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Apr 8, 2018 - ABSTRACT: C−X−C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a ...
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Article Cite This: J. Med. Chem. 2018, 61, 3685−3696

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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 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

‡

S Supporting Information *

ABSTRACT: 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−activityrelationship (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 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic β 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cellderived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenolinduced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

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INTRODUCTION C−X−C chemokine receptor type 7 (CXCR7), also known as atypical chemokine receptor 3 (ACKR3), is a seven-transmembrane-domain G-protein-coupled receptor (GPCR) expressed in monocytes, endothelial cells, B cells, and cardiomyocytes. Two ligands have been identified: stromalcell-derived factor 1α (SDF-1α), also known as C−X−C chemokine ligand 12 (CXCL12), and interferon-inducible Tcell α chemoattractant (I-TAC), also known as C−X−C chemokine ligand 11 (CXCL11). Unlike most chemokine receptors, CXCR7 does not participate in any G-protein signaling cascades. Rather, ligand binding induces the recruitment of β-arrestin and receptor internalization.1 CXCR7 is reported to be a “scavenger” receptor for SDF-1α, which is an 8 kDa, 89 amino acid peptide that also binds to C− X−C chemokine receptor type 4 (CXCR4).2,3 The scavenger activity of CXCR7 is thought to control the local levels of SDF1α, thereby modulating the activation state of CXCR4. Activation of CXCR4 by SDF-1α leads to G-protein signaling cascades involved in cardiomyocyte survival and angiogenesis. As such, SDF-1α has been reported to play a protective role after cardiac injuries, promoting repair processes, reducing fibrosis, and improving cardiac function.4 © 2018 American Chemical Society

Although SDF-1α gene therapy is being explored in the clinic as a treatment for patients with a prior myocardial infarctions (MI),5 we hereby propose a small-molecule modulator of CXCR7 as a novel approach to block its SDF-1α-scavenging activity, increase local SDF-1α concentrations, and promote the activation of CXCR4 to improve cardiac function in the setting of ischemic heart disease. Peptide GPCRs have been noted as being challenging drugdiscovery targets. Optimized ligands for peptide GPCRs often suffer from poor physiochemical properties, including low druglike properties (i.e., median MW = 518 and cLogP = 5).6 In addition, small-molecule chemical matter targeting chemokine receptors is often basic and lipophilic7 with undesired polypharmacology, making it challenging to identify suitable in vivo tools. A number of CXCR7 small-molecule8−22 and peptidic23−25 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 1,4-diazepine series with nanomolar affinities for CXCR7 and improved Received: February 5, 2018 Published: April 8, 2018 3685

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

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Scheme 1. Parallel Synthesis of the 1,4-Diazepanes 5a−5ha

physicochemical properties and off-target selectivities for use in proof-of-mechanism studies in a rodent model of heart failure.

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RESULTS AND DISCUSSION Synthesis and In Vitro Pharmacology. Estimates of binding affinities (Ki) for CXCR7 were determined by a radioligand competition-binding assay utilizing 125I-SDF-1α and membrane preparations from cells overexpressing the human25 or mouse CXCR7 receptor. As CXCR7 is not known to signal via G-proteins, we evaluated the functional effects of our compounds on the receptor using a β-arrestin recruitment assay from DiscoverX.25 The potency value reported for this assay is an EC50 or the concentration of the compound required to reach 50% of the maximum effect. Interestingly, all the compounds that bound to the receptor in a competitive manner with the native SDF-1α ligand also promoted the recruitment of β-arrestin. To ensure selectivity for CXCR7 over CXCR4, which shares the SDF-1α ligand, binding to the CXCR4 receptor was also monitored using a whole cell CXCR4 125I-SDF-1α competition-binding assay with Jurkat cells, an immortalized line of human T lymphocyte cells, which endogenously express the CXCR4 receptor. Under the present experimental conditions, the previously reported19 1,4-diazepane-based CXCR7 ligand, 1 (Figure 1),

a

Reaction conditions for the heteroaryl bromides: (a) Pd2dba3, RuPhos, NaOt-Bu, 1,4-dioxane, 95 °C. Reaction conditions for the heteroaryl chlorides: (b) i-Pr2NEt, DMF, 100 °C; (c) TFA, DCM, rt; (d) 2-phenylthiazole-4-carbaldehyde, NaBH(OAc)3, DCE, 50 °C.

desired N-aryl bond (4a−4h). Intermediates 4a−4h were used without further purification, wherein reductive amination with 2-phenylthiazole-4-carbaldehyde provided the substituted 1,4diazepanes, 5a−5h. Analogues 5a−5h were evaluated in the CXCR7-binding assay to understand the effects of the 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-headgroup replacements to pose a bigger challenge. For example, the naphthyridine analogues 5b (Ki = 730 nM) and 5c (Ki = 1900 nM) were considerably less potent, and the indazole 5d (Ki = 18 000 nM) virtually lost its binding affinity for CXCR7. The imidazopyridines (5e−5h), on the other hand, were found to be suitable replacements for the quinoline. Thus, 7-methoxy-imidazopyridine, 5e, demonstrated a good CXCR7binding affinity (Ki = 52 nM) with a high LipE. On the basis of the LipE value of 5e, we also made the 7-trifluoromethyl- and 7-methyl-imidazopyridine analogues (5f and 5g, respectively), both of which showed submicromolar potencies for CXCR7. In particular, 5g exhibited a good binding affinity (Ki = 29 nM) and a LipE of 4.8. In contrast, the corresponding 6-methylimidazopyridine isomer, 5h, exhibited an approximately 30-fold loss of binding affinity (Ki = 900 nM) in comparison with that of 5g. In addition to the CXCR7-binding-affinity measurements, we evaluated the HLM stabilities of these compounds and found that the majority of the analogues demonstrated high intrinsic clearances (CLint). Imidazopyridine 5e demonstrated the lowest metabolic clearance (CLint = 67 μL/min/mg) and was also the least lipophilic compound (sF log D = 1.7) of the analogues in Table 1. We hypothesized that lowering the lipophilicity (sF log D < 2) within this series would improve the overall metabolic clearance30 and also address the potential off-target pharmacology and affinity against the hERG channel, which are often associated with lipophilic and basic compounds.31 In order to lower the lipophilicity across the 1,4-diazepine series, we sought to replace the 2-phenylthiazole substituent with a saturated ring system, specifically a piperidine ring.32 Besides lowering the lipophilicities of the 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

Figure 1. Representative CXCR7 modulator.

revealed a high binding affinity for CXCR7 (Ki = 9 nM). However, 1 is lipophilic (shake flask (sF)log D26 = 3.4) and demonstrates inhibitory potency (Ki < 13 nM) in the hERGmembrane-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 1,4-diazepane 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 by the potential oxidative bioactivation of the 1,4diazepane-7-methoxyquinoline framework to a potentially electrophilic quinone-imine species.27 A number of analogues (5a−5h) of CXCR7 modulator 1 were synthesized in a parallel medicinal-chemistry fashion through robust C−N-bond formation and a reductive-amination synthetic sequence (Scheme 1). We focused our efforts on head groups (quinoline replacements) that either removed the methoxy substituent (i.e., 2a and 2b) or replaced the pyridine motif with different heterobicyclic systems (i.e., 2e). Heteroaryl bromides and chlorides (2a−2h) were treated with N-Boc homopiperazine (3), which was followed by N-Boc deprotection to form the 3686

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

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Table 1. In Vitro CXCR7-Binding Affinities, Physicochemical Properties, and Metabolic Intrinsic Clearances (CLint) of the 1,4Diazepinesa

a The assays were run using established protocols. References are provided where appropriate. bThe values were determined by a radioligand-binding assay and are reported as the geometric means of at least three independent experiments with the pKi (negative logarithm) ± SD in parentheses. c LipE, a measure of lipophilic efficiency, is the difference between pKi and sF log D. dThe HLM CLint values were generated following an incubation of the test compound (1 μM) in NADPH (1.3 mM)-supplemented HLM (protein concentration of 0.25 mg/mL) at 37 °C for 30 min.

CXCR7-binding affinity (Ki > 8500 nM) relative to that of 10a. Although the replacement of the cyclopentyl group in 10a with a tetrahydrofuran functionality (10b) led to a significant loss of CXCR7 potency (Ki > 9700 nM), the corresponding analogue demonstrated a marked improvement in its CLint in HLM (20 μL/min/mg), likely associated with its lower sF log D. On the basis of this observation, we decided to focus our attention on compounds with sF log D ∼ 1 in order to maintain low metabolic turnover, while improving upon the CXCR7-binding potency. Incorporation of exo-[2.2.1]-oxabicycle provided 10c with a favorable CXCR7 affinity (Ki = 520 nM) and an excellent LipE. The corresponding endo-[2.2.1]-oxabicycle, 10d, however, was 26-fold less potent (Ki = 14 000 nM) than 10c. We found that the homologation of the 7-methyl to a 7-ethyl group provided an 11-fold boost in potency for 11a, which was initially made as a racemic mixture. Synthesis of the enantiopure analogues identified 11c as the eutomer with a CXCR7 Ki of 13 nM, a LipE of 7.1, and importantly, a reduced CLint in HLM (46 μL/min/mg). In addition to its potent modulation of human CXCR7, we observed that 11c was active against the mouse form of the

selected 7-methyl and 7-ethyl imidazopyridine groups for further SAR evaluations because we found that the 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 C−N-bond formation is followed by reductive amination with t-butyl 4-formylpiperidine-1-carboxylate. Scheme 3 depicts the general parallel medicinal-chemistry strategy in which piperidines 7 and 8 were converted to the corresponding amides. The experimental data for a subset of the amides synthesized in the parallel medicinal fashion using commercially available carboxylic acids are shown in Table 2. The cyclopentyl piperidine amide, 10a, had a modest CXCR7-binding affinity (Ki = 950 nM) but was over one logarithm unit lower in lipophilicity compared with 5g (sF log D = 1.7 vs 2.8, respectively). Even though 10a had reduced CXCR7 affinity, the change from the 2-phenylthiazole to the piperidine amide was only one-half of a logarithm unit lower with regard to the LipE (4.3 vs 4.8, respectively). In contrast, the regioisomeric cyclopentyl piperidine amide, 12 (see the Experimental Section for the synthesis of 12), had a poor 3687

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Table 2. In Vitro CXCR7-Binding Affinities, Physicochemical Properties, and Metabolic Intrinsic Clearances (CLint) of the Piperidine Amidesa

a The assays were run using established protocols. References are provided where appropriate. bThe values were determined by a radioligand-binding assay and are reported as the geometric means of at least three independent experiments with the pKi ± SD in parentheses. cLipE, a measure of lipophilic efficiency, is the difference between pKi and sF log D dThe HLM CLint values were generated following an incubation of the test compound (1 μM) in NADPH (1.3 mM)-supplemented HLM (protein concentration of 0.25 mg/mL) at 37 °C for 30 min.

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 the data in the literature19 suggested that a substitution at that position could be tolerated. We found that substitution of the methylene linker in general led to CXCR7 affinities significantly lower than that of 11c (unpublished results). The exception was the acetamide, 18, which was equipotent. The synthesis of 18 was initiated with a palladium-catalyzed C−N-

receptor, which was not surprising considering the high species homology33 but was nonetheless important for use in a mouse model of cardiac fibrosis (Table 3). 11c was also found to have functional activity and to recruit β-arrestin, and it did not display binding to the related chemokine receptor CXCR4. However, further in vitro characterization of 11c revealed significant off-target 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 3688

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Table 3. In Vitro Pharmacology of 11c and 18a

Scheme 2. Representative Synthesis of a Piperidine Template in Support of the Parallel-Synthesis Effortsa

human CXCR7 Ki [nM]25,b β-arrestin EC50 [nM]25,c mouse CXCR7 Ki [nM]b human CXCR4 Ki [nM]b hERG patch-clamp IC50 [nM]d adrenergic α 1a Kb [nM]e adrenergic β 2 Kb [nM]e histamine 1 Kb [nM]e muscarinic 1 Kb [nM]e

11c

18

13 7 51 >6200 1300 520 2200 330 59

13 11 18 11 000 8300 >10 000 >10 000 >4400 >10 000

a

The assays were run using established protocols. References are provided where appropriate. bThe values were determined by radioligand-binding assays and are reported as the geometric means of at least three independent experiments. cThe values were determined by a β-arrestin-recruitment assay and are reported as the geometric means of at least two independent experiments. dMeasurements of potassium currents in human-embryonic-kidney 293 (HEK293) cells stably transfected with the hERG channel. eThe Kb values are the dissociation constants of the antagonists for the receptor and are reported as the geometric means of at least two independent experiments.

Reaction conditions: (a) Pd2dba3, RuPhos, NaOt-Bu, PhCH3, 95 °C, 6 h, 73%; (b) TFA, DCM, rt, 5 h; (c) t-butyl 4-formylpiperidine-1carboxylate, DCE, rt, 1 h, then NaBH(OAc)3, rt, 16 h, 59%.

a

cross-coupling reaction between bromoimidazopyridine, 13, with N-Boc homopiperazine, which introduced the core in good yield (Scheme 4). Following Boc-deprotection, the advanced intermediate, 14, was subjected to a Lewis acid mediated three-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 analogue with a lower affinity in comparison with that of 18 (data not shown). Following the deprotection of the piperidine, HATU-mediated 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, which was followed by HATU-mediated amidation with NH4Cl to generate the target tertiary β-amino amide, 18, after chromatography was performed. We were pleased to find that 18 demonstrated potent CXCR7-binding affinity (Ki = 13 nM) and β-arrestin 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 with 11c. The in vitro disposition characteristics of 18 are detailed in Table 4. Compound 18 exhibited moderate to high in vitro turnover in both NADPH-supplemented mouse-liver microsomes (MLM, 93 μL/min/mg) and hepatocytes (28 μL/min per million cells), showed poor passive absorptive permeability in the Madin−Darby-canine-kidney-cells (MDCK II)-permeability assay,35 and had good aqueous solubility. However, on the basis of 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 effects of CXCR7 modulation in a mouse heart-failure model. In Vivo Studies. Compound 18 was not suitable for oral administration because of its poor passive permeability combined with its high potential for hepatic first-pass extraction in mice. This was underlined by a single-dose oral pharmacokinetic study at 10 mg/kg in which the systemic exposure was low and variable (data not shown). Subcutaneous administration was selected as an alternative route, because it circumvents intestinal and hepatic first-pass extraction. Following subcutaneous administration to male C57BL6 mice at 5 mg/kg, 18 was rapidly absorbed with a mean maximal plasma concentration (Cmax) of 682 ng/mL, which occurred at 0.25 h (Tmax, Table 5). The corresponding mean area under the plasma-concentration-versus-time profile (AUC) was 740 ng/ mL·h. When corrected for the mouse-plasma unbound fraction ( f u,p,mouse = 0.34), the mean unbound Cmax and mean unbound average plasma concentration (Cave) were 446 and 97 nM, respectively, which offered favorable systemic coverages of mouse CXCR7 (Ki = 18 nM). 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)

Scheme 3. Parallel Synthesis of the Piperidine Amides

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Scheme 4. Synthesis of 18a

a Reaction conditions: (a) N-Boc homopiperazine, Pd2dba3, RuPhos, NaOt-Bu, 1,4-dioxane, 95 °C, 6 h, 69%; (b) 4 N HCl in dioxane, MeOH, rt, 3 h, then 4 N NaOH(aq), THF, rt, 16 h, 88%; (c) t-butyl 4-formylpiperidine-1-carboxylate, rt, 3 h, then t-butyl((1-methoxyvinyl)oxy) dimethylsilane, trimethylborate, DMSO, rt, 16 h, 62%; (d) chiral separation; (e) 4 N HCl in dioxane, MeOH, rt, 16 h; (f) carboxylic acid, (+)-9e, Et3N, HATU, THF, rt, 4 h (82% for two steps); (g) potassium trimethylsilanolate, THF, rt, 16 h; (h) NH4Cl, i-Pr2NEt, HATU, DMF, rt, 6 h (40% for two steps).

Table 4. Summary of the In Vitro Disposition Properties of 18a compd 18 HLM CLint [μL/min/mg]29 human hepatocytes CLint [μL/min per million cells]29 MLM CLint [μL/min/mg]29 mouse hepatocytes CLint [μL/min per million cells]29 mouse plasma fraction, unbound36 Papp [10−6 cm/s]35 solubility [μM]

60 6 93 28 0.34 1.1 517

a

The assays were run using established protocols. References are provided where appropriate.

Figure 2. Plasma SDF-1α levels in C57BL6 mice at 30 min after the single-dose administration of 18 at 30 mg/kg. Treatment with 18 resulted in a statistically significant increase in plasma SDF-1α levels compared with the vehicle (*p < 0.05, Student t test).

Table 5. Pharmacokinetic Profile of 18 in Mice Following Subcutaneous Administrationa compd

Tmax [h]

t1/2 [h]

Cmax [ng/mL]

18

0.25 (0.25, 0.25)

0.61 (0.64, 0.58)

682 (792, 572)

AUC(0−5) [ng/mL·h]

f u,p

740 (803, 677)

0.34

thus served as controls (n = 15). The fourth group (n = 5), which received both isoproterenol and compound 18, were used for blood sampling at 1 and 9 h postdose on days 1, 3, 6, and 9 (1 h only) to provide an overall estimate of CXCR7 coverage relative to mouse Ki. The exposures to compound 18 achieved in the BALB/c mice at the 30 mg/kg dose were approximately as expected, with the unbound Cave > 95% target coverage. As shown in Figure 3, the administration of isoproterenol for 9 days led to the development of cardiac fibrosis, as attested by the approximately 4-fold increase in collagen deposition relative to that in the control, which was detected by picrosirius-red staining. Treatment with compound 18 resulted in a statistically significant reduction in cardiac fibrosis, thereby demonstrating the protective role of CXCR7 modulation with compound 18 in an isoproterenol-induced cardiac injury. Consistent with the previous report that the deletion of CXCR4 exacerbates isoproterenol-induced cardiac dysfunction,37 this study suggests that increased SDF-1α bioavailability via the pharmacological modulation of CXCR7 and subsequent activation of the CXCR4 pathway may exert therapeutic benefits in the setting of heart failure.

a

The pharmacokinetic parameters were calculated from the plasmaconcentration−time data in male C57BL6 mice (n = 2/route) and are reported as the means with the individual values in parentheses. 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.

resulted in a statistically significant 2-fold increase in plasma SDF-1α levels at 30 min postdose compared with when only the vehicle was given (Figure 2), thereby demonstrating that the pharmacological modulation of CXCR7 with compound 18 increases the bioavailability of SDF-1α in vivo. The effect of chronic dosing with compound 18 on cardiac fibrosis was evaluated in a mouse model of an isoproterenolinduced cardiac injury. A total of 60 male BALB/c mice (8 weeks of age) 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, the mice in these two groups were further treated twice daily throughout the 9 day study duration with either compound 18 at 30 mg/kg (n = 20) or the vehicle (n = 20). The mice in the third group received PBS and the vehicle and 3690

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used without further purification. 1H 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), 3.70−3.34 (m, 8H), 2.58 (s, 3H), 2.32−2.09 (m, 2H); m/z = 231.6 [M + 1]+. Synthesis of 5a−5h. 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 NaOt-Bu (1.0 equiv) in 1,4-dioxane (0.2 M) was bubbled with nitrogen for 10 min prior to treatment with N-Boc 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 used without purification. Step 2: The N-Boc-protected 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 then evacuated. The crude salt was dissolved in MeOH (0.1 M), treated with an MP-carbonate resin, and then stirred for 30 min. The resin was filtered away and washed with MeOH, and all of the solvent was evacuated. The crude freebased 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 2phenylthiazole-4-carbaldehyde (1.2 equiv); this was followed by the addition of NaBH(OAc)3 (3.0 equiv). The reaction was heated to 50 °C for 16 h; the solvent was removed; and the target, 5, was purified 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 i-Pr2NEt (1.5 equiv), followed by N-Boc 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 Boc-protected 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 then evacuated. The crude salt was dissolved in MeOH (0.1 M), treated with an MPcarbonate resin, and then stirred for 30 min. The resin was filtered away and washed with MeOH, and all of the 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 2-phenylthiazole-4-carbaldehyde (1.2 equiv); this was followed by the addition of NaBH(OAc)3 (3.0 equiv). The reaction was heated to 50 °C for 16 h; the solvent was removed; and the target, 5, was purified by preparative HPLC. 2-Phenyl-4-((4-(7-(trifluoromethoxy)quinolin-8-yl)-1,4-diazepan1-yl)methyl)thiazole (5a). The compound was prepared according to general procedure A with 8-bromo-7-(trifluoromethoxy)quinoline (2a). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(6-Isopropyl-1,7-naphthyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5b). The compound was prepared according to general procedure B with 8-chloro-6-isopropyl-1,7naphthyridine (2b). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(1,7-Naphthyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5c). The compound was prepared according to general procedure B with 8-chloro-1,7-naphthyridine (2c). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(1-Methyl-1H-indazol-7-yl)-1,4-diazepan-1-yl)methyl)-2phenylthiazole (5d). The compound was prepared according to general procedure A with 7-bromo-1-methyl-1H-indazole (2d). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(7-Methoxyimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5e). The compound was prepared according to general procedure A with 8-bromo-7-methoxyimidazo[1,2-a]pyridine (2e). Purification by preparative HPLC afforded 5e

Figure 3. Cardiac fibrosis assessed by the histomorphometric analysis of picrosirius-red staining in the BALB/c mouse model of isoproterenol-induced 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 with daily dosing with the vehicle (*p < 0.05, ANOVA).

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CONCLUSION We have described the identification of a suitable in vivo tool compound for interrogating the effect of CXCR7 modulation on the SDF-1α−CXCR4 axis. By replacing the methoxyquinoline headgroup with a 7-ethyl-imidazopyridine moiety, we were able to eliminate the potential for the oxidative-bioactivation risk while maintaining the 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 off-target profile, ultimately leading to compound 18. Acute treatment of 18 at 30 mg/kg resulted in a roughly 2-fold increase in SDF-1α in mice. In addition, chronic BID treatment of 18 (30 mg/kg) in an isoproterenol-induced model of cardiac injury showed a reduction of cardiac fibrosis in comparison with that in 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

Synthetic Procedures. All the tested compounds were determined to have ≥95% purities as determined by HPLC analysis following compound purification. 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, which was followed by the addition of NaOt-Bu (1.37 g, 14.2 mmol), and then N-Boc homopiperazine, 3 (1.42 g, 7.11 mmol), was added to the suspension. The suspension was degassed and purged with nitrogen for an additional 0.25 h. The mixture was heated to 95 °C and stirred for 6 h under nitrogen. The reaction was cooled to rt and 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 (×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 N-Bocprotected material as a viscous oil (1.71 g, 5.16 mmol, 73%). 1H 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), 3.68−3.55 (m, 4H), 3.49−3.37 (m, 4H), 2.40−2.34 (m, 3H), 1.94−1.79 (m, 2H), 1.55−1.45 (m, 9H); m/ z = 331.2 [M + 1]+. To a solution of the purified, N-Boc-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 trifluoroacetate salt of 4g as a viscous oil, which was 3691

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

Journal of Medicinal Chemistry

Article

(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]+ (obsd). 2-Phenyl-4-((4-(7-(trifluoromethyl)imidazo[1,2-a]pyridin-8-yl)1,4-diazepan-1-yl)methyl)thiazole (5f). The compound was prepared according to general procedure A with 8-bromo-7-(trifluoromethyl)imidazo[1,2-a]pyridine (2f). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(7-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5g). The compound was prepared according to general procedure A with 8-bromo-7-methylimidazo[1,2-a]pyridine (2g). Purification by preparative HPLC afforded 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]+ (obsd). 4-((4-(6-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)-2-phenylthiazole (5h). The compound was prepared according to general procedure A with 8-bromo-6-methylimidazo[1,2-a]pyridine (2h). Purification by preparative HPLC afforded 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]+ (obsd). t-Butyl 4-((4-(7-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan1-yl)methyl)piperidine-1-carboxylate (6). A mixture of amine 4g (4.0 g, 12 mmol), t-butyl 4-formylpiperidine-1-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 the mixture was stirred 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 the crude product, which was purified by column chromatography (MeOH/CH2Cl2, 0− 10%) to give N-Boc-protected 6 as a yellow oil (3.0 g, 7.0 mmol, 59%). 1 H 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), 4.19−4.07 (m, 2H), 3.75−3.43 (m, 7H), 3.41−3.22 (m, 3H), 2.92−2.74 (m, 2H), 2.35 (s, 3H), 2.27−2.15 (m, 1H), 1.97−1.78 (m, 4H), 1.44 (s, 9H), 1.41−1.27 (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 N-Boc-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 crude trifluoroacetate salt of 7 as a viscous oil, which was used without further purification. 1H 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), 3.91−3.52 (m, 6H), 3.51−3.41 (m, 3H), 3.40−3.28 (m, 4H), 3.12−2.99 (m, 2H), 2.58 (s, 3H), 2.51−2.38 (m, 1H), 2.37−2.25 (m, 1H), 2.21−2.05 (m, 2H), 1.66−1.50 (m, 2H); m/z = 328.3 [M + 1]+. 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 were employed for the synthesis of 7, with 8-bromo-7-ethylimidazo[1,2-a]pyridine used in place of 8-bromo-7-methylimidazo[1,2-a]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), 3.48−3.33 (m, 4H), 3.25−3.15 (m, 2H), 2.96−2.77 (m, 6H), 2.74−2.62 (m, 2H), 2.50−2.41 (m, 2H), 1.99−1.82 (m, 4H), 1.71−1.55 (m, 1H), 1.33−1.15 (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 i-Pr2NEt (3.0 equiv), followed by HATU (1.5 equiv). The resulting mixture was stirred at rt for 16 h and 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). The compound was prepared according to general procedure C with 7 and cyclopentanecarboxylic acid (9a). Purification by preparative HPLC

afforded 10a (17 mg, 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]+ (obsd). (4-((4-(7-Methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)(tetrahydrofuran-3-yl)methanone (10b). The compound was prepared according to general procedure C with 7 and tetrahydrofuran-3-carboxylic acid (9b). Purification by preparative HPLC afforded 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]+ (obsd). ((rac-exo)-7-Oxabicyclo[2.2.1]heptan-2-yl)(4-((4-(7methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)methanone (10c). The compound was prepared according to general procedure C with 7 and (rac-exo)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (rac-9c). Purification by preparative HPLC afforded 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]+ (obsd). ((rac-endo)-7-Oxabicyclo[2.2. 1]heptan-2-yl)(4-((4-(7methylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)methanone (10d). The compound was prepared according to general procedure C with 7 and (rac-endo)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (rac-9d). Purification by preparative HPLC afforded 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]+ (obsd). (rac-exo)-7-Oxabicyclo[2.2.1]heptan-2-yl)(4-((4-(7-ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)methanone (11a). The compound was prepared according to general procedure C with 8 and (rac-exo)-7-oxabicyclo[2.2.1]heptane-2carboxylic acid (rac-9c). Purification by preparative HPLC afforded 11a (6.8 mg, 25%). HPLC (5−100%, 0.03% NH4OH in water/0.03% NH4OH in MeCN): tR = 1.03 min. LCMS for C27H39N5O2: 465.6 (calcd), 466.3 [M + H]+ (obsd). (( 1S,2 R,4R) -7 -O xabi cycl o[2 .2 . 1]hept an-2 -yl )(4 -( (4-(7 ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)methanone (11b). To a solution of compound 8 (0.20 g, 1.41 mmol) in DMF (5 mL) was added (1S,2R,4R)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (9f) (0.48 g, 1.41 mmol) and Et3N (0.59 mL, 4.23 mmol), followed by HATU (0.80 g, 2.12 mmol). The resulting mixture was stirred at rt for 16 h. The reaction mixture was diluted with additional DCM and washed with saturated NH4Cl(aq) and brine. The combined organic extracts were dried over Na2SO4, filtered, and evaporated to give the crude product, which was purified by column chromatography (0−20% MeOH in DCM) to give the desired product, 11b, as a white foam (0.35 g, 0.75 mmol, 54%). 1H NMR (CDCl3): δ 7.86 (d, J = 6.5 Hz, 1H), 7.51 (s, 1H), 7.46 (s, 1H), 6.60 (d, J = 7.0 Hz, 1H), 4.83−4.72 (m, 1H), 4.67−4.53 (m, 2H), 3.88−3.77 (m, 1H), 3.51−3.30 (m, 4H), 3.04−2.73 (m, 7H), 2.70− 2.64 (m, 1H), 2.60−2.33 (m, 3H), 2.15−2.04 (m, 1H), 1.98−1.85 (m, 3H), 1.81−1.65 (m, 5H), 1.52−1.42 (m, 2H), 1.19 (t, J = 7.6 Hz, 3H), 1.14−0.97 (m, 2H); LCMS for C27H39N5O2: 465.6 (calcd), 466.4 [M + H]+ (obsd). (( 1R, 2R ,4 S) -7 -O x abi c y cl o[2 .2 . 1]h e pt an-2 -yl )(4 -( (4- (7 ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)methyl)piperidin-1-yl)methanone (11c). To a solution of compound 8 (0.11 g, 0.77 mmol) in DMF (3 mL) was added (1R,2R,4S)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (9e) (0.26 g, 0.77 mmol) and Et3N (0.32 mL, 2.3 mmol), followed by HATU (0.44 g, 1.16 mmol). The resulting mixture was stirred at rt for 16 h. The reaction mixture was diluted with additional DCM and washed with saturated NH4Cl(aq) and brine. The combined organic extracts were dried over Na2SO4, filtered, and evaporated to give the crude product, which was purified by column chromatography (0−20% MeOH in DCM) to give the desired product, 11c, as a white foam (0.31 g, 0.54 mmol, 87%). 1H NMR (CDCl3): δ 7.87 (d, J = 7.0 Hz, 1H), 7.52 (s, 1H), 7.46 (s, 1H), 6.61 (d, J = 7.0 Hz, 1H), 4.84−4.75 (m, 1H), 4.67−4.54 (m, 2H), 3.89−3.77 (m, 1H), 3.48−3.31 (m, 4H), 3.04−2.72 (m, 7H), 2.71− 2.63 (m, 1H), 2.61−2.30 (m, 3H), 2.14−2.06 (m, 1H), 1.97−1.87 (m, 3H), 1.81−1.64 (m, 5H), 1.53−1.44 (m, 2H), 1.20 (t, J = 7.6 Hz, 3H), 3692

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

Journal of Medicinal Chemistry

Article

2H), 3.04−2.97 (m, 2H), 2.88 (q, J = 7.4 Hz, 2H), 1.98−1.89 (m, 2H), 1.25 (t, J = 7.4 Hz, 3H); m/z = 245.3 [M + 1]+. t-Butyl 4-(1-(4-(7-Ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan1-yl)-3-methoxy-3-oxopropyl)piperidine-1-carboxylate (15). To the free-based homopiperazine, 14 (13.2 g, 54 mmol), in DMSO (90 mL) was added a DMSO (90 mL) solution of t-butyl 4-formylpiperidine-1carboxylate (13.3 g, 59 mmol). The solution remained a brownish red and homogeneous. Trimethyl borate (12 mL, 108 mmol) was added dropwise to the homogeneous reaction, which was stirred at rt for 3 h. At this time, neat silyl ketene acetal (24 mL, 108 mmol) was added dropwise. The reaction was stirred at rt for 16 h. The reaction mixture was diluted with EtOAc and treated with brine. The aqueous layer was removed, and the organics were washed with additional brine (×3). The aqueous layers were combined and extracted with EtOAc (×3). All of the organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude material was purified using column chromatography, eluting with a gradient of 0− 100% EtOAc/heptane to give the racemic ester, 15 (20.8 g, 40.4 mmol, 62%). 1H NMR (MeOD): δ 8.15 (d, J = 7.0 Hz, 1H), 7.71 (s, 1H), 7.45 (s, 1H), 6.77 (d, J = 6.5 Hz, 1H), 4.14−4.03 (m, 2H), 3.71 (s, 3H), 3.38−3.22 (m, 6H), 3.04−2.82 (m, 7H), 2.81−2.66 (m, 2H), 2.61 (dd, J = 15.3, 7.0 Hz, 1H), 2.46 (dd, J = 15.0, 5.6 Hz, 1H), 2.21− 2.14 (m, 1H), 1.93−1.80 (m, 2H), 1.73−1.59 (m, 2H), 1.46 (s, 9H), 1.23 (t, J = 7.6 Hz, 3H); m/z = 514.5 [M + 1]+. Racemic compound 15 was purified by chiral chromatography to provide both enantiomers (ChiralTech AD-H 250 × 4.6 mm, 5 μm column; 5−60% CO2 with ethanol and 0.2% NH3; 3 mL/min). Enantiomerically pure compound 15 (>99% ee) was taken forward to the subsequent steps without further purification. See the Supporting Information for additional details. Methyl (S)-3-(4-(7-Ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan1-yl)-3-(piperidin-4-yl)propanoate (16). A solution of 15 (3.75 g, 7.3 mmol) in MeOH (50 mL) at rt was treated with 4 N HCl in 1,4dioxane (18 mL, 73 mmol). The reaction was stirred at rt for 16 h. All of the solvent was evaporated to give crude 16 as a tris-HCl salt (3.9 g). This material was used in the subsequent step without further purification. 1H NMR (MeOD): δ 8.61 (d, J = 7.0 Hz, 1H), 8.18 (s, 1H), 7.98 (s, 1H), 7.42 (d, J = 7.0 Hz, 1H), 4.02−3.90 (m, 1H), 3.87− 3.69 (m, 6H), 3.67 (s, 3H), 3.61−3.41 (m, 4H), 3.22−3.09 (m, 1H), 3.08−2.99 (m, 1H), 2.94 (q, J = 7.4 Hz, 2H), 2.87−2.72 (m, 1H), 2.69−2.49 (m, 1H), 2.31−2.03 (m, 2H), 1.97−1.76 (m, 1H), 1.73− 1.61 (m, 1H), 1.39 (t, J = 7.6 Hz, 3H); m/z = 414.3 [M + 1]+. Methyl (S)-3-(1-((1R,2R,4S)-7-Oxabicyclo[2.2.1]heptane-2carbonyl)piperidin-4-yl)-3-(4-(7-ethylimidazo[1,2-a]pyridin-8-yl)1,4-diazepan-1-yl)propanoate. To a flask containing the HCl salt of the amine (16, 3.78 g, 7.23 mmol) and chiral acid 9e (1.03 g, 7.23 mmol) in THF (36 mL) was added Et3N (6.02 mL, 43.4 mmol) and solid HATU (3.3 g, 8.67 mmol). The resulting mixture was stirred at rt for 4 h. The reaction was diluted with EtOAc and washed with brine. The aqueous fraction was re-extracted with EtOAc (×2). The combined organic extracts were dried over MgSO4, filtered, and evaporated to give a gummy solid. The crude material was purified by column chromatography using DCM/MeOH (0−15%) to give the desired amide (3.2 g, 5.95 mmol, 82%). 1H NMR (MeOD): δ 8.16 (d, J = 7.0 Hz, 1H), 7.72 (s, 1H), 7.46 (s, 1H), 6.77 (d, J = 7.0 Hz, 1H), 4.75−4.67 (m, 1H), 4.64−4.45 (m, 2H), 4.08−3.95 (m, 1H), 3.71 (s, 3H), 3.41−3.24 (m, 2H), 3.14−2.81 (m, 10H), 2.68−2.56 (m, 2H), 2.53−2.41 (m, 1H), 2.32−2.17 (m, 1H), 2.02−1.52 (m, 11H), 1.31− 1.14 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H); m/z = 538.6 [M + 1]+. (S)-3-(1-((1R,2R,4S)-7-Oxabicyclo[2.2.1]heptane-2-carbonyl)piperidin-4-yl)-3-(4-(7-ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)propanoic Acid (17). To a flask containing methyl (S)-3-(1((1R,2R,4S)-7-oxabicyclo[2.2.1]heptane-2-carbonyl)piperidin-4-yl)-3(4-(7-ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)propanoate (1.47 g, 2.73 mmol) in THF (30 mL) was added solid potassium trimethylsilanolate (1.05 g, 8.2 mmol). The homogeneous solution was stirred at rt for 16 h. The reaction was diluted with additional EtOAc and quenched by the dropwise addition of HCl (4 N in 1,4dioxane, 6.8 mL, 27.3 mmol), which caused the precipitation of a white solid. Stirring continued for 0.5 h, after which the solids were allowed

1.15−0.97 (m, 2H); LCMS for C27H39N5O2: 465.6 (calcd), 466.3 [M + H]+ (obsd). rac-Cyclopentyl(3-((4-(7-methylimidazo[1,2-a]pyridin-8-yl)-1,4diazepan-1-yl)methyl)piperidin-1-yl)methanone (12). A mixture of compound 6 (390 mg, 1.70 mmol) and t-butyl 3-formylpiperidine-1carboxylate (470 mg, 2.21 mmol) in DCE (20 mL) was stirred at 25 °C for 0.5 h; this was followed by the addition of NaBH(OAc)3 (1.1 g, 5.10 mmol). The mixture was stirred at rt for 16 h. At this time, the reaction mixture was diluted with DCM and washed with water. The organic layer was separated, dried over Na2SO4, and filtered. The filtrate was concentrated to afford the crude product, which was used without further purification. To a solution of the crude protected piperidine in DCM (3 mL) was added TFA (0.5 mL). The mixture was stirred at rt for 6 h. The mixture was concentrated to give the trifluoroacetate salt of the unprotected amine as a yellow oil. To a mixture of this crude intermediate compound (200 mg, 0.61 mmol), cyclopentanecarboxylic acid (77 mg, 0.67 mmol), and propylphosphonic anhydride (T3P, 291 mg, 0.91 mmol) in DCM (10 mL) was added Et3N (185 mg, 1.83 mmol). The mixture was stirred at rt for 16 h. The reaction mixture was diluted with additional DCM and washed with water. The organic layer was separated, dried over Na2SO4, and filtered. Following the evaporation of all of the volatiles, the crude product was purified by HPLC to give compound 12 (20 mg, 0.05 mmol, 3%). HPLC (5−95% MeOH (0.1% TFA)/water (0.1% TFA)): tR = 0.61 min. LCMS for C25H37N5O: 423.6 (calcd), 424.7 [M + H]+ (obsd). t-Butyl 4-(7-Ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepane-1carboxylate. To a 500 mL three-neck round-bottom flask was added the starting bromide, 13 (21.9 g, 89.5 mmol), in 1,4-dioxane (300 mL), followed by RuPhos (4.18 g, 8.95 mmol) and Pd2(dba)3 (4.10 g, 4.47 mmol), and the resulting solution was degassed by having nitrogen bubbled vigorously through it for 25 min. NaOt-Bu (21.5 g, 224 mmol) was added to the solution followed by amine 3 (19.4 mL, 98.4 mmol). The reaction was degassed with a steady stream of nitrogen for 20 min. The nitrogen purging needle was removed from the solution, and the nitrogen-flow rate was reduced to normal levels. The reaction was then heated to 95 °C (external temperature) for 6 h. The reaction was cooled to rt and added to saturated NH4Cl (400 mL); then, MTBE (200 mL) was added, and the mixture was stirred for 30 min before being filtered through Celite and washing with MTBE (150 mL). The layers were separated and the aqueous layer was extracted with MTBE two additional times (200 mL). The combined organic layers were washed with brine (200 mL). The layers were separated. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude material as a red oil. The crude oil was purified by column chromatography (10−100% EtOAc/heptanes) to give the desired product as a red-brown gum (21.24 g, 61.7 mmol, 69%). 1H NMR (CDCl3): δ 7.85−7.78 (m, 1H), 7.46 (s, 1H), 7.42−7.37 (m, 1H), 6.57−6.51 (m, 1H), 3.59−3.45 (m, 4H), 3.39−3.28 (m, 4H), 2.77− 2.67 (m, 2H), 1.84−1.71 (m, 2H), 1.47−1.36 (m, 9H), 1.16−1.06 (m, 3H); m/z = 345.4 [M + 1]+. 8-(1,4-Diazepan-1-yl)-7-ethylimidazo[1,2-a]pyridine (14). To the N-Boc compound t-butyl 4-(7-ethylimidazo[1,2-a]pyridin-8-yl)-1,4diazepane-1-carboxylate (21.2 g, 61.7 mmol) in MeOH (200 mL) was added 4 N HCl in 1,4-dioxane (93 mL, 370 mmol), and the reaction was stirred at rt for 3 h. All of the volatiles were removed in vacuo to give the crude HCl salt of the homopiperazine. The crude material was slurried into THF (250 mL), and this was followed by a treatment with 4 N NaOH(aq) (373 mL, 1490 mmol), which caused the solids to go into solution. The biphasic mixture was stirred at rt for 16 h. The reaction was diluted with additional THF (100 mL), and solid NaCl was added to saturate the aqueous layer. The mixture was transferred to a separatory funnel, and the organic extracts were removed. The aqueous layer was re-extracted with THF. All of the organic extracts were combined, dried over MgSO4, filtered, and evaporated to give the free-based homopiperazine, 14 (16.1 g, 65.7 mmol, 88%). This material was used without further purification. 1H NMR (MeOD): δ 8.18 (d, J = 7.0 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.47 (d, J = 1.2 Hz, 1H), 6.79 (d, J = 7.0 Hz, 1H), 3.44−3.32 (m, 4H), 3.10−3.05 (m, 3693

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

Journal of Medicinal Chemistry

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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. Serial blood samples were taken at 0.08, 0.25, 0.5, 1, 2, 5, and 24 h postdose, and the plasma was prepared by centrifugation. Plasma concentrations of 18 were determined by a specific HPLC-MS/MS method with a limit of determination of 1 ng/ mL. The LC-MS/MS system included a Waters Acquity UPLC system (Waters) and an API 5500 triple-stage quadrupole mass spectrometer (Sciex). Both instruments were controlled by Analyst 1.5.2 software (Applied Biosystems). Chromatography was performed on a reversephase column (Acquity HSS T3, 50 × 2.1 mm i.d., 1.8 μm particle size) at a flow rate of 0.650 mL/min under a gradient-elution method. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient started at 5% B for 0.1 min, ramped up to 95% B over 2.0 min, held at 95% B for 0.3 min, ramped down to 5% B over 0.1 min, and held at 5% B for 0.5 min before the next injection. The mass spectrometer was operated in positive-ionization mode using multiple-reaction monitoring at specific precursor-ion−product-ion transitions for both 18 (523.6 → 464.3) and the IS zolmitriptan (288.2 → 243.3) Analyst software was used for the data acquisition and chromatographic-peak integration. The Tmax, Cmax, AUC, and half-life values were determined using standard noncompartmental pharmacokinetic methods. Acute Treatment of 18 in Mice: Plasma SDF-1α Measurements. Compound 18 was administered to C57BL6 male mice (n = 9) subcutaneously at 30 mg/kg. The dose was formulated as a solution in a vehicle of 23% HPBCD in deionized water (w/v), and the dose volume was 3 mL/kg. Thirty minutes postdose, the animals were sacrificed, and the blood was collected. The plasma was prepared by centrifugation using EDTA-coated separators. Undiluted plasma samples were tested for SDF-1α levels using an ELISA kit (R&D Systems, #MCX120), following the manufacturer’s instructions. Briefly, in duplicate in the provided 96-well plate, 50 μL of the standard, control, or a plasma sample was added to a well that already had 50 μL of the assay diluent. The plate was incubated and shaken for 2 h. After 2 h, the plate was washed up to four times and incubated for an additional 2 h with 100 μL of the mouse SDF-1 conjugate. Subsequently, wash steps were performed again, and 200 μL of the substrate solution was added to each well, after which the plate was incubated for 30 min. All the incubations were conducted at rt. The final step involved adding 50 μL of the stop solution to each well, resulting in a color change from blue to yellow. The optical density of each well was determined using a microplate reader set to 450 nm. To analyze the data obtained, a standard curve was generated, and the SDF-1α concentration for each sample was calculated and plotted using GraphPad Prism 7.0. Statistical analysis was performed using unpaired t tests. Isoproterenol-Induced Cardiac Fibrosis in Mice. Cardiac fibrosis was induced in BALB/c mice via the subcutaneous administration of isoproterenol (Sigma, I5627; 5 mg/kg/day) for 9 consecutive days. At the end of the treatment period, the mice were euthanized by CO2 asphyxiation, and the hearts were collected, fixed in formalin, and processed for histological analysis. Mouse-heart sections stained with picrosirius red (PSR) were scanned on a Leica/Aperio AT2 whole-slide digital scanner using the 20× magnification setting. Image analysis was carried out using the Definiens Tissue Studio software, which quantified the PSR-stain percent areas in the mouseheart sections. Animal Care. All the procedures performed on animals in this study were in accordance with established guidelines and regulations and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. The Pfizer animal care facilities that supported this work are fully accredited by AAALAC International.

to settle. The supernatant was removed by pipet, and the solids were dried in vacuo to give a white solid. The solid material was slurried into EtOH, and the white precipitate was removed by filtration through a small pad of Celite (with additional EtOH washes). All of the solvent was evaporated to give the HCl salt of the carboxylic acid, 17, as a white solid, which was used crude in the next reaction without further purification (1.71 g, m/z = 524.4 [M + 1]+). (S)-3-(1-((1R,2R,4S)-7-Oxabicyclo[2.2.1]heptane-2-carbonyl)piperidin-4-yl)-3-(4-(7-ethylimidazo[1,2-a]pyridin-8-yl)-1,4-diazepan-1-yl)propanamide (18). To a flask containing the crude HCl salt of the carboxylic acid, 17 (1.56 g, 2.62 mmol), dissolved in DMF (30 mL) was added i-Pr2NEt (10 mL, 57.3 mmol) followed by a mixture of solid NH4Cl (2.3 g, 43.0 mmol) and solid HATU (1.26 g, 3.15 mmol). The mixture was stirred at rt for 6 h. The reaction was diluted with EtOAc and washed with brine (×4). The combined organic extracts were dried over MgSO4, filtered, and evaporated to give the crude amide product. The crude oil was dissolved in a minimal amount of DCM, and the solution was added dropwise to a flask containing excess diethyl ether, which facilitated the precipitation of a viscous dark-orange semisolid. This solid was purified by column chromatography (column conditions: 20% NH3 (2 M in MeOH) in DCM to 80% DCM with a gradient from 20 to 50%) to give a viscous oil, which when triturated with Et2O, provided the desired amide, 18, as an offwhite solid (0.570 g, 1.06 mmol, 40%). 1H NMR (MeOD): δ 8.15 (dd, J = 6.9, 1.7 Hz, 1H), 7.72 (dd, J = 2.5, 1.3 Hz, 1H), 7.49−7.41 (m, 1H), 6.77 (d, J = 6.9 Hz, 1H), 4.72 (dd, J = 13.3, 4.5 Hz, 1H), 4.62− 4.55 (m, 1H), 4.51 (t, J = 12.3 Hz, 1H), 4.08−3.95 (m, 1H), 3.40− 3.25 (m, 6H), 3.13−2.82 (m, 8H), 2.67−2.58 (m, 1H), 2.57−2.47 (m, 1H), 2.34−2.18 (m, 2H), 2.02−1.82 (m, 4H), 1.81−1.53 (m, 5H), 1.37−1.08 (m, 5H). 13C NMR (MeOD): δ 177.8, 173.6, 145.7, 140.1, 139.0, 132.2, 124.8, 116.0, 114.2, 79.9, 77.4, 68.8, 55.7, 54.0, 46.9, 46.6, 43.7, 43.4, 43.2, 40.8, 37.0, 32.3, 31.5, 31.1, 30.3, 30.2, 30.1, 24.6, 15.6. HRMS for C29H42N6O3: 522.6822 (calcd), 523.3383 [M + H]+ (obsd). SFC analysis: > 99% ee (see additional characterization information in the Supporting Information). CXCR7 Radiolabeled-Ligand-Binding Assay. The binding affinities of the test compounds for the human and mouse CXCR7 chemokine receptors were determined by their ability to displace 125ICXCL12 (PerkinElmer) from membranes overexpressing the receptor, as previously described.25 Membranes with the mouse receptor were obtained from CHO-K1 cells transiently transfected with the mouse CXCR7 receptor. Membranes with the human receptor were generated using CHO-K1 cells, obtained from DiscoverX, that stably express the human CXCR7 receptor. CXCR4 Radiolabeled-Ligand-Binding Assay. The binding affinities of the test compounds for the CXCR4 receptor were determined by their ability to displace 125I-CXCL12 (PerkinElmer) from Jurkat cells, an immortalized line of human T-lymphocyte cells that endogenously express the CXCR4 receptor. The test compounds were serialized in 100% DMSO and spotted into 96-well plates (NBS). Total-binding wells were spotted with diluent. Nonspecific wells were defined by the addition of cold CXCL12. To each well of the plate, 10 μL of 125I-CXCL12 at a final concentration of 100 pM was added, followed by 90 μL of the Jurkat cell suspension. The Jurkat cells and 125I-CXCL12 were diluted as needed in assay buffer (HBSS containing 10 mM HEPES and 0.2% BSA). After the addition of the Jurkat cells, the plates were incubated at rt for 2 h with shaking. The reactions were terminated by rapid filtration through poly(ethylenimine) (0.3%)-treated 96-well GF/C Unifilter plates (PerkinElmer). Unbound ligand was removed when the filters were washed with ice-cold wash buffer. The filters were allowed to dry thoroughly prior to the addition of the Ready Safe scintillation fluid (PerkinElmer). The amount of bound 125I-CXCL12 was quantitated by reading the plates on a Trilux luminometer (PerkinElmer). β-Arrestin-Recruitment Assay. The functional activities of the compounds were measured using the CXCR7 β-arrestin-recruitment assay from DiscoverX, which has been described previously.25 Pharmacokinetics. Compound 18 was administered to male C57BL6 mice (n = 2/route) by the subcutaneous route. The 5 mg/kg

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00190. 3694

DOI: 10.1021/acs.jmedchem.8b00190 J. Med. Chem. 2018, 61, 3685−3696

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Chiral separation of racemic compounds 15 and 18 (PDF) Molecular-formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 617 551 3517. E-mail: elnaz.menhaji-klotz@pfizer. com. ORCID

Elnaz Menhaji-Klotz: 0000-0003-3299-9480 David W. Piotrowski: 0000-0002-4659-6300 Markus Boehm: 0000-0002-7025-3287 Scott W. Bagley: 0000-0002-7365-7332 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Robert L. Dow, Stephen Jenkinson, Steven Coffey, Matt Dowling, David Ebner, and Kim McClure for their contributions to the project.

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ABBREVIATIONS USED CXCR7, C−X−C chemokine receptor type 7; ACKR3, atypical chemokine receptor 3; GPCR, seven-transmembrane-domain G-protein-coupled receptor; CXCR4, C−X−C chemokine receptor type 4; CXCL12, C−X−C chemokine ligand 12; ITAC, interferon-inducible T-cell α chemoattractant; CXCL11, C−X−C chemokine ligand 11; SDF-1α, stromal-cell-derived factor 1α; MI, myocardial infarction; EC50, half maximal effective concentration; Ki, binding affinity; Kb, dissociation constant; MDCKII-LE, low-efflux Madin−Darby canine kidney cell line; Papp, apparent permeability; sF, shake flask; LipE, lipophilic efficiency; HLM, human-liver microsome; f u,p, plasma unbound fraction; CLint, intrinsic clearance; AUC, area under the plasma-concentration-versus-time profile; Cmax, maximal plasma concentration; Cave, average plasma concentration; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

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