Toward Î²-Secretase-1 Inhibitors with Improved Isoform Selectivity

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Toward #-Secretase-1 Inhibitors with Improved Isoform Selectivity Patrik Johansson, Karin Kaspersson, Ian K Gurrell, Elisabeth Back, Susanna Eketjäll, Clay W Scott, Gvido Cebers, Philip Thorne, Michael J. McKenzie, Haydn Beaton, Paul Davey, Karin Kolmodin, Jorg Holenz, Mark E. Duggan, Samantha Budd Haeberlein, and Roland Werner Burli J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01716 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Journal of Medicinal Chemistry

Toward β-Secretase-1 Inhibitors with Improved Isoform Selectivity Patrik Johansson,1 Karin Kaspersson,1 Ian K. Gurrell,2 Elisabeth Bäck,1 Susanna Eketjäll,3 Clay W. Scott,4 Gvido Cebers,2,§ Philip Thorne,5 Michael J. McKenzie,5 Haydn Beaton,5 Paul Davey,6 Karin Kolmodin,7 Jörg Holenz,2,† Mark E. Duggan,2 Samantha Budd Haeberlein,2,‡ Roland W. Bürli2,* 1

Discovery Sciences, IMED Biotech Unit, AstraZeneca, S-43183 Mölndal, Sweden

2

Neuroscience, IMED Biotech Unit, AstraZeneca, Cambridge, CB21 6GH, UK

3

Cardiovascular and Metabolic Diseases, IMED Biotech Unit, AstraZeneca, 141 57 Huddinge,

Sweden 4

Discovery Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Waltham

MA 02451, USA 5

Charnwood Molecular, Loughborough, LE11 5DA, UK

6

Oncology Chemistry, IMED Biotech Unit, AstraZeneca, Cambridge, CB4 0WG, UK

7

Sprint Bioscience, 141 57 Huddinge, Sweden

KEYWORDS Alzheimer’s disease, β-secretase (BACE), structure-selectivity relationship, PMEL/Mβ processing, pigmentation.

ACS Paragon Plus Environment

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ABSTRACT

BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aβ production and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Co-crystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methyl-cyclohexyl element resulted in lead molecule 28, which exhibited ~50-fold selectivity. Compared to a non-selective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.

INTRODUCTION Cerebral formation of amyloid β peptides (Aβ) of 38 to 43 amino acid length and subsequent aggregation/plaque formation have long been established as a hallmark of Alzheimer’s disease (AD).1,2 These toxic Aβ peptides result from processing the amyloid precursor protein (APP) by proteases; specifically, cleavage of the APP protein by the β-site APP cleaving enzyme 1, commonly known as β-secretase 1 or BACE1, constitutes the first step in APP degradation leading to the membrane-bound intermediate called C99 and a soluble fragment (sAPPβ).3 Further fragmentation of C99 by ɣ-secretase then liberates the toxic Aβ peptides.4,5 This cascade


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of events leading to Aβ fragments, combined with incomplete Aβ removal, eventually leads to plaque deposits and ultimately degeneration of neuronal structure and function. Notably, other forms of secretases have been implicated in APP processing and the spatial and temporal interplay of activities of these individual proteases in different brain tissues at various time points is clearly complex and not fully understood.6,7 Supporting target validation for BACE1 is the fact that familial Alzheimer’s disease mutations like the Swedish mutations K670N, M671L and A673V, respectively, which are located proximal to the BACE1 cleavage site of APP, increase processing, thus leading to elevated Aβ production.8,9 In contrast, the A673T mutation impairs BACE1-mediated APP cleavage and appears to have a protective effect.10 Taken together, these genetic and mechanistic data form a strong hypothesis for BACE1 inhibition as a diseasemodifying therapy for patients who are at risk of developing AD. Thus, extensive research has been conducted since the unraveling of the role of the BACE1 protease in APP processing which has led to inhibitors with remarkable potency and brain permeability, several of which are subject to ongoing clinical efficacy studies.11,12 While the role of BACE1 has been extensively investigated and a number of substrates with biologically relevant functions at various developmental stages have been reported, less is known about the second isoform, β-secretase 2 (BACE2).13 However, there are several studies indicating that BACE2 adopts an important role in melanogenesis. In mice, it has been demonstrated that BACE2-mediated cleavage of the pigment cell-specific melanocyte protein (PMEL) is required to form functional (non-pathogenic) amyloid fibrils during melanogenesis.14 Consistent with this finding, BACE2 is highly expressed in pigment cells and genetic ablation of this enzyme leads to coat color defects in BACE2-/-, which has not been observed in BACE1-/- mice. It has recently been confirmed that wild type as well as BACE2+/- and BACE2-/- mice upon treatment with a


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non-selective BACE1/2 inhibitor display a reversible, dose-dependent appearance of depigmented hair which may indicate that in the absence of BACE2, BACE1 can partially compensate for its function in melanogenesis.15,16 Consistent with these data, a distinct phenotype has been documented for migration of zebra fish melanocytes carrying mutations in BACE2, but not in BACE1.17 Notably, other substrates for BACE2 have been discovered which may be affected by a non-selective BACE inhibitor.18 The majority of BACE inhibitors documented to date do not address subtype selectivity.11,12 While it is currently not fully understood if or to which extent the observations in BACE2impaired mice and zebra fish might translate to humans who are treated with a non-selective βsecretase inhibitor, it is encouraging that no changes in melanisation levels were observed in skin biopsies of human subjects following 12 days of daily lanabecestat (AZD3293, Figure 1) treatment at doses of up to 150 mg per day.15 Figure 1 We set out to investigate the feasibility of developing a BACE2-sparing inhibitor as it has yet to be shown in patients that BACE2 inhibition over a prolonged period will not result in a peripheral effect. As part of this strategy, selective inhibition of BACE1 needs to be combined with other properties such as good cell-based potency, blood-brain barrier (BBB) permeability, low metabolic clearance and minimal risk for off target activities.

RESULTS AND DISCUSSION


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To gain insights on which structural elements of non-peptido-mimetic BACE inhibitors may influence isoform selectivity, we analyzed biochemical inhibitory data of a total of 257 compounds for which we had activities for both human BACE isoforms. These inhibitors represent a diverse range of structures analogous to AZD3839 (Figure 1)19 with substitution patterns leading to variable selectivity characteristics. The data used for this comparison were based on testing the inhibitory effect of compounds on the human BACE1 or BACE2 forms. The same fluorescently labeled peptide substrate designed to span across the BACE cleavage site of APP was used in both protease assays. A large number of compounds displayed minimal desired selectivity or even a slight preference for BACE2, while about 55 inhibitors showed a greater than 10-fold preference for inhibiting the BACE1 isoform. Encouraged by this finding, we analyzed the structure-selectivity relationship and the active site pockets of the two proteases. BACE1 features a wide binding site crevice containing two catalytic residues D32 and D228. The pocket is lined by a long flexible β hairpin flap that shields the active side chains. Residue Y71 of the flap has been shown to play a critical role in the conformation of the hairpin both for substrate and inhibitor binding. In the BACE1 apo structure (1W50)20 as well as in many structures containing ligands, the hydroxy group of Y71 interacts with the indole nitrogen of W76, closing the flap and acting as a gate to the S2’ pocket.21 However, most larger ligands expand into the S2’ pocket, rotating the tyrosine side chain towards the tip of the hairpin.22 A second loop located on the other side of the wide substrate binding cleft, near S10, forms a deep water-filled crevice termed the S3 subpocket. The S10 loop has been shown to exhibit two discrete ligand-induced conformations connected to the complicated water network of the pocket. Compounds that do not disturb this hydrogen bond network retain the S3 subpocket in its open conformation (vide infra Figure 2) while even a


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minimal displacement of the top S3 water molecule causes the pocket to collapse. This conformational change is typically associated with a substantial potency gain, possibly due to the release of four well-coordinated H2O molecules into bulk solvent.23 Proximal to the two catalytic aspartate residues, the active sites of the BACE1 and BACE2 isoforms are structurally highly homologous. However, several differences exist within the second layer and towards the edge of the relatively large pockets. The two β-secretases exhibit four substitutions in between G66 and W76 of the flap hairpin of BACE1 (between D82 and W92 in BACE2), whereby the residues P70 and K75 located above the catalytic dyad are particularly important. Just beneath the flap, R128 is replaced by K144 in BACE2, decorating one of the sides of the S2’ pocket.24 The neighboring loop connecting β6 and β7 contains several substitutions including K107/N123, I110/L126, N111/P127 and S113/I129, shaping the rim above the S1 pocket differently in BACE1 compared to BACE2. However, despite of amino acid changes like Q12/R28, L152/M168 and L154/M170 proximal to the S10 loop, the BACE2 S3 subpocket features a complex water network which is very similar to that of BACE1. Of the above tested 257 inhibitors, we had crystal structures of 47 inhibitors bound to BACE1 available to probe for differences that could be correlated with the observed selectivity. To analyze these inhibitors, we categorized them into three groups according to their selectivity profile: red (10 fold). This comparison highlighted two distinct structural features influencing selectivity (Figure 2): compounds bearing a substituent that reaches into the S3 sub-pocket tend to display no or little selectivity whereas molecules that present an H-bond acceptor interacting with the top S3 water molecule show a trend for improved BACE1 selectivity. This observation might be attributed to the difference in property of the top S3 water molecules when bound to the two isoforms, combined with the large gain in


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desolvation energy when the C-ring substituent enters the S3 subpocket, releasing the signature H2O molecules. Figure 2 We also noted that many inhibitors with preference for inhibiting BACE1 tend to induce a conformational change to the flap loop and push it outward. In many of the co-structures of nonselective inhibitors bound to BACE1, Y71 is within hydrogen bonding distance of the backbone carbonyl of K107 in the loop between β6 and β7. Strikingly, the most selective compounds push the flap element outward by more than 2Å, shifting the Y71 side chain towards the backbone carbonyl of D106. In BACE2, D106 is replaced by E122 and K107 by N123, respectively. This change, combined with the G66/D82, Y68/T84, P70/K86, K75/S91 differences in the BACE1/2 hairpin, might lead to less flexibility in the BACE2 flap, thus rendering BACE2 less tolerant towards inhibitors that occupy the space close to Y71 and induce a conformational change. Notably, a recently published molecular dynamics study also arrived at the conclusion that the BACE1 active site cavity is more spacious in the flap region as compared to that of BACE2.25 Although this structure-selectivity relationship around the flap and the S3 subpocket did show promise, the most selective chemotypes in this analysis had functionalities that would likely hamper their further development due to lack of BBB permeability and/or other undesired properties. Our next goal was to incorporate the above described principles in the design of compounds with balanced properties and improved selectivity for BACE1 inhibition. The dispiro core motif of lanabecestat, our potent pan BACE1/2 inhibitor which is currently studied in advanced clinical trials, served as an excellent starting point for this study. In the design of new inhibitors, we aimed to probe the effect of intruding the S3 subpocket and increasing the steric


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demand of the cyclohexyl unit adjacent to the flap loop. Further, in line with the properties of lanabecestat as well as literature data, we intended to keep compounds weakly basic with a pKa value for the conjugate protonated form between 6 and 8 and their lipophilicity as well as polar surface areas within acceptable range.26,27 We decided to restrict the number of H-bond donors to two for the NH2 group in order to minimize the challenges with brain permeability. In this context, amino-oxazoline xanthenes have previously been reported to exhibit a low propensity for Pgp-mediated efflux.28,29 Thus, to optimize isoform selectivity and brain exposure, we elected to combine the Asp-interacting amino-oxazoline group with our previously reported di-spiro core motif of lanabecestat.12,30 Our initial compounds, the phenyl nitriles 18 and 20 displayed promising cell-based potency but no preference for inhibiting BACE1 over BACE2 (Table 1, Figure 3). As anticipated by modeling, the chlorophenyl nitrile 18 is ca. 100-fold more potent than its enantiomer 17. We synthesized the corresponding di-deuterio analogs 19 and 21 in anticipation that deuteration of the methylene group at the partially saturated oxazole unit may slow down metabolism.31 While with these match pairs, no significant difference in the metabolic turnover was found, in some instances, we observed a trend toward reduced efflux ratio in MDCK cells over-expressing Pglycoprotein 1 (also known as multidrug resistance gene 1, MDR1). For instance, the dihydrooxazole 20 showed a higher efflux ratio than the di-deutero analog 21 (3.1 vs. 1.8, respectively). An in vivo pharmacokinetic study further indicated that relative to their non-deuterated match pairs (18 and 20), the deuterated analogs 19 and 21 displayed improved Kpu,u values, i. e. ratios of unbound brain to plasma concentrations (Kpu,u: 0.21 for 18, 0.68 for 19, 0.07 for 20 and 0.27 for 21, respectively; for details, see Supporting Information). Encouraged by these preliminary data, we decided to keep the CD2 group intact for the subsequent structure-activity relationship


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studies. Following the design guidelines described above, the next step was to investigate compounds without a C-ring substituent to avoid occupancy of the S3 subpocket. For this, we introduced known heterocyclic C-rings such as pyridines and pyrimidines,19,28 which may present an H-bond acceptor functionality pointing toward the S3 subpocket. Indeed, the unsubstituted pyridine 22 and pyrimidine 24 displayed moderate preferences for inhibiting BACE1 whereas methylating the pyridine ring resulted in significant loss of biochemical activity (see 23), presumably due to an unproductive conformational change. Figure 3, Table 1 The compounds described in Table 1 were synthesized according to Scheme 1. Tebbe methylenation of the commercially available indanone 1 resulted in alkene 2.32 The corresponding di-deuterated alkene 3 was synthesized by a Wittig-type olefination using triphenyl(trideuteriomethyl)phosphonium iodide. The amino-oxazolines 4 and 5 were constructed in two steps by treating the corresponding olefins (2, 3) with in situ formed iodoisocyanate followed by NH4OH. Enantiomers were separated by supercritical fluid chromatography on a chiral stationary phase (→ 6 ̶ 9) and the resulting bromides converted to the final compounds 17 ̶ 24 under standard Pd-mediated coupling conditions. Scheme 1 A crystal structure of compound 23 bound to BACE1 led to an unambiguous assignment of the absolute configuration of 23, as well as the precursor 9 from which it derived (Figure 4).33 The absolute configuration of the other molecules was determined based on this assignment. Figure 4


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Notably, compared to the biochemical activity, most compounds displayed improved cellular potency as determined by analysis of sAPPβ release in SH-SY5Y cells. This observed ‘drop-on effect’ may be caused by several factors; on the one hand, the fluorescently labelled peptide used in the biochemical assay may be processed less efficiently by the protease than membranebound, full-length APP. On the other hand, the pH within the sub-cellular micro-environment where BACE1 processes APP may slightly differ from the biochemical assay, thus resulting in increased proteolytic activity of BACE.13 We reasoned that low nanomolar cellular potency will be the critical factor for a successful BACE1 inhibitor and used the biochemical activities to understand isoform selectivity. Based on our initial structure-selectivity relationship analysis, we hypothesized that substitution at the cyclohexyl moiety of the di-spiro unit may influence the conformation of the proteases in the flap region. As described above, such an induced fit might be more readily achieved in BACE1 than in BACE2, thus contributing to subtype selectivity. We therefore initiated a small but systematic study to explore the effect of one or two alkyl groups at the cyclohexane ring with the above described pyrimidyl unit as the preferred C-ring (Table 2, Figure 5). The dimethyl cyclohexane 25 (rac) showed low nanomolar cell-based activity and a promising 23-fold selectivity for BACE1. Unfortunately, this compound was very unstable in the presence of microsomes or hepatocytes, likely due to the added lipophilicity. The ethyl analogs 27 and 29 were also highly metabolized whereas the slightly less lipophilic methyl isomers 26 and 28 displayed improved resistance to metabolism. In accordance to our design hypothesis, all five compounds preferentially inhibited BACE1 over the other isoform. The methyl and ethyl substituted compounds 28 and 29, which place the alkyl group toward the R128 side chain near


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the flap loop, exhibited the most promising selectivity patterns with a 52- and 45-fold preference for BACE1. Figure 5, Table 2 Compounds bearing alkyl substituents at the cyclohexyl moiety were synthesized as illustrated in Scheme 2. A double 1,4-addition to the indanone 10 followed by a Dieckmann cyclization, methylation and decarboxylation resulted in racemic α-methyl ketone 11. Regio- and stereoselective reduction of this diketone (11) using NaBH4/CeCl3 provided alcohol 12 (rac) which was methylated to yield the corresponding ether 13 (rac). Dideuterio-methylenation under Wittig-type conditions as described above gave the olefin 14 (rac) which was further converted to the corresponding dideuterio-oxazole-2-amine and enantio-separation of the resulting products by supercritical fluid chromatography provided the key intermediates 15 and 16. The intermediates for the synthesis of compounds 25, 27, and 29, respectively, were synthesized in an analogous fashion as described above (documented in the Supporting Information). In the ultimate step, Pd-mediated coupling of pyrimidin-5-ylboronic acid to the corresponding bromointermediates provided final compounds 25−29, respectively. All final compounds were purified by HPLC and analyzed by 1H-NMR spectroscopy, high-resolution mass spectroscopy and analytical HPLC and displayed purities in excess of 95%. Scheme 2 A crystal structure of pyrimidine 28 bound to the active site of BACE1 allowed the assignment of the relative and absolute configuration of this compound as well as its precursor 15 (Figure 6).33 The configurations of the remaining final compounds derived from 15 were unambiguously assigned based on this information. As anticipated, the CH3-substituted cyclohexane of 28 was


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found to open the flap conformation, with the methyl group pointing outwards from the induced S2’ pocket. This places the CH3 group between the Cβ of Y71 and the side chain of R128, which is replaced by K144 in BACE2. The structure also demonstrates how the pyrimidine ring offers a ring nitrogen as an H-bond acceptor to the water molecule in the S3 subpocket. Figure 6 The calculated physicochemical properties of pyrimidine 28 are similar to those of lanabecestat: formal removal of the propynyl unit and introduction of a second nitrogen to the C-ring led to a lower molecular weight (378.5 vs. 412.5) and a higher polar surface area (75.6 vs. 66.2 Å2) while the predicted basicity of both inhibitors is very similar (pKa 6.7 vs. 6.9). As compound 28 displayed the best overall profile, we studied it in more detail in vitro and in vivo. Encouragingly, lead molecule 28 did not show propensity to interact with the human ether-a-go-go-related gene channel (hERG) up to 33 µM concentrations, a characteristic which has plagued previously reported BACE inhibitors.34 Further, compound 28 did not show any inhibitory activity for cathepsin D, cathepsin S, renin, and pepsin at concentrations up to 100 µM, indicating that it is a very selective protease inhibitor. We also set up an assay in human melanocytes to compare its BACE2-mediated activity with lanabecestat.12 Following a protocol by Rochin et al.,14 Mβ levels were quantified by western blotting to assess functional BACE2 activity.15 It has previously been demonstrated that Mβ, a cleavage product of PMEL, is further processed by BACE2; hence, its inhibition should result in Mβ accumulation. The western blot shown in Figure 7 indeed confirmed that lanabecestat gave rise to significant Mβ accumulation whereas treatment of the melanocytes with compound 28 led to a slight increase of Mβ only at much higher concentrations (~10 µM), which is consistent with its selectivity pattern.


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Figure 7 A pharmacokinetic study in rat indicated that the pyrimidine 28 distributed well into brain tissue with a Kpu,u of 0.37; however, it also exhibited high systemic clearance. The brain distribution of 28 was commensurate with its physicochemical properties and moderate efflux observed in the MDR1-MDCK assay. We subsequently performed an in vivo PK/PD study in C57BL/6 mice with the objective to investigate whether compound 28 has the potential for reducing Aβ levels in peripheral (blood) and central (brain) compartments. Figure 8 Following oral administration to C57BL/6 mice, 28 exhibited a robust, exposure-driven reduction in Aβ40 plasma concentrations (Figure 8). In brain tissue, we observed a less pronounced trend toward reduced Aβ40 and Aβ42 concentrations, when compared to vehicle control. This result is consistent with the finding that 28 is still a mild substrate for the Pglycoprotein efflux transporter (Pgp) and free brain concentrations are lower than free plasma exposure. At the 3-hour timepoint, compound 28 induced a ~30% reduction of Aβ42 with a free brain concentration of ~50 nM (28). This magnitude of Aβ42 reduction is in the range of that observed for AZD3839 (Figure 1), AstraZeneca’s initial clinical candidate.19

CONCLUSIONS An analysis of biochemical activity and structural data of BACE inhibitors with variable isoform selectivity revealed new design guidelines pointing to elements that can be modified to improve selectivity for BACE1 inhibition. We found that a hydrogen bond acceptor in proximity of the S3


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subpocket in combination with a small substituent at the cyclohexyl A-ring occupying the volume neighboring the flap region led to optimal BACE1 selectivity. It is conceivable that these ‘selectivity hotspots’ can be exploited in a similar manner using known BACE inhibitors such as amino-hydantoins or other scaffolds as structural starting points.11,36,37 A number of new inhibitors were investigated starting from a central dispiro scaffold and an amino-oxazoline head group. We replaced the CH2 element of this head group by CD2, initially with the aim to reduce metabolic vulnerability. While there was no marked change in metabolism between deuterated and non-deuterated analogs, a trend toward reduced Pgp-mediated efflux has been observed. The deuterio-methylene group was therefore kept intact for this study. Pyrimidine 28 displayed a 52fold selectivity for BACE1 inhibition, low nanomolar cellular activity (1.1 nM), and a good overall profile (incl. no hERG interaction and cathepsin D, renin, or pepsin inhibition). To demonstrate that a BACE1 selective inhibitor exhibits reduced potential for altering pigmentation, we set up a melanocyte assay. Compound 28 showed Mβ accumulation only at very high concentrations (~10 µM) while lanabecestat, a non-selective BACE1/2 inhibitor, displayed a significant effect at ~10 nM. In vivo, pyrimidine 28 is relatively highly cleared in rodents but showed an exposure-dependent trend toward Aβ40 and Aβ42 reduction in brain tissue. Further studies with 28 and analogs in mice and other species will be required to evaluate the potential of this chemotype for further development.

EXPERIMENTAL SECTION Commercially available anhydrous solvents were routinely used for reactions. Room temperature (RT) refers to 20–25 °C. Nuclear magnetic resonance (NMR) spectra were recorded on a 400


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MHz Jeol JNM-LA400 spectrometer fitted with a probe of suitable configuration. Spectra were recorded at RT unless otherwise stated. Chemical shifts are given in parts per million (ppm) with resonance multiplicities denoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) and app (apparent), respectively. The high-resolution mass spectroscopy (HRMS) analysis was carried using a Quattro Xevo Qtof in full scan ESI positive ion mode with Leucine Enkephalin as the lock mass. Mass spectrometry (MS) analysis was performed in positive or negative ion mode using electrospray ionization (ESI+/-) or atmospheric pressure chemical ionization (APCI+/-). High pressure liquid chromatography (HPLC) analysis was performed using a Waters system with Agilent Poroshell 120 SB-C18 column (2.7 mm, 4.6 mm id x 30 mm). Mobile phases consisted of CH3CN and H2O, both containing 0.1%v/v formic acid. Diode array data was captured from 210nm to 400nm. Final compounds displayed purities ≥95%. The optical rotations were determined on an Autopol III Automatic Polarimeter using a 100 mm cell at 26.0 °C (average of 5 x 5 scans). General procedure 1. Under N2 at RT, a mixture of arylbromide (1 eq), boronic acid (2 eq), 1,1bis(di-tert-butylphosphino)ferrocene palladium dichloride (0.1 eq) in 1,4-dioxane (3 ml) was treated with a solution of K2CO3 (3 eq) in H2O (1 ml) and heated at 100 ºC for 2 ̶ 4 h. The mixture was cooled to RT, treated with Deloxan (Pd scavenger resin), stirred for 15 min and concentrated in vacuo. The residue was suspended in CH2Cl2, filtered, concentrated and purified by HPLC (Gilson, 0.2% NH3 or 0.1% HCOOH or 0.1% TFA in H2O/CH3CN). Evaporation, and freeze drying afforded the title compound. (1r,4r)-6'-Bromo-4-methoxy-1'-methylene-1',3'-dihydrospiro[cyclohexane-1,2'-indene] (2). To a stirred solution of 6'-bromo-4-methoxy-spiro[cyclohexane-1,2'-indane]-1'-one 1 (6.18 g, 20.0 mmol) in THF (200 ml) at 0 °C under N2 was added a 1M solution of Tebbe's reagent (44


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ml, 22 mmol) in toluene over 5 min. The mixture was warmed to RT, stirred for 64 h and diluted with Et2O (100 ml) and 0.1M aq. NaOH solution (10 ml). The layers were separated and the organic phase dried (MgSO4), filtered (Celite) and evaporated. Flash chromatography (CH2Cl2) gave the title compound as a solid (5.86 g, 95%). 1H NMR (400 MHz, CDCl3) δ ppm 1.34 ̶ 1.44 (m, 2H), 1.48 ̶ 1.66 (m, 4H), 2.00 ̶ 2.08 (m, 2H), 2.84 (s, 2H), 3.18 ̶ 3.28 (m, 1H), 3.38 (s, 3H), 4.95 (s, 1H), 5.46 (s, 1H), 7.09 (d, J = 8.0, 1H), 7.31 (dd, J = 8.0, 1.8, 1H), 7.58 (d, J = 1.8, 1H). (1r,4r)-6'-Bromo-1'-(dideuteriomethylene)-4-methoxy-spiro[cyclohexane-1,2'-indane]

(3).

At −30 °C under N2, a mixture of triphenyl(trideuteriomethyl)phosphonium iodide (19.8 g, 48.5 mmol) in THF (1 l) was treated with a solution of n-BuLi (2.5M in hexane, 19.4 ml, 48.5 mmol) and stirred for 45 min at −30 °C. A solution of indanone 1 (10.0 g, 32.3 mmol) in THF (150 ml) was dropwise added and the mixture was allowed to warm to RT and stirred for 12 h. The mixture was concentrated in vacuo and the crude oil was vigerously stirred in Et2O (1 l). The resulting suspension was filtered and washed with Et2O. The filtrate was concentrated and purified by flash chromatography (CH2Cl2) to afford the desired product 3 as a white solid (8.51 g, 85%). 1H NMR (400 MHz, CDCl3) δ ppm 1.25 ̶ 1.39 (m, 2H), 1.42 ̶ 1.62 (m, 4H), 1.95 ̶ 2.03 (m, 2H), 2.76 (s, 2H), 3.12 ̶ 3.21 (m, 1H), 3.31 (s, 3H), 7.03 (d, J = 8.0, 1H), 7.24 (dd, J = 8.0, 1.8, 1H), 7.51 (d, J = 1.8, 1H). (1r,4r)-6'-Bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-2''amine (4). A solution of olefin 2 (5.86 g, 19.0 mmol) in THF (60 ml) and CH3CN (60 ml) at 22 o

C was treated with isocyanato-silver (8.58 g, 57.2 mmol) followed by iodine (7.26 g, 28.6

mmol), stirred at RT for 4 h, filtered through Celite and evaporated. The resulting yellow solids were dissolved in THF (90 ml), treated with NH4OH (10 ml) and stirred at RT for 18 h. The mixture was concentrated in vacuo and the residue partitioned between CH2Cl2 (200 ml) and


16

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H2O (200 ml). The aqueous layer was extracted with CH2Cl2 (2 x 40 ml) and the combined organic extracts dried (Na2SO4), filtered and evaporated. The residue was suspended in MeOH (200 ml), filtered (Celite) and evaporated. The resulting solids were triturated with Et2O (140 ml), collected by filtration and dried to afford 4 as a pale yellow solid (6.37 g, 91%). 1H NMR (400 MHz, CD3OD) δ ppm 1.22 ̶ 1.36 (m, 3H), 1.44 ̶ 1.59 (m, 3H), 1.96 ̶ 2.04 (m, 2H), 2.85 (q, J = 15.7, 2H), 3.15 ̶ 3.19 (m, 1H), 3.35 (s, 3H), 4.21 (d, J = 9.3, 1H), 4.64 (d, J = 9.3, 1H), 7.14 (d, J = 7.9, 1H), 7.35 (dd, J = 8.0, 1.8, 1H), 7.37 (d, J = 1.8, 1H). LCMS: rt = 2.36 min, m/z = 365/367 [M + H]+. (1r,4r)-6'-Bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-5'',5''d2-2''-amine (5). A solution of indane 3 (8.50 g, 27.0 mmol) in THF (80 ml) and CH3CN (80 ml) at RT was treated with isocyanatosilver (12.3 g, 81.9 mmol) followed by a portionwise addition of I2 (10.4 g, 40.9 mmol). The grey mixture was stirred for 4 h and the resulting suspension filtered through Celite and concentrated in vacuo. The resulting solids were dissolved in THF (200 ml), treated with NH4OH (40 ml) and stirred at RT for 48 h. The mixture was concentrated in vacuo and diluted with EtOAc (500 ml) and H2O (500 ml). The layers were separated and the aqueous phase extracted with EtOAc (250 ml). The combined organic extracts were dried (Na2SO4), filtered and evaporated to leave a pale-yellow solid which was triturated with Et2O (250 ml), filtered and dried to afford compound 5 a pale yellow solid (8.30 g, 82%) which was directly subjected to chiral SFC. (1r,1'R,4R)-6'-Bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]2''-amine

(6)

and

(1r,1'S,4S)-6'-bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-

indene-1',4''-oxazol]-2''-amine (7). Supercritical fluid chromatography (25cm x 20 mm ChromegaChiral CC4 column from ES Industries, West Berlin, NJ) of racemate 4 (5.50 g,


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dissolved in MeOH, CH2Cl2 9:1) applying an isocratic method (CH3CN/MeOH 3:1 plus 1% i

PropNH2 and CO2 as co-solvent (80 /min, 100 bar, 25 °C) yielded isomer 6 (second eluting

isomer, 2.2 g, analyt. SFC purity = 98%, ee = 100%) and isomer 7 (first eluting isomer, 2.3 g, analyt. SFC: purity = 92%, ee = 98.8%). (1r,1'S,4S)-6'-Bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]5'',5''-d2-2''-amine (8) and (1r,1'R,4R)-6'-bromo-4-methoxy-3'H,5''H-dispiro[cyclohexane1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine

(9).

Supercritical

fluid

chromatography

(Chiralpak AD 20 x 250 mm 5 um) of racemate 5 (5.0 g, dissolved in MeOH) applying an isocratic method (MeOH plus 0.2% DIEA) yielded isomer 8 (first eluting isomer, 2.21 g), 1H NMR (400 MHz, CDCl3) δ ppm 1.15 ̶ 1.60 (m, 6H), 1.94 ̶ 2.03 (m, 2H), 2.67 (d, J = 15.7, 1H), 2.84 (d, J = 15.7, 1H), 3.04 ̶ 3.14 (m, 1H), 3.33 (s, 3H), 4.35 (br. s, 2H), 7.03 (d, J = 8.0, 1H), 7.28 (dd, J = 7.9, 1.8, 1H), 7.33 (d, J = 1.8, 1H). LCMS: rt = 2.72 min, m/z = 367/369 [M + H]+, purity = 99% (ee = 98%); and isomer 9 (2.08 g), 1H NMR (400 MHz, CDCl3) δ ppm 1.15 ̶ 1.59 (m, 6H), 1.92 ̶ 2.03 (m, 2H), 2.66 (d, J = 15.7, 1H), 2.84 (d, J = 15.7, 1H), 3.03 ̶ 3.13 (m, 1H), 3.33 (s, 3H), 4.35 (br. s, 2H), 7.02 (d, J = 7.9, 1H), 7.28 (dd, J = 7.9, 1.8, 1H), 7.32 (d, J = 1.7, 1H). LCMS: rt = 2.72 min, m/z = 367/369 [M + H]+, purity = 100% (ee = 100%). (1S,3S)-6'-Bromo-3-methylspiro[cyclohexane-1,2'-indene]-1',4(3'H)-dione (11, rac). At 0 °C under N2, a mixture of indanone 10 (15.0 g, 71.1 mmol) and methyl acrylate (13.4 ml, 149 mmol) in THF (150 ml) was treated with KOtBu (9.47 g, 85 mmol) in small portions over a period of 30 min. The mixture was stirred for 3 h at RT, treated with DMF (40 ml) followed by CH3I (8.85 ml, 142 mmol) and stirred for 16 h. The resulting suspension was treated with 10% aq. citric acid (100 ml) and concentrated in vacuo to afford an orange oil which was washed with H2O/MeOH (9:1). The resulting semi-solid material was treated with toluene and evaporated (4 x


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500 ml) and the crude product used for the next step without further purification. A solution of this crude material in THF (450 ml) and H2O (450 ml) was treated with LiOH · H2O (11.9 g, 284 mmol), stirred at RT for 55 h, heated at 70 °C for 14 h and concentrated in vacuo. The resulting suspension was collected by filtration and the cake washed with H2O (400 ml) and MeOH resulting in a white solid (8.42 g). The filtrate was concentrated in vacuo and the resulting residue was stirred in Et2O (50 ml) to give second batch of the desired product (1.25 g), yielding racemate 11 (9.67 g, 44%). 1H NMR (400 MHz, CDCl3) δ ppm 1.05 (d, J = 6.5, 3H), 1.72 ̶ 1.82 (m, 2H), 1.93 (t, J = 13.4, 1H), 2.12 ̶ 2.24 (m, 1H), 2.46 ̶ 2.64 (m, 3H), 3.27 (s, 2H), 7.38 (d, J = 8.1, 1H), 7.72 (dd, J = 8.1, 1.9, 1H), 7.88 (d, J = 1.9, 1H). LCMS: rt = 3.26 min, m/z = 307/309 [M + H]+. (1S,3S,4S)-6'-Bromo-4-hydroxy-3-methyl-spiro[cyclohexane-1,2'-inden]-1'(3'H)-one

(12,

rac). A solution of cerium(III) chloride heptahydrate (1.17 g, 3.15 mmol) in MeOH (140 ml) under N2 at RT was treated with a mixture of racemic diketone 11 (9.67 g, 31.4 mmol) in THF (240 ml), cooled to −70 °C, stirred for 30 min and treated with NaBH4 (0.48 g, 12.6 mmol) in small portions. The mixture was stirred at −70 °C for 2 h and carefully treated with a sat. aq. NH4Cl (100 ml) and H2O (200 ml) whilst maintaining the internal temperature below −65 °C. The mixture was allowed to warm to RT and extracted with EtOAc (3 × 250 ml). The combined organic layers were dried (Na2SO4), filtered and concentrated and the residue purified by flash chromatography (10−50% EtOAc in toluene) to afford racemic alcohol 12 as a white foam (5.50 g, 56%). 1H NMR (400 MHz, CDCl3) δ ppm 1.03 (d, J = 5.9, 3H), 1.32 ̶ 1.58 (m, 4H), 1.71 (d, J = 4.5, 1H), 1.74 ̶ 1.86 (m, 1H), 1.98 ̶ 2.08 (m, 1H), 2.99 (s, 2H), 3.23 ̶ 3.36 (m, 1H), 7.32 (d, J = 8.1, 1H), 7.67 (dd, J = 8.2, 1.9, 1H), 7.85 (d, J = 1.9, 1H). LCMS rt = 3.10 min; m/z = 309/311 [M + H]+.


19


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(1S,3S,4S)-6'-Bromo-4-methoxy-3-methyl-spiro[cyclohexane-1,2'-indane]-1'-one (13, rac). At 0 °C under N2, a solution alcohol 12 (2.50 g, 8.09 mmol) in DMF (30 ml) was treated portionwise with NaH (60% dispersion in mineral oil, 0.39 g, 16.2 mmol), stirred for 2 h, treated with CH3I (1.41 ml, 22.6 mmol), allowed to warm to RT and stirred for 15 h. The mixture was treated with H2O (100 ml) and EtOAc (250 ml). The organic phase was separated, washed with H2O (2 × 150 ml), dried (Na2SO4) and evaporated to afford yellow solid. Purification of the crude material by flash chromatography (10−40% EtOAc in toluene) gave racemic methyl ether 13 as a white solid (1.76 g, 67%). 1H NMR (400 MHz, CDCl3) δ ppm 1.00 (d, J = 6.1, 3H), 1.20 ̶ 1.79 (m, 6H), 2.14 ̶ 2.24 (m, 1H), 2.75 ̶ 2.84 (m, 1H), 2.97 (s, 2H), 3.38 (s, 3H), 7.31 (d, J = 8.1, 1H), 7.67 (dd, J = 8.1, 1.9, 1H), 7.85 (d, J = 1.9, 1H). LCMS rt 3.53 min m/z 324 [M + H]+. (1S,3S,4S)-6'-Bromo-1'-(dideuteriomethylene)-4-methoxy-3-methyl-spiro[cyclohexane-1,2'indane] (14, rac). At −30 °C under N2, a mixture of n-BuLi (2.5M in hexane, 3.14 ml, 7.84 mmol) and THF (50 ml) was treated with a solution of triphenyl(trideuteriomethyl)phosphonium iodide (3.19 g, 7.84 mmol) in THF (ca. 20 ml), stirred for 45 min and treated dropwise with a solution of ketone 13 (1.69 g, 5.23 mmol) in THF (20 ml). The mixture was allowed to warm to RT, stirred for 12 h and concentrated in vacuo. The residue was purified by flash chromatography (60 ̶ 80% CH2Cl2 in hexane) to give the desired olefin 14 as a clear oil (1.28 g, 76%). 1H NMR (400 MHz, CDCl3) δ ppm 0.92 (d, J = 6.3, 3H), 1.16 ̶ 1.35 (m, 2H), 1.40 ̶ 1.65 (m, 4H), 1.98 ̶ 2.10 (m, 1H), 2.71 (br. td, J = 10.6, 4.2, 1H), 2.77 (s, 2H), 3.33 (s, 3H), 7.01 (d, J = 8.0, 1H), 7.24 (dd, J = 8.0 1.8, 1H), 7.51 (d, J = 1.8, 1H). (1S,1'R,3S,4S)-6'-Bromo-4-methoxy-3-methyl-3'H,5''H-dispiro[cyclohexane-1,2'-indene1',4''-oxazol]-5'',5''-d2-2''-amine (15) and (1R,1'R,3R,4R)-6'-bromo-4-methoxy-3-methyl3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine (16). At RT, a


20

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stirred solution of olefin 14 (1.60 g, 4.95 mmol) in THF (20 ml) and CH3CN (20 ml) was treated with isocyanatosilver (2.23 g, 14.8 mmol) followed by portionwise addition of I2 (1.88 g, 7.42 mmol). The mixture was stirred 4 h, filtered through Celite, washed with THF (50 ml) and evaporated. The residue was dissolved in THF (30 ml), treated with NH4OH (5 ml), stirred at RT for 18 h, and concentrated. The residue was partitioned between EtOAc (250 ml) and sat. aq. NaHCO3 (250 ml). The aqueous layer was extracted with EtOAc (200 ml) and the combined organic extracts were dried (Na2SO4) and evaporated to leave a beige solid (2.4 g). Chiral SFC (Lux C4 column, 250 mm x 20 mm, 5 um) applying an isocratic method (MeOH/CO2 40%) of the crude product (dissolved in MeOH) yielded isomer 15 (second eluting isomer, solids, 419 mg), 1H NMR (400 MHz, CDCl3) δ ppm 0.80 ̶ 0.92 (m, 4H), 1.05 ̶ 1.58 (m, 6H), 1.93 ̶ 2.03 (m, 1H), 2.56 ̶ 2.67 (m, 1H), 2.71 (s, 2H), 5.91 (br. s, 2H), 7.14 (d, J = 7.9, 1H), 7.19 (s, 1H), 7.28 (d, J = 7.8, 1H). LCMS: rt = 3.33 min, m/z = 381/383 [M + H]+, purity = 100% (ee = 97%); and isomer 16 (first eluting isomer, solids, 410 mg), 1H NMR (400 MHz, CDCl3) δ ppm 0.96 (d, J = 6.2, 3H), 1.14 ̶ 1.36 (m, 3H), 1.43 ̶ 1.62 (m, 3H), 2.02 ̶ 2.11 (m, 1H), 2.60 ̶ 2.73 (m, 2H), 2.88 (d, J = 15.6, 1H), 3.36 (s, 3H), 7.05 (d, J = 8.0, 1H), 7.31 (dd, J = 7.9, 1.9, 1H), 7.34 (d, J = 1.8, 1H). LCMS: rt = 3.33 min, m/z = 381/383 [M + H]+, purity = 99% (ee = 100%). {3-((1r,1'S,4S)-2''-Amino-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''oxazol]-6'-yl)-5-chlorobenzonitrile} (17). The title compound (50 mg, beige solid, 35%) was prepared according to General Procedure 1 using bromo-indane 6 (121 mg, 0.33 mmol) and 3chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (105 mg, 0.40 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.30 ̶ 1.48 (m, 3H), 1.50 ̶ 1.66 (m, 3H), 2.02 ̶ 2.12 (m, 2H), 3.02 (q, J= 16.3, 2H), 3.16 ̶ 3.27 (m, 1H), 3.37 (s, 3H), 4.65 (d, J = 9.5, 1H), 5.02 (d, J = 9.6, 1H), 7.42 (d, J = 7.8, 1H), 7.63 (dd, J = 7.8, 1.8, 1H), 7.68 (d, J = 1.4, 1H), 7.78 (t, J = 1.5, 1H),


21


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7.97 ̶ 8.01 (m, 2H), 2H not observed. HRMS (ESI) m/z 422.1633 (calcd 422.1635 for C24H24ClN3O2 [M + H]+). Analyt. HPLC: rt = 2.90 min (>99%). {3-((1r,1'R,4R)-2''-Amino-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''oxazol]-6'-yl)-5-chlorobenzonitrile} (18). The title compound (46 mg, beige solid, 33%) was prepared according to General Procedure 1 using bromo-indane 7 (121 mg, 0.33 mmol) and 3chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (105 mg, 0.40 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.29 ̶ 1.50 (m, 3H), 1.50 ̶ 1.68 (m, 3H), 2.02 ̶ 2.13 (m, 2H), 3.03 (q, J = 16.2, 2H), 3.16 ̶ 3.27 (m, 1H), 3.37 (s, 3H), 4.69 (d, J = 9.6, 1H), 5.06 (d, J = 9.6, 1H), 7.43 (d, J = 7.9, 1H), 7.66 (dd, J = 7.8, 1.8, 1H), 7.70 (d, J = 1.4, 1H), 7.78 (d, J = 1.5, 1H), 7.97 ̶ 8.01 (m, 2H), 2H not observed. HRMS (ESI) m/z 422.1633 (calcd 422.1635 for C24H24ClN3O2 [M + H]+). Analyt. HPLC: rt = 2.98 min (>99%). {3-((1r,1'R,4R)-2''-Amino-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''oxazol]-6'-yl-5'',5''-d2)-5-chlorobenzonitrile} (19). The title compound (65 mg, white solids, 22%) was prepared according to General Procedure 1 using bromo-indane 9 (250 mg, 0.68 mmol) and 3-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (360 mg, 1.37 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.28 ̶ 1.47 (m, 3H), 1.50 ̶ 1.68 (m, 3H), 2.02 ̶ 2.13 (m, 2H), 3.04 (q, J = 16.3, 2H), 3.18 ̶ 3.26 (m, 1H), 3.37 (s, 3H), 7.45 (d, J = 8.0, 1H), 7.68 (dd, J = 7.9, 1.8, 1H), 7.74 (d, J = 1.6, 1H), 7.79 (t, J = 1.5, 1H), 7.99 ̶ 8.01 (m, 2H), 2H not observed. HRMS (ESI) m/z 424.1761 (calcd 424.1765 for C24H22D2ClN3O2 [M + H]+). Analyt. HPLC: rt = 2.88 min (>99%). {3-((1r,1'R,4R)-2''-Amino-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''oxazol]-6'-yl)-5-methoxybenzonitrile} (20). The title compound (112 mg, white solids, 79%) was prepared according to General Procedure 1 using bromo-indane 7 (121 mg, 0.33 mmol) and


22

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3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (103 mg, 0.40 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.29 ̶ 1.45 (m, 3H), 1.50 ̶ 1.68 (m, 3H), 2.02 ̶ 2.12 (m, 2H), 3.01 (q, J = 16.2, 2H), 3.16 ̶ 3.24 (m, 1H), 3.37 (s, 3H), 3.90 (s, 3H), 4.61 (d, J = 9.5, 1H), 4.99 (d, J = 9.5, 1H), 7.26 (dd, J = 2.3, 1.2, 1H), 7.39 (d, J = 7.8, 1H), 7.45 (t, J = 1.6, 1H), 7.56 (t, J = 1.3, 1H), 7.59 (dd, J = 7.8, 1.6, 1H), 7.63 (s, 1H), 2H not observed. HRMS (ESI) m/z 418.2140 (calcd 418.2140 for C25H27N3O3 [M + H]+). Analyt. HPLC: rt = 2.84 min (95.1%). {3-((1r,1'R,4R)-2''-Amino-4-methoxy-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''oxazol]-6'-yl-5'',5''-d2)-5-methoxybenzonitrile} (21). The title compound (84 mg, white solids, 80%) was prepared according to General Procedure 1 using bromo-indane 9 (92 mg, 0.25 mmol) and 3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (64 mg, 0.25 mmol). 1

H NMR (400 MHz, CD3OD) δ ppm 1.31 ̶ 1.47 (m, 3H), 1.50 ̶ 1.70 (m, 3H), 2.04 ̶ 2.14 (m, 2H),

3.03 (q, J = 16.3, 2H), 3.18 ̶ 3.26 (m, 1H), 3.37 (s, 3H), 3.88 (s, 3H), 7.28 (dd, J = 2.4, 1.2, 1H), 7.42 (d, J = 7.8, 1H), 7.47 (dd, J = 2.4, 1.7, 1H), 7.59 (t, J = 1.4, 1H), 7.65 (dd, J = 7.8, 1.8, 1H), 7.70 (d, J = 1.5, 1H), 2H not observed. HRMS (ESI) m/z 420.2256 (calcd 420.2262 for C25H25D2N3O3 [M + H]+). Analyt. HPLC: rt = 2.95 min (>99%). {(1r,1'R,4R)-4-Methoxy-6'-(pyrimidin-3-yl)-3'H,5''H-dispiro[cyclohexane-1,2'-indene1',4''-oxazol]-5'',5''-d2-2''-amine} (22). The title compound (27 mg, white solids, 73%) was prepared according to General Procedure 1 using bromide 9 (37 mg, 0.10 mmol) and 3pyridylboronic acid (25 mg, 0.20 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.26 ̶ 1.43 (m, 3H), 1.50 ̶ 1.64 (m, 3H), 1.96 ̶ 2.08 (m, 2H), 2.92 (q, J = 15.6, 2H), 3.11 ̶ 3.21 (m, 1H), 3.36 (s, 3H), 7.35 (d, J = 7.5, 1H), 7.45 ̶ 7.52 (m, 3H), 8.07 (d, J = 7.9, 1H), 8.49 (d, J = 4.8, 1H), 8.76 (d, J = 2.2, 1H), 2 H not observed. HRMS (ESI) m/z 366.2151 (calcd 366.2155 for C22H23D2N3O2 [M + H]+). Analyt. HPLC: rt = 2.59 min (99.3%).


23


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{(1r,1'R,4R)-4-Methoxy-6'-(5-methyl-pyridin-3-yl)-3'H,5''H-dispiro[cyclohexane-1,2'indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (23). The title compound (14 mg, white solids, 16%) was prepared according to General Procedure 1 using bromide 9 (82 mg, 0.23 mmol) and (5methyl-3-pyridyl)boronic acid (31 mg, 0.23 mmol). 1H NMR (400 MHz, CD3OD) δ ppm 1.31 ̶ 1.46 (m, 3H), 1.51 ̶ 1.69 (m, 3H), 2.02 ̶ 2.12 (m, 2H), 2.43 (s, 3H), 3.03 (q, J = 16.1, 2H), 3.15 ̶ 3.26 (m, 1H), 3.37 (s, 3H), 7.42 (d, J = 7.8, 1H), 7.63 (d, J = 7.8, 1H), 7.68 (s, 1H), 7.94 (s, 1H), 8.36 (s, 1H), 8.59 (s, 1H), 2H not observed. HRMS (ESI) m/z 380.2307 (calcd 380.2289 for C23H25D2N3O2 [M + H]+). Analyt. HPLC: rt = 2.24 min (99.0%). {(1r,1'R,4R)-4-Methoxy-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane-1,2'-indene1',4''-oxazol]-5'',5''-d2-2''-amine} (24). The title compound (25 mg, off-white solids, 61%) was prepared according to General Procedure 1 using bromide 9 (40 mg, 0.11 mmol) and pyrimidin5-ylboronic acid (60 mg, 0.24 mmol). 1H NMR (400 MHz, CD3OD, 35 ºC) δ ppm 1.20 ̶ 1.45 (m, 3H), 1.50 ̶ 1.65 (m, 3H), 1.99 ̶ 2.00 (m, 2H), 2.95 (q, J = 16.0, 2H), 3.14 ̶ 3.16 (m, 1H), 3.36 (s, 3H), 7.38 (d, J = 8.0, 1H), 7.55 (d, J = 7.0, 2H), 9.03 (s, 2H), 9.09 (s, 1H), 2H not observed. HRMS (ESI) m/z 367.2103 (calcd 367.2113 for C21H22D2N4O2 [M + H]+). Analyt. HPLC: rt = 2.83 min (97.8%). {(1r,3R,4r,5S)-4-Methoxy-3,5-dimethyl-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (25, rac). The title compound (29 mg, solid) was prepared according to General Procedure 1 using (1r,3R,4r,5S)-6'-bromo-4-methoxy-3,5dimethyl-3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine (150 mg, see Supporting Information) and pyrimidin-5-ylboronic acid (95 mg). 1H NMR (400 MHz, CDCl3) δ ppm 0.99 ̶ 1.03 (m, 6H), 1.25 ̶ 1.35 (m, 2H), 1.46 ̶ 1.59 (m, 2H), 1.63 ̶ 1.70 (m, 2H), 2.35 (br. t, J = 9.8, 1H), 2.82 (br. d, J ≈ 15.2, 1H), 3.02 (br. d, J ≈ 16.2, 1H), 3.45 (s, 3H), 4.25 (br. s, 2H),


24

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7.33 (d, J = 6.2, 1H), 7.41 ̶ 7.43 (m, 2H), 8.93 (s, 2H), 9.18 (s, 1H). HRMS (ESI) m/z 395.2416 (calcd 395.2418 for C23H26D2N4O2 [M + H]+). Analyt. HPLC: rt = 3.52 min (96.8%). {(1R,1'R,3R,4R)-4-Methoxy-3-methyl-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (26). The title compound (28 mg, white solid) was prepared according to General Procedure 1 using bromide 16 (27 mg, 0.07 mmol) and pyrimidin-5-ylboronic acid (20 mg, 0.16 mmol). 1H NMR (400 MHz, CD3OD, 35 °C) δ ppm 1.01 (d, J = 6.3, 3H), 1.2 ̶ 1.5 (m, 3H), 1.55 (dq, J = 13.3, 3.3, 1H), 1.60 ̶ 1.70 (m, 2H), 2.15 ̶ 2.20 (m, 1H), 2.75 ̶ 2.80 (m, 1H), 3.07 (q, J = 17.1, 2H,), 3.38 (s, 3H), 7.50 (d, J = 7.8, 1H), 7.71 (dd, J = 7.9, 1.8, 1H), 7.79 (, d, J = 1.4, 1H), 9.07 (br. s, 2H), 9.13 (br. s, 1H), 2H not observed. HRMS (ESI) m/z 381.2260 (calcd 381.2276 for C22H24D2N4O2 [M + H]+). Analyt. HPLC: rt = 2.85 min (98.9%). {(1R,1'R,3R,4R)-3-ethyl-4-methoxy-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane-1,2'indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (27). The title compound (20 mg, solids) was prepared according to General Procedure 1 using (1R,1'R,3R,4R)-6'-bromo-3-ethyl-4-methoxy3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine (35 mg, 0.09 mmol, see Supporting Information) and pyrimidin-5-ylboronic acid (40 mg, 0.32 mmol). 1H NMR (400 MHz, CD3OD, 35 °C) δ ppm 0.83 (t, J = 7.5, 3H), 1.10 ̶ 1.50 (m, 5H), 1.55 (d, J = 9.3, 1H), 1.66 (d, J = 13.7, 1H), 1.75-1.85 (m, 1H), 2.13 (d, J = 9.2, 1H), 2.82 (td, J = 9.8, 4.3, 1H), 2.95 (q, J = 15.8, 2H), 3.37 (s, 3H), 7.39 (d, J = 8.2, 1H), 7.55 (m, 2H), 9.04 (s, 2H), 9.10 (s, 1H), 2H not observed. HRMS (ESI) m/z 395.2416 (calcd 395.2418 for C23H26D2N4O2 [M + H]+). Analyt. HPLC: rt = 3.11 min, (99.4%). {(1S,1'R,3S,4S)-4-Methoxy-3-methyl-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane-1,2'indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (28). The title compound (22 mg, white solid) was


25


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prepared according to General Procedure 1 using bromide 15 (27 mg, 0.07 mmol) and pyrimidin5-ylboronic acid (30 mg, 0.24 mmol). 1H NMR (400 MHz, CD3OD, 35 °C) δ ppm 1.00 (d, J = 6.2, 3H), 1.12 (t, J = 12.4, 1H), 1.40 (dq, J = 12.9, 4.2, 1H), 1.50 ̶ 1.70 (m, 4H), 2.15 ̶ 2.20 (m, 1H), 2.75 ̶ 2.80 (td, J = 6.3, 3.8, 1H), 3.07 (br. q, J = 16.8, 2H), 3.38 (s, 3H), 7.50 (d, J = 7.83, 1H), 7.71 (dd, J = 7.83, 1.6, 1H), 7.79 (d, J = 1.6, 1H), 9.07 (s, 2H), 9.13 (s, 1H), 2H not observed. HRMS (ESI) m/z 381.2260 (calcd 381.2276 for C22H24D2N4O2 [M + H]+). Analyt. 3

−1

−1

HPLC: rt = 3.42 min (98.4%). [α]26.0 D +65.0 ° cm g dm (c 1.00 g/100 ml in MeOH). {(1S,1'R,3S,4S)-3-Ethyl-4-methoxy-6'-(pyrimidin-5-yl)-3'H,5''H-dispiro[cyclohexane-1,2'indene-1',4''-oxazol]-5'',5''-d2-2''-amine} (29). The title compound (18 mg, white solids) was prepared according to General Procedure 1 using (1S,1'R,3S,4S)-6'-bromo-3-ethyl-4-methoxy3'H,5''H-dispiro[cyclohexane-1,2'-indene-1',4''-oxazol]-5'',5''-d2-2''-amine (35 mg, 0.09 mmol, see Supporting Information) and pyrimidin-5-ylboronic acid (30 mg, 0.24 mmol). 1H NMR (400 MHz, CD3OD, 35 °C) δ ppm 0.82 (t, J = 7.5, 3H), 0.97 (t, J = 12.8, 1H), 1.10 ̶ 1.20 (m, 1H), 1.20 ̶ 1.35 (m, 1H), 1.35 ̶ 1.65 (m, 4H), 1.70 ̶ 1.80 (m, 1H), 2.11 (dq, J = 12.5, 4.2, 1H), 2.82 (td, J = 10.4, 4.2, 1H), 2.95 (q, J = 15.7, 2H), 3.37 (s, 3H), 7.39 (d, J = 8.2, 1H), 7.55 (m, 2H), 9.04 (s, 2H), 9.10 (s, 1H), 2H not observed. HRMS (ESI) m/z 395.2416 (calcd 395.2418 for C23H26D2N4O2 [M + H]+). Analyt. HPLC: rt = 3.19 min (>99%). The intermediates leading to compounds 25, 27 and 29, respectively, have been synthesized by analogy to the intermediates described above (experimental details available in the Supporting Information). Detailed protocols for the in vitro biological and metabolism asssays, crystallography, and pharmacokinetics are described in the Supporting Information.


26

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SUPPORTING INFORMATION Supporting Information is available free of charge on the ACS Publications website. General methods (experimental details for analytical methods); protocols describing the preparation of intermediates for the synthesis of compounds 25, 27, 29; experimental details for the determination of the crystal structures of compounds 23 and 28; protocols for all biological, in vitro metabolic and in vivo pharmacokinetic assays; molecular formula strings.

AUTHOR INFORMATION Corresponding Author *R.W.B. Tel: +44 1223 898071. Email: [email protected] ORCID Roland W. Bürli: 0000-0001-7377-7628 Patrik Johansson: 0000-0002-7850-1169 Present Addresses §

G.C.: UCB Biopharma, Chemin du Foriest 1, 1420 Braine-l’Alleud, Belgium

†

J.H.: GSK Neuroscience, 1250 South Collegeville Rd, Collegeville, PA 19426, USA

‡

S.B.H.: Biogen, 225 Binney Street, Cambridge, MA 02142, USA

Notes The authors declare no competing financial interest.


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Contributions Experimental design: RWB, PJ, IG, JH, MD, GC, KK, SBH. Performed experiments: PT, MMK, HB, PD, PJ, KKa, EB, SE, CS. Data analysis: RWB, PJ, KKa, KK, SE, IG, GC, CS. Wrote the manuscript: RWB, PJ.

ACKNOWLEDGMENTS The authors would like to thank Frederik Martin and Lyn Rosenbrier-Ribeiro for enabling the off-target testing of compound 28 and to Stephane Turcotte for the determination of the optical rotation of 28.

ABBREVIATIONS USED APP, amyloid precursor protein; BACE, β-secretase; Clint, intrinsic clearance; HLM, human liver microsome; hERG, human ether-à-go-go-related gene; MDR1-MDCK (ER), multidrug resistant 1 transporter Madin-Darby canine kidney cells (efflux ratio, ratio of basolateral-to-apical/apicalto-basolateral flux); rHeps, rat hepatocytes.

ANCILLARY INFORMATION The coordinates and structure factors of compounds 23 and 28 have been deposited to the Protein Databank with PDB IDs 6EJ3 and 6EJ2, respectively. Authors will release the atomic coordinates and experimental data upon article publication.


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(37) Thomas, A. A., Hunt, K. W., Newhouse, B.,† Watts, R. J., Liu, X., Vigers, G., Smith, D., Rhodes, S. P., Brown, K. D., Otten, J. N., Burkar, M., Cox, A. A., Geck Do, M. K., Dutcher, D., Rana, S., DeLisle, R. K., Regal, K., Wright, A. D., Groneberg, R., Liao, J., Scearce-Levie, K., Siu, M., Purkey, H. E., Lyssikatos, J. P. 8‐Tetrahydropyran-2-yl chromans: highly selective βsite amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors. J. Med. Chem. 2014, 57, 10112−10129.


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Figures and Legends

F

NH2 NH 2

N N

N N

N

N O

N F

F

AZD3839

lanabecestat (AZD3293)

Ki (BACE1): 26 nM Ki (BACE2): 372 nM

IC50 (BACE1): 0.6 nM IC50 (BACE2): 0.9 nM

Figure 1. Structures and biochemical activities for AZ3839 and lanabecestat (AZD3293).12,19


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Figure 2. Crystal structures of BACE inhibitors with variable BACE1 selectivity bound to BACE1: red (10 fold). The most selective compounds were found to induce a conformational change of the BACE1 flap and/or to interact with a wellconserved H2O keeping the BACE1 S3 subpocket in an open conformation.

CN

NH2

O N

X

Cl

O

17

R R

NH2

O N

O

dideutero-oxazole scaffold

Figure 3. Left: structure of less active enantiomer 17; right: generic structure for compounds 18−24 (Table 1).

Figure 4. Co-crystal structure of compound 23 (PDB ID 6EJ3) bound to BACE1.


37


N N

D D

NH 2

O

N

N

N

D D

NH 2

O N

R1 OMe R2

OMe

25 (r ac)

Page 38 of 45

26 27 28 29

R1 = R1 = R1 = R1 =

CH 3 , R 2 = H CH 2 CH 3, R 2 = H H, R 2 = CH 3 H, R 2 = CH 2CH3

Figure 5. Structures 25−29 for Table 2.


38

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Figure 6. Co-crystal structure of compound 28 (PDB ID 6EJ2) bound to the active site of BACE1. Top: inhibitor 28 bound to the active site; middle: methyl-cyclohexyl inducing an open flap conformation; bottom: pyrimidyl nitrogen engaging in the H-bond network within the S3 subpocket.

Figure 7. Comparison of compound 28 with lanabecestat (AZD3293, IC50 (BACE1) 0.6 nM; IC50 (BACE2)

0.9 nM) on PMEL processing. Human melanocyte (MNT1) cells were incubated with

different concentrations of BACE inhibitors or vehicle (0.1% DMSO) and processed to identify parent PMEL (P1) and proteolytically cleaved Mβ fragment. Mβ fragment accumulates when BACE2 is inhibited.


39


Page 40 of 45

Figure 8. PK/PD study in mice following 50 mg/kg oral administration (PO). Top: free brain and plasma exposure of compound 28 over time; bottom: Aβ40 and Aβ42 levels in brain and peripheral Aβ40 levels over time.


40

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Schemes and Legends R

O Br

R

a (R = H) Br O b (R = D)

O

1

2 R=H 3 R=D c

R

R R

NH2

O N

Br

R R

NH2

O N

O

O

17-24 d

4 R=H 5 R=D

e

Br

R R

NH2

O N

+

Br

R R

NH2

O N

O

7 R=H 9 R=D

O

6 R=H 8 R=D

Scheme 1. Synthesis of compounds 17 ̶ 24 (Table 1). Reagents: (a) Tebbe’s reagent, THF, 0 ºC, 95%; (b) Ph3CD3PI, n-BuLi, ̶ 30 °C, 85%; (c) AgNCO, I2, THF, CH3CN, 22 °C, then THF, NH4OH, 84% (4), or 82% (5); (d) chiral SFC; (e) arylbromide, 1,1-bis(di-tertbutylphosphino)ferrocene palladium dichloride, K2CO3, H2O, 1,4-dioxane, 100 ºC.


41


Page 42 of 45

O

O Br

Br

a

O

11 (rac)

10

b X

O

Br

Br

c O

OH

13 (rac) X = O 14 (rac) X = CD2

d

12 (rac) e

D D R

NH2

O

D D

N R O

f

15 R = Br 28 R = 5-pyrimidinyl

NH2

O N

O

f

16 R = Br 26 R = 5-pyrimidinyl

Scheme 2. Synthesis of compounds shown in Table 2 & 3. Reagents: (a) methyl acrylate, CH3I, KOtBu, THF, then LiOH · H2O, THF, H2O, 44% (2 steps); (b) CeCl3 · 7 H2O, NaBH4, THF, ̶ 70 °C, 56%; (c) CH3I, NaH, DMF, 0 °C, 67%; (d) Ph3CD3PI, n-BuLi, THF, ̶ 30 °C, 76%; (e) AgNCO, I2, THF, CH3CN, 22 °C, then THF, NH4OH, 84%; (f) arylbromide, 1,1-bis(di-tertbutylphosphino)ferrocene palladium dichloride, K2CO3, H2O, 1,4-dioxane, 100 ºC.


42

Page 43 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Tables and Legends

entry

X

R

17

cell (IC50, nM)a

BACE1 (Ki, nM)b

BACE2 (Ki , nM)b

Ki (BACE2)/ Ki (BACE1)

MDR1MDCK (ER)c

HLM (CLint)d

rHeps (CLint)e

212 (75, n = 2)

2700 (2107, n = 3)

2193 (235, n = 3)

0.95

n.d.

n.d.

n.d.

18

H

3.4 (1.9, n = 2)

38 (8.8, n = 3)

13 (4.6, n = 3)

0.34

n.d. low recovery

11.1

11.6

19

D

4.8 (0.44, n = 2)

18 (15, n = 3)

21 (2.2, n = 3)

1.1

1.2

11.5

8.7

20

H

2.7 (2.4, n = 2)

23 (11, n = 3)

13 (0.32, n = 3)

0.58

3.1

13.9

23.9

21

D

2.9 (0.16, n = 2)

23 (13, n = 3)

13 (7, n = 3)

0.63

1.8

15.7

25.1

D

7 (1.3, n = 2)

430 (443, n = 3)

1478 (113, n = 3)

3.4

n.d.

n.d.

n.d.

D

5.9 (4.7, n = 5)

1179 (374, n = 6)

710 (80, n = 6)

0.6

n.d.

n.d.

n.d.

D

7 (n = 1)

134 (82, n = 3)

2077 (73, n = 3)

16

n.d.

n.d.

n.d.

N

22

23 N

24

Table 1. Cell-based potency as determined by sAPPβ release in SH-SY5Y cells,a biochemical inhibitory activity for BACE1 and BACE2,b respectively, efflux ratios (ER) as measured in MDR1 overexpressing MDCK cells,c metabolic stability in the presence of human liver microsome (HLM, µg/min/mg)d and rat hepatocytes (rHeps, µg/min/106 cells), n.d. not determined.e


43


Page 44 of 45

entry

cell (IC50, nM)a

BACE1 (Ki, nM)b

BACE2 (Ki, nM)b

Ki (BACE2)/ Ki (BACE1)

MDR1MDCK (ER)c

HLM (CLint)d

rHeps (CLint)e

hERGf

25

1.5 (0.04, n = 2)

45 (26, n = 7)

1011 (85, n = 7)

23

4.3

187

137

n.d.

26

0.9 (0.33, n = 2)

118 (73, n = 4)

1299 (387, n = 4)

11

4.5

52.4

82

>33 µM

27

0.91 (0.56, n = 3)

25 (11, n = 3)

443 (25, n = 3)

18

5.0

149

138

15%

28

1.1 (0.51, n = 10)

16 (7.6, n = 11)

817 (213, n = 11)

52

5

47

66

>33 µM

29

2.0 (0.09, n = 3)

45 (18, n = 3)

2022 (55, n = 3)

45

3.6

153

158

29%

Table 2. Cell-based potency as determined by sAPPβ release in SH-SY5Y cells,a biochemical inhibitory activity for BACE1 and BACE2,b respectively, efflux ratios (ER) as measured in MDR1 overexpressing MDCK cells,c metabolic stability in the presence of human liver microsome (HLM, µg/min/mg)d and rat hepatocytes (rHeps, µg/min/106 cells),e hERG activity:35 either percentage activity at 11.1 µM or IC50 value (µM).f


44

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Table of Contents graphic

N D D

NH 2

O

(a) Analysis of Xrays & SAR (b) Structure-based optimization

N

N N

D D

NH2

O N

OMe

OMe

Cl

BACE1 (Ki ) 18 nM BACE2 (Ki ) 21 nM cell (IC50 ) 4.8 nM

1x

BACE1 (Ki ) 16 nM BACE2 (Ki ) 817 nM cell (IC50) 1.1 nM

52x


45

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