DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4

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DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors Philip A. Harris,*,† Bryan W. King,‡ Deepak Bandyopadhyay,‡ Scott B. Berger,† Nino Campobasso,‡ Carol A. Capriotti,† Julie A. Cox,‡ Lauren Dare,† Xiaoyang Dong,† Joshua N. Finger,† LaShadric C. Grady,§ Sandra J. Hoffman,† Jae U. Jeong,† James Kang,† Viera Kasparcova,† Ami S. Lakdawala,‡ Ruth Lehr,‡ Dean E. McNulty,‡ Rakesh Nagilla,† Michael T. Ouellette,‡ Christina S. Pao,‡ Alan R. Rendina,‡ Michelle C. Schaeffer,‡ Jennifer D. Summerfield,§ Barbara A. Swift,† Rachel D. Totoritis,‡ Paris Ward,‡ Aming Zhang,∥ Daohua Zhang,† Robert W. Marquis,† John Bertin,†,⊥ and Peter J. Gough†,⊥ †

Pattern Recognition Receptor Discovery Performance Unit and ‡Platform Technology & Science, GlaxoSmithKline, Collegeville Road, Collegeville, Pennsylvania 19426, United States § Platform Technology & Science, GlaxoSmithKline, Winter Street, Waltham, Massachusetts 02451, United States ∥ Platform Technology & Science, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, United States S Supporting Information *

ABSTRACT: The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK’s DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK’481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

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apoptosis.3 Furthermore, in addition to its role downstream of TNF receptor 1 (TNFR1), RIP1 has also been shown to be a critical driver of inflammation downstream of various other pathways (FasL, TRAIL, TLR3 and TLR4).4 Thus, inhibition of RIP1 activation is likely to have broad therapeutic potential for multiple inflammatory diseases. Degterev et al.5 were first to identify a number of RIP1 inhibitor templates, which were termed “necrostatins”. They were initially identified from cellular screening through their

INTRODUCTION

Receptor interacting protein 1 (RIP1) kinase activity has recently emerged as an important driver of tumor necrosis factor (TNF)-mediated inflammation and pathology.1 This notion is strongly supported by genetic evidence in mice, where mutations that shunt signaling down the RIP1 kinase pathway result in spontaneous and robust inflammation.2 RIP1 kinase activity was originally thought to solely induce inflammation through control of a highly inflammatory form of cell death termed programmed necrosis, but subsequent work has now revealed that activated RIP1 can also directly regulate proinflammatory cytokine production and some forms of © 2016 American Chemical Society

Received: December 8, 2015 Published: February 8, 2016 2163

DOI: 10.1021/acs.jmedchem.5b01898 J. Med. Chem. 2016, 59, 2163−2178

Journal of Medicinal Chemistry

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selectivity and favorable developability profile make this series an excellent starting point for lead optimization.

ability to block necrotic death of monocytic U937 cells induced by treatment with TNF and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD.fmk).6 The most advanced series, containing an indole−hydantoin pharmacophore as represented by 1, known as Nec-1s or 7-Cl-O-Nec-1 (Figure 1), had excellent

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CHEMISTRY The synthesis of benzoxazepinones 9−11 was undertaken as outlined in Scheme 1. Nucleophilic aromatic substitution of 1Scheme 1

Figure 1. Structures of known RIP1 inhibitors.

kinase selectivity.7 The cocrystal structure of 1 bound in the RIP1 kinase domain showed the molecule occupying an allosteric lipophilic pocket at the back of the ATP binding site.8 However, a narrow structure−activity relationship (SAR) profile coupled with moderate potency and poor pharmacokinetic (PK) properties has limited their development in drug discovery.9 Recently, Degterev and co-workers further disclosed the potent and selective RIP1 inhibitor 2, which is a hybrid of 1 and ponatinib, the type-II kinase inhibitor of BCR-ABL.10 This hybrid increased the RIP1 potency compared with 1 while maintaining its high kinase selectivity, but the resulting high molecular weight makes this hybrid approach unfavorable for lead optimization. To identify starting points for our RIP1 drug discovery efforts, we initially screened the GSK kinase inhibitor set containing multiple chemotypes associated with historical GSK kinase programs. The screen identified a number of potent inhibitors of RIP1 belonging to the type-II class, targeting the “DFG-out” conformation of the kinase, as exemplified by 3.11 The developability profiles of these type-II inhibitors were challenging, with predominantly high molecular weights, high lipophilicities, and low aqueous solubilities. Additionally, these chemotypes possessed a range of off-target kinase activities that would need to be addressed during lead optimization. The developability limitations of the hits identified from traditional kinase inhibitor space led us to carry out a highthroughput screen of the GSK compound collection (∼2 million compounds). After some initial optimization, this effort led to the identification of 1-pivaloyl-5-phenyl-4,5-dihydropyrazole (4), known as GSK’963, a small, highly potent, and selective RIP1 inhibitor. Although an excellent tool compound to study RIP1 inhibition in vitro, 4 had minimal oral exposure in rodents, limiting its development.12 To further expand our search for truly novel chemotypes, we decided to screen RIP1 against GSK’s propriety collection of DNA-encoded small-molecule libraries. In this paper, we detail the discovery of a novel non-hinge-binding ATP-competitive RIP1 inhibitor series that originated from this screen. This benzoxazepinone series maintains the high potency and selectivity found with 4, combined with the capability to obtain good oral PK profiles in rodents. The exquisite kinase

fluoro-2-nitrobenzene with the sodium alkoxide of BOC-Lserine gave carboxylic acid 5 with the desired S stereochemistry. A two-step sequence involving nitro reduction of 5 followed by HATU-mediated lactam formation gave the benzoxazepinone core 7 in 85% overall yield. Removal of the BOC protecting group with HCl (or TFA) and reaction with the requisite carboyxlic acids under amide coupling conditions provided amides 9−11. To determine any stereochemical preference for RIP1, the R enantiomer of 9, benzoxazepinone 12 (see Table 1), was prepared in an analogous manner starting from Boc-Dserine. The synthesis of N-methylated benzoxazepinone 14 from this core was readily accomplished in three steps and 50% overall yield from intermediate 7 by way of methyl iodide Table 1. In Vitro Profiles of Initial Hits

IC50 (nM)a compd 1 9 10 11 12 14

RIP1 FP 630 32 40 320 >10000 10

± ± ± ±

b

280 5.8 16 140

± 4.8

ADP-Gloc 200 16 7.9 − − 1.6

± 60 ± 14 ± 0.73

± 1.1

U937 320 200 400 1600 >10000 10

± ± ± ±

120 37 83 400

± 3.9

a

The IC50 values are averages of at least two determinations. bThe lower limit of sensitivity was ca. 10 nM. cConventional data analysis was used for less potent inhibitors (IC50 > 10 nM), whereas tightbinding analyses were used for more potent inhibitors (IC50 < 10 nM). 2164



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alkylation, BOC deprotection, and amide formation (Scheme 2).

Scheme 4

Scheme 2

Photoaffinity label 21 was prepared starting from 1-(3bromophenyl)propan-2-one, which was converted to 5-(3bromobenzyl)-1H-pyrazole-3-carboxylic acid (16) as outlined in Scheme 3. Coupling of acid 16 with benzoxazepinone Scheme 3

sequent steps to convert the trifluoromethyl ketone hydrate functionality to the diazirine are similar to those for the first photoaffinity label, namely, conversion to tosylate 25, formation of diaziridine 26, and oxidation to give the desired trifluoromethyldiazirine 27.

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RESULTS AND DISCUSSION In an effort to identify novel RIP1 inhibitor chemotypes, we performed affinity selections of GSK’s propriety collection of DNA-encoded small-molecule libraries against the RIP1 kinase domain (1−375).14 The selections were conducted in parallel against a panel of libraries representing a diversity of chemotypes. Although the majority of hits identified had a high degree of similarity to known kinase hinge-binding motifs, a structurally intriguing family of less enriched warheads was observed from a three-cycle amino acid core library. This library was constructed in a similar fashion as previously reported libraries using a split-and-pool strategy with three cycles of building blocks (BBs) to provide a total warhead diversity of approximately 7.7 billion (Figure 2).14−16 Interpretation of the affinity selection output from this library in a cubic scatter plot, as shown in Figure 2, where each axis corresponds to a BB cycle, conveniently allowed the visualization of three related lines. The three lines contained enantiopure benzo[b][1,4]oxazepin-4-one, an atypical amino acid building block, as BB2 and one of three distinct but structurally related amine caps as BB3. The specificity of the selection experiment was quite remarkable, as only one out of 632 amino acids as BB2 and three out of 6594 amine caps as BB3s were featured. The appearance of the lines also indicated that there was no preference for BB1, suggesting that BB1 contributes very little to the binding. The unique and atypical nature of the purported chemotype outside the traditional kinase space coupled with common structural elements among the three BB3s drew our attention. Taking into account all of the information inferred from the data cube and truncating the carboxyl linker for BB2, we prepared benzoxazepinones 9−11 as preliminary representative exemplars of the BB2 + BB3-1−3 combinations. To determine any stereochemical preference for RIP1, benzoxazepinone 12, the R enantiomer of compound 9, was also prepared.

intermediate 13 yielded m-bromophenyl benzoxazepinone 17. At this stage the trifluoroacetyl moiety was introduced by initial halogen−metal exchange and subsequent addition of 1trifluoroacetylpiperidine to give 18, the gem-diol hydrate of the trifluoromethyl ketone. The oxime of 18 was formed by reaction with hydroxylamine and converted into tosylate 19, after which reaction with liquid ammonia in ether led to the formation of diaziridine 20. Oxidation of the diaziridine using iodine provided the desired diazirine 21. The synthesis of the second trifluoromethyldiazirine photoaffinity label, 27, started with (S)-3-amino-7-bromo-4,5dihydro-1H-benzo[b]azepin-2(3H)-one,13 which after BOC protection of the amine and methylation of the ε-lactam gave 7-bromobenzazepinone 22 as outlined in Scheme 4. Halogen− metal exchange and subsequent addition of ethyl trifluoroacetate gave 23, the gem-diol hydrate of the trifluoromethyl ketone. After deprotection of the amine, coupling with 5-benzylisoxazole-3-carboxylic acid provided intermediate 24. The sub2165



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dendrogram is shown in Figure 3. Details of both profiles are available in the Supporting Information.

Figure 2. (top) Three-cycle amino acid library and (bottom) cube view of the selection output.

The benzoxazepinone analogues 9−11 were evaluated in both a fluorescence polarization (FP) binding assay and an ADP-Glo functional biochemical assay, along with reference RIP1 inhibitor 1 for comparison, as shown in Table 1. Since expression of the full-length protein containing the death domain led to insoluble protein not suitable for assay development, the kinase domain of RIP1 (1−375) was employed. Excitingly, benzoxazepinones 9−11 were found to be potent biochemically, with two analogues having IC50 < 100 nM (Table 1). A 5−10-fold decrease in potency was observed upon testing in the human monocytic U937 cellular assay, which measures the ability to block the necrotic cell death induced by treatment with TNF and the caspase inhibitor zVAD.fmk. Comparison of enantiomers 9 and 12 demonstrated a clear stereochemical preference, as the R enantiomer 12 was completely inactive. Most encouragingly for an unoptimized hit, compound 9 exhibited good oral systemic exposure in rat, with an area under the curve (AUC) of 2.2 μg·h/mL, a maximum concentration (Cmax) of 810 ng/mL, and a half-life (t1/2) of 2.9 h at a dose of 2 mg/kg. The good enzymatic and cellular activities, combined with low molecular weights and evidence of good oral PK profiles, established this series as an excellent starting point for optimization. While inhibitors 9−11 served the intended purpose of enabling a quick confirmation of the benzoxazepinone chemotype, capturing its gross structure and some preliminary SAR, N-methylated benzoxazepinone 14 (GSK’481) more accurately reflected the combination of BB2 and BB3-1.13 Upon profiling in the RIP1 assays, benzoxazepinone 14 not only showed an increase in biochemical activity but also exhibited excellent translation in the U937 cellular assay with IC50 = 10 nM (Table 1). Remarkably 14 showed complete specificity for RIP1 kinase over all other kinases tested when profiled against both a P33 radiolabeled assay screen at Reaction Biology Corporation (318 kinases) and a KINOMEscan competition binding assay at DiscoveRx (456 kinases). In both assays, 14 was tested at a concentration of 10 μM, which represents an estimated >7500-fold selectivity window based on the RIP1 ADP-Glo potency of 1.3 nM. The DiscoveRx TREEspot kinase

Figure 3. Kinase selectivity profile of compound 14 as shown by DiscoveRx KINOMEscan against 456 kinases assayed at 10 μM in duplicate. Compound selectivity is represented in a TREEspot kinase dendrogram view of the human kinome phylogenetic tree. Inhibition of RIP1 kinase (100%) is shown by the red circle; all of the other kinases tested were inactive, as indicated by the green circles.

Benzoxazepinone 14 exhibited a shift to lower potency in tight-binding ADP-Glo IC50 determinations with increasing ATP concentration, corresponding to a competitive model with Ki = 0.8 nM (see the Supporting Information). We also examined the binding kinetics of this series in RIP1 by both FP competitive binding and stopped-flow kinetic studies (see the Supporting Information). In the FP competitive binding analysis, both the ligand and the inhibitor were simultaneously exposed to the enzyme. We examined the binding kinetics of a number of initial representative exemplars of this series, specifically ones in which the benzoxazepinone heterocycle (9, 14) was replaced by benzothiazepinone dioxide (28), benzothiazepinone (29), or benzodiazepinone (30, 31), as part of our initial exploration of the SAR. The on rates were high and could not be accurately determined, but the off rates were lower and could be measured; the results are listed in Table 2 along with those for reference RIP1 inhibitor 1 for comparison. In the stopped-flow kinetic studies, the inhibitor and enzyme were rapidly mixed in a stopped-flow spectrometer, and the intrinsic tryptophan fluorescence signal was quenched in simple single-exponential fashion upon binding of the inhibitor (see the Supporting Information). Replots of the observed rate constants versus inhibitor concentration were linear to 5 μM, consistent with a one-step binding mechanism with kon = slope and koff = Ki·kon. RIP1 inhibitor 1 showed similar quenching of the tryptophan fluorescence and a one-step binding mechanism. In contrast, representative type-II hinge-binding inhibitors all exhibited increasing tryptophan fluorescence upon binding, some with complex binding mechanisms. This differentiation suggested that the benzoxazepinone template 2166



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Table 2. Binding Kinetics of Benzoxazepinones, Benzothiazepinones, and Benzodiazepinones

compd 1 9 14 28 29 30 31

ADP-Glo IC50 (nM)a

estimated FP t1/2 (min)b

± ± ± ± ± ± ±

625

approximately equivalent RIP1 FP potencies against human and cynomolgus monkey RIP1 but was >100-fold less potent against nonprimate RIP1. This reduced biochemical potency also translated to reduced cellular efficacy, as evidenced by a 300-fold reduction in cellular potency for compound 14 in blockage of the necrotic death (induced by TNF and zVAD.fmk) of murine fibrosarcoma L929 cells (IC50 = 3.2 μM) compared with human monocytic U937 cells (IC50 = 0.01 μM). This species selectivity was not observed with any of the DFG-out hinge binders we profiled. For example, compound 3 had both comparable RIP1 human and mouse FP potencies (IC50 = 0.025 and 0.05 μM, respectively) and comparable human U937 and murine L929 cellular efficacies (IC50 = 0.5 and 0.4 μM, respectively). To develop an understanding of the origin of this species selectivity, we analyzed residues in the RIP1 kinase domain where the amino acid sequence for human matched that of cynomolgus monkey but diverged for nonprimate species (rabbit, minipig, mouse, and rat). As shown in Figure 10, the sequence differences in the N-terminal lobe where the inhibitor binds were found to cluster around the C-helix and the activation loop. This led to the hypothesis that the key domains 2169



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Figure 11. Compound 14 inhibition of RIP1 WT S166 phosphorylation (ELISA) in human vs mouse plasmids overexpressed in HEK293T cells.

the three but still was able to increase the potency of this mutant murine RIP1 to within 40-fold of that of human RIP1. This suggests that the activation loop is the key region in murine RIP1 that is responsible for the lower efficacy against this series. Presumably the differences in amino acid sequence cause murine RIP1 to have reduced flexibility in adopting the conformational changes in the activation loop that are required to potently bind this series. Additional data supporting an important role for the activation loop in binding of this series in human versus murine RIP1 using stopped-flow kinetics in rat RIP1 are available in the Supporting Information. Despite the lower nonprimate RIP1 activity, we had sufficient potency and exposure to examine this series in an acute (3 h) in vivo mouse model evaluating protection from TNF-induced lethal shock. Benzodiazepinone 31 exhibited moderate activity against both mouse RIP1 FP (IC50 = 1.0 μM) and murine L929 cellular efficacy (IC50 = 3.2 μM) in blockage of necrotic death induced by TNF plus zVAD.fmk. Compound 31 had an acceptable PK profile in rodents (see Table 4), allowing further evaluation in vivo using a mouse model of TNF-induced lethal shock. In this model, injection of TNF plus the caspase inhibitor zVAD.fmk leads to a systemic inflammatory response characterized by hypotension, hepatitis, hypothermia, and bowel necrosis. Duprez et al.20 reported that RIP1 kinase inhibitor 1, administered intravenously, showed significant protection from hypothermia and death in this model. In addition, we demonstrated that oral administration of a type-II RIP1 inhibitor was efficacious in this model.11 Benzodiazepinone 31 was dosed orally 15 min prior to TNF/zVAD.fmk injection and showed 8, 24, and 82% protection from body temperature loss over 3 h compared with TNF/zVAD.fmk alone at doses of 3, 10, and 50 mg/kg, respectively (Figure 12). Under the assumption that the efficacy is Cmax-driven, the calculated IC50 concentration of 640 ng/mL from this in vivo study correlates well with the observed in vitro murine cellular L929 IC50 of 1190 ng/mL. The protection was observed to be sustained at up to 8 h in the highest dose group.

Figure 10. RIP1 kinase domain sequence differences between primate and nonprimate.

in human RIP1 that undergo substantial shifts or refolding to accommodate the binding of compound 14, namely, the activation loop, the C-helix, and the glycine-rich loop, may have less flexibility to do so in nonprimates as a result of a combination of these amino acid differences. To explore this further, we examined whether the potency of compound 14 against murine RIP1 could be increased by selectively mutating the amino acid sequence differences in mouse RIP1 to those of primates. One mouse mutant prepared contained 15 point mutations: five mutations in the activation loop (V159L, T164M, Q176L, S180D, and S181G), eight mutations in the Chelix (K46T, T49K, R53C, A54I, Y56H, V59A, G64A, and H68N), and two mutations in the glycine-rich loop (K21A and D22E). A second mutant focused on just the five mutations in the activation loop (V159L, T164M, Q176L, S180D, and S181G), while the third contained only a single mutation at V159L in the activation loop. These three RIP1 mouse mutants, along with wild-type human and mouse RIP1, were transfected into HEK293T cells. Overexpression of RIP1 plasmids in this cell line leads to autophosphorylation at Ser166, which can be detected using a RIP1pSer166 polyclonal antibody by Western analysis and ELISA.2 Benzoxazepinone 14 is a potent inhibitor of S166 phosphorylation in wild-type human RIP1 (IC50 = 2.8 nM) but is ineffective at reducing S166 phosphorylation for wild-type mouse RIP1 (IC50 > 10 μM), as shown in Figure 11. Benzoxazepinone 14 inhibits Ser166 phosphorylation in all three mouse RIP1 mutants more potently than in wild-type mouse, as shown in Figure 11 for the 15-point mutation (mutant 1), five-point mutation (mutant 2), and single-point mutation (mutant 3). The activation loop five-point mutation (mutant 2) was most effective at increasing the potency compared with wild-type mouse and was approximately within 4-fold of that of human RIP1. The single-point mutation at V159L in the mouse activation loop was the least effective of

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CONCLUSION A novel kinase inhibitor chemotype was discovered from a DNA-encoded amino acid core library screen. This chemotype was found to be highly potent for the inhibition of RIP1 in a stereochemical-dependent manner and exhibited complete selectivity against >450 off-target kinases. A preliminary SAR observed directly from the screen was subsequently confirmed off-DNA and identified a potential avenue for further series optimization. The binding mode of this novel pharmacophore was elucidated by photoaffinity labeling, hydrogen−deuterium 2170



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Table 4. Mouse and Rat PK Profiles for Benzodiazepinone 31 species

dose (mg/kg)

route

AUC0−∞ (μg·h/mL)

mouse rat rat

30 1 2

oral iv oral

2.0 ± 0.2 0.31 ± 0.06 0.46 ± 0.08

bioavailability (%)

75 ± 14

volume (L/kg)

half-life (h)

57 ± 11

2.2 ± 0.2

1.5 ± 0.3 0.85 ± 0.3 1.9 ± 0.5

in parts per million relative to an internal solvent reference. Apparent peak multiplicities are described as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), or m (multiplet). Coupling constants (J) are reported in hertz. LC−MS analysis was conducted using one of the following methods: Agilent method A LC−MS was performed on an Agilent 1100 series system using a Zorbax SB-C8 column (4.6 mm × 150 mm, 5 μm), eluting with 5−100% CH3CN/H2O (with 0.02% TFA) over 12.5 min followed by a hold for 2.5 min at a flow rate of 1.5 mL/min at 40 °C. Agilent method B LC−MS was performed on an Agilent 1200 series system using a Zorbax XDB-C8 column (4.6 mm × 75 mm, 3.5 μm), eluting with 5−95% CH3CN/H2O (with 0.1% TFA) over 4 min followed by a hold for 1 min at a flow rate of 2 mL/min at 40 °C. Agilent method C LC−MS was performed on an Agilent 1100 series system using a Thermo Hypersil Gold column (C18, 20 mm × 2.1 mm, 1.9 μm), eluting with 2−100% CH3CN/H2O (with 0.02% TFA) over 2 min at a flow rate of 1.4 mL/min at 55 °C. Sciex LC−MS was performed on a PE Sciex single-quadrupole 150EX system using a Thermo Hypersil Gold column (C18, 20 mm × 2.1 mm, 1.9 μm), eluting with 4−95% CH3CN/H2O (with 0.02% TFA) over 2 min at a flow rate of 1.4 mL/min at 55 °C. Waters LC−MS was performed using the same column and conditions as for Sciex LC−MS, but a Waters Acquity SQD UPLC/MS system was employed. Retention times (tR) are expressed in minutes and were obtained using UV detection at 214 or 254 nm. All of the tested compounds were determined to be ≥95% pure by LC−MS. (S)-5-Benzyl-N-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3-carboxamide (9). A solution of NBOC-L-serine (1.0 g, 4.87 mmol) in DMF (2 mL) was added dropwise over 5 min to a suspension of sodium hydride (4.09 g, 10.23 mmol) in DMF (8 mL). Vigorous gas evolution was observed. Once gas evolution had ceased, 1-fluoro-2-nitrobenzene (0.51 mL, 4.87 mmol) was added dropwise neat. The reaction mixture was allowed to stir at rt for 3 h and then partitioned between ethyl acetate (40 mL) and 0.5 M HCl solution (40 mL). The layers were separated, and the organic layer was washed with water (3 × 20 mL) and brine (20 mL) and then concentrated under reduced pressure to provide the crude product. Purification of the crude material by silica gel chromatography (25− 55% EtOAc in hexane) afforded (S)-2-((tert-butoxycarbonyl)amino)3-(2-nitrophenoxy)propanoic acid (5) (1.23 g, 3.77 mmol, 77% yield) as a reddish -yellow semisolid. 1H NMR (400 MHz, CDCl3) δ: 7.88 (dd, J = 8.46, 1.64 Hz, 1H), 7.52−7.61 (m, 1H), 7.06−7.15 (m, 2H), 5.68 (br d, 1H), 4.75 (br s, 1H), 4.60−4.72 (m, 1H), 2.07 (s, 2H), 1.48 (s, 9H). Sciex LC−MS: m/z 327 ([M + H]+), 653 ([2M + H]+), tR 0.88 min A suspension of 5 (1.1 g, 3.4 mmol) and palladium on carbon (0.11 g) was exposed to an atmosphere of hydrogen (balloon) overnight (∼20 h). Analysis of the crude reaction mixture by LC−MS confirmed the formation of the desired product. The slurry was filtered through a 0.45 μm PTFE autovial, and the filtrate was concentrated under reduced pressure to give (S)-3-(2-aminophenoxy)-2-((tertbutoxycarbonyl)amino)propanoic acid (6) (0.98 g, 3.3 mmol, 98% yield) as a pale-brown semisolid. The residue was used in the next step without further purification. 1H NMR (DMSO-d6) δ: 7.42 (br s, 1H), 6.74 (d, J = 7.1 Hz, 1H), 6.64−6.70 (m, 1H), 6.57−6.62 (m, 1H), 6.47 (td, J = 7.6, 1.6 Hz, 1H), 4.40 (d, J = 4.3 Hz, 1H), 4.24 (dd, J = 9.5, 4.9 Hz, 1H), 4.00 (dd, J = 9.6, 3.5 Hz, 1H), 1.41 (s, 9H). Sciex LC−MS: m/z 297 ([M + H]+), 593 ([2M + H]+), tR 0.65 min. HATU (1.245 g, 3.27 mmol) was added portionwise over 2 min to a solution of 6 (0.97 g, 3.27 mmol) and DIPEA (0.63 mL, 3.6 mmol) in DMSO (12 mL). The reaction mixture was stirred at rt for 30 min. Addition of water (30 mL) resulted in the formation of a precipitate.

Figure 12. Evaluation of benzodiazepinone 31 in a mouse TNFinduced lethal shock model measuring the reduction in body temperature loss.

exchange analysis, and cocrystallization in a truncated RIP1 (1− 294). These studies revealed that the inhibitor sits deep in the ATP binding pocket with no hinge engagement, allowing the benzylic group to occupy the same allosteric lipophilic pocket as the “necrostatin” series. The enzyme is in an inactive conformation with several key structural movements required in the glycine-rich loop, the C-helix, and the activation loop to accommodate the inhibitor. The complete kinase specificity observed for this benzoxazepinone series as well as other chemotypes represented by 1 and 4 suggests that RIP1 kinase may be unique in possessing the flexibility to make all of the required accommodations to adopt this binding conformation. An unusual species selectivity for primate versus nonprimate RIP1 was investigated by designing specific mutations in mouse RIP1 to recover potency to primate RIP1 levels. This indicated that the loss in potency is a result of the inability of the activation loop in nonprimate RIP1 to adopt the required conformation to bind this template. Despite this reduced potency, we were able to demonstrate efficacy in an in vivo mouse model evaluating protection from TNF-induced lethal shock. Tractable SAR, excellent potency, extraordinary kinase specificity, oral bioavailability in rodents, and an efficient synthetic route make this a highly promising template for lead optimization. Efforts toward this end will be reported in due course.

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clearance (mL min−1 kg−1)

EXPERIMENTAL SECTION

General Methods. Unless otherwise noted, starting materials and reagents were purchased from commercial sources and used without further purification. Air- or moisture-sensitive reactions were carried out under a nitrogen atmosphere. Anhydrous solvents were obtained from Sigma-Aldrich. Microwave irradiation was carried out in a Personal Chemistry Emrys Optimizer microwave reactor. Flash chromatography was performed using silica gel (EM Science, 230− 400 mesh) under standard techniques or silica gel cartridges (RediSep normal-phase disposable flash columns) on an Isco CombiFlash system. Reversed-phase HPLC purification was conducted on a Gilson HPLC (monitoring at a wavelength of 214 or 254 nm) with a YMC ODS-A C18 column (5 μm, 75 mm × 30 mm), eluting with 5−90% CH3CN in H2O with 0.1% TFA unless otherwise noted. 1H NMR spectra were recorded on a Bruker Advance or Varian Unity 400 MHz spectrometer as solutions in perdeuterated dimethyl sulfoxide (DMSO-d6), unless otherwise stated. Chemical shifts (δ) are reported 2171



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The mixture was cooled in an ice bath for 15 min and then filtered. The collected solid was washed with water and dried in vacuo (high vacuum) to afford (S)-tert-butyl-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate (7) (0.76 g, 2.73 mmol, 84% yield) as an off-white solid. TLC (50% EtOAc in hexane): Rf 0.55. 1H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 6.99−7.21 (m, 5H), 4.17−4.45 (m, 3H), 1.36 (s, 9H). Sciex LC−MS: m/z 279 ([M + H]+), 556 ([2M]+), tR 0.87 min. A solution of 7 (32.9 g, 118 mmol) in DCM (100 mL) was treated with HCl (4 N in dioxane) (148 mL, 591 mmol). Evolution of gas was observed, and the reaction mixture was stirred at rt for 4 h. The resulting brown solid was filtered off, washed with DCM (50 mL), and dried to give (S)-3-amino-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)one hydrochloride (8) (26.1 g, 116 mmol, 98% yield). 1H NMR (400 MHz, DMSO-d6) δ: 10.5 (s, 1H), 8.62 (br s, 3H), 7.02−7.25 (m, 4H), 4.61 (dd, J = 10.61, 5.81 Hz, 1H), 4.36−4.47 (m, 1H), 4.23−4.36 (m, 1H). Agilent method C LC−MS: m/z 179 ([M + H]+), tR 0.24 min. HATU (0.042 g, 0.11 mmol) was added in one portion to a solution of 5-benzylisoxazole-3-carboxylic acid (0.024 g, 0.12 mmol) and DIPEA (0.048 mL, 0.274 mmol) in DMSO (0.75 mL). After 5 min of stirring at rt, a solution of (S)-3-amino-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one trifluoroacetic acid salt (8) (0.032 g, 0.11 mmol) in DMSO (0.75 mL) was added dropwise to the intermediate HOAt adduct. The reaction mixture was allowed to stir at rt for 45 min. Purification of the crude material using reversed-phase HPLC [35− 65% acetonitrile/water (0.1% NH4OH modifier), C18 50 mm × 30 mm Gemini column, 47 mL/min] gave the title compound 9 (0.014 g, 0.039 mmol, 35% yield) as a white solid. 1H NMR (400 MHz, DMSOd6) δ: 10.12 (s, 1H), 8.85 (d, J = 8.08 Hz, 1H), 7.23−7.47 (m, 5H), 7.06−7.23 (m, 4H), 6.57 (s, 1H), 4.81 (ddd, J = 10.74, 7.96, 6.82 Hz, 1H), 4.35−4.62 (m, 2H), 4.23 (s, 2H). Sciex LC−MS: m/z 364 ([M + H]+), tR 0.97 min, >99% purity. Chiral HPLC analysis using a Chiralpak AD-H analytical column (150 mm × 4.6 mm, 5 μm), eluting with CH3CN plus 0.1% isopropylamine as a modifier as the mobile phase for 20 min, provided good separation of a racemic standard. The R enantiomer (12) eluted at 3.64 min, and the S enantiomer (9) eluted at 4.93 min. This indicated that the chiral purity of 9 prepared by this method was >99.9% ee, with none of the R enantiomer detected. (S)-N-(4-Oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)3-phenoxybenzamide (10). HATU (0.077 g, 0.203 mmol) was added in one portion to a solution of 3-phenoxybenzoic acid (0.043 g, 0.203 mmol) and DIPEA (0.089 mL, 0.508 mmol) in DMSO (0.75 mL). After 5 min of stirring at rt, a solution of (S)-3-amino-2,3dihydrobenzo[b][1,4]oxazepin-4(5H)-one trifluoroacetic acid salt (8) (0.059 g, 0.203 mmol) in DMSO (0.75 mL) was added dropwise to the intermediate HOAt adduct. The reaction mixture was allowed to stir at rt for 30 min. Purification of the crude material using reversedphase HPLC [40−70% acetonitrile/water (0.1% NH4OH modifier), C18 50 mm × 30 mm Gemini column, 47 mL/min] gave the title compound 10 (0.029 g, 0.077 mmol, 38% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ: 10.07 (s, 1H), 8.75 (d, J = 8.34 Hz, 1H), 7.62−7.75 (m, 1H), 7.48−7.61 (m, 2H), 7.34−7.48 (m, 2H), 7.09−7.31 (m, 6H), 6.99−7.09 (m, 2H), 4.80−4.98 (m, 1H), 4.51 (t, J = 10.61 Hz, 1H), 4.42 (dd, J = 10.48, 6.95 Hz, 1H). Sciex LC−MS: m/ z 375 ([M + H]+), tR 1.01 min, >99% purity. (S)-N-(4-Oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)5-pentyl-1H-pyrazole-3-carboxamide (11). To a solution of 5pentyl-1H-pyrazole-3-carboxylic acid (0.035 g, 0.190 mmol) and DIPEA (0.083 mL, 0.475 mmol) in DMSO (0.75 mL) was added HATU (0.072 g, 0.190 mmol) in one portion. After 5 min of stirring at rt, a solution of (S)-3-amino-2,3-dihydrobenzo[b][1,4]oxazepin4(5H)-one trifluoroacetic acid salt (8) (0.056 g, 0.19 mmol) in DMSO (0.75 mL) was added dropwise to the intermediate HOAt adduct. The reaction mixture was allowed to stir at rt for 1 h. LC−MS analysis indicated clean formation of the desired product, but some starting material still remained. Additional DIPEA (0.06 mL) and HATU (0.06 g) were added, and the mixture was allowed to stir for 30 min. Purification of the crude material using reversed-phase HPLC [30−60% acetonitrile/water (0.1% NH4OH modifier), C18 50 mm ×

30 mm Gemini column, 47 mL/min] gave the desired product as a viscous oil. The material was lyophilized to provide the title compound 11 (0.009 g, 0.026 mmol, 14% yield) as a white amorphous solid. 1H NMR (DMSO-d6) δ: 13.01 (br s, 1H), 10.13 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.10−7.18 (m, 4H), 6.39 (d, J = 1.8 Hz, 1H), 4.76−4.83 (m, 1H), 4.39−4.49 (m, 2H), 2.60 (t, J = 7.6 Hz, 2H), 1.59 (quin, J = 7.5 Hz, 2H), 1.19−1.36 (m, 4H), 0.86 (t, J = 6.9 Hz, 3H). Sciex LC−MS: m/z 343 ([M + H]+), tR 0.92 min, 98% purity. (R)-5-Benzyl-N-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3-carboxamide (12). Following the same procedure as described above for the preparation of 7 from N-BOC-Lserine, the R enantiomer was prepared starting from the opposite enantiomer N-BOC-D-serine. Then HATU (0.175 g, 0.46 mmol) was added in one portion to a solution of 5-benzylisoxazole-3-carboxylic acid (0.093 g, 0.46 mmol) and DIPEA (0.201 mL, 1.15 mmol) in DMSO (1 mL). After 5 min of stirring at rt, a solution of (R)-3-amino2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one trifluoroacetic acid salt (0.134 g, 0.46 mmol) in DMSO (1 mL) was added dropwise to the intermediate HOAt adduct. The reaction mixture was allowed to stir at rt for 2 h. Additional DIPEA (0.2 mL) and HATU (0.11 g) were added, and the mixture was allowed to stir for an additional 2 h. Purification of the crude material using reversed-phase HPLC [35− 65% acetonitrile/water (0.1% NH4OH modifier), C18 50 mm × 30 mm Gemini column, 47 mL/min] gave the title compound 12 (0.106 g, 0.29 mmol, 63% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 10.12 (s, 1H), 8.85 (d, J = 8.08 Hz, 1H), 7.23−7.48 (m, 5H), 7.07−7.23 (m, 4H), 6.57 (s, 1H), 4.81 (ddd, J = 10.74, 7.96, 6.82 Hz, 1H), 4.51 (t, J = 10.61 Hz, 1H), 4.42 (dd, J = 10.48, 6.69 Hz, 1H), 4.23 (s, 2H). Sciex LC−MS: m/z 364 ([M + H]+), tR 0.96 min, >99% purity. Chiral HPLC analysis using a Chiralpak AD-H analytical column (150 mm × 4.6 mm, 5 μm), eluting with CH3CN with 0.1% isopropylamine as a modifier as the mobile phase for 20 min provided good separation of a racemic standard. The R enantiomer (12) eluted at 3.64 min, and the S enantiomer (9) eluted at 4.93 min. This indicated that the chiral purity of 12 prepared by this method was 99.2% ee. (S)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3-carboxamide (14). Methyl iodide (8.09 mL, 129 mmol) was added dropwise over 15 min to a solution of 7 (30 g, 108 mmol) and Cs2CO3 (49.2 g, 151 mmol) in DMF (300 mL) stirred under nitrogen at rt. The reaction mixture was stirred at rt for 16 h. TLC (30% EtOAc in hexane, Rf 0.4) showed that the reaction was complete. The reaction mixture was poured into cold water (1500 mL), which formed a solid; the resultant solid was filtered, and the filter cake was washed with water (two times) and dried in vacuo to afford the crude compound. This was triturated with 5% Et2O in hexane (300 mL) to afford (S)-tert-butyl (5-methyl-4-oxo-2,3,4,5tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate (19 g, 62.7 mmol, 58.1% yield) as a brown solid. 1H NMR (DMSO-d6) δ: 7.47 (dd, J = 7.7, 1.6 Hz, 1H), 7.23−7.33 (m, 2H), 7.14−7.21 (m, 2H), 4.25−4.41 (m, 3H), 3.28 (s, 3H), 1.34 (s, 9H). Waters LC−MS: m/z 193 (M − BOC), 315 ([M + Na]+), tR 0.81 min, >99% purity. HCl (4 M, 71.8 mL, 287 mmol) was added to a solution of (S)-tertbutyl (5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate (28 g, 96 mmol) in DCM (300 mL), and the reaction mixture was stirred under nitrogen at rt for 3 h. The solvents were evaporated to dryness to yield the crude compound, which was triturated with Et2O (200 mL), filtered, and dried in vacuo to afford (S)-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one hydrochloride (13) (22.2 g, 97 mmol, 101% yield) as a brown solid. Waters LC−MS: m/z 193 ([M + H]+), tR 0.25 min, >99% purity. Hunig’s base (0.356 mL, 2.04 mmol) was added to a solution of 5benzylisoxazole-3-carboxylic acid (0.173 g, 0.85 mmol) and HATU (0.323 g, 0.85 mmol) in DMSO (1.5 mL). After 10 min of stirring at rt, 13 (0.155 g, 0.68 mmol) in DMSO (1.5 mL) was added. After 1 h of stirring at rt, additional HATU (0.11 g, 0.29 mmol) and Hunig’s base (0.2 mL, 1.1 mmol) were added. After another 1 h of stirring at rt, the reaction was quenched with cold water, and a brownish sticky solid precipitated out. The reaction mixture was extracted with EtOAc (25 mL × 2). The combined organic solution was washed with brine (15 2172



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mL) and concentrated in vacuo to give the crude product. This was purified using silica gel chromatography (eluting with 5−25% EtOAc in hexane) to give the title compound 14 (0.22 g, 0.577 mmol, 85% yield) as a light-beige solid. 1H NMR (DMSO-d6) δ: 8.85 (d, J = 8.1 Hz, 1H), 7.51 (dd, J = 7.8, 1.8 Hz, 1H), 7.25−7.38 (m, 7H), 7.20− 7.25 (m, 1H), 6.54 (s, 1H), 4.83 (dt, J = 11.6, 8.0 Hz, 1H), 4.58 (dd, J = 11.6, 9.9 Hz, 1H), 4.39 (dd, J = 9.9, 7.8 Hz, 1H), 4.22 (s, 2H), 3.30 (s, 3H). Waters LC−MS: m/z 378 ([M + H]+), tR 0.96 min, >99% purity. Agilent method A HPLC: tR 8.89 min, >99% purity. (S)-N-(5-Methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzyl)-1H-pyrazole-3-carboxamide (21). 1-(3-Bromophenyl)propan-2-one (5.0 g, 23.5 mmol) was dissolved in toluene (90 mL), and then diethyl oxalate (3.9 mL, 28.2 mmol) was added under nitrogen, followed by KOtBu (3.42 g, 30.5 mmol). The mixture was maintained at rt for 16 h. The reaction mixture was quenched with water (50 mL), and the pH was adjusted to ∼3. The organic layer was separated, washed with brine (50 mL), dried over MgSO4, and concentrated to a yellow oil. This oil was purified by silica gel column chromatography (10%−50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give ethyl 5-(3-bromophenyl)-2,4-dioxopentanoate (15) (4.9 g, 15.7 mmol, 66% yield) as a yellow oil. Agilent method C LC−MS: m/z 313 ([M + H]+), tR 1.24 min, 95% purity. Compound 15 (4.0 g, 12.8 mmol) was dissolved in ethanol (50 mL), and then hydrazine (0.4 mL, 12.8 mmol) was added under nitrogen. The reaction mixture was maintained at 75 °C for 16 h and then concentrated, and the residue was purified by silica gel column chromatography (10%−50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give ethyl 5-(3-bromobenzyl)-1H-pyrazole-3-carboxylate (3.2 g, 10.5 mmol, 82% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 7.36−7.44 (m, 2H), 7.28 (s, 1H), 7.12−7.25 (m, 1H), 4.39 (q, J = 7.07 Hz, 2H), 4.03 (s, 2H), 1.40 (t, J = 7.20 Hz, 3H). Agilent method C LC−MS: m/z 309 ([M + H]+), tR 1.11 min, 95% purity. Ethyl 5-(3-bromobenzyl)-1H-pyrazole-3-carboxylate (1.5 g, 4.85 mmol) was dissolved in THF (20 mL), and then a solution of lithium hydroxide (0.263 g, 11 mmol) dissolved in H2O (50 mL) was added. The mixture was maintained at 50 °C for 4 h and then neutralized by addition of 4 N HCl in dioxane dropwise into the reaction mixture at rt. The organic layer was separated and concentrated to give 5-(3bromobenzyl)-1H-pyrazole-3-carboxylic acid (16) (1.0 g, 3.56 mmol, 73% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 13.26 (br s, 1H), 7.38−7.59 (m, 2H), 7.20−7.36 (m, 2H), 6.51 (s, 1H), 3.97 (s, 2H). Agilent method C LC−MS: m/z 281 ([M + H]+), tR 0.88 min, 95% purity. 4-Methylmorpholine (0.41 g, 4.0 mmol) and HATU (0.575 g, 1.5 mmol) followed by (S)-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (13) (0.24 g, 1.26 mmol) were added to a stirred solution of 16 (0.4 g, 1.26 mmol) in DMF (1 mL) at rt. The reaction mixture was allowed to stir at rt for 16 h and then concentrated, and the residue was purified by silica gel column chromatography (20%− 80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-5-(3-bromobenzyl)-N(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-1Hpyrazole-3-carboxamide (17) (0.5 g, 1.11 mmol, 88% yield) as an offwhite solid. 1H NMR (400 MHz, CDCl3) δ: 8.07 (d, J = 7.83 Hz, 1H), 7.95 (s, 2H), 7.25−7.31 (m, 1H), 7.16−7.22 (m, 2H), 7.11−7.16 (m, 1H), 7.02−7.09 (m, 2H), 6.44 (s, 1H), 5.07 (dt, J = 11.31, 7.61 Hz, 1H), 4.59 (dd, J = 9.85, 7.58 Hz, 1H), 4.25 (dd, J = 11.12, 10.11 Hz, 1H), 3.92 (s, 2H), 3.35 (s, 3H). Agilent method C LC−MS: m/z 455 ([M + H]+), tR 1.12 min, 95% purity. Compound 17 (0.5 g, 1.1 mmol) was dissolved in THF (100 mL), and then methylmagnesium bromide (2.4 mL, 2.4 mmol, 1.0 M in dibutyl ether) was added at 0 °C dropwise. The mixture was maintained at 0 °C for 10 min and then cooled to −78 °C. tertButyllithium (1.5 mL, 2.5 mmol, 1.7 M in pentane) was added to the mixture. The mixture was maintained at −78 °C for 10 min, and then 2,2,2-trifluoro-1-(piperidin-1-yl)ethanone (0.2 g, 1.1 mmol) was added. The reaction mixture was maintained at −78 °C for 10 min

and then was concentrated and purified by silica gel column chromatography (20%−80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin3-yl)-5-(3-(2,2,2-trifluoro-1,1-dihydroxyethyl)benzyl)-1H-pyrazole-3carboxamide (18) (0.1 g, 0.21 mmol, 19% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 8.39 (d, J = 8.08 Hz, 1H), 7.90−8.01 (m, 2H), 7.52−7.59 (m, 1H), 7.43−7.50 (m, 1H), 7.24−7.27 (m, 2H), 7.15−7.21 (m, 2H), 6.46 (s, 1H), 5.17 (dt, J = 11.49, 7.89 Hz, 1H), 4.58 (dd, J = 9.85, 7.58 Hz, 1H), 4.28−4.39 (m, 1H), 4.09 (s, 2H), 3.39 (s, 3H). Agilent method C LC−MS: m/z 491 ([M + H]+), tR 0.97 min, 80% purity. Compound 18 (0.1 g, 0.21 mmol) was dissolved in pyridine (1 mL), and then hydroxylamine hydrochloride (0.015 g, 0.21 mmol) was added. The mixture was maintained at 70 °C for 10 min. The reaction mixture was concentrated, and the residue was partitioned between 0.01 N HCl (10 mL) and EtOAc (10 mL). The organic layer was washed with brine (10 mL), dried over MgSO4, and concentrated to give (S,Z)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro-1-(hydroxyimino)ethyl)benzyl)1H-pyrazole-3-carboxamide (0.13 g, presumed 100% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 8.56−8.69 (m, 2H), 8.07−8.26 (m, 1H), 7.73 (tt, J = 7.61, 1.86 Hz, 1H), 7.16−7.40 (m, 6H), 6.47− 6.58 (m, 1H), 5.14 (dt, J = 11.56, 7.74 Hz, 1H), 4.53−4.67 (m, 1H), 4.25−4.35 (m, 1H), 3.86−3.99 (m, 2H), 3.38 (s, 3H). Agilent method C LC−MS: m/z 488 ([M + H]+), tR 1.09 min, 90% purity. (S,Z)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin3-yl)-5-(3-(2,2,2-trifluoro-1-(hydroxyimino)ethyl)benzyl)-1H-pyrazole-3-carboxamide (0.13 g of crude material) was dissolved in pyridine (1 mL), and then TsCl (0.061 g, 0.32 mmol) was added. The reaction mixture was maintained at 110 °C for 16 h and then concentrated and purified by silica gel column chromatography (20%− 50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S,Z)-N-(5-methyl-4-oxo2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro-1((tosyloxy)imino)ethyl)benzyl)-1H-pyrazole-3-carboxamide (19) (0.047 g, 0.073 mmol, 28% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 8.56−8.69 (m, 2H), 8.07−8.26 (m, 1H), 7.73 (tt, J = 7.61, 1.86 Hz, 1H), 7.16−7.40 (m, 6H), 6.47−6.58 (m, 1H), 5.14 (dt, J = 11.56, 7.74 Hz, 1H), 4.53−4.67 (m, 1H), 4.25−4.35 (m, 1H), 3.86−3.99 (m, 2H), 3.38 (s, 3H). Agilent method C LC−MS: m/z 641 ([M + H]+), tR 1.38 min, 90% purity. Compound 19 (0.047 g, 0.073 mmol) was dissolved in Et2O (1 mL), and the reaction solution was cooled to −78 °C. Ammonia (0.5 mL) was condensed into the reaction vial in a dry ice bath. The reaction solution was warmed to rt over 1 h, with the excess ammonia allowed to evaporate off. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (20%− 80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-N-(5-methyl-4-oxo2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(3(trifluoromethyl)diaziridin-3-yl)benzyl)-1H-pyrazole-3-carboxamide (20) (0.031 g, 0.064 mmol, 87% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 8.29 (d, J = 7.83 Hz, 1H), 7.46−7.54 (m, 2H), 7.36 (t, J = 7.71 Hz, 1H), 7.26−7.30 (m, 4H), 7.17−7.22 (m, 1H), 6.48 (s, 1H), 5.16 (dt, J = 11.56, 7.86 Hz, 1H), 4.60 (t, J = 8.72 Hz, 1H), 4.25−4.36 (m, 1H), 4.04 (s, 2H), 3.41 (s, 3H), 2.78 (d, J = 8.08 Hz, 1H), 2.38−2.50 (m, 1H). Agilent method C LC−MS: m/z 487 ([M + H]+), tR 1.02 min, 90% purity. Compound 20 (0.031 g, 0.064 mmol) was dissolved in DCM (10 mL), and TEA (0.027 mL, 0.19 mmol) was added at 0 °C, followed by iodine (0.018 g, 0.07 mmol). The solution was washed with 1 N NaOH (10 mL), water (10 mL), and brine (10 mL). The mixture was then filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (20%−80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give the desired product as a colorless oil, which upon lyophilization gave the title compound 21 (0.016 g, 0.033 mmol, 52% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 11.15 (br s, 1H), 8.26 (br s, 1H), 7.33−7.43 (m, 1H), 7.24−7.28 2173



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(m, 4H), 7.19−7.23 (m, 1H), 7.14 (d, J = 7.83 Hz, 1H), 6.50 (s, 1H), 7.01 (s, 1H), 5.17 (dt, J = 11.49, 7.64 Hz, 1H), 4.64 (dd, J = 9.85, 7.58 Hz, 1H), 4.29 (dd, J = 11.37, 9.85 Hz, 1H), 4.03 (s, 2H), 3.38 (s, 3H). Agilent method C LC−MS: m/z 485 ([M + H]+), tR 1.26 min, >99% purity. (S)-5-Benzyl-N-(1-methyl-2-oxo-7-(3-(trifluoromethyl)-3H-diazirin-3-yl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (27). TEA (1.1 mL, 7.6 mmol) and ditert-butyl dicarbonate (1.24 mL, 5.35 mmol) were added to a mixture of (S)-3-amino-7-bromo-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one (1.3 g, 5.1 mmol) in DCM (50 mL). The reaction mixture was maintained at rt for 2 h and then diluted with water (50 mL). The organic layer was separated and concentrated. The residue was purified by silica gel column chromatography (20%−80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-tert-butyl (7-bromo-2-oxo-2,3,4,5-tetrahydro1H-benzo[b]azepin-3-yl)carbamate (1.95 g, presumed 100% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 9.07 (s, 1H), 7.21−7.41 (m, 2H), 6.85 (d, J = 8.34 Hz, 1H), 5.63 (d, J = 7.83 Hz, 1H), 4.25 (dt, J = 11.62, 7.58 Hz, 1H), 2.76−3.00 (m, 1H), 2.52−2.73 (m, 2H), 1.93−2.08 (m, 1H), 1.40 (s, 9H). Agilent method C LC−MS: m/z 299/301 (M − tBu), tR 1.33 min, 90% purity. Iodomethane (0.38 mL, 6.11 mmol) was added to a mixture of cesium carbonate (2.32 g, 7.13 mmol) and (S)-tert-butyl (7-bromo-2oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)carbamate (1.95 g) in DMF (1 mL) and THF (50 mL). The reaction mixture was maintained at rt for 20 h and then filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (10%−50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-tert-butyl (7-bromo-1-methyl-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-yl)carbamate (22) (0.52 g, 1.4 mmol, 27% yield) as a white solid. Agilent method C LC−MS: m/z 313/315 (M − tBu), tR 1.17 min, 90% purity. TMEDA (0.41 mL, 2.7 mmol) was dissolved in THF (5 mL), and the solution was cooled to −78 °C. n-Butyllithium (2.5 M in hexane) (1.1 mL, 2.7 mmol) was then added dropwise at −78 °C. Compound 22 (0.4 g, 1.1 mmol) was dissolved in THF (2 mL), and this solution was cooled to −78 °C. The bromide solution was the added dropwise to the butyllithium/TMEDA solution at −78 °C. This mixture was maintained at −78 °C for 30 min, and then ethyl 2,2,2-trifluoroacetate (0.52 mL, 4.3 mmol) was added dropwise at −78 °C. This mixture was maintained at −78 °C for 1 h and then slowly warmed to rt. The reaction mixture was quenched with MeOH (10 mL) followed by saturated aqueous NH4Cl (10 mL). The organic layer was separated and concentrated. The residue was purified by silica gel column chromatography (20%−80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-tert-butyl (1-methyl-2-oxo-7-(2,2,2-trifluoro-1,1-dihydroxyethyl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)carbamate (23) (0.084 g, 0.22 mmol, 20% yield) as a yellow oil. Agilent method C LC−MS: m/z 404 ([M + H]+), tR 1.35 min, 90% purity. Compound 23 (0.084 g, 0.22 mmol) was dissolved in DCM (2 mL), and 4 N HCl in dioxane (0.54 mL, 2.2 mmol) was added. The yellow solution was maintained at rt for 3 h and then concentrated to a yellow residue. This residue was dissolved in MeOH (1 mL) and then loaded onto a 2 g bicarbonate solid-phase extraction cartridge to make the free base from the HCl salt. The organic washes were concentrated, and the residue was dissolved in DCM (5 mL). To this solution were added 5-benzylisoxazole-3-carboxylic acid (0.043 g, 0.21 mmol), 4-methylmorpholine (0.068 g, 0.67 mmol), and HATU (0.096 g, 0.25 mmol), and the reaction mixture was allowed to stir at rt for 16 h. The reaction mixture was then concentrated, and the residue was purified by silica gel column chromatography (20%−50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S)-5-benzyl-N-(1-methyl-2-oxo-7(2,2,2-trifluoro-1,1-dihydroxyethyl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (24) (0.07 g, 0.15 mmol, 71% yield) as a brown oil. Agilent method C LC−MS: m/z 490 ([M + H]+), tR 1.33 min, 90% purity.

Compound 24 (0.06 g, 0.13 mmol) was dissolved in pyridine (1 mL), and hydroxylamine hydrochloride (0.013 g, 0.19 mmol) was added. The mixture was maintained at 90 °C for 30 min. The reaction mixture was concentrated, and the residue was partitioned between 0.01 N HCl (5 mL) and EtOAc (5 mL). The organic layer was washed with brine (5 mL), dried over MgSO4, and concentrated to a yellow oil. The oil was purified by silica gel column chromatography (2%− 10% MeOH in DCM, with 10% TEA in the MeOH). The collected fractions containing the product were combined and concentrated to give (S,Z)-5-benzyl-N-(1-methyl-2-oxo-7-(2,2,2-trifluoro-1(hydroxyimino)ethyl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (0.01 g, 0.02 mmol, 16% yield) as a colorless oil. Agilent method C LC−MS: m/z 487 ([M + H]+), tR 1.21 min, 80% purity. (S,Z)-5 -Be nzyl-N-(1-methy l-2-oxo-7-(2,2,2-trifluo ro-1(hydroxyimino)ethyl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (0.01 g, 0.02 mmol) was dissolved in DCM (2 mL), and TEA (5.16 μL, 0.037 mmol) and DMAP (0.502 mg, 4.11 μmol) were added at 0 °C, followed by TsCl (4.7 mg, 0.025 mmol). The reaction mixture was warmed to rt and maintained at rt for 2 h. The reaction mixture was then concentrated, and the residue was purified by silica gel column chromatography (20%−50% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give (S,Z)-5-benzyl-N-(1-methyl-2oxo-7-(2,2,2-trifluoro-1-((tosyloxy)imino)ethyl)-2,3,4,5-tetrahydro1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (25) (0.007 g, 0.01 mmol, 53% yield) as a colorless oil. Agilent method C LC−MS: m/z 461 ([M + H]+), tR 1.49 min, 90% purity. Compound 25 (0.007 g, 11 μmol) was dissolved in Et2O (0.5 mL), and then the reaction solution was cooled to −78 °C. Ammonia (0.5 mL) was condensed into the reaction vial in a dry ice bath. The reaction solution was warmed to rt over 30 min with proper ventilation. The reaction solution was then concentrated to a white residue, and the residue was purified by silica gel column chromatography (10%−50% EtOAc/DCM). The collected fractions containing the product were combined and concentrated to give (S)-5benzyl-N-(1-methyl-2-oxo-7-(3-(trifluoromethyl)diaziridin-3-yl)2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (26) (0.0044 g, 0.01 mmol, 83% yield) as a colorless oil. Agilent method C LC−MS: m/z 486 ([M + H]+), tR 1.29 min, 95% purity. Compound 26 (0.0044 g, 9.1 μmol) was dissolved in DCM (1 mL), and then TEA (3.79 μL, 0.027 mmol) was added at 0 °C, followed by iodine (0.00253 g, 10 μmol). The solution was washed with 1 N NaOH (1 mL), water (1 mL), and brine (1 mL). The organic layer was separated and concentrated. The residue was purified by silica gel column chromatography (20%−80% EtOAc in hexane). The collected fractions containing the product were combined and concentrated to give the title compound 27 (0.004 g, 0.01 mmol, 91% yield) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.72 (d, J = 7.18 Hz, 1H), 7.31−7.36 (m, 2H), 7.29 (d, J = 6.42 Hz, 1H), 7.17−7.26 (m, 4H), 6.31 (s, 1H), 7.05 (s, 1H), 4.51−4.58 (m, 1H), 4.11 (s, 2H), 3.42 (d, J = 1.13 Hz, 3H), 2.89 (td, J = 12.84, 8.31 Hz, 1H), 2.73 (m, J = 12.65, 12.65, 6.80 Hz, 1H), 2.61−2.69 (m, 1H), 2.01−2.09 (m, 1H). Agilent method C LC−MS: m/z 484 ([M + H]+), tR 1.40 min, >99% purity. (R)-5-Benzyl-N-(1,1-dioxido-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)isoxazole-3-carboxamide (28). Sodium bicarbonate (5.72 g, 68.1 mmol) was added to a solution of (R)-2((tert-butoxycarbonyl)amino)-3-mercaptopropanoic acid (5.02 g, 22.69 mmol) in water (32 mL), and a solution of 1-fluoro-2nitrobenzene (3.20 g, 22.69 mmol) in ethanol (40 mL) was slowly added while stirring at 25 °C. The reaction mixture was stirred at reflux for 4 h and cooled to rt. The ethanol was removed under vacuum, and the resulting aqueous phase was diluted with water (50 mL) and washed with ether (2 × 100 mL). The aqueous layer was acidified to pH 4 with 1 N HCl and extracted with DCM (2 × 300 mL). The organic layers were combined, washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo to afford (R)-2-((tertbutoxycarbonyl)amino)-3-((2-nitrophenyl)thio)propanoic acid as a yellow solid (8 g, 20.4 mmol, 90% yield). 1H NMR (400 MHz, CDCl3) δ: 8.17 (d, J = 7.83 Hz, 1H), 7.59 (d, J = 6.57 Hz, 2H), 7.22− 2174



Article

1H), 4.86 (dt, J = 11.75, 7.01 Hz, 1H), 4.13 (s, 2H), 3.98 (dd, J = 11.12, 6.57 Hz, 1H), 3.04 (t, J = 11.37 Hz, 1H). Waters LC−MS: m/z 380 ([M + H]+), tR 0.91 min, 96% purity. Agilent method A HPLC: tR 8.4 min, 95% purity. 3-Chloroperoxybenzoic acid (0.025 g, 0.111 mmol) was added at 0 °C to a solution of (R)-5-benzyl-N-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)isoxazole-3-carboxamide (0.02 g, 0.037 mmol) in DCM (15 mL). The reaction mixture was stirred for 1 h at 0 °C, warmed to rt, and then stirred for 2 h at rt. The reaction was quenched with cold 1 N NaOH (15 mL). The layers were separated, and the aqueous phase was extracted with DCM (15 mL). The combined organic solution was washed with 2.5% aqueous Na2S2O3 (15 mL) and brine (15 mL). After drying over MgSO4, filtration, and concentration, the residue was subjected to Isco chromatography (eluting with 5− 60% EtOAc in hexanes) to provide the title compound 28 (0.008 g, 0.019 mmol, 53% yield) as a gum. Trituration with DCM and hexane afforded the product as a white solid. 1H NMR (MeOH-d4) δ: 8.06 (dd, J = 7.8, 1.3 Hz, 1H), 7.81 (d, J = 1.5 Hz, 1H), 7.48−7.68 (m, 1H), 7.15−7.45 (m, 8H), 6.41 (s, 1H), 4.97 (dd, J = 11.6, 7.3 Hz, 1H), 4.10−4.31 (m, 2H), 3.89−4.07 (m, 2H). Waters LC−MS: m/z 412 ([M + H]+), tR 1.03 min, 95% purity. (R)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)isoxazole-3-carboxamide (29). Cs2CO3 (0.33 g, 1.02 mmol) was added to a solution of (R)-tert-butyl (4-oxo-2,3,4,5tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (0.2 g, 0.68 mmol) in DMF (5 mL). The reaction mixture was stirred at rt for 5 min, and then MeI (0.05 mL, 0.815 mmol) was added. The solvent was removed on a rotary evaporator, and the crude residue was dissolved in EtOAc (200 mL). The solution was washed with water (100 mL) and brine (100 mL), and the organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford (R)-tert-butyl (5methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (0.2 g, 0.51 mmol, 75% yield). Waters LC−MS: m/z 309 ([M + H]+), tR 0.86 min, 79% purity. HCl (4 M in dioxane, 7.05 mL, 28.2 mmol) was added to a solution of (R)-tert-butyl (5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (0.29 g, 0.94 mmol) in DCM (3 mL). The reaction mixture was stirred at 25 °C for 3 h. The solvent was removed on a rotary evaporator, and the resulting solid was washed with ether and hexane to yield (R)-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]thiazepin-4(5H)-one hydrochloride (0.2 g, 0.82 mmol, 87% yield), which was used without further purification. Waters LC−MS: m/z 209 ([M + H]+), tR 0.32 min, >99% purity. N1-((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.13 g, 0.674 mmol) and 1H-benzo[d][1,2,3]triazol-1ol hydrate (0.103 g, 0.674 mmol) followed by 4-methylmorpholine (0.202 mL, 1.839 mmol) were added to a solution of 5benzylisoxazole-3-carboxylic acid (0.021 g, 0.919 mmol) in DCM (30 mL). The reaction mixture was stirred at rt for 5 min, and then (R)-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]thiazepin-4(5H)-one hydrochloride (0.15 g, 0.613 mmol) was added. The reaction mixture was stirred at 25 °C for 5 h. LC−MS showed the presence of product and that the reaction was complete. The solvent was removed on a rotary evaporator, and the crude residue was dissolved in EtOAc (200 mL). The solution was washed with water (100 mL), 0.1 N HCl (100 mL), aqueous NaHCO3 (100 mL), and brine (100 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford the crude product. Isco purification (eluting with 0− 50% EtOAc in hexane) gave an oil, which was triturated with ether and hexane to yield a solid. This was filtered, rinsed with hexane, and collected to give the title compound 29 (0.2 g, 83%). 1H NMR (400 MHz, DMSO-d6) δ: 8.96 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 7.3 Hz, 1H), 7.59 (d, J = 4.0 Hz, 2H), 7.44−7.19 (m, 6H), 6.52 (s, 1H), 4.62−4.43 (m, 1H), 4.21 (s, 2H), 3.52 (dd, J = 6.8, 11.4 Hz, 1H), 3.71−3.44 (m, 1H), 3.30 (s, 3H). Waters LC−MS: m/z 394 ([M + H]+), tR 0.98 min, 97% purity. (S)-5-Benzyl-N-(2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl)isoxazole-3-carboxamide (30). HATU (0.15 g, 0.340 mmol) and N-methylmorpholine (0.174 mL, 1.6 mmol) were added to a solution of (S)-3-amino-4,5-dihydro-1H-benzo[b][1,4]-

7.44 (m, 1H), 6.73 (br s, 2H), 4.69 (m, 1H), 3.61 (d, J = 9.35 Hz, 1H), 3.35−3.51 (m, 1H), 1.45 (s, 6H), 1.41 (s, 3H). Waters LC−MS: m/z 343 ([M + H]+), tR 0.80 min, 87% purity. Ammonium chloride (0.25 g, 4.67 mmol) and zinc (1.5 g, 23.4 mmol) at 25 °C were added to a solution of (R)-2-((tertbutoxycarbonyl)amino)-3-((2-nitrophenyl)thio)propanoic acid (0.8 g, 2.3 mmol) in MeOH (100 mL). After 1 h of stirring at rt, the mixture was heated to 75 °C for 2 h. The resulting mixture was then filtered through Celite, and the Celite was washed with hot MeOH (2 × 100 mL). The combined organic washes were partially concentrated under vacuum (25 mL), and the residue was allowed to stand overnight at rt. Solid salts were removed by filtration, and then DCM (100 mL) and water (100 mL) were added to the filtrate. The resulting organic phase was washed with water (3 × 100 mL), dried over Na2SO4, and concentrated in vacuo to afford (R)-3-((2aminophenyl)thio)-2-((tert-butoxycarbonyl)amino)propanoic acid (0.7 g, 2.2 mmol, 96% yield) as a solid. Waters LC−MS: m/z 313 ([M + H]+), tR 0.69 min, >99% purity. N1-((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (2.2 g, 11.62 mmol) was added to a solution of (R)-3((2-aminophenyl)thio)-2-((tert-butoxycarbonyl)amino)propanoic acid (3.3 g, 10.56 mmol) in DCM (100 mL). The reaction mixture was stirred at rt for 5 min, and then 4-methylmorpholine (1.742 mL, 15.85 mmol) was added. The reaction mixture was stirred at 25 °C for 5 h. The solvent was removed on a rotary evaporator, and the residue was dissolved in EtOAc (200 mL). The solution was washed with water (100 mL), 0.1 N HCl (100 mL), aqueous NaHCO3 (100 mL), and brine (100 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford the crude product. Isco purification (eluting with 0−70% EtOAc in hexane) afforded (R)-tertbutyl (4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (1.5 g, 5.1 mmol, 48% yield). 1H NMR (400 MHz, CDCl3) δ: 7.73− 7.57 (m, 1H), 7.39 (td, J = 1.4, 7.6 Hz, 2H), 7.27−7.03 (m, 2H), 5.58 (br s, 1H), 4.49 (dt, J = 7.2, 11.8 Hz, 1H), 3.85 (dd, J = 6.7, 11.0 Hz, 1H), 2.95 (t, J = 11.4 Hz, 1H), 1.42 (s, 9H). Waters LC−MS: m/z 295 ([M + H]+), tR 0.77 min, 97% purity. HCl (4 M in dioxane, 0.425 mL, 1.7 mmol) was added to a solution of (R)-tert-butyl (4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3yl)carbamate (0.1 g, 0.34 mmol) in dioxane (3 mL). The reaction mixture was stirred at 25 °C for 18 h. The solvent was removed on a rotary evaporator, and the resulting crude solid was washed with ether to yield (R)-3-amino-2,3-dihydrobenzo[b][1,4]thiazepin-4(5H)-one hydrochloride (0.07 g, 0.3 mmol, 88% yield), which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ: 10.61 (s, 1H), 8.34 (br s, 2H), 7.65 (d, J = 7.83 Hz, 1H), 7.50 (td, J = 7.71, 1.52 Hz, 1H), 7.22−7.36 (m, 1H), 7.19 (d, J = 7.83 Hz, 1H), 3.95 (m, 1H), 3.72 (dd, J = 11.37, 6.82 Hz, 1H), 3.09−3.28 (m, 1H). Waters LC− MS: m/z 195 ([M + H]+), tR 0.23 min, 95% purity. N1-((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.064 g, 0.334 mmol) and 1H-benzo[d][1,2,3]triazol1-ol hydrate (0.051 g, 0.334 mmol) followed by 4-methylmorpholine (0.083 mL, 0.759 mmol) were added to a solution of 5benzylisoxazole-3-carboxylic acid (0.1 g, 0.455 mmol) in DCM (30 mL). The reaction mixture was stirred at rt for 5 min, and then (R)-3amino-2,3-dihydrobenzo[b][1,4]thiazepin-4(5H)-one hydrochloride (70 mg, 0.3 mmol) was added. Sufficient DMF was then added to fully dissolve all of the starting materials, resulting in a clear solution. The reaction mixture was stirred at 25 °C overnight. The solvents were removed on a rotary evaporator, and the crude material was dissolved in EtOAc (200 mL). This solution was washed with water (100 mL), 0.1 N HCl (100 mL), aqueous NaHCO3 (100 mL), and brine (100 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to afford a crude product. Isco purification (eluting with 0−70% EtOAc in hexane) afforded an oil. The oil was triturated with ether and hexane to yield a solid, which was filtered and rinsed with hexane and collected to yield (R)-5-benzyl-N(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)isoxazole-3-carboxamide (80 mg, 0.211 mmol, 70% yield). 1H NMR (400 MHz, CDCl3) δ: 7.79 (d, J = 7.07 Hz, 1H), 7.70 (dd, J = 7.71, 1.39 Hz, 1H), 7.46−7.58 (m, 1H), 7.19−7.46 (m, 7H), 7.08−7.19 (m, 1H), 6.33 (s, 2175



Article

diazepin-2(3H)-one21 (0.07 g, 0.40 mmol) and 5-benzylisoxazole-3carboxylic acid (0.08 g, 0.40 mmol) in DMF (2 mL). The reaction mixture was stirred at rt overnight and then diluted with EtOAc (20 mL), washed with water (20 mL) and brine (20 mL), and dried over Na2SO4. The solvents were removed to provide the title compound 30 (0.091 g, 0.25 mmol, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.62 (d, J = 7.58 Hz, 1H), 7.17−7.77 (m, 5H), 6.90−6.97 (m, 2H), 6.82−6.87 (m, 1H), 6.72 (td, J = 7.45, 1.26 Hz, 1H), 5.85 (d, J = 5.31 Hz, 1H), 6.59 (s, 1H), 4.59 (ddd, J = 10.80, 7.14, 4.29 Hz, 1H), 4.23 (s, 2H), 3.59 (ddd, J = 11.24, 6.57, 4.42 Hz, 1H), 3.41−3.52 (m, 1H). Waters LC−MS: m/z 363 ([M + H]+), tR 1.05 min, >99% purity. (S)-5-Benzyl-N-(1-methyl-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b][1,4]diazepin-3-yl)isoxazole-3-carboxamide (31). (S)tert-Butyl (2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl)carbamate22 was converted to (S)-3-amino-1-methyl-4,5-dihydro-1Hbenzo[b][1,4]diazepin-2(3H)-one as described by Zhang et al.21 N1((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.21 g, 1.093 mmol) was added to a solution of 5benzylisoxazole-3-carboxylic acid (0.2 g, 0.994 mmol) in DCM (30 mL). This solution was stirred at rt for 5 min, and then (S)-3-amino-1methyl-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (0.19 g, 0.994 mmol) and 4-methylmorpholine (0.35 g, 3.48 mmol) were added. The reaction mixture was stirred at 25 °C for 15 h and then concentrated under reduced pressure to afford the crude product. Purification by Isco silica gel chromatography (eluting with 0−60% EtOAc in hexane) gave an oil, which was triturated with ether and hexane to yield a solid. This was filtered and rinsed with hexane to afford the title compound 31 (0.2 g, 0.531 mmol, 54% yield). 1H NMR (DMSO-d6, 400 MHz) δ: 8.66 (d, J = 7.8 Hz, 1H), 7.22−7.44 (m, 6H), 7.07−7.17 (m, 1H), 6.96−7.06 (m, 2H), 6.55 (s, 1H), 5.32 (d, J = 4.5 Hz, 1H), 4.65 (dt, J = 11.7, 7.0 Hz, 1H), 4.22 (s, 2H), 3.47−3.69 (m, 2H), 3.33 (s, 3H). Agilent method A LC−MS: m/z 377 ([M + H]+), tR 7.71 min, >99% purity. (S)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-1H-pyrazole-3-carboxamide (32). HATU (0.146 g, 0.385 mmol) was added to a solution of (S)-3-amino-5methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one hydrochloride (8) (0.08 g, 0.35 mmol), 3-benzyl-1H-pyrazole-5-carboxylic acid (0.074 g, 0.37 mmol), and Hunig’s base (0.153 mL, 0.875 mmol) in DMSO (1.3 mL). After 1 h of stirring at rt, the reaction mixture was diluted with MeOH (1 mL). After filtration through an Acrodisc CR 25 mm syringe filter with a 0.2 μm PTFE membrane, the solution was evaporated to give a crude product. HPLC purification (Waters Sunfire 30 mm × 150 mm column, eluting with CH3CN/30−70% H2O with 0.1% TFA at a flow rate of 50 mL/min) yielded the title compound 32 (0.05 g, 0.132 mmol, 38% yield). 1H NMR (400 MHz, DMSO-d6) δ: 8.11 (br s, 1H), 7.40−7.59 (m, 1H), 7.13−7.40 (m, 9H), 6.41 (br s, 1H), 4.83 (dt, J = 11.42, 7.84 Hz, 1H), 4.51 (t, J = 10.67 Hz, 1H), 4.27−4.44 (m, 1H), 3.98 (s, 2H), 3.31 (s, 3H). Agilent method B LC−MS: m/z 377 ([M + H]+), tR 2.07 min, >99% purity. (S)-5-Benzyl-N-(1-methyl-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-yl)isoxazole-3-carboxamide (33). A mixture of 5-benzylisoxazole-3-carboxylic acid (0.075 g, 0.371 mmol) and O-(7azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.153 g, 0.4 mmol) in CH3CN (4 mL) was stirred at rt for 2 h. N-Methylmorpholine (0.102 mL, 0.926 mmol) and (S)-3-amino-1methyl-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one hydrochloride (0.07 g, 0.309 mmol) were added, and the resulting mixture was stirred overnight. The reaction solution was concentrated on a rotary evaporator, and the resulting crude product was purified by Isco silica gel chromatography (0−40% EtOAc in hexanes). The pure-productcontaining fractions were combined and concentrated to yield the title compound 33 (0.071 g, 0.189 mmol, 61% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 8.70 (d, J = 7.83 Hz, 1H), 7.18−7.46 (m, 9H), 6.53 (s, 1H), 4.33 (dt, J = 11.18, 8.18 Hz, 1H), 4.21 (s, 2H), 3.30 (s, 3H), 2.60−2.81 (m, 2H), 2.10−2.37 (m, 2H). Agilent method A LC−MS: m/z 376 ([M + H]+), tR 8.92 min, 99% purity. Fluorescence Polarization Binding Assay. A fluorescencepolarization-based binding assay was developed to quantify the

potency of novel small-molecule inhibitors at the ATP binding pocket of RIP1 by competition with a fluorescently labeled ATP-competitive ligand, (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino)propyl]amino}2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6′]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate. Typically test compounds were prepared in 100% DMSO, and 100 nL aliquots were dispensed into individual wells of a 384-well plate. A 5 μL aliquot of RIP1 in assay buffer (50 mM HEPES, pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.5 mM DTT, and 0.02% CHAPS) was added to each well and incubated with compound for 10 min at room temperature. Following this preincubation, 5 μL of ligand prepared in assay buffer was added to each well, and the plate was incubated for 20 min at room temperature. Samples were read on an instrument capable of measuring fluorescence polarization (excitation 530 nm; emission 580 nm; 561 nm dichroic). The final concentrations of human RIP1 (1−375) enzyme and fluorescent ligand were 10 nM and 5 nM (approximately Kd), respectively. Non-human (rat, rabbit, dog, monkey, minipig, and mouse) RIP1 FP binding assays were performed following the same protocol as described above, with the exception of the enzyme concentrations used in the assays for minipig (30 nM) and mouse (100 nM) species. All of the FP binding IC50 data were normalized to the mean of 16 high and 16 low control wells on each plate. Test compound inhibition was expressed as percent inhibition of internal assay controls. Compounds were tested with at least n = 2 in the RIP1 FP binding assay, and the mean IC50 values are reported. A four-parameter curve fit was used to determine the potency of the compounds: Y=

(a − d) +d ⎡⎣1 + (x /c)b ⎤⎦

(1)

where Y is the % inhibition, x is the inhibitor concentration, a is the minimum value of Y, b is the Hill slope, c is the IC50 value, and d is the maximum value of Y. ADP-Glo Activity Assay. The catalytic activity of RIP1 was quantified by monitoring the conversion of ATP to ADP from both autophosphorylation and ATPase activity using the Promega ADP-Glo kinase kit. Test compounds were prepared in 100% DMSO, and 100 nL was dispensed into each well of a 384-well assay plate. Human RIP1 (1−375) and ATP were prepared in assay buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.02% CHAPS, and 0.5 mg/mL BSA) to achieve a final assay concentrations of 10 nM and 50 μM, respectively. Next, 5 μL of human RIP1 (1− 375) was added to the test compounds at twice the final assay enzyme concentration, immediately followed by the addition of 5 μL of ATP at twice the final assay concentration. Enzyme and ATP were incubated at room temperature for 4 h. Following the 4 h reaction, 5 μL of Promega ADP-Glo reagent I with 0.02% CHAPS was added to quench the reaction and deplete unconsumed ATP. The quenched reaction mixture was incubated at room temperature for 1 h. Next, 5 μL of Promega ADP-Glo detection reagent II with 0.02% CHAPS was added to each well, resulting in the conversion of ADP (the reaction product) to ATP, which activates a light-generating reaction between luciferase and luciferin. Following a 30 min incubation, the luminescence was detected on a PerkinElmer ViewLux microplate imager. ADP-Glo inhibition data were normalized to the mean of 16 high and 16 low control wells on each plate and expressed as fractional activity relative to the mean of the DMSO-treated high control. Test compound inhibition was determined using either a fourparameter curve fit (eq 1) or a tight-binding curve fit for compounds whose potencies were less than the detection limit of the assay (approximately half the enzyme concentration): 2176



Article

vi = v0 − ([I] − [E] + K iapp) +

SMILES strings and IC50 values for 1, 9−12, 14, 21, and 27−33 (CSV) ([I ] − [E] + K iapp)2 + 4[E]K iapp

Accession Codes

Coordinates and structure factors for the cocrystal structure of RIP1 (1−294, C34A, C127A, C233A, C240A) and benzoxazepinone 14 have been deposited in the Protein Data Bank with the accession number 5HX6.

2[E] (2) where vi/v0 is the fractional activity remaining relative to uninhibited controls, [I] is the total inhibitor concentration, [E] is the total is the IC50. ADP-Glo data are enzyme concentration, and Kapp i presented as mean IC50 values from at least n = 2 determinations. Biological in Vitro U937 Cell Assay. The efficacies of RIP1 inhibitors were tested in vitro using human monocytic leukemia U937 cells in a necroptosis assay.23 U937 cells were acquired from ATCC (cat. no. CRL-1593.2) and banked in liquid nitrogen. For the assay, frozen cells were thawed and diluted to 5 ×105 cells/mL in phenol redfree Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. (3S)-5-(2,6-Difluorophenoxy)-3[[(2S)-3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]4-oxopentanoic acid hydrate (QVD-OPh), a broad-spectrum caspase inhibitor, prepared as a 10 mM stock solution in neat DMSO, was added to the cell solution to achieve a final concentration of 25 μM. TNFα, prepared as a 100 μg/mL stock in phosphate-buffered saline containing 0.1% bovine serum albumin, was added to the cell solution to achieve a final concentration of 100 ng/mL. A 40 μL aliquot of cell suspension was added to each well of a luminescence-compatible white assay plate prestamped with 40 nL of compound in neat DMSO. Lidded plates were incubated for 24 h at 37 °C, 5% CO2. The next day, the cells were lysed, and the ATP content (viability) was measured via the addition of 20 μL of Cell Titer-Glo Luminescent Cell Viability reagent (Promega Corporation). Plates were incubated in the dark for 15 min followed by luminescence detection on an appropriately equipped plate reader. The ability of test compound to rescue cells from TNFα/QVD-induced necroptosis was expressed as percent survival relative to wells treated with 10 μM compound 1 as a positive control. For concentration response experiments, normalized data were fit and IC50 values determined using conventional techniques. All of the data are shown as mean ± standard error of the mean. Biological in Vitro L929 Cell Assay. Mouse L-cells NCTC 929 (L929) were acquired from ATCC (cat. no. ATCC CCL-1) and banked in liquid nitrogen. The assay protocol was the same as described for the U937 cell assay. Biological in Vivo Assay. The efficacy of RIP1 inhibitors was tested in mice in vivo using a TNF-driven systemic inflammatory response syndrome model.20 A total of seven mice per dose group were orally predosed with vehicle or compound 31 at doses of 3, 10, and 50 mg/kg 15 min before iv administration of mouse TNF (30 μg/ mouse) and zVAD (0.4 mg/mouse). Temperature loss in the mice was measured by rectal probe. The study was terminated after 3 h when the control group lost 7 °C, with the exception of the 50 mg/kg dose group which was continued out for 8 h. All of the data are shown as mean ± standard error of the mean. All studies involving the use of animals were conducted after review by the GlaxoSmithKline (GSK) Institutional Animal Care and Use Committee and in accordance with the GSK Policy on the Care, Welfare, and Treatment of Laboratory Animals.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: 610-917-6873. Author Contributions ⊥

J.B. and P.J.G. contributed equally to this work.

Notes

The authors declare the following competing financial interest(s): All of the authors are current employees and stockholders of GlaxoSmithKline.

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ABBREVIATIONS USED AMPK, AMP-activated protein kinase; BB, building block; BCR-ABL, breakpoint cluster region abelson murine leukemia viral oncogene homologue 1; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DFG, Asp-Phe-Gly; DIPEA, diisopropylethylamine; DTT, dithiothreitol; FasL, Fas ligand; FP, fluorescence polarization; HATU, 1-[bis(dimethylamino)methylene]-1H1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HDX-MS, hydrogen−deuterium exchange mass spectrometry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RIP1, receptor interacting protein 1; PTFE, polytetrafluoroethylene; QVD, quinolyl-Val-Asp-Oph; SMAC, second mitochondrial-derived activator of caspases; TEA, triethylamine; TLR3, toll-like receptor 3; TLR4, toll-like receptor 4; TNFR1, tumor necrosis factor receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; TSZ, TNF/SMAC-mimetic/ zVAD.fmk; zVAD.fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone

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REFERENCES

(1) Ofengeim, D.; Yuan, J. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death. Nat. Rev. Mol. Cell Biol. 2013, 14, 727−736. (2) Berger, S. B.; Kasparcova, V.; Hoffman, S.; Swift, B.; Dare, L.; Schaeffer, M.; Capriotti, C.; Cook, M.; Finger, J.; Hughes-Earle, A.; Harris, P. A.; Kaiser, W. J.; Mocarski, E. S.; Bertin, J.; Gough, P. J. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPINdeficient mice. J. Immunol. 2014, 192, 5476−5480. (3) Silke, J.; Rickard, J. A.; Gerlic, M. The diverse role of RIP kinases in necroptosis and inflammation. Nat. Immunol. 2015, 16, 689−697. (4) Pasparakis, M.; Vandenabeele, P. Necroptosis and its role in inflammation. Nature 2015, 517, 311−320. (5) Degterev, A.; Hitomi, J.; Germscheid, M.; Ch’en, I. L.; Korkina, O.; Teng, X.; Abbott, D.; Cuny, G. D.; Yuan, C.; Wagner, G.; Hedrick, S. M.; Gerber, S. A.; Lugovskoy, A.; Yuan, J. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 2008, 4, 313−321. (6) Degterev, A.; Huang, Z.; Boyce, M.; Li, Y.; Jagtap, P.; Mizushima, N.; Cuny, G. D.; Mitchison, T. J.; Moskowitz, M. A.; Yuan, J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 2005, 1, 112−119. (7) Christofferson, D. E.; Li, Y.; Hitomi, J.; Zhou, W.; Upperman, C.; Zhu, H.; Gerber, S. A.; Gygi, S.; Yuan, J. A novel role for RIP1 kinase in mediating TNFα production. Cell Death Dis. 2012, 3, e320.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01898. Details of enzyme preparations, mode of inhibition study, compound off and on rate constant determinations, kinase selectivity screening, photoaffinity labeling, HDXMS and crystallization procedures, and species-specific dose−response curves (PDF) 2177



Article

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DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4

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