(IAPs) Selective for the Second Baculovirus IAP Repeat (BIR2) Domain

Article pubs.acs.org/jmc

Benzazepinones and Benzoxazepinones as Antagonists of Inhibitor of Apoptosis Proteins (IAPs) Selective for the Second Baculovirus IAP Repeat (BIR2) Domain Andrew F. Donnell,*,† Christophe Michoud,† Kenneth C. Rupert,† Xiaochun Han,† Douglas Aguilar,‡ Karl B. Frank,§ Adrian J. Fretland,§ Lin Gao,‡ Barry Goggin,∥ J. Heather Hogg,† Kyoungja Hong,∥ Cheryl A. Janson,‡ Robert F. Kester,† Norman Kong,† Kang Le,† Shirley Li,‡ Weiling Liang,† Louis J. Lombardo,† Yan Lou,† Christine M. Lukacs,‡ Steven Mischke,† John A. Moliterni,† Ann Polonskaia,∥ Andrew D. Schutt,∥ Dave S. Solis,‡ Anthony Specian,† Robert T. Taylor,§ Martin Weisel,† and Stacy W. Remiszewski† †

Departments of Discovery Chemistry, ‡Discovery Technologies, §Non-clinical Safety, Early ADME, and ∥Discovery Oncology, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States S Supporting Information *

ABSTRACT: XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

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AVPI peptide sequence.12−15 This four-peptide motif has inspired substantial interest in compounds that target the IAPs,16−19 which has resulted in the discovery of several compounds that have entered clinical trials, those illustrated in Figure 1, SM-406 (1),20 GDC-0152 (2),21 and LCL161 (3),22,23 as well as one additional monovalent inhibitor and two bivalent inhibitors.24 These compounds are pan-IAP inhibitors that bind to XIAP, cIAP1, and cIAP2. They bind with high affinity to the BIR3 domain of these proteins and, in some cases, are selective for BIR3 over BIR2. In other cases, the BIR2 affinity has not been disclosed. It has been shown that they cause degradation of the cIAPs, leading to production of the cytokine TNFα via NF-κB pathways, and it has been suggested that their activity depends primarily on this process.25−27 We expected that competition with the terminal caspases by selective inhibitors of XIAP would present a distinct and attractive mechanism of action, and this could potentially be achieved by selectively targeting the BIR2 domain of XIAP. One recent paper describes a series of Smac-mimetic compounds with up to 7-fold selectivity for XIAP BIR2 versus XIAP BIR3.28 Herein, we report the discovery of a novel class of inhibitors that are highly selective for the BIR2 domain of XIAP over the BIR3 domain, and we describe the iterative

INTRODUCTION Apoptosis is a cell death program that is essential for normal physiological function, and the ability to avoid apoptosis is one of the characteristics of cancer cells.1 The inhibitor of apoptosis proteins (IAPs) are key regulators of the apoptosis pathways and are often overexpressed in cancer cells, rendering them potential targets for cancer therapy.2−5 Members of the IAP family include X-linked IAP (XIAP), cellular IAP 1 (cIAP1), cellular IAP 2 (cIAP2), and others. There are two major pathways for apoptotic signal transduction. The intrinsic pathway is activated by intracellular stress, such as that caused by chemotherapy and radiation, and leads to the activation of caspase 9. The extrinsic pathway is activated in response to extracellular signals mediated through the death receptors and leads to the activation of caspase 8. Both pathways converge by causing the activation of the effector caspases 3 and 7. XIAP regulates both pathways by directly binding to and inhibiting caspases 3, 7, and 9 via two of its three baculovirus IAP repeat (BIR) domains.6 The BIR3 domain of XIAP binds to and inhibits caspase 9,7,8 and the BIR2 domain is involved with caspase 3 and 7 binding.9−11 Second mitochondria-derived activator of caspases (Smac, also known as direct IAP-binding protein with a low pI, DIABLO) is an endogenous mitochondrial protein that binds with high affinity to the BIR3 domain of XIAP and the cIAPs and with lower affinity to the BIR2 domain via its N-terminal © 2013 American Chemical Society

Received: May 15, 2013 Published: October 1, 2013 7772

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CHEMISTRY Our studies of the benzazepinone scaffold included the exploration of a range of N-substituents of the azepinone amide. We also explored some modifications to the alanine group. The chemistry to prepare these analogues is outlined in Scheme 1. Starting with the amino benzazepinone 6, amide Scheme 1. Synthesis of Benzazepinones 12−23a

a

Reagents and conditions: (a) R1CO2H, HBTU, HOBt, Et3N, DMF, rt, 47%−quant; (b) R2CH2X, Cs2CO3, NaI, DMF, 60 °C; (c) HCl, MeOH, Et2O, rt or TFA, CH2Cl2, rt, 24−60% (2 steps).

coupling with either BOC-N-methyl-L-alanine or the amino acid of interest gave 7−11. These compounds were then alkylated with the appropriate alkyl halide or mesylate in the presence of cesium carbonate. Under these conditions, we observed exclusive alkylation of the aniline amide. The BOC group was then removed to provide the final compounds 12− 23. The compounds were typically isolated and tested as either the HCl or TFA salts. Construction of the benzoxazepinone scaffold commenced with the SNAr reaction of (S)-2-(tert-butoxycarbonylamino)-3hydroxypropanoic acid (24) and 1-fluoro-2-nitrobenzene to yield ether 25 (Scheme 2). Reduction of the nitro group,

Figure 1. IAP inhibitors.

synthetic explorations and structure-based design efforts leading to a highly potent and selective analogue that has good oral bioavailability. We show that this compound is 78-fold selective for the BIR2 versus the BIR3 domain of XIAP and it is 7-fold selective for XIAP versus cIAP1. We also demonstrate that it affects apoptotic signaling without promoting cIAP-related NFκB activation, supporting the utility of selective XIAP inhibition via the BIR2 domain as a potential approach for cancer therapy. A high-throughput screen of our corporate compound library led to the identification of benzazepinone 4 (Figure 2) as a

Scheme 2. Synthesis of Benzoxazepinone 27a

Figure 2. HTS hit and initial lead evolution.

BIR2-selective XIAP inhibitor. The relatively small size (MW 261) and low lipophilicity (cLogP 0.62) of 4 made it an appealing starting point for a medicinal chemistry campaign. Preliminary exploration of the SAR around this scaffold led to compounds such as 5, where benzazepinones and benzoxazepinones had comparable BIR2 potency, N-methylation of the L-alanine moiety was preferred, and there was room for expansion around the azepinone N-substituent. In particular, substituents containing aromatic groups were preferred in this position.

a

Reagents and conditions: (a) NaH, 1-fluoro-2-nitrobenzene, DMF, 0 °C, 97%; (b) H2, 10% Pd/C, EtOH, rt, 95%; (c) EDCI, DMF, rt, 62%; (d) TFA, CH2Cl2, 0 °C, quant; (e) BOC-N-methyl-L-alanine, HBTU, HOBt, Et3N, DMF, rt, 95%; (f) 6-bromo-1-(chloromethyl)-2methoxynaphthalene, Cs2CO3, NaI, DMF, 60 °C, 54%; (g) TFA, CH2Cl2, 0 °C, 56%. 7773

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Scheme 3. Synthesis of Spirocyclic Benzoxazepinone Analogues 33−40a

Reagents and conditions: (a) LDA, THF; dihydro-2H-pyran-4(3H)-one, −78 °C to rt, 50%; (b) KOH, H2O, MeOH, 60 °C, 92%; (c) 1-fluoro-2nitrobenzene, KHMDS, THF, rt, 92%; (d) Zn, NH4Cl, MeOH, 65 °C, quant; (e) EDCI, HOBt, DMF, rt, 74%; (f) H2, Pd(OH)2/C, MeOH, rt, quant; (g) BOC-N-methyl-L-alanine, HBTU, HOBt, Et3N, DMF, rt, separated diastereomers, 37%; (h) RCH2X, Cs2CO3, NaI, DMF, rt−70 °C; (i) for 33−35, 37, 38, HCl, MeOH, Et2O, rt; for 39, 40, TFA, CH2Cl2, 0 °C, 44−93% (2 steps); (j) 6-bromo-1-(chloromethyl)-2-methoxynaphthalene, Cs2CO3, NaI, DMF, 60 °C, 54%; (k) CO, MeOH, Pd(OAc)2, Xantphos, Et3N, 70 °C, 67%; (l) LiOH·H2O, THF−H2O, rt, 88%; (m) TFA, CH2Cl2, rt, 83%. a

bromo-2-methoxynaphthyl 19. None of these compounds had any significant activity at XIAP BIR3 at up to 54 μM. The 6-bromo-2-methoxynaphthyl analogue 19 displayed moderate clearance in our in vitro rat liver microsome and hepatocyte assays (17 and 38 mL/min/kg, respectively), however, when we advanced it into rat PK studies (singlecompound dosing, 2 mg/kg iv), we observed a very high in vivo clearance rate of 116 mL/min/kg, approximately two times hepatic blood flow. To help determine which structural features contributed to this clearance rate, we conducted an in vivo metabolite identification study in rats by administering a 4 mg/ kg iv dose of 19 and analyzing urine and plasma samples, where we observed significant quantities of the material derived from hydrolysis of the alanine amide. Initially, we sought to stabilize the amide against hydrolysis by modulating the steric properties of the amino acid substituent (Table 2). To efficiently profile the effect of these changes on the clearance values, we employed cassette dosing in our PK studies using groups of 3−4 compounds per study. Selected compounds were tested in both cassette and singlecompound PK studies, confirming that each method provided comparable values.29 The D-alanine analogue 20 was 10-fold less potent than the natural isomer. Some larger substituents were tolerated, such as those of the α-aminobutyric acid analogue 21 and the threonine analogue 22, although these groups did not improve the clearance, and in the case of the 22 it was considerably worse. Larger substituents were significantly less active at BIR2, as were analogues with a quaternary αcarbon (data not shown). Some modifications at the Nterminus were tolerated. For example, the methyl group could be replaced by ethyl (23), but this did not affect the clearance rate. These efforts were complicated by a continued lack of correlation between the clearance in rat liver microsomes or hepatocytes and that observed in vivo. Despite conducting the PK studies using cassette dosing, we still lacked sufficient throughput to rapidly build up the structure−property relationships necessary to address the clearance issue, so we aimed to develop an alternate in vitro screening protocol. We profiled selected compounds in several systems, including rat

cyclization under amide-coupling conditions, and acidic removal of the BOC group provided the amino benzoxazepinone core 26, which was subjected to chemistry similar to that described in Scheme 1 above to prepare compound 27. We also prepared benzoxazepinones with a spirocyclic tetrahydropyran substituent (Scheme 3). The synthesis of these analogues began with the aldol condensation of dibenzylglycine ethyl ester (28) and dihydro-2H-pyran4(3H)-one, which was followed by hydrolysis of the ester to provide acid 29. The ether linkage of 30 was formed by the reaction with 1-fluoro-2-nitrobenzene. Chemoselective reduction of the nitro group, cyclization, and removal of the benzyl protecting groups gave the amino benzoxazepinone scaffold 31. The amino acid moiety was installed and the diastereomers were separated using SFC to provide 32, and then the targeted analogues 33−40 were made via the same alkylation− deprotection sequence described above. This proved to be a versatile method for preparing spirocyclic benzoxazepinones, and we synthesized several novel analogues starting from a variety of cyclic ketones (not shown). Analogue 36, which has a carboxylic acid at the 6-position of the naphthalene, was prepared from the corresponding aryl bromide by palladiumcatalyzed carbonylation and hydrolysis of the resulting methyl ester.

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RESULTS AND DISCUSSION Our efforts commenced with a survey of azepinone Nsubstituents with the aim of improving the XIAP BIR2 potency while retaining selectivity over XIAP BIR3. Results are displayed in Table 1. The starting point was benzyl analogue 12. Extended linkers, such as that of 13, were disfavored. Naphthyl 14 was approximately equipotent with 12, while the analogue 15 that was attached at the 1-position of the naphthyl was more potent. We explored a range of substitutions on the naphthyl ring, and analogues such as 16 provided a modest 2fold improvement in potency compared with 15. Substitution at the 2-position of the naphthyl was preferred, and the 2methylnaphthyl analogue 17 had an IC50 of 0.489 μM. Further improvements were realized by replacing the methyl with a methoxy group (18), and the most promising analogue was 67774

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Table 1. XIAP Activity for N-Substituted Benzazepinone Analogues

Table 2. XIAP Activity for Benzazepinone Amino Acid Analogues

a

All compounds were HCl salts except 21 which was a TFA salt. Values that are the result of multiple determinations are reported as: mean ± SD (number of determinations). cCassette dosing, 0.5 mg/kg iv. dNot tested, the compound was not evaluated in this assay. b

synthetically tractable benzoxazepinone template. Many of the substitution patterns we examined brought improvements to blood stability, and the spiro-tetrahydropyran 33 was the most promising (Table 3). It had high blood stability, and when we profiled 33 in PK studies it had a substantially lower clearance than 27, although at 60 mL/min/kg its rate was still close to rat liver blood flow. While the spiro-tetrahydropyran may have effectively blocked the extrahepatic amide hydrolysis, other degradation pathways would also need to be addressed to bring the clearance of 33 to an acceptable range for in vivo applications. To determine potential sites of CYP-mediated oxidative metabolism, we incubated 33 with both rat and human hepatocytes and analyzed the metabolites formed. We detected two major metabolites: one derived from demethylation of the N-methylL-alanine moiety and the other from demethylation of the methoxy group of the naphthalene. To address the latter issue, we reexamined the naphthyl moiety (Table 4). Replacing the methoxy group with methyl caused a 10-fold loss of potency (compound 34). Attempts to stabilize the methoxy group with analogues such as the difluoromethoxy 35 likewise resulted in an unacceptable loss of potency. We did not evaluate the PK properties of these less potent analogues. In a different approach, we looked at other changes such as replacing the bromine with a carboxylic acid (36). The BIR2 potency of this compound was improved, but its clearance was worse. We prepared several heterocyclic analogues to modulate the electronic properties of the ring system. Quinoline 37 had an acceptable level of BIR2 activity, but its clearance was about 3fold higher than that of 33. Benzisoxazole 38 was significantly less active. To aid in our design of new analogues, we obtained a cocrystal structure of the carboxylic acid analogue 36 bound to the XIAP BIR2 domain (Figure 3). This also revealed some of the features that may contribute to the selectivity for BIR2 versus BIR3.30 For example, the fused benzene ring of the

All compounds were HCl salts. bValues are reported as: mean ± SD (number of determinations).

a

liver and kidney S9 fractions, plasma, and whole blood, and we observed a correlation between in vivo clearance and stability in rat whole blood. Compounds were incubated in rat blood for 4 h, and the percent of the compound remaining was quantitated. All compounds with 50% remaining, we observed a range of in vivo clearance values, suggesting that we were addressing one of multiple potential clearance pathways. Nevertheless, the blood stability assay proved to be a valuable filter to ease our reliance on resource-intensive PK studies. As we continued to modify the scaffold, only those analogues that passed the 50% blood-stability cutoff were profiled in vivo. Another approach we explored for blocking hydrolysis of the amide involved the introduction of substituents onto the azepinone ring. The benzazepinone and benzoxazepinone templates typically provided equivalent activity and carried similar metabolic liabilities (compare 19 and 27, Table 3), so most of these changes were evaluated using the more 7775

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Table 3. Comparison of in Vitro CL and Blood Stability with in Vivo CL across Scaffolds

XIAP BIR2 IC50 (μM)a XIAP BIR3 IC50 (μM)a rat liver microsomes CL (mL/min/kg) rat liver hepatocytes CL (mL/min/kg) rat blood stability (% remaining at 4 h) rat iv PK, CL (mL/min/kg)c

19

27

33

0.194 ± 0.124 (6) >54 (6) 17 38 40 155

0.101 ± 0.011 (2) >54 (2) 27 n.t.b 7 521

0.065 ± 0.042 (4) 23.5 ± 10.3 (4) 28 21 88 60

Values are reported as: mean ± SD (number of determinations). bNot tested, the compound was not evaluated in this assay. cCassette dosing, 0.5 mg/kg iv.

a

substrate Ac-DEVD-AFC, which is specific for caspase 3/7. Addition of full-length XIAP inhibited the caspase activity and addition of 40 restored it with an EC50 of 0.140 μM. This was similar behavior to that observed for 3. Compound 40 was screened for cell-based activity against several cancer cell lines. No single-agent activity was observed, but a significant effect on cell viability was observed in combination with the DR5 antibody conatumumab,32 which provides apoptotic pressure mediated through the extrinsic pathway. In that regard, its activity was compared to that of 3. Parts A and B of Figure 5 show the dose−response curves for conatumumab in a 5 day MTS assay in SW620 cells in combination with two concentrations of test compound. In the absence of test compound, conatumumab had little effect on cell viability at any of the tested concentrations. Modest sensitization was observed in the presence of 1.1 μM of test compound (Figure 5A) and greater sensitization was observed at 3.3 μM (Figure 5B). At the latter concentration, 40 was more effective than 3. Similar levels of activity were observed in other cancer cell lines (data is summarized in Figure 6; see Supporting Information Figure S1 for tabulated EC50 values). To confirm that this antiproliferative activity correlated with increased apoptotic signaling, we measured the caspase 3/7 activity in SW620 cells after treatment with conatumumab in combination with either 3 or 40 (Figure 7). Slightly elevated caspase 3/7 levels were observed upon treatment with conatumumab alone, and this was potentiated in a dosedependent manner by both 3 and 40. Again, 40 promoted greater levels of activity than 3. The BIR3-selective compound 3 exerts single agent activity in MDA-MB-231 cells that is dependent on cIAP binding and degradation, which leads to increased TNFα levels.33 Because 40 has some affinity for the BIR2 domain of cIAP1, we examined whether it would display a similar activity profile in this system. We treated MDA-MB-231 cells with either 3 or 40 and measured TNFα levels after 19 h (Figure 8). TNFα levels were significantly elevated in cells treated with 3, while cells treated with 40 were not substantially different from vehicle at up to 10 μM, confirming that 40 does not induce TNFα in this system. To further understand the activity of our inhibitor, we conducted an in vivo study using LOX xenograft-bearing nude mice. The mice were treated with a single dose of conatumumab (1 mg/kg ip), 3 (100 mg/kg po), or 40 (200

benzoxazepinone scaffold is situated in close proximity to His223. This is a larger tryptophan residue in BIR3 that would clash with the benzene ring. The naphthyl ring system extends into a hydrophobic pocket between the side chains of Lys206 and Lys208. This pocket is absent in BIR3, which has glycine and threonine at these positions. Additionally, the methoxy group of the naphthyl is in the cleft between His223 and Phe224; this cleft is much more restricted in BIR3 where these residues are tryptophan and tyrosine. The structural data indicated that relatively bulky groups could be added to the solvent-exposed edge of the naphthyl moiety, and we hypothesized that a 2-cyanophenyl group extending from the bicyclic system could accept a hydrogen bond from the backbone NH of Lys206. After modeling several possible arrangements, we expected a 5,6-bicycle such as indazole to optimally position this group (Figure 4). To test this, we prepared 39 and found that it did improve BIR2 potency compared to 38 (Table 4). Notably, this change also brought an improvement to the clearance. Because 2-position substituents on the naphthyl ring generally enhanced the BIR2 affinity, we prepared the analogous indole system of 40 with a nitrile in that position. This compound exhibited excellent BIR2 potency while maintaining an acceptable clearance rate. Interestingly, this compound also showed a greater level of XIAP BIR3 inhibition than the other analogues, but it was still 78-fold selective for BIR2. The PK properties of this compound were comparable in both rat and mouse and it had good oral exposure (Table 5). We felt that the potency and selectivity of this compound, coupled with its favorable PK profile, would allow us to more fully evaluate the biological properties of this class of XIAP BIR2 inhibitors. To further understand the selectivity profile of 40, we measured its activity against the BIR2 and BIR3 domains of cIAP1 (Table 6). This compound did not have any significant activity at the cIAP1 BIR3 domain, but it did have a submicromolar IC50 for cIAP1 BIR2 inhibition. Still, it was 7fold selective for XIAP BIR2. Table 6 also shows the IC50 values for the known BIR3-selective pan-IAP inhibitor 3 for comparison.31 To assess the functional competence of BIR2-selective compounds, we profiled 40 in an in vitro caspase reactivation assay (Table 6). Cellular lysates were treated with cytochrome c and ATP, resulting in activation of caspases. The caspase activity was monitored by the cleavage of the fluorogenic 7776

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Table 4. XIAP Activity of N-Substituted Derivatives of Spirocyclic Benzoxazepinone Core

a

All compounds were HCl salts except 36, which was a TFA salt and 39 and 40 which were free bases. bValues that are the result of multiple determinations are reported as mean ± SD (number of determinations). cCassette dosing, 0.5 mg/kg iv. dNot tested, the compound was not evaluated in this assay.

mg/kg po), or a combination of conatumumab with 3 or 4. After 8 h, the tumors were removed, and caspase 3/7 activity was determined using a fluorescence assay based on cleavage of DEVD-AMC (Figure 9). Additionally, the effect on protein

levels was examined by Western blot analysis (Figure 10). The caspase activity of conatumumab, 3, or 40 alone was not significantly different from vehicle, whereas in combination with the DR5 antibody, both 3 and 40 promoted substantial 7777

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Table 6. Biochemical Properties of 40 Compared to 3 property

value for 40a

XIAP activity BIR2 IC50 (μM) 0.039 ± 0.010 (2) BIR3 IC50 (μM) 3.06 ± 0.28 (2) cIAP1 activity BIR2 IC50 (μM) 0.271 ± 0.115 (2) BIR3 IC50 (μM) >55 (2) XIAP-caspase reactivation EC50 (μM) 0.140

value for 3a 1.05 ± 0.31 (2) 0.030 ± 0.009 (2) 2.72 ± 0.81 (2) 0.058 ± 0.004 (2) 0.283

a

Values that are the result of multiple determinations are reported as mean ± SD (number of determinations).

substantial levels of cIAP1 degradation, while this was not observed for 40. XIAP levels were not significantly impacted by either compound. Further profiling of 40 revealed significant in vitro safety liabilities. This compound was both a potent inhibitor and timedependent inhibitor of CYP3A4, and it also exhibited significant CYP3A4 induction. These liabilities limited the clinical utility of 40, however, we felt that we could apply some of the SAR lessons from this investigation to overcome these liabilities using a related benzodiazepinone scaffold. Those optimization efforts are the subject of a subsequent report.34 Despite its liabilities, 40 is a potent and selective XIAP BIR2 inhibitor with a PK profile suitable for in vivo studies, and it proved to be a valuable tool for investigating the biology of BIR2-selective XIAP inhibitors. In a separate report, we will describe additional studies of the biological utility of 40, including a demonstration of in vivo efficacy in a tumor xenograft study.35 These studies support the usefulness of targeting the BIR2-domain to achieve selectivity for XIAP and impact apoptotic pathways in a manner mechanistically differentiated from BIR3-selective pan-IAP inhibitors.

Figure 3. X-ray cocrystal structure of 36 bound to the peptide-binding groove of the BIR2 domain of XIAP, represented with an electrostatic surface. Hydrogen-bonding interactions between the ligand and the protein are indicated by dotted lines.

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Figure 4. The energy-minimized structure of 39 (green) overlaid with the crystal structure of 36 (yellow) bound to XIAP BIR2. The energy minimization of 39 was performed with MOE (Chemical Computing Group Inc., version 2011.10) using the MMFF94x forcefield.

Table 5. PK Properties of Compound 40 property

rat, iva

mouse, ivb

mouse, poc

AUC (ng·h/mL) Cmax (ng/mL) t1/2 (h) Vss (L/kg) CL (mL/min/kg) F (%)

196 190 1.7 4.1 41

543 495 6.9 8.6 33

2858 647 5.0

EXPERIMENTAL SECTION

Chemistry. General. All commercially available reactants, reagents, and solvents were used as received. Reactions using air- or moisturesensitive reagents were performed under an atmosphere of Ar or N2. Reactions were monitored by LC-MS or TLC. Flash chromatography was performed with Isco CombiFlash Companion or AnaLogix IntelliFlash chromatography systems using prepacked silica gel columns (40−60 μm particle size RediSep or 20−40 μm spherical silica gel RediSep Gold columns purchased from Teledyne Isco or comparable products from other vendors). Preparative HPLC was performed using a Waters HPLC system consisting of a 2767 sample manager, 2525 binary gradient module, and 2996 photodiode array detector with a SunFire Prep C18 OBD column (5 μm, 30 mm × 100 mm) eluted with a 10−100% MeCN−H2O linear gradient with 0.1% v/v TFA at a flow rate of 30 mL/min or using similar methods on a comparable system. NMR spectra were measured on Bruker 300 or 400 MHz spectrometers. Chemical shifts are reported in ppm downfield from TMS using residual nondeuterated solvent as an internal standard (CHCl3, 7.26 ppm; DMSO, 2.50 ppm; MeOH, 3.31 ppm). Data are reported in the form: chemical shift (multiplicity, coupling constants, integration). Multiplicities are recorded by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. HRMS data were recorded on a ThermoFisher OrbiTrap FTMS system using flow injection with electrospray ionization in positive mode. Analytical HPLC and LC-MS analyses were performed using either Waters or Agilent LC-MS systems. The Waters system was comprised of a ZQ mass spectrometer using multimode (ES/APCI) ionization with alternating ± switching and an ES Industries Chromegabond WR C18 column (3 μm, 120 Å, 30 mm × 3.2 mm) eluted with a 10−90% MeCN−H2O linear gradient with

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a

Cassette dosing, 0.5 mg/kg iv. bSingle-compound dosing, 1 mg/kg iv. c Single-compound dosing, 10 mg/kg po.

increases in caspase 3/7 activity. Western blot analysis showed cleavage of PARP and caspase 3 upon treatment with conatumumab in combination with either 3 or 40, consistent with the induction of apoptosis. Compound 3 promoted 7778

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Figure 5. (A,B) Comparison of the activity of 3 and 40 in combination with the DR5 antibody conatumumab in SW620 cells in a 5 day MTS cell proliferation assay. Each graph shows dose−response curves for conatumumab in the presence of the indicated concentration of test compound. Values are the mean of four determinations ± SD.

Figure 7. Caspase 3/7 activity in SW620 cells 4 h after treatment with the test compounds. Cells were incubated with conatumumab, 3, and/ or 40 at the indicated concentrations (combinations used 0.5 μg/mL conatumumab with the indicated concentrations of 3 or 40) for 4 h, and then they were lysed and the caspase 3/7 activity was measured by the cleavage of DEVD-AMC. Values are the mean of two determinations ± SD.

Figure 6. Cell line sensitivity to 40 and 3 in combination with DR5 agonistic antibody conatumumab. Each bar represents the EC50 of conatumumab, either alone (blue bars) or in the presence of 3 (3.3 μM) (red bars) or 40 (3.3 μM) (green bars) in a 5 day MTS assay measuring viability of the indicated cell line (for LOX cells, the assay was performed in a 3 day format). 0.1% v/v HCO2H. The Agilent system was comprised of a 6140 mass spectrometer using multimode (ES/APCI) ionization with alternating ± switching and an Agilent Zorbax SB C-18 column (3.5 μm, 30 mm × 2.1 mm) eluted with a 10−90% MeCN−H2O linear gradient with 0.1% v/v HCO2H. All tested compounds were evaluated on one of these analytical HPLC systems and determined to be ≥95% pure. General Procedure A: Amino Acid Coupling. To a rt solution of 6, 25, or 30 (1 equiv) in DMF (0.1−0.2 M) was added the appropriate amino acid (1.0−1.2 equiv), followed by Et3N (3 equiv), HBTU (1.2 equiv), and HOBt (1.2 equiv). The reaction was stirred at rt until complete, then it was diluted with H2O and extracted with EtOAc. The combined organic layers were washed with 1:1 satd aq NaHCO3− satd aq NaCl and then satd aq NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide product. General Procedure B: Alkylation of Core. To a rt solution of above material in DMF was added the appropriate alkyl halide or mesylate (1.4 equiv), Cs2CO3 (1.5 equiv), and NaI (1.5 equiv). The reaction was stirred at 60 °C overnight, then cooled to rt, diluted with H2O, and extracted with EtOAc. The combined organic layers were washed with satd aq NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide product.

Figure 8. Presence of TNFα in the supernate from MDA-MB-231 cells 19 h after treatment with the indicated concentration of BIR3selective compound 3 or BIR2-selective compound 40 in comparison with TNFα standards. Values are the mean of two determinations ± SD.

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tert-Butyl Methyl((R)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-ylamino)propan-2-yl)carbamate (8). According to general procedure A, racemic 6 (0.50 g, 2.8 mmol) and BOC-Nmethyl-D-alanine (692 mg, 3.4 mmol) were converted to 8 (0.409 g, 40%) as a white solid (the diastereomers were separated during flash chromatography). 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.88 (d, J = 7.86 Hz, 1H), 7.23−7.31 (m, 2H), 7.10−7.15 (m, 1H), 7.00 (d, J = 7.7 Hz, 1H), 4.32−4.68 (m, 1H), 4.15−4.24 (m, 1H), 2.70 (s, 3H), 2.63−2.77 (m, 2H), 2.20−2.33 (m, 1H), 2.02−2.14 (m, 1H), 1.39 (s, 9H), 1.13−1.21 (m, 3H). tert-Butyl Methyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-ylamino)butan-2-yl)carbamate (9). According to general procedure A, 6 (81.1 mg, 0.460 mmol) and (S)-2-(tertbutoxycarbonyl(methyl)amino)butanoic acid (100 mg, 0.460 mmol) were converted to 9 (121 mg, 70%) as a white foam. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.23−7.32 (m, 2H), 7.09−7.16 (m, 1H), 7.00 (d, J = 7.4 Hz, 1H), 4.10−4.48 (m, 2H), 2.68 (s, 3H), 2.64−2.75 (m, 2H), 2.20−2.33 (m, 1H), 2.02−2.13 (m, 1H), 1.73−1.87 (m, 1H), 1.46−1.61 (m, 2H), 1.38 (s, 9H), 0.76− 0.88 (m, 3H). MS m/z 376 [M + H]+. tert-Butyl (2S,3S)-3-Hydroxy-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylamino)butan-2-ylcarbamate (10). According to general procedure A, except 6 equiv Et3N were used, 6 (123 mg, 0.698 mmol) and BOC-L-allo-threonine dicyclohexylamine (336 mg, 0.838 mmol) were converted to 10 (263 mg, quant) as a white foam. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.23−7.33 (m, 2H), 7.11−7.18 (m, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.72 (d, J = 8.7 Hz, 1H), 4.88 (d, J = 4.0 Hz, 1H), 4.11−4.21 (m, 1H), 3.68−3.92 (m, 2H), 2.63−2.79 (m, 2H), 2.26−2.39 (m, 1H), 1.99−2.09 (m, 1H), 1.30−1.40 (m, 9H), 1.03 (d, J = 6.0 Hz, 3H). MS m/z 378 [M + H]+. tert-Butyl Ethyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-ylamino)propan-2-yl)carbamate (11). According to general procedure A, 6 (203 mg, 1.15 mmol) and (S)-2-(tertbutoxycarbonyl(ethyl)amino)propanoic acid (0.25 g, 1.2 mmol) were converted to 11 (0.205 mg, 47%) as a white foam. 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.65−7.79 (m, 1H), 7.22−7.32 (m, 2H), 7.09−7.17 (m, 1H), 7.00 (d, J = 7.7 Hz, 1H), 4.08−4.58 (m,

Figure 9. Caspase 3/7 activity in LOX melanoma xenografts. Tumorbearing nude mice were treated with a single dose of conatumumab (1 mg/kg ip), 40 (100 mg/kg po), 3 (200 mg/kg po), or a combination. After 8 h, xenografts were removed and caspase 3/7 activity was determined with a fluorescence assay measuring cleavage of DEVDAMC. Mean ± SD is indicated for each group (n = 3). General Procedure C: Deprotection. To a rt solution of above material in MeOH was added 2.0 M HCl in Et2O. The reaction was stirred at rt until complete, then concentrated in vacuo and lyophilized from MeCN−H2O to provide product. tert-Butyl Methyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b]azepin-3-ylamino)propan-2-yl)carbamate (7). According to general procedure A, 6 (0.419 g, 2.38 mmol) and BOC-Nmethyl-L-alanine (579 mg, 2.85 mmol) were converted to 7 (0.808 g, 94%) as a white foam. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.22−7.31 (m, 2H), 7.09−7.16 (m, 1H), 7.00 (d, J = 7.5 Hz, 1H), 4.29−4.62 (m, 1H), 4.10−4.24 (m, 1H), 2.69 (s, 3H), 2.66−2.75 (m, 2H), 2.19−2.36 (m, 1H), 2.00−2.14 (m, 1H), 1.36 (s, 9H), 1.22 (d, J = 7.2 Hz, 3H). MS m/z 362 [M + H]+.

Figure 10. Western blot analysis of LOX melanoma xenograft homogenates after 8 h treatment with a single dose of the DR5 antibody conatumumab (1 mg/kg ip) or a combination of conatumumab (1 mg/kg ip) with 3 (100 mg/kg po) or 40 (200 mg/kg po). Each lane is the result of a single xenograft. 7780

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

Article

(m, 2H), 1.95−2.09 (m, 1H), 1.61 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C26H30O2N3 [M + H]+ 416.23325, found 416.23212. (S)-N-((S)-1-((2-Methoxynaphthalen-1-yl)methyl)-2-oxo-2,3,4,5tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)propanamide Hydrochloride (18). According to general procedures B and C, 7 (0.10 g, 0.277 mmol) and 1-(chloromethyl)-2-methoxynaphthalene (0.086 g, 0.415 mmol) were converted to 18 (55.0 mg, 42% 2 steps) as a light-yellow solid. 1H NMR (300 MHz, methanol-d4) δ 8.24 (d, J = 8.5 Hz, 1H), 7.68−7.76 (m, 2H), 7.42−7.55 (m, 2H), 7.28−7.36 (m, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 9.1 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 6.90−6.95 (m, 1H), 6.04 (d, J = 14.5 Hz, 1H), 5.38 (d, J = 14.5 Hz, 1H), 4.25−4.38 (m, 1H), 3.88 (q, J = 6.9 Hz, 1H), 3.77 (s, 3H), 2.62 (s, 3H), 1.96−2.42 (m, 4H), 1.62 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C26H30O3N3 [M + H]+ 432.22817, found 432.22678. (S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)propanamide Hydrochloride (19). According to general procedures B and C, 7 (220 mg, 0.609 mmol) and 6-bromo-1-(chloromethyl)-2methoxynaphthalene (261 mg, 0.913 mmol) were converted to 19 (190 mg, 57% 2 steps) as a white solid. 1H NMR (400 MHz, DMSOd6) δ 8.95 (d, J = 7.9 Hz, 1H), 8.87 (br s, 2H), 8.17 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.52−7.60 (m, 2H), 7.25−7.33 (m, 2H), 7.04−7.09 (m, 1H), 6.98−7.02 (m, 1H), 5.94 (d, J = 14.5 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.15−4.24 (m, 1H), 3.80 (q, J = 6.9 Hz, 1H), 3.71 (s, 3H), 2.43 (s, 3H), 2.19−2.29 (m, 1H), 2.05−2.17 (m, 2H), 1.90−2.02 (m, 1H), 1.42 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C26H29O3N3Br [M + H]+ 510.13868, found 510.13901. (R)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)propanamide Hydrochloride (20). According to general procedure B, 8 (0.20 g, 0.55 mmol) and 6-bromo-1-(chloromethyl)-2-methoxynaphthalene (261 mg, 0.913 mmol) were reacted to provide tert-butyl (R)-1-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)methyl)-2-oxo2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (174 mg, 52%). A portion of this material (60 mg, 0.098 mmol) was reacted according to general procedure C to provide 20 (50.0 mg, 93%) as a white solid. 1H NMR (400 MHz, methanol-d4) δ 8.21 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.49−7.56 (m, 2H), 7.26 (t, J = 7.9 Hz, 1H), 7.22 (d, J = 9.1 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 6.7 Hz, 1H), 6.01 (d, J = 14.5 Hz, 1H), 5.37 (d, J = 14.5 Hz, 1H), 4.26 (dd, J = 11.8, 6.95 Hz, 1H), 3.82−3.91 (m, 1H), 3.75 (s, 3H), 2.71 (s, 3H), 2.14−2.37 (m, 3H), 1.96−2.08 (m, 1H), 1.46 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C26H29O3N3Br [M + H]+ 510.13868, found 510.13852. (S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)butanamide 2,2,2-trifluoroacetate (21). According to general procedure B, 9 (121 mg, 0.322 mmol) and 6-bromo-1-(chloromethyl)-2-methoxynaphthalene (138 mg, 0.483 mmol) were converted to tert-butyl (S)-1-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)methyl)2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylamino)-1-oxobutan2-yl(methyl)carbamate (88 mg, 44%). To a rt solution of this material in CH2Cl2 (2 mL) was added TFA (0.4 mL). The reaction was stirred at rt for 30 min, then concentrated in vacuo and lyophilized from MeCN−H2O to provide 21 (76.5 mg, 85%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 7.8 Hz, 1H), 8.86 (br s, 1H), 8.74 (br s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.4 Hz, 1H), 7.52−7.59 (m, 2H), 7.23−7.34 (m, 2H), 7.04− 7.09 (m, 1H), 6.99−7.02 (m, 1H), 5.93 (d, J = 14.5 Hz, 1H), 5.26 (d, J = 14.5 Hz, 1H), 4.20−4.31 (m, 1H), 3.68−3.74 (m, 1H), 2.39−2.46 (m, 3H), 1.70−2.29 (m, 6H), 0.99 (t, J = 7.4 Hz, 3H). HRMS m/z calcd for C27H31O3N3Br [M + H]+ 524.15433, found 524.15430. (2S,3S)-2-Amino-N-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-3-hydroxybutanamide hydrochloride (22). According to general procedures B and C, 10 (104 mg, 0.276 mmol) and 6-bromo-1(chloromethyl)-2-methoxynaphthalene (102 mg, 0.358 mmol) were converted to 22 (61 mg, 41% 2 steps) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (d, J = 7.9 Hz, 1H), 8.16 (d, J = 9.3 Hz, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.96 (br s, 3H), 7.80 (d, J = 9.3 Hz, 1H), 7.57

2H), 3.01−3.27 (m, 2H), 2.60−2.80 (m, 2H), 2.23−2.39 (m, 1H), 1.94−2.12 (m, 1H), 1.37 (s, 9H), 1.24 (d, J = 7.0 Hz, 3H), 1.02 (t, J = 6.9 Hz, 3H). MS m/z 398 [M + Na]+. (S)-N-((S)-1-Benzyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin3-yl)-2-(methylamino)propanamide Hydrochloride (12). According to general procedures B and C, 7 (0.10 g, 0.279 mmol) and benzyl bromide (0.071 g, 0.415 mmol) were converted to 12 (46.6 mg, 48% 2 steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.71 (d, J = 6.9 Hz, 1H), 7.30−7.40 (m, 2H), 7.16−7.27 (m, 7H), 5.29 (dd, J = 14.8, 2.4 Hz, 1H), 4.81−4.86 (m, 1H), 4.32−4.45 (m, 1H), 3.78−3.90 (m, 1H), 2.62 (d, J = 2.1 Hz, 3H), 2.54 (d, J = 5.4 Hz, 2H), 2.36 (d, J = 9.7 Hz, 1H), 2.01−2.20 (m, 1H), 1.49−1.63 (m, 3H). HRMS m/z calcd for C21H26O2N3 [M + H]+ 352.20195, found 352.20105. (S)-2-(Methylamino)-N-((S)-2-oxo-1-phenethyl-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (13). According to general procedure B, except the sodium iodide was omitted, and general procedure C, 7 (0.13 g, 0.36 mmol) and (2-bromoethyl)benzene (0.10 g, 0.54 mmol) were converted to 13 (52.4 mg, 36% 2 steps) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.09−9.33 (m, 1H), 8.81−8.95 (m, 1H), 8.65−8.79 (m, 1H), 7.33−7.41 (m, 2H), 7.08−7.32 (m, 7H), 4.30−4.50 (m, 1H), 4.05−4.23 (m, 1H), 3.71− 3.89 (m, 2H), 2.63−2.91 (m, 2H), 2.35−2.59 (m, 5H), 2.09−2.25 (m, 1H), 1.87−2.03 (m, 1H), 1.27−1.41 (m, 3H). HRMS m/z calcd for C22H28O2N3 [M + H]+ 366.21760, found 366.21683. (S)-2-(Methylamino)-N-((S)-1-(naphthalen-2-ylmethyl)-2-oxo2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (14). According to general procedure B, except the sodium iodide was omitted, and general procedure C, 7 (0.10 g, 0.279 mmol) and 2-(bromomethyl)naphthalene (0.092 g, 0.415 mmol) were converted to 14 (72.6 mg, 60% 2 steps) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.34 (br s, 1H), 8.97 (d, J = 7.6 Hz, 1H), 8.84 (br s, 1H), 7.76−7.89 (m, 3H), 7.70 (s, 1H), 7.43−7.51 (m, 3H), 7.30−7.38 (m, 2H), 7.14−7.25 (m, 2H), 5.44 (d, J = 15.1 Hz, 1H), 5.00 (d, J = 15.1 Hz, 1H), 4.22−4.37 (m, 1H), 3.75−3.86 (m, 1H), 3.45−3.63 (m, 1H), 2.47−2.60 (m, 1H), 2.42−2.46 (m, 3H), 2.03− 2.33 (m, 2H), 1.41 (d, J = 7.0 Hz, 3H). MS m/z 402 [M + H]+. (S)-2-(Methylamino)-N-((S)-1-(naphthalen-1-ylmethyl)-2-oxo2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (15). According to general procedures B and C, 7 (0.10 g, 0.279 mmol) and 1-(chloromethyl)naphthalene (0.073 g, 0.415 mmol) were converted to 15 (40.9 mg, 33% 2 steps) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.28 (br s, 1H), 9.00 (d, J = 7.6 Hz, 1H), 8.82 (br s, 1H), 8.21 (dd, J = 6.2, 3.5 Hz, 1H), 7.91 (dd, J = 6.2, 3.2 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.49−7.55 (m, 2H), 7.28−7.38 (m, 3H), 7.09−7.15 (m, 2H), 5.94 (d, J = 15.1 Hz, 1H), 5.21 (d, J = 15.4 Hz, 1H), 4.24−4.36 (m, 1H), 3.75− 3.86 (m, 1H), 3.35−3.48 (m, 1H), 2.41−2.47 (m, 3H), 1.97−2.38 (m, 3H), 1.42 (d, J = 6.6 Hz, 3H). HRMS m/z calcd for C25H28O2N3 [M + H]+ 402.21760, found 402.21646. (S)-2-(Methylamino)-N-((S)-1-((4-methylnaphthalen-1-yl)methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (16). According to general procedures B and C, 7 (0.10 g, 0.277 mmol) and 1-(chloromethyl)-4-methylnaphthalene (0.079 g, 0.415 mmol) were converted to 16 (30.5 mg, 24% 2 steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.22 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.44−7.57 (m, 3H), 7.28− 7.36 (m, 1H), 7.23−7.27 (m, 1H), 7.06−7.19 (m, 3H), 5.97 (d, J = 15.1 Hz, 1H), 5.17 (d, J = 15.1 Hz, 1H), 4.36−4.47 (m, 1H), 3.86 (q, J = 6.7 Hz, 1H), 2.63 (s, 3H), 2.60 (s, 3H), 2.21−2.43 (m, 3H), 2.01− 2.15 (m, 1H), 1.59 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C26H30O2N3 [M + H]+ 416.23325, found 416.23218. (S)-2-(Methylamino)-N-((S)-1-((2-methylnaphthalen-1-yl)methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (17). According to general procedures B and C, 7 (0.10 g, 0.277 mmol) and 1-(chloromethyl)-2-methylnaphthalene (0.079 g, 0.415 mmol) were converted to 17 (63.7 mg, 51% 2 steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.15−8.25 (m, 1H), 7.73−7.81 (m, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.37−7.46 (m, 2H), 7.07−7.20 (m, 4H), 7.01−7.06 (m, 1H), 5.91 (d, J = 14.8 Hz, 1H), 5.42 (d, J = 14.8 Hz, 1H), 4.24−4.34 (m, 1H), 3.86 (q, J = 6.9 Hz, 1H), 2.62 (s, 3H), 2.41 (s, 3H), 2.35−2.50 (m, 1H), 2.12−2.31 7781

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

Article

2.72 (m, 3H), 1.38 (s, 9H), 1.24 (d, J = 6.9 Hz, 3H). MS m/z 386 [M + Na]+. Step 2. According to general procedure B, tert-butyl methyl((S)-1oxo-1-((S)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3ylamino)propan-2-yl)carbamate (1.1 g, 3.03 mmol) and 6-bromo-1(chloromethyl)-2-methoxynaphthalene (951 mg, 3.33 mmol) were converted to tert-butyl (S)-1-((S)-5-((6-bromo-2-methoxynaphthalen1-yl)methyl)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (1.00 g, 54%) as a lightyellow foam. 1H NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 8.2 Hz, 1H), 7.99−8.07 (m, 2H), 7.78 (d, J = 9.1 Hz, 1H), 7.46−7.54 (m, 2H), 7.33 (d, J = 9.4 Hz, 1H), 7.02−7.17 (m, 2H), 6.89 (d, J = 7.6 Hz, 1H), 5.86 (d, J = 14.8 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.35−4.75 (m, 2H), 4.22−4.31 (m, 1H), 4.10−4.20 (m, 1H), 3.80 (s, 3H), 2.70 (s, 3H), 1.37 (s, 9H), 1.27 (d, J = 6.9 Hz, 3H). Step 3. To a 0 °C solution of tert-butyl (S)-1-((S)-5-((6-bromo-2methoxynaphthalen-1-yl)methyl)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (52 mg, 0.085 mmol) in CH2Cl2 (1 mL) was added TFA (1 mL). The reaction was stirred at 0 °C for 1 h, then concentrated in vacuo and triturated with Et2O to provide 27 (30 mg, 56%) as a light-brown solid. 1H NMR (300 MHz, DMSO-d6) δ 9.06 (d, J = 7.9 Hz, 1H), 8.67−8.90 (m, 2H), 8.00−8.07 (m, 2H), 7.79 (d, J = 9.1 Hz, 1H), 7.47−7.57 (m, 2H), 7.34 (d, J = 9.4 Hz, 1H), 7.05−7.20 (m, 2H), 6.92 (d, J = 6.6 Hz, 1H), 5.87 (d, J = 14.5 Hz, 1H), 5.26 (d, J = 14.5 Hz, 1H), 4.67−4.79 (m, 1H), 4.12−4.31 (m, 2H), 3.81−3.90 (m, 1H), 3.79 (s, 3H), 2.45−2.48 (m, 3H), 1.42 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for C25H27O4N3Br [M + H]+ 512.11795, found 512.11705. 2-(Dibenzylamino)-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetic Acid (29). Step 1. To a −78 °C solution of ethyl 2-(dibenzylamino)acetate (10 g, 35 mmol) in THF (100 mL) was added LDA (2.0 M solution, 38.8 mL, 77.6 mmol). After 30 min, a solution of dihydro2H-pyran-4(3H)-one (3.91 mL, 42.3 mmol) in THF (20 mL) was added. The reaction was warmed to rt and stirred for 1 h, then quenched with satd aq NH4Cl, concentrated in vacuo, poured into H2O (300 mL), and extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with H2O (2 × 200 mL) and satd aq NaCl (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide ethyl 2-(dibenzylamino)-2-(4-hydroxytetrahydro-2H-pyran-4yl)acetate (6.72 g, 50%) as a thick yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 7.32−7.38 (m, 8H), 7.22−7.30 (m, 2H), 4.57 (s, 1H), 4.10−4.29 (m, 4H), 3.36−3.58 (m, 6H), 3.09 (s, 1H), 1.77−1.86 (m, 1H), 1.49−1.59 (m, 1H), 1.38−1.48 (m, 1H), 1.29 (t, J = 7.0 Hz, 3H), 1.19−1.25 (m, 1H). MS m/z 384 [M + H]+. Step 2. To a rt solution of ethyl 2-(dibenzylamino)-2-(4hydroxytetrahydro-2H-pyran-4-yl)acetate (6.72 g, 17.5 mmol) in MeOH (150 mL) was added potassium hydroxide (5.9 g, 105 mmol) in H2O (50 mL). The mixture was stirred at 60 °C for 15 h, then cooled to rt and diluted with H2O (200 mL). The pH was adjusted to ∼4 by the addition of satd aq KHSO4, and the mixture was extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with H2O (2 × 200 mL) and satd aq NaCl (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo to provide 29 (5.72 g, 92%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.65 (br s, 1H), 7.32−7.38 (m, 8H), 7.23−7.29 (m, 2H), 4.12−4.21 (m, 2H), 3.46−3.58 (m, 4H), 3.29−3.41 (m, 2H), 3.01 (s, 1H), 1.76− 1.85 (m, 1H), 1.47−1.57 (m, 1H), 1.34−1.44 (m, 1H), 1.26−1.33 (m, 1H). MS m/z 356 [M + H]+. 2-(Dibenzylamino)-2-(4-(2-nitrophenoxy)tetrahydro-2H-pyran-4yl)acetic Acid (30). To a rt solution of 29 (5.72 g, 16.1 mmol) in THF (90 mL) was added KHMDS (1.0 M solution in THF, 35.4 mL, 35.4 mmol), followed by 1-fluoro-2-nitrobenzene (1.87 mL, 17.7 mmol). The reaction was stirred at rt for 2 h, then additional KHMDS (10 mL, 10 mmol) and 1-fluoro-2-nitrobenzene (1 mL, 9.5 mmol) were added and the reaction was stirred for an additional 2 h. The reaction was diluted with H2O (200 mL), acidified with satd aq KHSO4, and extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with H2O (2 × 300 mL) and satd aq NaCl (200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude

(dd, J = 9.1, 2.0 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 9.3 Hz, 1H), 7.26 (t, J = 7.1 Hz, 1H), 7.04−7.09 (m, 1H), 6.98−7.02 (m, 1H), 5.92 (d, J = 14.5 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 5.26 (d, J = 14.5 Hz, 1H), 4.12−4.25 (m, 2H), 3.81 (d, J = 4.1 Hz, 1H), 3.71 (s, 3H), 1.90−2.28 (m, 4H), 1.15 (d, J = 6.7 Hz, 3H). HRMS m/z calcd for C26H29O4N3Br [M + H]+ 526.13360, found 526.13416. (S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(ethylamino)propanamide Hydrochloride (23). According to general procedures B and C, 11 (205 mg, 0.545 mmol) and 6-bromo-1-(chloromethyl)-2methoxynaphthalene (234 mg, 0.818 mmol) were converted to 23 (206 mg, 67% 2 steps) as a light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.49 (br s, 1H), 9.00 (d, J = 7.8 Hz, 1H), 8.78 (br s, 1H), 8.17 (d, J = 9.4 Hz, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.58 (dd, J = 9.0, 2.0 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.24− 7.32 (m, 2H), 7.03−7.08 (m, 1H), 6.97−7.01 (m, 1H), 5.93 (d, J = 14.5 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.14−4.23 (m, 1H), 3.83− 3.93 (m, 1H), 3.71 (s, 3H), 2.71−2.86 (m, 2H), 1.90−2.29 (m, 4H), 1.44 (d, J = 6.64 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). HRMS m/z calcd for C27H31O3N3Br [M + H]+ 524.15433, found 524.15466. (S)-2-tert-Butoxycarbonylamino-3-(2-nitrophenoxy)propionic Acid (25). To a 0 °C suspension of NaH (60% w/w in mineral oil, 2.9 g, 72 mmol) in DMF (25 mL) was added a solution of (S)-2-(tertbutoxycarbonylamino)-3-hydroxypropanoic acid (7 g, 34 mmol) in DMF (25 mL). After 2 h, a solution of 1-fluoro-2-nitrobenzene (5.29 g, 37.5 mmol) in DMF (25 mL) was added and the resulting mixture was stirred at 0 °C for 3 h. The mixture was poured into 0 °C H2O (200 mL), acidified to pH 5.0 with 1 N aq HCl, and extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide 25 (10.9 g, 97%). MS m/z 349 [M + Na]+. (S)-3-Amino-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (26). Step 1. A mixture of 25 (7.20 g, 22.1 mmol) and 10% w/w palladium on carbon (720 mg, 0.677 mmol) in EtOH (250 mL) was stirred under hydrogen (1 atm) for 4 h. The reaction was filtered through Celite, and the filtrate was concentrated in vacuo to provide (S)-3-(2aminophenoxy)-2-(tert-butoxycarbonylamino)propionic acid (6.2 g, 95%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 7.44 (d, J = 8.5 Hz, 1H), 6.63−6.76 (m, 2H), 6.56−6.62 (m, 1H), 6.42−6.50 (m, 1H), 4.38−4.45 (m, 1H), 4.24 (dd, J = 9.5, 4.7 Hz, 1H), 3.94−4.03 (m, 1H), 1.40 (s, 9H). MS m/z 297 [M + H]+. Step 2. To a rt mixture of (S)-3-(2-aminophenoxy)-2-(tertbutoxycarbonylamino)propionic acid (5.8 g, 19.6 mmol) in DMF was added EDCI (3.75 g, 19.6 mmol). The reaction was stirred at rt for 1.5 h then diluted with EtOAc, washed with H2O and satd aq NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was triturated with hexanes to provide (S)-tert-butyl 4oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylcarbamate (3.4 g, 62%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 7.04−7.18 (m, 5H), 4.20−4.39 (m, 3H), 1.36 (s, 9H). MS m/z 301 [M + Na]+. Step 3. To a 0 °C solution of (S)-tert butyl 4-oxo-2,3,4,5tetrahydrobenzo[b][1,4]oxazepin-3-ylcarbamate (3.4 g, 7.2 mmol) in CH2Cl2 (20 mL) was added TFA (20 mL). The reaction was stirred for 1 h, then concentrated in vacuo. The residue was diluted with satd aq NaHCO3 and extracted with CH2Cl2 (4×). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to provide 26 (2.2 g, quant). 1H NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.02−7.11 (m, 4H), 4.28 (dd, J = 10.4, 6.2 Hz, 1H), 4.01 (t, J = 10.6 Hz, 1H), 3.60 (dd, J = 10.6, 6.3 Hz, 1H), 1.81 (br s, 2H). MS m/z 179 [M + H]+. (S)-N-((S)-5-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-4oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-2(methylamino)propanamide 2,2,2-Trifluoroacetate (27). Step 1. According to general procedure A, 26 (2.2 g, 12 mmol) and BOC-Nmethyl-L-alanine (2.48 g, 12.2 mmol) were converted to tert-butyl methyl((S)-1-oxo-1-((S)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylamino)propan-2-yl)carbamate (4.2 g, 95%) as a lightyellow foam. 1H NMR (300 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.05−7.16 (m, 4H), 4.24−4.71 (m, 4H), 2.68− 7782

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

Article

material was purified by flash chromatography to provide 30 (7.06 g, 92%) as a yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 13.00 (br s, 1H), 7.77 (dd, J = 8.2, 1.6 Hz, 1H), 7.33−7.41 (m, 9H), 7.26−7.32 (m, 2H), 7.16 (t, J = 7.6 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 4.08−4.17 (m, 2H), 3.89 (s, 1H), 3.60−3.69 (m, 2H), 3.30−3.50 (m, 2H), 3.14− 3.22 (m, 1H), 2.98−3.08 (m, 1H), 2.04−2.15 (m, 2H), 1.84−1.94 (m, 1H), 1.43−1.52 (m, 1H). MS 477 [M + H]+. 3-Amino-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine2,4′-pyran]-4(5H)-one (31). Step 1. A mixture of 30 (7.06 g, 14.8 mmol), ammonium chloride (1.59 g, 29.6 mmol), and zinc (15.0 g, 230 mmol) in MeOH (300 mL) was stirred at 65 °C for 15 h. The reaction was cooled to rt, filtered through Celite, concentrated in vacuo, then partitioned between satd aq NaOAc (400 mL) and EtOAc (400 mL). The aqueous layer was separated and extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with satd aq NaCl (3 × 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo to provide 2-(4-(2-aminophenoxy)tetrahydro-2H-pyran-4-yl)-2(dibenzylamino)acetic acid (6.75 g, quant) as a brown foam. 1H NMR (400 MHz, DMSO-d6) δ 7.35−7.48 (m, 4H), 7.14−7.30 (m, 6H), 6.68−6.76 (m, 2H), 6.50−6.59 (m, 1H), 6.34 (t, J = 7.4 Hz, 1H), 5.52 (br s, 2H), 4.11−4.41 (m, 2H), 3.71−3.97 (m, 3H), 3.35−3.46 (m, 1H), 3.10−3.23 (m, 1H), 2.84−2.98 (m, Hz, 1H), 2.34−2.46 (m, 1H), 2.24 (d, J = 8.6 Hz, 1H), 1.81−1.95 (m, 2H), 1.41−1.54 (m, 1H). MS m/z 447 [M + H]+. Step 2. A solution of 2-(4-(2-aminophenoxy)tetrahydro-2H-pyran4-yl)-2-(dibenzylamino)acetic acid (6.75 g, 15.1 mmol), HOBt (3.24 g, 21.2 mmol), and EDCI (2.9 g, 15.1 mmol) in DMF (100 mL) was stirred at rt for 1 h, then diluted with EtOAc (500 mL), washed with 1:1 satd aq NaHCO3−satd aq NaCl (3 × 200 mL) and satd aq NaCl (200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide 3(dibenzylamino)-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-4(5H)-one (4.77 g, 74%) as a beige solid. 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 7.30−7.36 (m, 8H), 7.22−7.29 (m, 2H), 7.18 (dd, J = 7.8, 1.2 Hz, 1H), 6.92−7.06 (m, 3H), 3.89 (d, J = 12.9 Hz, 2H), 3.65−3.74 (m, 1H), 3.57 (d, J = 13.3 Hz, 2H), 3.41−3.50 (m, 2H), 3.31−3.39 (m, 1H), 3.28 (s, 1H), 2.08− 2.14 (m, 1H), 1.45−1.55 (m, 1H), 1.35−1.42 (m, 1H), 1.12−1.22 (m, 1H). MS m/z 429 [M + H]+. Step 3. A mixture of 3-(dibenzylamino)-2′,3′,5′,6′-tetrahydro-3Hspiro[benzo[b][1,4]oxazepine-2,4′-pyran]-4(5H)-one (4.77 g, 11.1 mmol; equiv, 1.00) and 20% palladium hydroxide on carbon (2 g) in MeOH (150 mL) was stirred under H2 overnight. The reaction was treated with additional catalyst and H2 and stirred overnight, then filtered through Celite and concentrated in vacuo to provide 31 (2.75 g, quant) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 7.02−7.18 (m, 4H), 3.84−3.92 (m, 1H), 3.75−3.83 (m, 2H), 3.61−3.70 (m, 1H), 3.25 (s, 1H), 2.63−3.00 (m, 2H), 1.93−2.07 (m, 2H), 1.36−1.50 (m, 2H). MS m/z 249 [M + H]+. tert-Butyl Methyl((S)-1-oxo-1-((S)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)propan-2-yl)carbamate (32). A solution of HBTU (43.2 g, 114 mmol) and HOBt·H2O (17.4 g, 114 mmol) in DMF (300 mL) was added to a mixture of 3-amino-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-4(5H)-one hydrochloride (27 g, 94.8 mmol), BOC-N-methyl-L-alanine (23.1 g, 114 mmol), and Et3N (52.9 mL, 379 mmol) in DMF (300 mL). After 18 h, the mixture was poured into satd aq NaCl (750 mL) and extracted with EtOAc. The combined extracts were washed with 1:1 sat NaHCO3−satd aq NaCl and satd aq NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography and the diastereomers were separated by SFC (Daicel AD column (5 cm × 25 cm) eluted with 15% MeOH−CO2 at 200 mL/min; 220 nM detection; 100 bar backpressure; 35 °C oven). The first peak to elute from the column provided 32 (15.29 g, 37%) as an off-white foam. 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 7.06−7.24 (m, 4H), 4.60 (q, J = 7.2 Hz, 1H), 4.51 (d, J = 9.0 Hz, 1H), 3.62−3.95 (m, 4H), 2.74 (s, 3H), 2.01−2.06 (m, 1H), 1.50−1.61 (m, 3H), 1.38 (s, 9H), 1.20−1.30 (m, 3H). MS m/z 434 [M + H]+.

(S)-N-((S)-5-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-4oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine2,4′-pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride (33). According to general procedure B, 32 (332 mg, 0.766 mmol) and 6-bromo-1-(chloromethyl)-2-methoxynaphthalene (328 mg, 1.15 mmol) were converted to tert-butyl (S)-1-((S)-5-((6-bromo-2methoxynaphthalen-1-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3Hspiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2yl(methyl)carbamate (281 mg, 54%). According to general procedure C, a portion of this (92.3 mg, 0.135 mmol) was converted to 33 (67.7 mg, 81%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.10−9.19 (m, 1H), 9.08 (d, J = 9.29 Hz, 1H), 8.76−8.85 (m, 1H), 8.01−8.06 (m, 2H), 7.79 (d, J = 9.0 Hz, 1H), 7.56 (dd, J = 8.0, 1.3 Hz, 1H), 7.47 (dd, J = 9.0, 2.0 Hz, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.13− 7.18 (m, 1H), 7.05−7.10 (m, 1H), 6.84−6.88 (m, 1H), 5.90 (d, J = 14.8 Hz, 1H), 5.27 (d, J = 14.6 Hz, 1H), 4.54 (d, J = 9.3 Hz, 1H), 3.93−4.02 (m, 1H), 3.83 (s, 3H), 3.79−3.86 (m, 1H), 3.64−3.78 (m, 2H), 3.44 (t, J = 11.2 Hz, 1H), 2.45−2.49 (m, 3H), 2.11 (dt, J = 12.9. 5.3 Hz, 1H), 1.48−1.63 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.34−1.38 (m, 1H). HRMS m/z calcd for C29H33O5N3Br [M + H]+ 582.15981, found 582.15938. (S)-2-(Methylamino)-N-((S)-5-((2-methylnaphthalen-1-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-yl)propanamide Hydrochloride (34). According to general procedure B, except the reaction was carried out at rt, and general procedure C, 32 (100 mg, 0.231 mmol) and 1(chloromethyl)-2-methylnaphthalene (66.0 mg, 0.346 mmol) were converted to 34 (58.4 mg, 48% 2 steps) as a light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.13−9.23 (m, 1H), 9.10 (d, J = 8.9 Hz, 1H), 8.76−8.88 (m, 1H), 8.19−8.26 (m, 1H), 7.77 (dd, J = 6.3, 3.5 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.38−7.43 (m, 3H), 7.21 (d, J = 8.2 Hz, 1H), 7.00−7.08 (m, 2H), 6.89−6.94 (m, 1H), 5.97 (d, J = 15.2 Hz, 1H), 5.37 (d, J = 15.2 Hz, 1H), 4.55 (d, J = 9.0 Hz, 1H), 3.94− 4.04 (m, 1H), 3.67−3.89 (m, 3H), 3.45−3.59 (m, 1H), 2.49−2.51 (m, 3H), 2.46−2.49 (m, 3H), 2.06−2.19 (m, 1H), 1.66 (d, J = 13.3 Hz, 1H), 1.54 (td, J = 13.2, 5.7 Hz, 1H), 1.41 (d, J = 7.0 Hz, 3H), 1.35− 1.39 (m, 1H). MS m/z 488 [M + H]+. (S)-N-((S)-5-((2-(Difluoromethoxy)naphthalen-1-yl)methyl)-4oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine2,4′-pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride (35). To a rt solution of 32 in which the diastereomers had not been separated (118 mg, 0.272 mmol) was added (2(difluoromethoxy)naphthalen-1-yl)methyl methanesulfonate (98.7 mg, 0.327 mmol) and Cs2CO3 (115 mg, 0.354 mmol). The reaction was stirred at rt for 20 h, then diluted with EtOAc (25 mL), washed with H2O (25 mL) and satd aq NaCl (25 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography then by SFC to separate the diastereomers and provide tert-butyl (S)-1-((S)-5-((2-(difluoromethoxy)naphthalen-1yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (52.6 mg, 30%) as a white foam. This material was reacted according to general procedure C to provide 35 (46.3 mg, 98%) as a white solid. 1 H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 9.06 Hz, 1H), 8.78 (br s, 2H), 8.19 (d, J = 9.1 Hz, 1H), 7.82−7.93 (m, 2H), 7.43−7.56 (m, 3H), 7.27 (d, J = 8.7 Hz, 1H), 7.25 (t, J = 73.3 Hz, 1H), 7.04−7.18 (m, 2H), 6.87 (d, J = 7.2 Hz, 1H), 6.06 (d, J = 15.1 Hz, 1H), 5.27 (d, J = 15.1 Hz, 1H), 4.56 (d, J = 9.1 Hz, 1H), 3.90−4.02 (m, 1H), 3.63−3.85 (m, 3H), 3.43 (t, J = 11.9 Hz, 1H), 2.47−2.49 (m, 3H), 2.04−2.18 (m, 1H), 1.65 (d, J = 13.6 Hz, 1H), 1.46−1.59 (m, 1H), 1.39 (d, J = 6.4 Hz, 3H), 1.34−1.38 (m, 1H). HRMS m/z calcd for C29H32O5N3F2 [M + H]+ 540.23045, found 540.22913. 6-Methoxy-5-(((S)-3-((S)-2-(methylamino)propanamido)-4-oxo2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]5(4H)-yl)methyl)-2-naphthoic Acid 2,2,2-Trifluoroacetate (36). Step 1. A mixture of tert-butyl (S)-1-((S)-5-((6-bromo-2-methoxynaphthalen-1-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (0.17 g, 249 μmol), Pd(OAc)2 (2.24 mg, 9.96 μmol), 4,5bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (11.5 mg, 19.9 μmol), MeOH (0.10 mL, 2.5 mmol), and Et3N (0.5 mL) was 7783

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

Article

sparged with CO, sealed and stirred at 70 °C for 18 h. The reaction was cooled to rt, diluted with EtOAc, washed with 1 N aq HCl, H2O, and satd aq NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide methyl 5-(((S)-3-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2-naphthoate (111 mg, 67%) as a light-yellow foam. 1H NMR (400 MHz, DMSOd6) δ 8.48 (s, 1H), 8.28−8.40 (m, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 9.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.02−7.08 (m, 1H), 6.82 (d, J = 7.4 Hz, 1H), 5.93 (d, J = 14.8 Hz, 1H), 5.28 (d, J = 14.8 Hz, 1H), 4.63 (q, J = 7.0 Hz, 1H), 4.54 (d, J = 9.0 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.59−3.80 (m, 3H), 3.34−3.45 (m, 1H), 2.75 (s, 3H), 2.01−2.16 (m, 1H), 1.59 (d, J = 13.3 Hz, 1H), 1.43−1.52 (m, 1H), 1.38 (s, 9H), 1.23−1.32 (m, 4H). MS m/z 662 [M + H]+. Step 2. To a solution of methyl 5-(((S)-3-((S)-2-(tertbutoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2-naphthoate (111 mg, 0.167 mmol) in THF (3 mL) was added a solution of LiOH·H2O (35.0 mg, 835 μmol) in H2O (4 mL). The reaction was stirred at rt for 18 h, then diluted with 1 M aq HCl (20 mL) and extracted with EtOAc (2 × 15 mL). The combined organic extracts were washed with H2O (2 × 20 mL) and satd aq NaCl (20 mL), dried over Na2SO4, filtered, concentrated in vacuo, and lyophilized from MeCN−H2O to provide 5-(((S)-3-((S)-2(tert-butoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2-naphthoic acid (95.2 mg, 88%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.90 (br s, 1H), 8.44 (s, 1H), 8.30−8.39 (m, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 9.4 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.02−7.08 (m, 1H), 6.82 (d, J = 7.8 Hz, 1H), 5.93 (d, J = 14.8 Hz, 1H), 5.29 (d, J = 14.5 Hz, 1H), 4.63 (q, J = 7.4 Hz, 1H), 4.54 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H), 3.59−3.81 (m, 3H), 3.41 (t, J = 11.5 Hz, 1H), 2.75 (s, 3H), 2.02−2.15 (m, 1H), 1.60 (d, J = 13.3 Hz, 1H), 1.43−1.52 (m, 1H), 1.38 (s, 9H), 1.21−1.32 (m, 4H). MS m/z 648 [M + H]+. Step 3. To a rt solution of 5-(((S)-3-((S)-2-(tert-butoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2naphthoic acid (36.5 mg, 0.056 mmol) in CH2Cl2 (1 mL) was added TFA (0.2 mL). The reaction was stirred at rt for 1 h, then it was concentrated in vacuo and lyophilized from MeCN−H2O to provide 36 (31.0 mg, 83%) as an off-white solid. 1H NMR (300 MHz, DMSOd6) δ 12.93 (br s, 1H), 9.11 (d, J = 9.2 Hz, 1H), 8.66−8.90 (m, 2H), 8.45 (d, J = 1.3 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.80 (dd, J = 9.0, 1.5 Hz, 1H), 7.58 (dd, J = 7.9, 1.3 Hz, 1H), 7.37 (d, J = 9.2 Hz, 1H), 7.13−7.19 (m, 1H), 7.04−7.11 (m, 1H), 6.82− 6.88 (m, 1H), 5.94 (d, J = 14.7 Hz, 1H), 5.30 (d, J = 14.7 Hz, 1H), 4.55 (d, J = 9.2 Hz, 1H), 3.98 (d, J = 6.4 Hz, 1H), 3.86 (s, 3H), 3.63− 3.84 (m, 3H), 3.32−3.47 (m, 1H), 2.49−2.51 (m, 3H), 2.10 (td, J = 13.0, 5.3 Hz, 1H), 1.46−1.68 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.27− 1.37 (m, 1H). MS m/z 548 [M + H]+. ( S)-N-((S) -5-((3-Met ho xyqui nolin-4-yl)methyl)-4-oxo2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride (37). According to general procedures B, except that the reaction was conducted at rt, and general procedure C, 32 (100 mg, 0.231 mmol) and 4-(chloromethyl)-3-methoxyquinoline (71.9 mg, 0.346 mmol) were converted to 37 (75.9 mg, 57% 2 steps) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.25−9.34 (m, 1H), 9.08 (d, J = 9.4 Hz, 1H), 8.90 (s, 1H), 8.79−8.88 (m, 1H), 8.27 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.54−7.72 (m, 3H), 7.22 (t, J = 7.6 Hz, 1H), 7.11−7.17 (m, 1H), 6.93 (d, J = 7.8 Hz, 1H), 5.99 (d, J = 14.5 Hz, 1H), 5.31 (d, J = 14.8 Hz, 1H), 4.56 (d, J = 9.0 Hz, 1H), 3.95−4.01 (m, 1H), 3.95 (s, 3H), 3.65−3.82 (m, 3H), 3.43 (t, J = 11.3 Hz, 1H), 2.45 (t, J = 5.3 Hz, 3H), 2.08 (td, J = 13.0, 5.3 Hz, 1H), 1.45−1.59 (m, 2H), 1.39 (d, J = 6.6 Hz, 3H), 1.20−1.36 (m, 1H). HRMS m/z calcd for C28H33O5N4 [M + H]+ 505.24455, found 505.24310.

(S)-N-((S)-5-(Benzo[d]isoxazol-3-ylmethyl)-4-oxo-2′,3′,4,5,5′,6′hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-yl)-2(methylamino)propanamide Hydrochloride (38). To a rt solution of 32 in which the diastereomers had not been separated (156 mg, 0.360 mmol) was added 3-(bromomethyl)benzo[d]isoxazole (83.9 mg, 0.396 mmol), Cs2CO3 (141 mg, 0.432 mmol), and NaI (65 mg, 0.43 mmol). The reaction was stirred at rt for 18 h, then diluted with EtOAc (25 mL), washed with H2O (25 mL) and satd aq NaCl (25 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography then by SFC to separate the diastereomers and provide tert-butyl (S)-1-((S)-5(benzo[d]isoxazol-3-ylmethyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3Hspiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2yl(methyl)carbamate (54 mg, 27%) as a white foam. This material was reacted according to general procedure C to provide 38 (42 mg, 92%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 9.1 Hz, 1H), 8.92 (br s, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.59−7.74 (m, 3H), 7.23−7.39 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 5.71 (d, J = 15.5 Hz, 1H), 5.35 (d, J = 15.5 Hz, 1H), 4.65 (d, J = 9.1 Hz, 1H), 3.95 (q, J = 6.4 Hz, 1H), 3.70−3.89 (m, 3H), 3.54 (t, J = 11.3 Hz, 1H), 2.47 (s, 3H), 2.11 (td, J = 12.9, 5.1 Hz, 1H), 1.43−1.66 (m, 3H), 1.35 (d, J = 6.8 Hz, 3H). HRMS m/z calcd for C25H29O5N4 [M + H]+ 465.21325, found 465.21185. (S)-N-((S)-5-((1-(2-Cyanophenyl)-1H-indazol-3-yl)methyl)-4-oxo2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′pyran]-3-yl)-2-(methylamino)propanamide (39). According to general procedure B, 32 (55 mg, 0.127 mmol) and 2-(3-(bromomethyl)1H-indazol-1-yl)benzonitrile (78 mg, 0.25 mmol) were converted to (S)-2-amino-N-((S)-5-((1-(2-cyanophenyl)-1H-indazol-3-yl)methyl)4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)propanamide (60 mg, 71%). To a 0 °C solution of this material in CH2Cl2 (1 mL) was added TFA (1.5 mL). The reaction was stirred at 0 °C for 1 h, then diluted with satd aq NaHCO3 and extracted with CH2Cl2. The combined organic layers were concentrated in vacuo and the residue was purified by preparative TLC to provide 39 (48 mg, 94%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.31 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.86−7.94 (m, 2H), 7.63−7.79 (m, 3H), 7.45−7.57 (m, 2H), 7.19−7.31 (m, 3H), 7.06−7.11 (m, 1H), 5.50 (s, 2H), 4.63 (d, J = 9.0 Hz, 1H), 3.70−3.90 (m, 3H), 3.60 (t, J = 11.4 Hz, 1H), 2.96−3.07 (m, 1H), 2.18 (s, 3H), 1.91−2.06 (m, 1H), 1.66 (d, J = 13.4 Hz, 1H), 1.38−1.57 (m, 2H), 1.10 (d, J = 7.0 Hz, 3H); HRMS m/z calcd for C32H33O4N6 [M + H]+ 565.25578, found 565.25580. (S)-N-((S)-5-((2-Cyano-1-(2-cyanophenyl)-1H-indol-3-yl)methyl)4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine2,4′-pyran]-3-yl)-2-(methylamino)propanamide (40). Step 1. To a rt solution 32 (8.5 g, 19.6 mmol) and 3-(bromomethyl)-1-(2cyanophenyl)-1H-indole-2-carbonitrile (9.89 g, 29.4 mmol) in DMF (20 mL) was added Cs2CO3 (9.58 g, 29.4 mmol). The reaction was stirred at 70 °C for 1 h, then cooled to rt, diluted with satd aq NH4Cl, and extracted with EtOAc. The combined organic layers were washed with H2O, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography to provide tertbutyl (S)-1-((S)-5-((2-cyano-1-(2-cyanophenyl)-1H-indol-3-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (13 g, 96%) as a light-yellow solid. MS m/z 689 [M + H]+. Step 2. To a 0 °C solution of tert-butyl (S)-1-((S)-5-((2-cyano-1(2-cyanophenyl)-1H-indol-3-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (4 g, 5.81 mmol) in CH2Cl2 (50 mL) was added TFA (25 mL) dropwise over 2 h. The reaction was stirred for 30 min at 0 °C, then diluted with 1:1 satd aq NaHCO3 and H2O. The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuo to provide 40 (3.4 g, 97%) as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.13−8.24 (m, 1H), 7.76−7.91 (m, 3H), 7.62−7.68 (m, 1H), 7.46−7.52 (m, 1H), 7.33−7.45 (m, 2H), 7.16−7.31 (m, 3H), 7.01−7.06 (m, 1H), 6.91−6.97 (m, 1H), 5.85− 5.94 (m, 1H), 5.11−5.18 (m, 1H), 4.76−4.83 (m, 1H), 3.60−3.94 (m, 4H), 3.06−3.15 (m, 1H), 2.47−2.51 (m, 3H), 1.94−2.07 (m, 1H), 7784

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

Article

TNFα ELISA Assay. MDA-MB-231 cells (400,000 cells/well) were seeded into 24 well plates and incubated for 24 h. Media was aspirated from wells and replaced with either compound or media alone (400 μL/well) and further incubated at 37 °C with 5% CO2 for 19 h. Total media volume was then collected and centrifuged (1000g) at 4 °C to pellet debris. TNF of media was quantitated by ELISA (TNF Human ELISA kit, R&D Systems cat. no. DTA00B), and the results were plotted following subtraction of background values (media, no cells). in Vivo Caspase 3/7 Assay and Western Blot Analysis. A single dose of compound was administered as follows to female nude mice with LOX xenografts: conatumumab 1 mg/kg ip, 3 100 mg/kg po, 40 200 mg/kg po, or a combination. Xenografts were removed 8 h postdose, frozen (−80 °C), transferred to 15 mL conical tubes on dry ice, and treated with 1× lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TX-100, 5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM Na3VO4, 1 mM benzamidine, EMD proteinase inhibitor cocktail V). Tumors were homogenized at full speed, diluted with the same volume of lysis buffer, and kept on ice for 20 min. A portion of the homogenate (2 mL) was transferred to a microcentrifuge tube and thawed at full speed for 20 min at 4 °C. The supernatant was optionally centrifuged again. Protein was quantitated and samples were prepared for caspase assay and Western blot analysis (50 μg of total protein was used for caspase assay/60 μg of total protein was used for Western blot). Caspase 3/7 Assay. Tumor lysate (10 μL, 5 μg/μL) was added into white solid 96 well plate. Caspase-3/7 assay buffer (final concentration: 10 mM Tris, 5 mM DTT, 1 mM EDTA, 50 μM DEVD-AMC, 20 mM NaCl, 1% NP-40) (90 μL) was added into each well. The plate was incubated for 2 h at rt with shaking and read at 385/460 nM. Western Blot Analysis. Proteins were electrophoresed on 4−12% NuPAGE Bis-Tris midi-gel (Invitrogen) and transferred to PVDF membrane (Invitrogen). Membranes were blocked in 5% milk, incubated with primary antibodies, washed, and incubated with horseradish peroxidase linked secondary antibody (Cell Signaling Technology). The blots were stripped and reprobed. The following primary antibodies were used in the study: anti-XIAP (R&D Systems), anticaspase-3 (Santa Cruz), anti-cIAP1, anti-PARP.

1.55−1.78 (m, 3H), 1.31−1.35 (m, 3H). HRMS m/z calcd for C34H33O4N6 [M + H]+ 589.25578, found 589.25482. Biology. XIAP BIR2 and BIR3 TR-FRET Assay. Ten nanomolar of 6× histidine-tagged BIR2 (amino acids 124−240) or BIR3 (amino acids 241−356) domain of the XIAP protein was mixed with 20 nM of the peptide AVPIAQKSEK (ε-biotin)−OH 1:2 TFA in the presence of 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT), and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min incubation at 37 °C, europium−streptavidin and allophycocyanin conjugated anti-histidine antibody were added to a final concentration of 1.5 and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured after 1 h at rt. Compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each compound. Typically, Z′ = ∼0.9.36 A measure of the overall variability of the assay is provided by the positive control: BIR2 IC50 = 1.25 ± 0.91 μM (n = 160), BIR3 IC50 = 0.039 ± 0.025 μM (n = 168). cIAP1 BIR2 and BIR3 TR-FRET Assay. Thirty nanomolar of 6× histidine−thrombin−TEV tagged BIR2 (amino acids 174−256) or BIR3 (amino acids 260−352) domain of the cIAP protein was mixed with 50 nM of the peptide AVPIAQKSEK (ε-biotin)−OH 1:2 TFA in the presence of 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT), and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min incubation at 37 °C, europium−streptavidin and allophycocyanin conjugated anti-histidine antibody were added to a final concentration of 1.5 and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured after 1 h at rt. Compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each compound. Rat Blood Stability Assay. A 10 mM DMSO solution of test compound was diluted to 200 μM with MeCN and then further diluted to 1 μM (total volume 1.0 mL) with heparin-treated rat blood and then incubated at 37 °C with 5% CO2 for 4 h. Aliquots (50 μL) were removed at 0, 15, 60, and 240 min, then treated with 0.6 mL of 0.1% formic acid in MeCN with 2 mg/mL 7-hydroxycoumarin internal standard, vortexed, and centrifuged (4000 rpm) for 10 min. The supernatants (150 μL) were transferred to injection plates containing 150 μL of 0.1% formic acid in water, vortexed, and centrifuged (4000 rpm) for 5 min, then analyzed by LC-MS/MS. XIAP-Caspase Reactivation Assay. The caspase reactivation assay was performed in 20 mM HEPES buffer (pH 7.0), containing 1.5 mM MgCl2, 5 mM KCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM DTT, and 0.1 mg/mL BSA. S-100 cell extract served as the source of cellular caspases. Then 2 μM cytochrome C and 19.2 μM dATP were added to activate the caspases. The cleavage of a fluorogenic substrate specific for caspase-3 and -7, Ac-DEVD-AFC at 10 μM, was monitored at 530 nm after 4 h of incubation at rt. The addition of 0.75 nM glutathione S-transferase (GST)-tagged full length XIAP inhibited the caspase activation by ∼85%. The effect of serially diluted compounds to restore caspase activation was determined to generate an EC50 value for each compound. Cell-Based Assay. SW-620 cells (1500 cells/well) were seeded into a 96 well plate one day prior to treatment. Cells were treated with indicated compounds and/or antibody and further incubated at 37 °C with 5% CO2 for 5 days. Cell Titer 96 Aqueous One solution (25 μL/ well, Promega cat. no. G3580) was added, and the plates were further incubated for 2−3 h. Plate’s OD at 490 nm was determined using Envision 2101 plate reader (PerkinElmer), and EC50 values were determined for compounds or antibody. A similar protocol was used to evaluate the other cell lines. in Vitro Caspase 3/7 Assay. SW620 cells were treated with indicated compounds and/or antibody and incubated for 4 h. Cell lysate (25 μg) and caspase-3/7 assay buffer (1% NP40, 50 μM DEVDAMC (Biomol)) were added to a solid white 96 well plate. Lysates were incubated at rt for 20 min, and cleavage of DEVD-AMC substrate was measured with a plate reader (Molecular Devices Spectramax M5) at 385/460 nm.

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ASSOCIATED CONTENT

S Supporting Information *

EC50 values for activity in multiple cell lines, synthetic procedures for intermediates, crystallographic method, crystallographic data refinement and statistics. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

The PDB ID code for 36 bound to XIAP BIR2 is 4KJV.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (609) 785-1304. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Pamela Carroll and Dr. David Heimbrook for their support of this program. We also thank Charles Belunis for protein purification and Santina Russo and Joachim Diez of Expose for data collection at SLS. 7785

dx.doi.org/10.1021/jm400731m | J. Med. Chem. 2013, 56, 7772−7787

Journal of Medicinal Chemistry

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ABBREVIATIONS USED IAP, inhibitor of apoptosis protein; XIAP, X-linked inhibitor of apoptosis; cIAP, cellular inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; Smac, second mitochondria-derived activator of caspases; DIABLO, direct IAP-binding protein with a low pI; DR5, death receptor 5; EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; HOBt, hydroxybenzotriazole, HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; PARP, poly ADP ribose polymerase

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