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-4-ones as highly potent and mono-selective receptor interacting protein 1 (RIP1) 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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01898 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 14, 2016

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Journal of Medicinal Chemistry

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DNA-encoded library screening identifies benzo[b][1,4]oxazepin-4-ones as highly potent and mono-selective receptor interacting protein 1 (RIP1) 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 DPU 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.

ACS Paragon Plus Environment

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ABSTRACT. The recent discovery of the role of RIP1 kinase in 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 mono-kinase selectivity for RIP1, plus unique species selectivity for primate versus non-primate 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 over known RIP1 inhibitors in combining high potency and kinase selectivity with good PK 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.

INTRODUCTION Receptor interacting protein 1 (RIP1) kinase activity has recently emerged as an important driver of TNF-mediated inflammation and pathology.1 This notion is strongly supported by genetic evidence in mice, where mutations which 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 pro-inflammatory cytokine production and some forms of apoptosis.3 Furthermore, in addition to its role downstream of TNFR1, RIP1 has also been shown to be a critical driver of inflammation downstream of various other pathways (TNFR1, FasL, TRAIL,



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TLR3 and TLR4).4 Thus, inhibition of RIP1 activation is likely to have a broad therapeutic potential for multiple inflammatory diseases. Degterev et al were first to identify a number of RIP1inhibitor templates which were termed “Necrostatins”.5 They were initially identified from cellular screening through their ability to block necrotic death of monocytic U937 cells induced by treatment with TNF and the caspase inhibitor 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), had excellent kinase selectivity.7 The co-crystal 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 SAR profile coupled with moderate potency and poor pharmacokinetic properties has limited their development in drug discovery.9 Recently, Degterev et al further disclosed the potent and selective RIP1 inhibitor 2, which is a hybrid of 1 and the type II kinase inhibitor of BCR-ABL Ponatinib.10 This hybrid increased the RIP1 potency compared to 1 whilst 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 addressing during lead-optimization.


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The developability limitations of the hits identified from traditional kinase inhibitor space led us to carry out a high-throughput screen of the ~2 million GSK compound collection. After some initial optimization, this effort led to the identification of 1-pivaloyl-5-phenyl 4,5dihydropyrazole (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 RIP1against 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 selectivity and favorable developability profile made this series an excellent starting point for lead optimization.

Figure 1. Structure of known RIP1 inhibitors.

CHEMISTRY



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Synthesis of benzoxazepinones 9-11 was undertaken as outlined in Scheme 1. Nucleophilic aromatic substitution of 1-fluoro-2-nitrobenzene with the sodium alkoxide of Boc-L-serine 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 the amides 9-11. To determine any stereochemical preference for RIP1, the corresponding (R) enantiomer benzoxazepinone (12) (Table 1) was prepared in an analogous manner starting from Boc-Dserine.

Scheme 1.

Synthesis of the N-methylated benzoxazepinone 14 from this core was readily accomplished in three steps and 50% overall yield from intermediate 13 by way of methyl iodide alkylation, BOC deprotection, and amide formation (Scheme 2).

Scheme 2.


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Photoaffinity label 21 was prepared starting from 1-(3-bromophenyl)propan-2-one, which was converted to 2-(5-(3-bromobenzyl)-1H-pyrazol-3-yl)-2-oxoacetic acid 16 as outlined in Scheme 3. Coupling of acid 16 with benzoxazepinone intermediate 13 yielded the meta-bromo phenyl benzoxazepinone 17. At this stage the trifluoroacetyl moiety was introduced by first halogenmetal exchange and subsequent addition of 1-trifluoroacetyl piperidine to give the gem-diol hydrate of the trifluoromethylketone 18. The oxime of 18 was formed from reaction with hydroxylamine and converted into the 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.

Scheme 3.



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The synthesis of the second trifluoromethyl diazirine photoaffinity label 27 started with (S)-3amino-7-bromo-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one,13 which after BOC protection of the amine and methylation of the ε-lactam gave 7-bromo-benzazepinone 22, as outline in Scheme 4. Halogen-metal exchange and subsequent addition of ethyl trifluoroacetate gave the gem-diol hydrate of the trifluoromethylketone 23. After deprotection of the amine, coupling with 5benzyl-isoxazole acid-3-carboxylic acid provided intermediate 24. The subsequent steps to convert the trifluoromethylketone hydrate functionality to the diazirine are similar to that for the first photoaffinity label; namely, conversion to the tosylate 25, formation of diaziridine 26 and oxidation to the desired trifluoromethyl diazirine 27.

Scheme 4.


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RESULTS AND DISCUSSION In an effort to identify novel RIP1 inhibitor chemotypes, we performed an affinity selection of GSK’s propriety collection of DNA-encoded small-molecule libraries against the RIP1 kinase domain (1-375).14 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 lower enriched warheads was observed from a three-cycle amino acid core library. This library was constructed in a similar fashion to previously reported libraries using a split-and-pool strategy with three cycles of building blocks (BBs) to provide approximately 7.7 billion in total warhead diversity (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 for the visualization of three related lines. All three lines contained the enantiopure benzo[b][1,4]oxazepin-4-one as BB2, an atypical amino acid building block, and three distinct, but structurally related, amine-capping BB3s. The specificity of the selection experiment was quite remarkable as only 1 of 632 amino acid BB2s, in conjunction with 3 of 6594 amine-



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capping BB3s, were featured. The appearance of the lines also indicated there was no preference for BB1, suggesting that BB1 contributed very little to binding. Figure 2. Three-Cycle Amino Acid Library and Cube View of Selection Output.

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 the information inferred from the data cube and truncating the carboxyl linker for BB2, benzoxazepinones 9-11 were prepared 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. The benzoxazepinone analogs 9-11 were evaluated in both a fluorescence polarization (FP) binding assay and an ADP-Glo functional biochemical assay as shown in Table 1 along with reference RIP1 inhibitor 1 for comparison. 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


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biochemically with two analogs having IC50’s < 100 nM, as shown in Table 1. A 5-10 fold decrease in potency was observed upon testing in the human monocytic U937 cellular assay, measuring 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 the rat, with an AUC of 2.2 µg.h/mL, a Cmax of 810 ng/mL and a half-life of 2.9 hours, at a dose of 2 mg/kg. The good enzymatic and cellular activities, combined with low molecular weights and evidence of good oral PK, established this series as an excellent starting point for optimization.

Table 1. In vitro profiles of initial hitsa

RIP1 FPb

ADP-Gloc

U937

IC50 (nM)

IC50 (nM)

IC50 (nM)

1

630 ± 280

200 ± 60

320 ± 120

9

32 ± 5.8

16 ± 14

200 ± 37

10

40 ± 16

7.9 ± 0.73

400 ± 83

11

320 ± 140

-

1,600 ± 400

12

>10,000

_

>10,000

Cpd



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14

10 ± 4.8

1.6 ± 1.1

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10 ± 3.9

a

IC50 values are the average of at least two determinations. bLower limit of sensitivity is ca. 10 nM. cConventional data analysis was used for less potent inhibitors (IC50 > 10 nM); whereas tight binding analyses was used for more potent inhibitors (IC50 < 10 nM).

While inhibitors 9-11 served the intended purpose of allowing for a quick confirmation of the benzoxazepinone chemotype, capturing its gross structure and some preliminary SAR, Nmethylated 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 an IC50 = 10 nM (Table 1). Remarkably benzoxazepinone 14 showed complete specificity for RIP1 kinase over all other kinases tested when profiled against both a P33 radiolabeled assay screen at Reaction Biology Corp (318 kinases) and a competition binding assay KINOMEscan at DiscoveRx (456 kinases). In both assays benzoxazepinone 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 dendrogram is shown in Figure 3. Details on both profiles are available in the Supporting Information section.

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 other kinases tested were inactive, as indicated by green circles.


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Benzoxazepinone 14 exhibited a shift to lower potency in tight binding ADP-Glo IC50 determinations with increasing ATP concentrations, corresponding to a competitive model with Ki = 0.8 nM (see Supporting Information). We also examined the binding kinetics of this series in RIP1 by both fluorescence polarization (FP) competitive binding and stopped-flow kinetic studies (see Supporting Information). In the FP competitive binding analysis, both the ligand and inhibitor were simultaneously exposed to enzyme. We examined the binding kinetics of a number of intial representative exemplars of this series; specifically where the benzoxazepinone heterocycle (9¸ 14) was replaced by benzothiazepinone dioxide (28), benzothiazepinone (29), and benzodiazepinone (30, 31), as part of our initial exploration of the SAR. The on-rates were fast and could not be accurately determined; whereas the slower off-rates could be measured and are listed in Table 2 along with reference RIP1 inhibitor 1 for comparison. With the stopped-flow kinetics, 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 Supporting Information). Re-plots of the observed rate constants



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versus inhibitor concentration were linear to 5 µM consistent with a one-step binding mechanism with kon = slope and koff calculated from Ki times kon. RIP1 inhibitor 1 showed similar quenching of the tryptophan fluorescence and 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 was binding to a RIP1 conformation similar to 1, in contrast to type II hinge-binders (see Supporting Information). Although on-rate constants vary by up to 5 fold, in general, the highly potent inhibitors (compounds 14, 29-31) have slower off-rates than their less potent analogs (compounds 1, 9 and 28).

Table 2. Binding Kinetics of Benzoxazepinone/Benzothiazepinones/Benzodiazepinones

ADP-Glo

Estimated FP

Stopped-flow

Calculated

IC50 (nM)a

t1/2 (min.)b

kon (µM-1s-1)c

t1/2 (min.)b

1

200 ± 60

10

>625

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 non-primate 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 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 non-primates due to 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: 5 mutations at the activation loop (V159L, T164M, Q176L, S180D, S181G), 8 mutations at the C-Helix (K46T, T49K, R53C, A54I, Y56H, V59A, G64A, H68N) and 2 at the glycine-rich loop (K21A, D22E). A second mutant focused just on the 5 mutations at the activation loop (V159L, T164M, Q176L,


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S180D, S181G), whilst the third contained only a single mutation at V159L in the activation loop.

Figure 10. RIP1 kinase domain sequence differences between primate versus non-primate.

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 by 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 3 mouse RIP1 mutants more potently than wild-type mouse as shown in Figure 11 for the 15 point mutation (Mutant 1), 5 point mutation (Mutant 2) and single point mutation (Mutant 3). The activation loop 5 point mutation



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(Mutant 2) was most effective at increasing potency compared to 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 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 activation loop conformational changes required to potently bind this series. Additional data supporting an important role for the activation loop in binding this series in human versus murine RIP1 using stopped-flow kinetics in rat RIP1 is available in the Supporting Information.

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

Despite the lower non-primate RIP1 activity, we had sufficient potency and exposure to examine this series in an acute (3 hour) 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


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Table 4) allowing for 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 have reported that the RIP1 kinase inhibitor 1, administered intravenously, showed significant protection from hypothermia and death in this model.20 In addition, we have demonstrated that oral administration of a type II RIP1 inhibitor was efficacious in this model.11 Benzodiazepinone 31 was dosed orally 15 minutes prior to TNF/ zVAD.fmk injection and showed 8, 24 and 82% protection from body temperature loss over 3 hours, compared to TNF/ zVAD.fmk alone, at doses of 3, 10 and 50 mg/kg, respectively (Figure 12). Assuming the efficacy is Cmax driven, the calculated IC50 concentration of 640 ng/mL from this in vivo study correlated 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 hours in the highest dose group.

Table 4. Mouse and Rat PK for Benzodiazepinone 31

AUC 0-inf Bioavailability Clearance (%) (mL/min./kg) (µg.h/mL)

Species

Dose (mg/kg)

Route

Mouse

30

Oral

2.0 ± 0.2

Rat

1

IV

0.31 ± 0.06

Rat

2

Oral

0.46 ± 0.08

Volume (L/kg)

Half-life (h) 1.5 ± 0.3

57 ± 11 75 ± 14

2.2 ± 0.2

0.85 ± 0.3 1.9 ± 0.5

Figure 12. Evaluation of benzodiazepinone 31 in mouse TNF induced lethal shock model measuring reduction in body temperature loss.



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CONCLUSION In 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. Some 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 exchange analysis and co-crystallization in a truncated RIP1 (1-294). These studies revealed that the inhibitor sits deep in the ATP binding pocket with no hinge engagement allowing for 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 the required accommodations to adopt this binding conformation. An unusual species selectivity for primate versus non-primate RIP1 was investigated by designing specific mutations in mouse RIP1 to recover potency to primate RIP1 levels. This indicated the loss in potency is a result of the inability of the activation loop in non-primate RIP1 to adopt the


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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, made this a highly promising template for leadoptimization. Efforts towards this end will be reported in due course.

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. Flash chromatography was performed using silica gel (EM Science, 230-400mesh) under standard techniques or using silica gel cartridges (RediSep normal phase disposable flash columns) on an Isco CombiFlash. Reverse 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 H20 with 0.1% TFA unless otherwise noted. 1H NMR spectra were recorded on a Bruker Advance or Varian Unity 400MHz spectrometer as solutions in DMSO-d6 (unless otherwise stated). Chemical shifts (δ) are reported in ppm 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 (Hz) after the integration. LCMS analysis was conducted on the following methods: Agilent Method A LCMS was performed on a Agilent 1100 series, using a Zorbax SB-C8 column (4.6x 150 mm, 5 µm), 5-100% CH3CN:H2O (with



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0.02% TFA) over 12.5 min. and hold for 2.5 min., flow rate = 1.5 mL/min. at 40 °C. Agilent Method B LCMS was performed on a Agilent 1200 series, using a Zorbax XDB-C8 column (4.6x 75 mm, 3.5 µm), 5-95% CH3CN:H2O (with 0.1% TFA) over 4 min. and hold for 1 min., flow rate = 2 mL/min. at 40 °C. Agilent Method C LCMS was performed on a Agilent 1100 series, using a Thermo Hypersil Gold (C18, 20 x 2.1 mm, 1.9 u particle diam.), 2-100% CH3CN:H2O (with 0.02% TFA) over 2 min., flow rate = 1.4 mL/min. at 55 °C. Sciex LCMS was performed on a PE Sciex Single Quadrupole 150EX, using a Thermo Hypersil Gold (C18, 20 x 2.1 mm, 1.9 u particle diam.), 4-95% CH3CN:H2O (with 0.02% TFA) over 2 min., flow rate = 1.4 mL/min. at 55 °C. Waters LCMS was performed using the same column and conditions as for Sciex except using a Waters Acquity SQD UPLC/MS system. The retention time (Rt) is expressed in minutes at a UV detection of 214 or 254 nm. All tested compounds were determined to be ≥95% purity by LCMS . ((S)-5-Benzyl-N-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3carboxamide (9). A solution of N-Boc-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. The reaction mixture was partitioned between ethyl acetate (40 mL) and 0.5 M HCl solution (40 mL). The layers were separated, the organic layer was washed with water (3 x 20 mL), brine (20 mL), and 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-((tertbutoxycarbonyl)amino)-3-(2-nitrophenoxy)propanoic acid 5 (1.23 g, 3.77 mmol, 77 % yield) as a


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reddish yellow semi-solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.88 (dd, J = 8.46, 1.64 Hz, 1 H), 7.52 - 7.61 (m, 1 H), 7.06 - 7.15 (m, 2 H), 5.68 (br. d., 1 H), 4.75 (br. s., 1 H), 4.60 - 4.72 (m, 1 H), 2.07 (s, 2 H), 1.48 (s, 9 H). Sciex LCMS (m/z) 327 (M+H+), 653 (2M+H+), Rt 0.88 min. A suspension of (S)-2-((tert-butoxycarbonyl)amino)-3-(2-nitrophenoxy)propanoic acid 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 by LCMS confirmed the formation of the desired product. The slurry was filtered through a 0.45 micron PTFE autovial and the filtrate concentrated

under

reduced

pressure

to

give

(S)-3-(2-aminophenoxy)-2-((tert-

butoxycarbonyl)amino)propanoic acid 6 (0.98 g, 3.3 mmol, 98 % yield) as a pale brown semi solid. 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 LCMS (m/z) 297 (M+H+), 593 (2M+H+), Rt 0.65 min. HATU (1.245 g, 3.27 mmol) was added portion wise over 2 min to a solution of (S)-3-(2aminophenoxy)-2-((tert-butoxycarbonyl)amino)propanoic acid 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. The addition of water (30 mL) resulted in the formation of a precipitate. The mixture was cooled in an ice-bath for 15 min, then was 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 offwhite solid. TLC: 50% EtOAc in Hexane; Rf: 0.55. 1H NMR (400 MHz, DMSO-d6) d ppm 9.92 (s, 1 H), 6.99 - 7.21 (m, 5 H), 4.17 - 4.45 (m, 3 H), 1.36 (s, 9 H). Sciex LCMS (m/z) 279 (M+H+), 556 (2M+), Rt 0.87 min.



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A solution of (S)-tert-butyl (4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate 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 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-2,3dihydrobenzo[b][1,4]oxazepin-4(5H)-one hydrochloride 8 (26.1 g, 116 mmol) with 98% yield. 1

H NMR (400 MHz, DMSO-d6) δ ppm 10.5 (s, 1 H), 8.62 (br. s., 3 H), 7.02 - 7.25 (m, 4 H), 4.61

(dd, J =,10.61, 5.81 Hz, 1 H) 4.36 - 4.47 (m, 1 H), 4.23 - 4.36 (m, 1 H). Agilent Method C LCMS (m/z) 179 (M+H+), Rt 0.24 min. HATU (0.042 g, 0.11 mmol) was added in one portion to a solution of 5-benzylisoxazole-3carboxylic acid (0.024 g, 0.12 mmol) and DIPEA (0.048 mL, 0.274 mmol) in DMSO (0.75 mL). After stirring at rt for 5 min., a solution of (S)-3-amino-2,3- dihydrobenzo[b][1,4]oxazepin4(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 was allowed to stir at rt for 45 min. Purification of the crude material using reverse phase HPLC [35-65% acetonitrile:water (0.1% NH4OH modifier), C18 50x30 mm Gemini column, 47 mL/min.] gave the titled compound 9 (0.014 g, 0.039 mmol) as a white solid (35% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.12 (s, 1 H), 8.85 (d, J = 8.08 Hz, 1 H), 7.23 - 7.47 (m, 5 H), 7.06 - 7.23 (m, 4 H), 6.57 (s, 1 H), 4.81 (ddd, J = 10.74, 7.96, 6.82 Hz, 1 H), 4.35 - 4.62 (m, 2 H), 4.23 (s, 2 H). Sciex LCMS (m/z) 364 (M+H+), Rt 0.97 min., >99% purity. Chiral HPLC analysis using a Chiralpak AD-H analytical column (150 mm, 4.6mm, 5u) 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


Page 33 of 62

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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-phenoxy benzamide (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 stirring at rt for 5 min., a solution of (S)-3-amino-2,3- dihydrobenzo[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 was allowed to stir at rt for 30 min. Purification of the crude material using reverse phase HPLC [40-70% acetonitrile:water (0.1% NH4OH modifier), C18 50x30 mm Gemini column, 47 mL/min.] gave the titled compound 10 (0.029 g, 0.077 mmol) as an off-white solid (38% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.07 (s, 1 H) 8.75 (d, J = 8.34 Hz, 1 H) 7.62 - 7.75 (m, 1 H) 7.48 - 7.61 (m, 2 H) 7.34 - 7.48 (m, 2 H) 7.09 7.31 (m, 6 H) 6.99 - 7.09 (m, 2 H) 4.80 - 4.98 (m, 1 H) 4.51 (t, J = 10.61 Hz, 1 H) 4.42 (dd, J = 10.48, 6.95 Hz, 1 H). Sciex LCMS (m/z) 375 (M+H+), Rt 1.01 min., >99% purity. (S)-N-(4-Oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-pentyl-1H-pyrazole-3carboxamide (11). To a solution of 5-pentyl-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

stirring

at

rt

for

5

min.,

a

solution

of

(S)-3-amino-2,3-

dihydrobenzo[b][1,4]oxazepin-4(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 was allowed to stir at rt for 1 h. LCMS analysis indicated clean formation of desired product, however some starting material still remained. An additional amount of DIPEA (0.06 mL) and



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Page 34 of 62

HATU (0.06 g) were added and the reaction allowed to stir for 30 min. Purification of the crude material using reverse phase HPLC [30-60% acetonitrile:water (0.1% NH4OH modifier), C18 50x30 mm Gemini column, 47 mL/min.] gave the desired product as a viscous oil. The material was lyophilized to provide the titled compound 11 (0.009 g, 0.026 mmol) as a white amorphous solid (14 % yield). 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 LCMS (m/z) 343 (M+H+), Rt 0.92 min., 98% purity. (R)-5-Benzyl-N-(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3carboxamide (12). Following the same procedure described above for the preparation of

(S)-3-amino-2,3-

dihydrobenzo[b][1,4]oxazepin-4(5H)-one (7) from N-Boc-L-serine, the (R) enantiomer was prepared starting from the opposite enantiomer N-Boc-D-serine. HATU (0.175 g, 0.46 mmol) was added in one portion to a solution of 5-benzylisoxazole-3carboxylic acid (0.093 g, 0.46 mmol) and DIPEA (0.201 mL, 1.15 mmol) in DMSO (1 mL). After stirring at rt for 5 min., a solution of (R)-3-amino-2,3-dihydrobenzo[b][1,4]oxazepin4(5H)-one, trifluoroacetate (0.134 g, 0.46 mmol) in DMSO (1 mL) was added dropwise to the intermediate HOAt adduct. The reaction was allowed to stir at rt for 2 h. An additional amount of DIPEA (0.2 mL) and HATU (0.11 g) were added and the reaction allowed to stir for an additional 2 h. Purification of the crude material using reverse phase HPLC [35-65% acetonitrile:water (0.1% NH4OH modifier), C18 50x30 mm Gemini column, 47 mL/min.] gave the titled compound 12 (0.106 g, 0.29 mmol, 63% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.12 (s, 1 H), 8.85 (d, J = 8.08 Hz, 1 H), 7.23 - 7.48 (m, 5 H), 7.07 - 7.23 (m,


Page 35 of 62

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4 H), 6.57 (s, 1 H), 4.81 (ddd, J = 10.74, 7.96, 6.82 Hz, 1 H), 4.51 (t, J = 10.61 Hz, 1 H), 4.42 (dd, J =10.48, 6.69 Hz, 1 H), 4.23 (s, 2 H). Sciex LCMS (m/z) 364 (M+H+), Rt 0.96 min., >99% purity. Chiral HPLC analysis using a Chiralpak AD-H analytical column (150 mm, 4.6mm, 5u) eluting with CH3CN as the mobile phase, with 0.1% isopropylamine as a modifier, 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 (R)-5-benzyl-N(4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)isoxazole-3-carboxamide (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-3carboxamide (14). Methyl iodide (8.09 mL, 129 mmol) was added dropwise during 15 min to a solution of (S)tert-butyl (4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate 7 (30 g, 108 mmol) and Cs2CO3 (49.2 g, 151 mmol) in DMF (300 mL) stirred under nitrogen at room temp. 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 was poured into cold water (1500 mL) which formed a solid, the resultant solid was filtered, 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-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate (19g, 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 LCMS (m/z): 193 (M-BOC), 315 (M+Na+), Rt 0.81 min., >99% purity. 4M HCl (71.8 mL, 287 mmol) was added to a solution of (S)-tert-butyl (5-methyl-4-oxo2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)carbamate (28 g, 96 mmol) ) in DCM (300 mL)



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Page 36 of 62

and the reaction stirred under nitrogen at room temp 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 LCMS (m/z): 193 (M+H+), Rt 0.25 min., >99% purity. Hunig's base (0.356 mL, 2.04 mmol) was added to a solution of 5-benzylisoxazole-3carboxylic acid (0.173 g, 0.85 mmol) and HATU (0.323 g, 0.85 mmol) in DMSO (1.5 mL) . After stirring for 10 min. at rt, (S)-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)one hydrochloride 13 (0.155 g, 0.68 mmol) in DMSO (1.5 mL) was added. After stirring for 1 h 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 stirring at rt, the reaction was quenched with cold water and a brownish sticky solid was precipitated out. The reaction mixture was extracted with EtOAc (25 mL x2). The combined organic solution was washed with brine (15 mL) and concentrated in vacuo to give the crude product. This was purified using silica gel chromatography (eluent: 5%-25% EtOAc in hexane) to give the titled compound 14 (0.22 g, 0.577 mmol, 85 % yield) as a light beige solid. 1

H 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 LCMS (m/z): 378 (M+H+), Rt 0.96 min., >99% purity. Agilent Method A HPLC Rt 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,


Page 37 of 62

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30.5 mmol). The mixture was maintained at rt for 16 h. The reaction mixture was quenched with water (50 mL), and 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 LCMS (m/z) 313 (M+H+), Rt 1.24 min., 95% purity. Ethyl 5-(3-bromophenyl)-2,4-dioxopentanoate 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 mixture was maintained at 75 °C for 16 h. The reaction mixture was 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-(3bromobenzyl)-1H-pyrazole-3-carboxylate (3.2 g, 10.5 mmol, 82 % yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.36 - 7.44 (m, 2 H), 7.28 (s, 1 H), 7.12 - 7.25 (m, 1 H), 4.39 (q, J = 7.07 Hz, 2 H), 4.03 (s, 2 H), 1.40 (t, J = 7.20 Hz, 3 H). Agilent Method C LCMS (m/z) 309 (M+H+), Rt 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 was neutralized by adding 4N HCl in dioxane dropwise into the reaction mixture at rt. The organic layer was separated and concentrated to give 5-(3-bromobenzyl)-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 ppm 13.26 (br. s., 1 H), 7.38 -



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Page 38 of 62

7.59 (m, 2 H), 7.20 - 7.36 (m, 2 H), 6.51 (s, 1 H), 3.97 (s, 2 H). Agilent Method C (m/z) 281 (M+H+), Rt 0.88 min., 95% purity. 4-Methylmorpholine (0.41 g, 4.0 mmol) and HATU (0.575 g, 1.5 mmol), followed by (S)-3amino-5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one 13 (0.24 g, 1.26 mmol) was added to a stirred solution of 5-(3-bromobenzyl)-1H-pyrazole-3-carboxylic acid 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. 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)-5-(3-bromobenzyl)-N-(5-methyl-4-oxo-2,3,4,5-

tetrahydrobenzo[b][1,4]oxazepin-3-yl)-1H-pyrazole-3-carboxamide 17 (0.5 g, 1.11 mmol, 88% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.07 (d, J = 7.83 Hz, 1 H), 7.95 (s, 2 H), 7.25 - 7.31 (m, 1 H), 7.16 - 7.22 (m, 2 H), 7.11 - 7.16 (m, 1 H), 7.02 - 7.09 (m, 2 H), 6.44 (s, 1 H), 5.07 (dt, J = 11.31, 7.61 Hz, 1 H), 4.59 (dd, J = 9.85, 7.58 Hz, 1 H), 4.25 (dd, J = 11.12, 10.11 Hz, 1 H), 3.92 (s, 2 H), 3.35 (s, 3 H). Agilent Method C LCMS (m/z) 455 (M+H+), Rt 1.12 min., 95% purity. (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, 1.1 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 was 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).


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The collected fractions containing the product were combined and concentrated to give (S)-N-(5methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro-1,1dihydroxyethyl)benzyl)-1H-pyrazole-3-carboxamide 18 (0.1 g, 0.21 mmol, 19% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 8.39 (d, J = 8.08 Hz, 1 H), 7.90 - 8.01 (m, 2 H), 7.52 - 7.59 (m, 1 H), 7.43 - 7.50 (m, 1 H), 7.24 - 7.27 (m, 2 H), 7.15 - 7.21 (m, 2 H), 6.46 (s, 1 H), 5.17 (dt, J = 11.49, 7.89 Hz, 1 H), 4.58 (dd, J = 9.85, 7.58 Hz, 1 H), 4.28 - 4.39 (m, 1 H), 4.09 (s, 2 H), 3.39 (s, 3 H). Agilent Method C LCMS (m/z) 491 (M+H+), Rt 0.97 min., 80% purity. (S)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro1,1-dihydroxyethyl)benzyl)-1H-pyrazole-3-carboxamide 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 0.01N HCl (10 mL) and EtOAc (10 mL). The organic layer was washed by brine (10 mL), dried over MgSO4, 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(hydroxyimino)ethyl)benzyl)-1H-pyrazole-3-carboxamide (0.13 g, presumed 100% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 8.56 - 8.69 (m, 2 H), 8.07 - 8.26 (m, 1 H), 7.73 (tt, J = 7.61, 1.86 Hz, 1 H), 7.16 - 7.40 (m, 6 H), 6.47 - 6.58 (m, 1 H), 5.14 (dt, J = 11.56, 7.74 Hz, 1 H), 4.53 - 4.67 (m, 1 H), 4.25 - 4.35 (m, 1 H), 3.86 -3.99 (m, 2 H), 3.38 (s, 3 H). Agilent Method C LCMS (m/z) 488 (M+H+), Rt 1.09 min., 90% purity. (S,Z)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro1-(hydroxyimino)ethyl)benzyl)-1H-pyrazole-3-carboxamide (0.13 g crude material) was dissolved in pyridine (1 mL), and then TsCl (0.061 g, 0.32 mmol) was added. The mixture was



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maintained at 110 °C for 16h. The reaction mixture was 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-oxo-2,3,4,5-

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 ppm 8.56 - 8.69 (m, 2 H), 8.07 - 8.26 (m, 1 H), 7.73 (tt, J = 7.61, 1.86 Hz, 1 H), 7.16 - 7.40 (m, 6 H), 6.47 - 6.58 (m, 1 H), 5.14 (dt, J = 11.56, 7.74 Hz, 1 H), 4.53 - 4.67 (m, 1 H), 4.25 - 4.35 (m, 1 H), 3.86 - 3.99 (m, 2 H), 3.38 (s, 3 H). Agilent Method C LCMS (m/z) 641 (M+H+), Rt 1.38 min., 90% purity. (S,Z)-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(2,2,2-trifluoro1-((tosyloxy)imino)ethyl)benzyl)-1H-pyrazole-3-carboxamide 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 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-(5methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-5-(3-(3-(trifluoromethyl diaziridin3-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 ppm 8.29 (d, J = 7.83 Hz, 1 H), 7.46 - 7.54 (m, 2 H), 7.36 (t, J = 7.71 Hz, 1 H), 7.26 - 7.30 (m, 4 H), 7.17 - 7.22 (m, 1 H), 6.48 (s, 1 H), 5.16 (dt, J = 11.56, 7.86 Hz, 1 H), 4.60 (t, J = 8.72 Hz, 1 H), 4.25 - 4.36 (m, 1 H), 4.04 (s, 2 H), 3.41 (s, 3 H), 2.78 (d, J = 8.08 Hz, 1 H), 2.38 - 2.50 (m, 1 H). Agilent Method C LCMS (m/z) 487 (M+H+), Rt 1.02 min., 90% purity.


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(S)-N-(5-methyl-4-oxo-2,3,4,5-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) 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 1N 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 lypholization gave the titled compound 21 0.016 g, 0.033 mmol, 52% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 11.15 (br. s., 1 H), 8.26 (br. s., 1 H), 7.33 - 7.43 (m, 1 H), 7.24 - 7.28 (m, 4 H), 7.19 - 7.23 (m, 1 H), 7.14 (d, J = 7.83 Hz, 1 H), 6.50 (s, 1 H), 7.01 (s, 1 H), 5.17 (dt, J = 11.49, 7.64 Hz, 1 H), 4.64 (dd, J = 9.85, 7.58 Hz, 1 H), 4.29 (dd, J = 11.37, 9.85 Hz, 1 H), 4.03 (s, 2 H), 3.38 (s, 3 H). Agilent Method C LCMS (m/z) 485 (M+H+), Rt 1.26 min., >99% purity. (S)-5-Benzyl-N-(1-methyl-2-oxo-7-(3-(trifluoromethyl)-3H-diazirin-3-yl)-2,3,4,5tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (27). TEA (1.1 mL, 7.6 mmol) and di-tert-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-2,3,4,5tetrahydro-1H-benzo[b]azepin-3-yl)carbamate (1.95 g, presumed 100% yield) as a white solid. 1

H NMR (400 MHz, CDCl3) δ ppm 9.07 (s, 1 H), 7.21 - 7.41 (m, 2 H), 6.85 (d, J = 8.34 Hz, 1



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H), 5.63 (d, J = 7.83 Hz, 1 H), 4.25 (dt, J = 11.62, 7.58 Hz, 1 H), 2.76 - 3.00 (m, 1 H), 2.52 2.73 (m, 2 H), 1.93 - 2.08 (m, 1 H), 1.40 (s, 9 H). Agilent Method C LCMS (m/z) 299/301 (MtBu), Rt 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-2-oxo-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. The reaction mixture was 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-1methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)carbamate 22 (0.52 g, 1.4 mmol, 27% yield) as a white solid. Agilent Method C LCMS (m/z) 313/315 (M-tBu), Rt 1.17 min., 90% purity. TMEDA (0.41 mL, 2.7 mmol) was dissolved in THF (5 mL) and then cooled to -78 °C. nButyllithium (2.5M in hexane) (1.1 mL, 2.7 mmol) was then added dropwise at -78 °C. (S)-tertbutyl (7-bromo-1-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)carbamate 22 (0.4 g, 1.1 mmol) was dissolved in THF (2 mL) and was then cooled to -78 °C. The bromide solution was added dropwise to the butyllithium and 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 1hour, and then was slowly warmed up 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-


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methyl-2-oxo-7-(2,2,2-trifluoro-1,1-dihydroxyethyl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3yl)carbamate 23 (0.084 g, 0.22 mmol, 20% yield) as a yellow oil. Agilent Method C LCMS (m/z) 404 (M+H+), Rt 1.35 min., 90% purity. (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) was dissolved in DCM (2 mL) and 4N HCl in dioxane (0.54 mL, 2.2 mmol) was added. The yellow solution was maintained at rt for 3h before concentrating to a yellow residue. This residue was dissolved in MeOH (1mL) and then loaded on to a 2g bicarbonate SPE cartridge to make the free base from the HCl salt. The organic washes were concentrated and the residue was dissolved in DCM (5 mL) and 5-benzylisoxazole3-carboxylic acid (0.043 g, 0.21 mmol), 4-methylmorpholine (0.068 g, 0.67 mmol), and HATU (0.096 g, 0.25 mmol) were added. 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-1,1dihydroxyethyl)-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 LCMS (m/z) 490 (M+H+), Rt 1.33 min., 90% purity. (S)-5-benzyl-N-(1-methyl-2-oxo-7-(2,2,2-trifluoro-1,1-dihydroxyethyl)-2,3,4,5-tetrahydro1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide 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 0.01N HCl (5 mL) and EtOAc (5 mL). The organic layer was washed by brine (5 mL), dried over MgSO4 and concentrated to a yellow oil. The oil was



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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-tetrahydro1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide (0.01 g, 0.02 mmol, 16% yield) as a colorless oil. Agilent Method C LCMS (m/z) 487 (M+H+), Rt 1.21 min., 80% purity. (S,Z)-5-benzyl-N-(1-methyl-2-oxo-7-(2,2,2-trifluoro-1-(hydroxyimino)ethyl)-2,3,4,5tetrahydro-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-tetrahydro-1H-benzo[b]azepin-3yl)isoxazole-3-carboxamide 25 (0.007 g, 0.01 mmol, 53% yield) as a colorless oil. Agilent Method C LCMS (m/z) 461 (M+H+), Rt 1.49 min., 90% purity. (S,Z)-5-benzyl-N-(1-methyl-2-oxo-7-(2,2,2-trifluoro-1-((tosyloxy)imino)ethyl)-2,3,4,5tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3-carboxamide 25 (0.07 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/CH2Cl2). The collected fractions containing the product were combined and concentrated to give (S)-5-benzylN-(1-methyl-2-oxo-7-(3-(trifluoromethyl)diaziridin-3-yl)-2,3,4,5-tetrahydro-1H-benzo[b]azepin-


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3-yl)isoxazole-3-carboxamide 26 (0.0044 g, 0.01 mmol, 83% yield) as a colorless oil. Agilent Method C LCMS (m/z) 486 (M+H+), Rt 1.29 min., 95% purity. (S)-5-benzyl-N-(1-methyl-2-oxo-7-(3-(trifluoromethyl)diaziridin-3-yl)-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-yl)isoxazole-3-carboxamide 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 by 1N 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 titled compound 27 (0.004 g, 0.01 mmol, 91% yield) as a white solid. 1H NMR (600 MHz, CDCl3) δ ppm 7.72 (d, J=7.18 Hz, 1 H), 7.31 - 7.36 (m, 2 H), 7.29 (d, J=6.42 Hz, 1 H), 7.17 - 7.26 (m, 4 H), 6.31 (s, 1 H) 7.05 (s, 1 H), 4.51 - 4.58 (m, 1 H), 4.11 (s, 2 H), 3.42 (d, J=1.13 Hz, 3 H), 2.89 (td, J=12.84,8.31 Hz, 1 H), 2.73 (m, J=12.65, 12.65, 6.80 Hz, 1 H), 2.61 - 2.69 (m, 1 H), 2.01 - 2.09 (m, 1 H). Agilent Method C LCMS (m/z) 484 (M+H+), Rt 1.40 min., >99% purity. (R)-5-Benzyl-N-(1,1-dioxido-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3yl)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-2-nitrobenzene (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 4h and cooled to rt. The ethanol was removed under vacuum and the resulting aqueous phase was diluted with water (50 mL), washed with ether (2 x 100 mL). The aqueous was acidified to pH 4 with 1N HCl and extracted with DCM (2 x 300 mL). The organic layers were combined, washed with brine, dried



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over Na2SO4 and concentrated in vacuo to afford (R)-2-((tert-butoxycarbonyl)amino)-3-((2nitrophenyl)thio)propanoic acid as yellow solid (8 g, 20.4 mmol, 90% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.17 (d, J = 7.83 Hz, 1 H), 7.59 (d, J = 6.57 Hz, 2 H), 7.22 - 7.44 (m, 1 H), 6.73 (br. s., 2 H), 4.69 (m, 1 H), 3.61 (d, J = 9.35 Hz, 1 H), 3.35 - 3.51 (m, 1 H), 1.45 (s, 6 H), 1.41 (s, 3 H). Waters LCMS (m/z) 343 (M+H+), Rt 0.80 min., 87% purity. Ammonium chloride (0.25 g, 4.67 mmol) and zinc (1.5 g, 23.4 mmol) at 25 °C was added to a solution of (R)-2-((tert-butoxycarbonyl)amino)-3-((2-nitrophenyl)thio)propanoic acid (0.8 g, 2.3 mmol) in MeOH (100 mL) . After stirring at rt for 1h, 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 x 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 eliminated by filtration, then DCM (100 mL) and water (100 mL) was added to the filtrate. The resulting organic phase was washed with water (3x100 mL), dried over Na2SO4 and concentrated in vacuo to afford (R)3-((2-aminophenyl)thio)-2-((tert-butoxycarbonyl)amino)propanoic acid as a solid (R)-3-((2aminophenyl)thio)-2-((tert-butoxycarbonyl)amino)propanoic acid (0.7 g, 2.2 mmol, 96 % yield). Waters LCMS (m/z) 313 (M+H+), Rt 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 (3.3g, 10.56 mmol) in DCM (100 mL) . The reaction was stirred at rt for 5 min., then 4-methylmorpholine (1.742 mL, 15.85 mmol) was added. The reaction mixture was stirred at 25 °C for 5h. The solvent was removed on a rotavapor and the residue dissolved in EtOAc (200 mL) and the solution was washed with water (100 mL), 0.1N HCl (100 mL), aqueous NaHCO3 (100 mL) and brine (100 mL). The organic phase was dried


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over Na2SO4 and concentrated under reduced pressure to afford crude product. Isco purification (eluting

with

0-70%

of EtOAc in

hexane)

afforded

(R)-tert-butyl

(4-oxo-2,3,4,5-

tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (1.5 g, 5.1 mmol, 48 % yield). 1H NMR (400MHz, CDCl3) δ = 7.73 - 7.57 (m, 1 H), 7.39 (td, J = 1.4, 7.6 Hz, 2 H), 7.27 - 7.03 (m, 2 H), 5.58 (br. s., 1 H), 4.49 (dt, J = 7.2, 11.8 Hz, 1 H), 3.85 (dd, J = 6.7, 11.0 Hz, 1 H), 2.95 (t, J = 11.4Hz, 1 H), 1.42 (s, 9 H). Waters LCMS (m/z) 295 (M+H+), Rt 0.77 min., 97% purity. HCl (0.425 mL, 1.7 mmol, 4M in dioxane) was added to a solution of (R)-tert-butyl (4-oxo2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (0.1 g, 0.34 mmol) in dioxane (3 mL) . The reaction mixture was stirred at 25 °C for 18h. The solvent was removed on a rotavapor, the resulting crude solid washed with ether to yield (R)-3-amino-2,3-dihydrobenzo[b][1,4]thiazepin4(5H)-one hydrochloride (0.07g, 0.3 mmol, 88% yield), which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.61 (s, 1 H), 8.34 (br. s., 2 H), 7.65 (d, J =,7.83 Hz, 1 H), 7.50 (td, J = 7.71, 1.52 Hz, 1 H), 7.22 - 7.36 (m, 1 H), 7.19 (d, J =,7.83 Hz, 1 H), 3.95 (m, 1 H), 3.72 (dd, J = 11.37, 6.82 Hz, 1 H), 3.09 - 3.28 (m, 1 H). Waters LCMS (m/z) 195 (M+H+), Rt 0.23 min., 95% purity. N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.064 g, 0.334 mmol) and 1Hbenzo[d][1,2,3]triazol-1-ol hydrate (0.051 g, 0.334 mmol), followed by 4methylmorpholine (0.083 mL, 0.759 mmol) was added to a solution of 5-benzylisoxazole-3carboxylic acid (0.1 g, 0.455 mmol) in DCM (30 mL). The reaction was stirred at rt for 5 min., then (R)-3-amino-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 starting materials to result in a clear solution. The reaction mixture was stirred at 25 °C overnight. The solvents were removed on a rotavapor and the crude dissolved in EtOAc (200 mL). This solution was washed



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with water (100 mL), 0.1N 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% of 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 ppm 7.79 (d, J = 7.07 Hz, 1 H), 7.70 (dd, J = 7.71, 1.39 Hz, 1 H), 7.46 - 7.58 (m, 1 H), 7.19 7.46 (m, 7 H), 7.08 - 7.19 (m, 1 H), 6.33 (s, 1 H), 4.86 (dt, J = 11.75, 7.01 Hz, 1 H), 4.13 (s, 2 H), 3.98 (dd, J = 11.12, 6.57 Hz, 1 H), 3.04 (t, J = 11.37 Hz, 1 H). Waters LCMS (m/z) 380 (M+H+), Rt 0.91 min., 96% purity. Agilent Method A HPLC Rt 8.4 min., 95% purity. 3-Chlorobenzoperoxoic acid (0.025 g, 0.111 mmol) was added at 0 °C to a solution of (R)-5benzyl-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 and warmed up to rt, then stirred for 2 h at rt. The reaction was quenched with cold 1N NaOH (15 mL). The layers were separated and the aqueous phase 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 the Isco chromatography (eluent 5% to 60% EtOAc in hexanes) to provide the titled compound 28 (0.008 g, 0.019 mmol, 53 % yield) as 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 LCMS (m/z) 412 (M+H+), Rt 1.03 min., 95% purity.


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(R)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-3-yl)isoxazole3-carboxamide (29). Cs2CO3 (0.33 g, 1.02 mmol) was added to a solution of

(R)-tert-butyl (4-oxo-2,3,4,5-

tetrahydrobenzo[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., then MeI (0.05 mL, 0.815 mmol) was added. The solvent was removed on a rotavapor and the crude residue dissolved in EtOAc (200 mL) and the solution was washed with water (100 mL) and brine (100 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to (R)-tert-butyl (5-methyl-4-oxo-2,3,4,5tetrahydrobenzo[b][1,4]thiazepin-3-yl)carbamate (0.2 g, 0.51 mmol, 75 % yield). Waters LCMS (m/z) 309 (M+H+), Rt 0.86 min., 79% purity. Hydrogen chloride (7.05 mL, 28.2 mmol, 4M in dioxane) was added to a solution of (R)-tertbutyl (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 3h. The solvent was removed on a rotavapor and the resulting solid was washed with ether and hexane to yield (R)-3amino-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 LCMS (m/z) 209 (M+H+), Rt 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-1-ol hydrate (0.103 g, 0.674 mmol), then 4methylmorpholine (0.202 mL, 1.839 mmol) were added to a solution of 5-benzylisoxazole-3carboxylic acid (0.021 g, 0.919 mmol) in DCM (30 mL) . The reaction was stirred at rt for 5 min., 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 5h. LCMS



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showed product and that the reaction was completed. The solvent was removed on a rotavapor and the crude residue dissolved in EtOAc (200 mL) and the solution was washed with water (100 mL), 0.1N 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 crude product. Isco purification (eluting with 0-50% of EtOAc in hexane ) gave an oil, which was triturated with ether and hexane to yield a solid. This was filtered and rinsed with hexane and collected to give the titled compound 29 (0.2 g, 83%). 1H NMR (400MHz, DMSO-d6) δ = 8.96 (d, J = 7.8 Hz, 1 H), 7.67 (d, J = 7.3 Hz, 1 H), 7.59 (d, J = 4.0 Hz, 2H), 7.44 - 7.19 (m, 6 H), 6.52 (s, 1 H), 4.62 4.43 (m, 1 H), 4.21 (s, 2 H), 3.52 (dd, J = 6.8, 11.4 Hz, 1 H), 3.71- 3.44 (m, 1 H), 3.30 (s, 3 H). Waters LCMS (m/z) 394 (M+H+), Rt 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-3carboxamide (30). HATU (0.15 g, 0.340 mmol) and N-methylmorpholine (0.174 mL, 1.6 mmol) was added to a solution of (S)-3-amino-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one21 (0.07 g, 0.40 mmol), and 5-benzylisoxazole-3-carboxylic acid (0.08 g, 0.40 mmol) in DMF (2 mL) . The reaction mixture was stirred at rt overnight. The reaction was diluted with EtOAc (20 mL) and washed with water (20 mL) and brine (20 mL), dried over Na2SO4. The solvents were removed solvent to provide the titled compound 30 (0.091 g, 0.25 mmol, 63 % yield). Waters 1H NMR (400 MHz, DMSO-d6) δ ppm 9.92 (s, 1 H), 8.62 (d, J=7.58 Hz, 1 H), 7.17 - 7.77 (m, 5 H), 6.90 - 6.97 (m, 2 H), 6.82 - 6.87 (m, 1 H), 6.72 (td, J=7.45, 1.26 Hz, 1 H), 5.85 (d, J=5.31 Hz, 1 H), 6.59 (s, 1 H), 4.59 (ddd, J=10.80, 7.14, 4.29 Hz, 1 H), 4.23 (s, 2 H), 3.59 (ddd, J=11.24, 6.57, 4.42 Hz, 1 H), 3.41 - 3.52 (m, 1 H). LCMS (m/z) 363 (M+H+), Rt 1.05 min., >99% purity.


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(S)-5-Benzyl-N-(1-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3yl)isoxazole-3-carboxamide (31). (S)-Tert-butyl converted to

(2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl)carbamate22

was

(S)-3-amino-1-methyl-4,5-dihydro-1H-benzo[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 5-benzylisoxazole-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-1-methyl-4,5dihydro-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 to the reaction. The reaction mixture was stirred at 25 °C for 15 h. The reaction mixture was concentrated under reduced pressure to afford the crude product. Isco silica gel chromatography purification (eluting with 0-60% of 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, 400MHz): δ = 8.66 (d, J = 7.8 Hz, 1 H), 7.22 - 7.44 (m, 6 H), 7.07 - 7.17 (m, 1 H), 6.96 - 7.06 (m, 2 H), 6.55 (s, 1 H), 5.32 (d, J = 4.5 Hz, 1 H), 4.65 (dt, J = 11.7, 7.0 Hz, 1 H), 4.22 (s, 2 H), 3.47 - 3.69 (m, 2 H), 3.33 ppm (s, 3 H). Agilent Method A LCMS (m/z) 377 (M+H+), Rt 7.71 min., >99% purity. (S)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-1Hpyrazole-3-carboxamide (32). HATU (0.146 g, 0.385 mmol) was added to a solution of (S)-3-amino-5-methyl-2,3dihydrobenzo[b][1,4]oxazepin-4(5H)-one hydrochloride 8 (0.08 g, 0.35 mmol), 3-benzyl-1Hpyrazole-5-carboxylic acid (0.074 g, 0.37 mmol), and Hunig's base (0.153 mL, 0.875 mmol) in



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DMSO (1.3 mL) . After stirring for 1 hr at rt, the reaction mixture was diluted with MeOH (1 mL). After filtration through Acrodisc CR 25 mm syringe filter with 0.2 uM PTFE membrane, the solution was evaporated to give a crude product. HPLC purification (Waters Sunfire 30 x150 mm column, eluting with CH3CN : 30-70% H2O with 0.1% TFA, flow rate: 50 mL/min.) of yielded the titled compound 32 (0.05 g, 0.132 mmol, 38 % yield). 1H NMR (400 MHz, DMSOd6) d ppm 8.11 (br. s., 1 H), 7.40 - 7.59 (m, 1 H), 7.13 - 7.40 (m, 9 H), 6.41 (br. s., 1 H), 4.83 (dt, J = 11.42, 7.84 Hz, 1 H), 4.51 (t, J = 10.67 Hz, 1 H), 4.27 - 4.44 (m, 1 H), 3.98 (s, 2 H), 3.31 (s, 3 H). Agilent Method B LCMS (m/z) 377 (M+H+), Rt 2.07 min., >99% purity. (S)-5-Benzyl-N-(1-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)isoxazole-3carboxamide (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-1-methyl-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one hydrochloride (0.07 g, 0.309 mmol) were added, followed by stirring overnight. The reaction solution was concentrated on a rotavapor and the resulting crude product was purified by Isco silica gel chromatography (0% 40% EtOAc in hexanes). The pure product containing-fractions were combined and concentrated to yield the titled compound 33 (0.071 g, 0.189 mmol, 61 % yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.70 (d, J = 7.83 Hz, 1 H), 7.18 - 7.46 (m, 9 H), 6.53 (s, 1 H), 4.33 (dt, J =,11.18, 8.18 Hz, 1 H), 4.21 (s, 2 H), 3.30 (s, 3 H), 2.60 - 2.81 (m, 2 H), 2.10 - 2.37 (m, 2 H). Agilent Method A LCMS (m/z) 376 (M+H+), Rt 8.92 min., 99% purity. Fluorescence polarization (FP) binding assay.


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A fluorescent polarization based binding assay was developed to quantify 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-2sulfonate. Typically test compounds were prepared in 100% DMSO and 100 mL was dispensed into individual wells of a 384 well plate. 5 µL 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 the plates and incubated with compound for 10 minutes at room temperature. Following this pre-incubation, 5 µL of ligand prepared in assay buffer was added to the plate and the plate incubated for 20 minutes at room temperature.

Samples were read on an instrument capable of measuring

fluorescent 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 RIP1 (rat, rabbit, dog and monkey) FP binding assays were performed following the same protocol described above with the exception of enzyme concentration used in the assay for minipig (30 nM) and mouse (100 nM) species. All 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 reported. A four parameter curve fit was used to determine the potency of the compounds, where Y is % inhibition, x is inhibitor concentration, ‘a’ is the minimum, ‘b’ is the Hill slope, ‘c’ is the IC50 and ‘d’ is the maximum: Y=(a-d)/(1+(x/c)b)+d.



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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 a 384 well assay plate. Human RIP1 (1-375) and ATP were prepared in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.02% CHAPS, 0.5 mg/mL BSA to achieve a final assay concentration of 10 nM and 50 µM, respectively. Next, 5 µL hRIP1 (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 hours. Following the 4 hour 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 was incubated at room temperature for 1 hour. Next 5 µL of Promega ADPGlo detection reagent II with 0.02% CHAPS was added to the plate, resulting in the conversion of ADP (reaction product) to ATP which activates a light generating reaction between luciferase and luciferin. Following a 30 minute incubation, the luminescence was detected on a Perkin Elmer ViewLux. 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 four parameter curve fit (eq 1) or a tight binding curve fit for compounds whose potency was less than the detection limit of the assay (~ half the enzyme concentration). − ([ I ] − [ E ] + appK i ) + ([ I ] − [ E ] + appK i ) 2 + 4[E] * appK i vi = vo 2[E]


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In eq 2 vi/vs is the fractional activity remaining relative to uninhibited controls, I is the total inhibitor concentration, E is the total enzyme concentration and the app Ki is the IC50. ADP-Glo data are presented as the mean IC50 from at least n=2 determinations. Biological in vitro U937 cell assay The efficacy of RIP1 inhibitors were tested in vitro using human monocytic leukemia U937 cells in a necroptosis assay.23 U937 cells were acquired from ATCC (catalog# CRL-1593.2) and banked in liquid nitrogen. For the assay, frozen cells were thawed and diluted to 5e5 cells/ml in phenol red free Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Q-VD-OPh ((3S)-5-(2,6difluorophenoxy)-3-[[(2S)-3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxopentanoic acid hydrate), a broad spectrum caspase inhibitor, prepared as a 10 mM stock 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 PBS containing 0.1% bovine serum albumin was added to the cell solution to achieve a final concentration of 100 ng/mL. Forty (40) µL of cell suspension was added to each well of a luminescence compatible white assay plate pre-stamped with 40 nL of compound in neat DMSO. Lidded plates were incubated for 24 hours at 37°C, 5% CO2. The next day, cells were lysed and ATP content (viability) was measured via the addition of 20 µL Cell Titer-Glo Luminescent Cell Viability reagent (Promega Corporation).

Plates were

incubated in the dark for 15 minutes 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 10 µM compound 1 treated positive control wells. For concentration response experiments, normalized data were fit and IC50 values



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determined using conventional techniques. All data are shown as mean ± standard deviation of the mean. Biological in vitro L929 cell assay Mouse L-cells NCTC 929 (L929) were acquired from ATCC (catalog# ATCC CCL-1) and banked in liquid nitrogen. The assay protocol is as described for the U937 cell assay. Biological in vivo assay. The efficacy of RIP1 inhibitors can be tested in mice in vivo using a TNF-driven systemic inflammatory response syndrome model.20 A total of 7 mice per dose group were orally predosed with vehicle or compound 31 at doses of 3, 10 and 50 mg/kg 15 minutes before i.v. 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 hours when the control group lost 7 degrees with the exception of the 50 mg/kg dose group which was continued out for 8 hours. All data are shown as means ± 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.

ASSOCIATED CONTENT Supporting Information The Supporting Information contains details on enzyme preparations, mode of inhibition study, compound off and on rate constant determinations, kinase selectivity screening, photoaffinity labeling, HDX-MS and crystallization procedures, and species selectivity dose response curves.


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

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Telephone: 610-917-6873 Author Contributions: John Bertin and Peter J. Gough contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes The authors declare the following competing financial interests: All authors are current employees and stockholders of GlaxoSmithKline.

ABREVIATIONS USED AMPK, AMP-activated protein kinase; BB, building blocks; BCR-ABL, breakpoint cluster region abelson murine leukemia viral oncogene homolog 1; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate; DFG, Asp-Phe-Gly; DPU discovery performance unit; DIPEA, diisopropylethylamine; DTT, dithiothreitol; FasL, Fas ligand; FP, fluorescence polarization; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid 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,



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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

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.; Cookm 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 SHARPIN-deficient 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.


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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. 8. Xie, T.; Peng, W.; Liu, Y.; Yan, C.; Maki, J.; Degterev, A.; Yuan, J.; Shi Y. Structural basis of RIP1 inhibition by necrostatins. Structure 2013, 21, 493-499. 9. Teng, X.; Degterev, A.; Jagtap, P.; Xing, X.; Choi, S.; Denu, R.; Yuan, J.; Cuny, G. D. Structure-activity relationship study of novel necroptosis inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 5039-5044. 10. Najjar, M.; Suebsuwong, C.; Ray, S. S.; Thapa, R. J.; Maki, J. L.; Nogusa, S.; Shah, S.; Saleh, D.; Gough, P. J.; Bertin, J.; Yuan, J.; Balachandran, S.; Cuny, G. D.; Degterev, A. Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1. Cell Rep. 2015, 10, 1850-1860. 11. Harris, P. A.; Bandyopadhyay, D.; Berger, S. B.; Campobasso, N.; Capriotti, C. A.; Cox, J. A.; Dare, L.; Finger, J. N.; Hoffman, S. J.; Kahler, K. M.; Lehr, R.; Lich, J. D.; Nagilla, R.; Nolte, R. T.; Ouellette, M. T.; Pao, C. S.; Schaeffer, M. C.; Smallwood, A.; Sun, H. H.; Swift, B. A.; Totoritis, R. D.; Ward, P.; Marquis, R. W.; Bertin, J.; Gough, P. J. Discovery of small molecule RIP1 kinase inhibitors for the treatment of pathologies associated with necroptosis. ACS Med Chem Lett. 2013, 4, 1238-1243.



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Preparation of

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

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