Spirocyclic β-Site Amyloid Precursor Protein ... - ACS Publications

Article pubs.acs.org/jmc

Spirocyclic β‑Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitors: From Hit to Lowering of Cerebrospinal Fluid (CSF) Amyloid β in a Higher Species Kevin W. Hunt,*,† Adam W. Cook,† Ryan J. Watts,‡ Christopher T. Clark,†,§ Guy Vigers,† Darin Smith,† Andrew T. Metcalf,† Indrani W. Gunawardana,† Michael Burkard,† April A. Cox,†,∥ Mary K. Geck Do,†,⊥ Darrin Dutcher,†,# Allen A. Thomas,† Sumeet Rana,† Nicholas C. Kallan,†,∞ Robert K. DeLisle,† James P. Rizzi,†,¶ Kelly Regal,†,× Douglas Sammond,†,○ Robert Groneberg,†,△ Michael Siu,‡ Hans Purkey,‡ Joseph P. Lyssikatos,‡ Allison Marlow,† Xingrong Liu,‡ and Tony P. Tang† †

Array BioPharma, 3200 Walnut Street, Boulder, Colorado 80301, United States Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States

‡

ABSTRACT: A hallmark of Alzheimer’s disease is the brain deposition of amyloid beta (Aβ), a peptide of 36−43 amino acids that is likely a primary driver of neurodegeneration. Aβ is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer’s disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aβ in vivo, despite efflux. Starting with spirocycle 1a, we explore structure−activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood−brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aβ lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aβ in rodents and in monkey.

■

INTRODUCTION Alzheimer’s disease (AD) is an irreversible neurodegenerative disease that affects more than 18 million patients worldwide. The cost of care for AD patients exceeds $172 billion in the U.S. alone, despite over 15 million family members providing unpaid care. By 2050, the U.S. cost of care is predicted to exceed $1 trillion.1 The consequences of AD (principally the projected stresses upon health care systems) have spurred greater government funding for AD by France and the United States.2 Further investment from government agencies is timely, as many pharmaceutical companies are divesting CNS research.3 Modifying the course of AD may be possible by decreasing the production of amyloid β peptides (Aβ), the presumptive molecules central to the amyloid hypothesis resulting from metabolism of the amyloid precursor protein (APP).4 This hypothesis is based upon the increased production of Aβ in patients with dominantly inherited forms of AD, the high Aβ1−40,42 content in plaques from post-mortem brains of AD patients, and experiments demonstrating the neuronal toxicity of Aβ. Further, Down syndrome patients, who often develop AD pathology by age 50, have increased Aβ levels from birth and show plaque deposition as early as their preteen years.5 The amyloid hypothesis has been modified slightly to focus on the greater in vivo neurotoxicity of Aβ oligomers vs monomers.6 A corollary to the amyloid hypothesis is that decreasing Aβ production will be disease modifying, at least in © XXXX American Chemical Society

the early stages of the disease. This view was bolstered by the recent discovery of a coding mutation in the APP gene that protects against AD and cognitive decline in the elderly. The mutation is near the aspartyl protease β-site of APP and reduces in vitro Aβ production by approximately 40%.7 The enzyme that initiates the cascade of Aβ production is the β-site APP cleaving enzyme 1 (BACE1), also known as membrane-associated aspartic protease 2 (memapsin 2) and aspartyl protease 2 (ASP2).8 BACE1 is most highly expressed in the brain where it cleaves APP to release the amino-terminal fragment of APP (sAPPβ) and the membrane bound Cterminal fragment C99. Cleavage of the C99 fragment by γsecretase releases Aβ.9 The therapeutic potential for BACE1 inhibition coupled with the inferred safety from the viability of BACE1−/− mice10 has resulted in numerous publications about the discovery and characterization of BACE1 inhibitors.11 Recently, Eli Lilly researchers published phase I clinical results for a BACE1 inhibitor yielding important proof of mechanism data for this pathway in humans. While the Lilly compound effectively lowered CSF Aβ levels, advancement of the molecule was halted because of retinal pathology identified in a 3-month rat toxicology study.12 The observed retinal pathology of the Lilly compound is consistent with observations from animals Received: February 13, 2013

A

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Aminohydantoinsa

deficient in the aspartyl protease cathepsin D (CatD).13 These data suggest that selectivity for BACE1 over CatD inhibition is essential. The efficacy of a BACE1 inhibitor is equally dependent upon blood−brain barrier (BBB) permeability as it is upon potency. While protecting the brain from exposure to pollutants and toxins, the BBB also limits the entry of potentially beneficial xenobiotics such as pharmaceutical agents.14 Fortunately, limiting total polar surface area (tPSA < 90),15 molecular weight (MW < 450), lipophilicity (cLogP < 4), hydrogen bond donors (65% CSF Aβ1−40 reduction at the 8 h time point. Efforts to optimize BACE1 potency by utilizing P2′ interactions, increase CatD selectivity with non-aniline amides, and eliminate efflux will be published in due course. H

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 5. Single Enantiomer Profiles

a Mean IC50 of at least two independent experiments. bMean EC50 of at least two independent experiments for cellular Aβ production. cA to B in parental LLC-PK1 cell line. dEfflux ratio is the (B to A)/(A to B) value derived from LLC-PK1 cells transfected with human MDR1 (Pgp). e Compounds (1 μM) incubated for 20 min at 37 °C in the presence of NADPH with mouse (M), rat (R), guinea pig (GP), cynomolgus monkey (C), or human (H) liver microsomes (LM).

Table 6. Guinea Pig Pharmacodynamics (Aβ1−40 Lowering) compda

formulation

(R)-34

1% CMC/0.5% Tween 80

(R)-45 (R)-46

1% CMC/0.5% Tween 80 0.5% HPMCd/0.5% Tween 80

(R)-50

1% CMC/0.5% Tween 80

(R)-63

40/10/50 PEG400/ethanol/H2O

dose (po) (mg/kg)

time point (h)

Cfree,plasma (nM)

Cfree,brain (nM)

free (b/p)

CCSF (nM)

coverage ratiob

CSF Aβ1−40 reduction (%)c

25 50 100 200 400 150 75 150 10 30 100 60 60 60 60 60 50 150

5 5 5 5 5 5 5 5 3 3 3 1 3 5 8 12 3 3

33 224 1209 2891 4962 122 304 562 15 74 913 5380 1940 534 127 66 3019 6673

6.3 32 105 224 456 NDe 11 NDe 22 53 230 146 176 124 48 41 NDe NDe

0.19 0.14 0.087 0.077 0.091

10 86 456 1321 2384 195 20 247 27 125 1620 2300 920 440 66 47 888 5609

0.50 2.3 7.5 16 33

31 51 53 71 72 29 0 50 0 23 55 20 43 50 35 23 30 70

0.037 0.68 0.72 0.25 0.027 0.091 0.232 0.378 0.622

1.2 0.81 2.0 8.5 5.2 6.3 4.4 1.7 1.5

a Purity of >95% by 1H NMR and HPLC. bCfree,brain/(cell IC50). cReduction vs vehicle control. dHydroxypropylmethylcellulose (HPMC). eNot determined.

■

protein was concentrated to 10 mg/mL and frozen for crystallographic studies. Crystals were grown by hanging drop vapor diffusion with a precipitant of 20% PEG200, 0.1 M sodium acetate, pH 4.5−5.6, and 0−2 mM cobalt chloride. Crystals grew in space group C2221 in approximately 1 week. Crystals were then transferred to a soaking solution (27−30% PEG200, 0.1 M NaOAc, pH 5.0−5.6, 0−2 mM CoCl2, and 10% DMSO) with compound at a final concentration of 1 mM. Crystals were soaked for 18−24 h and either flash-frozen in liquid nitrogen or mounted directly for data collection. The soaking solution also served as a cryprotectant for data collection at 100 K.

EXPERIMENTAL SECTION

X-ray Crystal Structure. Human BACE1 (residues 57−453) was expressed in E. coli with a noncleavable 6-His tag at the C-terminus. The protein was refolded and purified in a manner similar to that from Tomasselli et al.41 Briefly, cells were lysed using mechanical disruption and inclusion bodies recovered by differential centrifugation. Inclusion body proteins were solubilized in urea and refolded by dilution into water. Following a 3 week refold period, BACE protein was recovered by Q-Sepharose anion-exchange chromatography. Fractions containing active protein were pooled and further purified over Source-Q anionexchange and Sephadex 200 size-exclusion columns. Purified BACE I

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 7. Rat Pharmacodynamics (Aβ1‑40 Lowering) compda

formulation

dose (po) (mg/kg)

time point (h)

Cfree,plasma (nM)

Cfree,brain (nM)

free (b/p)

CCSF (nM)

coverage ratiob

CSF Aβ1−40 reduction (%)c

(R)-34

1% CMC/0.5% Tween 80

150

(R)-50

1% CMC/0.5% Tween 80

60

1 3 5 8 1 3 5 8 12

1166 1023 1254 1353 2442 1733 561 57 24

42 53 56 68 78 90 50 39 26

0.036 0.052 0.044 0.050 0.032 0.052 0.089 0.67 1.09

8330 15600 24200 2270 269 236 47 15 11

3.0 3.8 4.0 4.9 2.9 3.3 1.9 1.4 1.0

12 29 20 18 16 17 24 NDd 13

a

Purity of >95% by 1H NMR and HPLC. bCfree,brain/(cell IC50). cReduction vs vehicle control. dNot determined. calculated from the fluorescence increase. The final concentrations were 10 nM enzyme and 2 μM substrate. Cell-Based Assay. Human embryonic kidney cells (HEK293) stably expressing APPwt were plated at a density of 35K cells/well in 96-well collagen coated plates (BD-Biosciences). The cells were cultivated overnight at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS. The following day the medium was changed and the cells were incubated for 48 h with test compounds at concentrations ranging from 0.0008 to 16 μM. Following incubation with the test compounds the conditioned medium was collected and the Aβ1−40 levels were determined using an HTRF assay (CisBio). The IC50 was calculated from the percent of control Aβ40 as a function of the concentration of the test compound. The HTRF to detect Aβ1−40 was performed in 384-well microtiter plates (Corning). The antibody pair that was used to detect Aβ1−40 from cell supernatants was a pair of monoclonal antibodies, one labeled with cryptate and one labeled with XL655 as supplied by the manufacturer (CisBio). Conditioned medium was incubated with antibody pair overnight at 4 °C. The plates were measured for time-resolved fluorescence on a Victor2 plate reader (Wallac). Permeability and Efflux Assay. The LLC-PK1 cell line, stably transfected with human MDR1 cDNA, was obtained from BD Biosciences (Franklin Lakes, NJ). Both parental LLC-PK1 and MDR1 transfected LLC-PK1 cells were cultured and plated according to the manufacturer’s recommendations with the exception that the passage medium contained only 2% fetal bovine serum to extend passage time out to 7 days. The cells were plated into Millicell-96 cell culture insert plates at a density of 50 000 and 40 000 cells/well for the MDR1 and parental LLC-PK1 cell lines, respectively. The plates were incubated at 37 °C and in an atmosphere of 5% CO2 with saturating humidity. The medium was replaced on day 2 of culture. Permeability experiments were conducted on culture day 6. A trans-epithelial electrical resistance (TEER) value of at least 500 Ω as determined by the Millipore Millicell-ERS TEER device (Millipore, Billerica, MA) was required for monolayer acceptance. Transport buffer was prepared using Hanks balanced salt solution (HBSS), 1% DMSO, and 10 mM HEPES. The pH of the assay buffer was adjusted to 7.4, using 1.0 M KOH. Stock solutions for the test compounds were prepared in DMSO for final concentration of 1 μM. Final organic concentration in the assay was 1%. Bidirectional permeability was assessed by adding either 75 or 250 μL of buffer containing compound to the apical or basolateral compartments, respectively. Buffer only was added to the receiver compartment. All tests were performed in triplicate, and compounds were assayed for both apical to basolateral (A to B) and basolateral to apical (B to A) transport. After a 2 h incubation on an orbital plate shaker (VWR, West Chester, PA) at 50 rpm and 37 °C with 5% CO2, the culture plates were removed and an amount of 50 μL was sampled from the apical and basolateral portion of each well and added to a plate containing 150 μL of 1 μM labetalol (internal standard) in 2:1 acetonitrile (ACN)/H2O, v/v. The plates were read using a Molecular Devices (Sunnyvale, CA) Gemini fluorometer to evaluate the lucifer yellow concentrations at excitation/emission wavelengths of 425/535 nm. These values were accepted when found to be below 5% for apical to basolateral and basolateral to apical flux across the MDR1

Figure 3. Monkey pharmacodynamics of (R)-50 (Aβ1−40 lowering). CSF concentration of Aβ1−40 (baseline normalized, left Y-axis, broken line) and (R)-50 (right Y-axis, solid line) following single 100 mg/kg oral dose of (R)-50 in cynomolgus monkeys. Data points represent mean and SD from 12 animals. Diffraction data were collected on a Rigaku FR-E generator and Raxis IV++ detector (Rigaku Inc., 9009 New Trails Drive, The Woodlands, TX 77381-5209, U.S.). Data were processed in Moslfm,42 and the initial structure was solved by molecular replacement from 1FNK.pdb using Molrep. All crystal structures were refined in Refmac, all three of these programs being part of the CCP4 suite (Winn et al., 2011).43 Model building was performed in Coot.44 BACE1 Enzymatic Assay. The activity of purified recombinant human BACE1, expressed in CHO cells, was determined using a custom synthesized biotinylated peptide based upon sAPP. Uncleaved substrate was detected via HTRF whereby europium-labeled anti-Aβ (amino acids 1−17, clone 6E10) was combined with D2-labeled streptavidin. Specifically, 20 nM enzyme was incubated with 150 nM peptide (biotin-Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu-Asp-AlaGlu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu, representing P10−P17′) for 6 h at 22 °C in an assay buffer consisting of 50 mM sodium acetate (pH 4.4) and 0.1% CHAPS. In IC50 studies, compounds were serially diluted (1:2.5) in DMSO (11 points), compound and enzyme were preincubated for 15 min at room temperature, and the enzymatic reaction was initiated with peptide. The detection solution consisted of 43 nM streptavidin-D2 and 1.1 nM antibody in 200 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), 0.1% BSA, and 0.8 M KF. Following incubation for 2 h at 22 °C, the samples were excited at 320 nm and the emission ratio of 665 nm/615 nm was determined. CatD Enzymatic Assay. The catalytic activity of cathepsin D, purified from human spleen (Calbiochem), was assessed in a FRET peptide substrate assay using 50 mM sodium acetate (pH 4.4) and 0.1% CHAPS. In IC50 studies, compounds were serially diluted (1:2.5) in DMSO (11 points). The substrate (Anaspec catalog no. 72097) contains a 5-FAM/QXL 520 pair whereby cleavage results in an increase of 5-FAM fluorescence that is measured using an excitation of 485 nm and an emission of 535 nm. The fluorescence was monitored continuously for 1 h at room temperature, and initial rates were J

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

transfected LLC-PK1 cell monolayers. The compound concentrations were determined from the ratio of the peak areas of the compound to labetalol in comparison to the dosing solution. The LC−MS/MS system comprised an HTS-PAL autosampler (Leap Technologies, Carrboro, NC), an HP1200 HPLC (Agilent, Palo Alto, CA), and a MDS Sciex 4000 Q trap system (Applied Biosystems, Foster City, CA). Mass spectrometric detection of the analytes was accomplished using the ion spray positive mode. Analyte responses were measured by multiple reaction monitoring (MRM) of transitions unique to each compound in comparison to labetalol. Permeability (Papp) was calculated in BioAssay, version 9.0 (Cambridge Soft, Cambridge, MA) using the following equation: Papp (×10−6 cm/s) =

concentration in rat and guinea pig CSF samples was analyzed by MSD 96-well MULTI-ARRAY human/rodent (4G8) Aβ 40 ultrasensitive assay (rat) or human (6E10) Aβ 40 ultrasensitive kit (Meso Scale Discovery, Gaithersburg, MD). The 96-well MULTI-SPOT Aβ1−40 peptide plate was incubated with 1% BSA solution at room temperature with shaking for 1 h and washed per manufacturer’s instructions with Tris wash buffer in deionized water. The detection antibody solution (25 μL/well, sulfo-tag 4G8 in 1% BSA) and CSF samples or standards (25 μL/well) were added to the plate for incubation at room temperature for 2 h. The plate was washed three times before addition of MSD read buffer and was read on Sector Imager 2400 (Meso Scale Discovery, Gaithersburg, MD) immediately afterward. The Aβ1−40 levels obtained as pg/mL were analyzed by twotailed Student’s t test to compare mean percentage Aβ1−40 reduction in vehicle-treated control animals and compound-treated animals. Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Anhydrous solvents were obtained from EM Science and used directly. All reactions involving air- or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere. All final compounds were purified to ≥95% purity as determined by high-performance liquid chromatography (HPLC). Purity was measured using either system A (Varian ProStar with UV detection at 220 and 254 nm, a Waters YMC ODS-AQ C18 3.0 mm × 50 mm, 3.0 μm, 5−95% CH3CN in H2O with 0.1% NH4OAc for 5 min at 1.0 mL/min) or system B (Varian ProStar with UV detection at 220 and 254 nm, a Waters YMC ODS-AQ C18 3.0 mm × 50 mm, 3.0 μm, 5−95% CH3CN in H2O with 0.1% formic acid for 5 min at 1.0 mL/min). Silica gel columns were used with prepacked silica gel cartridges (Biotage or Teledyne Isco). 1H NMR spectra were recorded on a Varian Mercury 400 (400 MHz) spectrometer or Varian Inova 400 (500 MHz) spectrometer at 25 °C. NMR spectra were processed using VNMRj, version 2.1b. All observed protons are reported as parts per million (ppm) downfield from tetramethylsilane (TMS) or other internal reference in the appropriate solvent indicated. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and number of protons. Low-resolution mass spectrometry (MS) data were determined on an Agilent 1100 series LCMS with UV detection at 220 and 254 nm, a Halo C18 2.1 mm × 30 mm, 2.7 μm, 5−95% CH3CN in H2O with 0.1% NH4OAc for 3 min at 1.0 mL/min, and atmospheric-pressure chemical ionization (APCI). 2′-Amino-6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-1a). Step A. 6-Bromo-2,2-dimethylchroman-4-one (18.2 g, 71.3 mmol), KCN (9.29 g, 143 mmol), and ammonium carbonate (48.0 g, 499 mmol) were diluted with formamide (200 mL). The mixture was bubbled with argon for 15 min, sealed, heated to 70 °C, and stirred for 24 h. The mixture was then heated to 110 °C and stirred for 48 h. The mixture was allowed to cool and poured into ice−water (1L). The pH was adjusted to 5 with concentrated HCl, and the mixture was stirred for 1 h. The precipitate was filtered and rinsed with water. The solid was air-dried for 5 h and placed under vacuum for 15 h to afford 6-bromo-2,2dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (21 g, 64.6 mmol, 90.5% yield) as a tan solid. 1H NMR (400 MHz, CDCl3-d) δ 7.34 (dd, J1 = 9.4 Hz, J2 = 2.3 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 6.76 (d, J = 9.4 Hz, 1H), 6.35 (br s, 1H), 2.6 (d, J = 14.9, 1H), 2.1 (d, J = 14.9 Hz, 1H), 1.51 (s, 3H), 1.31 (s, 3H); LCMS (APCI−) m/z 323, 325 (M − 1). Step B. To a suspension of NaH (246 mg, 6.15 mmol) in DMF (6 mL) was added dropwise a solution of 6-bromo-2,2-dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (1.0 g, 3.08 mmol) in DMF (8 mL). After the mixture was stirred for 1 h, CH3I (0.198 mL, 3.08 mmol) was added, and the mixture was stirred for 16 h. The mixture was diluted with DCM and washed with 1 N HCl, water, brine, dried over MgSO4, filtered, and concentrated in vacuo. The isolated residue was purified on silica gel, eluting with 5−100% ethyl acetate/hexanes to yield 6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazolidine]2′,5′-dione (1.03 g, 3.04 mmol, 98.7% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.04 (d, J = 2.3 Hz,

CdV (1 × 106) (t )(0.12 cm 2)(C0)

where Cd, V, t, and C0 are the detected concentration (μM), the volume on the dosing side (mL), the incubation time (s), and the initial dosing concentration (μM), respectively. The calculations for Papp were made for each replicate and then averaged. The efflux ratio (ER) was calculated from the basolateral to apical transport divided by the apical to basolateral transport: ER =

Papp(B→A) Papp(A→B)

Microsomal Stability Assay. Compounds were prepared in a 100 mM potassium phosphate buffer solution (pH 7.4) for a final concentration of 1 μM and incubated in the presence of 1 mg/mL human, monkey, rat, guinea pig, or mouse liver microsomes. The final concentration of DMSO in the incubation was less than 0.1%. Metabolism was initiated with the addition of a 10 μL aliquot of NADPH-regenerating solution (20 mM MgCl2 buffer containing NADP (17 mg/mL stock in 100 mM KH2PO4), glucose 6-phosphate (78 mg/mL stock in 100 mM KH2PO4), and glucose 6-phosphate dehydrogenase (150 U/mL stock in 100 mM KH2PO4)) for a total incubation volume of 50 μL. After 20 min of incubation at 37 °C and 100% relative humidity, metabolism was stopped with the addition of 150 μL of 60% acetonitrile containing internal standard (0.25 μM labetalol). The plate was immediately spun in a centrifuge at 2095g for 7 min at room temperature. A 200 μL aliquot of the supernatant was transferred from each well to a 96-well shallow plate, sealed, and analyzed by LC−MS/MS. Pharmacodynamic Assays. Male Sprague−Dawley rats and male Hartley guinea pigs (6−8 weeks old, 250−350 g) were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories (Kingston, NY), respectively. The animals were housed in groups of 3 at controlled temperature and humidity in an alternating 12 h light and dark cycle with free access to food and water in accordance with Array BioPharma Institutional Animal Care and Use Committee Guidelines and in harmony with the Guide for Laboratory Animal Care and Use. Rats and guinea pigs (6−8 per group) received orally either vehicle or compound at appropriate dose at a volume of 4−5 mL/kg. The rats were euthanized by CO2 inhalation, and guinea pigs were euthanized by euthasol (250 mg/kg) via intraperitoneal injection at predetermined time points after dosing. The whole blood samples were collected post-mortem via cardiac puncture in tubes containing 1.5% EDTA, centrifuged at 14 000 rpm for 10 min at 4 °C. The plasma was stored at −20 °C until drug concentration analysis. The CSF samples (40−100 μL) were drawn post-mortem via cisterna magna puncture with a 25 gauge needle (Becton Dickinson, Franklin Lakes, NJ) into tubes containing 50 μL of 10% BSA in water. The CSF samples were snap frozen in liquid nitrogen and stored at −80 °C until Aβ1−40 analysis. Sample weights were recorded to determine the actual volume of CSF collected. The post-mortem animals were perfused with 20 mL of sterile saline. Brain hemispheres were dissected (brain stem and cerebellum were not collected), halved longitudinally, and weighed, and only the left half of the brain was placed in a 15 mL conical tube and snap frozen in liquid nitrogen. All tissues were stored at −80 °C until drug concentration determination. CSF Aβ1−40 K

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 6.76 (d, J = 8.6 Hz, 1H), 6.1 (br s, 1H), 3.1 (s, 3H), 2.6 (d, J = 14.8, 1H), 2.1 (d, J = 14.8 Hz, 1H), 1.51 (s, 3H), 1.31 (s, 3H). Step C. 6-Bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (3.0 g, 8.8 mmol) and Lawesson’s reagent (2.1 g, 5.3 mmol) were diluted with toluene (50 mL) and heated under reflux with a condenser and drying tube for 12 h. The mixture was allowed to cool, diluted with ethyl acetate, and washed with 10% aqueous sodium carbonate, water, and brine. The organic layer was separated, dried (MgSO4), filtered, and concentrated. The residue obtained was purified on silica gel, eluting with 5−50% ethyl acetate/hexanes to yield 6-bromo-1′,2,2-trimethyl-2′-thioxospiro[chroman-4,4′-imidazolidin]-5′-one (2.0 g, 5.6 mmol, 64% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.35 (dd, J1 = 9.0 Hz, J2 = 2.3 Hz, 1H), 7.34 (br s, 1H), 6.94 (d, J = 2.3 Hz, 1H), 6.76 (d, J = 9.0 Hz, 1H), 3.34 (s, 3H), 2.56 (d, J = 14.8, 1H), 2.1 (d, J = 14.8 Hz, 1H), 1.51 (s, 3H), 1.35 (s, 3H). Step D. A solution of 6-bromo-1′,2,2-trimethyl-2′-thioxospiro[chroman-4,4′-imidazolidin]-5′-one (2.0 g, 5.63 mmol) in methanol (40 mL) was treated with tert-butyl hydroperoxide (11.7 mL, 84.4 mmol) followed by concentrated NH4OH (19.7 mL, 563 mmol). The mixture was heated to 40 °C and stirred for 2 h. The mixture was removed from the heat and stirred for an additional 2 h. The mixture was diluted with water and extracted with DCM. The combined organics were dried (MgSO4), filtered, and concentrated in vacuo. The residue obtained was purified on silica gel, eluting with 1−10% methanol/DCM with 1% NH4OH to yield 2′-amino-6-bromo-1′,2,2trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (1.5 g, 4.44 mmol, 78.8% yield). 1H NMR (400 MHz, CD3OD-d4) δ 7.27 (d, J = 8.6 Hz, 1H), 6.88 (s, 1H), 6.74 (d, J = 8.6 Hz, 1H), 3.14 (s, 3H), 2.31 (d, J = 14.1 Hz, 1H), 1.93 (d, J = 14.1 Hz, 1H), 1.45 (s, 3H), 1.36 (s, 3H); LCMS (APCI+) m/z 338.1 (M + 1). (2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-11). 2′-Amino-6bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (50 mg, 0.15 mmol) (from step D of example 1a), phenylboronic acid (28 mg, 0.18 mmol), and tetrakis(triphenylphosphine)palladium(0) (8.5 mg, 0.0074 mmol) were combined in a vial and diluted with dioxane (1 mL). Then 2 M aqueous Na2CO3 (370 μL, 0.74 mmol) was added, and the vial was sealed and the mixture heated to 95 °C and stirred overnight. The mixture was allowed to cool and loaded onto silica gel, eluting with 1−10% MeOH/DCM with 1% NH4OH to yield 2′amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′imidazol]-5′(1′H)-one (25 mg, 0.067 mmol, 46% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.40 (m, 5H), 7.30 (d, J = 8.6 Hz, 1H), 6.97 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 3.18 (s, 3H), 2.55 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 336.2 (M + 1). 2′-Amino-6-(3-chlorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-12). was synthesized in the same fashion as compound 11, substituting 3-chlorophenylboronic acid for phenylboronic acid to afford 2′-amino-6-(3-chlorophenyl)1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (8 mg, 0.022 mmol, 29% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.35 (m, 5H), 6.93 (m, 2H), 3.20 (s, 3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 370.2 (M + 1) 3-(2′-Amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-6-yl)benzonitrile (rac-13). rac-13 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with 3-cyanophenylboronic acid to afford 3-(2′amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-6-yl)benzonitrile (0.031 g, 0.0860 mmol, 58.2% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.71 (s, 1H), 7.66 (d, J1 = 7.83 Hz 1H), 7.56 (d, J1 = 7.83 Hz 1H), 7.48 (t, J1 = 7.63 Hz 1H), 7.38 (dd, J1 = 8.61 Hz, J2 = 1.97 Hz, 1H), 6.96 (d, J1 = 8.61 Hz, 1H), 6.93 (s, 1H), 3.23 (s, 3H), 2.55 (d, J1 = 13.7 Hz, 1H), 1.99 (d, J1= 14.1 Hz, 1H), 1.53 (s, 3H), 1.44 (s, 3H); LCMS (APCI+) m/z 361.1 (M + 1) 2′-Amino-6-(3-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-14). rac-14 was synthesized in the same fashion as compound 11, substituting 3methoxyphenylboronic acid for phenylboronic acid to afford 2′-

amino-6-(3-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (60 mg, 0.164 mmol, 25.9% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.40 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.98 (m, 2H), 6.91 (d, J = 8.6 Hz, 1H), 6.84 (dd, J1 = 8.2 Hz, J2 = 2.7 Hz, 1H), 3.84 (s, 3H), 3.17 (s, 3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1). 2′-Amino-6-(2-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-15). rac-15 was synthesized in the same fashion as compound 11, substituting 2methoxyphenylboronic acid for phenylboronic acid to afford 2′amino-6-(2-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (10 mg, 0.027 mmol, 19% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.38 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.27 (m, 1H), 7.22 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.03 (br d, J = 1.9 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 3.76 (s, 3H), 3.17 (s, 3H), 2.6 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1). 2′-Amino-6-(4-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-16). rac-16 was synthesized in the same fashion as compound 11, substituting 4methoxyphenylboronic acid for phenylboronic acid to afford 2′amino-6-(4-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.049 mmol, 33% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.35 (m, 3H), 6.91 (m, 4H), 3.82 (s, 3H), 3.19 (s, 3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.51 (s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1). 2′-Amino-6-(3,5-dichlorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-17). rac-17 was synthesized in the same fashion as compound 11, substituting 3,5dichlorophenylboronic acid for phenylboronic acid to afford 2′amino-6-(3,5-dichlorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (20 mg, 0.05 mmol, 32% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.29 (d, J = 1.9 Hz, 2H), 7.27 (d, J = 1.9 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.91 (br s, 1H), 3.20 (s, 3H), 2.54 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 404.1 (M + 1) 2′-Amino-6-(3-fluoro-5-methoxyphenyl)-1′,2,2trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-18). rac-18 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-fluoro-5-methoxyphenyl)boronic acid to afford 2′-amino-6-(3-fluoro-5-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (19 mg, 0.050 mmol, 34% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.37 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 6.95 (s, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.75 (m, 2H), 6.55 (m, 1H), 3.82 (s, 3H), 3.19 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 384.2 (M + 1). 2′-Amino-6-(2-fluoro-5-methoxyphenyl)-1′,2,2trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-19). rac-19 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (2-fluoro-5-methoxyphenyl)boronic acid to afford 2′-amino-6-(2-fluoro-5-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (15 mg, 0.039 mmol, 26% yield). 1H NMR (400 MHz, CDCl3) δ 7.37 (m, 1H), 6.99 (m, 2H), 6.91 (d, J = 8.6 Hz, 1H), 6.83 (m, 1H), 6.76 (m, 1H), 3.79 (s, 3H), 3.16 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 384.2 (M + 1). 2′-Amino-6-(3-chloro-5-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-20). rac-20 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-chloro-5-fluorophenyl)boronic acid to afford 2′-amino-6(3-chloro-5-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (25 mg, 0.064 mmol, 44% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.35 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.21 (m, 1H), 7.01 (m, 2H), 6.92 (m, 2H), 3.20 (s, 3H), 2.54 (d, J = 14.1 Hz, 1H), 1.96 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 388.2 (M + 1). L

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2′-Amino-6-(5-chloro-2-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-21). rac-21 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-chloro-6-fluorophenyl)boronic acid to afford 2′-amino-6(5-chloro-2-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (25 mg, 0.064 mmol, 44% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.34 (dm, J1 = 8.59 Hz, J2 = 2.05 Hz, 1H) 7.29 (dd, J1 = 6.83 Hz, J2 = 2.73 Hz, 1H) 7.20 (m, 1H), 7.02 (dd, J1 = 9.96 Hz, J2 = 8.78 1H), 6.95 (s, 1H), 6.92 (d, J1 = 8.59 Hz, 1H), 3.18 (s, 3H), 2.56 (d, J1 = 14.3 Hz, 1H), 1.98 (d, J1 = 14.3 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 388.2 (M + 1). 2′-Amino-6-(3-isopropoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-22). rac-22 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-isopropoxyphenyl)boronic acid to afford 2′-amino-6-(3isopropoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (27 mg, 0.069 mmol, 46% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.39 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 6.99 (m, 3H), 6.90 (d, J = 8.6 Hz, 1H), 6.81 (dd, J1 = 8.2 Hz, J2 = 2.3 Hz, 1H), 4.58 (qn, J = 5.9 Hz, 1H), 3.19 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H), 1.35 (s, 3H), 1.34 (s, 3H); LCMS (APCI+) m/z 394.2 (M + 1). 2′-Amino-6-(3-ethoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-23). rac-23 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-ethoxyphenyl)boronic acid to afford 2′-amino-6-(3ethoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)one (11 mg, 0.029 mmol, 20% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.0 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 6.99 (m, 3H), 6.90 (d, J = 8.6 Hz, 1H), 6.81 (dd, J1 = 8.2 Hz, J2 = 2.3 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.19 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (t, J = 7.0 Hz, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 380.2 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(3-(trifluoromethyl)phenyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-24). rac-24 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with 3-(triflouromethyl)phenylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(3-(trifluoromethyl)phenyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (13 mg, 0.032 mmol, 22% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.66 (s, 1H), 7.60 (d, J1= 7.61 Hz, 1H), 7.54 (d, J1 = 7.61 Hz, 1H), 7.49 (d, J1 = 7.61 Hz, 1H), 7.40 (dd, J1 = 8.59 Hz, J2 = 2.15 Hz, 1H), 6.97 (s, 1H), 6.95 (d, J1 = 8.59 Hz, 1H), 3.19 (s, 3H), 2.56 (d, J1 = 14.1 Hz, 1H), 1.98 (d, J1 = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 404.2 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(3-(methylthio)phenyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-25). rac-25 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-(methylthio)phenylboronic acid to afford 2′-amino-1′,2,2trimethyl-6-(3-(methylthio)phenyl)spiro[chroman-4,4′-imidazol]5′(1′H)-one (23 mg, 0.0619 mmol, 42% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.38 (dd, J1 = 8.59 Hz, J2 = 2.15 Hz, 1H), 7.31 (s, 1H), 7.28 (d, J1 = 7.61 Hz, 1H), 7.26 (s, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 6.96 (d, J1 = 1.95 Hz, 1H), 6.91 (d, J1 = 8.59 Hz, 1H), 3.17 (s, 3H), 2.56 (d, J1 = 14.25 Hz, 1H), 2.49 (s, 3H), 1.97 (d, J1 = 14.25 Hz, 1H), 1.51 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 382.1 (M + 1). 2′-Amino-6-(3-(difluoromethoxy)phenyl)-1′,2,2trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-26). rac-26 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (3-(difluoromethoxy)phenyl)boronic acid to afford 2′-amino-6-(3-(difluoromethoxy)phenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (11 mg, 0.03 mmol, 19% yield). 1 H NMR (400 MHz, CDCl3) δ 7.36 (m, 2H), 7.27 (m, 1H), 7.18 (m, 1H), 7.03 (m, 1H), 6.94 (m, 1H), 6.53 (t, J = 74 Hz, 1H), 3.19 (s, 3H), 2.54 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 402.1 (M + 1) 2′-Amino-6-(3-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-27). rac-27 was synthesized in the same fashion as compound 11 substituting 3fluorophenylboronic acid for phenylboronic acid to afford 2′-amino-

6-(3-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (8 mg, 0.023 mmol, 31% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.45 (m, 2H), 7.30 (m, 2H), 7.12 (m, 1H), 6.88 (m, 2H), 3.05 (s, 3H), 2.2 (d, J = 14.1 Hz, 1H), 1.80 (d, J = 14.1 Hz, 1H), 1.43 (s, 3H), 1.39 (s, 3H); LCMS (APCI+) m/z 354.2 (M + 1). 2′-Amino-6-cyclohexyl-1′,2,2-trimethylspiro[chroman-4,4′imidazol]-5′(1′H)-one (rac-28). A mixture of 2′-amino-6-bromo1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (0.050 g, 0.148 mmol), cyclohexylzinc(II) bromide (0.44 mmol), and bis(tritert-butylphosphine)palladium(0) (0.014 mmol) in THF (1 mL) was heated in a sealed vial overnight at 90 °C. MS showed that the reaction was completed. The sample was then purified on silica gel, eluting with 2−10% MeOH/DCM + 1% NH4OH to give 2′-amino-6-cyclohexyl1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (13.7 mg, 0.041 mmol, 27%). 1H NMR (400 MHz, CDCl3-d) δ 7.04 (dd, J1 = 8.59 Hz, J2 = 2.15 Hz, 1H), 6.76 (d, J1 = 8.40, 1H), 6.61 (d, J1 = 1.95 Hz, 1H), 3.20 (s, 3H), 2.53 (d, J1 = 14.1 Hz, 1H), 2.34 (m, 1H), 1.94 (d, J1 = 14.3 Hz, 1H), 1.75 (m, 5H), 1.47 (s, 3H), 1.35 (s, 3H), 1.25 (m, 5H), LCMS (APCI+) m/z 342.2 (M + 1). 2′-Amino-6-isobutoxy-1′,2,2-trimethylspiro[chroman-4,4′imidazol]-5′(1′H)-one (rac-29). Step A. A mixture of 2′-amino-6bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (2.0 g, 5.91 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (9.0 g, 35.5 mmol), Pd(PPh3)4 (0.683 g, 0.59 mmol), 3 M KHF2 (5 mL, 15 mmol), and 2 M Na2CO3 (5.91 mL, 11.8 mmol) in dioxane (9 mL, 5.91 mmol) was heated in a sealed vial at 90 °C overnight. The mixture was cooled to ambient temperature, filtered through a Celite plug, and the filtrate collected was concentrated in vacuo. The residue obtained was purified by flash chromatography on silica gel, eluting with 1−10% MeOH/EtOAc + 1% TEA to provide (rac)-2′-amino1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (1.65 g, 72%) as a light yellow solid contaminated with a small amount of dioxaboralane. 1H NMR (400 MHz, CDCl3-d) δ 7.64 (dd, J1 = 8.589 Hz, J2 = 1.562 Hz, 1H), 7.29 (br s, 1H), 6.83 (d, J = 8.21 Hz, 1H), 3.21 (s, 3H), 2.55 (d, J = 14.445 Hz, 1H), 1.96 (d, J = 14.055 Hz, 1H), 1.49 (s, 3H), 1.36 (s, 3H), 1.29 (s, 12H); LCMS (APCI+) m/z 286.2 (M + 1), retention time 2.19 min. Step B. To a solution of 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]5′(1′H)-one (0.25 g, 0.649 mmol) in MeOH (5 mL) and CH2Cl2 at 0 °C was added 50% hydrogen peroxide in H2O (1.1 mL, 16.2 mmol). The reaction mixture was stirred at 0 °C for 45 min and then diluted with EtOAc/H2O, and the layers were separated. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo to provide 2′amino-6-hydroxy-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (0.113 g, 0.410 mmol, 63.3% yield) as a white foamy solid. 1H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.789 (brs, 1H), 6.55 (m, 1H), 6.11 (s, 1H), 3.92 (s, 2H), 3.02 (s, 3H), 2.14 (d, J = 13.66 Hz, 1H), 1.77 (d, J = 12.664 Hz, 1H), 1.36 (s, 3H), 1.29 (s, 3H); LCMS (APCI+) m/z 276.2 (M + 1), retention time 0.78 min. Step C. To a solution of 2′-amino-6-hydroxy-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.0654 mmol) in DMF (0.5 mL) were added Cs2CO3 (21.3 mg, 0.0654 mmol) and 1-bromo2-methylpropane (0.00782 mL, 0.0719 mmol). The reaction mixture was stirred at ambient temperature. After 18 h, an additional 3 equiv of Cs2CO3 was added, stirred at room temperature for 2 h, and then heated at 60 °C overnight. The mixture was cooled to ambient temperature and partitioned between brine and EtOAc. The combined aqueous layers were back-extracted with EtOAc. The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The residue obtained was purified by flash chromatography on silica gel (10 g Snap cartridge, on Biotage SP1) using a gradient of 2− 15% MeOH/CH2Cl2 + 2% NH4OH to provide 2′-amino-6-isobutoxy1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (6.2 mg, 28.6%) as a pale yellow foamy solid. 1H NMR (400 MHz, CDCl3d) δ 6.78−6.77 (m, 2H), 6.37−6.36 (m, 1H), 3.59−3.58 (m, 2H), 3.19 (s, 3H), 2.52 (d, J = 14.09 Hz, 1H), 2.04−1.96 (m, 1H), 1.94 (d, J = 14.09 Hz, 1H), 1.47 (s, 3H), 1.34 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H). M

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2′-Amino-1′,2,2-trimethyl-6-(piperidin-1-yl)spiro[chroman4,4′-imidazol]-5′(1′H)-one formate (rac-30). A solution of 1 N lithium bis(trimethylsilyl)amide in THF (1.892 mL, 1.892 mmol) and piperidine (0.07018 mL, 0.7096 mmol) was injected into a vial containing Pd2dba3 (10.83 mg, 0.011 83 mmol), 2′-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2-amine (5.585 mg, 0.014 19 mmol), and 2′-amino-6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (200 mg, 0.5914 mmol). The mixture was stirred at 65 °C for 15 h. The mixture was cooled to room temperature and concentrated in vacuo. The residue obtained was purified first by silica gel chromatography, eluting with a gradient of 10−100% MeOH/ DCM containing 1% NH4OH, then by C-18 reverse phase chromatography (10−100% CH3CN/H2O (both eluents with 0.5% TFA) to provide 2′-amino-1′,2,2-trimethyl-6-(piperidin-1-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one formate (0.8 mg, 0.002 059 mmol, 0.35% yield). 1H NMR (400 MHz, CD3OD-d4) δ 6.98 (dd, J1 = 8.98 Hz, J2 = 2.73 Hz, 1H), 6.76 (d, J1 = 8.98 Hz, 1H), 6.49 (d, J1 = 2.73 1H), 3.23 (s, 3H), 2.92 (t, J1 = 4.57 Hz, 4H), 2.37 (d, J1 = 14.4 Hz, 1H), 2.14 (d, J1 = 14.4 Hz, 1H), 1.67 (m, 4H), 1.51 (m, 2H), 1.43 (s, 3H), 1.32 (s, 3H); MS (APCI+/ESI+ multimode) m/z 343.2 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(pyridin-3-yl)spiro[chroman4,4′-imidazol]-5′(1′H)-one (rac-31). rac-31 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with pyridin-3-ylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(pyridin3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.05351 mmol, 24.13% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J1 = 1.96 Hz, 1H), 8.50 (dd, J1 = 4.70 Hz, J2 = 1.57 Hz, 1H), 7.87 (dt, J1 = 8.61 Hz, J2 = 1.96 Hz 1H), 7.49 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.42 (dd, J1 = 7.83 Hz, J2 = 4.70 Hz, 1H), 6.94 (d, J1 = 2.35 Hz 1H), 6.89 (d, J1 = 8.61 Hz, 1H), 6.52 (s, 2H), 3.04 (s, 3H), 2.22 (d, J1 = 14.1 Hz, 1H), 1.84 (d, J1 = 13.7 Hz, 1H), 1.46 (s, 3H), 1.39 (s, 3H); MS (APCI+) m/z 337.1 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(pyridin-2-yl)spiro[chroman4,4′-imidazol]-5′(1′H)-one (rac-32). Step A. A mixture of 2′-amino6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (2.0 g, 5.91 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (9.01 g, 35.5 mmol), Pd(PPh3)4 (0.683 g, 0.591 mmol), and Na2CO3 (5.91 mL, 11.8 mmol) in dioxane (9 mL, 5.91 mmol) was heated in a sealed vial overnight at 90 °C. The mixture was filtered through Celite 545 and concentrated in vacuo. The residue obtained was purfied on silica gel, eluting with 1−10% MeOH/EtOAc (1% TEA) to provide 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (1.65 g, 72.4%). Step B. A solution of 4:1 dioxane/water (1 mL) was sparged with N2 for 30 min. The sparged solution was added to a pressure vessel containing 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (100 mg, 0.2596 mmol), 2-chloropyridine (0.036 54 mL, 0.3893 mmol), Pd(PPh3)4 (29.99 mg, 0.025 96 mmol), and 2 M Na2CO3 (82.53 mg, 0.7787 mmol). The contents were sealed and heated to 80 °C for 15 h. The mixture was concentrated and purified by silica gel chromatography, eluting with a gradient of 0−30% MeOH/EtOAc containing 6% NH4OH to provide 2′-amino-1′,2,2-trimethyl-6(pyridin-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.05351 mmol, 20.62% yield) as an off-white powder. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J1 = 4.70 Hz, 1H), 7.80 (m, 3H), 7.42 (s, 1H), 7.25 (m, 1H), 6.88 (d, J1 = 8.61 Hz, 1H), 6.55 (s, 2H), 3.05 (s, 3H), 2.23 (d, J1 = 13.3 Hz, 1H), 1.83 (d, J1 = 14.5 Hz 1H), 1.47 (s, 3H), 1.39 (s, 3H); MS (APCI+) m/z 337.1 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(pyridin-4-yl)spiro[chroman4,4′-imidazol]-5′(1′H)-one (rac-33). rac-33 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with (pyridin-4-ylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(pyridin-4-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.05351 mmol, 18.1% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.55 (d, J1 = 6.26 Hz 2H), 7.59 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz 1H), 7.52 (dd, J1 = 4.70 Hz, J2 = 1.57 Hz, 2H), 7.02 (d, J1 = 2.35 Hz 1H), 6.92 (d, J1 = 8.61 Hz 1H), 6.55 (s, 2H), 3.05 (s, 3H), 2.22 (d, J1 = 14.1 Hz, 1H),

1.83 (d, J1 = 14.1 Hz, 1H), 1.43 (s, 3H), 1.39 (s, 3H); MS (APCI/ESI, multimode) m/z 337.2. (M + 1). 2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-34). rac-34 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with 5-chloropyridin-3-ylboronic acid to afford 2′-amino-6-(5chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (25 mg, 0.067 mmol, 46% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.57 (d, J1 = 1.95 Hz, 1H), 8.48, (d, J1 = 2.34 Hz, 1H), 7.71 (t, J1 = 2.15 Hz, 1H), 7.37 (dd, J1 = 8.59 Hz, J2 = 2.34 Hz, 1H), 6.97 (d, J1 = 8.59 Hz, 1H), 6.94 (s, 1H), 3.21 (s, 3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.97 (d, J1 = 14.1 Hz, 1H), 1.53 (s, 3H), 1.43 (s, 3H); LCMS (APCI+) m/z 371.2 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(5-(trifluoromethyl)pyridin-3yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-35). rac-35 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with 5-(trifluoromethyl)pyridin-3-ylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(5-(trifluoromethyl)pyridin-3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (33 mg, 0.082 mmol, 55% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.88 (s, 1H), 8.79 (s, 1H), 7.94 (s, 1H), 7.41 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.0 (d, J1 = 8.61 Hz, 1H), 6.97 (d, J1 = 1.96 Hz, 1H), 3.20 (s, 3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.98 (d, J1 = 14.1 Hz, 1H), 1.54 (s, 3H), 1.44 (s, 3H); LCMS (APCI+) m/z 405.2 (M + 1). 2′-Amino-1′,2,2-trimethyl-6-(5-(prop-1-ynyl)pyridin-3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-36). A resealable glass pressure tube was charged with 2′-amino-1′,2,2-trimethyl-6(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (30 mg, 0.078 mmol), 3-bromo-5-(prop-1-ynyl)pyridine (18 mg, 0.093 mmol), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (3.2 mg, 0.0039 mmol), 20% aqueous Na2CO3 (144 μL, 0.27 mmol), and 1,4-dioxane (779 μL, 0.078 mmol). The mixture was sparged with N2 for 5 min, capped, and stirred at 90 °C for 1 h. The solvents were removed in vacuo, and the residue obtained was suspended in 1.5 mL of MeOH and filtered through a 45 μm filter. The filtrate collected was purified by C-18 reverse phase preparative HPLC on Gilson UniPoint instrument using a 5−95% CH3CN/water + 0.5% formic acid gradient. The fractions containing the product were pooled, concentrated in vacuo, evaporated from CH3CN (2 × 5 mL), and dried under high vacuum for 48 h to give 2′-amino-1′,2,2-trimethyl-6(5-(prop-1-ynyl)pyridin-3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)one (23 mg, 0.061 mmol, 79% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.55 (d, J1 = 10.96 Hz, 2H), 7.72 (s, 1H), 7.42 (dd, J1 = 8.61 Hz, J2 = 1.96 Hz, 1H), 6.99 (d, J1 = 8.61 Hz, 1H), 6.95 (d, J1 = 2.35 Hz, 1H), 3.30 (s, 3H), 2.57 (d, J1 = 14.5 Hz, 1H), 2.12 (d, J1 = 14.5 Hz, 1H), 2.09 (s, 3H), 1.54 (s, 3H), 1.46 (s, 3H); LCMS (APCI+) m/z 375.2 (M + 1). 2′-Amino-6-(2-fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-37). rac-37 was synthesized in the same fashion as compound 32, substituting 2chloropyridine for 2-fluoro-3-iodopyridine to afford 2′-amino-6-(2fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (15 mg, 0.04233 mmol, 32.61% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J1 = 7.70 Hz, 1H), 7.90 (m, 1H), 7.40 (d, J1 = 8.61 Hz 1H), 7.35 (m, 1H), 7.05 (s, 1H), 6.91 (d, J1 = 8.61 Hz, 1H), 2.79 (s, 3H), 2.36 (d, J1 = 11.74 Hz, 1H), 1.89 (m, 1H), 1.47 (s, 3H), 1.45 (s, 3H); MS (APCI+/ESI+ multimode) m/z 55.2 (M + 1). 2′-Amino-6-(5-fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-38). rac-38 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with 5-fluoropyridin-3-yl boronic acid (21 mg, 0.15 mmol) to afford 2′-amino-6-(5-fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (10 mg, 0.028 mmol, 19% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.53 (s, 1H), 8.38 (d, J1 = 2.54 Hz, 1H), 7.44 (dt, J1 = 9.57 Hz, J2 = 2.25 Hz, 1H), 7.38 (dd, J1 = 8.59 Hz, J2 = 2.34 Hz, 1H), 6.97 (d, J1 = 8.59 Hz, 1H), 6.96 (s, 1H), 3.20 (s, 3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.97 (d, J1 = 14.3 Hz, 1H), 1.53 (s, 3H), 1.43 (s, 3H); LCMS (APCI+) m/z 355.2 (M + 1). N

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2′-Amino-1′,2,2-trimethyl-6-(pyrimidin-5-yl)spiro[chroman4,4′-imidazol]-5′(1′H)-one (rac-39). rac-39 was synthesized in the same fashion as compound 11, replacing phenylboronic acid with pyrimidin-5-yl boronic acid to afford 2′-amino-1′,2,2-trimethyl-6(pyrimidin-5-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (0.037 g, 0.110 mmol, 74.2% yield). 1H NMR (400 MHz, CDCl3-d) δ 9.13 (s, 1H), 8.83 (s, 2H), 7.39 (d, J1 = 6.65 Hz, 1H), 7.00 (d, J1 = 8.61 Hz, 1H), 6.95 (s, 1H), 3.19 (s, 3H), 2.54 (d, J1 = 14.5 Hz, 1H), 1.97 (d, J1 = 14.1 Hz, 1H), 1.53 (s, 3H), 1.44 (s, 3H); LCMS (APCI+) m/z 338.3 (M + 1). 2′-Amino-6-(isothiazol-5-yl)-1′,2,2-trimethylspiro[chroman4,4′-imidazol]-5′(1′H)-one (rac-40). A glass pressure tube was charged with a mixture of 2′-amino-6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (50 mg, 0.15 mmol), 5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)isothiazole (62 mg, 30 mmol), Pd(PPh3)4 (8.5 mg, 0.0074 mmol), 2 M Na2CO3 (0.15 mL, 0.30 mmol), and dioxane (1.5 mL, 0.15 mmol). The tube was sealed and heated at 90 °C for 18 h. The crude reaction mixture was directly purified by flash chromatography on silica gel, eluting with a gradient of 2−10% MeOH/DCM containing 1% NH4OH to provide 2′-amino6-(isothiazol-5-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (9 mg, 18%) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 8.395 (d, J = 1.56 Hz, 1H), 7.414 (dd, J1 = 8.589 Hz, J2 = 1.56 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 1.952 Hz, 1H), 6.91 (d, J = 8.589 Hz, 1H), 3.21 (s, 3H), 2.54 (d, 14.06 Hz, 1H), 1.96 (d, J = 14.45 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 343.1 (M + 1), retention time 1.996 min. N-(2′-Amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-6-yl)-5-methylfuran-2-carboxamide (rac-41). Step A. A mixture of 2′-amino-6-bromo-1′,2,2trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (2.0 g, 5.91 mmol), tert-butyl carbamate (6.93 g, 59.1 mmol), and Cs2CO3 (3.85 g, 11.8 mmol) in THF (20 mL, 5.91 mmol) was purged with Ar for 5 min. Pd2dba3 (542 mg, 0.591 mmol) and XPHOS (564 mg, 1.18 mmol) were added, and the mixture was heated to 90 °C overnight in a sealed vial. The mixture was then cooled to ambient temperature and filtered through Celite. The filtrate collected was concentrated in vacuo, and the residue obtained was purified by flash chromatography on silica gel, eluting with CH2Cl2/MeOH/NH4OH (90:9:1). The product isolated was taken up in DCM/TFA (1:3, 20 mL) and stirred at ambient temperature for 3 h. The mixture was concentrated in vacuo, and the resulting oil was evaporated from EtOAc and hexanes to provide 2′,6-diamino-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one (1.15 g, 71% yield) as a light brown solid which was used for the next reaction directly. LCMS (APCI+) m/z 275.1 (M + 1). Step B. To a mixture of 2′,6-diamino-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (0.032 g, 0.0862 mmol) and 5methylfuran-2-carbonyl chloride (0.0125 g, 0.0862 mmol) in DCM (0.007 32 g, 0.0862 mmol) was added TEA (d, 0.726) (0.0120 mL, 0.0862 mmol), and the mixture was stirred for 20 min at room temperature. The mixture was concentrated in vacuo, and the residue obtained was purified on reverse phase C-18 HPLC to give N-(2′amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-6-yl)-5-methylfuran-2-carboxamide (15 mg, 0.0384 mmol, 45% yield). 1H NMR (400 MHz, CDCl3-d) δ 11.72 (br s, 1H), 11.09 (br s, 1H), 8.14 (s, 1H), 7.55 (s, 1H), 7.35 (d, J = 9.39 Hz, 1H), 7.06 (d, J = 3.13 Hz, 1H), 6.89 (d, J = 8.61 Hz, 1H), 6.15 (d, J = 3.13 Hz, 1H), 3.30 (s, 3H), 2.56 (d, J = 14.86 Hz, 1H), 2.37 (s, 3H), 2.17 (d, J = 14.08 Hz, 1H), 1.52 (s, 3H), 1.39 (s, 3H). LCMS (APCI+) m/z 483.2 (M + 1), retention time 1.956 min. 2-Amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one (rac42). Step A. 6-Bromo-2,2-dimethylthiochroman-4-one (7.456 mL, 7.456 mmol) in absolute ethanol (1M, 7.4 mL) was treated sequentially with (NH4)2CO3 (7.165 g, 74.56 mmol), potassium cyanide (1.457 g, 22.37 mmol), and sodium acid sulfite (0.1552 g, 1.491 mmol) in a steal bomb containing a stir bar. The bomb was sealed and heated in an oil bath at 150 °C for 16 h. The mixture was cooled to ambient temperature and saw about >75% conversion by LC. Therefore an additional 4 equiv of (NH4)2CO3 and 1 equiv of

KCN were added to the mixture. The bomb was sealed and heated back to 150 °C for a further 24 h. The bomb mixture was cooled to ambient temperature, and the contents were poured into water. The rapidly stirred suspension was carefully acidified with 5 M hydrochloric acid (37.28 mL, 186.4 mmol). The mixture was diluted further with water (50 mL), and the resulting solids were filtered, washing with water. The solid collected was first air-dried and then vacuum-dried for 24 h to provide 6′-bromo-2′,2′-dimethylspiro[imidazolidine-4,4′thiochroman]-2,5-dione (1.8 g, 7.75% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.34−7.32 (m, 2H), 7.05 (J = 8.61 Hz, 1H), 2.68 (d, J = 14.08 Hz, 1H), 2.29 (d, J = 14.86 Hz, 1H), 1.47 (s, 3H), 1.45 (s, 3H). MS (neg) m/z 339.0, 341.0 (M − 1). Step B. 6′-Bromo-2′,2′-dimethylspiro[imidazolidine-4,4′-thiochroman]-2,5-dione (1.75 g, 5.13 mmol) in DMF (0.4 M, 9.8 mL) was treated with K2CO3 (709 mg, 5.13 mmol) followed by iodomethane (0.319 mL, 5.13 mmol) at ambient temperature. After 30 min, the reaction was diluted with water (50 mL), and the resulting solid was filtered, air-dried, and dried under high vacuum for 16 h to provide 6′bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′-thiochroman]-2,5dione (1.63 g, 4.59 mmol, 89.5% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 7.35−7.32 (m, 1H), 7.18−7.17 (m, 1H), 7.07 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 3.17 (s, 3H), 2.68 (d, J = 14.86 Hz, 1H), 2.12 (d, J = 14.86 Hz, 1H), 0.01 (s, 6H); MS (neg) m/z 352.1, 354.9 (M − 1)− . Step C. 6′-Bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′-thiochroman]-2,5-dione (1.60 g, 4.50 mmol) in THF was concentrated to a paste. The paste was suspended in toluene (9 mL, 0.5 M) and heated to reflux under a reflux condenser fitted with a drying tube. When the mixture became a solution, Lawesson’s reagent (1.09 g, 2.70 mmol) was added and heating at reflux was continued for 16 h. The mixture was cooled to ambient temperature and partitioned between EtOAc and water. The aqueous layer was extracted a second time with ethyl acetate. The combined organics were dried (Na2SO4), filtered, and concentrated in vacuo. The residue isolated was purified by flash chromatography on silica gel, eluting with 5−50% ethyl acetate− hexanes to provide 6′-bromo-1,2′,2′-trimethyl-2-thioxospiro[imidazolidine-4,4′-thiochroman]-5-one (1.04 g, 2.80 mmol, 62.2% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 7.37−7.34 (m, 1H), 7.10−7.06 (m, 2H), 3.39 (s, 3H), 2.62 (d, J = 14.87 Hz, 1H), 2.26 (d, J = 14.87 Hz, 1H), 1.50 (s, 3H), 1.46 (s, 3H); MS (neg) m/z 368.9, 371.1 (M − 1). Step D. 6′-Bromo-1,2′,2′-trimethyl-2-thioxospiro[imidazolidine4,4′-thiochroman]-5-one (35 mg, 0.094 mmol) in DCM (0.5 mL) was treated with 7 N ammonia in methanol (673 μL, 4.7 mmol) at ambient temperature. Then 70% tert-butyl hydroperoxide in water (52 μL, 0.38 mmol) was added, and the mixture was stirred at ambient temperature. After 16 h, another 2 equiv of tBu-OOH was added, and the mixture was stirred at room temperature for another 12 h. The reaction mixture was transferred to separatory funnel and partitioned between brine and ethyl acetate. The phases were separated, and the aqueous layer was extracted with ethyl acetate (1×). The combined organics were dried (Na2SO4), filtered, concentrated, and purified by flash chromatography on silica gel, eluting with 4−20% methanol− dichloromethane to provide 2-amino-6′-bromo-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one (14 mg, 0.040 mmol, 42% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 7.26 (m, 1H), 7.04−7.01 (m, 1H), 3.18 (s, 3H), 2.61−2.56 (m, 1H), 2.09−2.05 (m, 1H), 1.50 (s, 3H), 1.46 (s, 3H); MS (APCI+) m/z 354.2, 356.0 (M + 1). Step E. 2-Amino-6′-bromo-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one (42 mg, 0.119 mmol) in toluene (0.1 M) was treated with 3-chloro-5-fluorophenylboronic acid (26.9 mg, 0.154 mmol), 20% aqueous sodium carbonate (157 μL, 0.296 mmol), and tetrakis(triphenylphosphine)palladium(0) (6.85 mg, 0.005 93 mmol). The mixture was sparged with Ar for 2 min, capped, and heated to reflux for 16 h. The mixture was applied directly to silica gel, eluting with a gradient of 4−20% methanol−dichloromethane to provide 2amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole4,4′-thiochroman]-5(1H)-one (30 mg, 0.0743 mmol, 62.7% yield) as a tan solid. 1H NMR (500 MHz, CDCl3-d) δ 7.33 (dd, J1 = 8.321 Hz, J2 O

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 2.61 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, J = 14.08 Hz, 1H), 1.82 (dd, J1 = 14.08 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H), 1.02 (s, 3H). MS (positive) m/z 387 (M + 1)+. Analytical HPLC purity = 93% (220 nm), retention time 3.546 min. 3-(2-Amino-1,2′,2′-trimethyl-5-oxo-1,5-dihydrospiro[imidazole-4,4′-thiochroman]-6′-yl)benzonitrile (rac-44). 2Amino-6′-bromo-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]5(1H)-one (51 mg, 0.144 mmol) and 3-cyanophenylboronic acid (31.7 mg, 0.216 mmol) were suspended in dioxane (0.75 mL), and 20% aqueous sodium carbonate (237 μL, 0.446 mmol) was added with rapid stirring. The suspension was sparged with argon for 2 min, at which point dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (6 mg, 0.0072 mmol) was added. The reaction vial was sealed and heated to 100 °C for 16 h. The reaction mixture was partitioned between EtOAc and water. The aqueous layer was extracted a second time with ethyl acetate. The combined organics were dried (Na2SO4), filtered, concentrated, and purified on silica gel, eluting with 1−15% MeOH/DCM (containing 2% ammonia) to provide 3-(2-amino-1,2′,2′-trimethyl-5-oxo-1,5dihydrospiro[imidazole-4,4′-thiochroman]-6′-yl)benzonitrile (32 mg, 0.0850 mmol, 59% yield) as a solid. 1H NMR (400 MHz, CDCl3-d) δ 7.94 (m, 1H), 7.80 (d, J = 1.56 Hz, 1H), 7.79 (m, 1H), 7.65 (t, J = 7.83 Hz, 1H), 7.52−7.49 (m, 1H), 7.19 (d, J = 7.83 Hz, 1H), 6.98 (brs, 1H), 6.54 (brs, 2H), 3.05 (s, 3H), 2.35 (d, J = 14.09 Hz, 1H), 1.97− 1.89 (m, 1H), 1.51 (s, 3H), 1.42 (m, 3H); MS (APCI+) m/z 377.2 (M + 1); analytical HPLC purity 95 area %, retention time 3.324 min. 3-(2-Amino-1,3′,3′-trimethyl-5-oxo-1,3′,4′,5-tetrahydro-2′Hspiro[imidazole-4,1′-naphthalene]-7′-yl)benzonitrile (rac-45). 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2’H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and 3cyanophenylboronic acid (28 mg, 0.19 mmol) were processed according to the method described for the synthesis of 3-(2-amino1,2′,2′-trimethyl-5-oxo-1,5-dihydrospiro[imidazole-4,4′-thiochroman]6′-yl)benzonitrile to provide 3-(2-amino-1,3′,3′-trimethyl-5-oxo1,3′,4′,5-tetrahydro-2′H-spiro[imidazole-4,1′-naphthalene]-7′-yl)benzonitrile (34 mg, 0.095 mmol, 64% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.74 (d, J = 1.56 Hz, 1H), 7.69 (d, J = 7.83 Hz, 1H), 7.61−7.58 (m, 1H), 7.50 (t, J = 7.83 Hz, 1H), 7.39 (dd, J1 = 7.83 Hz, J2 = 2.25 Hz, 1H), 7.21 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H), 3.21 (s, 3H), 2.83 (d, J = 15.65 Hz, 1H), 2.61 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, J = 14.09 Hz, 1H), 1.81 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H0, 1.02 (s, 3H); LCMS (APCI+) m/z 359 (M + 1), retention time 2.263 min. 2-Amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac46). 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and 2fluoropyridin-3-ylboronic acid (27 mg, 0.19 mmol) were processed according to the method described for the synthesis of 2′-amino-6-(2fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]5′(1′H)-one to yield 2-amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (37 mg, 0.10 mmol, 71% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.16 (dd, J1 = 7.04 Hz, J2 = 1.56 Hz, 1H), 7.80−7.75 (m, 1H), 7.42 (dt, J1 = 7.83 Hz, J2 = 1.56 Hz,1H), 7.26−7.23 (m, 1H), 7.27 (d, J = 7.82 Hz, 1H), 7.10 (s, 1H), 3.20 (s, 3H), 2.82 (d, J = 15.65 Hz, 1H), 2.62 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09 Hz, 1H), 1.83 (dd, J1 = 14.09 Hz, J2 = 3.13 Hz, 1H), 1.17 (s, 3H), 1.03 (s, 3H); 13C NMR (400 MHz, CDCl3-d) δ 178.4 (C), 160 (C), 156.0 (C), 146.4 (CH), 140.5 (CH), 137.4 (C), 134.2 (C), 132.6 (C), 130.5 (CH), 128.8 (CH), 127.8 (CH), 123.4 (C), 121.8 (CH), 65.6 (C), 46.8 (CH2), 43.4 (CH2), 31.3−25.8 (CH3), 29.6 (C), 25.8 (CH3); LCMS (APCI+) m/z 353.2 (M + 1), retention time 2.099 min. 5-Amino-6′-(5-chloropyridin-3-yl)-1,2′,2′-trimethylspiro[pyrrole-3,4′-thiochroman]-2(1H)-one (rac-47). rac-47 was synthesized in the same fashion as 2-amino-6′-(3-chloro-5-fluorophenyl)1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one, substituting 5-chloropyridin-3-ylboronic acid (14.4 mg, 0.0915 mmol) for 3chloro-5-fluorophenylboronic acid to afford 5-amino-6′-(5-chloropyridin-3-yl)-1,2′,2′-trimethylspiro[pyrrole-3,4′-thiochroman]-2(1H)-one

= 1.664 Hz, 1H), 7.25 (m, 2H), 7.04 (m, 3H), 3.22 (s, 3H), 2.65 (d, J = 14.146 Hz, 1H), 2.14 (d, J = 14.146 Hz, 1H), 1.56 (s, 3H), 1.50 (s, 3H). 13C NMR (500 MHz, CDCl3-d) 155.8 (C), 143.6 (C), 136.0 (C), 135.3 (C), 132.1 (C), 131.9 (C), 131.9 (C), 128.9 (CH), 127.3 (CH), 125.9 (CH), 122.9 (CH), 113 (CH), 113 (CH), 112 (C), 67.4 (C), 49.5 (CH2), 41.4 (C), 31.5−29.2 (CH3), 26.1 (CH3). LCMS (APCI+) m/z 404.1 (M + 1), retention time = 1.10 min. 2-Amino-7′-(3-chloro-5-fluorophenyl)-1,3′,3′-trimethyl3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-43). Step A. 7-Bromo-3,3-dimethyl-3,4-dihydronaphthalen1(2H)-one (3.00 g, 11.9 mmol) in EtOH (12 mL) was treated with sodium bisulfite (100 mg), KCN (2.32 g, 35.6 mmol), and (NH4)2CO3 (7.97 g, 83.0 mmol) as described for the synthesis of 6′-bromo-2′,2′-dimethylspiro[imidazolidine-4,4′-thiochroman]-2,5dione to provide 7′-bromo-3′,3′-dimethyl-3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (1.5 g, 4.64 mmol, 39% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.01 (br s, 1H), 8.58 (s, 1H), 7.46 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.14−7.12 (m, 2H), 2.59−2.52 (m, 2H), 2.10 (d, J = 14.09 Hz, 1H), 1.79 (dd, J1 = 14.09 Hz, J2 = 1.57 Hz, 1H), 1.07 (s, 3H), 0.86 (s, 3H). Step B. 7′-Bromo-3′,3′-dimethyl-3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (15.0 g, 46.41 mmol) in DMF (60 mL) was treated with K2CO3 (6.735 g, 48.73 mmol) and CH3I (3.04 mL, 48.73 mmol) according to the procedure described for the synthesis of 6′-bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′thiochroman]-2,5-dione to provide 7′-bromo-1,3′,3′-trimethyl-3′,4′dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (15.6 g, 46.26 mmol, 99.67% yield). 1H NMR (CDCl3-d) δ 7.36 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.12 (d, J = 2.35 Hz, 1H), 6.99 (d, J = 8.61 Hz, 1H), 6.15 (br s, 1H), 3.13 (s, 3H), 2.72 (s, J = 16.43 Hz, 1H), 2.52 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.32 (d, J = 14.09 Hz, 1H), 1.82 (dd, J1 = 14.08 Hz, J2 = 3.13 Hz, 1H), 1.61 (s, 3H), 0.95 (s, 3H). Analytical HPLC purity 99%, retention time 3.780 min. Step C. 7′-Bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (874 mg, 2.59 mmol) was treated with Lawesson’s reagent (629 mg, 1.56 mmol), as described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 7′bromo-1,3′,3′-trimethyl-2-thioxo-3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalen]-5-one (440 mg, 1.25 mmol, 48% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.38 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.34 (brs, 1H), 7.02−6.99 (m, 2H), 3.37 (s, 3H), 2.72 (d, J = 16.43 Hz, 1H), 2.53 (dd, J1 = 16.43 Hz, J2 = 3.13 Hz, 1H), 2.27 (d, J = 14.08 Hz, 1H), 1.85 (dd, J1 = 14.08 Hz, J2 = 2.35 Hz, 1H), 1,17 (s, 3H), 0.98 (s, 3H). LCMS (neg) m/z 351, 352 (M − 1); retention time 1.666 min. Step D. 7′-Bromo-1,3′,3′-trimethyl-2-thioxo-3′,4′-dihydro-2′Hspiro[imidazolidine-4,1′-naphthalen]-5-one (2.44 g, 6.91 mmol) in THF (17 mL) was treated with 7 N NH3 in MeOH (19.7 mL, 138 mmol) and 70% tert-butyl hydroperoxide in water (9.88 mL, 69.1 mmol) as described for the synthesis of 2-amino-6′-bromo-1,2′,2′trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one to provide 2amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole4,1′-naphthalen]-5(1H)-one (1.71 g, 5.09 mmol, 73.6% yield) as a white powder. 1H NMR (400 MHz, CDCl3-d) δ 7.32 (dd, J1 = 7.83 Hz, J2 = 1.56 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H), 6.97 (d, J = 8.61 Hz, 1H), 3.18 (s, 3H), 2.71 (d, J = 15.65 Hz, 1H), 2.50 (dd, J1 = 16.43 Hz, J2 = 3.13 Hz, 1H), 2.23 (d, J = 14.08 Hz, 1H), 1.75 (dd, J1 = 14.08 Hz, J2 = 2.35 Hz, 1H), 1.14 (s, 3H), 0.963 (s, 3H). LCMS (APCI+) m/z 336, 338 (M + 1), retention time 0.983 min. Step E. 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′Hspiro[imidazole-4,1′-naphthalen]-5(1H)-one (56 mg, 0.17 mmol) and 3-chloro-5-fluorophenylboronic acid (38 mg, 0.22 mmol) were processed as described for the synthesis of 2-amino-6′-(3-chloro-5fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]5(1H)-one to yield 2-amino-7′-(3-chloro-5-fluorophenyl)-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)one (37 mg, 0.096 mmol, 58% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.37 (dd, J1 = 7.83 Hz, J2 = 1.56 Hz, 1H), 7.24 (s, 1H), 7.18 (d, J = 8.61 Hz, 1H), 7.07−7.03 (m, 3H), 3.22 (s, 3H), 2.82 (d, J = 16.42 Hz, P

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(14 mg, 46% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.58 (m, 1H), 8.51 (m, 1H), 7.73 (m, 1H), 7.35−7.30 (m, 1H), 7.28 (m, 1H), 7.04 (m, 1H), 3.20 (s, 3H), 2.64 (d, J = 14.08 Hz, 1H), 2.13 (d, J = 14.08 Hz, 1H), 1.56 (s, 3H), 1.49 (s, 3H); MS (APCI+) m/z 387.2 (M + 1). 2-Amino-7′-(5-chloropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac48). 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (112 mg, 0.333 mmol) and 5-chloropyridin-3-ylboronic acid (68 mg, 0.433 mmol) were processed according to the method described for the synthesis of 5-amino-6′-(5chloropyridin-3-yl)-1,2′,2′-trimethylspiro[pyrrole-3,4′-thiochroman]2(1H)-one to provide 2-amino-7′-(5-chloropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)one (50 mg, 0.136 mmol, 41% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.60 (d, J = 2.35 Hz, 1H), 8.51 (d, J = 2.35 Hz, 1H), 7.45 (m, 1H), 7.39 (dd, J1 = 7.83 Hz, J2 = 1.56 Hz, 1H), 7.22 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H), 3.20 (s, 3H), 2.84 (d, J = 16.43 Hz, 1H), 2.62 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09 Hz, 1H), 1.81 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H), 1.02 (s, 3H). MS (APCI+) m/z 369 (M + 1). 5-Amino-1,2′,2′-trimethyl-6′-(pyrimidin-5-yl)spiro[pyrrole3,4′-thiochroman]-2(1H)-one (rac-49). rac-49 was synthesized in the same fashion as 2-amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one substituting pyrimidin-5-yl boronic acid (13.4 mg, 0.108 mmol) for 3-chloro-5fluorophenylboronic acid to afford 5-amino-1,2′,2′-trimethyl-6′-(pyrimidin-5-yl)spiro[pyrrole-3,4′-thiochroman]-2(1H)-one (19 mg, 56.5% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.96 (s, 2H), 7.57 (m, 1H), 7.24 (m, 1H), 7.07 (m, 1H), 3.04 (s, 3H), 2.35 (m, 1H), 1.92 (m, 1H), 1.51 (s, 3H), 1.42 (s, 3H); MS (APCI+) m/z 354.2 (M + 1); analytical HPLC retention time 2.967 min. 2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-50). 2Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and pyrimidin-5-yl boronic acid (24 mg, 0.19 mmol) were processed as described for the synthesis of 5-amino-1,2′,2′-trimethyl-6′-(pyrimidin-5-yl)spiro[pyrrole-3,4′-thiochroman]-2(1H)-one to yield 2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′naphthalen]-5(1H)-one (38 mg, 0.11 mmol, 76% yield). 1H NMR (400 MHz, CDCl3-d) δ 9.16 (s, 1H), 8.85 (s, 2H), 7.42 (dd, J = −7.83 Hz, J2 = 2.35 Hz, 1H), 7.09 (d, J = 1.56 Hz, 1H), 3.22 (s, 3H), 2.86 (d, J = 15.65 Hz, 1H), 2.63 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, 14.08 Hz, 1H), 1.84 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.18 (s, 3H), 1.03 (s, 3H); 13C NMR (400 MHz, CDCl3-d) δ 179.7 (C), 157.4 (CH), 155.3 (C), 154.8 (CH), 154.8 (CH), 138.1 (C), 135.4 (C), 133.9 (C), 133.0 (C), 131.1 (CH), 126.9 (CH), 125.3 (CH), 66.0 (C), 46.9 (CH2), 43.5 (CH2), 31.2−25.9 (CH3), 29.6 (C), 26.0 (CH3); LCMS (APCI+) m/z 336.2 (M + 1)+, retention time 1.905 min (5 min run). 2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione (rac-51). Step A. A solution of 2-amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) in acetic acid (124 μL, 0.15 mmol) was cooled to 0 °C and treated with a solution of CrO3 (33 mg, 0.33 mmol) in acetic acid (124 μL, 0.15 mmol) and water (124 μL, 0.15 mmol). The resulting mixture was allowed to warm to ambient temperature overnight. The reaction mixture was diluted with water and extracted with DCM. The organic extracts were washed with NaHCO3, brine, dried, and concentrated in vacuo to provide crude 2-amino-7′-bromo-1,3′,3′-trimethyl-2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-trifluoroacetate (26 mg, 38% yield), which was used for the next reaction directly. LCMS (APCI+) m/z 350, 352 (M + 1)+, retention time 1.005 min. Step B. 2-Amino-7′-bromo-1,3′,3′-trimethyl-2′H-spiro[imidazole4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-trifluoroacetate (26 mg, 0.056 mmol) and pyrimidin-5-yl-boronic acid (9.7 mg, 0.078 mmol) were processed according to the synthesis of 2-amino-6′-(3-chloro-5-

fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]5(1H)-one to provide 2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione (22 mg, 0.0475 mmol, 85% yield). 1H NMR (400 MHz, CD3OD-d4) δ 9.21 (s, 1H), 9.12 (s, 2H), 8.22 (d, J = 8.22 Hz, 1H), 7.97 (d, J = 8.22 Hz, 1H), 7.74 (s, 1H), 3.03 (s, 3H), 2.65 (d, J = 14.48 Hz, 1H), 2.42 (d, J = 14.86 Hz, 1H), 1.38 (s, 3H), 1.33 (s, 3H). LCMS (APCI+) m/z 350.1 (M + 1)+, retention time 0.911 min. 2-Amino-4′,4′-difluoro-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-52). Step A. To a solution of 2-amino-7′-bromo-1,3′,3′-trimethyl2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-trifluoroacetate (17 mg, 0.0366 mmol) in dichloroethane (183 μL, 0.0366 mmol) at 0 °C in a plastic Falcon tube was added Deoxo-Fluor (20.3 μL, 0.110 mmol), and the resulting mixture was stirred at 0 °C for 15 min while warming to room temperature. The crude reaction mixture was purified by Gilson C18 HPLC to provide TFA salt of 2amino-7′-bromo-4′,4′-difluoro-1,3′,3′-trimethyl-3′,4′-dihydro-2′Hspiro[imidazole-4,1′-naphthalen]-5(1H)-one (3 mg, 22%), which was used for the next reaction directly. Step B. 2-Amino-7′-bromo-4′,4′-difluoro-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one 2,2,2-trifluoroacetate (3 mg, 0.00617 mmol) and pyrimidin-5-ylboronic acid (1.07 mg, 0.00864 mmol) were processed according to the method described for the synthesis of 2-amino-6′-(3-chloro-5-fluorophenyl)1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one and purified by reverse phase C-18 HPLC (5−95% gradient of CH3CN/ water) to provide 2-amino-4′,4′-difluoro-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)one (2.6 mg, 0.005 36 mmol, 86.8% yield). 1H NMR (400 MHz, CD3OD-d4) δ 9.19 (s, 1H), 9.06 (s, 2H), 7.97−7.94 (m, 2H), 7.66 (m, 1H), 4.85 (s, 3H), 2.60 (d, J = 14.87 Hz, 1H), 2.31 (dd, J1 = 14.87 Hz, J2 = 4.34 Hz, 1H), 1.29 (s, 3H), 1.21 (s, 3H); LCMS (APCI+) m/z 372.1 (M + 1)+, retention time 1.045 min. 2′-Amino-6-(3-chlorophenyl)-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-53). Step A. To a mixture of 6-bromo-2,2-dimethylspiro[chroman-4,4′-imidazolidine]2′,5′-dione (200 mg, 0.6151 mmol) and K2CO3 (850 mg, 0.6151 mmol) in DMF (4 mL) was added a solution of ethyl iodide (0.049 65 mL, 0.6151 mmol) in DMF (0.5 mL). The mixture was heated at 45 °C for 2 h and then at 60 °C for 18 h. Then an additional 0.3 equiv of ethyl iodide and K2CO3 were added, and the mixture was heated at 60 °C for an additional 2 h. The mixture was concentrated in vacuo, and the residue obtained was partitioned between ethyl acetate and 2 M aqueous Na2CO3. The organic phases were combined and washed with 2 M aqueous Na2CO3 (2×), water (1×), and brine (1×). The organic layer was dried (Na2SO4), filtered, and concentrated in vacuo. The residue obtained was purified on silica gel, eluting with a gradient of 8−50% EtOAc/hexanes (10 CV) on Biotage SP1 unit to give 6bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′dione (172 mg, 0.4870 mmol, 79% yield) as a pale yellow solid. Analytical HPLC, >92 area % pure, retention time 3.808 min. FIMS (APCI−) m/z 351/353 (M − 1) with Br isotope. Step B. 6-Bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (172 mg, 0.487 mmol) and Lawesson’s reagent (138 mg, 0.341 mmol) were processed according to the method described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 6bromo-1′-ethyl-2,2-dimethyl-2′-thioxospiro[chroman-4,4′-imidazolidin]-5′-one (108 mg, 0.292 mmol, 60% yield) as a white solid. FIMS (APCI−) m/z 367, 369 (M − 1) with Br isotope; analytical HPLC purity 97 area %, retention time 4.35 min. Step C. To a solution of 6-bromo-1′-ethyl-2,2-dimethyl-2′thioxospiro[chroman-4,4′-imidazolidin]-5′-one (108 mg, 0.292 mmol) in MeOH (2 mL) and THF (0.7 mL) were added sequentially tert-butyl hydroperoxide (70% aqueous) (607 μL, 4.39 mmol) and 30% NH4OH (1.14 mL, 8.77 mmol), and the reaction mixture was heated at 40 °C. After 2 h, the mixture was concentrated under a stream of N2, and the resulting residue was partitioned between saturated aqueous NH4Cl and DCM (2×). The organic layers were Q

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

combined, dried (Na2SO4), filtered, and concentrated in vacuo. The residue obtained was purified by silica gel flash chromatography to provide 2′-amino-6-bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′imidazol]-5′(1′H)-one (41 mg, 0.116 mmol, 39.8% yield) as a white foam. FIMS (APCI−) m/z 352/354 (M − 1) with Br isotope, retention time 1.020 min. Step D. The compound was synthesized in the same fashion as 2amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole4,4′-thiochroman]-5(1H)-one, substituting 3-chlorophenylboronic acid (22 mg, 0.140 mmol) for 3-chloro-5-fluorophenylboronic acid to provide 2′-amino-6-(3-chlorophenyl)-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (17 mg, 0.0443 mmol, 38% yield) as a white powder. 1H NMR (CDCl3-d) δ 7.44−7.35 (m, 2H), 7.33−7.27 (m, 3H), 6.70−6.91 (m, 2H), 3.75−3.67 (m, 2H), 2.56 (d, J = 14.09 Hz, 1H), 1.98 (d, J = 14.87 Hz, 1H), 1.51 (s, 3H), 1.41 (s, 3H), 1.33 (t, J = 7.04 Hz, 3H); FIMS (APCI+) m/z 384 (M + 1); analytical HPLC purity >99 area %, retention time 3.415 min. 2′-Amino-6-(3-chlorophenyl)-2,2-dimethyl-1′-(2,2,2trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac54). Step A. To a mixture of 6-bromo-2,2-dimethylspiro[chroman4,4′-imidazolidine]-2′,5′-dione (200 mg, 0.615 mmol) and K2CO3 (850 mg, 0.615 mmol) in DMF (1.5 mL) at ambient temperature was added a solution of 1,1,1-trifluoro-2-iodoethane (129 mg, 0.615 mmol) in DMF (1 mL). The reaction mixture was heated at 60 °C for 18 h and then at 85 °C and stirred for another 5 days. The mixture was cooled to ambient temperature and partitioned between saturated NH4Cl solution and DCM (2×). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The crude was purified on silica gel (Snap 50 cartridge), eluting with a gradient of 10−50% EtOAc/hexane (8 CV), followed by 50% EtOAc/hexanes (2 CV) to give 6-bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazolidine]-2′,5′-dione (155 mg, 0.381 mmol, 62% yield) as a white foam. FIMS (APCI−) m/z 405/407 (M − 1) with Br isotope, retention time 4.054 min Step B. 6-Bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazolidine]-2′,5′-dione (155 mg, 0.381 mmol) and Lawesson’s reagent (108 mg, 0.266 mmol) were processed according to the procedure described for the synthesis of 6′-bromo-1,2′,2′trimethyl-2-thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to give 6-bromo-2,2-dimethyl-2′-thioxo-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazolidin]-5′-one (50 mg, 0.118 mmol, 31% yield) as a clear colorless residue. LC/MS (ESI−) m/z 421/423 (M − 1) with Br isotope; analytical HPLC purity 96 area %, retention time 4.379 min. Step C. To a mixture of 6-bromo-2,2-dimethyl-2′-thioxo-1′-(2,2,2trifluoroethyl)spiro[chroman-4,4′-imidazolidin]-5′-one (50 mg, 0.118 mmol) in MeOH (0.6 mL) and THF (0.15 mL) was added sequentially 70% aqueous tert-butyl hydroperoxide (0.245 mL, 1.77 mmol) and 30% NH4OH (0.460 mL, 3.54 mmol), and the reaction mixture was heated at 40 °C with stirring. After 2 h, the reaction mixture was diluted with water (2 mL), and the organics were removed in vacuo. The resulting mixture was partitioned between saturated aqueous NH4Cl and DCM (2×). The combined organic extracts were dried (Na2SO4), filtered, and concentrated in vacuo. The crude was purified on silica gel (Snap 10g), eluting with a gradient of 1−8% MeOH/DCM (15 CV) followed by 8% MeOH/DCM (5 CV) on Biotage SP1 unit to provide 2′-amino-6-bromo-2,2-dimethyl-1′(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (12 mg, 25% yield) as a white solid. LC/MS (APCI+) m/z 406/408 (M + 1) with Br isotope. Step D. 2′-Amino-6-bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (12 mg, 0.0295 mmol) and 3-chlorophenylboronic acid (7 mg, 0.0443 mmol) were processed according to the method described for the synthesis of 2-amino-6′-(3chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one to provide 2′-amino-6-(3-chlorophenyl)-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (11 mg, 0.0251 mmol, 85% yield) as a white solid. 1H NMR (CDCl3d) δ 7.68−7.63 (m, 1H), 7.48−7.46 (m, 1H), 7.43−7.40 (m, 2H), 7.31−7.29 (m, 2H), 6.94 (d, J = 8.61 Hz, 1H), 4.40−4.19 (m, 2H),

2.58 (d, J = 14.09 Hz, 1H), 2.02 (d, J = 14.86 Hz, 1H), 1.53 (s, 3H), 1.43 (s, 3H); FIMS (APCI+) m/z 438 (M + 1), retention time 3.630 min. 2′-Amino-6-(3-chlorophenyl)-1′-(2-methoxyethyl)-2,2dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-55). Step A. To a mixture of 6-bromo-2,2-dimethylspiro[chroman-4,4′imidazolidine]-2′,5′-dione (200 mg, 0.6151 mmol) and K2CO3 (85 mg, 0.6151 mmol) in DMF (4 mL) was added a solution of 1-bromo2-methoxyethane (86 mg, 0.615 mmol) in DMF (0.5 mL). The mixture was heated at 45 °C for 2 h and at 60 °C overnight. The mixture was cooled to ambient temperature and treated with an additional 0.3 equiv of 1-bromo-2-methoxyethane and K2CO3 and heated at 60 °C. After 20 h (total time), DMF was removed in vacuo, and the residue obtained was partitioned between EtOAc and 2 M aqueous Na2CO3. The organic layers were combined, washed with water (1×), brine (1×), dried (Na2SO4), filtered, and concentrated in vacuo. The residue obtained was purified on silica gel, eluting with a gradient of 8−66% EtOAc/hexanes (10 CV) followed by 66% EtOAc/ hexanes (2 CV) to give 6-bromo-1′-(2-methoxyethyl)-2,2dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (196 mg, 0.511 mmol, 83% yield) as a yellow solid. FIMS (APCI−) m/z 381/383 (M − 1) with Br isotope; HPLC purity >90 area%, retention time 3.702 min. Step B. 6-Bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro[chroman4,4′-imidazolidine]-2′,5′-dione (196 mg, 0.511 mmol) and Lawesson’s reagent (145 mg, 0.358 mmol) were processed according to the method described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 6bromo-1′-(2-methoxyethyl)-2,2-dimethyl-2′-thioxospiro[chroman4,4′-imidazolidin]-5′-one (104 mg, 0.260 mmol, 51% yield) as a white solid. FIMS (APCI−) m/z 397/399 (M − 1) with Br isotope; HPLC purity 96 area %, retention time 4.211 min. Step C. 6-Bromo-1′-(2-methoxyethyl)-2,2-dimethyl-2′-thioxospiro[chroman-4,4′-imidazolidin]-5′-one (104 mg, 0.260 mmol), tert-butyl hydroperoxide (70% aqueous) (0.541 mL, 3.91 mmol), and 30% NH4OH (1.01 mL, 7.81 mmol) were processed according to the method described for the synthesis of 2′-amino-6-bromo-2,2-dimethyl1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one to provide 2′-amino-6-bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (90 mg, 0.235 mmol, 90% yield) as a white foam. FIMS (APCI−) m/z 382/384 (M − 1) with Br isotope. Step D. 2′-Amino-6-bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (90 mg, 0.235 mmol) and 3chlorophenylboronic acid (44 mg, 0.283 mmol) were processed according to the method described for the synthesis of 2-amino-6′-(3chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one to provide 2′-amino-6-(3-chlorophenyl)-1′-(2-methoxyethyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (23 mg, 0.0556 mmol, 24% yield) as a yellow powder. 1H NMR (CDCl3-d) δ 7.40 (s, 1H), 7.36 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.30−7.25 (m, 2H), 7.24−7.21 (m, 1H), 6,92 (d, J = 8.61 Hz, 1H), 6.88 (s, 1H), 3.94−3.85 (m, 1H), 3.80−3.72 (m, 1H), 3.64−3.54 (m, 2H), 3.42 (s, 3H), 2.49 (d, J = 14.09 Hz, 1H), 1.94 (d, J = 14.09 Hz, 1H), 1.52 (s, 3H), 1.47 (s, 3H); FIMS (APCI+) m/z 414 (M + 1), retention time 3.480 min. 1′-((1-Acetylpiperidin-4-yl)methyl)-2′-amino-6-(3-chlorophenyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-56). Step A. To a solution of 2′-amino-6-bromo-2,2dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (100 mg, 0.308 mmol) in DMF (1.2 mL) was added DMF dimethylacetal (0.131 mL, 0.925 mmol). The reaction mixture was stirred at ambient temperature for 18 h and concentrated in vacuo. The resulting residue was dissolved in minimal DCM, and this solution was added dropwise to vigorously stirring hexanes, causing precipitation. The solids were isolated by vacuum filtration (0.2 μm nylon filter membrane), rinsed with hexanes, air-dried, and dried in vacuo to give (E)-N′-(6-bromo2,2-dimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′yl)-N,N-dimethylformimidamide (80 mg, 0.211 mmol, 68.4% yield) as a white powder. 1H NMR (DMSO-d6) δ 9.39 (s, 1H),8.78 (s, 1H), R

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.31 (dd, J1 = 9.39 Hz, J2 = 2.35 Hz, 1H), 6.80 (d, J = 2.35 Hz, 1H), 6.75 (d, J = 8.61 Hz, 1H), 3.22 (s, 3H), 3.10 (s, 3H), 2.27 (d, J = 14.09 Hz, 1H), 1.94 (d, J = 14.09 Hz, 1H),1.41 (s, 3H), 1.27 (s, 3H); LC/ MS (APCI+) m/z 379/381 (M + 1). Step B. To a mixture of (E)-N′-(6-bromo-2,2-dimethyl-5′-oxo-1′,5′dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimidamide (47 mg, 0.124 mmol) and K2CO3 (26 g, 0.186 mmol) in DMF (0.6 mL) was added tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (41.4 mg, 0.149 mmol). The mixture was heated to 60 °C and stirred overnight. After 16 h, additional K2CO3 (26 mg, 0.186 mmol) and tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (42 mg, 0.149 mmol) were added, and the reaction mixture was heated to 80 °C for 5 h and then at 90 °C for 2 more days. The reaction mixture was cooled to ambient temperature and concentrated under a nitrogen stream. The resulting residue was suspended in DCM, sonicated, and the resulting solids were removed by filtration. The filtrate collected was concentrated in vacuo, and the isolated crude was purified by preparative TLC, eluting with 9:1 DCM/MeOH to give (E)-tert-butyl 4-((6-bromo-2′-((dimethylamino)methyleneamino)-2,2-dimethyl-5′oxospiro[chroman-4,4′-imidazole]-1′(5′H)-yl)methyl)piperidine-1carboxylate (50 mg, 0.0867 mmol, 70% yield) as a clear colorless residue. LCMS (APCI+) m/z 576/578 (M + 1) with Br isotope; LC/ MS purity 96 area %, retention time 1.347 min. Step C. To a solution of (E)-tert-butyl 4-((6-bromo-2′((dimethylamino)methyleneamino)-2,2-dimethyl-5′-oxospiro[chroman-4,4′-imidazole]-1′(5′H)-yl)methyl)piperidine-1-carboxylate (43 mg, 0.0746 mmol) in DCM (0.7 mL) at ambient temperature was added neat TFA (0.115 mL, 1.49 mmol), and the mixture was stirred for 1 h. The mixture was then concentrated under a nitrogen stream to provide the crude (E)-N′-(6-bromo-2,2-dimethyl-5′-oxo-1′-(piperidin4-ylmethyl)-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimidamide (43 mg, 0.0750 mmol, 101% yield), which was used for the next reaction directly. Step D. To a solution of crude (E)-N′-(6-bromo-2,2-dimethyl-5′oxo-1′-(piperidin-4-ylmethyl)-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimidamide (53 mg, 0.0924 mmol) and TEA (0.0515 mL, 0.370 mmol) in DCM (0.8 mL) at ambient temperature was added acetyl chloride (0.00923 mL, 0.129 mmol), and the mixture was stirred for 2 h. The reaction mixture was concentrated under a nitrogen stream, and the crude was purified by preparative TLC (1 mm plate, 19:1 DCM/MeOH) to give (E)-N′-(1′((1-acetylpiperidin-4-yl)methyl)-6-bromo-2,2-dimethyl-5′-oxo-1′,5′dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimidamide (23 mg, 0.0444 mmol, 48.0% yield) as a white residue. LCMS (APCI+) m/z 518/520 (M + 1) with Br isotope. Step E. A sealable glass tube was charged with a mixture of (E)-N′(1′-((1-acetylpiperidin-4-yl)methyl)-6-bromo-2,2-dimethyl-5′-oxo1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimidamide (18 mg, 0.034 72 mmol) and 7 N NH3 in MeOH (0.4960 mL, 3.472 mmol). The tube was capped and heated at 50 °C. After 3 days, the mixture was cooled to ambient temperature and treated with additional 7 N NH3 in MeOH (100 equiv) and heated at 60 °C with stirring. After 28 h, the mixture was cooled to ambient temperature, concentrated in vacuo, and the residue obtained was purified by preparative TLC (1 mm, 9:1 DCM/7 N NH3/MeOH) to give 1′-((1acetylpiperidin-4-yl)methyl)-2′-amino-6-bromo-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (14 mg, 0.03021 mmol, 87% yield) as a white residue. LCMS (APCI+) m/z 463/465 (M + 1) with Br isotope. Step F. A sealable glass vial was charged with a mixture of 1′-((1acetylpiperidin-4-yl)methyl)-2′-amino-6-bromo-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (14 mg, 0.0302 mmol), 3chlorophenylboronic acid (6.14 mg, 0.0393 mmol), PdCl2(dppf)·DCM (2.47 mg, 0.003 02 mmol), dioxane (0.3 mL), and 2 M aqueous Na2CO3 (0.0604 mL, 0.121 mmol). The mixture was purged with nitrogen, vial capped, and the reaction mixture was heated at 90 °C and stirred for 90 min. The reaction mixture was diluted with DCM (0.2 mL) and purified by preparative TLC plate (1 mm plate, 9:1 DCM/7 N NH3/MeOH) to give 1′-((1-acetylpiperidin-4-yl)methyl)-2′-amino-6-(3-chlorophenyl)-2,2-dimethylspiro[chroman-

4,4′-imidazol]-5′(1′H)-one (11.6 mg, 0.0234 mmol, 77% yield) as a white residue. LC/MS (APCI+) m/z 495 (M + 1), retention time 1.024 min. 7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-oxazol]-2′-amine (rac-57). rac-57 was synthesized in the same fashion as compound 62 (below), replacing 2-fluoropyridin-3-ylboronic acid with (5-chloropyridin-3-yl)boronic acid to afford 7-(5-chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (8 mg, 0.023 mmol, 29% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.69 (d, J = 2 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 7.82 (m, 1H), 7.44 (d, J = 2 Hz, 1H), 7.35 (dd, J1 = 7.8 Hz, J2 = 2 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 2.7 (d, 16 Hz, 1H), 2.57 (dd, J1 = 16 Hz, J2 = 1.2 Hz, 1H), 2.06 (d, J = 14 Hz, 1H), 1.86 (dd, J1 = 14 Hz, J2 = 1.6 Hz, 1H), 1.1 (s, 3H), 0.98 (s, 3H); LCMS (APCI+) m/z 342.1 (M + 1). 7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-thiazol]-2′-amine (rac-58). rac-58 was synthesized in the same fashion as compound 57, substituting silver cyanate with silver thiocyanate to afford 7-(5-chloropyridin-3-yl)-3,3dimethyl-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-thiazol]-2′amine (10 mg, 0.028 mmol, 45% yield). 1H NMR (400 MHz, CDCl3d) δ 8.69 (d, J = 2 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 7.84 (t, J = 2 Hz, 1H), 7.63 (d, J = 2 Hz, 1H), 7.35 (dd, J1 = 7.8 Hz, J2 = 2 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 3.65 (dd, J1 = 11 Hz, J2 = 10 Hz, 2H), 2.7 (d, 16 Hz, 1H), 2.6 (dd, J1 = 16 Hz, J2 = 1.2 Hz, 1H), 2.07 (dd, J1 = 14 Hz, J2 = 1.6 1H), 1.9 (d, J1 = 14 Hz, 1H), 1.09 (s, 3H), 1.07 (s, 3H); LCMS (APCI+) m/z 358.1 (M + 1). 7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine (rac-59). Step A. A solution of vinyl magnesium bromide 1 M in THF (39.5 mL, 39.5 mmol) was added dropwise to a solution of 7-bromo-3,3-dimethyl-3,4dihydronaphthalen-1(2H)-one (5 g, 19.8 mmol) in THF at 0 °C under N2. After 2 h, the reaction mixture was poured into ice cold saturated NH4Cl solution (150 mL) and extracted into EtOAc (2 × 100 mL). The combined organic layers were washed with brine, then dried (MgSO4) and concentrated in vacuo to provide the crude 7-bromo3,3-dimethyl-1-vinyl-1,2,3,4-tetrahydronaphthalen-1-ol as a liquid (5.7 g, 83% yield). 1H NMR (400 MHz, CDCl3-d) δ 7.40 (d, J = 1.96 Hz, 1H), 7.33 (dd, J1 = 8.22 Hz, J2 = 2.35 Hz, 1H), 7.04 (d, J = 8.22 Hz, 1H), 5.91 (dd, J1 = 16.82 Hz, J2 = 10.17 Hz, 1H), 5.27 (dd, J1 = 16.84 Hz, J2 = 1.96 Hz, 1H), 5.06 (dd, J1 = 10.17 Hz, J2 = 1.96 Hz, 1H), 5.03 (s, 1H, exchanged with D2O), 2.45 (m, 2H), 1.74 (d, J = 14.06 Hz, 1H), 1.68 (d, J = 14.06 Hz, 1H), 0.98 (s, 3H), 0.96 (s, 3H). Step B. To a solution of crude 7-bromo-3,3-dimethyl-1-vinyl1,2,3,4-tetrahydronaphthalen-1-ol (5.55g, 19.7 mmol) in CH3CN (50 mL) at 0 °C was added thionyl chloride (2.88 mL, 39.5 mmol). The reaction mixture was stirred at 0 °C for 10 min and treated with thiourea (3.0 g, 39.5 mmol) in one portion. The mixture was then allowed to stir at ambient temperature for 10 min and then at 50 °C for 1 h. The resulting mixture was allowed to stir at ambient temperature over the weekend. The solid formed was filtered and washed with additional CH3CN (3 × 5 mL) and dried to provide (E)2-(7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)ethyl carbamimidothioate hydrochloride (7.9 g, 90.5% yield) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 2.35 Hz, 1H), 7.37 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz, 1H), 7.09 (d, J = 8.22 Hz, 1H), 6.31 (t, J = 8.22 Hz, 1H), 4.10 (d, J = 7.83 Hz, 2H), 2.58 (s, 2H), 2.3 (s, 2H), 0.93 (s, 6H); LCMS (APCI+) m/z 400 (M + 1)+; analytical HPLC purity 91 area % (254 nM), retention time 3.276 min. Step C. A solution of (E)-2-(7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)ethyl carbamimidothioate hydrochloride (7.8 g, 21 mmol) in 2,2,2-trifluoroacetic acid (21 mL, 21 mmol) at 0 °C was treated dropwise with methanesulfonic acid (10 mL, 21 mmol). The resulting mixture was stirred at 0 °C for 30 min and then at ambient temperature overnight. The mixture was then cooled to 0 °C and slowly poured into ice cold saturated Na2CO3 solution (200 mL). The resulting slurry was stirred at ambient temperature for 30 min. The solid formed was filtered, washed with copious amount of water, then triturated with hot MeOH and filtered. The filtrate collected was concentrated in vacuo and dried to provide 7-bromo-3,3S

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

internal temperature below −74 °C. The resulting mixture was stirred at −78 °C for 1 h and treated dropwise with a solution of (E)-N-(7bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)-2-methylpropane-2-sulfinamide (4.15 g, 11.6 mmol) in THF (70 mL) at a rate such that the internal temperature did not excess −74 °C. Once the addition was complete, the mixture was stirred at −78 °C for 3 h. The mixture was then poured into a saturated NaHCO3 solution (100 mL) and extracted into EtOAc (3 × 50 mL). The organic layers were combined, dried (MgSO4), and concentrated in vacuo. The crude isolated was purified by flash chromatography on silica gel (Ready Sep 220 g), eluting with a step gradient of 20% EtOAc/hexane (to recover unreacted SM) and then with 30%EtOAc/hexane followed by 50% EtOAc/hexane to provide methyl 2-((1R)-7-bromo-1-(1,1-dimethylethylsulfinamido)-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)acetate as a white solid (2.28 g, 45% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.65 (d, J = 2.35 Hz, 1H), 7.36 (dd, J1 = 7.82 Hz, J2 = 1.96 Hz, 1H), 7.06 (d, J = 8.22 Hz, 1H), 5.36 (s, 1H), 3.60 (s, 3H), 3.05 (d, J = 15.65 Hz, 1H), 2.86 (d, J = 16.04 Hz, 1H), 2.54 (d, J = 15.65 Hz, 1H), 2.48−2.46 (m, 1H), 2.36-d, J = 14.87 Hz, 1H), 1.94 (d, J = 14.87 Hz, 1H), 1.12 (s, 9H), 0.98 (s, 3H), 0.92 (s, 3); FIMS (APCI +) m/z 429.8, 431.8; analytical HPLC purity 100 area %, retention time 4.473 min. Step C. A solution of methyl 2-(7-bromo-1-(1,1-dimethylethylsulfinamido)-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)acetate (2.27 g, 5.27 mmol) in dioxane (10 mL) was treated with 4 M HCl in dioxane (6.59 mL, 26.4 mmol). The resulting solution was stirred at room temperature for 2.5 h. The solvent was removed in vacuo, and the residue was evaporated from DCM. The residue obtained was dried under high vacuum for 30 min to provide methyl 2-(1-amino-7bromo-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)acetate (1.7 g, 5.21 mmol, 98.8% yield) as a pale yellow gum. 1H NMR (400 MHz, DMSO-d6) δ 7.75 (d, J = 1.96 Hz, 1H), 7.31 (dd, J1 = 1.96 Hz, J2 = 7.83 Hz, 1H), 7.02 (d, J = 8.22 Hz, 1H), 3.51 (s, 3H), 2.79 (d, J = 15.26 Hz, 1H), 2.71 (d, J = 14.87 Hz, 1H), 2.45−2.41 (m, 2H), 1.97 (d, J = 14.06 Hz, 1H), 1.63 (dd, J1 = 14.06 Hz, J2 = 1.57 Hz, 1H), 0.99 (s, 3H), 0.89 (s, 3H); LCMS (APCI+) m/z 325.8, 327.7 (M + 1). Step D. A suspension of methyl 2-(1-amino-7-bromo-3,3-dimethyl1,2,3,4-tetrahydronaphthalen-1-yl)acetate (500 mg, 1.53 mmol), EDCI (529 mg, 2.76 mmol), and methyl carbamothioylcarbamate (10) (437 mg, 2.30 mmol) in N,N-dimethylformamide (7663 μL, 1.53 mmol) was treated with N-ethyl-N-isopropylpropan-2-amine (1303 μL, 7.66 mmol) and stirred at ambient temperature for 18 h. The mixture was poured into water (50 mL) and extracted with EtOAc (3 × 40 mL). The organic layers were combined, dried (MgSO4), and concentrated in vacuo. The residue obtained was crystallized from MeOH to provide tert-butyl 7-bromo-1′,3,3-trimethyl-6′-oxo-3,4,5′,6′-tetrahydro1′H,2H-spiro[naphthalene-1,4′-pyrimidine]-2′-ylcarbamate (480 mg, 69.5% yield) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 9.962 (s, 1H), 7.91 (d, J = 1.96 Hz, 1H), 7.50 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz, 1H), 7.15 (d, J = 8.22 Hz, 1H), 3.48 (d, J = 15.65 Hz, 1H), 3.16 (s, 3H), 2.11 (dd, J1 = 15.65 Hz, J2 = 1.56 Hz, 1H), 1.92 (d, J = 14.08 Hz, 1H), 1.76 (d, J = 14.09 Hz, 1H), 1.36 (s, 2H), 0.99 (s, 3H), 0.87 (s, 3H); LCMS (APCI+) m/z 450, 451 (M + 1) with bromine isotope. Step E. tert-Butyl 7-bromo-1′,3,3-trimethyl-6′-oxo-3,4,5′,6′-tetrahydro-1′H,2H-spiro[naphthalene-1,4′-pyrimidine]-2′-ylcarbamate (63 mg, 0.14 mmol), 5-chloropyridin-3-ylboronic acid (26 mg, 0.17 mmol), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (5.8 mg, 0.0070 mmol), 20% aqueous Na2CO3 (259 μL, 0.49 mmol), and 1,4-dioxane (1399 μL, 0.14 mmol) were processed according to the method described for the synthesis of 7-(5-chloropyridin-3-yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine to provide deprotected 2′-amino-7-(5-chloropyridin-3-yl)-1′,3,3-trimethyl-3,4-dihydro1′H,2H-spiro[naphthalene-1,4′-pyrimidin]-6′(5′H)-one (42 mg, 0.11 mmol, 78% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.95 (d, J = 1.96 Hz, 1H), 8.64 (d, J = 2.34 Hz, 1H), 8.33 (t, J = 1.96 Hz, 1H), 8.16 (d, J = 1.96 Hz, 1H), 7.80 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz, 1H), 7.33 (d, J = 7.83 Hz, 1H), 3.77 (d, J = 16.04 Hz, 1H), 3.28 (s, 3H), 2.81 (d, J = 16.82 Hz, 1H), 2.67 (d, J = 16.04 Hz, 1H), 2.60 (d, J = 16.44 Hz, 1H), 2.04 (dd, J1 = 14.09 Hz, J2 = 1.57 Hz,

dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]thiazin]2′-amine (5.2 g, 74% yield) as a solid. 1H NMR (DMSO-d6) δ 9.99 (br s, 1H), 7.70 (s, 1H), 7.50 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz, 1H), 7.16 (d, J = 7.83 Hz, 1H), 3.39 (td, J1 = 12.91 Hz, J2 = 3.91 Hz, 1H), 3.25 (d,t, J1 = 12.91 Hz, J2 = 3.91 Hz, 1H), 2.55 (d, J = 5.87 Hz, 2H), 2.29−2.30 (m, 1H), 2.29−2.14 (m, 1H), 1.85 (s, 2H), 1.05 (s, 3H), 0.87 (s, 3H); LCMS (APCI+) m/z 340 (M + 1); analytical HPLC purity 96 area %, retention time 3.032 min. Step D. A resealable glass pressure tube was charged with 7-bromo3,3-dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine (40 mg, 0.12 mmol), 5-chloropyridin-3-ylboronic acid (22 mg, 0.14 mmol), Pd(dppf)·DCM complex (4.7 mg, 0.0059 mmol), 20% aqueous Na2CO3 (219 μL, 0.41 mmol), and 1,4-dioxane (1179 μL, 0.12 mmol). The mixture was sparged with N2 for 5 min, capped, and stirred at 90 °C for 15 h, and allowed to cool temperature. The mixture was diluted with EtOAc (10 mL) and washed with brine (2 × 5 mL). The organic layer was separated, dried (MgSO4), and concentrated in vacuo. The residue obtained was passed through a silica plug (2 g), eluting with 10% MeOH/DCM + 1% NH3. The semipure material obtained was purified by C-18 reverse phase HPLC (Gilson Unipoint), eluting with a gradient of 5−95% CH3CN/water containing 0.1% TFA to provide the TFA salt of 7-(5-chloropyridin-3yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine (8 mg, 0.022 mmol, 18% yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 11.66 (s, 1H), 6.30 (m, 2H), 7.92 (s, 1H), 7.44 (m, 2H), 7.28 (m, 1H), 3.37−3.30 (m, 1H), 3.13−3.01 (m, 1H), 2.37−2.30 (m, 1H), 2.23−2.14 (m, 3H), 2.12 (d, J = 13.69 Hz, 1H), 1.85 (d, J = 14.09 Hz, 1H), 1.17 (s, 3H), 1.01 (s, 3H); LCMS (APCI+) m/z 372 (M + 1), retention time 2.334 min. 2′-Amino-7-(5-chloropyridin-3-yl)-1′,3,3-trimethyl-3,4-dihydro-1′H,2H-spiro[naphthalene-1,4′-pyrimidin]-6′(5′H)-one (rac-60). Step A. To a solution of 7-bromo-3,3-dimethyl-3,4dihydronaphthalen-1(2H)-one (5 g, 19.8 mmol) and 2-methylpropane-2-sulfinamide (3.11 g, 25.7 mmol) in THF (65.8 mL, 19.8 mmol) was added freshly distilled Ti(OEt)4 (9.01 g, 39.5 mmol) in one portion. The resulting mixture was refluxed for 18 h. The mixture was cooled to ambient temperature and poured into saturated NaHCO3 (500 mL) solution. The resulting suspension was shaken with EtOAc (300 mL) and filtered through a pad of Celite 545. The solid particles in the filter funnel were crushed with a spatula and washed well with EtOAc (∼200 mL) until all the yellow color disappeared from the solids. The filtrate was transferred to a separatery funnel, and the layers were separated. The aqueous layer was extracted once with EtOAc (100 mL). The combined organic layers were washed with brine (1 × 100 mL), dried (MgSO4), filtered, and concentrated in vacuo. The residue obtained was purified by flash chromatography on silica gel (Ready Sep 120 g), eluting with 15% EtOAc (2 L) followed by 20% EtOAc/hexane (500 mL) to provide (E)-N-(7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)2-methylpropane-2-sulfinamide (4.2 g, 11.8 mmol, 59.7% yield) as a yellow solid. The unreacted SM 7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-one was also recovered from this reaction. 1H NMR (400 MHz, CDCl3-d) δ 8.22 (d, J = 1.96 Hz, 1H), 7.50 (dd, J1 = 8.88 Hz, J2 = 2.35 Hz, 1H), 7.04 (d, J = 8.22 Hz, 1H), 3.14 (d, J = 16.82 Hz, 1H), 2.87 (dd, J1 = 16.82 Hz, J2 = 0.783 Hz, 1H), 2.72 (d, J = 16.43 Hz, 1H), 2.61 (d, J = 16.43 Hz, 1H), 1.33 (s, 9H), 1.07 (s, 3H), 1.01 (s, 3H); LCMS (APCI+) m/z 356, 359 (M + 1); analytical HPLC purity 97 area %, retention time 4.691 min. Step B. A round-bottom flask equipped with a N2 inlet, rubber septum, and an internal temperature probe was charged with a solution of diisopropylamine (3.44 mL, 24.5 mmol) in THF (25 mL). The solution was cooled to −78 °C and treated dropwise with nbutyllithium 2.5 M in hexanes (9.78 mL, 24.5 mmol). Once the addition was complete, the cooling bath was replaced with an ice bath, and the mixture was allowed to stir at 0 °C for 40 min. Meanwhile a separate round-bottom flask equipped with a N2 inlet, rubber septum, and an internal temperature probe was charged with a solution of methyl acetate (2.04 mL, 25.6 mmol) in THF (20 mL). The mixture was cooled to −78 °C. Then the above-prepared LDA solution in THF was slowly added to this solution via a cannula, maintaining T

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Hz, 2H), 2.7 (d, 16 Hz, 1H), 2.57 (dd, J1 = 16 Hz, J2 = 1.2 Hz, 1H), 2.06 (d, J = 14 Hz, 1H), 1.86 (dd, J1 = 14 Hz, J2 = 1.6 Hz, 1H), 1.1 (s, 3H), 0.98 (s, 3H); LCMS (APCI+) m/z 326.1 (M + 1). (R)-2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one ((R)-34). The racemic compound 34 was separated by SFC chromatography using a Chrialpak AD-H (2 cm × 15 cm) 901061 column. The column was eluted with 15% ethanol (0.2% DEA)/CO2 at 100 mL/min (100 bar). This afforded complete separation of the single enantiomer (R)-34 (>99% ee). 1H NMR (400 MHz, CDCl3-d) δ 8.57 (d, J1 = 1.95 Hz, 1H), 8.48, (d, J1 = 2.34 Hz, 1H), 7.71 (t, J1 = 2.15 Hz, 1H), 7.37 (dd, J1 = 8.59 Hz, J2 = 2.34 Hz, 1H), 6.97 (d, J1 = 8.59 Hz, 1H), 6.94 (s, 1H), 3.21 (s, 3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.97 (d, J1 = 14.1 Hz, 1H), 1.53 (s, 3H), 1.43 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ 179.5 (C), 155.7 (C), 154.5 (C), 146.6 (CH), 145.7 (CH), 137.3 (C), 133.6 (CH), 132.0 (C), 129.2 (C), 128.8 (CH), 125.9 (CH), 121.4 (C), 119.6 (CH), 74.3 (C), 64.5 (C), 43.9 (CH2), 29.8−25.0 (CH3), 26.1 (CH3); LCMS (APCI+) m/z 371.2 (M + 1). (R)-3-(2-Amino-1,3′,3′-trimethyl-5-oxo-1,3′,4′,5-tetrahydro2′H-spiro[imidazole-4,1′-naphthalene]-7′-yl)benzonitrile ((R)45). The racemic compound 45 was separated by SFC chromatography using a Chiralcel OD-H (3 cm × 15 cm) 07-8754 column, eluting with 40% methanol (0.1% DEA)/CO2 at 100 bar at a flow rate of 80 mL/min (injection volume 2 mL, 53 mg/mL methanol). This afforded the single enantiomer (R)-45 (>99% ee). 1H NMR (400 MHz, CDCl3-d) δ 7.74 (d, J = 1.56 Hz, 1H), 7.69 (d, J = 7.83 Hz, 1H), 7.61−7.58 (m, 1H), 7.50 (t, J = 7.83 Hz, 1H), 7.39 (dd, J1 = 7.83 Hz, J2 = 2.25 Hz, 1H), 7.21 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H), 3.21 (s, 3H), 2.83 (d, J = 15.65 Hz, 1H), 2.61 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, J = 14.09 Hz, 1H), 1.81 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H0, 1.02 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ 178.5 (C), 156.0 (C), 141.8 (C), 137.8 (C), 137.4 (C), 134.5 (C), 131.5 (CH), 130.7 (CH), 130.7 (CH), 130.7 (CH), 129.6 (CH), 127.3 (CH), 125.3 (CH), 118.8 (C), 112.9 (C), 65.8 (C), 47.0 (CH2), 43.4 (CH2), 31.1−25.9 (CH3), 29.6 (C), 25.9 (CH3). LCMS (APCI+) m/z 359 (M + 1), retention time 2.263 min. (R)-2-Amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one ((R)46). The title compound was prepared according to the procedures of compound (R)-50, in which 2-fluoropyridin-3-ylboronic acid was used in place of pyrimidin-5-ylboronic acid in step B. 1H NMR (400 MHz, CDCl3-d) δ 8.16 (dd, J1 = 7.04 Hz, J2 = 1.56 Hz, 1H), 7.80−7.75 (m, 1H), 7.42 (dt, J1 = 7.83 Hz, J2 = 1.56 Hz,1H), 7.26−7.23 (m, 1H), 7.27 (d, J = 7.82 Hz, 1H), 7.10 (s, 1H), 3.20 (s, 3H), 2.82 (d, J = 15.65 Hz, 1H), 2.62 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09 Hz, 1H), 1.83 (dd, J1 = 14.09 Hz, J2 = 3.13 Hz, 1H), 1.17 (s, 3H), 1.03 (s, 3H). 13C NMR (500 MHz, CDCl3-d) δ 177.1 (C), 160 (C), 157.8 (C), 146.4 (CH), 140.5 (CH), 137.7 (C), 132.7 (C), 132.7 (C), 130.5 (CH), 129.3 (CH), 126.8 (CH), 123 (C), 121.8 (CH), 65.1 (C), 46.8 (CH2), 43.2 (CH2), 31.2−25.8 (CH3), 29.5 (C), 25.8 (CH3). LCMS (APCI+) m/z 353.2 (M + 1), retention time 2.099 min. (R)-2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one. ((R)-50). Step A. SFC separation of racemic 2-amino-7′-bromo-1,3′,3′trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)one (133 g, 397 mmol) was performed on a Lux Cellulose-4 (3 × 25 cm) column, eluting with 35% methanol (0.1%NH4OH)/CO2 at 100 bar at a flow rate of 200 mL/min (injection volume 2 mL, 309 mg/mL methanol). The peaks isolated were analyzed on a Lux Cellulose-4 (0.46 cm × 5 cm, 3 μm) column, eluting with 25% methanol (0.1% NH4OH)/CO2 at 120 bar (flow rate 5 mL/min, 210 nm). From this separation, (R)-2-amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′Hspiro[imidazole-4,1′-naphthalen]-5(1H)-one (peak 1, 46.55 g, chemical purity >99%, >99% ee) was isolated. Step B. (R)-2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′Hspiro[imidazole-4,1′-naphthalen]-5(1H)-one (1.00 g, 2.97 mmol), pyrimidin-5-ylboronic acid (0.479 g, 3.87 mmol), and Pd(PPh3)4 (0.0859 g, 0.0744 mmol) were combined with 15 mL of dioxane and 2 M Na2CO3 (3.72 mL, 7.44 mmol) (both degassed with nitrogen sparge for 30 min prior to use), and the reaction mixture was heated in

1H), 1.82 (d, J = 14.09 Hz, 1H), 1.03 (s, 3H), 0.94 (s, 3H); LCMS (APCI+) m/z 283 (M + 1). 7-(2-Fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-oxazol]-2′-amine (rac-62). Step A. NaH (94.8 mg, 3.95 mmol) was added to DMSO (6 mL) in a round-bottom flask. The mixture was heated to 75 °C and stirred for 30 min. The mixture was cooled to 0 °C, and methyltriphenylphosphonium bromide (1411 mg, 3.95 mmol) was added dropwise in warm DMSO (4 mL). After the mixture was stirred for 15 min, 7-bromo-3,3dimethyl-3,4-dihydronaphthalen-1(2H)-one (500 mg, 1.98 mmol) in DMSO (4 mL) was added dropwise. The mixture was allowed to warm to ambient temperature and stirred overnight. The mixture was diluted with ethyl acetate and water. The layers were separated, and the organics were washed with water and brine, dried over MgSO4, filtered, and concentrated. The material was purified on silica gel. Elution with hexanes to yield 7-bromo-3,3-dimethyl-1-methylene1,2,3,4-tetrahydronaphthalene (300 mg, 1.19 mmol, 60.5% yield). Step B. 7-Bromo-3,3-dimethyl-1-methylene-1,2,3,4-tetrahydronaphthalene (300 mg, 1.19 mmol) was diluted with diethyl ether (3 mL) followed by the addition of silver cyanate (537 mg, 3.58 mmol). The mixture was placed under nitrogen and cooled to 0 °C. I2 (303 mg, 1.19 mmol) was added, and the mixture was stirred for 1 h, warming to ambient temperature. The mixture was filtered through glass microfiber paper and concentrated. The residue was diluted with acetone (3 mL) and NH4OH (600 μL). The mixture was stirred overnight. The mixture was diluted with ethyl acetate and water. The layers were separated, and the organics were dried over MgSO4, filtered, and concentrated. The material was purified on reverse phase HPLC, eluting with 5−95% acetonitrile/water (0.1% TFA) monitoring at 220 nm to afford 7-bromo-3,3-dimethyl-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-oxazol]-2′-amine (70 mg, 0.226 mmol, 19% yield). Step C. 7-Bromo-3,3-dimethyl-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (25 mg, 0.081 mmol) and 2fluoropyridin-3-ylboronic acid (23 mg, 0.16 mmol) were diluted with dioxane (500 μL) followed by the addition of Pd(PPh3)4 (0.93 mg, 0.000 81 mmol) and Na2CO3 (121 μL, 0.24 mmol). The mixture was purged with argon, sealed, heated to 85 °C, and stirred for 12 h. The mixture was loaded onto silica gel and eluted with 1−10% methanol/ DCM (1% NH4OH) to yield 7-(2-fluoropyridin-3-yl)-3,3-dimethyl3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (25 mg, 0.077 mmol, 95% yield). 1H NMR (400 MHz, CDCl3-d) δ 8.17 (m, 1H), 7.85 (m, 1H), 7.44 (s, 1H), 7.35 (m, 1H), 7.23 (m, 1H), 7.11 (d, J = 7.8 Hz, 1H), 4.37 (s, 2H), 2.7 (d, J = 16 Hz, 1H), 2.56 (d, J = 16 Hz, 1H), 2.05 (d, J = 13.7 Hz, 1H), 1.84 (d, J = 13.7 Hz, 1H), 1.1 (s, 3H), 0.97 (s, 3H); LCMS (APCI+) m/z 326.1 (M + 1). 3,3-Dimethyl-7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (rac-63). rac-63 was synthesized in the same fashion as compound 62, replacing 2-fluoropyridin-3ylboronic acid with pyrimidin-5-ylboronic acid to afford 3,3-dimethyl7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (18 mg, 0.058 mmol, 72% yield). 1H NMR (400 MHz, CDCl3-d) δ 9.2 (s, 1H), 8.9 (s, 2H), 7.46 (d, J = 1.96 Hz, 1H), 7.37 (dd, J1 = 7.8 Hz, J2 = 1.96 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 2.7 (d, 16.4 Hz, 1H), 2.57 (dd, J1 = 16.4 Hz, J2 = 1.6 Hz, 1H), 2.06 (d, J = 13.7 Hz, 1H), 1.86 (dd, J1 = 13.7 Hz, J2 = 1.6 Hz, 1H), 1.1 (s, 3H), 0.98 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ 160.2 (C), 157.3 (CH), 154.8 (CH), 154.8 (CH), 142.5 (C), 136.8 (C), 134.3 (C), 132.8 (C), 130.2 (CH), 125.8 (CH), 125.7 (CH), 83.0 (CH2), 69.6 (C), 50.6 (CH2), 44.1 (CH2), 30.5 (C), 30.3−27.5 (CH3); LCMS (APCI+) m/z 309.1 (M + 1). 7-(5-Fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-oxazol]-2′-amine (rac-61). rac-61 was synthesized in the same fashion as compound 62, substituting 2fluoropyridin-3-ylboronic acid for (5-fluoropyridin-3-yl)boronic acid to afford 7-(5-fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (10 mg, 0.031 mmol, 38% yield). 1 H NMR (400 MHz, CDCl3-d) δ 8.65 (m, 1H), 8.42 (d, J = 2.7 Hz, 1H), 7.55 (m, 1H), 7.45 (d, J1 = 1.56 Hz, 1H), 7.35 (dd, J1 = 7.8 Hz, J2 = 1.9 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz, J2 = 4.3 U

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a 100 °C reaction block and stirred for 17 h. The reaction mixture was then concentrated, and the residue was combined with ethyl acetate and water. The mixture was extracted 2× with ethyl acetate, and the combined extracts were dried (Na2SO4), filtered, and concentrated. The crude was purified on silica gel (5−20% MeOH in dichloromethane gradient) to give (R)-2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)one (0.755 g, 2.25 mmol, 75.7% yield) as a white powder. 1H NMR (400 MHz, CDCl3-d) δ 9.16 (s, 1H), 8.85 (s, 2H), 7.42 (dd, J = −7.83 Hz, J2 = 2.35 Hz, 1H), 7.09 (d, J = 1.56 Hz, 1H), 3.22 (s, 3H), 2.86 (d, J = 15.65 Hz, 1H), 2.63 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, 14.08 Hz, 1H), 1.84 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.18 (s, 3H), 1.03 (s, 3H). 13C NMR (500 MHz, CDCl3-d) δ 178.0 (C), 157.5 (CH), 155.7 (C), 154.8 (CH), 154.8 (CH), 138.1 (C), 135.3 (C), 133.8 (C), 133.1 (C), 131.1 (CH), 126.9 (CH), 125.3 (CH), 66.0 (C), 46.8 (CH2), 43.4 (CH2), 31.1−26.0 (CH3), 29.6 (C), 26.0 (CH3); LCMS (APCI+) m/z 336.2 (M + 1)+, retention time 1.905 min (5 min run). (R)-3,3-Dimethyl-7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′Hspiro[naphthalene-1,4′-oxazol]-2′-amine ((R)-63). The racemic compound 63 was separated by SFC chromatography using a chiral column (Chiralpak AD-H, 2 cm × 15 cm), eluting with 35% methanol (20 nM NH3)/CO2, 100 bar, 60 mL/min and monitored at 220 nm to afford the title compound (>99% ee). 1H NMR (400 MHz, CDCl3-d) δ 9.2 (s, 1H), 8.9 (s, 2H), 7.46 (d, J = 1.96 Hz, 1H), 7.37 (dd, J1 = 7.8 Hz, J2 = 1.96 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 2.7 (d, 16.4 Hz, 1H), 2.57 (dd, J1 = 16.4 Hz, J2 = 1.6 Hz, 1H), 2.06 (d, J = 13.7 Hz, 1H), 1.86 (dd, J1 = 13.7 Hz, J2 = 1.6 Hz, 1H), 1.1 (s, 3H), 0.98 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ 160.2 (C), 157.3 (CH), 154.8 (CH), 154.8 (CH), 142.5 (C), 136.8 (C), 134.4 (C), 132.7 (C), 130.2 (CH), 125.8 (CH), 125.6 (CH), 83.0 (CH2), 69.5 (C), 50.6 (CH2), 44.0 (CH2), 30.5 (C), 30.3−27.4 (CH3); LCMS (APCI+) m/z 309.1 (M + 1).

■

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Andrew Allen (Array BioPharma) for multidimensional NMR spectroscopy support and chiral HPLC methods development. We thank Chris Hamman (Genentech) and Mengling Wong (Genentech) for chiral separation.

■

ABBREVIATIONS USED CatD, cathepsin D; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; LLC-PK1, Lilly Laboratories cell porcine kidney; HTRF, homogeneous time-resolved fluorescence; f u, fraction unbound; CL, clearance; CLu, unbound clearance; Cfree, unbound concentration; Pi (e.g., P3, P2′), amino acid residue from N to C terminus of the polypeptide substrate; Si (e.g., S3, S2′), binding subsite

■

ASSOCIATED CONTENT

Accession Codes

The PDB accession codes for the X-ray cocrystal structures of BACE1 + 1a, BACE1 + 12, BACE1 + 13, and BACE1 + (R)-50 are 4JOO, 4JP9, 4JPC, and 4JPE, respectively.

■

REFERENCES

(1) Alzheimer’s Association.. Alzheimer’s disease facts and figures. Alzheimer’s Dementia 2012, 8, 131−168. (2) (a) Comer, M. French lessons: leadership in Alzheimer’s disease. Alzheimer’s Dementia 2009, 5, 61−65. (b) The response of the United States is markedly more modest: U.S. Department of Health & Human Services. National Plan To Address Alzheimer’s Disease (2nd draft). http://aspe.hhs.gov/daltcp/napa/NatlPlan.shtml. (3) Jarvis, L. M. Tough times for neuroscience R&D. Chem. Eng. News 2012, 90, 22−25. (4) (a) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: the amyloid cascade hypothesis. Science 1992, 256, 184−185. (b) Karran, E.; Mercken, M. M.; Strooper, B. D. The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discovery 2011, 10, 698−712. (5) Lemere, C. A.; Blusztajn, J. K.; Yamaguchi, H.; Wisniewski, T.; Saido, T. C.; Selkoe, D. J. Sequence of deposition of heterogeneous amyloid beta-peptides and APOE in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol. Dis. 1996, 3, 16− 32. (6) Brouillette, J.; Caillierez, R.; Zommer, N.; Alves-Pires, C.; Benilova, I.; Blum, D.; Strooper, B.; Buée, L. Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloidβ1−42 oligomers are revealed in vivo by using a novel animal model. J. Neurosci. 2012, 32, 7852−7861. (7) Jonsson, T.; Atwal, J. K.; Steinberg, S.; Snaedal, J.; Jonsson, P. V.; Bjornsson, S.; Stefansson, H.; Sulem, P.; Gudbjartsson, D.; Maloney, J.; Hoyte, K.; Gustafson, A.; Liu, Y.; Lu, Y.; Bhangale, T.; Graham, R. R.; Huttenlocher, J.; Bjornsdottir, G.; Andreassen, O. A.; Jönsson, E. G.; Palotie, A.; Behrens, T. W.; Magnusson, O. T.; Kong, A.; Thorsteinsdottir, U.; Watts, R. J.; Stefansson, K. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 2012, 488, 96−99. (8) (a) Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.; Meek, T. D.; Chapman, C.; Gloger, I. S.; Murphy, K. E.; Southan, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D. L.; Walsh, F. S.; Dingwall, C.; Christie, G. Identification of a novel aspartic protease (Asp 2) as betasecretase. Mol. Cell. Neurosci. 1999, 14, 419−427. (b) Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.; Davis, D.; Doan, M.; Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-Croak, K.; Jewett, N.; Keim, P.; Knops, J.; Lieberburg, I.; Power, M.; Tan, H.; Tatsuno, G.; Tung, J.; Schenk, D.; Seubert, P.; Suomensaari, S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue, L.; Varghese, J. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 1999, 402, 537−540. (c) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers, G.; Citron,

AUTHOR INFORMATION

Corresponding Author

*Phone: 303-386-1153. Fax: 303-386-1130. E-mail: kevin. [email protected]. Present Addresses

§ For C.T.C.: 1200 Mercer Street No. 610, Seattle, WA 98109, United States. ∥ For A.A.C.: Clinical Sciences Building, Room 309, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29403, United States. ⊥ For M.K.G.D.: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1956, Houston, TX 77030, United States. # For D.D.: Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877, United States. ∞ For N.C.K.: Department of Chemistry, Regis University, 3333 Regis Boulevard, D4, Denver, CO 80221, United States. ¶ For J.P.R.: Peloton Therapeutics, 2330 Inwood Road, Suite 226, Dallas, TX 75235, United States. × For K.R.: ProPharma Services, 3195 E. Yarrow Circle, Superior, CO 80027, United States. ○ For D.S.: 7484 Old Post Rd., Boulder, CO 80301, United States. △ Agilent Technologies, Inc., 5555 Airport Blvd. Suite 100, Boulder, CO 80301, United States.

V

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

M. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the trans-membrane aspartic protease BACE. Science 1999, 286, 735−741. (d) Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.; Pauley, A. M.; Brashier, J. R.; Stratman, N. C.; Mathews, W. R.; Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.; Heinrikson, R. L.; Gurney, M. E. Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature 1999, 402, 533−537. (e) Lin, X.; Koelsch, G.; Wu, S.; Downs, D.; Dashti, A.; Tang, J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1456−1460. (9) De Strooper, B.; Vassar, R.; Golde, T. The secretases: enzymes with therapeutic potential in Alzheimer’s disease. Nat. Rev. Neurol. 2010, 6, 99−107. (10) (a) Cai, H.; Wang, Y.; McCarthy, D.; Wen, H.; Borchelt, D. R.; Price, D. L.; Wong, P. C. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat. Neurosci. 2001, 4, 233− 234. (b) Luo, Y.; Bolon, B.; Kahn, S.; Bennett, B. D.; Babu-Khan, S.; Denis, P.; Fan, W.; Kha, H.; Zhang, J.; Gong, Y.; Martin, L.; Louis, J. C.; Yan, Q.; Richards, W. G.; Citron, M.; Vassar, R. Mice deficient in BACE1, the Alzheimer’s beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat. Neurosci. 2001, 4, 231−232. (c) Roberds, S. L.; Anderson, J.; Basi, G.; Bienkowski, M. J.; Branstetter, D. G.; Chen, K. S.; Freedman, S. B.; Frigon, N. L.; Games, D.; Hu, K.; Johnson-Wood, K.; Kappenman, K. E.; Kawabe, T. T.; Kola, I.; Kuehn, R.; Lee, M.; Liu, W.; Motter, R.; Nichols, N. F.; Power, M.; Robertson, D. W.; Schenk, D.; Schoor, M.; Shopp, G. M.; Shuck, M. E.; Sinha, S.; Svensson, K. A.; Tatsuno, G.; Tintrup, H.; Wijsman, J.; Wright, S.; McConlogue, L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer’s disease therapeutics. Hum. Mol. Genet. 2001, 10, 1317−1324. (11) (a) Ghosh, A. K.; Brindisis, M.; Tang, T. Developing β-secretase inhibitors for treatment of Alzheimer’s disease. J. Neurochem. 2012, 120 (Suppl. 1), 71−83. (b) Stachel, S. J. Progress toward the development of a viable BACE-1 inhibitor. Drug. Dev. Res. 2009, 70, 101−110. (c) Silvestri, R. Boom in the development of non-peptidic beta-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease. Med. Res. Rev. 2009, 29, 295−338. (d) Ghosh, A. K.; Venkateswara Rao, K.; Yadav, N. D.; Anderson, D. D.; Gavande, N.; Huang, X.; Terzyan, S.; Tang, J. Structure-based design of highly selective β-secretase inhibitors: synthesis, biological evaluation, and protein−ligand X-ray crystal structure. J. Med. Chem. 2012, 55, 9195− 9207. (e) Kaller, M. R.; Harried, S. S.; Albrecht, B.; Amarante, P.; Babu-Khan, S.; Bartberger, M. D.; Brown, J.; Brown, R.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.; Graceffa, R.; Hickman, C.; Judd, T.; Kriemen, C.; La, D.; Li, V.; Lopez, P.; Luo, Y.; Masse, C.; Monenschein, H.; Nguyen, T.; Pennington, L. D.; San Miguel, T.; Sickmier, E. A.; Wahl, R. C.; Weiss, M. M.; Wen, P. H.; Williamson, T.; Wood, S.; Xue, M.; Yang, B.; Zhang, J.; Patel, V.; Zhong, W.; Hitchcock, S. A potent and orally efficacious, hydroxyethylamine-based inhibitor of β-secretase. ACS Med. Chem. Lett. 2012, 3, 886−891. (f) Brodney, M. A.; Barreiro, G.; Ogilvie, K.; Hajos-Korcsok, E.; Murray, J.; Vajdos, F.; Ambroise, C.; Christoffersen, C.; Fisher, K.; Lanyon, L.; Liu, J.; Nolan, C. E.; Withka, J. M.; Borzilleri, K. A.; Efremov, I.; Oborski, C. E.; Varghese, A.; O’Neill, B. T. Spirocyclic sulfamides as β-secretase 1 (BACE-1) inhibitors for the treatment of Alzheimer’s disease: utilization of structure based drug design, WaterMap, and CNS penetration studies to identify centrally efficacious inhibitors. J. Med. Chem. 2012, 55, 9224−9239. (12) May, P. C.; Dean, R. A.; Lowe, S. L.; Martenyi, F.; Sheehan, S. M.; Boggs, L. N.; Monk, S. A.; Mathes, B. M.; Mergott, D. J.; Watson, B. M.; Stout, S. L.; Timm, D. E.; Smith Labell, E.; Gonzales, C. R.; Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.; Calligaro, D. O.; Cocke, P. J.; Greg Hall, D.; Friedrich, S.; Citron, M.; Audia, J. E. Robust central reduction of amyloid-β in humans with an orally available, non-peptidic β-secretase inhibitor. J. Neurosci. 2011, 31, 16507−16516.

(13) (a) Zhang, D.; Brankov, M.; Makhija, M. T.; Robertson, T.; Helmerhorst, E.; Papadimitriou, J. M.; Rakoczy, P. E. Correlation between inactive cathepsin D expression and retinal changes in mcd2/ mcd2 transgenic mice. Invest. Ophthalmol. Visual Sci. 2005, 46, 3031− 3038. (b) Koike, M.; Nakanishi, H.; Saftig, P.; Ezaki, J.; Isahara, K.; Ohsawa, Y.; Schulz-Schaeffer, W.; Watanabe, T.; Waguri, S.; Kametaka, S.; Shibata, M.; Yamamoto, K.; Kominami, E.; Peters, C.; von Figura, K.; Uchiyama, Y. Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons. J. Neurosci. 2000, 15, 6898−6906. (c) Saftig, P.; Hetman, M.; Schmahl, W.; Weber, K.; Heine, L.; Mossmann, H.; Köster, A.; Hess, B.; Evers, M.; von Figura, K.; Peters, C. Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells. EMBO J. 1995, 14, 3599−3608. (14) Fischer, H.; Gottschlich, R.; Seelig, A. Blood−brain barrier permeation: molecular parameters governing passive diffusion. J. Membr. Biol. 1998, 165, 201−211. (15) Properties estimated with ChemDraw Ultra, version 12.0. (16) (a) Hitchcock, S. A.; Pennington, L. D. Structure−brain exposure relationships. J. Med. Chem. 2006, 49, 7559−7583. (b) Pajouhesh, H.; Lenz, G. R. Medicinal chemical properties of successful central nervous system drugs. NeuroRx 2005, 2, 541−553. (c) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615−2623. (17) (a) Edwards, P. D.; Albert, J. S.; Sylvester, M.; Aharony, D.; Andisik, D.; Callaghan, O.; Campbell, J. B.; Carr, R. A.; Chessari, G.; Congreve, M.; Frederickson, M.; Folmer, R. H.; Geschwindner, S.; Koether, G.; Kolmodin, K.; Krumrine, J.; Mauger, R. C.; Murray, C. W.; Olsson, L. L.; Patel, S.; Spear, N.; Tian, G. Application of fragment-based lead generation to the discovery of novel, cyclic amidine β-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency. J. Med. Chem. 2007, 50, 5912− 5925. (b) Malamas, M. S.; Erdei, J.; Gunawan, I.; Barnes, K.; Johnson, M.; Hui, Y.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.; Olland, A.; Bard, J.; Robichaud, A. J. Aminoimidazoles as potent and selective human βsecretase (BACE1) inhibitors. J. Med. Chem. 2009, 52, 6314−6323. (c) Swahn, B.-M.; Holenz, J.; Kihlström, J.; Kolmodin, K.; Lindström, J.; Plobeck, N.; Rotticci, D.; Sehgelmeble, F.; Sundström, M.; von Berg, S.; Fälting, J.; Georgievska, B.; Gustavsson, S.; Neelissen, J.; Ek, M.; Olsson, L.-L.; Berg, S. Aminoimidazoles as BACE-1 inhibitors: the challenge to achieve in vivo brain efficacy. Bioorg. Med. Chem. Lett. 2012, 22, 1854−1859. (d) Huang, H.; La, D. S.; Cheng, A. C.; Whittington, D. A.; Patel, V. F.; Chen, K.; Dineen, T. A.; Epstein, O.; Graceffa, R.; Hickman, D.; Kiang, Y.-H.; Louie, S.; Luo, Y.; Wahl, R. C.; Wen, P. H.; Wood, S.; Fremeau, R. T., Jr. Structure- and propertybased design of aminooxazoline xanthenes as selective, orally efficacious, and CNS penetrable BACE inhibitors for the treatment of Alzheimer’s disease. J. Med. Chem. 2012, 55, 9156−9169. (e) Cheng, Y.; Judd, T. C.; Bartberger, M. D.; Brown, J.; Chen, K.; Fremeau, R. T., Jr.; Hickman, D.; Hitchcock, S. A.; Jordan, B.; Li, V.; Lopez, P.; Louie, S. W.; Luo, Y.; Michelsen, K.; Nixey, T.; Powers, T. S.; Rattan, C.; Sickmier, E. A.; St. Jean, D. J., Jr.; Wahl, R. C.; Wen, P. H.; Wood, S. From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). J. Med. Chem. 2011, 54, 5836−5857. (f) Gravenfors, Y.; Viklund, J.; Blid, J.; Ginman, T.; Karlström, S.; Kihlström, J.; Kolmodin, K.; Lindström, J.; von Berg, S.; von Kieseritzky, F.; Slivo, C.; Swahn, B.-M.; Olsson, L.-L.; Johansson, P.; Eketjäll, S.; Fälting, J.; Jeppsson, F.; Strömberg, K.; Janson, J.; Rahm, F. New aminoimidazoles as β-secretase (BACE-1) inhibitors showing amyloid-β (aβ) lowering in brain. J. Med. Chem. 2012, 55, 9297−9311. (g) Mandal, M.; Zhu, Z.; Cumming, J. N.; Liu, X.; Strickland, C.; Mazzola, R. D.; Caldwell, J. P.; Leach, P.; Grzelak, M.; Hyde, L.; Zhang, Q.; Terracina, G.; Zhang, L.; Chen, X.; Kuvelkar, R.; Kennedy, M. E.; Favreau, L.; Cox, K.; Orth, P.; Buevich, A.; Voigt, J.; Wang, H.; Kazakevich, I.; McKittrick, B. A.; Greenlee, W.; Parker, E. M.; Stamford, A. W. Design and validation of bicyclic iminopyrimidinones as beta amyloid cleaving enzyme-1 (BACE1) inhibitors: W

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

conformational constraint to favor a bioactive conformation. J. Med. Chem. 2012, 55, 9331−9345. (h) Swahn, B.-M.; Kolmodin, K.; Karlström, S.; von Berg, S.; Söderman, P.; Holenz, J.; Berg, S.; Lindström, J.; Sundström, M.; Turek, D.; Kihlström, J.; Slivo, C.; Andersson, L.; Pyring, D.; Rotticci, D.; Ohberg, L.; Kers, A.; Bogar, K.; von Kieseritzky, F.; Bergh, M.; Olsson, L.-L.; Janson, J.; Eketjäll, S.; Georgievska, B.; Jeppsson, F.; Fälting, J. Design and synthesis of β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of β-amyloid peptides. J. Med. Chem. 2012, 55, 9346−9361. (i) Cumming, J. N.; Smith, E. M.; Wang, L.; Misiaszek, J.; Durkin, J.; Pan, J.; Iserloh, U.; Wu, Y.; Zhu, Z.; Strickland, C.; Voigt, J.; Chen, X.; Kennedy, M. E.; Kuvelkar, R.; Hyde, L. A.; Cox, K.; Favreau, L.; Czarniecki, M. F.; Greenlee, W. J.; McKittrick, B. A.; Parker, E. M.; Stamford, A. W. Structure based design of iminohydantoin BACE1 inhibitors: identification of an orally available, centrally active BACE1 inhibitor. Bioorg. Med. Chem. Lett. 2012, 22, 2444−2449. (j) Malamas, M. S.; Erdei, J.; Gunawan, I.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.; Chopra, R.; Olland, A.; Bard, J.; Jacobsen, S.; Magolda, R. L.; Pangalos, M.; Robichaud, A. J. Design and synthesis of 5,5′-disubstituted aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors. J. Med. Chem. 2010, 53, 1146−1158. (18) (a) Ishikawa, M.; Hashimoto, Y. Improvement in aqueous solubility in small molecule drug discovery programs by disruption of molecular planarity and symmetry. J. Med. Chem. 2011, 54, 1539− 1554. (b) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland: increasing saturation as an approach to improving clinical success. J. Med. Chem. 2009, 52, 6752−6756. (19) In a recent patent review, spirocyclic molecules from a Vitae patent application were disclosed: Probst, G.; Xu, Y. Z. Small-molecule BACE1 inhibitors: a patent literature review (2006−2011). Expert. Opin. Ther. Pat. 2012, 22, 511−540. (20) (a) Buckle, D. R.; Arch, J. R.; Fenwick, A. E.; Houge-Frydrych, C. S.; Pinto, I. L.; Smith, D. G.; Taylor, S. G.; Tedder, J. M. Relaxant activity of 4-amido-3,4-dihydro-2H-1-benzopyran-3-ols and 4-amido2H-1-benzopyrans on guinea pig isolated trachealis. J. Med. Chem. 1990, 33, 3028−3034. (b) Vuligonda, V.; Standeven, A. M.; Escobar, M.; Chandraratna, R. A. A new class of potent RAR antagonists: dihydroanthracenyl, benzochromenyl and benzothiochromenyl retinoids. Bioorg. Med. Chem. Lett. 1999, 9, 743−748. (c) Ashwood, V. A.; Cassidy, F.; Evans, J. M.; Gagliardi, S.; Stemp, G. Synthesis and antihypertensive activity of pyran oxygen and amide nitrogen replacement analogues of the potassium channel activator cromakalim. J. Med. Chem. 1991, 34, 3261−3267. (21) Wittekind, R. R.; Rosenau, J. D.; Poos, G. I. Ring cleavage reactions of trans-2-amino-3a,4,5,6,7,7a-hexahydrobenzoxazole. J. Org. Chem. 1960, 26, 444−446. (22) Cohen, N.; Banner, B. L. Synthesis of 2-amino-5,6-didydro-4H1,3-thiazines and related compounds by acid catalyzed cyclization of allylic isothiuronium salts. J. Heterocycl. Chem 1977, 14, 717−23. (23) Tang, T. P.; Ellman, J. A. Asymmetric synthesis of α-amino acid derivatives incorporating a broad range of substitution patterns by enolate additions to tert-butanesulfinyl imines. J. Org. Chem. 2002, 67, 7819−7832. (24) The preference for phenyl meta substitution and the 3-pyridyl interaction with the Ser229 bridging water molecule parallels the findings of biaryl and nonspirocyclic cores. See ref 17. (25) We found several (but not all) literature compounds to be much more potent against CatD in our assay than reported. Furthermore, outsourced CatD assays were found to be unreliable, as analogue (R)34 (Table 5, 1.7 μM vs CatD) was found to be inactive at 30 μM at two contract research organizations. (26) Fernandes, J.; Gattass, C. R. Topological polar surface area defines substrate transport by multidrug resistance associated protein 1 (MRP1/ABCC1). J. Med. Chem. 2009, 52, 1214−1218. (27) Stamford, A. W.; Scott, J. D.; Li, S. W.; Babu, S.; Tadesse, D.; Hunter, R.; Wu, Y.; Misiaszek, J.; Cumming, J. N.; Gilbert, E. J.; Huang, C.; McKittrick, B. A.; Hong, L.; Guo, T.; Zhu, Z.; Strickland, C.; Orth, P.; Voigt, J. H.; Kennedy, M. E.; Chen, X.; Kuvelkar, R.; Hodgson, R.; Hyde, L. A.; Cox, K.; Favreau, L.; Parker, E. M.;

Greenlee, W. J. Discovery of an orally available, brain penetrant BACE1 inhibitor that affords robust CNS Aβ reduction. ACS Med. Chem. Lett. 2012, 3, 897−902. (28) The pKa of the 2-aminoheterocycles may play a role: (a) Rajapakse, H. A.; Nantermet, P. G.; Selnick, H. G.; Barrow, J. C.; McGaughey, G. B.; Munshi, S.; Lindsley, S. R.; Young, M. B.; Ngo, P. L.; Holloway, M. K.; Lai, M. T.; Espeseth, A. S.; Shi, X. P.; Colussi, D.; Pietrak, B.; Crouthamel, M. C.; Tugusheva, K.; Huang, Q.; Xu, M.; Simon, A. J.; Kuo, L.; Hazuda, D. J.; Graham, S.; Vacca, J. P. SAR of tertiary carbinamine derived BACE1 inhibitors: role of aspartate ligand amine pKa in enzyme inhibition. Bioorg. Med. Chem. Lett. 2010, 20, 1885−1889. (b) Falgueyret, J. P.; Desmarais, S.; Oballa, R.; Black, W. C.; Cromlish, W.; Khougaz, K.; Lamontagne, S.; Massé, F.; Riendeau, D.; Toulmond, S.; Percival, M. D. Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against offtarget cathepsins and reduced functional selectivity. J. Med. Chem. 2005, 48, 7535−7543. (29) Compounds with medium to high permeability (Papp > 4) demonstrated acceptable pharmacokinetics. Compounds with low permeability (Papp < 4) were not generally evaluated in pharmacokinetic studies. (30) Preliminary experiments indicate a role for aldehyde oxidase in the CL of pyridine and pyrimidine moieties. (31) Beck, M.; Bigl, V.; Rossner, S. Guinea pigs as a nontransgenic model for APP processing in vitro and in vivo. Neurochem. Res. 2003, 28, 637−644. (32) (a) Yamakawa, H.; Yagishita, S.; Futai, E.; Ishiura, S. β-Secretase inhibitor potency is decreased by aberrant β-cleavage location of the “Swedish mutant” amyloid precursor protein. J. Biol. Chem. 2010, 285, 1634−1642. (b) Haass, C.; Lemere, C. A.; Capell, A.; Citron, M.; Seubert, P.; Schenk, D.; Lannfelt, L.; Selkoe, D. J. The Swedish mutation causes early-onset Alzheimer’s disease by β-secretase cleavage within the secretory pathway. Nat. Med. 1995, 1, 1291−1296. (33) The γ-secretase inhibitor semagacestat was utilized for assay validation, providing 58% CSF Aβ reduction at 3 h with a 60 mg/kg oral dose in guinea pig. (34) (a) Schechter, I.; Ziv, E. Cathepsins S, B and L with aminopeptidases display β-secretase activity associated with the pathogenesis of Alzheimer’s disease. Biol. Chem. 2011, 392, 555−69. (b) Hook, V.; Funkelstein, L.; Wegrzyn, J.; Bark, S.; Kindy, M.; Hook, G. Cysteine Cathepsins in the secretory vesicle produce active peptides: cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer’s disease. Biochim. Biophys. Acta 2012, 1824, 89−104. (35) Lin, J. H. CSF as a surrogate for assessing CNS exposure: an industrial perspective. Curr. Drug Metab. 2008, 9, 46−59. (36) Maurer, T. S.; Debartolo, D. B.; Tess, D,A.; Scott, D. O. Relationship between exposure and nonspecific binding of thirty-three central nervous system drugs in mice. Drug. Metab. Dispos. 2005, 33, 175−181. (37) Sarasa, M.; Pedro, P. Natural non-transgenic animal models for research in Alzheimer’s disease. Curr. Alzheimer Res. 2009, 6, 171−178. (38) The γ-secretase inhibitor semagacestat was utilized for assay validation, providing 40% CSF Aβ reduction at 3 h with a 60 mg/kg oral dose in rat. (39) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in central nervous system drug discovery. J. Med. Chem. 2013, 56, 2−12. (40) Sankaranarayanan, S.; Holahan, M. A.; Colussi, D.; Crouthamel, M. C.; Devanarayan, V.; Ellis, J.; Espeseth, A.; Gates, A. T.; Graham, S. L.; Gregro, A. R.; Hazuda, D.; Hochman, J. H.; Holloway, K.; Jin, L.; Kahana, J.; Lai, M. T.; Lineberger, J.; McGaughey, G.; Moore, K. P.; Nantermet, P.; Pietrak, B.; Price, E. A.; Rajapakse, H.; Stauffer, S.; Steinbeiser, M. A.; Seabrook, G.; Selnick, H. G.; Shi, X. P.; Stanton, M. G.; Swestock, J.; Tugusheva, K.; Tyler, K. X.; Vacca, J. P.; Wong, J.; Wu, G.; Xu, M.; Cook, J. J.; Simon, A. J. First demonstration of cerebrospinal fluid and plasma Aβ lowering with oral administration of a β-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates. J. Pharmacol. Exp. Ther. 2009, 328, 131−140. X

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(41) Tomasselli, A. G.; Paddock, D. J.; Emmons, T. L.; Mildner, A. M.; Leone, J. W.; Lull, J. M.; Cialdella, J. I.; Prince, D. B.; Fischer, H. D.; Heinrikson, R. L.; Benson, T. E. High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms. Protein Pept. Lett. 2008, 15, 131− 143. (42) Leslie, A. G. W.; Powell, H. R. Processing Diffraction Data with Mosfim. In Evolving Methods for Macromolecular Crystallography; Read, R. J., Sussman, J. L., Eds.; NATO Science Series II: Mathematics, Physics, and Chemistry 245; Springer: New York, NY, 2007; pp 41−51. (43) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, D67, 235−242. (44) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132.

Y

dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX