Orphanin FQ

Article pubs.acs.org/jmc

Discovery of a Novel Series of Orally Active Nociceptin/Orphanin FQ (NOP) Receptor Antagonists Based on a Dihydrospiro(piperidine4,7′-thieno[2,3‑c]pyran) Scaffold Miguel A. Toledo,*,† Concepción Pedregal,†,§ Celia Lafuente,† Nuria Diaz,† Maria Angeles Martinez-Grau,† Alma Jiménez,† Ana Benito,† Alicia Torrado,† Carlos Mateos,† Elizabeth M. Joshi,‡ Steven D. Kahl,‡ Karen S. Rash,‡ Daniel R. Mudra,‡ Vanessa N. Barth,‡ David B. Shaw,‡ David McKinzie,‡ Jeffrey M. Witkin,‡ and Michael A. Statnick‡ †

Centro de Investigación Lilly, Avenida de la Industria 30, 28108-Alcobendas, Madrid, Spain Eli Lilly & Co., Lilly Research Laboratories, Indianapolis, Indiana 46285, United States

‡

S Supporting Information *

ABSTRACT: Nociceptin/OFQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the ORL1/NOP receptor. Nociceptin appears to regulate a host of physiological functions such as biological reactions to stress, anxiety, mood, and drug abuse, in addition to feeding behaviors. To develop tools to study the function of nociceptin and NOP receptor, our research effort sought to identify orally available NOP antagonists. Our effort led to the discovery of a novel chemical series based on the dihydrospiro(piperidine-4,7′thieno[2,3-c]pyran) scaffold. Herein we show that dihydrospiro(piperidine-4,7′-thieno[2,3-c]pyran)-derived compounds are potent NOP antagonists with high selectivity versus classical opioid receptors (μ, δ, and κ). Moreover, these compounds exhibit sufficient bioavailability to produce a high level of NOP receptor occupancy in the brain following oral administration in rats.

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receptor antagonist with potency and selectivity.8 In fact this molecule shows high affinity for NOP receptor with greater than 600-fold selectivity over the other opioid receptors. Compound 1 has become an important pharmacological tool for the elucidation of the different physiological functions related to NOP receptor. Further optimization of this scaffold delivered 2, disclosed by Banyu as the first clinical candidate for multiple indications using an experimental medicine approach.9 3 (JTC-801) is the only NOP antagonist with potent antinociceptive effects in animal models of acute pain.10 Although 3 has entered clinical trials as a potentially novel analgesic,4,6 data have not been released. Phenylpiperidine 4 (SB-612111) was reported as a potent and selective NOP receptor antagonist.11a,b It was found to inhibit food intake and reduce immobility in forced swim and tail suspension assays.11c 4 was also recently proposed for the treatment of Parkinson’s disease.11d Spiropiperidines (5−8) represent a very common structural motif for NOP antagonists.12 Compounds 9,13 10,14 and 1115 are additional representative scaffolds reported in the literature. Finally, 12 (MK-1925) characterizes a new chemical scaffold and was selected by Banyu as the second clinical candidate.16 When dosed orally in mice, 12 exhibited dosedependent antagonist activity against the reduction in locomotor activity induced by a NOP receptor agonist, thus reflecting target engagement.16

INTRODUCTION ORL-1 or NOP receptor is the most recently discovered member of the opioid receptor family.1 It belongs to the class A GPCR, and the 17 amino acid peptide nociceptin/Orphanin FQ (N/OFQ) is the endogenous ligand.2 Activation of NOP receptor is coupled to inhibition of adenylate cyclase as well as to voltage-gated Ca2+ and K+ channels.3 Although the four opioid receptors are structurally related, N/OFQ does not have significant affinity for the classical opioid receptors, and the opioid peptides (enkephalins, endorphin, and dynorphin) do not bind to NOP receptor. This receptor is widely expressed in the CNS, the peripheral nervous system, and some non-neural tissues including gastrointestinal tract, smooth muscle, and immune system. The activation of NOP receptor by its endogenous ligand modulates several physiological functions and behaviors such as anxiety, depression, stress, feeding, bradycardia, locomotor activity, body temperature, substance abuse, diuresis, memory, pain, and inflammatory bowel diseases.4−6 As a result, NOP receptor represents a very promising target to study different physiological mechanisms, and the identification of both agonists and antagonists is an area of interest in the discovery of novel drug therapies.4 Many pharmaceutical companies have devoted research programs to identify small molecules as potent and selective NOP agonists and antagonists.7 As a consequence, several NOP antagonists have been reported in the literature (Figure 1) with Banyu as one of the most active companies in the field. In 1999 they reported 1 (J-113397) as the first small molecule NOP © 2014 American Chemical Society

Received: January 22, 2014 Published: March 28, 2014 3418

dx.doi.org/10.1021/jm500117r | J. Med. Chem. 2014, 57, 3418−3429

Journal of Medicinal Chemistry

Article

Figure 1. Reported NOP receptor antagonists.

In a paper published in 2012, Stevens reported the crystal structure of the human nociceptin/orphanin FQ receptor in complex with the peptide mimetic antagonist compound 5.17 The X-ray structure showed substantial conformational differences in the pocket regions between NOP and the classical opioid receptors and provides new structural avenues for the design of novel NOP receptor antagonists. Early in our medicinal chemistry effort we discovered a new series of high-affinity small molecule NOP receptor antagonists based on a 3-[2′-halo-4′,5′-dihydrospiro(piperidine-4,7′-thieno[2,3-c]pyran)-1-yl]-2-(2-halobenzyl)-N-alkylpropanamide scaffold (Figure 2A).18 As previously reported, this chemotype provided very high NOP receptor binding affinity, selectivity, rapid brain penetration, and favorable kinetics for imaging and 11 C-labeling. The spiropiperidine moiety was part of our initial hit, and the medicinal chemistry effort was developed in parallel to the discovery of NOP receptor PET tracer B (Figure 2). Herein, we describe the discovery of a new series of small molecule NOP receptor antagonists based on the 2′-halo-4′,5′dihydrospiro(piperidine-4,7′-thieno[2,3-c]pyran) scaffold. These derivatives exhibit high NOP receptor affinity and antagonist potency, high selectivity versus the classic opioid receptors, optimal oral bioavailability and PK parameters, and very high levels of receptor occupancy maintained up to 24 h.

Figure 2. 3-(2′-Halo-4′,5′-dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran]-1-yl)-2-(2-halobenzyl)-N-alkylpropanamide scaffold (A) and PET tracer for NOP receptor (B).

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CHEMISTRY The synthesis of the 2-chloro- and 2-fluorothienospiropiperidine ligands 15−21 is outlined in Scheme 1. Starting from the previously reported 2-chloro- and 2-fluorospiro(4,5dihydrothieno[2,3-c]pyran-7,4′-piperidine) intermediates 13 and 14,18 the corresponding final compounds 15−21 were 3419



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16 as a very potent and selective NOP receptor antagonist (Table 1). Table 1 shows the compounds that were prepared and tested for NOP receptor binding and antagonist functional activity. Compounds 15−21 were found to have very high binding affinity, represented by subnanomolar Ki values, to human recombinant NOP receptor using [3H]-nociceptin as radioligand. In a functional assay of receptor-mediated G-protein activation using [35S]-GTPγS binding, these ligands were found to be very potent antagonists of the NOP receptor with subnanomolar Kb values. 4-Pyrazole with a methyl substituent at position 3 was the optimal heterocycle to maintain in vitro potency. After extensive SAR in the distal aromatic ring, it was found that 2,6-disubstituted phenyl and 6-substituted 2-pyridine were preferred for high in vitro potency. As it was shown with the initial carboxamide series, the [2,3]-fused thiophene with 2chloro or 2-fluoro substituents produced equally high potent NOP receptor antagonists (Table 1). Compounds containing a thiophene functionality have been reported to potentially form reactive intermediates due to its πexceeding character.20 Given this concern, in vitro trapping assays were conducted with GSH before progressing further the SAR with this chemical series. Compounds 17 and 20 were incubated with rat hepatic microsomes, and metabolite profiling by LC/MS demonstrated the presence of GSH conjugates (results not shown). Based on these results, modifications to the SAR were incorporated with two goals in mind: (1) block potential benzylic oxidation; (2) decrease the electron density of the thiophene to avoid the formation of reactive intermediates. To this end, we decided to introduce a gemdifluoro group at the benzylic position of the spiro moiety. This approach yielded compound 37 which did not produce GSHrelated conjugates when incubated in the GSH trapping assay within rat hepatic microsomes. Based on the positive results, additional gem-difluoro substituted analogues were prepared and evaluated. Table 2 contains NOP receptor binding and functional antagonism data on compounds with the gem-difluoro group. Ligands 27−32 and 35−37 were found to have very high binding affinitiy to human recombinant NOP receptor as represented by subnanomolar Ki values. These compounds were also very potent antagonists at NOP receptor with subnanomolar Kb potencies. Selectivity versus mu, kappa, and delta opioid receptors was also excellent (Ki > 375 nM). These novel NOP antagonists exhibited excellent in vitro activity and selectivity at NOP receptor, and they were tested in the receptor occupancy assay, previously published, to demonstrate in vivo target engagement.18 Table 3 shows RO data for spiropiperidines 27−32 and 35−37 measured 24 h following oral administration at 10 mg/kg in rats. All compounds showed sustained receptor occupancy, higher than 60% for most cases, proving brain penetration not to be an issue. Two pairs of phenyl/pyridine analogues (27/28 and 31/36) were selected to evaluate the pharmacokinetic profile in rats (Table 4). All compounds show good oral bioavailability. However, as we expected, the lipophilicity reduction provided by the pyridine had significant impact in both the iv and the po PK parameters. Clearance and volume of distribution were reduced for pyridines 28 and 36 in comparison with 27 and 31, respectively. AUC after oral administration was also significantly higher for pyridines 28 and 36. Pyridine compounds 28

Scheme 1. Synthesis of 2-Chloro- and 2Fluorothienospiropiperidines as NOP Receptor Antagonistsa

a

Reagents and conditions: (a) Appropriate pyrazole-4-carbaldehyde, NaBH(OAc)3, 1,2-dichloroethane or THF, rt.

prepared by reductive amination with the appropriate pyrazole4-carbaldehyde using classical conditions. Scheme 2 describes the preparation of the NOP receptor antagonists containing a gem-difluoro group at the benzylic position of the 2-chlorospiro(4,5-dihydrothieno[2,3-c]pyran7,4′-piperidine). These derivatives were all prepared from the key intermediate 25. Benzylic bromination of the previously reported intermediate 2218 with N-bromosuccinimide followed by oxidation under Kornblum conditions19 with sodium bicarbonate gave a mixture of secondary alcohol and ketone 23. This mixture was subjected to a TEMPO/NaClO oxidation to convert the secondary alcohol to the ketone 23. Fluorination with bis(2-methoxyethyl)aminosulfur trifluoride yielded the gem-difluoro derivative 24. This intermediate was deprotected under standard acidic conditions to give the common intermediate 25. Reductive amination with the corresponding pyrazole-4-carbaldehyde took place using classical experimental conditions to deliver key intermediates 26 and 33 and final compounds 27−30. Reduction of the ester 26 with lithium aluminiun hydride gave the benzylic alcohol 31 that was further converted to the secondary amine 32 in a two-step sequence involving oxidation with manganese dioxide and reductive amination with methylamine. Copper-catalyzed reaction of the pyrazole 33 with the appropriate bromopyridine gave rise to the final product 37 and the intermediate aldehyde 34. Intermediate 34 was further transformed to the imidazole 35 or the benzylic alcohol 36 using glyoxal with ammonia or sodium borohydride, respectively.

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RESULTS AND DISCUSSION Starting from our initial hit series (A, Figure 2), the medicinal chemistry effort focused first on modifying substitution on the piperidine nitrogen to optimize the overall performance characteristics of molecules. We envisioned the replacement of the carboxamide by five-membered heterocycles to eliminate the chiral center and reduce the number of rotatable bonds. After the preparation of a few series of compounds with different heterocycles and several patterns of substitution, we identified the 2,6-difluorophenyl substituted 3-methylpyrazole 3420



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Scheme 2. Synthesis of gem-Difluorothienospiropiperidines as NOP Receptor Antagonistsa

a Reagents and conditions: (a) (1) NBS, ℏν, chlorobenzene, 45 °C, (2) DMSO, NaHCO3, rt, (3) KBr, 2,2,6,6-tetramethylpiperidine N-oxide, 6% NaClO (adjusted at pH = 9 with NaHCO3), DCM, 5 °C to rt; (b) (bis(2-methoxyethyl)amino)sulfur trifluoride, THF, 70 °C; (c) (1) 37% HCl, iPrOH, 45 °C, (2) 6 N NaOH; (d) appropriate aldehyde, NaBH(OAc)3, 1,2-dichloroethane or THF, rt; (e) LiAlH4, THF, −20 °C; (f) (1) MnO2, DCM, reflux, (2) 40% MeNH2 in water, NaBH4, EtOH, rt; (g) ArBr, CuI, K2CO3, trans-N,N′-dimethyl-1,2-cyclohexanediamine, toluene, 110 °C; (h) 40% glyoxal in water, 2 N ammonia in MeOH, MeOH, 0 to 40 °C; (i) NaBH4, DCM/MeOH, 0 °C to rt.

NOP receptor binding and functional activity, high selectivity over classical opioid receptors, oral bioavailability, and excellent brain penetration to guarantee high receptor occupancy that was maintained at 10 mg/kg up to 24 h in rats. Consistent with the in vivo target engagement at NOP receptor, a dosedependently pharmacodynamic action preventing agonistinduced hypothermia was demontrated with compound 36.

and 36 improved the overall pharmacokinetic profile due to the reduced lipophilicity (Table 4). To demonstrate a pharmacodynamic action of the current series, reversal of the hypothermic effect induced by the NOP agonist Ro64-619821 was studied in vivo in rat. The hypothermic effects of NOP receptor activation are well documented in the literature.22 Consistent with its in vitro antagonist properties, 36 reversed the NOP agonist-induced hypothermia in a dose-dependent manner, as demonstrated by a significant reversal of NOP-induced hypothermia at 0.1 and 0.3 mg/kg [F(4,35) = 48.03, p < 0.001; Dunnett posthoc comparisons with alpha set at p < 0.05]. Moreover, 36 had no effect on basal body temperature as measured prior to Ro646198 administration [F(3,34) = 0.12, p > 0.95; data not shown]. Therefore, 36 is a potent and selective NOP receptor antagonist with CNS activity in rats following oral administration.

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EXPERIMENTAL SECTION

Materials and Methods. All reagents used were obtained from commercial sources (Sigma-Aldrich, unless otherwise stated). All solvents were of an analytical grade. The NOP agonist ligand Ro646198 was synthesized as reported.21 [35S]-GTPγS was obtained commercially. Flash chromatography was performed in ISCO automated systems using SiO2 cartridges. All final compounds analyzed were >95% pure unless otherwise indicated. 1H NMR (300 MHz) spectra were acquired at 20 °C on a Bruker Avance DPX 300 MHz spectrometer. Abbreviations s, d, dd, ddd, m, and br denote singlet, doublet, double doublet, double double doublet, multiplet, and broad, respectively. High-resolution mass spectrometry (HRMS) was performed on an Agilent 6200 Series ToF with Agilent MassHunter Software (TOF Method, 2 μL injection, 0.2 mL/min isocratic flow rate, 70/30 water +0.1% formic acid/acetonitrile +0.1% formic acid, and 1.5 min run time). LC-MS was performed with an 1100 Series LC-

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SUMMARY We have identified orally bioavailable NOP receptor antagonists based on the dihydrospiro(piperidine-4,7′-thieno[2,3c]pyran) scaffold. These compounds display very potent 3421



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Table 1. Binding Affinity and Functional Activity of the Novel NOP Receptor Antagonists 15−21a

compoundb nociceptin 15 16 17 18 19 20 21

R

X

NOP binding Ki (nM)c

Y

e

F F F F Cl Cl F

N CF CF CF CF N N

0.214 (0.037, n = 9) 0.139 (0.045, n = 3) 0.0711 (0.0080, n = 3) 0.0954 (0.0240, n = 4) 0.0648 (0.0433, n = 3) 0.122 (0.016, n = 3) 0.0908 (0.0246, n = 3) 0.0939 (0.0207, n = 3)

F F CONH2 CH2NMe2 CH2NMe2 CH2OMe CH2OMe

NOP antagonism Kb (nM)d 2.59 (0.26, n = 9)f 0.136 (0.011, n = 3) 0.0494 (0.0034, n = 3) 0.0954 (0.0354, n = 3) 0.0865 (0.0183, n = 3) 0.104 (0.010, n = 3) 0.0805 (0.0080, n = 3) 0.0815 (0.0025, n = 3)

a Shown are geometric means (SEM, n) for Ki and Kb values. b(L)-Tartaric acid salt. c[3H]-Nociceptin was used as a reference NOP radioligand in a binding assay with human recombinant NOP receptor overexpressed in CHO cells. dFunctional activity was determined in an assay of receptormediated G-protein activation using [35S]-GTPγS and membranes from CHO cells overexpressing the human NOP receptor. See Materials and Methods for assay details. eNumber of replicates for nociceptin control correspond to the same individual binding assays where compounds were tested. fNociceptin response in agonist mode of assay (EC50); number of replicates correspond to the same invidual functional assays where compounds were tested.

In Vitro Functional Blockade of NOP Receptor AgonistMediated G-Protein Activation: GTPγ-[35S] Binding. Experimental details were previously disclosed.18a In Vivo Receptor Occupancy (RO) Assays. Male Sprague− Dawley rats weighing between 220 and 300 g were housed on a 12 h light:dark cycle (testing during light phase) and received free access to normal rat chow and water. Animals received either oral vehicle or a single dose of a NOP receptor compound (10 mg/kg). Twenty-four hours later animals received a lateral iv tail vein bolus injection of the nonlabeled tracer (3 μg/kg). Forty minutes after tracer delivery, animals were sacrificed by cervical dislocation and brain tissues (hypothalamus and striatum) were harvested. Tissue samples were weighed and placed in conical centrifuge tubes on ice. Samples were then homogenized in four volumes (w/v) of acetonitrile (ACN) containing 0.1% formic acid and centrifuged at 14 000 rpm for 16 min. Supernatant liquid was diluted by adding sterile water to samples for subsequent LC-MS/MS analysis.18a Rat Pharmacokinetics. Male Sprague−Dawley rats (n = 3 per treatment), fasted for a period between 4 and 24 h and water provided ad libitum, were dosed by intravenous (iv) bolus administration (1 mL/kg) at 1 mg/kg or per oral (po) administration (5 or 10 mL/kg) at 3 or 10 mg/kg of compound 27, 28, 31, or 36. Both iv and po doses were formulated in 20% (w/w) Captisol (Ligand Pharmaceuticals, La Jolla, CA) in phosphate buffer (25 mM, pH 2). Blood samples (between 0.15 and 0.20 mL) were drawn via an arterial catheter into disodium EDTA-containing tubes (BD, Franklin Lakes, NJ) at 0, 0.08, 0.25, 0.50, 1, 2, 4, 8, 12, and 16 or 24 h postdose. Whole blood samples were stored on wet-ice until centrifuged (3000 rpm, 10 min, 4 °C), and plasma supernatant was collected and stored (−20 to −70 °C) until processed for bioanalysis. Plasma proteins were precipitated by mixing with an internal standard (IS) solution (10 ng/mL in methanol:acetonitrile, 1:1) and centrifuged (4000 rpm 10 min, and 4 °C) and the resulting supernatants analyzed for compounds 27, 28, 31, and 36. Analytes were separated by reverse-phase chromatography with biphasic gradient elution (1 M NH4HCO3 mobile phase A; MeOH/1 M NH4HCO3 (200:1, v/v) mobile phase B) on a Betasil C18 column (2.1 × 20 mm, 5 μm; Thermo Fisher Scientific, Waltham, MA) and detected by selective reaction monitoring in positive ion mode (Sciex API 4000; Applied Biosystems/MDS, Foster City, CA). Compounds 27, 28, 31, and 36 were quantified against analyte-in-

MSD single quadrupole instrument (Agilent; Santa Clara, CA) with an ESI interface. The samples were analyzed by the following described methods: Method 1. Used a heated (50 °C) XBridge C18 column (3.5 μm; 2.1 mm × 50 mm) eluted at 1.0 mL/min with a gradient of A (aq 10 mM (NH4)HCO3; pH = 9) and B (acetonitrile), with B increased linearly from 10% to 100% over 7 min. Ions in the range of m/z 100− 800 were captured after electrospray ionization of the eluted test sample. Method 2. Used a heated (50 °C) XTerra-C18 column (3.5 μm; 2.1 mm × 50 mm), eluted at 1.0 mL/min with a gradient of A (aq 10 mM (NH4)HCO3; pH = 9) and B (acetonitrile), with B increased linearly from 10% to 100% over 3 min. Ions in the range of m/z 100−800 were captured after electrospray ionization of the eluted test sample. Statistics. Values are given as means ± the standard deviation of the mean, unless otherwise stated. For the NOP agonist-induced hypothermia experiment, a between-group analysis of variance (ANOVA) with Dunnett posthoc analyses was conducted using vehicle and NOP agonist groups as controls. Reversal of Nociceptin Agonist-Induced Hypothermia. Subjects were male Sprague−Dawley rats weighing 225 g and housed 2 per cage. Rats were maintained on a 12 h light:dark cycle (testing during light phase) and received free access to normal rat chow and water. On test day, rats were weighed, returned to their home cage, and allowed to acclimate in the procedure room for at least 90 min before onset of the study. For each rat, an initial body temperature was taken by rectal probe followed immediately by oral gavage with vehicle or compound 36 (0.03, 0.1, or 0.3 mg/kg). Sixty minutes later, a second body temperature was taken and followed immediately by a 6 mg/kg subcutaneous injection of vehicle or the NOP agonist Ro646198. Finally, a third body temperature was taken 120 min later, a time period in which pilot studies determined that robust hypothermic effects of Ro64-6198 were present. All experiments with rats were performed in accord with the National Research Council Guide under protocols approved by the Animal Care and Use Committee of Eli Lilly and Company. In Vitro NOP Receptor Binding. Experimental details were previously disclosed.18a In Vitro Opioid Receptor Binding. Experimental details were previously disclosed.18a 3422



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clearance, volume of distribution, T1/2, and bioavailability calculated by the ratio of dose normalized (dn) plasma exposures, dnAUCpo/ dnAUCiv. Chemistry. General Procedure for Reductive Amination (Method A). A solution of the corresponding spiropiperidine derivative and the appropriate aldehyde was stirred at room temperature in the appropriate solvent (0.15−0.55 M tetrahydrofuran or 1,2-dichloroethane) for 10−60 min (t1). Then sodium triacetoxyborohydride (1.2−2.0 equiv) was added and the reaction mixture was stirred at room temperature until it was completed (2−16 h, t2). The reaction mixture was treated with ice/saturated solution of sodium bicarbonate, and the organic phase was extracted with ethyl acetate, dichloromethane, or tert-butyl ethyl ether (S2). The organic layer was separated, washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by chromatography. General Procedure for (L)-Tartrate Salt Formation (Method B). The corresponding spiropiperidine compound (1 equiv) was dissolved in methanol (0.2 M), and a solution of (L)-tartaric acid (1 equiv) in methanol (0.2 M) was added. The mixture was stirred for 15 min at room temperature. The solvent was evaporated, and the residue was dried in vacuo overnight to give the final product in high yield (>90%). 2-Fluoro-1′-[[1-(3-fluoro-2-pyridyl)-3-methylpyrazol-4-yl]methyl]spiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)-Tartrate (15). The free base of the title compound was essentially prepared following the general method A using 13 (225 mg, 0.99 mmol), 1-(3fluoro-2-pyridyl)-3-methylpyrazole-4-carbaldehyde (284 mg, 1.38 mmol), and sodium triacetoxyborohydride (420 mg, 1.98 mmol) using 6.6 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S = ethyl acetate). The crude product was purified by reverse phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 40% solvent B isocratic, 2 → 9 min, 40% to 60% solvent B gradient, 9 → 10 min, 60% to 99% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/min, from minute 2 to end: 25 mL/min) to get 157.2 mg of the free base of the title compound (38%). LC-MS (ESI) m/z: 417 (M + H)+, tR = 3.51 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.32 (dt, J = 4.8, 1.5 Hz, 1H), 8.23 (broad s, 1H), 7.95 (ddd, J = 11.5, 8.2, 1.5 Hz, 1H), 7.47− 7.41 (m, 1H), 6.45 (d, J = 1.8 Hz, 1H), 3.85 (t, J = 5.5 Hz, 2H), 3.33 (s, 2H), 2.67−2.60 (m, 5H), 2.35−2.25 (m, 4H), 1.95 (d, J = 12.4 Hz, 2H), 1.65 (td, J = 12.8, 4.1 Hz, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 99% yield. LC-MS (ESI) m/z: 417 (M + H)+, tR = 3.51 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.35−8.30 (m, 2H), 7.97 (ddd, J = 11.6, 8.3, 1.4 Hz, 1H), 7.49−7.43 (m, 1H), 6.48−6.46 (m, 1H), 4.18 (s, 2H), 3.86 (t, J = 5.3 Hz, 2H), 3.64 (s, 2H), 3.57−3.51 (m, 2H), 2.84−2.79 (m, 2H), 2.54−2.48 (m, 4H), 2.27 (s, 3H), 2.06−1.98 (m, 2H), 1.78− 1.66 (m, 2H). HRMS (ESI): calcd for C21H22F2N4OS, 416.1482; found, 416.1492. 1′-[[1-(2,6-Difluorophenyl)-3-methylpyrazol-4-yl]methyl]-2fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)-Tartrate (16). Triethylamine (0.064 mL, 0.46 mmol) was added to a suspension of 2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] hydrochloride (13·HCl) (121 mg, 0.46 mmol) in 1,2dichloroethane (1.15 mL). To the resulting solution was added 1(2,6-difluorophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde (153 mg, 0.69 mmol) followed by sodium triacetoxyborohydride (420 mg, 1.98 mmol). The resulting reaction mixture was stirred at room temperature overnight. The solvent was evaporated, and the residue was purified by SCX (methanol and 2 M ammonia in methanol). The fractions containing the product were collected and evaporated. This crude product was further purified by reverse phase HPLC (XTerra MS C18:5 μm, 10 × 100 mm. pH = 8. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 50% solvent B isocratic, 2 → 9 min, 50% to 70% solvent B gradient, 9 → 10 min, 70% to 88% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/min, from minute 2 to end: 25 mL/min) to get 92.2 mg of the free base of the title compound (46%) as an oil. LC-MS (ESI) m/z: 434 (M + H)+, tR

Table 2. Binding affinity and functional activity of the novel NOP receptor antagonists 27-32 and 35-37a

compoundb

X

27

CF

F

28

N

F

29

CF

CH2NMe2

30

N

CH2NMe2

31

CF

CH2OH

32

CF

CH2NHMe

35

N

2-imidazol

36

N

CH2OH

37

N

CH2OMe

R

NOP binding Ki (nM)c

NOP antagonism Kb (nM)d

0.172 (0.034, n = 3) 0.176 (0.025, n = 4) 0.145 (0.033, n = 3) 0.371 (0.030, n = 3) 0.114 (0.017, n = 3) 0.0970 (0.0421, n = 3) 0.0916 (0.0122, n = 4) 0.105 (0.036, n = 3) 0.185 (0.007, n = 3)

0.198 (0.080, n 0.273 (0.061, n 0.221 (0.019, n 1.20 (0.32,

= 3) = 4) = 3) n = 3)

0.0840 (0.0177, n = 3) 0.190 (0.019, n = 3) 0.202 (0.031, n = 4) 0.166 (0.035, n = 3) 0.252 (0.070, n = 2)

a Shown are geometric means (SEM, n) for Ki and Kb values. Radioligand binding with the nonselective opioid antagonist, [3H]diprenorphine, using membranes prepared from CHO cells overexpressing the recombinant human kappa, mu, or delta opioid receptors gave Ki values >375 nM. Human NOP EC50 agonist activity was >10000 nM for molecules tested (compounds 30, 32−37). See Materials and Methods for assay details. b(L)-Tartaric acid salt except free base for compound 35. c[3H]-Nociceptin was used as a reference NOP radioligand in a binding assay with human recombinant NOP receptor overexpressed in CHO cells. dFunctional activity was determined in an assay of receptor-mediated G-protein activation using [35S]-GTPγS and membranes from CHO cells overexpressing the human NOP receptor.

Table 3. Receptor Occupancy (RO) of NOP Antagonists in Rats after 24 h of Oral Administration

a

compounda

RO (%) 10 mg/kg

27 28 29 30 31 32 35 36 37

49 55 97 72 87 100 88 62 90

(L)-Tartaric acid salt.

plasma standard curve (prepared between 1 and 50 000 ng/mL) prepared under the same conditions as the test samples, and total plasma concentration was determined by analyte:IS chromatographic peak area ratios. Concentration−time data were analyzed by noncompartmental pharmacokinetic methods to determine plasma 3423



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Table 4. In Vivo Pharmacokinetic Data for NOP Receptor Antagonists in Rats after Oral and Intraveous Administrationa intravenous administration

a

compd

clogP

dose (mg/kg)

27 28 31 36

5.1 4.4 4.3 3.5

1 1 1 1

Cl (mL/min/kg) 52.9 19.8 60.5 23.9

± ± ± ±

0.3 5.8 8.4 5.9

oral administration

Vd (L/kg) 28.2 13.6 12.7 5.3

± ± ± ±

3.2 3.9 0.9 0.8

t1/2 (h)

dose (mg/kg)

± ± ± ±

3 3 10 10

10.8 12.1 3.9 3.8

1 1.8 0.2 0.5

po AUC0‑inf (ng·h/mL)

F (%)

± ± ± ±

49 62 51 57

438 1815 1279 4103

110 1213 469 1418

Cl, clearance; Vd, volume of distribution; t1/2, plasma elimination half-life; AUC, area under the curve; F, bioavailability.

Figure 3. NOP receptor antagonist 36 prevents NOP receptor agonist-induced hypothermia in rats. Ro64-6198 (6 mg/kg, sc) was the NOP receptor agonist used in this experiment. V = Vehicle treatment. #p < 0.05 versus V/NOP agonist group; *p < 0.05 versus V/V group. = 4.10 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.86 (s, 1H), 7.60−7.52 (m, 1H), 7.36−7.29 (m, 2H), 6.45 (d, J = 1.8 Hz, 1H), 3.86 (t, J = 5.3 Hz, 2H), 3.34−3.32 (m, 4H), 2.7−2.6 (m, 2H), 2.35−2.20 (m, 5H), 1.95 (d, J = 12.4 Hz, 2H), 1.65 (td, J = 12.9, 4.1 Hz, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 97% yield. LC-MS (ESI) m/z: 434 (M + H)+, tR = 4.13 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.94 (s, 1H), 7.61−7.51 (m, 1H), 7.37−7.30 (m, 2H), 6.47 (d, J = 1.8 Hz, 1H), 4.19 (s, 2H), 3.87 (t, J = 5.5 Hz, 2H), 3.64−3.54 (m, 2H), 2.83−2.76 (m, 2H), 2.52−2.45 (m, 4H), 2.24 (s, 3H), 2.00 (d, J = 13.2 Hz, 2H), 1.73 (td, J = 13.0, 3.8 Hz, 2H). HRMS (ESI): calcd for C22H22F3N3OS, 433.1436; found, 433.1488. 3-Fluoro-2-[4-[(2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]benzamide (L)-Tartrate (17). The free base of the title compound was essentially prepared following the general method A using 13 (5.26 g, 23.14 mmol), 3-fluoro-2-(4-formyl-3-methylpyrazol-1-yl)benzamide (5.20 g, 21.03 mmol), and sodium triacetoxyborohydride (5.35 g, 25.24 mmol) using 52 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S = ethyl acetate). The crude product was purified by silica gel eluting with CH2Cl2/MeOH (5%) to get 8.5 g (87%) of the free base of the title compound as a white foam. LC-MS (ESI) m/z: 459 (M + H)+, tR = 2.73 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 7.70 (d, J = 8.0 Hz, 1H), 7.52−7.45 (m, 2H), 7.35−7.28 (m, 1H), 6.50 (broad s, 1H), 6.11 (d, J = 1.4 Hz, 1H), 5.80 (broad s, 1H), 3.91 (t, J = 5.5 Hz, 2H), 3.47 (s, 2H), 2.70 (d, J = 11.0 Hz, 2H), 2.54 (t, J = 5.4 Hz, 2H), 2.37− 2.33 (m, 5H), 2.00 (d, J = 12.6 Hz, 2H), 1.80 (td, J = 12.9, 4.1 Hz, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 99% yield. LC-MS (ESI) m/z: 459 (M + H)+, tR = 2.78 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.83 (s, 1H), 7.64 (s, 1H), 7.57−7.46 (m, 2H), 7.41−7.36 (m, 2H), 6.47 (d, J = 1.9 Hz, 1H), 4.16 (s, 2H), 3.87 (t, J = 5.1 Hz, 2H), 3.65 (broad s, 2H), 2.93−2.82 (m, 2H), 2.60−2.46 (m, 4H), 2.21 (s, 3H), 2.02 (d, J = 13.2 Hz, 2H), 1.81−1.73 (m, 2H). HRMS (ESI): calcd for C23H24F2N4O2S, 458.1588; found, 458.1596. 1-[3-Fluoro-2-[4-[(2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]phenyl]-N,N-dimethylmethanamine (L)-Tartrate (18). 1-[2-(Dimethylaminomethyl)-6-fluorophenyl]-3-methylpyrazole-4-carbaldehyde (214 mg, 0.82

mmol) was added to a solution of 13 (186 mg, 0.82 mmol) in 1,2dichloroethane (4 mL). Then sodium triacetoxyborohydride (348 mg, 1.64 mmol) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated, and the crude material was purified using a 5 g SCX column (methanol and 2 M ammonia in methanol). The basic fraction was evaporated, and the material was further purified by reserve phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 40% solvent B isocratic, 2 → 9 min, 40% to 65% solvent B gradient, 9 → 10 min, 65% to 99% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/min, from minute 2 to end: 25 mL/min) to give 246 mg (64%) of the free base of the title compound. LC-MS (ESI) m/z: 473 (M + H)+, tR = 3.96 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.73 (s, 1H), 7.53−7.43 (m, 1H), 7.40−7.25 (m, 2H), 6.45 (d, J = 1.8 Hz,1H), 3.85 (t, J = 5.1 Hz, 2H), 3.30 (s, 2H), 3.14 (s, 2H), 2.71−2.59 (m, 2H), 2.57−2.50 (m, 2H), 2.32−2.21 (m, 2H), 2.20 (s, 3H), 2.01 (s, 6H), 1.99−1.89 (m, 2H), 1.72−1.57 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 100% yield. LC-MS (ESI) m/z: 473 (M + H)+, tR = 3.82 min, method 1. 1H NMR (300 MHz, MeOD) δ: 8.38 (d, J = 2.2 Hz, 1H), 7.81−7.69 (m, 3H), 6.47 (d, J = 1.8 Hz, 1H), 4.53 (s, 2H), 4.24 (d, J = 12.1 Hz, 3H), 4.16 (t, J = 5.5 Hz, 2H), 3.54 (s, 2H), 3.46 (d, J = 1.5 Hz, 1H), 3.31−3.21 (m, 2H), 2.92 (s, 6H), 2.78 (t, J = 5.3 Hz, 2H), 2.62 (s, 3H), 2.40−2.25 (m, 4H). HRMS (ESI): calcd for C25H30F2N4OS, 472.2108; found, 472.212. 1-[2-[4-[(2-Chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-N,N-dimethylmethanamine (L)-Tartrate (19). The free base of the title compound was essentially prepared following general method A using 14 (200 mg, 0.82 mmol), 1-[2-(dimethylaminomethyl)-6-fluorophenyl]-3-methylpyrazole-4-carbaldehyde (257 mg, 0.98 mmol), and sodium triacetoxyborohydride (348 mg, 1.64 mmol) using 2 mL of 1,2-dichloroethane (t1 = 60 min, t2 = 16 h, S = dichloromethane). The crude product was purified by reverse phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 60% solvent B isocratic, 2 → 9 min, 60% to 80% solvent B gradient, 9 → 10 min, 80% to 99% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow 3424



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rate: first 2 min: 15 → 25 mL/min, from minute 2 to end: 25 mL/ min) to give 204 mg (51%) of the free base of the title compound. LCMS (ESI) m/z: 489 (M + H)+, tR = 4.28 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.81 (s, 1H), 7.52−7.44 (m, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.30 (dt, J = 8.1, 1.5 Hz, 1H), 6.85 (s, 1H), 3.84 (t, J = 5.4 Hz, 2H), 3.33 (s, 2H), 3.14 (s, 2H), 2.70−2.60 (m, 2H), 2.57−2.50 (m, 2H), 2.34−2.22 (m, 2H), 2.21 (s, 3H), 2.01 (s, 6H), 1.99−1.90 (m, 2H), 1.75−1.61 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 100% yield. LCMS (ESI) m/z: 489 (M + H)+, tR = 4.14 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.85 (s, 1H), 7.55−7.45 (m, 1H), 7.38−7.30 (m, 2H), 6.87 (s, 1H), 4.15 (s, 2H), 3.85 (t, J = 5.2 Hz, 2H), 3.65− 3.58 (m, 2H), 3.25 (s, 2H), 3.16 (s, 2H), 2.87−2.77 (m, 2H), 2.58− 2.48 (m, 2H), 2.24 (s, 3H), 2.08 (s, 6H), 2.06−1.96 (m, 2H), 1.86− 1.70 (m, 2H). HRMS (ESI): calcd for C25H30ClFN4OS, 488.1813; found, 488.1821. 2-Chloro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazol4-yl]methyl]spiro[4,5-dihydrothieno [2,3-c]pyran-7,4′-piperidine] (L)-Tartrate (20). The free base of the title compound was essentially prepared following the general method A using 14 (200 mg, 0.82 mmol), 1-(3-methoxymethy1−2-pyridy1)-3-methy1-pyrazole-4-carbaldehyde (0.228 g, 0.984 mmol), and sodium triacetoxyborohydride (348 mg, 1.64 mmol) using 3 mL of 1,2-dichloroethane (t1 = 60 min, t2 = 16 h, S = dichloromethane). The crude product was purified by reverse phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 40% solvent B isocratic, 2 → 9 min, 40% to 60% solvent B gradient, 9 → 10 min, 60% to 99% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/ min, from minute 2 to end: 25 mL/min) to get the free base of the title compound (286 mg, 76% yield). LC-MS (ESI) m/z: 459 (M + H)+, tR = 4.31 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.38 (dd, J = 4.5, 1.8 Hz, 1H), 8.27 (s, 1H), 8.02 (m, 1H), 7.38 (dd, J = 7.8, 4.8 Hz, 1H), 6.85 (s, 1H), 4.79 (s, 2H), 3.82 (m, 2H), 3.41 (s, 2H), 3.33 (s, 3H), 2.66 (m, 2H), 2.54 (m, 2H), 2.28 (m, 2H), 2.25 (s, 3H), 1.96 (m, 2H), 1.68 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 98% yield. LCMS (ESI) m/z: 459 (M + H)+; tR = 4.35 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.39 (m, 1H), 8.36 (s, 1H), 8.04 (m, 1H), 7.40 (dd, J = 7.5, 4.8 Hz, 1H), 6.87 (s, 1H), 4.80 (s, 2H), 4.19 (s, 2H), 3.85 (m, 2H), 3.63 (s, 2H), 3.35 (s, 3H), 2.83 (m, 2H), 2.58−2.45 (m, 4H), 2.28 (br s, 3H), 2.02 (m, 2H), 1.76 (m, 2H). HRMS (ESI): calcd for C23H27ClN4O2S, 458.1543; found, 458.1547. 2-Fluoro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazol4-yl]methyl]spiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)Tartrate (21). The free base of the title compound was essentially prepared following general method A using 13 (225 mg, 0.99 mmol), 1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazole-4-carbaldehyde (320 mg, 1.48 mmol), and sodium triacetoxyborohydride (420 mg, 1.98 mmol) using 6 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S = ethyl acetate). The crude product was purified by reverse phase HPLC in basic conditions (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient 40−70% B in 8 min) to get 203.4 mg of the free base of the title compound (43%). LC-MS (ESI) m/z: 443 (M + H)+, tR = 3.94 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.38 (dd, J = 1.5, 4.8 Hz, 1H), 8.26 (s, 1H), 8.03−8.01 (m, 1H), 7.38 (dd, J = 4.8, 7.7 Hz, 1H), 6.44 (d, J = 1.5 Hz, 1H), 4.79 (s, 2H), 3.85 (t, J = 5.3 Hz, 2H), 3.40 (s, 3H), 2.65 (d, J = 10.6 Hz, 2H), 2.50 (dd, J = 1.8, 3.7 Hz, 11H), 2.27−2.25 (m, 5H), 1.94 (d, J = 12.8 Hz, 2H), 1.65 (td, J = 12.8, 3.8 Hz, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 99% yield. LC-MS (ESI) m/z: 443 (M + H)+, tR = 3.94 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.40−8.37 (m, 2H), 8.04 (d, J = 7.5 Hz, 1H), 7.40 (dd, J = 4.7, 7.7 Hz, 1H), 6.46 (d, J = 1.8 Hz, 1H), 4.79 (s, 2H), 4.17 (s, 1H), 3.86 (t, J = 5.3 Hz, 2H), 3.66 (s, 1H), 3.35 (s, 3H), 2.85 (d, J = 11.1 Hz, 1H), 2.60−2.57 (m, 2H), 2.53−2.50 (m, 3H), 2.28 (s, 3H), 2.04− 2.00 (m, 1H), 1.80−1.74 (m, 1H). tert-Butyl 2′-Chloro-4′-oxospiro[piperidine-4,7′-thieno[2,3-c]pyran]-1-carboxylate (23). N-Bromosuccinimide (115.02 g, 646.23

mmol) was added to a solution of tert-butyl 2′-chloro-4′,5′dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran]-1-carboxylate (22) (200 g, 581.61 mmol) in chlorobenzene (1.6 L) at rt. The resulting suspension was irradiated with three 100 W bulb lamps situated almost in contact with the external reactor wall, and reactor T was set to 45 °C. After 4 h, N-bromosuccinimide (26.14 g, 146.87 mmol) was added and T was maintained at 40 °C for 15 h. The reaction mixture was cooled to 0 °C and MTBE (500 mL) was added. The solid was filtered. The solution was concentrated to about 1000 mL solution in chlorobenzene. Then MTBE (1000 mL) was added, the solids were filtered, and the filtrate was concentrated to afford a 600 mL chlorobenzene solution of the bromide. DMSO (806.47 mL, 11.35 mol) was added at room temperature, and then sodium bicarbonate (95.38 g, 1.14 mol) was added. After stirring 24 h at room temperature, H2O/ice (1000 mL) was added and the phases were separated. The organic phase was washed with H2O (2 × 1 L) and concentrated to afford a solution in chlorobenzene. Then dichloromethane (1.2 L) was added, and the mixture was cooled to 5 °C (ice/ H2O bath). Potassium bromide (20.27 g, 170.31 mmol) and 2,2,6,6tetramethylpiperidine N-oxide (4.43 g, 28.38 mmol) were added. Then a solution of 6% sodium hypochlorite in H2O (644.40 mL, 567.68 mmol) adjusted to pH = 9 with NaHCO3 (s) was added to the reaction mixture at 5 °C, and the resulting mixture was stirred 1 h from 5 °C to rt. H2O (1 L) was added, and the phases were separated. The organic phase was washed with H2O (2 × 0.5 L) and was cooled with ice/H2O bath. Then potassium bromide (2.03 g, 17.03 mmol), 2,2,6,6-tetramethylpiperidine N-oxide (50 mg; 0.32 mmol), and a solution of 6% sodium hypochlorite in H2O (128.88 mL, 113.54 mmol) adjusted to pH = 9 with NaHCO3 (s) was added to the reaction mixture at 5 °C. The resulting mixture was stirred 1 h from 5 °C to room temperature. Then H2O (1L) was added, and the phases were separated. The organic phase was washed with H2O (2 × 1 L) dried and concentrated to afford a dark brown solid. The solid was triturated with hexane (500 mL), methyl tert-butyl ether/hexane 5% (250 mL), and methyl tert-butyl ether/hexane 10% (250 mL) to obtain the title compound as a light brown solid (135 g, 65% yield). LC-MS (ESI) m/z: 258 (M+ − 101 (− CO2tBu)). tR = 2.54 min, method 2. 1H NMR (300 MHz, CDCl3) δ: 7.18 (s, 1H), 4.32 (s, 2H), 4.19−3.90 (m, 2H), 3.12 (t, J = 11.5 Hz, 2H), 2.17−2.03 (m, 2H), 1.83−1.69 (m, 2H), 1.47 (s, 9H). tert-Butyl 2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′piperidine]-1′-carboxylate (24). In a 500 mL PFA flask was added (bis(2-methoxyethyl)amino)sulfur trifluoride (183.62 g, 829.94 mmol) to tetrahydrofuran (81.00 mL), and then 23 (135 g, 377.24 mmol) was added. The resulting suspension was stirred at 70 °C for 24 h. Then it was cooled to room temperature and slowly poured over a mixture of ice and a saturated solution of sodium bicarbonate (4 L) with stirring (gas evolution). Methyl tert-butyl ether was used to transfer the remaining material from flasks. After gas evolution ceased, sodium bicarbonate (solid) was added with stirring until pH = 8 was reached. The resulting mixture was extracted with methyl tert-butyl ether (3 × 500 mL) until no product was detected by TLC in the aqueous phase. Combined organics were washed with H2O (3 × 500 mL) and brine (500 mL), dried over Na2S04, and concentrated to afford a dark thick oil (250 g). Crude material was filtered though a SiO2 plug (dissolved in dichloromethane), eluting with methyl tert-butyl ether/hexane 10% (6 L) and methyl tert-butyl ether/hexane 20% (4 L). Fractions were collected until no product was detected by TLC (20% methyl tertbutyl ether/hexane, UV, Rf = 0.5). The filtrate was concentrated to get the title compound as a light brown solid (93g, 65% yield). LC-MS (ESI) m/z: 324 (M+ − 57 (− tBu)). tR = 2.83 min, method 2. 1H NMR (300 MHz, CDCl3) δ: 6.98 (s, 1H), 4.09−3.99 (m, 4H), 3.11 (t, J = 12.6 Hz, 2H), 2.06 (d, J = 12.9 Hz, 2H), 1.72 (td, J = 13.3, 4.8 Hz, 2H), 1.48 (s, 9H). HRMS (ESI): calcd for C16H20ClF2NO3S 379.082, found 379.0828. 2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine (25). Hydrochloric acid (37%) in H2O (74.12 mL, 789.78 mmol) was added to a solution of 24 (60 g, 157.96 mmol) in isopropyl alcohol (420 mL) at 45 °C. The resulting solution was stirred at 45 °C 15 h. The reaction mixture was concentrated to 1/4 volume to afford a 3425



Article

white suspension. Then H2O (100 mL) was added, and the suspension was basified with 6 N sodium hydroxide to obtain a two-layer mixture. It was extracted with methyl tert-butyl ether (3 × 100 mL). Combined organics were washed with brine (50 mL), dried over Na2SO4, and concentrated to afford the title compound as a light brown solid (42 g, 95% yield). LC-MS (ESI) m/z: 280 (M + H)+; tR = 1.92 min, method 2. 1H NMR (300 MHz, CDCl3) δ: 6.97 (s, 1H), 4.06 (t, J = 10.2 Hz, 2H), 3.10−2.97 (m, 4H), 2.14−2.07 (m, 2H), 1.86−1.76 (m, 2H). HRMS (ESI): calcd for C11H12ClF2NOS 279.0296, found 279.0305. Methyl 2-[4-[(2-chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorobenzoate (26). The title compound was essentially prepared following general method A using 25 (19.95 g, 71.31 mmol), methyl 3-fluoro-2(4-formyl-3-methylpyrazol-1-yl)benzoate (17 g, 64.83 mmol) and sodium triacetoxyborohydride (16.49 g, 77.79 mmol) using 170 mL of tetrahydrofuran (t1 = 60 min, t2 = 16 h, S = ethyl acetate). The crude product was dissolved in methyl tert-butyl ether, and a beige solid precipitated (excess of spiropiperidine). The solid was filtered, and the solution was concentrated and purified by ISCO (330 g cartridge), eluting with hexane/ethyl acetate (80:20 to 0:100 ratio) to obtain the title compound as a beige solid (25 g, 73% yield). LC-MS (ESI) m/z: 526 (M + H)+; tR = 2.60 min, method 2. 1H NMR (300 MHz, CDCl3) δ: 7.58 (m, 2H), 7.43−7.31 (m, 2H), 6.96 (s, 1H), 4.04 (t, J = 10.2 Hz, 2H), 3.70 (s, 3H), 3.49 (s, 2H), 2.81−2.77 (m, 2H), 2.43−2.35 (m, 2H), 2.30 (s, 3H), 2.16−2.04 (m, 2H), 1.89 (m, 2H). HRMS (ESI): calcd for C24H23ClF3N3O3S 525.1101, found 525.1098. 2-Chloro-1′-[[1-(2,6-difluorophenyl)-3-methylpyrazol-4-yl]methyl]-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine (L)Tartrate (27). The free base of the title compound was essentially prepared following general method A using 25 (7.14 g, 25.52 mmol), 1-(2,6-difluorophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde (5.40 g, 24.30 mmol), and sodium triacetoxyborohydride (9.27 g, 43.75 mmol) using 54 mL of 1,2-dichloroethane (t1 = 30 min, t2 = 2 h, S = tertbutyl methyl ether). The crude product was purified by silica gel chromatography eluting with dichloromethane/methanol (97:3) to give 7.1 g (60% yield) of the free base of the title compound. LC-MS (ESI) m/z: 486 (M + H)+; tR = 4.66 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 7.54 (s, 1H), 7.37−7.28 (m, 1H), 7.08−7.01 (m, 2H), 6.96 (s, 1H), 4.05 (t, J = 10.3 Hz, 2H), 3.49 (s, 2H), 2.84−2.80 (m, 2H), 2.41 (m, 2H), 2.35 (s, 3H), 2.13−2.06 (m, 2H), 1.98−1.81 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 96% yield. LC-MS (ESI) m/z: 486 (M + H)+. tR = 4.67 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.92 (s, 1H), 7.61−7.51 (m, 1H), 7.36−7.30 (m, 3H), 4.24 (s, 2H), 4.18 (t, J = 10.7 Hz, 1H), 3.54 (s, 2H), 2.80 (d, J = 11.1 Hz, 2H), 2.47−2.37 (m, 2H), 2.23 (s, 3H), 2.19−2.12 (m, 2H), 1.88−1.76 (m, 2H). HRMS (ESI): calcd for C22H20ClF4N3OS, 485.0952; found, 485.0963. 2-Chloro-4,4-difluoro-1′-[[1-(3-fluoro-2-pyridyl)-3-methylpyrazol4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (L)-Tartrate (28). The free base of the title compound was essentially prepared following general method A using 25 (90% purity) (19.99 g, 64.33 mmol), 1-(3-fluoro-2-pyridyl)-3-methylpyrazole-4-carbaldehyde (12 g, 58.48 mmol), and sodium triacetoxyborohydride (18.59 g, 87.72 mmol) using 120 mL of 1,2-dichloroethane (t1 = 15 min, t2 = 15 h, S = tert-butyl methyl ether). The crude product was purified by silica gel chromatography, eluting with mixtures of dichloromethane/methanol to give 18 g of a thick oil which was triturated with hexane and 10% tert-butyl methyl ether/hexane to afford 16.5 g (61% yield) of the free base of the title compound. LC-MS (ESI) m/z: 469 (M + H)+; tR = 4.27 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.33−8.31 (m, 1H), 8.24 (s, 1H), 7.95 (ddd, J = 11.5, 8.2, 1.5 Hz, 1H), 7.47−7.41 (m, 1H), 7.33 (s, 1H), 4.16 (t, J = 10.8 Hz, 2H), 3.44 (s, 2H), 2.70 (d, J = 11.7 Hz, 2H), 2.28 (m, 2H), 2.26 (s, 3H), 2.12−2.04 (m, 2H), 1.76 (td, J = 12.9, 3.8 Hz, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 97% yield. LCMS (ESI) m/z: 469 (M + H)+. tR = 4.28 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.33 (d, J = 4.3 Hz, 1H), 8.28 (s, 1H), 7.97− 7.93 (m, 1H), 7.48−7.43 (m, 1H), 7.35 (s, 1H), 4.24 (s, 2H), 4.18 (t, J = 10.9 Hz, 2H), 3.55 (s, 2H), 2.81−2.77 (m, 2H), 2.42−2.39 (m, 2H),

2.27 (s, 3H), 2.18−2.11 (m, 2H), 1.80−1.77 (m, 2H). HRMS (ESI): calcd for C21H20ClF3N4OS, 468.0998; found, 468.0997. 1-[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]N,N-dimethylmethanamine (L)-Tartrate (29). The free base of the title compound was essentially prepared following general method A using 25 (151 mg, 0.54 mmol), 1-[2-(dimethylaminomethyl)-6fluorophenyl]-3-methylpyrazole-4-carbaldehyde (128 mg, 0.49 mmol), and sodium triacetoxyborohydride (208 mg, 0.98 mmol) using 3 mL of tetrahydrofuran (t1 = 60 min, t2 = 16 h, S = ethyl acetate). The crude product was purified by Isco chromatography, eluting with dichloromethane/2 M ammonia in methanol from 3% to 7% to get the free base of the title compound (256 mg, 100%). LC-MS (ESI) m/z: 525 (M + H)+; tR = 2.67 min, method 2. The title compound (tartrate salt) was essentially prepared following general method B with 94% yield. LC-MS (ESI) m/z: 525 (M + H)+; tR = 4.47 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.81 (s, 1H), 7.51 (td, J = 7.9, 5.7 Hz, 1H), 7.40−7.33 (m, 3H), 4.19−4.14 (m, 3H), 3.49−3.40 (m, 5H), 2.81−2.71 (m, 2H), 2.52−2.50 (m, 1H), 2.25− 2.33 (m, 1H), 2.23 (s, 3H), 2.18−2.07 (m, 8H), 1.86−1.70 (m, 2H). 1-[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-pyridyl]-N,N-dimethylmethanamine (L)-Tartrate (30). The free base of the title compound was essentially prepared following general method A using 25 (140 mg, 0.51 mmol), 1-(3-dimethylaminomethylpyridin-2-yl)-3methyl-1H-pyrazole-4-carbaldehyde (0.12 g, 0.51 mmol), and sodium triacetoxyborohydride (220 mg, 1.02 mmol) using 2.5 mL of tetrahydrofuran (t1 = 60 min, t2 = 6 h, S = ethyl acetate). The crude product was purified by reverse phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. gradient 40−70% B in 8 min) to give 106 mg (40%) of the title compound as the free base. LC-MS (ESI) m/z: 508 (M + H)+. tR = 4.08 min, method 1. 1H NMR (300 MHz, DMSOd6) δ: 8.37 (dd, J = 1.8, 4.8 Hz, 1H), 8.16 (s, 1H), 8.03 (dd, J = 1.8, 7.7 Hz, 1H), 7.41−7.34 (m, 1H), 7.34 (s, 1H), 4.20−4.13 (m, 2H), 3.72 (s, 2H), 3.43−3.40 (m, 2H), 2.71 (d, J = 11.0 Hz, 2H), 2.28−2.25 (m, 2H), 2.25 (s, 3H), 2.10−2.08 (m, 2H), 2.08 (s, 6H), 1.83−1.72 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B (97% yield). LC-MS (ESI) m/z: 508 (M + H)+. tR = 3.97 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.46 (dd, J = 1.6, 4.8 Hz, 1H), 8.31 (s, 1H), 8.04 (dd, J = 1.6, 7.7 Hz, 1H), 7.42 (dd, J = 4.8, 7.7 Hz, 1H), 7.34 (s, 1H), 4.21−4.03 (m, 5H), 3.47 (s, 2H), 3.16 (s, 2H), 2.74−2.71 (m, 2H), 2.40−2.30 (m, 5H), 2.29 (m, 8H), 2.18−2.09 (m, 2H), 1.83−1.73 (m, 2H). [2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]methanol (L)-Tartrate (31). A solution of 1 M lithium aluminum hydride in tetrahydrofuran (45.63 mL, 45.63 mmol) was added to a solution of 26 (30 g, 57.04 mmol) in tetrahydrofuran (240 mL) under nitrogen at −20 °C. The cold bath was removed, allowing the reaction mixture to reach 0 °C in 30 min. Water (2 mL) was added dropwise carefully, followed by 2 N sodium hydroxide (2 mL) and water (6 mL). The resulting suspension was stirred at room temperature for 30 min, filtered over Celite, and solid washed with ethyl acetate (20 mL). The filtrate was dried over sodium sulfate, concentrated, and purified by Isco (330 g cartridge), eluting with 2-propanol/dichloromethane (3:97 to 6:94 ratio) to get the free base of the title compound as a colorless oil (23.75 g, 84% yield). LC-MS (ESI) m/z: 498 (M + H)+; tR = 4.10 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 7.65 (d, J = 3.9 Hz, 1H), 7.30 (m, 1H), 7.29 (s, 1H), 7.17 (m, 1H), 6.96 (s, 1H), 5.29 (br s, 1H), 4.37 (br s, 2H), 4.04 (t, J = 10.2 Hz, 2H), 3.47 (s, 2H), 2.78 (m, 2H), 2.39 (m, 2H), 2.33 (s, 3H), 2.1 (m, 2H), 1.86 (m, 2H). HRMS (ESI): calcd for C23H23ClF3N3O2S 497.1152, found 497.1151. The title compound (tartrate salt) was essentially prepared following general method B with 95% yield. LC-MS (ESI) m/z: 498 (M + H)+; tR = 4.14 min, method 1. 1H NMR (500 MHz, acetone-d6) δ: 8.04 (br s, 1H), 7.54−7.47 (m, 2H), 7.27 (m, 1H), 7.18 (s, 1H), 4.44 (s, 2H), 4.40 (s, 2H), 4.22 (t, J = 10.2 Hz, 2H), 4.06 (s, 2H), 3.35−2.96 (m, 4H), 2.37 (s, 3H), 2.32 (m, 4H). HRMS (ESI): calcd for C23H23ClF3N3O2S 497.1152, found 497.1148. 3426



Article

[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-Nmethylmethanamine (L)-Tartrate (32). Manganese(IV) oxide (13.89 g, 159.77 mmol) was added to a solution of 31 (28 g, 56.23 mmol) in dichloromethane (224 mL) at room temperature, and the resulting suspension was stirred at reflux for 2.5 h. Additional manganese(IV) oxide (33.33 g, 383.38 mmol) was added, and the mixture was stirred at reflux for 4 h and then at room temperature for 15 h. More manganese(IV) oxide (8.33 g, 95.82 mmol) was added, and stirring was continued for 7 h at room temperature. The reaction mixture was left stand for 1 h without stirring and filtered through Celite eluting with dichloromethane. The filtrate was concentrated to provide the intermediate aldehyde (17 g) that was used without further purification. Methylamine (40%) in water (3.25 mL, 37.71 mmol) was added to a solution of the intermediate aldehyde (17 g, 34.28 mmol) in ethanol (170 mL) at 0 °C. The resulting mixture was stirred at room temperature for 15 h and then cooled with an ice/water bath. Sodium borohydride (0.778 g, 20.57 mmol) was added, and the mixture was stirred at room temperature for 3 h. An aqueous solution of 5% HCl was added dropwise at 0 °C until no gas evolution was observed (pH = 6, around 20 mL), and the mixture was concentrated to 1/4 of the volume. A saturated aqueous solution of sodium bicarbonate was added (100 mL), and the resulting suspension was extracted with ethyl acetate (3 × 100 mL). Combined organic layers were dried over sodium sulfate and concentrated to afford a crude mixture that was purified by Isco (330 g cartridge), eluting with 7 N ammonia in methanol/dichloromethane (3:97 to 5:95 ratio) to get the free base of the title compound (14 g, 80% yield). LC-MS (ESI) m/z: 511 (M + H)+; tR = 4.05 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 7.49 (d, J = 2.1 Hz, 1H), 7.33 (m, 1H), 7.25 (m, 1H), 7.11 (m, 1H), 6.96 (s, 1H), 4.04 (t, J = 10.2 Hz, 2H), 3.53 (s, 2H), 3.48 (s, 2H), 2.78 (m, 2H), 2.39 (m, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 2.1 (m, 2H), 1.86 (m, 2H). HRMS (ESI): calcd for C24H26ClF3N4OS 510.1468, found 510.1469. The title compound (tartrate salt) was essentially prepared following general method B with 95% yield. LC-MS (ESI) m/z: 511 (M + H)+; tR = 4.09 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.92 (d, J = 2.1 Hz, 1H), 7.58−7.43 (m, 3H), 7.35 (s, 1H), 4.18 (t, J = 10.8 Hz, 2H), 3.98 (s, 2H), 3.75 (s, 2H), 3.46 (s, 2H), 2.74 (m, 2H), 2.42 (s, 3H), 2.32 (m, 2H), 2.26 (s, 3H), 2.1 (m, 2H), 1.79 (m, 2H). HRMS (ESI): calcd for C24H26ClF3N4OS 510.1468, found 510.1466. 2-Chloro-4,4-difluoro-1′-[(3-methyl-1H-pyrazol-4-yl)methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (33). The title compound was essentially prepared following general method A using 25 (105 g, 375 mmol), 3-methyl-1H-pyrazole-4-carbaldehyde (43.40 g, 394.12 mmol), and sodium triacetoxyborohydride (95.46 g, 450.42 mmol) using 1.58 L of tetrahydrofuran (t1 = 60 min, t2 = 15 h, S = ethyl acetate). A 140 g amount of the title compound was obtained (100% yield). LC-MS (ESI) m/z: 374 (M + H)+. tR = 2.09 min, method 2. 1H NMR (300 MHz, CDCl3) δ: 7.50 (s, 1H), 6.95 (s, 1H), 4.03 (t, J = 10.3 Hz, 2H), 3.47 (s, 2H), 2.86−2.78 (m, 2H), 2.40 (t, J = 11.4 Hz, 2H), 2.30 (s, 3H), 2.11−2.04 (m, 2H), 1.96−1.84 (m, 2H). HRMS (ESI): calcd for C16H18ClF2N3OS 373.0827, found 373.0834. 2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]pyridine-3-carbaldehyde (34). To a 250 mL flask were added copper(I) iodide (1.91 g, 10.03 mmol), 33 (25 g, 66.87 mmol), potassium carbonate (19.60 g, 140.43 mmol), toluene (50 mL), and a stir bar. The reaction mixture was degassed by five vacuum/refill cycles. Then 2-bromo-3formylpyridine (18.66 g, 100.31 mmol) and (±)-trans-N,N′dimethyl-1,2-cyclohexanediamine (3.16 mL, 20.06 mmol) were added. The reaction was stirred at room temperature for 5 min. Then it was immersed in a preheated oil bath at 115 °C and stirred for 15 h at that temperature. The reaction mixture was cooled to room temperature, diluted with 300 mL of ethyl acetate, and filtered through Celite. It was washed with ethyl acetate (100 mL) and aqueous solution of ammonium hydroxide (3%) (4 × 100 mL) to remove copper traces, and then it was washed with water (50 mL) and brine (50 mL). The solution was dried over sodium sulfate. The solvent was evaporated in vacuo to give a tan solid that was filtered through a pad of silica gel, eluting with 2-propanol/dichloromethane (3% to 5% of 2-

propanol) to give 22 g of the title compound (69% yield). LC-MS (ESI) m/z: 479 (M + H)+. tR = 2.72 min, 88% purity, method 2. 1H NMR (300 MHz, CDCl3) δ: 10.75 (s, 1H), 8.55 (dd, J = 1.8, 4.8 Hz, 1H), 8.42 (s, 1H), 8.24 (dd, J = 1.9, 7.7 Hz, 1H), 7.30−7.28 (m, 1H), 6.96 (s, 1H), 4.04 (t, J = 10.3 Hz, 2H), 3.48 (s, 2H), 2.80 (d, J = 11.3 Hz, 2H), 2.46−2.37 (m, 2H), 2.34 (s, 3H), 2.10 (dd, J = 2.3, 14.4 Hz, 2H), 1.92−1.82 (m, 2H). 2-Chloro-4,4-difluoro-1′-[[1-[3-(1H-imidazol-2-yl)-2-pyridyl]-3methylpyrazol-4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (35). A solution of 40% in water of ethanedial (1.69 mL, 14.70 mmol) and ammonium hydroxide (32% in water) (1.79 mL, 14.70 mmol) was added to a solution of 34 (1.76 g, 3.67 mmol) in methanol (37 mL) at 0 °C. The reaction mixture was stirred for 30 min at 0 °C and then heated at 50 °C with stirring for 16 h. The solvent was removed under reduced pressure. The crude mixture was purified by column chromatography with silica gel, eluting with 2 N ammonia in methanol/dichloromethane (1:99 to 10:90 ratio) to get the title compound (821 mg, 43% yield). LC-MS (ESI) m/z: 518 (M + H)+; tR = 3.75 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 12.08− 12.04 (m, 1H), 8.52 (dd, J = 1.8, 4.8 Hz, 1H), 8.22 (dd, J = 1.8, 7.7 Hz, 1H), 7.96 (s, 1H), 7.49 (dd, J = 4.8, 7.7 Hz, 1H), 7.34 (s, 1H), 7.19− 7.15 (m, 1H), 6.99 (s, 1H), 4.21−4.14 (m, 2H), 3.37 (m, 2H), 2.69 (d, J = 10.6 Hz, 2H), 2.28−2.18 (m, 2H), 2.14−2.08 (m, 5H), 1.79−1.69 (m, 2H). HRMS (ESI): calcd for C24H23ClF2N6OS, 516.1311; found, 516.1325. [2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-pyridyl]methanol (L)-Tartrate (36). To a solution of 34 (12.5 g, 26.10 mmol) in dichloromethane (125 mL) at 0 °C were added sodium tetrahydroborate (395 mg, 10.44 mmol) and methanol (37.50 mL). The ice bath was removed, and the reaction was stirred at room temperature for 30 min. Water (50 mL) was added, and the mixture was concentrated to half volume to precipitate a white sticky solid. Dichloromethane (100 mL) was added, and the biphasic mixture was separated. The organic layer was washed with water (50 mL), dried over sodium sulfate, and concentrated to half volume (around 100 mL). Methyl tert-butyl ether (100 mL) was added, and when the solution was concentrated under vacuum, a solid precipitated that was filtered and dried under vacuum to give 12 g (96% yield) of the title compound as the free base. LC-MS (ESI) m/z: 481 (M + H)+. tR = 4.13 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 8.41 (s, 1H), 8.37 (dd, J = 1.5, 4.8 Hz, 1H), 7.76 (dd, J = 1.6, 7.5 Hz, 1H), 7.18 (dd, J = 4.8, 7.3 Hz, 1H), 6.96 (s, 1H), 6.06 (t, J = 7.7 Hz, 1H), 4.66 (d, J = 7.7 Hz, 2H), 4.04 (t, J = 10.2 Hz, 2H), 3.48 (s, 2H), 2.79 (d, J = 11.7 Hz, 2H), 2.41−2.36 (m, 5H), 2.10 (d, J = 12.1 Hz, 2H), 1.91−1.81 (m, 2H). The title compound (tartrate salt) was essentially prepared following general method B with 100% yield. LC-MS (ESI) m/z: 481 (M + H)+. tR = 4.14 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.36−8.35 (m, 2H), 8.13 (d, J = 7.4 Hz, 1H), 7.42−7.35 (m, 2H), 4.79 (s, 2H), 4.24−4.14 (m, 4H), 3.55 (s, 2H), 2.82−2.75 (m, 2H), 2.50−2.35 (m, 2H), 2.27 (s, 3H), 2.14 (d, J = 12.9 Hz, 2H), 1.84−1.75 (m, 2H). 2-Chloro-4,4-difluoro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3methylpyrazol-4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (L)-Tartrate (37). To a screw-cap test tube were added copper(I) iodide (1.15 g, 6.02 mmol), 33 (15 g, 40.12 mmol), potassium carbonate (11.76 g, 85.09 mmol), 15 mL of toluene (previously bubbled with nitrogen for 20 min), and a stir bar. The reaction mixture was bubbled with nitrogen for additional 10 min, and then 2-bromo-3methoxymethylpyridine (10.54 g, 52.16 mmol) and (±)-trans-N,N′dimethyl-1,2-cyclohexanediamine (1.9 mL, 12.04 mmol) were added. The reaction tube was quickly sealed (caution: buildup of pressure possible; use a safety shield), stirred at room temperature for 5 min, and immersed in a preheated oil bath at 115 °C for 24 h. The sample was cooled down to room temperature, diluted with ethyl acetate, and filtered through Celite. The solvent was evaporated in vacuo. The residue was purified by normal phase Isco chromatography using hexanes/ethyl acetate (30−70%). The desired fractions were collected and evaporated. Some impure fractions (

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