950 nM) but was
over one log unit lower in lipophilicity compared to 5g (sFlogD = 1.7 vs. 2.8, respectively).
Even though 10a had reduced CXCR7 affinity, the change to a piperidine amide from a 2ꢀ
phenylthiazole was only a half log unit lower with regard to LipE (4.3 vs. 4.8, respectively). In
contrast, regioisomeric cyclopentyl piperidine amide 12 (see Experimental Section for synthesis
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of 12) had poor CXCR7 binding affinity (Ki > 8,500 nM) relative to 10a.
Although
replacement of the cyclopentyl group in 10a with a tetrahydrofuran functionality (10b) led to a
significant loss of CXCR7 potency (Ki > 9,700 nM), the corresponding analogue demonstrated a
marked improvement in CLint in HLM (20 ꢀL/min/mg), which is likely associated with its lower
sFlogD. Based on this observation, we decided to focus our attention on compounds with
sFlogD ~ 1 in order to maintain low metabolic turnover, while improving upon the CXCR7
binding potency. Incorporation of exoꢀ[2.2.1]ꢀoxabicycle provided 10c with favorable CXCR7
affinity (Ki = 520 nM) and excellent LipE. The corresponding endoꢀ[2.2.1]ꢀoxabicycle 10d,
however, was 26ꢀfold less potent (Ki = 14,000 nM) than 10c. We found that homologation of the
7ꢀmethyl to a 7ꢀethyl group provided an 11ꢀfold boost in potency for 11a, which was initially
made as a racemic mixture. Synthesis of enantiopure analogues identified 11c as the eutomer
with CXCR7 Ki = 13 nM, LipE = 7.1, and importantly, reduced CLint (46 ꢀL/min/mg) in HLM.
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Table 2. CXCR7 In Vitro Binding Affinity, Physicochemical Properties and Metabolic Intrinsic
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Clearance (CLint) of Piperidines Amides.
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9
HLM CLint
CXCR7 Ki
[nM]25, a
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Compd.
10a
Structure
sFlogD26 LipE28,b
[ꢀL/min/mg]29, c
950
1.7
0.2
4.3
4.8
198
20
(6.0±0.6)
>9,700
10b
(5.0±0.4)
520
0.4
0.7
0.7
5.9
4.2
6.6
22
68
44
10c
10d
11a
(6.3±0.2)
14,000
(4.9±0.3)
47
(7.3±0.4)
760
0.8
0.8
5.3
7.1
25
46
11b
11c
(6.1±0.2)
13
(7.9±0.2)
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>8,500
1.8
(5.1±0.5)
3.3
6.8
148
60
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1.1
(7.9±0.3)
Assays were run using established protocols with references provided where appropriate.
aValues were determined by a radioligand binding assay and are reported as geometric mean of
at least three independent experiments with pKi ± SD in parentheses. bLipE, a measure of
lipophilic efficiency, is the difference between pKi and sFLogD. cHLM CLint values were
generated following incubation of the test compound (1 ꢀM) in NADPH (1.3 mM)ꢀsupplemented
HLM (protein concentration = 0.25 mg/mL) at 37 °C for 30 min.
Table 3. In Vitro Pharmacology of 11c and 18.
11c
13
18
13
Human CXCR7 Ki [nM]25, a
βꢀarrestin EC50 [nM]25, b
Mouse CXCR7 Ki [nM]d
Human CXCR4 Ki [nM]c
hERG patch clamp IC50 [nM]e
Adrenergic α1a Kb [nM]f
7
11
51
18
>6,200
1,300
520
11,000
8,300
>10,000
Adrenergic β 2 Kb [nM] f
Histamine 1 Kb [nM] f
Muscarinic 1 Kb [nM] f
2200
330
59
>10,000
>4,400
>10,000
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Assays were run using established protocols with references provided where appropriate.
aValues were determined by a radioligand binding assay and are reported as geometric mean of
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at least three independent experiments. Values were determined by a βꢀarrestin recruitment
c
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assay and are reported as geometric mean of at least two independent experiments. Values were
determined by a radiolabeled binding assay and are reported as a geometric mean of at least three
d
independent experiments. Values were determined by a radiolabeled binding assay and are
e
reported as a geometric mean of at least three independent experiments. Measurement of
potassium current in human embryonic kidney 293 (HEK293) cells stably transfected with the
hERG channel. fKb value is the dissociation constant of antagonist for the receptor and are
reported as geometric mean of at least two independent experiments.
In addition to potent modulation of human CXCR7, we observed that 11c was active against
the mouse form of the receptor which was not surprising considering the high species
homology33 but nonetheless important for use in a mouse model of cardiac fibrosis (Table 3).
11c was also found to have functional activity and recruit βꢀarrestin and did not display binding
to the related chemokine receptor CXCR4. However, further in vitro characterization of 11c
revealed significant offꢀtarget pharmacology against several aminergic GPCR targets (Table 3);
the most potent being antagonist activity against muscarinic 1 receptor (Kb = 59 nM). Potency
against the adrenergic α 1a and β 2 receptors was of particular concern with respect to
cardiovascular liabilities34 in addition to the aforementioned risk of hERG activity.
We sought to incorporate additional polarity at the methylene linker to improve the
polypharmacology of 11c because literature data19 suggested that subsititution at that position
could be tolerated. We found that substitution of the methylene linker in general led to
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significantly lower CXCR7 affinity than 11c (unpublished results). The exception was
acetamide 18 which was equipotent. The synthesis of 18 was initiated with a palladiumꢀ
catalyzed C−N crossꢀcoupling reaction between bromoimidazopyridine 13 with NꢀBoc
homopiperazine, which introduced the core in good yield (Scheme 4). Following Bocꢀ
deprotection, the advanced intermediate 14 was subjected to a Lewis acidꢀmediated threeꢀ
component Mannich reaction to furnish the racemic ester 15, which necessitated a chiral
separation to achieve the requisite enantiomeric excess. Of the two isolated isomers, only one
was taken forward because the undesired isomer led to an analog with lower affinity in
comparison to 18 (data not shown). Following deprotection of the piperidine, a HATUꢀmediated
amide bond formation with the optimal chiral bicyclic carboxylic acid (+)-9e was executed in
good yield. The pendant ester was hydrolyzed to the corresponding carboxylic acid with
potassium trimethylsilanolate, followed by a HATUꢀmediated amidation with NH4Cl to generate
the target tertiary βꢀamino amide 18 after chromatography. We were pleased to find that 18
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demonstrated potent CXCR7 binding affinity (Ki = 13 nM) and βꢀarrestin activity (EC50 = 11
nM). Compound 18 also exhibited improved selectivity in the GPCR panel and an improved
therapeutic index in the hERG patch clamp assay in comparison to 11c.
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Scheme 4. Synthesis of 18. Reaction conditions: (a) NꢀBoc homopiperazine, Pd2dba3, RuPhos,
o
NaOtꢀBu, 1,4ꢀdioxane, 95 C, 6 h, 69%; (b) 4N HCl in dioxane, MeOH, rt, 3 h then 4N NaOH
(aq), THF, rt, 16 h, 88%; (c) tꢀbutyl 4ꢀformylpiperidineꢀ1ꢀcarboxylate, rt, 3 h then tꢀbutyl((1ꢀ
methoxyvinyl)oxy) dimethylsilane, trimethylborate, DMSO, rt, 16 h, 62%; (d) chiral separation;
(e) 4N HCl in dioxane, MeOH, rt; 16 h (f) Carboxylic acid (+)-9e, Et3N, HATU, THF, rt, 4 h
(82% for 2 steps); (g) potassium trimethylsilanolate, THF, rt, 16 h (h) NH4Cl, iꢀPr2NEt, HATU,
DMF, rt, 6 h (40% for 2 steps).
The in vitro disposition characteristics of 18 are detailed in Table 4. Compound 18 exhibited
moderate to high in vitro turnover in both NADPHꢀsupplemented mouse liver microsomes
(MLM) (93 ꢀL/min/mg) and hepatocytes (28 ꢀL/min/million cells) and showed poor passive
absorptive permeability in the MadinꢀDarby canine kidney cells (MDCK II) permeability assay35
and good aqueous solubility. However, based on its propensity to modulate CXCR7 in vitro,
compound 18 was selected for a preliminary pharmacokinetic evaluation to ascertain its utility as
an in vivo tool to interrogate the effect of CXCR7 modulation in a mouse heart failure model.
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Table 4. Summary of In Vitro Disposition Properties of 18
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18
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9
HLM CLint [ꢀL/min/mg]29
60
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Human hepatocytes CLint [ꢀL/min/million cells]29
MLM CLint [ꢀL/min/mg]29
93
Mouse hepatocytes CLint [ꢀL/min/million cells]29
Mouse plasma fraction unbound36
28
0.34
1.1
P
app [10ꢀ6 cm/s]35
Solubility [ꢀM]
517
Assays were run using established protocols with references provided where appropriate.
In vivo studies
Compound 18 was not suitable for oral administration due to poor passive permeability
combined with high potential for hepatic firstꢀpass extraction in mice. This was underlined by a
single dose oral pharmacokinetic study at 10 mg/kg where systemic exposure was low and
variable (data not shown). Subcutaneous administration was selected as an alternative route,
since it circumvents intestinal and hepatic firstꢀpass extraction. Following subcutaneous
administration to male C57BL6 mice at 5 mg/kg, 18 was rapidly absorbed with mean maximal
plasma concentration (Cmax) of 682 ng/mL, which occurred at 0.25 hours (Tmax). The
corresponding mean area under the plasma concentration versus time profile (AUC) was 740
ng/mL×h. When corrected for mouse plasma unbound fraction (fu,p, mouse = 0.34), the mean
unbound Cmax and mean unbound average plasma concentration (Cavg) were 446 nM and 97 nM,
respectively, which offered favorable systemic coverage of the mouse CXCR7 Ki of 18 nM.
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Table 5: Pharmacokinetic Profile of 18 in Mice Following Subcutaneous Administrationa
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6
7
8
AUC(0ꢀ5)
9
Compound
Tmax [h]
0.25
t1/2 [h]
0.61
Cmax [ng/mL]
fu,p
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[ng/mL×h]
740
682
0.34
18
(0.25, 0.25) (0.64, 0.58)
(792, 572)
(803, 677)
aPharmacokinetic parameters were calculated from plasma concentrationꢀtime data in male
C57BL6 mice (n = 2/route) and are reported as the mean with individual values in parenthesis.
The 5 mg/kg subcutaneous dose was formulated as a solution in a vehicle of
DMSO/PEG400/water (10/30/60 v/v) and administered in a dose volume of 3 mL//kg.
For the acute pharmacology study, we selected a higher subcutaneous dose of 18 (30 mg/kg) to
maximize CXCR7 target engagement. Treatment of male C57BL6 mice (n = 9) resulted in a
statistically significant twoꢀfold increase in plasma SDFꢀ1α levels compared with vehicle at 30
minutes postꢀdose (Figure 2), thereby demonstrating that pharmacological modulation of
CXCR7 with compound 18 increases the bioavailability of SDFꢀ1α in vivo.
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Figure 2. Plasma SDFꢀ1α in C57BL6 mice at 30 minutes after a single dose administration of 18
at 30 mg/kg. Treatment with 18 resulted in a statistically significant increase in plasma SDFꢀ1α
compared to the vehicle (*p<0.05, Student tꢀtest)
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The effect of chronic dosing with compound 18 on cardiac fibrosis was evaluated in a mouse
model of isoproterenolꢀinduced cardiac injury. A total of 60 eightꢀweek old male BALB/c mice
were randomized into four study groups. In two groups, isoproterenol (5 mg/kg) was
administered subcutaneously once daily for 9 days to induce cardiac fibrosis. In addition, mice
in these two groups were further treated twice daily throughout the 9ꢀday study duration with
either compound 18 at 30 mg/kg (n = 20) or vehicle (n = 20). Mice receiving PBS and vehicle in
the third group served as controls (n = 15). The fourth group (n = 5) receiving both isoproterenol
and compound 18 were used for blood sampling at 1 and 9 hour postꢀdose on days 1, 3, 6 and 9
(1 hour only) to provide an overall estimate of CXCR7 coverage relative to mouse Ki. The
exposures of compound 18 achieved in Balb/c mice at the 30 mg/kg dose were approximately as
expected with unbound Cave > 95% target coverage.
As shown in Figure 3, administration of isoproterenol for 9 days led to the development of
cardiac fibrosis as attested by approximately fourꢀfold increased collagen deposition relative to
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control, detected by picrosirius red staining. Treatment with compound 18 resulted in a
statistically significant reduction in cardiac fibrosis, thereby demonstrating a protective role of
CXCR7 modulation with compound 18 in isoproterenolꢀinduced cardiac injury. Consistent with
the previous report that deletion of CXCR4 exacerbates isoproterenolꢀinduced cardiac
dysfunction,37 this study suggests that increased SDFꢀ1α bioavailability via pharmacological
modulation of CXCR7 and subsequent activation of the CXCR4 pathway may exert therapeutic
benefit in the setting of heart failure.
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Figure 3. Cardiac fibrosis assessed by histomorphometric analysis of picrosirius red staining in
the BALB/c mouse model of isoproterenolꢀinduced cardiac injury. Daily dosing with compound
18 for 9 consecutive days at 30 mg/kg resulted in a statistically significant reduction in cardiac
fibrosis compared to vehicle (*p<0.05, ANOVA)
CONCLUSION
We have described the identification of a suitable in vivo tool compound to interrogate the
effect of CXCR7 modulation on the SDFꢀ1α/CXCR4 axis. By replacing the methoxyquinoline
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head group with a 7ꢀethylꢀimidazopyridine moiety, we were able to eliminate the potential for
oxidative bioactivation risk while maintaining CXCR7 binding affinity and LipE. Incorporation
of a piperidine amide afforded analogues with LipE > 6 and improvements in metabolic
clearance. The amide side chain optimized the offꢀtarget profile, ultimately leading to compound
18. Acute treatment of 18 at 30 mg/kg resulted in a roughly twoꢀfold increase in SDFꢀ1α in
mice. In addition, chronic BID treatment of 18 (30 mg/kg) in an isoproterenolꢀinduced model of
cardiac injury showed a reduction of cardiac fibrosis in comparison to the vehicle group. Further
work to improve the passive permeability and metabolic stability within this series will be
required to identify orally available compounds suitable for clinical testing.
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EXPERIMENTAL SECTION
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Synthetic Procedures
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All tested compounds were determined to be ≥95% purity as determined by HPLC analysis
following compound purification.
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8-(1,4-Diazepan-1-yl)-7-methylimidazo[1,2-a]pyridine (4g): To a solution of 2g (1.50 g, 7.11
mmol) in PhCH3 (36 mL) was added Pd2(dba)3 (342 mg, 0.355 mmol) and RuPhos (339 mg,
0.711 mmol). The suspension was degassed and purged with nitrogen for 0.25 h followed by
addition of NaOtꢀBu (1.37 g, 14.2 mmol) and NꢀBoc homopiperazine 3 (1.42 g, 7.11 mmol) was
added to the suspension. The suspension was degassed and purged with nitrogen for an
o
additional 0.25 h. The mixture was heated to 95 C and was stirred for 6 h under nitrogen. The
reaction was cooled to rt and was then poured into saturated NH4Cl (aq). The mixture was
diluted with MTBE, the organic layer was removed, and the aqueous layer was extracted with
MTBE (x 3). The organic extracts were combined, washed with brine, dried over Na2SO4,
filtered and concentrated to give the crude reaction. The residue was purified by column
chromatography (heptanes/EtOAc) to give the intermediate NꢀBoc protected material as a
1
viscous oil (1.71 g, 5.16 mmol, 73%). H NMR (CDCl3): δ 7.84 (dd, J = 6.6, 3.5 Hz, 1H), 7.54
(s, 1H), 7.48 (s, 1H), 6.60 (dd, J = 6.6, 3.9 Hz, 1H), 3.68ꢀ3.55 (m, 4H), 3.49ꢀ3.37 (m, 4H), 2.40ꢀ
2.34 (m, 3H), 1.94ꢀ1.79 (m, 2H), 1.55ꢀ1.45 (m, 9H); m/z = 331.2 [M+1]+. To a solution of the
purified NꢀBoc protected material in DCM (100 mL) was added TFA (4 mL). The mixture was
stirred at rt for 5 h. At this time, the reaction mixture was concentrated to give the crude
1
trifluoroacetate salt of 4g as a viscous oil, which was used without further purification. H NMR
(MeOD): δ 8.54 (d, J = 7.0 Hz, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.33 (J =
6.6 Hz, 1H), 3.70ꢀ3.34 (m, 8H), 2.58 (s, 3H), 2.32ꢀ2.09 (m, 2H); m/z = 231.6 [M+1]+.
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Synthesis of 5a-5h.
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General Procedure A (Parallel Format; Scheme 1). Step 1: A suspension of heteroaryl
bromide (2) (1.0 equiv), RuPhos (20 mol %), Pd2(dba)3 (12 mol %), and NaOtꢀBu (1.0 equiv) in
1,4ꢀdioxane (0.2 M) was bubbled with nitrogen for 10 min prior to treatment with NꢀBoc
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homopiperazine (3; 1.0 equiv). The reaction was stirred under nitrogen at 95 C for 5 h. At this
time, the reaction was filtered through a short silica plug, evacuated, and was used without
purification. Step 2: The NꢀBocꢀprotected intermediate (1.0 equiv) was dissolved in DCM (0.1
M) and was treated with TFA (10 equiv). The reaction was stirred at rt for 1 h and was then
evacuated. The crude salt was dissolved in MeOH (0.1 M) and was treated with MPꢀCarbonate
resin followed by stirring for 30 min. The resin was filtered away, washed with MeOH, and all
solvent was evacuated. The crude free based amine 4 was used directly without further
purification. Step 3: To a solution of amine 4 (1.0 equiv) in DCE (0.1 M) was added 2ꢀ
phenylthiazoleꢀ4ꢀcarbaldehyde (1.2 equiv) followed by addition of NaBH(OAc)3 (3.0 equiv).
The reaction was heated to 50 oC for 16 h, followed by removal of solvent and purification of the
target 5 by preparative HPLC.
General Procedure B (Parallel Format; Scheme 1). Step 1: A solution of the appropriate
heteroaryl chloride (2) (1.0 equiv) in DMF (0.25 M) was treated with iꢀPr2NEt (1.5 equiv),
followed by NꢀBoc homopiperazine (3; 1.0 equiv). The reaction was stirred at 100 °C for 16 h
and then evaporated to the crude product, which was used directly in the next step. Step 2: The
Bocꢀprotected intermediate (1.0 equiv) was dissolved in DCM (0.1 M) and treated with TFA (10
equiv). The reaction was stirred at rt for 1 h and was then evacuated. The crude salt was
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dissolved in MeOH (0.1 M) and was treated with MPꢀCarbonate resin followed by stirring for 30
min. The resin was filtered away, washed with MeOH, and all solvent was evacuated. The crude
free based amine 4 was used directly without further purification. Step 3: To a solution of amine
4 (1.0 equiv) in DCE (0.1 M) was added 2ꢀphenylthiazoleꢀ4ꢀcarbaldehyde (1.2 equiv) followed
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by addition of NaBH(OAc)3 (3.0 equiv). The reaction was heated to 50 C for 16 h, followed by
removal of solvent and purification of the target 5 by preparative HPLC.
2-Phenyl-4-((4-(7-(trifluoromethoxy)quinolin-8-yl)-1,4-diazepan-1-yl)methyl)thiazole (5a).
Prepared according to general procedure A with 8ꢀbromoꢀ7ꢀ(trifluoromethoxy)quinoline (2a).
Purified by preparative HPLC to afford 5a (31 mg, 36%). HPLC (30−100%; 0.05% TFA in
water/0.05% TFA in MeCN): tR = 3.07 min. LCMS for C25H23F3N4OS: 484.5 (calcd); 485.2
[M+H]+ (observed).
4-((4-(6-Isopropyl-1,7-naphthyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5b).
Prepared according to general procedure B with 8ꢀchloroꢀ6ꢀisopropylꢀ1,7ꢀnaphthyridine (2b).
Purified by preparative HPLC (to afford 5b (25 mg, 12%). HPLC (5−100%; 0.05% TFA in
water/0.05% TFA in MeCN): tR = 2.24 min. LCMS for C26H29N5S: 443.6 (calcd); 444.2 [M+H]+
(observed).
4-((4-(1,7-Naphthyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5c). Prepared
according to general procedure B with 8ꢀchloroꢀ1,7ꢀnaphthyridine (2c). Purified by preparative
HPLC to afford 5c (5.9 mg, 9.7%). HPLC (35−100%; 0.03% NH4OH in water/0.03% NH4OH in
MeCN): tR = 1.88 min. LCMS for C23H23N5S: 401.5 (calcd); 402.2 [M+H]+ (observed).
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4-((4-(1-Methyl-1H-indazol-7-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5d). Prepared
according to general procedure A with 7ꢀbromoꢀ1ꢀmethylꢀ1Hꢀindazole (2d). Purified by
preparative HPLC to afford 5d (31 mg, 24%). HPLC (20−100%; 0.05% TFA in water/0.05%
TFA in MeCN): tR = 2.60 min. LCMS for C23H25N5S: 403.5 (calcd); 404.1 [M+H]+ (observed).
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4-((4-(7-Methoxyimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole
(5e). Prepared according to general procedure A with 8ꢀbromoꢀ7ꢀmethoxyimidazo[1,2ꢀa]pyridine
(2e). Purified by preparative HPLC to afford 5e (18 mg, 13%). HPLC (5−100%; 0.05% TFA in
water/0.05% TFA in MeCN): tR = 1.95 min. LCMS for C23H25N5OS: 419.5 (calcd); 420.2
[M+H]+ (observed).
2-Phenyl-4-((4-(7-(trifluoromethyl)imidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-
yl)methyl)thiazole (5f). Prepared according to general procedure A with 8ꢀbromoꢀ7ꢀ
(trifluoromethyl)imidazo[1,2ꢀa]pyridine (2f). Purified by preparative HPLC to afford 5f (18 mg,
39%). HPLC (5−100%; 0.05% TFA in water/0.05% TFA in MeCN): tR = 2.49 min. LCMS for
C23H22F3N5S: 457.5 (calcd); 458.2 [M+H]+ (observed).
4-((4-(7-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5g).
Prepared according to general procedure A with 8ꢀbromoꢀ7ꢀmethylimidazo[1,2ꢀa]pyridine (2g).
Purified by preparative HPLC to afford 5g (11 mg, 38%). HPLC (5−100%; 0.05% TFA in
water/0.05% TFA in MeCN): tR = 1.76 min. LCMS for C23H25N5S: 403.5 (calcd); 404.2 [M+H]+
(observed).
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4-((4-(6-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole
(5h). Prepared according to general procedure A with 8ꢀbromoꢀ6ꢀmethylimidazo[1,2ꢀa]pyridine
(2h). Purified by preparative HPLC to afford 5h (21 mg, 46%). HPLC (5−100%; 0.05% TFA in
water/0.05% TFA in MeCN): tR = 1.93 min. LCMS for C23H25N5S: 403.5 (calcd); 404.1 [M+H]+
(observed).
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t-Butyl 4-((4-(7-methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidine-1-
carboxylate (6): A mixture of amine 4g (4.0 g, 12 mmol), tꢀbutyl 4ꢀformylpiperidineꢀ1ꢀ
carboxylate (2.97 g, 13.9 mmol) and Et3N (2.35 g, 23.2 mmol) in DCE (40 mL) was stirred at rt
for 1 h. Then to the mixture was added NaBH(OAc)3 (6.16 g, 29.0 mmol) and continued stirring
at rt for 16 h. The reaction mixture was diluted with DCM (20 mL) and washed with water. The
organic layer was separated, dried over Na2SO4 and filtered. The filtrate was concentrated to
afford crude product, which was purified by column chromatography (MeOH:CH2Cl2; 0ꢀ10%) to
give the NꢀBoc protected 6 as a yellow oil (3.0 g, 7.0 mmol, 59%). 1H NMR (MeOD): δ 8.19 (d,
J = 6.8 Hz, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 4.19ꢀ
4.07 (m, 2H), 3.75ꢀ3.43 (m, 7H), 3.41ꢀ3.22 (m, 3H), 2.92ꢀ2.74 (m, 2H), 2.35 (s, 3H), 2.27ꢀ2.15
(m, 1H), 1.97ꢀ1.78 (m, 4H), 1.44 (s, 9H), 1.41ꢀ1.27 (m, 2H); m/z = 428.3 [M+1]+.
7-Methyl-8-(4-(piperidin-4-ylmethyl)-1,4-diazepan-1-yl)imidazo[1,2-a]pyridine (7): To a
solution of the purified NꢀBoc protected material in DCM (30 mL) was added TFA (2 mL). The
mixture was stirred at rt for 5 h. At this time, the reaction mixture was concentrated to give the
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crude trifluoroacetate salt of 7 as a viscous oil, which was used without further purification. H
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NMR (MeOD): δ 8.54 (d, J = 6.6 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.94 (br s, 1H), 7.33 (d, J =
7.0 Hz, 1H), 3.91ꢀ3.52 (m, 6H), 3.51ꢀ3.41 (m, 3H), 3.40ꢀ3.28 (m, 4H), 3.12ꢀ2.99 (m, 2H), 2.58
(s, 3H), 2.51ꢀ2.38 (m, 1H), 2.37ꢀ2.25 (m, 1H), 2.21ꢀ2.05 (m, 2H), 1.66ꢀ1.50 (m, 2H); m/z =
328.3 [M+1]+.
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7-Ethyl-8-(4-(piperidin-4-ylmethyl)-1,4-diazepan-1-yl)imidazo[1,2-a]pyridine (8).
Compound 8 was prepared as a yellow foam using the same procedures and reaction sequence
that was employed for the synthesis of 7, where 8ꢀbromoꢀ7ꢀethylimidazo[1,2ꢀa]pyridine was
used in place of 8ꢀbromoꢀ7ꢀmethylimidazo[1,2ꢀa]pyridine. 1H NMR (CDCl3): δ 7.88 (d, J = 6.9
Hz, 1H), 7.55 (d, J = 1.3 Hz, 1H), 7.48 (d, J = 1.3 Hz, 1H), 6.63 (d, J = 6.9 Hz, 1H), 3.48ꢀ3.33
(m, 4H), 3.25ꢀ3.15 (m, 2H), 2.96ꢀ2.77 (m, 6H), 2.74ꢀ2.62 (m, 2H), 2.50ꢀ2.41 (m, 2H), 1.99ꢀ1.82
(m, 4H), 1.71ꢀ1.55 (m, 1H), 1.33ꢀ1.15 (m, 5H); m/z = 341.9 [M+1]+.
Synthesis of 10a-10d and 11a-11c.
General Procedure C (Parallel Format; Scheme 3): To a solution of compound 7 or 8 (1.0
equiv) in DMF (0.25 M) was added the corresponding carboxylic acid (9; 1.2 equiv) and iꢀ
Pr2NEt (3.0 equiv), followed by HATU (1.5 equiv). The resulting mixture was stirred at rt for 16
h and was then evaporated to afford crude 10 or 11, which was purified by preparative HPLC.
Cyclopentyl(4-((4-(7-methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-
yl)methyl)piperidin-1-yl)methanone (10a). Prepared according to general procedure C with 7
and cyclopentanecarboxylic acid (9a). Purified by preparative HPLC to afford 10a (17 mg,
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40%). HPLC (5−95% MeOH (0.1% TFA)/water (0.1% TFA)): tR = 0.60 min. LCMS for
C25H37N5O: 423.6 (calcd); 424.4 [M+H]+ (observed).
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(4-((4-(7-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-
yl)(tetrahydrofuran-3-yl)methanone (10b). Prepared according to general procedure C with 7
and tetrahydrofuranꢀ3ꢀcarboxylic acid (9b). Purified by preparative HPLC to afford 10b (12 mg,
27%). HPLC (5−100% MeCN (0.04% TFA)/water (0.02% TFA)): tR = 1.94 min. LCMS for
C24H35N5O2: 425.6 (calcd); m/z = 426.6 [M+H]+ (observed).
((Rac-exo)-7-oxabicyclo[2.2.1]heptan-2-yl)(4-((4-(7-methylimidazo[1,2-a]pyridin-8-yl)-1,4-
diazepan-1-yl)methyl)piperidin-1-yl)methanone (10c). Prepared according to general
procedure C with 7 and (rac-exo)ꢀ7ꢀoxabicyclo[2.2.1]heptaneꢀ2ꢀcarboxylic acid (rac-9c).
Purified by preparative HPLC to afford 10c (6.9 mg, 15%). HPLC (5−100% MeCN/water
(0.05% NH4OH)): tR = 1.90 min. LCMS for C26H37N5O2: 451.6 (calcd); 452.3 [M+H]+
(observed).
((Rac-endo)-7-oxabicyclo[2.2.1]heptan-2-yl)(4-((4-(7-methylimidazo[1,2-a]pyridin-8-yl)-1,4-
diazepan-1-yl)methyl)piperidin-1-yl)methanone (10d). Prepared according to general
procedure C with 7 and (rac-endo)ꢀ7ꢀoxabicyclo[2.2.1]heptaneꢀ2ꢀcarboxylic acid (rac-9d).
Purified by preparative HPLC to afford 10d (11 mg, 26%). HPLC (5−100% MeCN/water
(0.05% NH4OH)): tR = 2.91 min. LCMS for C26H37N5O2: 451.6 (calcd); 452.4 [M+H]+
(observed).
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