Identification of a novel orally bioavailable phosphodiesterase 10A (PDE10A) inhibitor with efficacy in animal models of schizophrenia.

Article pubs.acs.org/jmc

Identification of a Novel Orally Bioavailable Phosphodiesterase 10A (PDE10A) Inhibitor with Efficacy in Animal Models of Schizophrenia. José Manuel Bartolomé-Nebreda,† Sergio A. Alonso de Diego,† Marta Artola,† Francisca Delgado,† Ó scar Delgado,† María Luz Martín-Martín,† Carlos M. Martínez-Viturro,† Miguel Á ngel Pena,† Han Min Tong,† Michiel Van Gool,† José Manuel Alonso,‡ Alberto Fontana,‡ Gregor J. Macdonald,§ Anton Megens,∥ Xavier Langlois,∥ Marijke Somers,⊥ Greet Vanhoof,# and Susana Conde-Ceide*,† †

Neuroscience Medicinal Chemistry and ‡Discovery Sciences Analytical Chemistry, Janssen Research & Development, Calle Jarama 75, Polígono Industrial, Toledo 45007, Spain § Neuroscience Medicinal Chemistry, ∥Neuroscience Biology, ⊥Discovery Sciences ADME/Tox, and #Discovery Sciences Cellular Pharmacology, Janssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium S Supporting Information *

ABSTRACT: We report the continuation of a focused medicinal chemistry program aimed to further optimize a series of imidazo[1,2-a]pyrazines as a novel class of potent and selective phosphodiesterase 10A (PDE10A) inhibitors. In vitro and in vivo pharmacokinetic and pharmacodynamic evaluation allowed the selection of compound 25a for its assessment in preclinical models of psychosis. The evolution of our medicinal chemistry program, structure− activity relationship (SAR) analysis, as well as a detailed pharmacological profile for optimized lead 25a are described.

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to five different molecules have progressed into clinical evaluation to confirm this hypothesis.8 Our research group has recently reported the design, synthesis, and preliminary SAR of a series of imidazo[1,2-a]pyrazine derivatives originated from a high-throughput screening campaign resulting in the identification of compound 1 (Figure 1) as a novel, potent, selective, and orally efficacious

INTRODUCTION Cyclic nucleotides, such as cAMP and cGMP, play a prominent role as second messengers that activate several neuronal cellular signaling pathways.1 Phosphodiesterases (PDEs) are enzymes that metabolically inactivate these intracellular second messengers as such regulating and compartmentalizing the cyclic nucleotide signaling cascades. The PDE family comprises 11 members (PDE1−11) encoded by distinct genes with each of them encoding for several splice variants. Such diversity in enzyme structure outside the conserved catalytic core suggests that gene duplication and divergence have allowed for PDE specialization in different tissues. Among the different PDEs, PDE10A has one of the most restricted expression profiles almost exclusively limited to the brain and testes in different mammalian species. 2 In the brain, PDE10A is highly expressed in striatal medium spiny neurons (MSNs) which are crucial for transmission and control of glutamatergic and dopaminergic input within the basal ganglia, and hence it is believed that inhibition of PDE10A contributes to the regulation of cyclic nucleotide signaling in the corticostriato-thalamic circuit, which plays a key role in cognitive and motor processes and in emotional behaviors.3 In light of these features, during the past decade several pharmaceutical companies have initiated research programs with the aim to identify selective PDE10A inhibitors in order to evaluate their potential as novel central nervous system (CNS) therapeutic agents.4 These efforts have culminated in the identification of several structurally different PDE10A inhibitors with proved efficacy in a wide variety of preclinical models of schizophrenia5 and other neurological disorders such as Huntington’s disease (HD)6 and Lesch−Nyhan disease.7 Up © XXXX American Chemical Society

Figure 1. Compound 1 (initial hit) and compound 2.

PDE10A inhibitor.9 Encouraged by its overall promising profile, we considered to expand our SAR exploration by seeking for alternatives to the N-(methoxyethyl)pyrazole moiety present in 1, aiming to further improve its PDE10A inhibitory efficacy. For this purpose, and based on our previous SAR findings,9 we explored close analogues of the 8-morpholine-2-methylimidazo[1,2-a]pyrazine. The most relevant findings of this exploration are summarized herein. Received: October 28, 2014

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dx.doi.org/10.1021/jm501651a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Compound 9a

Reagents and conditions: (a) ethyl 2-chloroacetoacetate, EtOH, 90 °C, 18 h, 82%; (b) isopropylmagnesium chloride, N,O-dimethylhydroxylamine hydrochloride, DCM, THF, −20 °C to rt, 21 h, 68%; (c) methylmagnesium bromide, THF, −78 °C to rt, 16 h, 92%; (d) trimethylsilyl trifluoromethanesulfonate, DIPEA, DCM, 0 °C to rt, 16 h, 97%; (e) NBS, NaHCO3, THF, −78 °C, 1 h, 67%; (f) isovaleramide, 1,4-dioxane, DMF, 90 °C, 18 h, 38%.

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Scheme 2. Synthesis of Compounds 11 and 13a

CHEMISTRY As a first step, we targeted the synthesis of a small set of analogues in which the pyrazole ring was replaced by other fivemembered heterocycles such as oxazole, thiazole, or pyrrole. Prompted by synthetic accessibility, and supported by the in vitro PDE10A inhibitory activity of the corresponding (isobutyl)pyrazole-holding prototype 2,9 an isobutyl side chain was incorporated on the oxazole and thiazole containing analogues. The synthesis of the oxazole derivative, outlined in Scheme 1, commenced with the reaction of commercially available 2-amino-4-morpholinepyrazine 3 with ethyl 2-chloroacetoacetate to give the corresponding 2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine-3-carboxylic acid ethyl ester 4. Activation of the ester via the Weinreb amide 5, followed by treatment with methylmagnesium bromide, gave ketone 6, which was then selectively monobrominated at α position after its conversion into the corresponding trimethylsilyl enol ether 7 and subsequent reaction with N-bromosuccinimide (NBS). Finally, the reaction of bromo ketone 8 with isovaleramide led to the desired compound 9. The thiazole-containing derivative 11 was prepared via C−H activation and coupling of the 2-isobutylthiazole with precursor bromoheteroarene 1010 catalyzed by palladium acetate and in the presence of tert-butyldicyclohexylphosphine (Scheme 2). The preparation of the corresponding pyrrole analogue 13 was accomplished via Suzuki−Miyaura cross-coupling of 10 with the 1-(triisopropylsilyl)pyrrole-3-boronic acid and subsequent alkylation with 2-bromoethyl methyl ether to yield compound 13 (Scheme 2). We next turned our attention to pyridines as possible replacements for the pyrazole ring. The key iodo intermediate 15 was prepared in two steps in a similar way as previously described for the synthesis of bromo derivative 10,9 selective iodination of 1411 at 3-position with N-iodosuccinimide (NIS),12 followed by nucleophilic substitution with morpholine. From this common intermediate different synthetic strategies were implemented depending upon the substitution on the pyridine derivative targeted (see Schemes 3 and 5). Thus, Suzuki−Miyaura cross-coupling of iodide 15 with 2-chloropyridyl-4-boronic acid yielded compound 16, and subsequent Suzuki−Miyaura cross-coupling with vinylboronic acid pinacolester followed by addition of sodium methoxide afforded compound 17 (Scheme 3).

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Reagents and conditions: (a) 2-isobutylthiazole, Pd(OAc)2 , K3PO4, tert-butyldicyclohexylphosphine, NMP, 125 °C, 18 h, 29%; (b) 1-(triisopropylsilyl)pyrrole-3-boronic acid, PdCl 2 (PPh 3 ) 2 , Na2CO3, 1,4-dioxane, 100 °C, 16 h, 82%; (c) 2-bromoethyl methyl ether, Cs2CO3, DMF, 160 °C, 30 min, microwave irradiation, 73%.

The synthetic route followed to prepare compound 22 was slightly different. In this case, the Wittig reaction of the commercially available 6-bromonicotinaldehyde 18 with (methoxymethyl)triphenylphosphonium chloride followed by hydrogenation of the double bond formed in the presence of rhodium as catalyst afforded 20. The transformation of bromopyridine 20 into stannanyl derivative 21 followed by Stille coupling yielded the desired analogue 22 (see Scheme 4). To further expand the exploration of regioisomeric C-alkylated pyridines, a similar approach as reported for the synthesis of compound 17 was followed. Suzuki−Miyaura cross-coupling of iodo derivative 15 with 2-chloropyridine-5boronic acid afforded intermediate 23, key in order to prepare a variety of analogues as depicted in Scheme 5. Chloropyridine 23 was coupled with the vinylboronic acid pinacolester, affording compound 24 that was reacted with MeOH in the presence of potassium hydrogen sulfate to yield 25a, was reacted with a variety of different alcohols in basic media to obtain compounds 25b−c, was treated with potassium hydrogen sulfate and water to afford the hydroxy derivative 25d, or was hydrogenated in the presence of Pd/C to yield the corresponding ethyl analogue 25e. Analogues 25f−k were prepared via B



Article

Scheme 3. Synthesis of 17a

a Reagents and conditions: (a) NIS, DCM, 0 °C, 2 h, 97%; (b) morpholine, DIPEA, ACN, 80 °C, 16 h, 80%; (c) 2-chloropyridine-4-boronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 90−110 °C, 10.5 h, 83%; (d) vinylboronic acid pinacolester, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 2 h, 78%; (e) MeONa, MeOH, 100 °C, 18 h, 25%.

Scheme 4. Synthesis of 22a

Reagents and conditions: (a) (methoxymethyl)triphenylphosphonium chloride, nBuLi, THF, 0 °C to rt, 16 h, 73%; (b) Rh/C 5% cartridge, EtOH, full H2 mode, 70 °C, H-Cube, 21%; (c) Bu3SnCl, nBuLi, THF, −78 °C to rt, 1 h, 100%; (d) 21, Pd(PPh3)4, CuBr, 1,4-dioxane, 160 °C, 20 min, microwave irradiation, 11%. a

terms of their extraction ratio (ER) values and intrinsic clearance (Clint), and those considered sufficiently metabolically stable (ER < 0.5 and Clint < 40 μL/min/mg at 1 μM concentration) were then tested in vivo for their ability to revert the apomorphine-induced stereotypy in rats, a model that has been extensively used to assess potential antipsychotic efficacy.16 Among the 5-member ring heterocycles evaluated as possible replacements of the pyrazole ring, the oxazole and thiazole containing analogues 9 and 11 maintained comparable in vitro potency to prototype 2 while the pyrrole derivative 13 showed decreased activity compared to lead 1. Unfortunately, neither 9 nor 11 were found metabolically stable enough to be considered for in vivo evaluation (see Table 1). In the case of the replacement of the methoxyethyl pyrazole moiety by pyridine, the three regioisomeric derivatives 17, 22, and 25a showed comparable in vitro activity with the initial lead 1 and displayed acceptable metabolic stability (Table 1) to be considered for in vivo evaluation. The 3-pyridine analogue 25a was found the most potent within the set with an in vivo activity ∼3.5 fold higher than 1 (ED50 = 1.02 mg/kg). With this promising result in hand, the possibility to explore diversity on the 3-pyridine was considered attractive and SAR development continued along those lines. As in the case of the pyrazole analogues previously reported,9 a wide range of variations of the methoxyethyl side chain was allowed (Table 1).17 The elongation of the methoxy group to an ethoxy afforded compound 25b that showed a similar profile to 25a while the incorporation of an isopropoxy group yielded compound 25c that was not metabolically stable enough to be further profiled. Compound 25d, where the methoxy group was replaced by a hydroxy group while being much more stable metabolically lost in vivo activity. The introduction of two gem-methyls on α position to the methoxy moiety did not have a detrimental impact on the in vitro profile and 25f presented similar in vivo

Suzuki−Miyaura cross-coupling of the corresponding bromo intermediate 10 with the corresponding pyridinylboronic ester derivatives.13 Alternatively, other side chains were introduced on the pyridine by reaction with various Grignard derivatives (compounds 25l,m), through Suzuki−Miyaura cross-coupling reaction (compound 25n) or via nucleophilic substitution with different amines (compounds 25o−p,r) or Buchwald type reaction (compound 25q). To further explore pyridine derivatives, we synthesized analogues modified at 2- and 8-position of the central scaffold. These analogues, 28a−g, were prepared via Suzuki−Miyaura cross-coupling of the corresponding bromo derivatives 26a−g14 with the 2-(2-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (27)15 (Scheme 6). Finally, derivatives modified at C-6 of the scaffold were prepared starting from the 3-chloro-5-iodo-pyrazin-2-amine (29) as outlined in Scheme 7. Reaction with 2-chloroacetone followed by bromination at the 3-position with NBS afforded bicycle 30. Selective substitution of the chlorine atom by morpholine yielded 31. The introduction of a methyl group at the 6-position was accomplished by palladium catalyzed reaction of the iodo derivative 31 with methylindium generated in situ. The trifluoromethyl group was introduced via coppermediated cross coupling of 31 with methyl 2,2-difluoro-2(fluorosulfonyl)acetate (MDFA). Finally, Suzuki−Miyaura cross-coupling of 26h and 26i with pyridine 27 yielded, respectively, compounds 28h and 28i.

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RESULTS AND DISCUSSION The most relevant findings of this exploration are summarized in Tables 1 and 2. Compounds were first evaluated for their in vitro PDE10A inhibitory activity. Compounds with a pIC50 > 6.5 were selected to assess their in vitro metabolic stability in rat liver microsomes preparations. Compounds were ranked in C



Article

Scheme 5. Synthesis of 25a−ra

Reagents and conditions: (a) 2-chloropyridine-5-boronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 23 h, 93%; (b) vinylboronic acid pinacolester, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C, 1 h, 90%; (c) for 25a, KHSO4, MeOH, 80 °C, 3 days, 63%; for 25b, EtONa, EtOH, 100 °C, 16 h, 47%; for 25c, 2-propanol, NaH, 100 °C, 18 h, 25%; for 25d, KHSO4, water, 80 °C, 16 h, 18%; for 25e, Pd/C 10%, H2 atm, EtOH/EtOAc/ DCM, rt, 16 h, 91%; (d) heteroarylboronic derivative, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 140−150 °C, 15−20 min, microwave irradiation or 90 °C, 18 h, 11−94%; (e) For 25l, isopropylmagnesium chloride, Ni(dppf)2, THF, 0 °C to rt, 30 min, 56%; for 25m, isobutylmagnesium chloride, Ni(dppf)2, THF, 0 °C to rt, 3 h, 56%; for 25n, cyclopropylboronic acid, 2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl, Pd(OAc)2, K3PO4, toluene, 80 °C, 22 h, 29%; for 25o, pyrrolidine, 130 °C, 30 min, microwave irradiation, 60%; for 25p, (i) (R)-3-pyrrolidinol, 120 °C, 3 h, (ii) NaH, MeI, THF, rt, 3 days, 19%; for 25q, isopropylamine, Pd(OAc)2, Binap, Cs2CO3, toluene, 50 °C, 16 h, 42%; for 25r, piperazine, 120 °C, 24 h, 22%. a

but did not show in vivo activity in the apomorphine model at the dose tested. The relatively unresponsive in vitro pyridine side chain SAR observed in the reported analogues could be explained based on the fact that the R group of the pyridine, as in the case of the pyrazole series reported in the previous paper, gets oriented toward the solvent with no significant interactions with the protein. To determine levels of PDE10A occupancy, we evaluated the most potent compounds from the induced stereotypy model (25a, 25b, 25f, and 25i) in a central in vivo occupancy assay that measures the displacement of the selective PDE10A ligand [3H]MP-10.18 The specific binding was determined as the difference between [3H]MP-10 binding quantified in the striatum (a brain area showing a high density of PDE10A enzyme) and in the cortex (a brain area where PDE10A is virtually absent). Occupancy ED50 was calculated as the inhibition of specific [3H]MP-10 binding in drug treated animals relative to vehicle-treated animals. Gratifyingly, occupancy data for compounds 25a, 25b, and 25f confirmed target engagement in the brain at comparable ED50s to our behavioral model. Unfortunately, and due to unknown reasons, PDE10A occupancy could not be confirmed for the in vivo most potent analogue, 25i, and therefore it was discarded for additional profiling. Out of the remaining derivatives tested, compound 25a proved to be the most promising analogue with

potency to compound 25a. Elongation of the side chain by one carbon atom (compound 25g) or introduction of an oxygen linker on the β-position with regard to the pyridine ring (compound 25h) had no significant impact on the measured in vitro properties, but both compounds lacked in vivo activity at the dose tested. Truncation of the side chain to a methyl (compound 25k) or an ethyl (compound 25e) or the incorporation of other small alkylic groups, such as an isoPr (compound 25l), a cycloPr (compound 25n), or an isoBu compound (25m), had little effect on in vitro primary activity. These compounds were not further profiled due to lack of adequate metabolic stability (25k−m) or in vitro potency (25n). Additional SAR was carried out by the introduction of an oxygen atom as linker. Ether 25i resulted in an attractive compound with an in vivo ED50 of 0.79 mg/kg, while analogue 25j possessed much attenuated in vitro metabolic stability under microsomal incubation and was not further profiled. Likewise, the incorporation of nitrogen containing side chains in pyrrolidine analogues 25o and 25p resulted in different degrees of metabolic stability. The methoxy-substituted pyrrolidine, 25p, is more stable than the unsubstituted pyrrolidine, 25o. Unfortunately, 25p lacked relevant in vivo efficacy. Isopropylamine derivative 25q was highly unstable, while piperazine analogue 25r, sterically similar to the pyrrolidine 25o, was highly stable in microsomes incubation D



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Scheme 6. Synthesis of 28a−ga

increase of in vitro activity vs 25a (0.3 log units), an effect that translated into a similar in vivo potency and occupancy compared with 25a. Unfortunately, compound 28c showed undesirable side effects in rats after administration of a single dose of 40 mg/kg sc (passivity, hypotonia, flat body posture, hypothermia, sedation) and was not further progressed. In a similar fashion as previously described for our initial hit 1,9 we also explored the possibility to replace the morpholine moiety by other substituents. In this case, replacement by pyrrolidine (28e), 4-pyridine (28f), or 3-pyridine (28g) was found detrimental for the in vitro potency. Finally, introduction of small groups, such as methyl (28h) or trifluoromethyl (28i) on the 6-position of the central scaffold was not well-tolerated and led to a decrease in in vitro potency. At this point in time and because none of the modifications tried was successful, compound 25a was selected to be further profiled. Further profiling of 25a confirmed a high degree of PDE10A selectivity (79-fold toward PDE1B, more than 400-fold toward others PDEs), high selectivity against a selection of GPCRs, ion channels, and transporters (CEREP radioligand binding panel, IC50 > 10 μM), and no relevant inhibition (>50% inhibition at 10 μM) of a selection of kinases in Millipore panel.19 In vitro ADME evaluation revealed a clean CYP inhibition profile (IC50 all tested CYPs > 10 μM), a low binding to plasma proteins (hPPB 58% and rPPB 60% bound) and to brain tissue (hBTB 69% and rBTB 85% bound) in the different species tested. We also explored its pharmacokinetic properties in rats. Absorption of 25a following oral administration of a single dose (10 mg/kg) resulted in an absolute bioavailability of 45%. Compound 25a showed moderate clearance (34 mL/min/kg), low volume of distribution (1.9 L/kg), and a moderate short terminal half-life (0.8 h). Additionally, 25a showed good brain exposure, reaching total levels of 1212 ng/g in the brain after 1 h (10 mg/kg sc). On the basis of these data, the compound was deemed an excellent candidate for evaluation in acute behavioral studies in rats.20

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Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, 1,4-dioxane, 150 °C, 15 min, microwave irradiation, 48−90%.

CONCLUSION In conclusion, SAR expansion of our initial lead 1 focused on the replacement of its pyrazole moiety by other heterocycles, resulting in the identification of 25a. Compound 25a shows improved efficacy in animal models believed to mimic the positive symptoms of schizophrenia than the initial lead 1 and confirmed PDE10A engagement in the brain. Selectivity profiling, in vitro ADME, and in vivo PK evaluation in rats confirmed 25a as an excellent candidate for extensive efficacy and side effect profiling evaluation in acute behavioral studies in rats.20

improved in vivo potency and occupancy over compound 1 (see Table 1) Table 2 summarizes some results of our subsequent efforts aimed to further increase the potency of compound 25a. Keeping the methoxyethylpyridine fixed at the 3-position, we first evaluated the influence of the introduction of small groups (CF3, isoPr, cycloPr, OMe) on the 2-position of the scaffold. Among them, the best results were obtained by the introduction of a cyclopropyl substituent (28c), resulting in a moderate Scheme 7. Synthesis of 28h,ia

Reagents and conditions: (a) 2-chloroacetone, NaI, 90 °C, 24 h, 28%; (b) NBS, DCM, rt, 3 h, 83%; (c) morpholine, DIPEA, ACN, 160 °C, 30 min, microwave irradiation, 83%; (d) For 26h, InCl3, MeLi, THF, −78 °C, 30 min then Pd(PPh3)4, 80 °C, 16 h, 78%; for 26i, MDFA, CuI, DMF, 90 °C, 16 h, 60%; (e) 27, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 150 °C, 15−30 min, microwave irradiation, 82%.

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Table 1. PDE10A Inhibitory Activity of the 5 and 6-Member Ring Heterocyclic Systems

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Table 1. continued

Values are mean ± SD of at least two experiments. bData refer to compound metabolized after incubation of tested compound with rat liver microsomes, at 1 μM concentration across a time course (typically 0, 5, 10, 20, 40, and 60 min) (n = 1 per dose). cWistar rats (n = 3 per dose) were pretreated with test compound (sc) or solvent and after 30 min, apomorphine (1.0 mg/kg, iv) induced stereotypy was scored every 5 min over the first hour after injection of apomorphine. dCompound 25i was administered oral (po). eDose−response experiments to measure PDE10 occupancy were performed 1 h after sc administration (n = 3 per dose). a

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Labstation (Milestone, Inc.). Nuclear magnetic resonance (NMR) spectra were recorded with either a Bruker DPX-400 or a Bruker AV-500 spectrometer (Bruker AG) with standard pulse sequences operating at 400 and 500 MHz, respectively, using CDCl3 and DMSOd6 as solvents. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane (δ = 0). Coupling constants are reported in hertz. Splitting patterns are defined by s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), tt (triplet of triplet), spt (septuplet), dquin (doublet of quintet), dt (doublet of triplet),

EXPERIMENTAL SECTION

General Procedures. Unless otherwise noted, all reagents and solvents were obtained from commercial suppliers and used without further purification. Thin layer chromatography (TLC) was carried out on silica gel 60 F254 plates (Merck). Flash column chromatography was performed on silica gel, particle size 60 Å, mesh of 230−400 (Merck), under standard techniques. Microwave assisted reactions were performed in a single-mode reactor, Biotage Initiator Sixty microwave reactor (Biotage), or in a multimode reactor, MicroSYNTH G



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Table 2. PDE10A Inhibitory Activity of Compounds 28a−i

Values are mean ± SD of at least two experiments. bData refer to compound metabolized after incubation of tested compound with rat liver microsomes, at 1 μM concentration across a time course (typically 0, 5, 10, 20, 40, and 60 min) (n = 1 per dose). cWistar rats (n = 3 per dose) were pretreated with test compound (sc) or solvent and after 30 min, apomorphine (1.0 mg/kg, iv) induced stereotypy was scored every 5 min over the first hour after injection of apomorphine. dDose−response experiments were performed to measure PDE10A occupancy 1 h after sc administration (n = 3 per dose).. a

br (broad signal), or m (multiplet). Purities of all new compounds were determined by analytical reverse phase RP (HPLC or UPLC) coupled to a mass spectrometry detector, using the area percentage method on the DAD trace, and were found to have ≥95% purity unless otherwise specified. The HPLC or UPLC measurement was performed using either an HP1100 (Agilent Technologies) system or an Acquity UPLC system (Waters) comprising a pump (quaternary or binary) with degasser, an autosampler, a column oven, a diode array detector (DAD), and a column as specified in the respective methods. The MS detector (LCT, SQD, MSD) was configured with an electrospray ionization source. Nitrogen was used as the nebulizer gas. Data

acquisition was performed with MassLynx-Openlynx software or Chemstation-Agilent Data Browser software. Compounds are described by their experimental retention times (tR) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M + H]+ (protonated molecule). For molecules with multiple isotopic patterns (Br, Cl, ...) the reported value is the one obtained for the lowest isotope mass unless otherwise specified. All results were obtained with experimental uncertainties that are commonly associated with the method used. Low-resolution MS is given for the compounds except for the ones analyzed on the LCT detector which are validated by high-resolution MS. Detailed H



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information about the different LCMS methods employed can be found in the Supporting Information. Melting point (mp) values are peak values and were obtained with experimental uncertainties that are commonly associated with this analytical method. Melting points were determined in open capillary tubes performing the experiment up to a maximum temperature of 300 °C, measuring them either on a Mettler apparatus (FP62 or FP 81HT/FP90 with a temperature gradient of 10 °C/min) or on a Shanghai Precision/Scientific Instrument Co. Ltd. apparatus (WRS-2A with a temperature gradient of 0.2−5.0 °C/min). 2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine-3-carboxylic Acid Ethyl Ester (4). A mixture of 3-morpholinopyrazin-2-amine 3 (2 g, 11.1 mmol) and ethyl 2-chloroacetoacetate (7.7 mL, 55.5 mmol) in EtOH (8 mL) was stirred at 90 °C for 18 h. The mixture was cooled down to rt and diluted with Et2O. The solid formed was filtered off and dried in vacuo to yield intermediate 4 (2.98 g, 82%) as a white solid (hydrochloride salt). MS: m/z 291 [M + H]+. tR = 1.25 min (method 8). 1H NMR (500 MHz, CDCl3) δ ppm 1.46 (t, J = 7.2 Hz, 3 H), 2.69 (s, 3 H), 3.86−4.00 (m, 4 H), 4.47 (q, J = 7.1 Hz, 2 H), 4.50 (br s, 4 H), 7.56 (d, J = 4.9 Hz, 1 H), 8.65 (d, J = 4.9 Hz, 1 H). 2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine-3-carboxylic Acid Methoxy-methyl-amide (5). A 2 M solution of isopropylmagnesium chloride in THF (10.33 mL, 20.67 mmol) was added over 15 min to a stirred suspension of intermediate 4 (2 g, 6.89 mmol) and N,Odimethylhydroxylamine hydrochloride (1.0 g, 10.33 mmol) in a mixture of THF (15 mL) and DCM (8 mL) at −20 °C under nitrogen. The mixture was stirred at −5 °C for 1 h and then allowed to warm to rt and stirred for a further 16 h. The mixture was cooled to −20 °C and further N,O-dimethylhydroxylamine hydrochloride (1 g, 10.33 mmol) and a 2 M solution of isopropylmagnesium chloride in THF (10.33 mL, 20.67 mmol) were added. The mixture was stirred at −20 °C for 5 min, allowed to warm to rt, and then stirred for a further 5 h. The mixture was cooled to −10 °C, and a saturated solution of ammonium chloride was added. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 50/50). The desired fractions were collected and evaporated in vacuo to yield intermediate 5 (1.43 g, 68%) as a pink solid. MS: m/z 306 [M + H]+. tR = 1.99 min (method 5). 1H NMR (400 MHz, CDCl3) δ ppm 2.50 (s, 3 H), 3.41 (s, 3 H), 3.54 (s, 3 H), 3.88 (t, J = 4.9 Hz, 4 H), 4.21 (t, J = 4.9 Hz, 4 H), 7.42 (d, J = 4.6 Hz, 1 H), 7.83 (d, J = 4.4 Hz, 1 H). 1-(2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-ethanone (6). A 1.4 M solution of methylmagnesium bromide in THF (4.3 mL, 5.96 mmol) was added to a stirred solution of intermediate 5 (1.4 g, 4.59 mmol) in THF (30 mL) at −78 °C, under nitrogen. The mixture was allowed to warm to rt and then stirred for 16 h. A saturated solution of ammonium chloride was added, and the mixture was extracted with EtOAc. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 40/60). The desired fractions were collected and evaporated in vacuo to yield intermediate 6 (1.1 g, 92%, 71% pure) as a white solid. MS: m/z 261 [M + H]+. tR = 1.98 min (method 5). 1H NMR (400 MHz, CDCl3) δ ppm 2.62 (s, 3 H), 2.76 (s, 3 H), 3.88 (t, J = 4.9 Hz, 4 H), 4.20 (dd, J = 5.1, 4.6 Hz, 4 H), 7.57 (d, J = 4.6 Hz, 1 H), 8.95 (d, J = 4.6 Hz, 1 H). 2-Methyl-8-morpholin-4-yl-3-(1-trimethylsilanyloxy-vinyl)imidazo[1,2-a]pyrazine (7). Trimethylsilyl trifluoromethanesulfonate (2.23 mL, 12.3 mmol) and N,N-diisopropyl-ethylamine (2.84 mL, 16.3 mmol) were added to a stirred solution of intermediate 6 (0.8 g, 3.1 mmol) in DCM (12 mL). The mixture was stirred at 0 °C for 1.5 h, allowed to warm to rt, and then stirred for a further 16 h. The mixture was partitioned between a cold saturated solution of sodium hydrogen carbonate and DCM. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo to yield intermediate 7 (0.99 g, 97%) as a colorless oil which was used in next step without further purification. 2-Bromo-1-(2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)ethanone (8). N-Bromosuccinimide (0.224 g, 1.26 mmol) and sodium

hydrogen carbonate (0.192 g, 2.29 mmol) were added to a stirred solution of intermediate 7 (0.38 g, 1.14 mmol) in THF (8 mL). The mixture was stirred at −78 °C for 1 h and then diluted with Et2O and extracted with a cold saturated solution of sodium hydrogen carbonate. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 10/90). The desired fractions were collected and evaporated in vacuo to yield intermediate 8 (0.26 g, 67%) as a pale-yellow solid. MS: m/z 339 [M + H]+. tR =2.33 min (method 6). 1H NMR (500 MHz, CDCl3) δ ppm 2.82 (s, 3 H), 3.87 (t, J = 4.9 Hz, 4 H), 4.22 (br t, J = 4.6, 4.6 Hz, 4 H), 4.35 (s, 2 H), 7.63 (d, J = 4.6 Hz, 1 H), 8.91 (d, J = 4.3 Hz, 1 H). 3-(2-Isobutyl-oxazol-4-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (9). Isovaleramide (0.082 g, 0.81 mmol) was added to a stirred solution of intermediate 8 (0.25 g, 0.74 mmol) in 1,4-dioxane (5 mL). The mixture was stirred at 90 °C for 18 h under nitrogen, and then the solvent was evaporated in vacuo and DMF (5 mL) and further isovaleramide (0.082 g, 0.81 mmol) were added. The mixture was stirred at 90 °C for a further 24 h, further isovaleramide (0.082 g, 0.81 mmol) was added and the mixture was stirred at 90 °C for a further 24 h. The mixture was diluted with Et2O and extracted with water. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 50/50). The desired fractions were collected and evaporated in vacuo to yield compound 9 (95 mg, 38%) as a white solid. MS: m/z 342 [M + H]+. tR = 3.85 min (method 5). 1H NMR (400 MHz, CDCl3) δ ppm 1.04 (d, J = 6.7 Hz, 6 H), 2.24 (spt, J = 6.8 Hz, 1 H), 2.51 (s, 3 H), 2.75 (d, J = 7.2 Hz, 2 H), 3.88 (t, J = 4.9 Hz, 4 H), 4.22 (t, J = 4.6 Hz, 4 H), 7.40 (d, J = 4.6 Hz, 1 H), 7.76 (s, 1 H), 8.30 (d, J = 4.4 Hz, 1 H). Regioselectivity confirmed by NOE between methyl group on imidazopyrazine core and CH of the oxazole ring; mp 121 °C. 3-(2-Isobutyl-thiazol-5-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (11). Palladium(II) acetate (0.01 g, 0.05 mmol) and tert-butyldicyclohexylphosphine (0.027 mL, 0.1 mmol) were added to a stirred solution of intermediate 10 (0.3 g, 1.01 mmol), 2isobutylthiazole (0.142 g, 1.01 mmol), and potassium phosphate (0.428 g, 2.01 mmol) in NMP (4 mL). The mixture was stirred at rt for 15 min under nitrogen and then at 125 °C for 18 h. The mixture was diluted with Et2O and washed with a 1% solution of potassium hydroxide. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 40/60). The desired fractions were collected and the solvents evaporated in vacuo. Then the crude product was purified by RP HPLC (0.1% solution of ammonium bicarbonate/ammonium hydroxide buffer pH 9 in ACN 80/20 to 0/100). The desired fractions were collected and evaporated in vacuo to yield compound 11 (0.10 g, 29%) as a yellow solid. ESI-HRMS: m/z for C18H24N5OS 358.1707 [M + H]+ calcd, 358.1701; found, 358.1707 (1.7 ppm). tR = 4.68 min (method 2). 1H NMR (400 MHz, CDCl3) δ ppm 1.06 (d, J = 6.5 Hz, 6 H), 2.19 (spt, J = 6.7 Hz, 1 H), 2.46 (s, 3 H), 2.95 (d, J = 7.2 Hz, 2 H), 3.88 (br t, J = 4.9 Hz, 4 H), 4.26 (br t, J = 4.9 Hz, 4 H), 7.39 (d, 1 H), 7.48 (d, J = 4.4 Hz, 1 H), 7.71 (s, 1 H). Regioselectivity confirmed by HSQC, CH of thiazole ring was detected at 143 ppm; mp 84.1 °C. 2-Methyl-8-morpholin-4-yl-3-(1H-pyrrol-3-yl)-imidazo[1,2-a]pyrazine (12). Dichlorobis(triphenylphosphine)palladium(II) (0.008 g, 0.012 mmol) was added to a stirred solution of intermediate 10 (0.071 g, 0.24 mmol) and (triisopropylsilyl)pyrrole-3-boronic acid (0.096 g, 0.36 mmol) in a mixture of 1,4-dioxane (2 mL) and a 1 M solution of sodium carbonate (0.72 mL, 0.72 mmol). The mixture was stirred at 100 °C for 16 h, and then the solid formed was filtered off and the filtrate was evaporated. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 40/60). The desired fractions were collected and evaporated in vacuo to yield compound 12 (0.056 g, 82%) as a white solid. ESI-HRMS: m/z for C15H18N5O [M + H]+ calcd, 284.1511; found, 284.1483 (−9.9 ppm). tR = 2.74 min (method 1). 1H NMR (500 MHz, DMSO-d6) δ ppm 2.38 (s, 3 H), 3.74 (t, J = 4.6 Hz, 4 H), 4.14 (t, J = 4.9 Hz, 4 H), 6.33 (q, J = 2.3 Hz, 1 H), 6.98 (q, I



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organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 20/80 to 70/30). The desired fractions were collected and evaporated in vacuo to yield 2-methyl-8morpholin-4-yl-3-(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine (0.23 g, 78%, 87% pure) as a white solid. MS: m/z 322 [M + H]+. tR =2.28 min (method 5). Step 2: Synthesis of 17. Sodium methoxide (0.22 g, 4.04 mmol) was added to a stirred solution of 2-methyl-8-morpholin-4-yl-3(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine (0.23 g, 0.67 mmol) in MeOH (8 mL). The mixture was stirred at 100 °C for 18 h in a sealed tube and then poured into a saturated solution of sodium hydrogen carbonate and extracted with DCM. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH and EtOAc in DCM 0/50/50 to 10/90/0). The desired fractions were collected and the solvents evaporated in vacuo to yield compound 17 (60 mg, 25%) as a white solid. MS: m/z 354 [M + H]+. tR = 1.92 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.49 (s, 3 H), 3.14 (t, J = 6.4 Hz, 2 H), 3.38 (s, 3 H), 3.83 (t, J = 6.4 Hz, 2 H), 3.86−3.93 (m, 4 H), 4.23−4.30 (m, 4 H), 7.22 (dd, J = 5.2, 1.5 Hz, 1 H), 7.31 (s, 1 H), 7.37 (d, J = 4.6 Hz, 1 H), 7.54 (d, J = 4.6 Hz, 1 H), 8.69 (d, J = 5.1 Hz, 1 H). 2-Bromo-5-(2-methoxy-vinyl)-pyridine (19). A 2.5 M solution of n-butyllithium in hexanes (9.94 mL, 24.8 mmol) was added dropwise to a stirred solution of (methoxymethyl)triphenylphosphonium chloride (8.51 g, 24.8 mmol) in THF (150 mL) at 0 °C, and then 6-bromonicotinaldehyde (3.3 g, 17.7 mmol) was slowly added to the red mixture. The mixture was stirred at rt for 16 h and then diluted with Et2O and washed with water. The aqueous layer was extracted with DCM, and the organic layer was dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; DCM in heptane 0/100 to 50/50). The desired fractions were collected and the solvents evaporated in vacuo to yield intermediate 19 (2.8 g, 73%, 81% pure) as a mixture 57/43 of E and Z isomers. MS: m/z 214 [M + H]+. tR = 2.13/2.23 min (sterochemistry not assigned) (method 5). 2-Bromo-5-(2-methoxy-ethyl)-pyridine (20). A solution of intermediate 19 (2.3 g, 10.7 mmol) in EtOH (100 mL) was hydrogenated in a H-cube reactor (1.5 mL/min, Rh/C 5% cartridge, full H2 mode, 70 °C, 3 cycles). The solvent was evaporated in vacuo and the crude product purified by flash column chromatography (silica; EtOAc in heptane and DCM 0/50/50 to 0/0/100 to 20/0/80). The desired fractions were collected and evaporated in vacuo to yield intermediate 20 (0.48 g, 21%) as a colorless oil. MS: m/z 216 [M + H]+. tR = 1.83 min (method 5). 5-(2-Methoxy-ethyl)-2-tributylstannanyl-pyridine (21). A 2.5 M solution of n-butyllithium in hexanes (1.1 mL, 2.72 mmol) was added dropwise to a solution of intermediate 20 (0.245 g, 1.13 mmol) in THF (10 mL). The mixture was stirred at −78 °C for 1 h, and then tributyltin chloride (0.74 mL, 2.72 mmol) was slowly added. The mixture was allowed to warm to rt over 1 h, and then a saturated solution of ammonium chloride was added. The mixture was extracted with Et2O and EtOAc. The combined organic layers were dried (Na2SO4), filtered, and the solvents evaporated in vacuo to yield intermediate 21 (0.72 g, quantitative, 67% pure), which was used in the next step without any further purification. ESI-HRMS (120Sn): m/z for C20H38NOSn [M + H]+ calcd, 428.1975; found, 428.1945 (7.0 ppm). tR = 3.91 min (method 2). 3-[5-(2-Methoxy-ethyl)-pyridin-2-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (22). Intermediate 10 (0.392 g, 1.32 mmol), tetrakis(triphenylphosphine)palladium (0) (0.038 g, 0.032 mmol), and copper(I) bromide (0.010 g, 0.066 mmol) were added to a stirred solution of 5-(2-methoxy-ethyl)-2-tributylstannanyl-pyridine (21) (0.468 g, 1.1 mmol) in 1,4-dioxane (20 mL). The mixture was stirred at 160 °C for 20 min in a sealed tube under nitrogen and under microwave irradiation, and then the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 40/60 to 90/10). The desired fractions were collected, and the solvents evaporated in vacuo. The crude product was purified again by flash column chromatography (silica; 7 M solution of

J = 2.5 Hz, 1 H), 7.12 (q, J = 1.7 Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H), 7.71 (d, J = 4.6 Hz, 1 H), 11.30 (br s, 1 H). 3-[1-(2-Methoxy-ethyl)-1H-pyrrol-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (13). 2-Bromoethyl methyl ether (0.024 mL, 0.254 mmol) and cesium carbonate (0.088 g, 0.271 mmol) were added to a stirred solution of compound 12 (0.048 g, 0.169 mmol) in DMF (3 mL). The mixture was stirred at 160 °C for 30 min under nitrogen and under microwave irradiation, and then further 2-bromoethyl methyl ether (0.072 mL, 0.762 mmol) was added. The mixture was stirred at 160 °C for a further 30 min under microwave irradiation, and then the solid formed was filtered off and the filtrate evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 40/60). The desired fractions were collected and evaporated in vacuo to yield compound 13 (0.042 g, 73%) as an oil. ESI-HRMS: m/z for C18H24N5O2 [M + H]+ calcd, 342.1930; found, 342.1983 (15.5 ppm). tR = 3.48 min (method 1). 1H NMR (500 MHz, DMSO-d6) δ ppm 2.38 (s, 3 H), 3.27 (s, 3 H), 3.66 (t, J = 5.3 Hz, 2 H), 3.74 (br t, J = 4.6 Hz, 4 H), 4.04−4.25 (m, 6 H), 6.30 (dd, J = 2.6, 1.7 Hz, 1 H), 6.99 (t, J = 2.3 Hz, 1 H), 7.16 (t, J = 1.7 Hz, 1 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.74 (d, J = 4.6 Hz, 1 H). 3-Iodo-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (15). Step 1: Synthesis of 3-iodo-8-chloro-2-methyl-imidazo[1,2-a]pyrazine. N-Iodosuccinimide (14.1 g, 62 mmol) was added to a stirred solution of intermediate 14 (9.58 g, 57 mmol) in a mixture of DCM and acetic acid at 0 °C. The mixture was allowed to warm to rt and then stirred for 16 h. The mixture was diluted with further DCM and washed with a saturated solution of sodium carbonate and sodium thiosulfite. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was precipitated from diisopropylether to yield intermediate 3-iodo-8-chloro-2-methylimidazo[1,2-a]pyrazine (16 g, 97%) as a pale-brown solid which was used in the next step without further purification. Step 2: Synthesis of 15. A mixture 3-iodo-8-chloro-2-methyl-imidazo[1,2-a]pyrazine (50 g, 170.36 mmol), morpholine (17.8 g, 204.43 mmol), and N,Ndiisopropylethylamine (44 g, 340.72 mmol) in ACN (500 mL) was stirred at 80 °C for 16 h. The precipitate was filtered and washed with petroleum ether. The resulting solid was dried to give intermediate 15 (46.8 g, 80%) as a white solid. MS: m/z 345 [M + H]+. tR = 2.43 min (method 6). 1H NMR (500 MHz, CDCl3) δ ppm 7.46 (d, J = 4.6 Hz, 1 H), 7.44 (d, J = 4.6 Hz, 1 H), 4.20−4.25 (m, 4 H), 3.84−3.88 (m, 4 H), 2.45 (s, 3 H); mp 135.3−136.7 °C. 3-(2-Chloro-pyridin-4-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (16). Tetrakis(triphenylphosphine)palladium (0) (0.165 g, 0.143 mmol) was added to a stirred solution of intermediate 15 (1.645 g, 4.780 mmol) and 2-chloropyridine-5-boronic acid (0.83 g, 5.26 mmol) in a mixture of 1,4-dioxane (44 mL) and a saturated solution of sodium hydrogen carbonate (11 mL). The mixture was stirred at 90 °C for 3 h, at 100 °C for 2.5 h and at rt for 48 h. Then further 1,4-dioxane (10 mL) and a saturated solution of sodium hydrogen carbonate (2.5 mL) were added. The mixture was stirred at 110 °C for a further 5 h and then diluted with EtOAc and water and filtered over diatomaceous earth. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 20/80). The desired fractions were collected and evaporated in vacuo to yield intermediate 16 (1.3 g, 83%, 92% pure) as a white solid. MS: m/z 330 [M + H]+. tR = 2.67 min (method 5). 1 H NMR (500 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.86−3.91 (m, 4 H), 4.25−4.31 (m, 4 H), 7.33 (d, J = 4.3 Hz, 1 H), 7.37 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 8.1 Hz, 1 H), 7.74 (dd, J = 8.1, 2.3 Hz, 1 H), 8.49 (d, J = 1.7 Hz, 1 H). 3-[2-(2-Methoxy-ethyl)-pyridin-4-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (17). Step 1: Synthesis of 2-methyl-8morpholin-4-yl-3-(2-vinyl-pyridin-4-yl)-imidazo[1,2-a]pyrazine. Tetrakis(triphenylphosphine)palladium (0) (0.031 g, 0.027 mmol) was added to a stirred solution of intermediate 16 (0.30 g, 0.909 mmol) and vinylboronic acid pinacolester (0.17 g, 1.09 mmol) in a mixture of 1,4-dioxane (6 mL) and a saturated solution of sodium carbonate (3 mL). The mixture was stirred at 80 °C for 3 h under nitrogen and then diluted with DCM and extracted with water. The J



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ammonia in MeOH in DCM 0/100 to 1/99). The desired fractions were collected and the solvents evaporated in vacuo to yield compound 22 (0.044 g, 11%) as a white solid. MS: m/z 354 [M + H]+. tR = 2.29 min (method 7). 1H NMR (500 MHz, CDCl3) δ ppm 2.60 (s, 3 H), 2.95 (t, J = 6.5 Hz, 2 H), 3.39 (s, 3 H), 3.68 (t, J = 6.5 Hz, 2 H), 3.87−3.92 (m, 4 H), 4.19−4.25 (m, 4 H), 7.40 (d, J = 4.6 Hz, 1 H), 7.47 (d, J = 7.8 Hz, 1 H), 7.71 (dd, J = 8.1, 2.0 Hz, 1 H), 8.45 (d, J = 4.6 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H). 3-(6-Chloro-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (23). Tetrakis(triphenylphosphine)palladium (0) (1.5 g, 1.3 mmol) was added to a stirred solution of intermediate 15 (11.5 g, 33.42 mmol) and 2-chloropyridine-5-boronic acid (6.1 g, 3.87 mmol) in a mixture of 1,4-dioxane (200 mL) and a saturated solution of sodium hydrogen carbonate (50 mL). The mixture was stirred at 100 °C for 18 h under nitrogen, and then further tetrakis(triphenylphosphine)palladium (0) (0.35 g, 0.3 mmol) and 2-chloropyridine-5-boronic acid (0.6 g, 2.8 mmol) were added. The mixture was stirred at 100 °C for further 5 h and then concentrated in vacuo and partitioned between DCM, water, and a saturated solution of sodium carbonate. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was precipitated from MeOH to yield intermediate 23 (10.3 g, 93%) as a white solid. MS: m/z 330 [M + H]+. tR = 1.78 min (method 7). 1H NMR (500 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.86−3.91 (m, 4 H), 4.25−4.31 (m, 4 H), 7.33 (d, J = 4.3 Hz, 1 H), 7.37 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 8.1 Hz, 1 H), 7.74 (dd, J = 8.1, 2.3 Hz, 1 H), 8.49 (d, J = 1.7 Hz, 1 H). 2-Methyl-8-morpholin-4-yl-3-(6-vinyl-pyridin-3-yl)-imidazo[1,2a]pyrazine (24). Tetrakis(triphenylphosphine)palladium (0) (0.623 g, 0.54 mmol) was added to a stirred solution of intermediate 23 (8.9 g, 26.99 mmol) and vinylboronic acid pinacolester (5.91 mL, 35.08 mmol) in a mixture of 1,4-dioxane (60 mL) and a saturated solution of sodium carbonate (30 mL). The mixture was stirred at 100 °C for 1 h under nitrogen and then diluted with DCM and extracted with water. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98). The desired fractions were collected and the solvents evaporated in vacuo to yield intermediate 24 (7.8 g, 90%, 91% pure) as a white solid. MS: m/z 322 [M + H]+. tR = 1.72 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.45 (s, 3 H), 3.86−3.92 (m, 4 H), 4.25−4.30 (m, 4 H), 5.59 (dd, J = 10.8, 1.0 Hz, 1 H), 6.32 (dd, J = 17.5, 1.0 Hz, 1 H), 6.90 (dd, J = 17.5, 10.8 Hz, 1 H), 7.35 (d, J = 4.6 Hz, 1 H), 7.40 (d, J = 4.6 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.73 (dd, J = 8.1, 2.3 Hz, 1 H), 8.66 (d, J = 1.6 Hz, 1 H). 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25a). Potassium hydrogensulfate (12 g, 88.13 mmol) was added to a stirred solution of intermediate 24 (6 g, 18.67 mmol) in MeOH (120 mL). The mixture was stirred at 80 °C for 3 days and then poured onto a saturated solution of sodium carbonate and extracted with DCM. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by open column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 1.5/98.5). The impure fractions were collected and evaporated in vacuo and the crude product purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98). The combined desired fractions were collected and the solvents evaporated in vacuo and triturated with heptane to yield compound 25a (4.13 g, 63%) as a white solid. MS: m/z 354 [M + H]+. tR = 1.93 min (method 7). 1H NMR (500 MHz, CDCl3) δ ppm 2.44 (s, 3 H), 3.16 (t, 2 H), 3.40 (s, 3 H), 3.86 (t, 2 H), 3.89 (br t, J = 4.9 Hz, 4 H), 4.27 (br t, J = 4.9 Hz, 4 H), 7.34 (d, 1 H), 7.39 (d, J = 4.6 Hz, 1 H), 7.40 (d, J = 8.1 Hz, 1 H), 7.68 (dd, J = 8.1, 2.3 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H); mp >300 °C. 3-[6-(2-Ethoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25b). 3-[6-(2-Ethoxy-ethyl)-pyridin-3-yl]-2methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to compound 17 (step 2) from intermediate 24 and sodium ethoxide. Flash column chromatography (silica; EtOAc in DCM 50/50 to 100/0) then flash column

chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98) and precipitation from heptane, yielded compound 25b as a white solid (47%). MS: m/z 368 [M + H]+. tR = 2.27 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.22 (t, J = 7.1 Hz, 3 H), 2.44 (s, 3 H), 3.16 (t, J = 6.7 Hz, 2 H), 3.56 (q, J = 6.9 Hz, 2 H), 3.84−3.92 (m, 6 H), 4.24−4.30 (m, 4 H), 7.32−7.39 (m, 2 H), 7.41 (d, J = 8.1 Hz, 1 H), 7.68 (dd, J = 7.9, 2.3 Hz, 1 H), 8.61 (d, J = 2.1 Hz, 1 H); mp 87.6 °C. 3-[6-(2-Isopropoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25c). 3-[6-(2-Isopropoxy-ethyl)-pyridin-3-yl]2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to compound 17 (step 2) from intermediate 24 and sodium isopropoxide. Flash column chromatography (silica; EtOAc in DCM 0/100 to 100/0) and precipitation from heptane yielded compound 25c as a white solid (25%). MS: m/z 382 [M + H]+. tR = 2.62 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.17 (d, J = 6.2 Hz, 6 H), 2.43 (s, 3 H), 3.14 (t, J = 6.7 Hz, 2 H), 3.63 (spt, J = 6.1 Hz, 1 H), 3.83−3.93 (m, 6 H), 4.24−4.31 (m, 4 H), 7.32−7.39 (m, 2 H), 7.42 (d, J = 7.9 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2 Hz, 1 H), 8.61 (d, J = 2.3 Hz, 1 H). 2-[5-(2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-pyridin-2-yl]-ethanol (25d). 2-[5-(2-Methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazin-3-yl)-pyridin-2-yl]-ethanol was prepared according to a protocol analogous to compound 25a from intermediate 24, potassium hydrogensulfate, and water. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 3/97) and flash column chromatography (silica; MeOH in EtOAc 0/100 to 2/98) yielded compound 25d as a white solid (18%). MS: m/z 340 [M + H]+. tR = 1.36 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.44 (s, 3 H), 3.13 (t, J = 5.4 Hz, 2 H), 3.83−3.98 (m, 5 H), 4.11 (t, J = 5.4 Hz, 2 H), 4.25−4.30 (m, 4 H), 7.33−7.38 (m, 3 H), 7.70 (dd, J = 7.9, 2.3 Hz, 1 H), 8.59 (d, J = 2.1 Hz, 1 H). 3-(6-Ethyl-pyridin-3-yl)-2-methyl-8-morpholin-4-yl-imidazo[1,2a]pyrazine (25e). Palladium 10% on charcoal (0.042 g) was added to a suspension of intermediate 24 (0.25 g, 0.78 mmol) in a mixture of EtOH (3 mL), EtOAc (2 mL), and DCM (1 mL). The mixture was hydrogenated (atmospheric pressure) at rt for 16 h and then filtered through a pad of diatomaceous earth. The filtrate was evaporated in vacuo and the crude product purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98). The desired fractions were collected, and the solvents evaporated in vacuo and triturated with Et2O to yield compound 25e (0.23 g, 91%) as a white solid. MS: m/z 324 [M + H]+. tR = 2.32 min (method 7). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.7 Hz, 3 H), 2.36 (s, 3 H), 2.85 (q, J = 7.5 Hz, 2 H), 3.73−3.78 (m, 4 H), 4.14−4.20 (m, 4 H), 7.35 (d, J = 4.6 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H), 7.60 (d, J = 4.6 Hz, 1 H), 7.89 (dd, J = 7.9, 2.5 Hz, 1 H), 8.63 (d, J = 1.7 Hz, 1 H); mp 80.3 °C. 3-[6-(2-Methoxy-2-methyl-propyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (25f). 3-[6-(2-Methoxy-2-methylpropyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to intermediate 16 from intermediate 10 and 2-(2-methoxy-2-methyl-propyl)pyridine-5-boronic acid pinacol ester at 140 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; EtOAc in DCM 50/50 to 80/20), flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98), and precipitation from heptane yielded compound 25f as a white solid (71%). MS: m/z 382 [M + H]+. tR = 2.40 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.26 (s, 6 H), 2.44 (s, 3 H), 3.07 (s, 2 H), 3.34 (s, 3 H), 3.86−3.93 (m, 4 H), 4.23−4.31 (m, 4 H), 7.32−7.41 (m, 2 H), 7.42 (d, J = 8.1 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2 Hz, 1 H), 8.61 (d, J = 2.1 Hz, 1 H) (regioselectivity confirmed by NOE between gem-dimethyl group and CH2 attached to 6-position of pyridine ring); mp 133.2 °C. 3-[6-(3-Methoxy-propyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25g). 3-[6-(3-Methoxy-propyl)-pyridin-3-yl]2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to intermediate 16 from intermediate 10 and 2-(1-methoxy-propyl)pyridine-5-boronic acid pinacol ester at K



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150 °C for 20 min and under microwave irradiation. Flash column chromatography (silica; EtOAc in heptane 50/50 to 80/20) yielded compound 25g as a white solid (11%). MS: m/z 368 [M + H]+. tR = 2.20 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.03−2.14 (m, 2 H), 2.44 (s, 3 H), 2.93−3.00 (m, 2 H), 3.38 (s, 3 H), 3.49 (t, J = 6.4 Hz, 2 H), 3.86−3.92 (m, 4 H), 4.25−4.30 (m, 4 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.34 (d, J = 7.9 Hz, 1 H), 7.38 (d, J = 4.4 Hz, 1 H), 7.67 (dd, J = 7.9, 2.3 Hz, 1 H), 8.61 (d, J = 2.1 Hz, 1 H). 3-(6-Ethoxymethyl-pyridin-3-yl)-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25h). 3-(6-Ethoxymethyl-pyridin-3-yl)-2methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to intermediate 16 from intermediate 10 and 2-ethoxymethylpyridine-5-boronic acid pinacol ester at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98) then RP HPLC (0.1% solution of ammonium bicarbonate/ ammonium hydroxide buffer pH 9 in EtOAc 80/20 to 0/100) yielded compound 25h as a white solid (48%). MS: m/z 354 [M + H]+. tR = 2.23 min (method 7). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.23 (t, J = 6.9 Hz, 3 H), 2.38 (s, 3 H), 3.63 (q, J = 7.1 Hz, 2 H), 3.76 (br t, J = 4.9 Hz, 4 H), 4.18 (br t, J = 4.9 Hz, 4 H), 4.64 (s, 2 H), 7.37 (d, J = 4.6 Hz, 1 H), 7.63 (d, J = 8.1 Hz, 1 H), 7.64 (d, J = 4.6 Hz, 1 H), 8.00 (dd, J = 8.1, 2.3 Hz, 1 H), 8.68 (d, J = 1.7 Hz, 1 H); mp >300 °C (dec). 4-[2-Methyl-3-(6-methyl-3-pyridyl)imidazo[1,2-a]pyrazin-8-yl]morpholine (25i). 4-[2-Methyl-3-(6-methyl-3-pyridyl)imidazo[1,2-a]pyrazin-8-yl]morpholine was prepared according to a protocol analogous to intermediate 16 from intermediate 15 and 2-picoline-5boronic acid pinacol ester at 90 °C for 18 h. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98) yielded compound 25i as a white solid (48%, 92% pure). MS: m/z 310 [M + H]+. tR = 2.12 min (method 5). 1H NMR (500 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 2.66 (s, 3 H), 3.87−3.92 (m, 4 H), 4.24−4.30 (m, 4 H), 7.32−7.37 (m, 3 H), 7.65 (dd, J = 7.8, 2.3 Hz, 1 H), 8.58 (d, J = 2.0 Hz, 1 H); mp 87.3 °C. 3-[6-(2-Methoxy-ethoxy)-pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25j). 3-[6-(2-Methoxy-ethoxy)-pyridin-3-yl]2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine was prepared according to a protocol analogous to intermediate 16 from intermediate 10 and 2-picoline-5-boronic acid pinacol ester at 140 °C for 20 min and under microwave irradiation. Flash column chromatography (silica; MeOH in DCM 5/95) yielded compound 25j as a white solid (43%). MS: m/z 370 [M + H]+. tR = 2.34 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 3.32 (s, 3 H), 3.67−3.73 (m, 2 H), 3.75 (br t, J = 4.9 Hz, 4 H), 4.17 (br t, J = 4.9 Hz, 4 H), 4.40−4.53 (m, 2 H), 7.04 (d, J = 8.6 Hz, 1 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.55 (d, J = 4.6 Hz, 1 H), 7.88 (dd, J = 8.7, 2.4 Hz, 1 H), 8.30 (d, J = 2.3 Hz, 1 H). 4-[3-(6-Isopropoxy-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]morpholine (25k). 4-[3-(6-Isopropoxy-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]morpholine was prepared according to a protocol analogous to intermediate 16 from intermediate 10 and 2isopropoxypyridine-5-boronic acid pinacol ester at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98) and ion exchange chromatography using an ISOLUTE SCX2 cartridge (eluting with MeOH then 7 M solution of ammonia in MeOH) yielded compound 25k as a white solid (94%). ESI-HRMS: m/z for C19H24N5O2 [M + H]+ calcd, 354.1930; found, 354.1922 (−2.3 ppm). tR = 3.85 min (method 2). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.35 (d, J = 6.2 Hz, 6 H), 2.34 (s, 3 H), 3.71−3.79 (m, 4 H), 4.13− 4.21 (m, 4 H), 5.33 (spt, J = 6.2 Hz, 1 H), 6.94 (dd, J = 8.6, 0.7 Hz, 1 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.55 (d, J = 4.6 Hz, 1 H), 7.84 (dd, J = 8.6, 2.5 Hz, 1 H), 8.30 (dd, J = 2.5, 0.7 Hz, 1 H); mp 57.8 °C. 4-[3-(6-Isopropyl-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8yl]morpholine (25l). [1,3-Bis(diphenylphosphino)propane]dichloronickel(II) (0.014 g, 0.027 mmol) was added to a solution of intermediate 23 (0.18 g, 0.545 mmol) in THF (4 mL), and the resulting mixture was cooled to 0 °C. A 2 M solution of isopropylmagnesium chloride in Et2O (0.54 mL, 1.09 mmol) was slowly added, and the stirring was continued for 30 min. The reaction

was quenched by addition of a saturated solution of ammonium chloride. Then 1 M aqueous solution of hydrochloride acid and DCM were added and the product extracted into the aqueous layer. The organic layer was washed once more with diluted hydrochloride acid. The combined aqueous layers were basified with saturated solution of sodium carbonate and the product extracted with DCM. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 40/60). The desired fractions were collected and the solvents evaporated in vacuo to yield compound 25l (0.10 g, 56%) as a solid. MS: m/z 338 [M + H]+. tR = 3.37 min (method 5). 1H NMR (500 MHz, CDCl3) δ ppm 1.38 (d, J = 6.9 Hz, 6 H), 2.44 (s, 3 H), 3.16 (spt, J = 6.9 Hz, 1 H), 3.86−3.92 (m, 4 H), 4.24−4.31 (m, 4 H), 7.32−7.36 (m, 2 H), 7.39 (d, J = 4.3 Hz, 1 H), 7.68 (dd, J = 7.9, 2.2 Hz, 1 H), 8.62 (d, J = 1.7 Hz, 1 H). 4-[3-(6-Isobutyl-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]morpholine (25m). 4-[3-(6-Isobutyl-3-pyridyl)-2-methyl-imidazo[1,2-a]pyrazin-8-yl]morpholine was prepared according to a protocol analogous to compound 25l from intermediate 23 and isobutylmagnesium bromide. Flash column chromatography (silica; EtOAc in heptane 60/40) and triturated with Et2O yielded compound 25m (56%). MS: m/z 352 [M + H]+. tR = 3.11 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.94 (d, J = 6.7 Hz, 6 H), 2.13 (dquin, J = 13.6, 6.8 Hz, 1 H), 2.36 (s, 3 H), 2.70 (d, J = 7.2 Hz, 2 H), 3.72−3.78 (m, 4 H), 4.14− 4.20 (m, 4 H), 7.35 (d, J = 4.4 Hz, 1 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.59 (d, J = 4.6 Hz, 1 H), 7.88 (dd, J = 8.1, 2.3 Hz, 1 H), 8.63 (d, J = 1.8 Hz, 1 H); mp >300 °C (dec). 3-(6-Cyclopropyl-pyridin-3-yl)-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (25n). Palladium(II) acetate (0.036 g, 0.16 mmol) and 2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl (0.131 g, 0.32 mmol) were added to a stirred mixture of intermediate 23 (0.35 g, 1.06 mmol), cyclopropylboronic acid (0.137 g, 1.59 mmol), and potassium phosphate (0.451 g, 2.12 mmol) in toluene (5 mL). The mixture was stirred at 80 °C for 22 h under nitrogen and then diluted with DCM and extracted with water. The organic layer was separated, dried (MgSO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 100/0 to 3/97). The desired fractions were collected and the solvents evaporated in vacuo and the crude product purified again by flash column chromatography (silica; EtOAc in heptane 50/50 to 100/0). The desired fractions were collected and the solvents evaporated in vacuo and triturated with diisopropylether to yield compound 25n (0.103 g, 29%) as a palebrown solid. MS: m/z 336 [M + H]+. tR = 2.65 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.00−1.19 (m, 4 H), 2.05−2.19 (m, 1 H), 2.42 (s, 3 H), 3.89 (br t, J = 4.9 Hz, 4 H), 4.27 (br t, J = 4.6 Hz, 4 H), 7.31 (d, J = 8.3 Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H), 7.35 (d, J = 4.4 Hz, 1 H), 7.59 (dd, J = 8.1, 2.1 Hz, 1 H), 8.51 (d, J = 2.3 Hz, 1 H); mp > 300 °C (dec). 4-[2-Methyl-3-(6-pyrrolidin-1-yl-3-pyridyl)imidazo[1,2-a]pyrazin8-yl]morpholine (25o). A mixture of intermediate 23 (250 mg, 0.758 mmol) in pyrrolidine (0.314 g, 3.64 mmol) was stirred at 130 °C for 30 min under microwave irradiation. The solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; MeOH in DCM 0/100 to 5/95). The desired fractions were collected and the solvents evaporated in vacuo. The product triturated with diisopropylether to yield compound 25o (0.167 g, 60%) as a solid. MS: m/z 365 [M + H]+. tR = 2.88 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.00−2.12 (m, 4 H), 2.40 (s, 3 H), 3.50−3.57 (m, 4 H), 3.85−3.93 (m, 4 H), 4.22−4.29 (m, 4 H), 6.50 (d, J = 8.8 Hz, 1 H), 7.30 (d, J = 4.4 Hz, 1 H), 7.35 (d, J = 4.6 Hz, 1 H), 7.46 (dd, J = 8.7, 2.4 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H); mp 132.2 °C. (S)-3-[6-(3-Methoxy-pyrrolidin-1-yl)-pyridin-3-yl]-2-methyl-8morpholin-4-yl-imidazo[1,2-a]pyrazine (25p). A mixture of intermediate 23 (0.15 g, 0.45 mmol) and (S)-3-hydroxypyrrolidine (0.159 g, 1.82 mmol) was stirred at 120 °C for 3 h, and then the mixture was diluted with water and extracted with EtOAc. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was dissolved in THF (3 mL), and a 60% dispersion of sodium hydride in mineral oils (0.020 g, 0.5 mmol) was L



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The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; MeOH in DCM 0/100 to 2/98). The desired fractions were collected and the solvents evaporated in vacuo to yield intermediate 26h (0.86 g, 78%) as a white solid. MS: m/z 311 [M + H]+. tR = 2.62 min (method 5). 1H NMR (400 MHz, CDCl3) δ ppm 2.32 (d, J = 0.9 Hz, 3 H), 2.39 (s, 3 H), 3.83−3.88 (m, 4 H), 4.19−4.26 (m, 4 H), 7.26−7.28 (m, 1 H). 3-Bromo-2-methyl-6-trifluoromethyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (26i). Copper(I) iodide (0.18 g, 0.95 mmol) and MDFA (0.12 mL, 0.95 mmol) were added to a stirred solution of intermediate 31 (0.20 g, 0.47 mmol) in DMF (2 mL). The mixture was stirred at 90 °C for 16 h in a sealed tube under nitrogen and then diluted with Et2O and washed with a saturated solution of ammonium hydroxide. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 50/50). The desired fractions were collected and the solvents evaporated in vacuo to yield intermediate 26i (0.15 g, 60%, 69% pure) as a pale-brown solid. MS: m/z 365 [M + H]+. tR = 2.94 min (method 6). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.36 (s, 3 H), 3.72−3.77 (m, 4 H), 4.17−4.27 (m, 4 H), 8.01 (s, 1 H). 2-(2-Methoxyethyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan2-yl)-pyridine (27). Step 1: Synthesis of 2-(5-bromo-pyridin-2-yl)ethanol. A 2.5 M solution of n-butyllithium in pentane (6.97 mL, 17.44 mmol) was added dropwise to a solution of N,N-diisopropylamine (3.29 mL, 23.25 mmol) in THF (50 mL). The mixture was stirred at 0 °C for 30 min, cooled down to −78 °C, and then a solution of 5-bromo-2-methylpyridine (2.0 g, 11.63 mmol) in THF (50 mL) was added. The mixture was stirred at −78 °C for a further 2 h, and then DMF (8.5 g, 116.26 mmol) was added dropwise. The mixture was stirred at −78 °C for 2 h, at 0 °C for 30 min, and finally allowed to warm to rt. MeOH (25 mL) and sodium borohydride (0.439 g, 11.6 mmol) were added, and the mixture was stirred at rt for further 30 min. A saturated solution of ammonium chloride was added, and the organic layer was separated. The aqueous layer was extracted with EtOAc, and the combined organic extracts were dried (Na2SO4) and filtered, and the solvents evaporated in vacuo to yield 2-(5-bromopyridin-2-yl)-ethanol (2.8 g, 87%, 73% pure) as a colorless oil. MS: m/z 202 [M + H]+. tR = 1.07 min (method 5). Step 2: Synthesis of 5-bromo-2-(2-methoxy-ethyl)-pyridine. A 60% dispersion of sodium hydride in mineral oils, (0.43 g, 11.1 mmol) was added portionwise to a stirred solution of 2-(5-bromo-pyridin-2-yl)-ethanol (2.8 g, 10.1 mmol) in THF (50 mL). The mixture was stirred at 0 °C for 30 min and at rt for 16 h. A saturated solution of ammonium chloride was added, and the organic layer was separated. The aqueous layer was extracted with DCM, and the combined organic extracts were dried (Na2SO4), filtered, and the solvents evaporated in vacuo to yield 5-bromo-2-(2-methoxy-ethyl)-pyridine (0.9 g, 41%) as a colorless oil. MS: m/z 216 [M + H]+. tR = 2.25 min (method 5). Step 3: Synthesis of 27. [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.061 g, 0.083 mmol) was added to a stirred suspension of 5-bromo2-(2-methoxy-ethyl)-pyridine (0.6 g, 2.77 mmol), 4,4,4′,4′,5,5,5′,5′octamethyl-2,2′-bi-1,3,2-dioxaborolane (0.846 g, 3.33 mmol), and potassium acetate (0.817 g, 8.33 mmol) in a mixture of 1,4-dioxane (9 mL) and DMF (1.2 mL). The mixture was stirred at 150 °C for 40 min in a sealed tube under nitrogen and under microwave irradiation. The mixture was filtered through a pad of diatomaceous earth and the filtrate diluted with DCM and washed with water. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo to yield intermediate 27 (1.1 g, 64%, 43% pure) used in the next step without further purification. ESI-HRMS (11B): m/z for C14H23BNO3 [M + H]+ calcd, 264.1771; found, 264.1819 (18.2 ppm). tR = 2.37 min (method 2). 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-yl-2-trifluoromethyl-imidazo[1,2-a]pyrazine (28a). 28a was prepared according to a protocol analogous to intermediate 16 from intermediate 26a and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH and EtOAc in DCM 3/0.3/96.7) then flash column

added. The mixture was stirred at rt for 5 min, and then iodomethane (0.07 g, 0.49 mmol) was added. The mixture was stirred at rt for a further 3 days and then extracted with a saturated solution of ammonium chloride. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98 to 10/90). The desired fractions were collected and the solvents evaporated in vacuo to yield compound 25p (0.033 g, 19%) as a white solid. MS: m/z 395 [M + H]+. tR = 2.44 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.08− 2.29 (m, 2 H), 2.40 (s, 3 H), 3.40 (s, 3 H), 3.56−3.74 (m, 4 H), 3.85− 3.92 (m, 4 H), 4.14 (tt, J = 4.8, 2.6 Hz, 1 H), 4.22−4.29 (m, 4 H), 6.51 (d, J = 8.8 Hz, 1 H), 7.30 (d, J = 4.6 Hz, 1 H), 7.33 (d, J = 4.6 Hz, 1 H), 7.47 (dd, J = 8.8, 2.3 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H). N-Isopropyl-5-(2-methyl-8-morpholino-imidazo[1,2-a]pyrazin-3-yl)pyridin-2-amine (25q). Palladium(II) acetate (0.010 g, 0.045 mmol) and racemic-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.042 g, 0.068 mmol) were added to a stirred solution of intermediate 23 (0.30 g, 0.91 mmol), isopropylamine (0.78 mL, 9.09 mmol), and cesium carbonate (0.74 g, 2.27 mmol) in toluene (4 mL). The mixture was stirred at 60 °C for 4 days and then diluted with DCM, washed with water, and the organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 40/60 to 100/0). The desired fractions were collected, the solvents evaporated in vacuo and then triturated from diisopropylether to yield a compound that was washed with a saturated solution of sodium carbonate and then diluted with DCM, the organic layer was separated, dried (Na2SO4), and filtered and the solvents evaporated in vacuo. The resulting residue was purified by flash column chromatography (silica; EtOAc in heptane 30/70 to 70/30). The desired fractions were collected, the solvents evaporated in vacuo to yield compound 25q (0.19 g, 59%) as a white solid. MS: m/z 353 [M + H]+. tR = 2.57 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 1.30 (d, J = 6.2 Hz, 6 H), 2.40 (s, 3 H), 3.84−3.92 (m, 4 H), 3.92−4.02 (m, 1 H), 4.21− 4.30 (m, 4 H), 4.61 (br d, J = 7.9 Hz, 1 H), 6.50 (d, J = 8.6 Hz, 1 H), 7.30 (d, J = 4.6 Hz, 1 H), 7.35 (d, J = 4.4 Hz, 1 H), 7.43 (dd, J = 8.8, 2.3 Hz, 1 H), 8.12 (d, J = 2.3 Hz, 1 H); mp 105.2 °C. 2-Methyl-8-morpholin-4-yl-3-(6-piperazin-1-yl-pyridin-3-yl)imidazo[1,2-a]pyrazine (25r). A mixture of intermediate 23 (0.3 g, 0.91 mmol) and piperazine (0.314 g, 3.64 mmol) was stirred at 120 °C for 24 h. The mixture was diluted with EtOAc and extracted with water and a 1N solution of sodium hydroxide. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 10/90). The desired fractions were collected and evaporated in vacuo and the crude product purified again by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 3/97) and by RP-HPLC (0.1% solution of ammonium bicarbonate/ammonium hydroxide buffer pH 9 in ACN 80/20 to 0/100). The desired fractions were collected and the solvents evaporated and the crude product triturated with diisopropylether to yield compound 25r (0.074 g, 22%) as a white solid. MS: m/z 380 [M + H]+. tR = 1.19 min (method 7). 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.32 (s, 3 H), 2.81 (br t, J = 5.1 Hz, 4 H), 3.32 (br s, 1 H), 3.51 (dd, J = 5.3, 4.9 Hz, 4 H), 3.74 (br t, J = 4.9 Hz, 4 H), 4.16 (dd, J = 4.9, 4.4 Hz, 4 H), 6.96 (d, J = 8.8 Hz, 1 H), 7.32 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 4.6 Hz, 1 H), 7.64 (dd, J = 8.8, 2.3 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H). 3-Bromo-2,6-dimethyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (26h). A 1.6 M solution of methyllithium in THF (2.66 mL, 4.25 mmol) was added dropwise to a solution of indium(III) chloride (0.35 g, 1.59 mmol) in THF (35 mL) at −78 °C. The mixture was stirred at −78 °C for 30 min and then allowed to warm to rt. The trimethylindium pale-white solution obtained was transferred via cannula to a stirred solution of intermediate 31 (1.5 g, 3.55 mmol) and tetrakistriphenylphosphine palladium (0) (0.21 g, 0.18 mmol) in THF (20 mL). The mixture was stirred at 80 °C for 16 h, and then the solvent was evaporated in vacuo. The crude product was dissolved in DCM and washed with a saturated solution of ammonium chloride. M



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2.53 (s, 3 H), 3.10 (t, J = 6.6 Hz, 2 H), 3.29 (s, 3 H), 3.79 (t, J = 6.6 Hz, 2 H), 7.56 (d, J = 7.9 Hz, 1 H), 8.03 (dd, J = 8.1, 2.3 Hz, 1 H), 8.06 (d, J = 4.6 Hz, 1 H), 8.44 (d, J = 4.6 Hz, 1 H), 8.68−8.72 (m, 2 H), 8.76 (d, J = 2.1 Hz, 1 H), 8.80−8.84 (m, 2 H); mp 279.0 °C. 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-pyridin-3-ylimidazo[1,2-a]pyrazine (28g). 28g was prepared according to a protocol analogous to intermediate 16 from intermediate 26g and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 1/99 then EtOAc in DCM 0/100 to 100/0) and precipitation from Et2O yielded compound 28g as a white solid (48%). MS: m/z 346 [M + H]+. tR = 1.68 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.52 (s, 3 H), 3.09 (t, J = 6.6 Hz, 2 H), 3.29 (s, 3 H), 3.79 (t, J = 6.6 Hz, 2 H), 7.56 (d, J = 8.1 Hz, 1 H), 7.63 (dd, J = 8.1, 4.9 Hz, 1 H), 8.02 (dd, J = 5.8, 2.3 Hz, 1 H), 8.03 (d, J = 4.4 Hz, 1 H), 8.38 (d, J = 4.6 Hz, 1 H), 8.74 (dd, J = 4.6, 1.6 Hz, 1 H), 8.76 (d, J = 2.3 Hz, 1 H), 9.04 (dt, J = 8.0, 1.9 Hz, 1 H), 9.85 (d, J = 2.1 Hz, 1 H); mp > 300 °C (dec). 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2,6-dimethyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine (28h). 28h was prepared according to a protocol analogous to intermediate 16 from intermediate 26h and intermediate 27 at 150 °C for 30 min and under microwave irradiation. Flash column chromatography (silica; MeOH in DCM 4/96) yielded compound 28h as a white solid (82%). MS: m/z 368 [M + H]+. tR = 2.39 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.22−2.25 (m, 3 H), 2.41 (s, 3 H), 3.15 (t, J = 6.5 Hz, 2 H), 3.41 (s, 3 H), 3.81− 3.92 (m, 6 H), 4.23−4.32 (m, 4 H), 7.18−7.23 (m, 1 H), 7.39 (d, J = 7.9 Hz, 1 H), 7.67 (dd, J = 8.0, 2.2 Hz, 1 H), 8.59−8.64 (m, 1 H); mp 112.2 °C. 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-morpholin-4-yl6-trifluoromethyl-imidazo[1,2-a]pyrazine (28i). 28i was prepared according to a protocol analogous to intermediate 16 from intermediate 26i and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; EtOAc in DCM 0/100 to 100/0) and trituration with diisopropylether/heptane yielded compound 28i as a solid (41%, 93% pure by RP-LCMS, 98% by NMR). MS: m/z 422 [M + H]+. tR = 3.24 min (method 7). 1H NMR (400 MHz, CDCl3) δ ppm 2.43 (s, 3 H), 3.17 (t, J = 6.5 Hz, 2 H), 3.41 (s, 3 H), 3.82−3.93 (m, 6 H), 4.30−4.46 (m, 4 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.67 (dd, J = 8.1, 2.3 Hz, 1 H), 7.71 (s, 1 H), 8.61 (d, J = 2.3 Hz, 1 H); mp 118.4 °C. 3-Bromo-8-chloro-6-iodo-2-methyl-imidazo[1,2-a]pyrazine (30). Step 1: Synthesis of 8-chloro-6-iodo-2-methyl-imidazo[1,2-a]pyrazine. A mixture of intermediate 29 (2.5 g, 9.78 mmol), sodium iodide (2.93 g, 19.57 mmol), and 2-chloroacetone (4.67 mL, 58.72 mmol) was stirred at 90 °C for 24 h in a sealed tube protected from light. After cooling to rt, Et2O was added and the solid formed was suspended in a saturated solution of sodium hydrogen carbonate and extracted with DCM. The organic layer was dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by open column chromatography (silica; DCM). The desired fractions were collected and the solvents evaporated in vacuo to yield 8-chloro6-iodo-2-methyl-imidazo[1,2-a]pyrazine (0.85 g, 28%) as a white solid (hydroiodide). MS: m/z 294 [M + H]+. tR = 2.01 min (method 4). Step 2: Synthesis of 30. N-Bromosuccinimide (1.091 g, 6.133 mmol) was added to a stirred solution of 8-chloro-6-iodo-2-methyl-imidazo[1,2-a]pyrazine (1.9 g, 6.47 mmol) in DCM (50 mL). The mixture was stirred at rt for 3 h and then diluted with further DCM and washed with a saturated solution of sodium carbonate. The organic layer was separated, dried (Na2SO4), filtered, and the solvent evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 40/60). The desired fractions were collected and the solvents evaporated in vacuo to yield intermediate 30 (2 g, 83%) as a white solid. MS: m/z 372 [M + H]+. tR = 2.40 min (method 6). 1H NMR (500 MHz, CDCl3) δ ppm 2.55 (s, 3 H), 8.25 (s, 1 H). 3-Bromo-6-iodo-2-methyl-8-morpholin-4-yl-imidazo[1,2-a]pyrazine (31). Morpholine (0.241 mL, 2.75 mmol) was added to a stirred solution of intermediate 30 (790 mg, 2.12 mmol) and N,Ndiisopropylethylamine (0.554 mL, 3.18 mmol) in ACN (10 mL).

chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 0.5/99.5) and freeze-drying yielded compound 28a as a white solid (50%). MS: m/z 408 [M + H]+. tR = 2.80 min (method 7). 1 H NMR (400 MHz, CDCl3) δ ppm 3.17 (t, J = 6.5 Hz, 2 H), 3.40 (s, 3 H), 3.83−3.91 (m, 6 H), 4.30−4.37 (m, 4 H), 7.24 (d, J = 4.6 Hz, 1 H), 7.40−7.44 (m, 2 H), 7.70 (dd, J = 8.0, 2.2 Hz, 1 H), 8.63 (d, J = 2.1 Hz, 1 H); mp 118.4 °C. 2-Isopropyl-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-ylimidazo[1,2-a]pyrazine (28b). 28b was prepared according to a protocol analogous to intermediate 16 from intermediate 26b and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98) and ion exchange chromatography using an ISOLUTE SCX2 cartridge (eluting with MeOH then 7 M solution of ammonia in MeOH) yielded compound 28b as a clear syrup (52%). MS: m/z 382 [M + H]+. tR = 2.90 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.24 (d, J = 6.7 Hz, 6 H), 2.95−3.05 (m, 1 H), 3.07 (t, J = 6.6 Hz, 2 H), 3.28 (s, 3 H), 3.74−3.80 (m, 6 H), 4.16− 4.23 (m, 4 H), 7.33 (d, J = 4.6 Hz, 1 H), 7.49 (d, J = 4.6 Hz, 1 H), 7.51 (d, J = 8.1 Hz, 1 H), 7.86 (dd, J = 7.9, 2.3 Hz, 1 H), 8.58 (d, J = 2.3 Hz, 1 H). 2-Cyclopropyl-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin4-yl-imidazo[1,2-a]pyrazine (28c). 28c was prepared according to a protocol analogous to intermediate 16 from intermediate 26c and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 4/96) and precipitation with Et2O yielded compound 28c as a brown solid (43%). MS: m/z 380 [M + H]+. tR = 2.67 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.86− 0.97 (m, 4 H), 1.90−2.01 (m, 1 H), 3.07 (t, J = 6.6 Hz, 2 H), 3.27 (s, 3 H), 3.70−3.75 (m, 4 H), 3.77 (t, J = 6.7 Hz, 2 H), 4.07−4.18 (m, 4 H), 7.34 (d, J = 4.6 Hz, 1 H), 7.52 (d, J = 7.9 Hz, 1 H), 7.59 (d, J = 4.6 Hz, 1 H), 7.94 (dd, J = 8.1, 2.3 Hz, 1 H), 8.69 (d, J = 1.6 Hz, 1 H); mp 103.9 °C. 2-Methoxy-3-[6-(2-methoxy-ethyl)-pyridin-3-yl]-8-morpholin-4-ylimidazo[1,2-a]pyrazine (28d). 28d was prepared according to a protocol analogous to intermediate 16 from intermediate 26d and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 2/98), flash column chromatography (silica; EtOAc in heptane 30/70 to 100/0), and freeze-drying yielded compound 28d as a brown solid (50%). MS: m/z 370 [M + H]+. tR = 2.35 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.02 (t, J = 6.6 Hz, 2 H), 3.26 (s, 3 H), 3.73 (t, J = 6.5 Hz, 2 H), 3.74−3.78 (m, 4 H), 4.00 (s, 3 H), 4.07−4.12 (m, 4 H), 7.45 (d, J = 8.3 Hz, 1 H), 7.46 (d, J = 4.6 Hz, 1 H), 7.85 (d, J = 4.6 Hz, 1 H), 7.92 (dd, J = 8.1, 2.3 Hz, 1 H), 8.70 (d, J = 2.1 Hz, 1 H); mp > 300 °C (dec). 3-[6-(2-Methoxyethyl)-3-pyridyl]-2-methyl-8-pyrrolidin-1-ylimidazo[1,2-a]pyrazine (28e). 28e was prepared according to a protocol analogous to intermediate 16 from intermediate 26e and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 1/99), flash column chromatography (silica; EtOAc in heptane 0/100 to 100/0), and precipitation from diisopropylether yielded compound 28e as a solid (56%). MS: m/z 338 [M + H]+. tR = 2.35 min (method 7). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.53 (s, 3 H), 3.10 (t, J = 6.6 Hz, 2 H), 3.29 (s, 3 H), 3.79 (t, J = 6.6 Hz, 2 H), 7.56 (d, J = 7.9 Hz, 1 H), 8.03 (dd, J = 8.1, 2.3 Hz, 1 H), 8.06 (d, J = 4.6 Hz, 1 H), 8.44 (d, J = 4.6 Hz, 1 H), 8.70 (dd, J = 4.6, 1.6 Hz, 2 H), 8.76 (d, J = 2.1 Hz, 1 H), 8.82 (dd, J = 4.4, 1.6 Hz, 2 H); mp 111.0 °C. 3-[6-(2-Methoxy-ethyl)-pyridin-3-yl]-2-methyl-8-pyridin-4-ylimidazo[1,2-a]pyrazine (28f). 28f was prepared according to a protocol analogous to intermediate 16 from intermediate 26f and intermediate 27 at 150 °C for 15 min and under microwave irradiation. Flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 2/98) and precipitation from Et2O yielded compound 28f as a white solid (90%). ESI-HRMS: m/z for C20H20N5O [M + H]+ calcd, 346.1668; found, 346.1663 (−1.4 ppm). tR = 2.44 min (method 2). 1H NMR (400 MHz, DMSO-d6) δ ppm N



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The mixture was stirred at 160 °C for 30 min under microwave irradiation. The mixture was diluted with DCM and washed with a saturated solution of ammonium chloride. The organic layer was separated, dried (Na2SO4), filtered, and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; 7 M solution of ammonia in MeOH in DCM 0/100 to 1/99). The desired fractions were collected and the solvents evaporated in vacuo and the crude product precipitated from Et2O to yield intermediate 31 (750 mg, 83%, 90% pure) as a white solid. MS: m/z 423 [M + H]+. tR =3.49 min (method 3). 1H NMR (400 MHz, CDCl3) δ ppm 2.39 (s, 3 H), 3.78−3.87 (m, 4 H), 4.22−4.32 (m, 4 H), 7.68 (s, 1 H); mp 181.2−182.1 °C. Biology Experimental. The Institutional Ethical Committee on Animal Experimentation approved the experimental protocols, in compliance with the Belgian law (Royal Decree on the Protection of Laboratory Animals, April 6, 2010). In Vitro Assay PDE10A. Rat recombinant PDE10A (rPDE10A) was expressed in Sf9 cells using a recombinant rPDE10A baculovirus construct. Cells were harvested after 48 h of infection, and the rPDE10A protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100-fold of the final concentration in the assay. Compound dilutions (0.4 μL) were added in 384-well plates to 20 μL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). Then 10 μL of rPDE10A enzyme in incubation buffer was added, and the reaction was started by addition of 10 μL substrate to a final concentration of 60 nM cAMP and 0.008 μCi [3H]cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 μL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min, the luminescence was measured in a PerkinElmer Topcount scintillation counter and results were expressed as cpm. For blank values the enzyme was omitted from the reaction and replaced by incubation buffer. Control values were obtained by addition of a final concentration of 1% DMSO instead of compound. A best fit curve was fitted by a minimum sum of squares method to the plot of % of control value subtracted with blank value versus compound concentration and the half-maximal inhibitory concentration (IC50) value was derived from this curve. PDE10A Occupancy. Dose−response or single dose experiments were performed to measure PDE10A occupancy 1 h after oral (sc or po) administration. Male Wistar rats (200 g) were treated by sc or po administration of various PDE10 inhibitors. The PDE10 radioligand [3H]MP-10 (10 μCi/animal) was injected intravenously (iv) 30 min before sacrifice. Brains were immediately removed from the skull and rapidly frozen. Then 20 μm thick brain sections were cut using a cryostat-microtome, thaw-mounted on microscope slides and loaded in a β-imager (Biospace Laboratory, Paris, France) for 8 h to quantify PDE10A occupancy in the striatum. Digital autoradiograms were quantified using the Beta Vision Program (Biospace Laboratory). The specific binding was determined as the difference between [3H]MP-10 binding quantified in the striatum (a brain area showing a high density of PDE10A) and in the cortex (a brain area where PDE10A is virtually absent). Occupancy was calculated as the inhibition of specific [3H]MP-10 binding in drug-treated animals relative to vehicle-treated animals. Apomorphine-Induced Stereotypy in Rats. Apomorphine (1.0 mg/kg, iv) induced stereotypy (compulsive sniffing, licking, chewing) was scored every 5 min over the first hour after injection of apomorphine. The score system was: (3) pronounced, (2) moderate, (1) slight, and (0) absent. Criteria for drug-induced inhibition of stereotypy: fewer than 6 scores of 3 (0.16% false positives), fewer than 6 scores of ≥2 (0.0% false positives), or fewer than 7 scores of ≥1 (0.81% false positives). ED50 for Functional Effects. All-or-none criteria for drug-induced effects were defined by analyzing a frequency distribution of a series of historical control data, aiming for less than 5% responders in the control population. The fraction of animals responding to these criteria in animals pretreated with test compound was determined per dose level (n ≥ 3 in the relevant dose range; at least 3 doses). ED50s and corresponding 95% confidence limits were determined according

to the modified Spearman−Kaerber estimate, using theoretical probabilities instead of empirical ones.21 This modification allows the determination of the ED50 and its confidence interval as a function of the slope of the log dose−response curve.22

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ASSOCIATED CONTENT

S Supporting Information *

Pharmacokinetics experimental details, and analytical methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 925232331. Fax: +34 925245771. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank our co-worker Herman Hendrickx from the Neuroscience Discovery team. ABBREVIATIONS USED ACN, acetonitrile; cAMP, cyclic adenosine monophosphate; APO, apomorphine; BTB, brain tissue binding; calcd, calculated; Clint, intrinsic clearance; CNS, central nervous system; CYPs, cytochromes; D2, dopamine 2; DCM, dichloromethane; DAD, diode array; dec, decomposition; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH, ethanol; ER, extraction ratio; cGMP, cyclic guanosine monophosphate; h, hours; HPLC, high-performance liquid chromatography; iv, intravenous; MSD, mass selective detector; MSNs, medium spiny neurons; QTOF, quadrupole time-of-flight detector; [M + H]+, the protonated mass of the free base of the compound; MDFA, methyl 2,2-difluoro-2-(fluorosulfonyl)acetate; MeOH, methanol; min, minutes; mp, melting point; MS, mass spectrum; NBS, N-bromosuccinimide; NIS, N-iodosuccinimide; NMP, N-methyl pyrrolidine; NMR, nuclear magnetic resonance; PCP, phencyclidine; PDE, phosphodiesterase; PET, positron emission tomography; rPPB, rat plasma protein binding; po, per oral; rt, room temperature; RP-HPLC, reverse phase high performance liquid chromatography; sc, subcutaneous; SAR, structure−activity relationship; SQD, single quadrupole detector; THF, tetrahydrofuran; TLC, thin layer chromatography; TOF, time-of-flight detector; tR, retention time; UPLC, ultraperformance liquid chromatography

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REFERENCES

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Discovery of Phosphodiesterase 10A (PDE10A) PET Tracer AMG 580 to Support Clinical Studies.

Characterization of binding and inhibitory properties of TAK-063, a novel phosphodiesterase 10A inhibitor.

Addressing phototoxicity observed in a novel series of biaryl derivatives: discovery of potent, selective and orally active phosphodiesterase 10A inhibitor ASP9436.

Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: A therapeutic approach for schizophrenia with improved side effect profile.

Discovery of [¹¹C]MK-8193 as a PET tracer to measure target engagement of phosphodiesterase 10A (PDE10A) inhibitors.

Discovery of 1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (TAK-063), a highly potent, selective, and orally active phosphodiesterase 10A (PDE10A) inhibitor.

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer.

Discovery of PF-04620110, a Potent, Selective, and Orally Bioavailable Inhibitor of DGAT-1.

A structurally novel inhibitor of cGMP phosphodiesterase with vasodilator activity.

RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models.

Discovery of PF-04449913, a Potent and Orally Bioavailable Inhibitor of Smoothened.

Identification of Tetrahydropyrido[4,3-d]pyrimidine Amides as a New Class of Orally Bioavailable TGR5 Agonists.

Biologic activity of the novel orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 against canine melanoma cell lines.