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Discovery of Clinical Candidate 1‑(4-(3-(4-(1H‑Benzo[d]imidazole-2carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (AMG 579), A Potent, Selective, and Efficacious Inhibitor of Phosphodiesterase 10A (PDE10A) Essa Hu,*,† Ning Chen,† Matthew P. Bourbeau,† Paul E. Harrington,† Kaustav Biswas,† Roxanne K. Kunz,† Kristin L. Andrews,‡ Samer Chmait,‡ Xiaoning Zhao,⊥ Carl Davis,§ Ji Ma,∇ Jianxia Shi,∇ Dianna Lester-Zeiner,# Jean Danao,# Jessica Able,# Madelyn Cueva,# Santosh Talreja,# Thomas Kornecook,∥ Hang Chen,# Amy Porter,∥ Randall Hungate,† James Treanor,∥ and Jennifer R. Allen† †

Department of Medicinal Chemistry, ‡Department of Molecular Structure and Characterization, §Department of Pharmacokinetics and Drug Metabolism, ∥Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 93012-1799, United States ⊥ Department of Molecular Structure and Characterization, #Department of Neuroscience, ∇Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States S Supporting Information *

ABSTRACT: We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the inhibitory activity of 4 but not 6. In the in vivo LC−MS/MS target occupancy (TO) study at 10 mg/kg, 3, 5, and 6 achieved 86−91% occupancy of PDE10A in the brain. Furthermore, both CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.

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INTRODUCTION Of the 11 isoforms of phosphodiesterase (PDE) identified to date, PDE10A has recently received much attention in the field of neuroscience research.1 Because PDE10A has the highest expression level in the striatum,2−4 modulation of this CNS target offers an opportunity to regulate signal transmissions in a region of the brain associated with various neurological conditions such as schizophrenia and Huntington’s disease.5−13 At the preparation of this manuscript, two PDE10A inhibitors have been reported to advance into clinical studies for treatment of schizophrenia.14,15 Other inhibitors have also been described in the literature.16−26 The function of PDEs is to reduce levels of cyclic nucleotide monophosphates (cNMP) by cleavage of their phosphodiester bonds.27 Thus, inhibition of PDE10A enzyme would increase the levels of intracellular secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).28 It has been hypothesized that this mechanism effectively replicates the downstream functional effect of current standard of care (SOC) which targets dopamine receptors (D1 and D2) and NMDA receptors.29−32 Intolerable adverse events that contributed to compliance issues in schizophrenic patients © 2014 American Chemical Society

treated with typical and atypical antipsychotics have been linked to either direct interaction with the dopaminergic receptors or polypharmacology.33,34 Therefore, we hypothesized targeting PDE10A would provide a promising approach, using a single selective molecule, to both treat schizophrenia and circumvent the adverse events. We recently reported a detailed structure−activity relationship (SAR) study that led to the identification of keto-benzimidazoles 1 and 2 as novel and potent inhibitors of PDE10A with mitigated P-gp efflux (Figure 1).35 Both analogues achieved single-digit inhibitory activity against PDE10A enzyme in the biochemical assay (IC50 = 4.5 and 5.1 nM, respectively). Despite their similar in vitro potencies and structures, their difference in in vivo efficacies in our rodent LC−MS/MS target occupancy (TO) assay was pronounced. At 10 mg/kg PO, morpholine 1 only produced 21.3% TO of PDE10A in the CNS while N-acetyl piperazine 2 afforded a significantly higher TO of 57.1%. The realization that a small structural difference between 1 and 2 could have such a dramatic impact on in vivo efficacy prompted Received: May 8, 2014 Published: July 25, 2014 6632

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Article

3 did change the preferred binding orientation of the saturated rings relative to the pyrazine rings, however. Even though both rings were freely rotating, the densities of the co-crystal structures captured the more favored binding conformation. For 1, the morpholine ring was nearly coplanar with the pyrazine ring, presumably due to the stabilizing delocalization of the nitrogen lone pair into the anti-p-orbitals on the pyrazine ring. In contrast, the tetrahydropyran ring on 3 preferred a nonplanar alignment with the pyrazine ring, presumably to minimize the steric interactions between the two ring systems. We postulated that the greater torsion angle between the two rings on 3 enabled the tetrahydropyran to establish more hydrophobic contacts with the hydrophobic residues in the binding pocket, resulting in an increase in binding affinity. This computational prediction of the binding conformation of 3 was later supported by an X-ray cocrystal structure of pyridyl analogue 4, which showed the tetrahydropyran in a nonplanar orientation relative to the pyridine ring analogous to the conformation predicted for 3. We then discovered a second single atom replacement that produced a significant effect (Table 1). With a pyridine ring

Figure 1. Promising keto-benzimidazole leads 1 and 2.

us to explore additional changes to seek further optimization. We sought to further increase the in vitro potency and in vivo efficacy of our PDE10A inhibitors in order to deliver a clinical candidate with a low projected human dose and minimal drug burden on patients.

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RESULTS AND DISCUSSION Of all the small structural changes we had explored, the first breakthrough discovery was a simple nitrogen atom to carbon atom replacement on the saturated rings. As shown in Figure 2,

Table 1. Structure−Activity Relationships of Ketobenzimidazoles 1−6

Figure 2. Single atom change resulted in 3 with increased potency.

tetrahydropyran 3 showed PDE10A IC50 = 0.8 nM, a 5-fold increase in potency from morpholine 1. To analyze the structural impact of this single atom change, we compared our previously reported X-ray co-crystal structure of 135 in human PDE10A catalytic domain with computational model of 336 in human PDE10A catalytic domain (Figure 3). As expected, the ketobenzimidazole region on 3 was bound to PDE10A catalytic domain in a manner identical to the keto-benzimidazole region on 1. Both pyrazine rings were sandwiched between Phe719 and Phe686 stabilized by π-stacking interactions. The conserved Gln 716 further anchored the scaffold by forming a hydrogen bonding interaction with one of the pyrazine nitrogens. Meanwhile, the phenyl keto-benzimidazole region was locked into a planar binding conformation by a second hydrogen bonding interaction between Tyr683 and one of the nitrogens on the benzimidazole ring. The one atom difference between 1 and

compd

X

Y

Z

PDE10A IC50 (nM)a

1 3 4 2 5 6

N N C N N C

N C C N C C

O O O NAc NAc NAc

4.5 0.8 0.1 5.1 0.1 0.1

HLM RLM (μL/min/mg)b (μL/min/mg)b 55 50 100 34 20 29

74 119 >399 108 41 463

a

Each IC50 value reported is an average of at least two independent experiments with 22-point dose response curve in duplicates at each concentration. bIn vitro microsomal stability studies were conducted in the presence of NADPH at 37 °C for 30 min at final compound concentration of 1 μM of compound.

instead of pyrazine ring, 4 experienced an 8-fold improvement in PDE10A inhibitory activity (IC50 = 0.1 nM). Compared to initial morpholine pyrazine lead 1, N-acetyl piperidine pyridine 4 represented 45-fold improvement in potency. Further profiling showed each single atom exchange had a different impact on in

Figure 3. Comparison of X-ray co-crystal structures of 1 (left), computational model of 3 (center), and co-crystal structure of 4 (right) in human PDE10A catalytic domain. 6633

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Scheme 1. Synthesis of Keto-benzimidazoles 3a

a Reagents and conditions: (a) cesium carbonate, NMP, 100 °C, 70% yield; (b) 5 mol % PdCl2(P(t-Bu)2(PhNMe2))2, potassium carbonate, acetonitrile, water 80 °C, 91% yield; (c) 5 mol % Pd(OH)2/C, hydrogen, ethanol, 65% yield; (d) (i) 13, HC(Oi-Pr)3, toluene, reflux, (ii) LDA, THF, −78 °C, 12, 84% yield.

Scheme 2. Synthesis of Keto-benzimidazoles 4, 5, and 6a

a Reagents and conditions: (a) sodium carbonate, Pd(PPh3)2Cl2, ether, water, 80 °C, 89% yield; (b) 9.5 mol % Pd(OH)2, hydrogen, ethanol, 96% yield; (c) I2, Ph3P, imidazole, THF, 84% yield; (d) (i) zinc dust, TMSCl, 1,2-dibromoethane, DMA, (ii) 3 mol % PdCl2dppf, CuI, DMA, 80 °C, two step yields 85% (20a) and 70% (20b); (e) Pd(OH)2, hydrogen, toluene, acetic anhydride, 91% (21a), 92% (21b); (f) para-methoxybenzyl chloride, potassium carbonate, NaI, DMF, 98% yield; (g) (i) HC(OEt)3, benzenesulfonic acid, toluene, (ii) LDA, 66% yield; (h) TFA, dichloromethane, 99% yield; (i) cesium carbonate, NMP, 120 °C, 48% yield (4), 87% yield (5), 66% yield (6).

modifications on N-acetyl piperazine lead 2 was not entirely the same. Similar to 3, one nitrogen atom to carbon atom change made N-acetyl piperidine 5 over 50-fold more potent than Nacetyl piperazine 2 (IC50 = 0.1 vs 5.1 nM, respectively) without

vitro clearance. Pyrazine 3 retained human and rat in vitro metabolic stability comparable to 1. However, rat metabolic stability of pyridine 4 deteriorated significantly. Interestingly, compared to morpholine lead 1, the SAR of these same 6634

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impacting its rat and human in vitro microsomal clearance (HLM = 20 μL/min/mg; RLM = 41 μL/min/mg). Unlike 4, further improvement in potency was not observed when the pyrazine ring on 5 was replaced with a pyrdine ring (6). As with 4, pyridine 6 also experienced a dramatic decrease in its RLM to 463 μL/ min/mg. Scheme 1 depicts the synthetic route used to obtain ketobenzimidazoles 3. The synthesis began with SNAr displacement of dichloropyrazine 7 with ethyl 4-hydroxybenzoate 8. Suzuki coupling of ethyl 4-((3-chloropyrazin-2-yl)oxy)benzoate 9 with 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 10 using PdCl2(P(t-Bu)2(PhNMe2))2 produced ethyl 4-((3-(3,6-dihydro-2H-pyran-4-yl)pyrazin-2-yl)oxy)benzoate 11 in 91% yield.37 Palladium catalyzed hydrogenation reduced the olefin and afforded ethyl 4-((3-(tetrahydro-2H-pyran-4yl)pyrazin-2-yl)oxy)benzoate 12. A two-step, one-pot protocol beginning with in situ N−H protection of benzimidazole 13 followed by LDA deprotonation formed (1H-benzo[d]imidazol2-yl)(4-((3-(tetrahydro-2H-pyran-4-yl)pyrazin-2-yl)oxy)phenyl)methanone 3 in 84% yield. Syntheses of keto-benzimidazoles 4, 5, and 6 were accomplished by a convergent route illustrated in Scheme 2. Two routes were developed to access the three left-hand fragments. Dihydropyran pyridine 15 was synthesized by Suzuki coupling of 10 to 3-bromo-2-fluoropyridine 14. Palladium catalyzed hydrogenation of the olefin afforded 16 in 96% yield. For the N-acetyl piperidine fragments (pyrazine 21a and pyridine 21b), our synthesis began with iodination of benzyl 4hydroxypiperidine-1-carboxylate 17 to form benzyl 4-iodopiperidine-1-carboxylate 18 using iodine and triphenylphosphine in 84% yield. In a key transformation, Negishi coupling of 18 to pyrazine 19 or pyridine 14 via zinc activation38 and palladium catalysis produced N-Cbz piperidine pyrazine 20a and pyridine 20b. A one-pot Cbz deprotection and acetylation afforded Nacetyl piperidines 21a and 21b. Preparation of the right-hand fragment, keto-benzimidazole 23, began with para-methoxybenzyl (PMB) protection of ethyl 4-hydroxybenzoate 8 to form protected phenol ester 22. Ketone formation using a similar onepot benzimidazole protection and deprotonation protocol followed by trifluoroacetic acid promoted removal of the PMB protecting group resulted in (1H-benzo[d]imidazol-2-yl)(4hydroxyphenyl)methanone 23. Final coupling of tetrahydropyran pyridine 16, N-acetyl piperidines 21a or 21b, to keto-phenol 23 was accomplished using cesium carbonate to give ketobenzimidazoles 4, 5, and 6 in 48%, 87%, and 66% yield, respectively. The synthetic routes developed allowed for compounds 3−6 to be prepared on a large scale in order to enable rodent efficacy assessment as well as rodent and nonrodent pharmacokinetic and exploratory preclinical toxicology studies. To determine whether these structural changes resulted in improvements in in vivo efficacies, keto-benzimidazoles 3, 4, 5, and 6 were advanced into LC−MS/MS target occupancy (TO) measurement study and phencyclidine induced-locomotor activity (PCP-LMA) model (Table 2). We recently reported the development of an in vivo LC−MS/MS TO assay platform and a selective PDE10A tracer 5-(6,7-dimethoxycinnolin-4-yl)N-isopropyl-3-methylpyridin-2-amine (AMG 7980, 24) to measure PDE10A target occupancy of drug candidates in rat brain.39 By measuring compound levels in brain regions with high levels of PDE10A (striatum) and low expression levels of PDE10A (thalamus) and measuring PDE10A tracer level changes in the striatum due to compound binding, this LC−

Table 2. PDE10A in Vivo Target Occupancy and Behavioral PD Readouts of Keto-benzimidazoles 1−6a compd

single dose TOb (at 10 mg/kg) (%)

dose response TOc EC50 (nMtotal)

reversal of PCP-LMAd (MED) (mg/kg)

1 3 4

21.3 91.0 69.2

117

1.0

2 5 6

57.1 86.3 86.5

711 965

0.3 0.3

a

Dose and methods: LC−MS/MS target occupancy measurements were taken 1 h post PO dosing of compounds. bDose and methods: at single dose of 10 mg/kg, n = 8. cDose and methods: at doses 0.1, 0.3, 1, 3, 10, and 30 mg/kg n = 4. dReversal of PCP-induced locomotor activity was assessed at doses 0.1, 0.3, 1, and 3 mg/kg, n = 10. Compounds were dosed PO 1 h prior to administration of PCP. Number of laser beam breaks post PCP dosing was collected at 10 min intervals and compiled over 2 h period. MED was determined at the lowest dose which a statistically significant reduction of locomotor activity occurred.

MS/MS technology platform has enabled us to identify compounds with high in vivo PDE10A specificity and coverage efficacy. Measurements of CNS PDE10A target occupancy were conducted 1 h post PO dosing of inhibitors and 10 min post IV dosing of tracer. TO was determined by comparing the tracer levels in the striatum, a region of the brain most highly expressed with PDE10A, versus the thalamus, a control region for nonspecific binding. At a single dose of 10 mg/kg PO, ketobenzimidazole 3 achieved 91% TO, a significant improvement from 21% TO of 1. Even though 4 was biochemically more potent than 3, 4 exhibited reduced 69.2% TO, presumably due to its reduced metabolic stability in rat. Meanwhile, N-acetyl piperidine 5 showed enhanced 86.3% TO at 10 mg/kg compared to 57.1% TO of 2. Pyridine 6 also afforded comparable TO of 86.5% despite its higher rat in vitro clearance. Compounds 3, 5, and 6 were advanced into dose response LC−MS/MS TO study at doses 0.1, 0.3, 1, 3, 10, and 30 mg/kg, and all three compounds exhibited dose-dependent occupancy of PDE10A receptors in the brain (Figure 4). On the basis of these data, TO EC50 was

Figure 4. Dose dependent in vivo target occupancy with ketobenzimidazole 5.

calculated to be 117 nM for 3, 711 nM for 5, and 965 nM for 6 (total plasma). High occupancy PDE10A inhibitors 3, 5, and 6 were then tested in the PCP-LMA model, a widely used preclinical rodent model to mimic positive symptoms of schizophrenia (Figure 5).40 Compounds were dosed at 0.1, 0.3, 1, and 3 mg/kg PO 1 h prior to administration of PCP. The 6635

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ethanone 5 passed all our preclinical toxicological studies and advanced as AMG 579, our PDE10A clinical candidate for treatment of schizophrenia. Starting with promising keto-benzimidazole leads 1 and 2, we discovered single atom replacements from nitrogen atom to carbon atom produced significant improvements in both PDE10A enzyme potency and in vivo efficacy in the LC−MS/ MS TO assay. The first single atom modification of the saturated rings from N-linked to C-linked (1 to 3 and 2 to 5) increased the biochemical potency by 5-fold and 50-fold, respectively. X-ray co-crystal structures of 1 and 3 in the human PDE10A enzyme catalytic domain showed the single atom replacement changed the preferred orientation of the saturated ring relative to the pyrazine ring. A second nitrogen atom to carbon atom change from pyrazine ring to pyridine ring (3 to 4 and 5 to 6) resulted in 8-fold increase of PDE10A activity for 4 but no potency improvement for 6. Both pyrazines (3 and 5) maintained their in vitro metabolic stability, while both pyridines (4 and 6) showed much higher in vitro clearance in rat. It is worth noting that these modifications had minimal impact on the permeability, efflux ratio, or PDE selectivity of these keto-benzimidazole analogues compared to original leads 1 and 2. In vivo LC−MS/MS TO studies at 10 mg/kg showed 3, 5, and 6 achieved 86−91% TO. All three compounds were efficacious in the PCP-LMA model, a widely used model of positive schizophrenic behaviors in preclinical species. Both occupancy of CNS PDE10A receptors and efficacy in PCP-LMA model with keto-benzimidazole 5 were found to occur in a dose dependent manner. Keto-benzimidazole 5 was selected as our clinical candidate upon comparison of in vivo efficacies and in vivo PK profiles of compounds 3, 5, and 6 in rat, dog, and nonhuman primate (Figure 6). This molecule was also highly selective with IC50 > 30 μM against all other PDE isoforms. Advanced preclinical and clinical data will be disclosed in separate publications in the near future.

Figure 5. Dose dependent reversal of PCP-induced locomotor activity model with keto-benzimidazole 5.

magnitude of rat locomotor activity was quantified as number of beam breaks over a 2 h period. Compared to the untreated group, all three inhibitors showed statistically significant reduction of PCP induced behavior in rats over the 2 h period. Minimum effective doses (MED) for efficacy in PCP-LMA model were determined to be at 1 mg/kg for 3, 0.3 mg/kg for 5, and 0.3 mg/ kg for 6. In line with CNS target occupancy, all compounds were effective in reversing PCP-induced locomotor activity in a dose dependent manner. It is also worth noting that compared to the vehicle treated group, none of our compounds impacted the locomotor activity of these animals prior to PCP challenge. All three compounds (3, 5, and 6) that achieved greater than 80% TO in the single dose LC−MS/MS assay and demonstrated efficacy in the PCP-LMA behavioral model were also profiled in rat in vivo PK (Table 3). Both 3 and 5 showed modest in vivo clearance in rat (0.62 and 0.54 L/h/kg, respectively) compared to high rat clearance observed with 6 (3.03 L/h/kg). However, oral bioavailability of 3 was low (22% F) compared to 5 (49% F) and 6 (63% F). Higher species in vivo PK with 5 and 6 showed acceptable in vivo clearance in both dog and nonhuman primate. However, in dog, 5 exhibited superior oral bioavailability of 72% compared to 45% with 6. With a clearly more robust metabolic profile across multiple preclinical species and a low projected human dose of 0.4 mg/kg QD, 1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-

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EXPERIMENTAL METHODS

Chemistry, Materials, and General Methods. Unless otherwise noted, all reagents and solvents were obtained from commercial suppliers such as Aldrich, Sigma, Fluka, Acros, EMD Sciences, etc., and used without further purification. Dry organic solvents (dichloromethane, acetonitrile, DMF, etc.) were purchased from Aldrich packaged under nitrogen in Sure/Seal bottles. All reactions involving

Table 3. In Vivo PK Data for Keto-benzimidazoles 3, 5, and 6 compd 3

5

species rat

rat dog NHP

6

rat dog NHP

dose (mg/kg)/route

Cl (L/h/kg)

Vss (L/kg)

AUC (μM·h)

T1/2 (h)

%F

2, IVa 10, POb

0.62

2.27

7.75 7.93

4.26 3.99

22

2, IVa 10, POb 0.5, IVb 1, POb 0.5, IVd 0.5, POc

0.54

0.81

0.074

0.77

0.34

0.83

8.31 19.80 13.70 16.90 3.41 2.40

3.16 3.35 6.89 11.0 1.09 2.48

2, IVa 10, POb 0.5, IVc 0.5, POb 0.5, IVd

3.03

2.78 29.50 21.50 9.59 1.61

23.1 1.72 7.41 5.07 1.26

31.2

0.052

0.50

0.82

1.62

49 72 71

63 45

a

Dosing vehicle: 100% DMSO. bDosing vehicles: Tween 80, 2% HPMC, 97% water, methanesulfonic acid pH 2.0. cDosing vehicles: 30% captisol, 70% water, methanesulfonic acid, pH 4.0. dDosing vehicle: 30% hydroxypropyl β-cyclodextrin, 70% water. NHP = nonhuman primate. 6636

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Article

Bu)PPhNMe2)2 (0.07 g, 0.11 mmol), and potassium acetate (0.52 g, 5.3 mmol) was added acetonitrile (7.0 mL), water (0.70 mL), and 10 (0.52 g, 2.5 mmol). The reaction mixture was degassed by bubbling nitrogen through the solution for 5 min and heated to 80 °C. After 2.5 h, the reaction was diluted with EtOAc, water, and brine. The aqueous phase was extracted with EtOAc (1×). The combined organic extracts were washed with brine (1×), dried over MgSO4, filtered, and concentrated. Purification by flash column chromatography on silica gel (eluted with 10−50% EtOAc in hexanes) gave 11 (0.60 g, 91% yield) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.40 (t, J = 7.14 Hz, 3 H), 2.76 (dd, J = 4.40, 2.64 Hz, 2 H), 3.97 (t, J = 5.48 Hz, 2 H), 4.36−4.43 (m, 4 H), 7.03 (br s, 1 H), 7.18 (d, J = 8.61 Hz, 2 H), 7.92 (d, J = 2.35 Hz, 1 H), 8.12 (d, J = 8.80 Hz, 2 H), 8.30 (d, J = 2.54 Hz, 1 H). LC−MS m/z [M + H] 327.2. Step 3. Ethyl 4-(3-(Tetrahydro-2H-pyran-4-yl)pyrazin-2-yloxy)benzoate (12). To a mixture of 11 (3.0 g, 9.2 mmol) and palladium hydroxide (20% by weight palladium on carbon, 0.32 g, 0.46 mmol) under argon was added EtOH (18 mL). The argon atmosphere was replaced by hydrogen from a double balloon, and the reaction mixture was stirred at room temperature. After 3 h, the solution was filtered and concentrated. Purification by flash column chromatography on silica gel (eluted with 10−40% EtOAc in hexanes) gave 12 (2.0 g, 65% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.40 (t, J = 7.14 Hz, 3 H), 1.88 (d, J = 13.30 Hz, 2 H), 2.02−2.13 (m, 2 H), 3.43 (tt, J = 11.70, 3.30 Hz, 1 H), 3.61 (t, J = 11.83 Hz, 2 H), 4.13 (dd, J = 11.25, 3.81 Hz, 2 H), 4.39 (q, J = 7.24 Hz, 2 H), 7.18 (d, J = 8.61 Hz, 2 H), 7.94 (d, J = 2.74 Hz, 1 H), 8.12 (d, J = 8.61 Hz, 2 H), 8.26 (d, J = 2.74 Hz, 1 H). LC−MS m/z [M + H] 329.2. Step 4. (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-pyran-4yl)pyrazin-2-yloxy)phenyl)methanone (3). A suspension of 13 (5.9 g, 50 mmol) and triisopropyl orthoformate (63.6 mL, 287 mmol) in toluene (150 mL) in a round-bottomed flask fitted with a Dean−Stark trap topped with a reflux condenser was heated to reflux under nitrogen (bath temperature 140 °C). After 1 h, the reaction was cooled to ambient temperature and was concentrated in vacuo. The crude orthoester amide was dried under high vacuum for 6 h and diluted with tetrahydrofuran (150 mL). Compound 12 (15.7 g, 47.9 mmol) was azeotroped with dry toluene (3×15 mL), dissolved in tetrahydrofuran (100 mL), and added via cannula to the reaction flask containing the benzimidazole orthoester amide. The resulting mixture was then cooled to −78 °C under nitrogen. Lithium diisopropylamide (27.5 mL of a 2 M solution in heptane/tetrahydrofuran/ethylbenzene, 55.0 mmol) was added in a dropwise fashion over 75 min. After 2 h, the reaction was removed from the acetone−dry ice bath and quenched with via slow addition of 2 N HCl (25 mL). The solution was stirred at 23 °C for 45 min and neutralized to pH = 7.0 with solid sodium carbonate. The suspension was diluted with EtOAc (500 mL) and washed with saturated sodium bicarbonate solution (2 × 150 mL) and brine (150 mL), dried over magnesium sulfate, concentrated in vacuo, and slurried with ethanol (100 mL). The residue was filtered and dried under vacuum to afford 3 (16 g, 84% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.52 (1 H, br s), 8.70 (2 H, d, J = 8.8 Hz), 8.41 (1 H, d, J = 2.5 Hz), 8.11 (1 H, d, J = 2.5 Hz), 7.87 (1 H, br s), 7.64 (1 H, br s), 7.20−7.48 (4 H, m), 3.82−4.13 (2 H, m), 3.52 (2 H, td, J = 11.2, 3.1 Hz), 3.36−3.45 (1 H, m), 1.63−2.12 (4 H, m). LC−MS m/z [M + H] 401.1. HRMS (ES+) calcd for [C23H21N4O3]+ 401.1614; found 401.1611. 13C NMR (101 MHz, DMSO-d6) δ ppm 30.21, 36.55, 66.87, 121.03, 132.16, 132.96, 138.95, 139.06, 147.94, 149.68, 156.55, 157.80, 182.13. Synthesis of Common Intermediate Benzyl 4-Iodopiperidine-1carboxylate (18) Used in the Syntheses of Keto-benzimidazoles 5 and 6. Benzyl 4-Iodopiperidine-1-carboxylate (18). To a mixture of imidazole (11.5 g, 168 mmol), triphenylphosphine (43.9 g, 167 mmol), and 17 (32.9 g, 140 mmol) was added THF (100 mL). The solution was cooled to 0 °C, and a solution of iodine (42.7 g, 168 mmol) in THF (100 mL) at 0 °C was added dropwise over 20 min. After the addition, the reaction mixture was warmed to room temperature. After 18 h, the reaction was quenched with saturated Na2SO3 and diluted with hexanes (200 mL). The aqueous phase was extracted with hexanes (3×). The combined organic extracts were washed with brine (1×), dried over

Figure 6. Profile of clinical candidate 5. air or moisture sensitive reagents were performed under a nitrogen or argon atmosphere. Silica gel chromatography was performed using prepacked silica gel cartridges (Biotage or RediSep). Microwave assisted reactions were performed in Biotage Initiator Sixty microwave reactor. 1 H NMR spectra were recorded on a Bruker DRX 300 MHz, Bruker DRX 400 MHz, Bruker AV 400 MHz, Varian 300 MHz, or a Varian 400 MHz spectrometer at ambient temperature. Chemical shifts are reported in parts per million (ppm, δ units). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and number of protons. Reactions were monitored using Agilent 1100 series LC/MSD SL high performance liquid chromatography (HPLC) systems with UV detection at 254 nm and a low resonance electrospray mode (ESI). All final compounds were purified to >95% purity, as determined by high performance liquid chromatography (HPLC). HPLC methods used the following: Agilent 1100 spectrometer, Zorbax SB-C18 column (50 mm × 3.0 mm, 3.5 μm) at 40 °C with a 1.5 mL/min flow rate; solvent A of 0.1% TFA in water, solvent B of 0.1% TFA in acetonitrile; 0.0−3.0 min, 5−95% B; 3.0−3.5 min, 95% B; 3.5−3.51 min, 5% B. Flow from UV detector was split (50:50) to the MS detector, which was configured with APIES as ionizable source. All high resolution mass spectrometry (HRMS) data were acquired on a Synapt G2 HDMS instrument (Waters Corporation, Manchester, UK) operated in positive electrospray ionization mode. The sample was diluted to 10 μM in 50% acetonitrile (v/v), 0.1% formic acid (v/v), and infused into the mass spectrometer at a flow rate of 5 μL/min through an electrospray ionization source operated with a capillary voltage of 3 kV. The sample cone was operated at 30 V. The time-of-flight analyzer was operated at a resolution (fwhm) of 30000 at m/z 785 and was calibrated over the m/z range 50−1200 using a 1 μM sodium iodide (NaI) solution (50% v/v acetonitrile solution). To obtain accurate mass measurements, an internal lock-mass correction was applied using leucine enkephalin (m/z 556.2771). Collision induced fragmentation (CID) was performed using an injection voltage of 28 V. Instrument control was performed through the software suite MassLynx, version 4.1. Synthesis of (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2Hpyran-4-yl)pyrazin-2-yloxy)phenyl)methanone (3). Step 1. Ethyl 4(3-Chloropyrazin-2-yloxy)benzoate (9). To a mixture of cesium carbonate (72.8 g, 223 mmol) and 8 (30.1 g, 181 mmol) was added NMP (200 mL) and 7 (33.0 mL, 317 mmol). The reaction mixture was degassed by bubbling nitrogen through the solution for 5 min and heated to 100 °C. After 15 min, the mixture was diluted with EtOAc. The organic phase was washed with water (2×), brine (1×), dried over MgSO4, filtered, and concentrated. The concentrate was diluted with hexanes (50 mL), and the mixture was allowed to stand at −15 °C for 15 h. The precipitate that formed was collected by filtration, washed with hexanes, and dried under high vacuum to give 9 (35.3 g, 70% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (t, J = 7.14 Hz, 3 H), 4.33 (q, J = 7.20 Hz, 2 H), 7.41 (d, J = 8.41 Hz, 2 H), 8.05 (d, J = 8.41 Hz, 2 H), 8.22 (d, J = 2.54 Hz, 1 H), 8.29 (d, J = 2.54 Hz, 1 H). LC− MS m/z [M + H] 279.1. Step 2. Ethyl 4-(3-(3,6-Dihydro-2H-pyran-4-yl)pyrazin-2-yloxy)benzoate (11). To a mixture of 9 (0.56 g, 2.0 mmol), PdCl2((t6637

dx.doi.org/10.1021/jm500713j | J. Med. Chem. 2014, 57, 6632−6641

Journal of Medicinal Chemistry

Article

MgSO4, filtered, and concentrated. The concentrate was diluted with DCM (50 mL) followed by hexanes (200 mL). The solution was concentrated to half the original volume, and the precipitate that formed was removed and the filtrate was concentrated. Purification by flash column chromatography on silica gel (eluted with 0−20% EtOAc in hexanes) gave 18 (40.4 g, 84% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 2.03 (br s, 4 H), 3.40 (dt, J = 13.60, 5.72 Hz, 2 H), 3.66 (dt, J = 13.45, 5.31 Hz, 2 H), 4.46 (quin, J = 5.80 Hz, 1 H), 5.13 (s, 2 H), 7.31−7.39 (m, 5 H). LC−MS m/z [M + H] 346.1. Synthesis of Common Intermediate (1H-Benzo[d]imidazol-2yl)(4-hydroxyphenyl)methanone (23) Used in Syntheses of Ketobenzimidazoles 4, 5, and 6. Step 1. Ethyl 4-((4-Methoxybenzyl)oxy)benzoate (22). To a stirred solution of 8 (467 g, 2.8 mol) in DMF (5.3 L) were added potassium carbonate powder (1 kg, 7.2 mol), NaI (42 g, 0.28 mol), and PMB-Cl (460 mL, 3.37 mol) at ambient temperature. The reaction mixture was heated at 50 °C overnight under a nitrogen atmosphere. The reaction mixture was cooled to ambient temperature. Ice−water (8 L) was added, and the resulting mixture was stirred for 2 h. The precipitate was filtered and washed with water (4 × 2 L). The solid was then slurried in water (2 × 3.5 L) for 30 min, filtered, and dried at 45 °C under vacuum for 3 days to obtain 22 (794 g, 98% yield). Step 2. (1H-Benzo[d]imidazol-2-yl)(4-((4-methoxybenzyl)oxy)phenyl)methanone. A solution of 13 (112.8 g, 0.95 mol), triethyl orthoformate (317 mL, 1.9 mol), and benzenesulfonic acid (4.1 g, 25.8 mmol) in toluene (1 L) was heated to reflux, and 500 mL was removed by distillation. Toluene (750 mL) was added again, and 500 mL was removed by slow distillation. The reaction mixture was cooled and neutralized with diisopropylamine (13.4 mL, 95.4 mmol). To the reaction mixture was added THF (1 L) and 22 (300 g, 1.0 mol). The mixture was cooled to −78 °C before addition of LDA (572 mL, 2 M in THF/hexane/ethylbenzene, 1.1 mol) dropwise over 4 h. After the addition, the reaction mixture was stirred at −78 °C for 2 h and then allowed to gradually warm to ambient temperature. A solution of 2 N aqueous HCl (3.9 L) was added, and the resulting mixture was stirred overnight. The mixture was diluted with an additional 2 L of water, and the layers were separated. The organic layer was washed with 10% aqueous NaHCO3 solution (2 L) and brine (1.5 L), dried over Na2SO4, and filtered. The filtrate was concentrated. Purification by flash chromatography on silica gel (0% to 50% DCM/EtOAc) to afford product that was further triturated with MTBE to give (1Hbenzo[d]imidazol-2-yl)(4-((4-methoxybenzyl)oxy)phenyl)methanone (229 g, 66% yield). Step 3. (1H-Benzo[d]imidazol-2-yl)(4-hydroxyphenyl)methanone (23). To a slurry of compound (1H-benzo[d]imidazol-2-yl)(4-((4methoxybenzyl)oxy)phenyl)methanone (220 g, 0.61 mol) in DCM (2.2 L) at ambient temperature was added TFA (235 mL, 3.1 mol) slowly using an addition funnel over a period of 2.5 h. The reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was neutralized with saturated aqueous NaHCO3 (4 L) and stirred for an additional 30 min. The resulting solid was filtered and washed with water (3 × 1 L) and air-dried. The material was further dried under vacuum at 45 °C to remove water. The material was washed with hexanes:EtOAc (3:2; 3×500 mL) and dried under vacuum at 45 °C to give 23 (136 g, 99% yield). 1H NMR (300 MHz, DMSO-d6) δ 13.39 (s, 1H), 10.62 (s,1H), 8.62 (d, J = 9 Hz., 2H), 7.87 (d, J = 8.1 Hz, 1H), 7.6 (d, J = 5.7 Hz, 1H), 7.32−7.42 (m, 2H), 6.98 (d, J = 9 Hz, 2H). LC−MS m/z [M + H] 239.1. Synthesis of (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2Hpyran-4-yl)pyridin-2-yloxy)phenyl)methanone (4). Step 1. 3-(3,6Dihydro-2H-pyran-4-yl)-2-fluoropyridine (15). To a 500 mL roundbottom flask was added 14 (8.7 g, 49 mmol), 10 (8.0 g, 38 mmol), and disodium carbonate (12.1 g, 114 mmol) in DME (75 mL) and water (25 mL). Argon was bubbled through the resulting solution for 3 min. Bis(triphenylphosphine)palladium(II) chloride (1.5 g, 2.1 mmol) was added while still keeping the reaction under argon. The resulting mixture was heated to 80 °C. After overnight heating, the reaction mixture was evaporated by rotovap to remove DME. The residue was diluted with water and extracted with EtOAc (2×). The organic layers were combined and washed with brine and dried over sodium sulfate. Purification by flash column chromatography on silica gel (eluted with

0−20% EtOAc in hexanes) gave 15 (6.09 g, 89% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.13 (td, J = 1.69, 4.84 Hz, 1H), 7.89−8.00 (m, 1H), 7.37 (ddd, J = 2.15, 4.99, 7.34 Hz, 1H), 6.21−6.30 (m, 1H), 4.24 (q, J = 2.74 Hz, 2H), 3.82 (t, J = 5.38 Hz, 2H), 2.39−2.47 (m, 2H). LC−MS m/ z [M + H] 180.2. Step 2. 2-Fluoro-3-(tetrahydro-2H-pyran-4-yl)pyridine (16). To a mixture of palladium hydroxide (20% by weight palladium on carbon, 0.98 g, 1.4 mmol) and 15 (2.6 g, 15 mmol) was added EtOH (45 mL). The argon atmosphere was replaced by hydrogen from a double balloon, and the reaction mixture was stirred at room temperature. After 4 h, the reaction mixture was filtered and concentrated to give 16 (2.5 g, 96% yield) as a pale-yellow solid that was used without additional purification. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.76−1.87 (m, 4 H), 3.01−3.11 (m, 1 H), 3.50−3.62 (m, 2 H), 4.09 (dt, J = 11.35, 3.03 Hz, 2 H), 7.17 (ddd, J = 7.19, 5.14, 1.56 Hz, 1 H), 7.64 (td, J = 8.42, 1.56 Hz, 1 H), 8.07 (d, J = 4.89 Hz, 1 H). LC−MS m/z [M + H] 182.2. Step 3. (1H-Benzo[d]imidazol-2-yl)(4-(3-(tetrahydro-2H-pyran-4yl)pyridin-2-yloxy)phenyl)methanone (4). A mixture of 16 (1 g, 5.5 mmol), 23(2.6 g, 11.0 mmol), and cesium carbonate (3.6 g, 11.0 mmol) in NMP (11 mL) was heated in a microwave reactor at 180 °C for 1 h and at 200 °C for 2 h. The mixture was partitioned between water (20 mL) and DCM (30 mL). The layers were separated, and the aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification by flash chromatography on silica gel (2− 6% MeOH/DCM). The isolated material was then recrystallized from ethanol to deliver 4 (1.1 g, 2.7 mmol, 48.0% yield). 1H NMR (300 MHz, DMSO-d6) δ 13.49 (br s, 1H), 8.68 (d, J = 8.92 Hz, 2H), 8.06 (d, J = 4.58 Hz, 1H), 7.86 (d, J = 7.02 Hz, 1H), 7.31 (d, J = 8.92 Hz, 4H), 7.22 (d, J = 2.78 Hz, 1H), 3.97 (d, J = 11.40 Hz, 2H), 3.39−3.56 (m, 2H), 3.08−3.25 (m, 1H), 1.71−1.88 (m, 4H). LC−MS m/z [M + H] 400.1. HRMS (ES +) calcd for [C24H22N4O3]+ 400.1661; found 400.1658. 13C NMR (75 MHz, DMSO-d6) δ 182.6, 160.2, 159.7, 148.5, 145.4, 143.7, 137.8, 134.6, 133.5, 131.8, 130.0, 126.1, 123.6, 121.7, 121.0, 120.9, 113.3, 67.8, 35.1, 32.2. Synthesis of 1-(4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (5). Step 1. Benzyl 4(3-Fluoropyrazin-2-yl)piperidine-1-carboxylate (20a). To zinc dust (0.26 g, 4.0 mmol) at room temperature was added dimethylacetamide (2.5 mL), and 0.08 mL of a 7:5 v/v mixture of TMSCl/1,2dibromoethane was added over 1 min. After 2 min, the solution became warm and bubbled. The solution was stirred for an additional 15 min, and 18 (1.3 g, 3.3 mmol) was added dropwise over 5 min. The mixture was stirred for 30 min and added via cannula to a degassed mixture of PdCl2dppf (0.01 g, 0.01 mmol), CuI (0.02 g, 0.24 mmol), and 19 (0.51 g, 2.3 mmol) in dimethylacetamide (3 mL) at room temperature. The reaction mixture was heated to 80 °C for 75 min and diluted with EtOAc and 1 M NaH2PO4. The aqueous phase was extracted with EtOAc (2×), and the combined organic extracts were washed with brine (1×), dried over MgSO4, filtered, and concentrated. Purification by flash column chromatography on silica gel (eluted with 5−30% EtOAc in hexanes) gave 20a (0.61 g, 85% yield) as an orange oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.81−1.94 (m, 4 H), 2.96 (br s, 2 H), 3.19 (quin, J = 7.60 Hz, 1 H), 4.35 (br s, 2 H), 5.15 (s, 2 H), 7.29−7.39 (m, 5 H), 8.05 (t, J = 2.15 Hz, 1 H), 8.41 (dd, J = 4.11, 2.54 Hz, 1 H). LC−MS m/z [M + H] 316.2. Step 2. 1-(4-(3-Fluoropyrazin-2-yl)piperidin-1-yl)ethanone (21a). To a mixture of palladium hydroxide (20% by weight palladium on carbon, 19.4 g, 13.8 mmol) and 20a (57.0 g, 181 mmol) was added PhMe (700 mL) and acetic anhydride (100 mL, 1.1 mol). The argon atmosphere was replaced by hydrogen from a double balloon. Hydrogen was bubbled directly into the solution via a needle for 10 min, and the reaction mixture was stirred at room temperature. After 9 h, the solution was filtered. The filtrate was diluted with brine and EtOAc, and the pH was adjusted to 9 with 5 M NaOH. The aqueous phase was extracted with EtOAc (5×), and the combined organic extracts were washed with brine (1×), dried over MgSO4, filtered, and concentrated. Purification by flash column chromatography on silica gel (eluted with 30−80% EtOAc (10% MeOH)) gave 21a (36.8 g, 91% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.75−1.97 (m, 4 H) 2.14 (s, 3 H) 6638

dx.doi.org/10.1021/jm500713j | J. Med. Chem. 2014, 57, 6632−6641

Journal of Medicinal Chemistry

Article

2.74 (td, J = 13.10, 2.74 Hz, 1 H), 3.20−3.29 (m, 2 H), 3.97 (dm, J = 13.30 Hz, 1 H), 4.77 (dm, J = 13.11 Hz, 1 H), 8.07 (t, J = 2.15 Hz, 1 H), 8.42 (dd, J = 4.21, 2.64 Hz, 1 H). LC−MS m/z [M + H] 224.2. Step 3. 1-(4-(3-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (5). To a mixture of cesium carbonate (7.4 g, 22.6 mmol), 23 (5.3 g, 22.4 mmol), and 21a (2.5 g, 11.2 mmol) was added NMP (22 mL). The mixture was degassed by bubbling argon through the solution for 5 min and heated to 120 °C. After 5 h, the reaction was diluted with EtOAc, saturated NH4Cl, and water. The aqueous phase was extracted with EtOAc (4×). The combined organic extracts were washed with brine (1×), dried over MgSO4, filtered, and concentrated. Purification by flash column chromatography on silica gel (eluted with 50−100% EtOAc in hexanes) gave 5 (4.3 g, 87% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.81−2.06 (m, 4 H), 2.16 (s, 3 H), 2.78 (td, J = 12.86, 2.64 Hz, 1 H), 3.23−3.32 (m, 1 H), 3.38−3.51 (m, 1 H), 4.00 (dm, J = 14.28 Hz, 1 H), 4.81 (dm, J = 13.11 Hz, 1 H), 7.31 (d, J = 8.80 Hz, 2 H), 7.39 (td, J = 7.20, 1.17 Hz, 1 H), 7.45 (td, J = 7.20, 0.98 Hz, 1 H), 7.60 (d, J = 8.22 Hz, 1 H), 7.95−7.99 (m, 2 H), 8.28 (d, J = 2.74 Hz, 1 H), 8.89 (d, J = 8.10 Hz, 2 H), 10.50 (br s, 1 H). LC−MS m/z [M + H] 442.3. HRMS (ES+) calcd for [C25H24N5O3]+ 442.1879; found 442.1877. 13C NMR (101 MHz, CDCl3-d) δ ppm 21.55, 29.86, 30.15, 37.99, 41.75, 46.61, 112.00, 120.98, 122.27, 123.82, 126.50, 132.20, 133.14, 133.58, 138.99, 139.02, 144.02, 147.81, 149.91, 156.94, 158.19, 168.94, 182.19. Synthesis of 1-(4-(2-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyridin-3-yl)piperidin-1-yl)ethanone (6). Step 1. Benzyl 4(2-Fluoropyridin-3-yl)piperidine-1-carboxylate (20b). To an ovendried 25 mL round-bottomed flask was charged dry dimethylacetamide (5 mL) and zinc dust (2.1 g, 32 mmol). The reaction mixture was stirred at room temperature, while a mixture of chlorotrimethylsilane (0.33 mL, 2.6 mmol) and 1,2-dibromoethane (0.22 mL, 2.6 mmol) was added slowly. The resulting slurry was aged for 15 min. A solution of 18 (8.9 g, 25.8 mmol) in dimethylacetamide (13 mL) was then added slowly to the above mixture. After stirring for 30 min, the resulting milky solution was cooled to room temperature (solution A). To an oven-dried flask were charged 14 (3.3 g, 18.6 mmol), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (0.46 g, 0.56 mmol), copper(I) iodide (0.21 g, 1.1 mmol), and dimethylacetamide (25 mL). The resulting mixture was degassed with alternating vacuum/nitrogen purges. Then solution A from above was filtered through a fitted funnel into the reaction mixture. The resulting mixture was degassed one more time and then heated to 80 °C with stirring for 16 h. After cooling to room temperature, the reaction mixture was partitioned between EtOAc and 1 N ammonium chloride solution. The aqueous layer was back extracted with EtOAc (2×), and the combined organic layers were washed with 1 N ammonium chloride solution and brine, dried over sodium sulfate, and concentrated. The crude material as purified by chromatography using a Redi-Sep prepacked silica gel column (120 g), eluting with a gradient of 0−40% EtOAc in hexane, to provide 20b (4.1 g, 13.0 mmol, 70% yield) as light-yellow oil. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.58−1.72 (m, 2 H) 1.87 (d, J = 12.32 Hz, 2 H) 2.82− 3.03 (m, 3 H) 4.36 (m, 2 H) 5.16 (s, 2 H) 7.15 (ddd, J = 7.19, 5.13, 1.56 Hz, 1 H) 7.29−7.42 (m, 5 H) 7.59 (ddd, J = 9.59, 7.63, 1.76 Hz, 1 H) 8.07 (dt, J = 4.74, 1.44 Hz, 1 H). LC−MS m/z [M + H] 315.2. Step 2. 1-(4-(2-Fluoropyridin-3-yl)piperidin-1-yl)ethanone (21b). To a solution of 20b (2.9 g, 9.1 mmol) in tetrahydrofuran (46 mL) was added glacial acetic acid (1.1 mL, 18.3 mmol), acetic anhydride (4.3 mL, 46 mmol), and 10% palladium on carbon (0.97 g, 0.91 mmol). The reaction mixture was hydrogenated under 1 atm of hydrogen for 4 h. The reaction mixture was filtered through a pad of Celite and washed with tetrahydrofuran (3×). The filtrate was concentrated in vacuo. To the residue were added EtOAc and 1N NaOH. The aqueous layer was back extracted with EtOAc (3×), and the combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 21b (1.9 g, 8.4 mmol, 92% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.57−1.72 (m, 2 H) 1.85−2.01 (m, 2 H) 2.14 (s, 3 H) 2.65 (td, J = 12.96, 2.64 Hz, 1 H) 3.03 (tt, J = 12.23, 3.52 Hz, 1 H) 3.21 (td, J = 13.11, 2.54 Hz, 1 H) 3.95 (dt, J = 13.69, 1.96 Hz, 1 H) 4.83 (dt, J = 13.35, 2.13 Hz, 1 H) 7.16 (ddd, J = 7.14, 5.09, 1.66 Hz, 1 H) 7.60 (ddd,

J = 9.59, 7.63, 1.76 Hz, 1 H) 8.04−8.13 (m, 1 H). LC−MS m/z [M + H] 223.1. Step 3. 1-(4-(2-(4-(1H-Benzo[d]imidazole-2-carbonyl)phenoxy)pyridin-3-yl)piperidin-1-yl)ethanone (6). To a mixture of cesium carbonate (2.4 g, 7.3 mmol), 23 (1.7 g, 7.0 mmol), and 21b (0.89 g, 4.0 mmol) was added NMP (8 mL). The reaction mixture was degassed and heated to 145 °C for 3 h, 150 °C for 12 h, 180 °C for 1 h, and then at 200 °C for 2 h. The reaction mixture was diluted with EtOAc and water. The pH was adjusted to 7 using concentrated hydrogen chloride solution. The aqueous phase was extracted with EtOAc (3×), and the combined organic extracts were washed with brine (1×), dried over magnesium sulfate, filtered, and concentrated. Purification by flash column chromatography on silica gel (30−100% of 10% MeOH/EtOAc and hexanes) gave the product. The residual NMP was removed by dissolving the product in EtOAc (200 mL). The organic phase was washed with water (3×) and brine (1×), dried over magnesium sulfate, filtered, concentrated, and dried under high vacuum to give 6 (1.2 g, 2.7 mmol, 68% yield) as a white solid. 1H NMR (300 MHz, CDCl3-d) δ ppm 1.62−1.79 (m, 2 H) 1.93−2.10 (m, 2 H) 2.15 (s, 3 H) 2.69 (td, J = 12.93, 2.63 Hz, 1 H) 3.16−3.31 (m, 2 H) 3.97 (d, J = 13.74 Hz, 1 H) 4.80−4.91 (m, 1 H) 7.10 (dd, J = 7.53, 4.90 Hz, 1 H) 7.24−7.26 (m, 1 H) 7.27−7.29 (m, 1 H) 7.35−7.50 (m, 2 H) 7.56−7.64 (m, 2 H) 7.97 (d, J = 8.33 Hz, 1 H) 8.10 (dd, J = 4.82, 1.75 Hz, 1 H) 8.83−8.91 (m, 2 H) 10.38 (br s, 1 H). LC−MS m/z [M + H] 441.2. HRMS (ES+) calcd for [C26H25N4O3]+ 441.1927; found 441.1924. 13C NMR (101 MHz, CDCl3-d) δ ppm 21.57, 31.03, 32.32, 35.97, 42.13, 47.02, 112.01, 120.13−120.32, 122.20, 123.69, 126.32, 129.38, 131.31, 133.19, 133.59, 136.56, 144.00, 145.50, 147.98, 159.89, 169.01, 182.26. Previously Reported Biological Assays. Detailed descriptions of our PDE10A biochemical assay, permeability and transcellular transport assay, rat and human liver microsomal assay, and LC−MS/MS target occupancy assay have been reported in refs 35 and 39. Ex vivo Target Occupancy Assays. Animals. Adult male Sprague−Dawley rats weighing 180−225 g (Harlan, San Diego) were cared for in accordance to the Guide for the Care and Use of Laboratory Animals, 8th edition. Animals were group-housed at an Association for Assessment and Accreditation of Laboratory Animal Committee, internationally accredited facility in nonsterile ventilated microisolator housing on corn cob bedding. All research protocols were approved by the Amgen, Thousand Oaks Institutional Animal Care and Use Committee (IACUC). Animals had ad libitum access to pelleted feed (Harlan Teklad 2020X, Indianapolis, IN) and water (on-site generated reverse osmosis) via automatic watering system. Animals were maintained on a 12−12 h light−dark cycle in rooms at (70 ± 5 °F, 50 ± 20% RH) and had access to enrichment opportunities (nesting materials and plastic domes). All animals were sourced from approved vendors who meet or exceed animal health specifications for the exclusion of specific pathogens (i.e., mouse parvovirus, Helicobacter). Rats were allowed at least 3 days of acclimation prior to any procedures. Ex Vivo RO Assay with PO Administration. PDE10A inhibitors were dissolved in 2% hydroxypropylmethylcellulose (HPMC), 1% Tween-80, pH 2.2 with methanesulfonic acid. Four rats per group were dosed orally with either vehicle or 3 mg/kg PDE10A inhibitors and then returned to their home cage to allow for absorption of the compounds. After 4 h, rats were sacrificed by CO2 inhalation. Sample Analysis and Target occupancy Determination. Blood was obtained by heart puncture, and plasma was frozen and stored at −80 °C for exposure analysis. Brains were removed and immediately frozen in chilled methylbutane and stored at −80 °C until cutting. Three coronal brain slices per brain containing the striatum were cut at 20 μm using a cryostat and placed onto microscope slides, air-dried, and stored at −20 °C. For radioligand binding experiments, slides were thawed at room temperature and then incubated with 1 nM 3H-24 in binding buffer (150 mM phosphate-buffered saline containing 2 mM MgCl2 and 100 mM DTT, pH 7.4) for 1 min at 4 °C. To assess nonspecific binding, slides containing adjacent brain sections were incubated in the same solution with addition of 10 mM of test compound. Afterward, slides were washed 3 times in ice-cold binding buffer, dipped into distilled water to remove buffer salts, and dried under a stream of cold air. Emission of beta particles from the sections was counted for 8 h in a Beta Imager 6639

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2000 (Biospace, Paris, France) and digitized and analyzed using M3 Vision software (Biospace, Paris, France). Total binding radioactivity in the striatum was measured as cpm/mm2 in hand-drawn regions of interest and averaged across the three sections per brain. Nonspecific binding was subtracted to obtain specific binding values and percent occupancy was calculated by setting vehicle specific binding as 0% occupancy. Behavioral Model. Animals. All experiments were conducted under approved research protocols by Amgen’s Animal Care and Use Committee (IACUC) and in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC). Adult male Sprague−Dawley rats (250−280 g) were purchased from Harlan (Harlan, Indianapolis). Rats were group-housed on a filtered, forced air isolation rack and maintained on sterile wood chip bedding in a quiet room on a 12−12 h light−dark cycle, with food and water available ad libitum. Animals were allowed a minimum 3 days of adaptation to the laboratory conditions prior to being utilized in the experiments. Reversal of PCP-Induced Hyperlocomotor Activity. To measure locomotor activity, group-housed animals were taken from the animal colony room, weighed and numbered, and placed individually into a new cage with bedding. The cage was then placed in a 4 × 8 photobeam frame connected to a computer for activity monitoring (Home Cage Photo-Beam Activity System, San Diego Instruments, San Diego, CA). The number of photobeam breaks is correlated with locomotor activity, and total photobeam breaks were measured and recorded every 5 min. Assessment of locomotor activity occurred over 3 phases: In phase 1, animals were acclimated to the novel testing environment in the absence of any drug treatment for 60 min. To begin phase 2, animals were administered the test article (5, n = 10; risperidone, n = 9; or vehicle, n = 9), returned to the test cage, and activity was recorded for another 60 min period. Finally, in phase 3, animals were injected subcutaneously with PCP (8 mg/kg), and locomotor activity was recorded for an additional 120 min. Statistical Analysis. Behavioral results are expressed as the mean ± SEM (standard error of mean). A one-way analysis of variance (ANOVA) was used to test for a significant treatment effect on PCPinduced hyperlocomotion followed by Dunnett’s posthoc analyses comparing each drug-treated group to the Vehicle control group. All statistical analyses were performed on GraphPad Prism software, version 5 (GraphPad Inc., San Diego, CA).

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chromatography−tandem mass spectroscopy; LDA, lithium diisopropylamide; NaI, sodium iodide; NHP, nonhuman primate; NMP, N-methyl-2-pyrrolidone; PCP-LMA, phencyclidine induced locomotor activity; PDE, phosphodiesterase; QD, once a day dosing; RLM, rat liver microsome; SAR, structure− activity relationship; SOC, standard of care; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSCl, trimethylsilyl chloride; TO, target occupancy

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(1) Chappie, T. A.; Helal, C. J.; Hou, X. Current landscape of Phosphodiesterase 10A (PDE10A) inhibition. J. Med. Chem. 2012, 55, 7299−7331. (2) Soderling, H.; Bayuga, J.; Beavo, A. Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 7071−7076. (3) Seeger, T. F.; Bartlett, B.; Coskran, T. M.; Culp, J. S.; James, L. C.; Krull, D. L.; Lanfear, J.; Ryan, A. M.; Schmidt, C. J.; Strick, C. A.; Varghese, A. H.; Williams, R. D.; Wylie, P. G.; Menniti, F. S. Immunohistochemical localization of PDE10A in the rat brain. Brain Res. 2003, 985, 113−126. (4) Lakics, V.; Karran, E. H.; Boess, F. G. Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues. Neuropharmacology 2010, 59, 367−374. (5) Nishi, A.; Kuroiwa, M.; Miller, D. B.; O’Callaghan, J. P.; Bateup, H. S.; Shuto, T.; Sotogaku, N.; Fukuda, T.; Heintz, N.; Greengard, P.; Snyder, G. L. Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum. J. Neurosci. 2008, 28, 10460− 10471. (6) Siuciak, J. A.; McCarthy, S. A.; Chapin, D. S.; Fujiwara, R. A.; James, L. C.; Williams, R. D.; Stock, J. L.; McNeish, J. D.; Strick, C. A.; Menniti, F. S.; Schmidt, C. J. Genetic deletion of the striatum-entriched phosphodiesterase PDE10A: evidence for altered striatal function. Neuropharmacology 2006, 51, 374. (7) Siuciak, J.; McCarthy, S.; Chapin, D.; Martin, A.; Harms, J.; Schmidt, C. Behavioral characterization of mice deficient in the phosphodiesterase-10A (PDE10A) enzyme on a C57/Bl6N congenic background. Neuropharmacology 2007, 54, 417−427. (8) Grauer, S. M.; Pulito, V. L.; Navarra, R. L.; Kelly, M. P.; Kelley, C.; Graf, R.; Langen, B.; Logue, S.; Brennan, J.; Jiang, L.; Charych, E.; Egerland, U.; Liu, F.; Marquis, K. L.; Malamas, M.; Hage, T.; Comery, T. A.; Brandon, N. J. Phosphodiesterase 10A inhibitor activity in preclinical models of the positive, cognitive and negative symptoms of schizophrenia. J. Pharmacol. Exp. Ther. 2009, 331, 574−590. (9) Kehler, J.; Ritzén, A.; Greve, D. The potential therapeutic use of phosphodiesterase 10 inhibitors. Expert Opin. Ther. Pat. 2007, 17, 147− 158. (10) Simpson, E. H.; Kellendonk, C.; Kandel, E. A possible role for the striatum in the pathogenesis of the cognitive symptoms of schizophrenia. Neuron 2010, 65, 585−596. (11) Hebb, A. L.; Robertson, H. A. Role of phosphodiesterases in neurological and psychiatric disease. Curr. Opin. Pharmacol. 2007, 7, 86−92. (12) Menniti, F. S.; Faraci, W. S.; Schmidt, C. J. Phosphodiesterases in the CNS: targets for drug development. Nature Rev. Drug Discovery 2006, 5, 661−670. (13) Kleiman, R. J.; Kimmel, L. H.; Bove, S. E.; Lanz, T. A.; Harms, J. F.; Romegialli, A.; Miller, K. S.; Willis, A.; des Etages, S.; Kuhn, M.; Schmidt, C. J. Chronic suppression of phosphodiesterase 10A alters striatal expression of genes responsible for neurotransmitter synthesis, neurotransmission, and signaling pathways implicated in Huntingont’s Disease. J. Pharmacol. Exp. Ther. 2011, 336, 64−76. (14) As of the preparation of this manuscript, clinicaltrial.gov reported two PDE10A inhibitors, PF-02545920 and RO5545965, have entered into clinical trials. (15) Verhoest, P. T.; Chapin, D. S.; Corman, M.; Fonseca, K.; Harms, J. F.; Hou, X.; Marr, E. S.; Menniti, F. S.; Nelson, F.; O’Connor, R.; Pandit,

ASSOCIATED CONTENT

* Supporting Information S

Details of computational model of 3, methods for determination of X-ray co-crystal structure of 4 with PDE10A, and summary of data collection are provided. Methods and data for co-crystal structure of 1 were previously reported in ref 35. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

The PDB ID codes for coordinates of 1 and 4 with PDE10A are 4MVH and 4PHW, respectively.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: 805-313-5300. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; cNMP, cyclic nucleotide monophosphate; DMA, dimethylacetamide; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; HLM, human liver microsome; HPMC, hydroxyproply methcellulos; LC−MS/MS, liquid 6640

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