Substituted Indazoles as Nav1.7 Blockers for the ... - ACS Publications

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Substituted Indazoles as Nav1.7 Blockers for the Treatment of Pain Jennifer M. Frost,*,† David A. DeGoey,† Lei Shi,† Rebecca J. Gum,† Meagan M. Fricano,† Greta L. Lundgaard,† Odile F. El-Kouhen,† Gin C. Hsieh,† Torben Neelands,† Mark A. Matulenko,† Jerome F. Daanen,† Madhavi Pai,† Nayereh Ghoreishi-Haack,‡ Cenchen Zhan,† Xu-Feng Zhang,† and Michael E. Kort† †

Research and Development, AbbVie, 1 North Waukegan Road, North Chicago, Illinois 60064, United States Aptinyx, 1801 Maple Avenue, No. 4300, Evanston, Illinois 60201, United States

J. Med. Chem. 2016.59:3373-3391. Downloaded from pubs.acs.org by AUCKLAND UNIV OF TECHNOLOGY on 01/29/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: The genetic validation for the role of the Nav1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Nav blockers is often limited due to adverse cardiovascular and CNS side effects. We sought more selective Nav1.7 blockers with oral activity, improved selectivity, and good druglike properties. The work described herein focused on a series of 3- and 4-substituted indazoles. SAR studies of 3substituted indazoles yielded analog 7 which demonstrated good in vitro and in vivo activity but poor rat pharmacokinetics. Optimization of 4-substituted indazoles yielded two compounds, 27 and 48, that exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both 27 and 48 demonstrated robust activity in the acute rat monoiodoacetate-induced osteoarthritis model of pain, and subchronic dosing of 48 showed a shift to a lower EC50 over 7 days.

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INTRODUCTION Voltage-gated sodium channels are involved in the initiation and propagation of action potentials in excitable tissues such as nerve and muscle.1,2 Nav1.7, one of nine sodium channel isoforms, is of particular interest as a target for the treatment of pain based on strong genetic evidence. Specifically, loss-offunction mutations in SCN9A, the gene that encodes Nav1.7, result in congenital insensitivity to pain (CIP), a condition characterized by the total absence of the ability to perceive any kind of noxious stimulus as painful.3−6 In contrast, gain-offunction SCN9A mutations can result in painful conditions such as inherited erythromelalgia and paroxysmal extreme pain disorder.7,8 Furthermore, knockout studies in which Nav1.7 was ablated in both sensory and sympathetic neurons resulted in the ablation of both inflammatory and neuropathic pain.9 Clinically, nonselective sodium channel blockers approved for use as local anesthetics, anticonvulsants, or tricyclic antidepressants have proven useful in the management of pain, although their utility may be hindered by their lack of subtype selectivity leading to unwanted side effects.10 Given the high level of validation, there have been significant efforts toward identifying selective Nav1.7 blockers for the treatment of pain.11−16 In addition to subtype selectivity, sodium channel blockers can bind preferentially to the inactivated state of the channel which is thought to be of particular importance in the hyperexcitable neurons present in chronic pain.17,18 Identification of state dependent blockers that can target channels in different kinetic states and tissues could thus impart improved functional selectivity. © 2016 American Chemical Society

Our efforts described herein were aimed at discovering novel Nav1.7 blockers that demonstrated robust efficacy in pain models while lacking CNS and cardiovascular side effects and also exhibiting good druglike properties (cLogP, MW, pharmacokinetics). Our initial work focused on a 3-substituted indazole series which led to several active analogs with moderate to poor selectivity versus the cardiac sodium channel Nav1.5 and generally poor pharmacokinetics profiles (Figure 1).

Figure 1. General structures of 3- and 4-substituted indazoles.

We then focused on 4-substituted indazole analogs which exhibited improved selectivity versus Nav1.5 and improved pharmacokinetic properties. Select compounds were interrogated in the rat monoiodoacetate-induced osteoarthritis model of pain, as we viewed the treatment of osteoarthritis pain as a significant unmet need. This clinical target of Nav1.7 in Received: January 14, 2016 Published: March 25, 2016 3373

DOI: 10.1021/acs.jmedchem.6b00063 J. Med. Chem. 2016, 59, 3373−3391

Journal of Medicinal Chemistry

Article

Scheme 1. Preparation of 3-Hydroxyindazolesa

(a) (i) CDl, i-Pr2EtN, THF, 92% yield, (ii) NaNO2, HCl, EtOH, H2O, 76 °C, 59% yield; (b) (a) RX, NaH, DMF, 16−76% yield; (c) (i) methyl 2bromopropanoate, Cs2CO3, CH3CN, 76% yield, (ii) NaOH, THF/MeOH, 94% yield; (d) oxalyl chloride, CH2Cl2, morpholine, pyridine, 94% yield; (e) 1,1′-(azodicarbonyl)dipiperidine, R1OH, n-Bu3P, PhCH3, 80 °C, 75−80% yield; (f) Mel, NaH, DMF, 82% yield; (g)1,1′-(azodicarbonyl)dipiperidine, 2-(3-oxazolidine)ethanol, n-Bu3P, PhCH3, 80 °C, 42% yield; (h) CDl, DBU, THF, 95% yield. a

Scheme 2. Preparation of 3-Hydroxyindazoles 15 and 16a

(a) (i) NaNO2, Na2SO3, 2 N HCl, H2O, conc HCl, 80 °C, 68% yield; (ii) Boc2O, DMAP, CH3CN, quantitative yield; (b) (i) 1,1′(azodicarbonyl)dipiperidine, 1-(2-hydroxyethyl)imidazolidin-2-one, n-Bu3P, PhCH3, 80 °C, 25% yield, (ii) TFA, CH2Cl2, 65% yield; (c) aryl iodine, Cul, trans-N1,N2-dimethylcyclohexane-l,2-diamine, K3PO4, 1,4-dioxane. a

imidazole to give 7 or 9, respectively. Analog 7 was then alkylated with methyl iodide and NaH to form methylated analog 10. Mitsunobu coupling of 3 and commercially available 2-(3-oxazolidine)ethanol gave intermediate 11 which was then cyclized in the presence of carbonyldiimidazole (CDI) and 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) to generate oxazolidinone 12. Analogs 15 and 16 were obtained as shown in Scheme 2. The 3-hydroxyindazole intermediate 13 was obtained upon treatment of 1 with NaNO2 and Na2SO3 in the presence of acid. Boc-protection of the resulting indazole nitrogen followed by Mitsunobu coupling with 1-(2-hydroxyethyl)-2-imidazolidinone, and removal of the Boc group resulted in intermediate 14. Buchwald coupling 22,23 of 14 with 2,4-difluoro-1iodobenzene and 1-fluoro-4-iodobenzene gave 15 and 16, respectively. The synthetic routes to the 4-substituted indazole analogs are shown in Schemes 3−8. As shown in Scheme 3, the coupling of commercially available 17 with 1-fluoro-2-iodobenzene in the presence of CuI gave the intermediate N-arylated product which was then debenzylated with ammonium formate and

osteoarthritis pain is supported by the reported knockout studies and additional literature.9,19,20

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CHEMISTRY The preparation of the 3-hydroxy substituted indazoles is shown in Schemes 1 and 2 and began with the coupling of commercially available acid 1 and hydrazine 2 in the presence of carbonyldimidazole (CDI) and Hünig’s base to give the intermediate hydrazide which when treated with NaNO2 and HCl at 76 °C gave 3-hydroxyindazole 3 as shown in Scheme 1. Intermediate 3 was then alkylated with commercially available 2-bromo-1-morpholinoethanone or 1-(2-chloroethyl)-1,3-dihydro-2H-imidazol-2-one in the presence of NaH to give 4 and 8, respectively. Alkylation of 3 with methyl 2-bromopropanoate followed by base hydrolysis gave carboxylic acid 6. Acid 6 was treated with oxalyl chloride to form the intermediate acid chloride which was then coupled with morpholine to give analog 5. The 3-hydroxyindazole 3 was also subjected to Mitsunobu coupling conditions21 in the presence of commercially available 1-(2-hydroxyethyl)-2-imidazolidinone or 1-(2-hydroxyethyl)3374



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Scheme 3. Preparation of 4-Substituted Indazole 19a

Piperazinone analog 38 was prepared as described in Scheme 5. Coupling of piperazin-2-one with bromoindazole intermediate 21 using Pd2(dba)3 and Nolan’s catalyst gave 36. Alkylation with ethyl 2-iodoacetate in the presence of NaH followed by hydrolysis resulted in carboxylic acid 37. Amide formation with HATU and Hünig’s base gave piperazinone analog 38. Bicyclic analog 42 was prepared as described in Scheme 6 starting from commercially available materials 39 and 40. Cyclization of 39 and 40 occurred in the presence of catalytic TFA and was followed by removal of the Boc-protecting group to give 41 in good yield. Intermediate 41 then underwent the CuI-catalyzed Buchwald coupling with 21 to generate analog 42. The chiral bicycle 48 was also prepared starting with 39 and using commercially available α-methylbenzyl analog 43 in the presence of catalytic TFA to give an approximate 1:1 mixture of isomers 44 and 45 which was separated via silica gel chromatography (Scheme 7). The resulting single enantiomer 45 next underwent Boc-removal in the presence of excess TFA, and the resulting lactam was coupled with indazole bromide 21 using CuI-Buchwald conditions to give 46. The α-methylbenzyl analog 46 was then treated with chloroethyl chloroformate to give free amine 47 which then underwent amide coupling with 3-hydroxy-3-methylbutanoic acid to give desired analog 48. Related analog 55 was prepared as shown in Scheme 8. Literature precedence24 describes the racemic synthesis of intermediate 52 with the literature analog having an N-benzyl group in place of the α-methylbenzyl group shown. But-3-en-1ol (49) was treated with MsCl to give the intermediate mesylate which then underwent displacement with (S)-1phenylethanamine followed by alkylation with methyl bromoacetate to give 50. Sequential treatment of 50 with lithium diisopropylamide and zinc bromide followed by iodine gave the

a (a) (i) 1-fluoro-2-iodobenzene, Cul, trans-N1,N2-dimethylcyclohexane-1,2-diamine, K3PO4, 1,4-dioxane, 88% yield, (ii) ammonium formate, 10% Pd/C, MeOH, 75% yield; (b) tert-butyl (2-bromoethyl)carbamate, NaH, DMA, 88% yield.

palladium on carbon to give 18. Alkylation of 18 with tert-butyl (2-bromoethyl)carbamate and NaH resulted in Boc-amine 19. As shown in Scheme 4, imidazolidinone analogs 24, 26, and 32−34 were prepared by first coupling commercially available 2-bromo-6-fluorobenzaldehyde (20) and (2-fluorophenyl)hydrazine (2) in the presence of Cs2CO3 in N-methyl-2pyrrolidinone at 140 °C to give intermediate 21. Aryl bromide 21 then underwent Pd-catalyzed coupling with imidazolidin-2one giving 22 which was then alkylated with various alkyl halides in the presence of NaH to give analogs 24, 26, 32−34 in moderate to good yield. Intermediate 22 was also Bocprotected giving 23 and was N-arylated with 4-iodopyridine in the presence of CuI to give analog 35. Intermediate 22 was also treated with tert-butyl bromoacetate and NaH, and the resulting tert-butyl ester was then hydrolyzed with TFA to give carboxylic acid intermediate 30. The acid was then coupled with various amines using HATU conditions to give amides 25, 27−29, and 31 in moderate to good yield. Scheme 4. Preparation of 4-Substituted Indazolesa

(a) Cs2CO3, NMP, 140 °C, 91% yield; (b) imidazolidin-2-one, CS2CO3, Xantphos, Pd2(dba)3, DME, 80 °C, 51% yield; (c) alkyl halide, NaH, DMF, 24−76% yield; (d) Boc2O, DMAP, CH3CN, 78% yield; (e) 4-iodopyridine, Cul, trans-N1,N2-dimethylcyclohexane-1,2-diamine, K3PO4, 1,4dioxane, 83% yield; (f) (i) tert-butyl 2-bromoacetate, NaH, DMF, 81% yield, (ii) TFA, CH2Cl2, 99% yield; (g) amine, HATU, i-Pr2EtN, THF 54− 96% yield. a

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(a) Piperazin-2-one, cesium carbonate, Nolan’s catalyst, Pd2(dba)3, dioxane, 80 °C, 22% yield; (b) (i) ethyl 2-iodoacetate, NaH, DMF, (ii) NaOH (aq), quantitative yield; (c) (S)-3-fluoropyrrolidine, HATU, i-Pr2EtN, THF, 21% yield.

a


a

(a) (i) trifluoroacetic acid (cat.), CH2Cl2, 86% yield, (ii) trifluoroacetic acid (8 equiv), CH2Cl2, 73% yield; (b) 21, Cul, trans-N1,N2dimethylcyclohexane-1,2-diamine,K3PO4, 1,4-dioxane, 83% yield.


(a) Trifluoroacetic acid, CH2Cl2, 40% yield 45 separated from ∼1:1 mixture of 44 and 45; (b) (i) TFA, CH2Cl2, quantitative yield; (ii) 21, Cul, trans-N1,N2-dimethylcyclohexane-1,2-diamine, K3PO4, 1,4-dioxane, 87% yield; (c) 1-chloroethyl chloroformate, CH2Cl2, 50 °C; MeOH, reflux, 73% yield; (d) 3-hydroxy-3-methylbutanoic acid, HATU, i-Pr2EtN, THF, 68% yield.

a

cyclized iodo intermediate which was reacted with sodium azide to give 51. Staudinger reduction of the azide to the corresponding amine resulted in lactam formation. To facilitate purification, the lactam was Boc-protected and the isomers

were separated via silica gel chromatography which, after removal of the Boc-group, gave the desired single enantiomer 52 in good yield. The lactam 52 was then coupled with indazole bromide 21 again using CuI-Buchwald conditions to give 53 3376



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a (a) (i) MsCl, Et3N, CH2Cl2, 92% yield, (ii) (S)-1-phenylethanamine, CH3CN, 85 °C, 20% yield, (iii) methyl bromoacetate, i-Pr2EtN, DMSO, 85% yield; (b) (i) LDA, Et2O, −78 °C; ZnBr2, Et2O, −74 °C to rt; iodine, Et2O, 0 °C, 96% yield; (ii) NaN3, DMF, 50 °C, quantitative yield; (c) (i) Ph3P, 1:1 MeTHF/H2O, 74 °C; (ii) Boc2O, DMAP (cat.), i-Pr2EtN, DMF; minor isomer separated out; 71% yield; (iii) TFA, CH2Cl2, 99% yield; (d) 21, Cul, trans-N1,N2-dimethylcyclohexane-1,2-diamine, K3PO4, 1,4-dioxane, 100 °C, 77% yield; (e) 20% Pd(OH)2 on carbon (wet), 30 psi, trifluoroethanol, quantitative yield; (f) 3-hydroxy-3-methylbutanoic acid, HATU, Et3N, DMF, 62% yield.

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RESULTS AND DISCUSSION Our work began with the 3-hydroxyindazole series (Table 1) with 4 as the lead compound, which was identified from a high throughput screen. While similar analogs have been reported in the literature,27 the analogs described here lack the basic amine side chain pharmacophore of the previously reported structures. Although 4 showed reasonable in vitro activity at Nav1.7 in both the FRET (IC50 = 0.76 μM) and electrophysiology assays (IC50 = 1.4 μM) along with moderate rat and human microsomal stability, its bioavailability in rat PK (F = 22%) was low primarily due to plasma instability (1% remaining, 4 h in plasma). Our goal for this series was to improve plasma stability while maintaining Nav1.7 activity and good microsomal stability. We also sought improved selectivity against the cardiac sodium channel Nav1.5. Analog 5 with an additional methyl group on the glycolate amide demonstrated improved Nav1.7 activity (FRET IC50 = 0.11 μM; EP IC50 = 0.62 μM) and stability in plasma (>80% remaining, 4 h in plasma) but poor metabolic stability (hClint,u = 19 L h−1 kg−1). Replacement of the glycolate amide with a cyclic urea provided compound 7 which exhibited moderate activity at Nav1.7 in FRET (IC50 = 1.9 μM) and electrophysiology assays (IC50 = 2.7 μM) and had moderate metabolic stability (hClint,u = 11 L h−1 kg−1). Rat PK studies demonstrated that 7 had similar bioavailability (F = 17%) to 4 (F = 22%) but was stable in plasma (>80% remaining, 4 h in plasma), although 7 did exhibit a short halflife in rat (t1/2 = 0.4 h). Metabolite identification studies in human and rat liver microsomes suggested that metabolism was primarily occurring at the 4-position of the N-aryl group with additional metabolites resulting from oxidation of the cyclic urea ring being observed. Cyclic urea 7 did exhibit robust activity in our osteoarthritis model of pain (vide infra), and as such, efforts were undertaken to improve the pharmacokinetic properties of the series. Most attempts to modify the cyclic urea moiety resulted in analogs with good to moderate activity at Nav1.7 but generally poor metabolic stability. For example, unsaturated analog 8 maintained activity at Nav1.7 (FRET IC50 = 0.96 μM) and was somewhat selective versus Nav1.5 (IC50 =

which underwent hydrogenation to provide amine 54. Amide coupling with 3-hydroxy-3-methylbutanoic acid resulted in formation of desired analog 55.

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BIOLOGICAL EVALUATION The indazole analogs were first evaluated in a fluorescence resonance energy transfer (FRET)-based membrane potential functional assay using HEK293 cells that were stably expressing recombinant human Nav1.7. Selected analogs were additionally tested in an electrophysiology (EP) assay using HEK293 cell lines stably expressing human Nav1.7 utilizing a voltage protocol to assess activity at resting and partially inactivated states of the channel (holding potentials of −100 mV and −65 mV, respectively) to evaluate the state-dependence of compounds. Data shown in the Tables 1 and 2 depict the −65 mV value which is thought to be representative of pain states.16 Virtually no activity was observed at the resting state (−100 mV) for all analogs, and as such, all were determined to be state-dependent. The Nav1.7 FRET and electrophysiology data generally correlated; the expected variations that were observed may be attributable to significant differences in the in vitro assay conditions. Compounds were further evaluated for selectivity versus the cardiac sodium channel Nav1.5 via an electrophysiology assay using HEK293 cell lines stably expressing human Nav1.5 from a holding potential of −90 mV and a voltage protocol designed to mimic cardiac myocyte physiological activity.25 ADME properties were gauged using metabolic stability in human and rat liver microsomes and in rat PK on select compounds. The P-gp efflux ratio was determined using MDCK cells transfected with the MDR1 gene which encodes human P-gp grown on Transwell inserts, and directional differences in cell monolayer permeability of test compounds are reported as a ratio. Finally, select analogs were tested in the rat monoiodoacetate-induced osteoarthritis model (MIA-OA, grip force end point) wherein an intra-articular injection of monoiodoacetate causes osteoarthritis-like knee joint lesions. Analogs were then administered orally, and the rats were tested for grip force deficit of hind limbs.26 3377



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Table 1. In Vitro Activity of 3-Hydroxyindazoles

Nav1.7 IC50 values were determined by least-squares fitting of a logistic equation to data from full eight-point, half-log concentration−response curves using a FRET-membrane potential assay as described in the Experimental Section. Data are shown with standard error (SEM) and represent the mean of two or more separate determinations. bNav1.7 EP, data were collected using a slow inactivated state QPatch protocol with V1/2 = −65 mV. cNav1.5, data were collected using an inactivated state protocol with V1/2 = −90 mV with standard error (SEM) representing the mean of two or more separate determinations. dHuman and rat liver microsomal stability data, rat bioavailability and half-life were obtained as described in the Experimental Section. a

5.1 μM); however, the half-life in rat (t1/2 = 0.4 h) was not improved. Imidazole 9 also showed an insufficient improvement in PK properties relative to 7. Methylated analog 10 and oxazolidinone 12 exhibited acceptable activity at Nav1.7, but both analogs had poor metabolic stability. The identification of the 4-position of the N-phenyl group as the primary site of metabolism for 7 led to an investigation of fluorination as a blocking strategy with analogs 15 and 16. Both analogs demonstrated only moderate Nav1.7 activity; however, 15 did exhibit somewhat improved rat PK properties (F = 71%, t1/2 = 0.94 h). With consistently poor metabolic stability for the 3substituted indazole series and narrow SAR, adequate progress was not made toward achieving the desired potency and pharmacokinetic profile. We postulated that other indazole connectivities may allow for similar binding to the receptor but also tolerate increased functional group diversity, particularly the 4-position of the indazole which would project the side

chain with a similar vector to the 3-position. We discovered that the 4-substituted indazoles did indeed provide more tolerant SAR in increased metabolic stability, and we thus shifted our focus to this series (Table 2). We first looked at linear side chain analog 19 which we were pleased to note maintained activity at Nav1.7 (FRET IC50 = 0.58 μM) but exhibited poor microsomal stability in human (hClint,u = 43 L h−1 kg−1) and rat liver microsomes (hClint,u = 513 L h−1 kg−1). Cyclization of the side chain to the cyclic urea resulted in 23 which again maintained activity at Nav1.7 (FRET IC50 = 0.73 μM; EP IC50 = 2.3 μM). Importantly, the metabolic stability of 23 was significantly better than linear analog 19 and the PK properties of 23 were also markedly improved relative to 3-hydoxyindazole 7. We next made a series of related amides (24−29, 31) which all demonstrated good selectivity for Nav1.7 versus Nav1.5. In the series of amides described, the (S)-3-fluoropyrrolidine 27 exhibited good activity at Nav1.7 (FRET IC50 = 0.37 μM, EP 3378



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Table 2. In Vitro Activity of 4-Hydroxyindazoles

Nav1.7 IC50 values were determined by least-squares fitting of a logistic equation to data from full eight-point, half-log concentration−response curves using a FRET-membrane potential assay as described in the Experimental Section. Data are shown with standard error (SEM) and represent the mean of two or more separate determinations. bNav1.7 EP, data were collected using a slow inactivated state QPatch protocol with V1/2 = −65 mV. cNav1.5, data were collected using an inactivated state protocol with V1/2 = −90 mV with standard error (SEM) representing the mean of two or more separate determinations. An asterisk (∗) denotes an n = 1 where no SEM can be reported. dHuman and rat liver microsomal stability data, rat bioavailability and half-life, P-gp efflux, and brain and plasma levels were obtained as described in the Experimental Section. a

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IC50 = 0.48 μM; manual EP IC50 = 0.57 μM), good selectivity versus Nav1.5 (>33 μM), and good pharmacokinetic properties (F = 83%; t1/2 = 1.7 h). This analog was further tested in vivo (vide infra). It was also noted that the corresponding (R)-3fluoropyrrolidine 28, the unsubstituted pyrrolidine 26, and (S)3-hydroxypyrrolidine 29 offered no improvement in Nav1.7 activity or overall properties relative to 27. The (S)-3fluoropiperidine 31, although active at Nav1.7 (FRET IC50 = 1.1 μM), had weaker potency than 27 and a shorter half-life in rats (t1/2 = 0.83 h). The acetamide 24 maintained Nav1.7 activity (FRET IC50 = 0.84 μM; EP IC50 = 2.5 μM) and selectivity but showed unfavorable rat PK properties. Reported Nav1.7 knockout studies found that ablation of sensory neurons was adequate to abolish mechanical pain and inflammatory pain, while ablation of both sensory and sympathetic neurons was required for the abolishment of neuropathic pain.9 This observation suggests that analogs with CNS penetration would be beneficial for neuropathic pain modulation, but CNS penetration may not be necessary for treatment of osteoarthritis (viewed as our primary indication) which may be peripherally driven. While both centrally penetrant and peripherally restricted analogs were of interest, we postulated that the CNS penetrant analogs may exhibit broader efficacy in pain models as suggested by the reported knockout studies9,28 and that peripherally restricted analogs may also be active in peripheral pain models, offering the opportunity to limit potential CNS side effects. Interestingly, we observed that the 4-indazole substituent influenced the extent to which an analog was a substrate for Pgp mediated efflux. We further noted that the P-gp efflux ratio (ratio of ≥2 suggested a P-gp efflux substrate) correlated well with observed brain impairment (BI) ([plasma]free/[brain]free ≥ 3) (Table 2). We observed that while carbamate 23 was not an P-gp efflux substrate, all of the amides tested (24−29, 31, 38, 42, 48, 55) were efflux substrates. We also noted that analogs with ketone or aryl substituents on the cyclic urea were not Pgp efflux substrates (32−35) (Table 2). Ketone 32 was of interest in that it showed good Nav1.7 activity in the FRET assay (IC50 = 0.57 μM). Notably, 32 was not an efflux substrate and as such was not brain impaired. A series of aryl substituted cyclic ureas (33−35) was investigated, and these analogs were also found not to be P-gp efflux substrates. While all aryl analogs were active at Nav1.7, methylpyrazine 34 was the most potent (FRET IC50 = 0.27 μM) and was significantly more selective versus Nav1.5 than oxazole 33 or pyridine 35. Once again, however, the rat pharmacokinetic profile was deemed too unfavorable for further in vivo characterization. Finally, we looked at replacements of the cyclic urea with other conformationally constrained analogs such as 38, 42, 48, and 55 (Table 2). Piperazinone 38 exhibited robust activity at Nav1.7 (FRET IC50 = 0.19 μM; EP IC50 = 0.54 μM), but selectivity verus Nav1.5 was diminished relative to analog 27. Bicyclic analog 42 also demonstrated good activity at Nav1.7 (FRET IC50 = 0.58 μM) but had relatively poor selectivity versus Nav1.5 (IC50 = 2.5 μM) and very poor human and rat metabolic stability. Further investigation of the bicyclo[3.3.0] core led to analogs 48 and 55. Although 48 demonstrated more moderate activity at Nav1.7 in the FRET assay (FRET IC50 = 3.9 μM), it exhibited no activity at Nav1.5, excellent metabolic stability, and good PK properties. As such, 48 was tested in Nav1.7 manual electrophysiology where 48 gave an IC50 of 0.34 μM (Table 2). Given the activity, the promising in vitro

metabolism and PK data, 48 was further tested in vivo (vide infra). The regiomeric analog 55 was also synthesized, and although 55 exhibited improved activity at Nav1.7 in the FRET assay (IC50 = 1.35 μM) relative to 48, increased activity at Nav1.5 was noted as was decreased metabolic stability. We further noted that, as was the case with amides in the cyclic urea series, both 48 and 55 were efflux substrates and as such exhibited decreased CNS drug levels as indicated by the brain impairment ratio in Table 2. On the basis of activity at Nav1.7, selectivity versus Nav1.5, and overall druglike properties (metabolic stability, rat PK), analogs 27 and 48 were selected for further testing in the rat monoiodoacetate-induced osteoarthritis model (MIA-OA, grip force end point). Centrally penetrant analog 7 did not have a favorable rat PK profile, but it was chosen to exemply the 3hydroxyindazole series. Although we were primarily focused on selectivity in vivo (pain model activity vs cardiovascular and CNS side effects), these analogs were also tested in the Nav1.2, Nav1.3, and Nav1.6 FRET assays to better assess their potential CNS liabilities (Table 3). While the 3-substituted indazole 7 Table 3. Selectivity versus Nav1.2, Nav1.3, and Nav1.6a IC50 (μM)

tetracaine 7 27 48

FRET hNav1.7

FRET hNav1.2

FRET hNav1.3

FRET hNav1.6

0.056 1.9 0.37 3.9

0.49 2.6 4.0 >20

0.74 3.4 7.1 >20

0.52 5.0 8.6 >20

a Nav1.7, 1.2, 1.3, and 1.6 IC50 values were determined by least-squares fitting of a logistic equation to data from full eight-point, half-log concentration−response curves using a FRET-membrane potential assay as described in the Experimental Section.

exhibited moderate to poor selectivity, the peripherally restricted 4-substituted indazole analogs 27 and 48 demonstrated better selectivity relative to 7. In this model, an intra-articular injection of monoiodoacetate causes osteoarthritis-like knee joint lesions with analogs being tested in these rats approximately 3 weeks after injection of the monoiodoacetate. The rats were then tested for grip force deficit of hind limbs.22 The NSAID diclofenac was used as a positive control and typically produced about an 80% increase in grip force relative to vehicle. Centrally active, 3-hydroxyindazole analog 7 (BI = 0.83) exhibited a robust dose response effect in this model at 3, 10, and 30 mg/kg (Figure 2A). Similarly, analog 27 also demonstrated robust activity in the MIA-OA model (Figure 2B) with significant effects at 3, 10, and 30 mg/kg. Interestingly, the efficacious plasma levels (total) for the more peripherally restricted analog 27 (EC50 of 2.2 μg/mL) were higher than the centrally active analog 7 (EC50 = 500 ng/ mL). Finally, [3.3.0] bicyclic diamine analog 48 also exhibited good activity in the acute rat monoiodoacetate-induced osteoarthritis model at 10 and 30 mg/kg (EC50 = 2.5 μg/ mL) as shown in the solid line in Figure 2C. Again, we observed that the peripherally restricted analog 48 required higher plasma concentrations than the centrally active analog 7. On the basis of our observations, CNS penetration was not required for efficacy in the MIA-OA model of OA pain; however higher plasma levels of peripherally restricted agents were required for full efficacy. Interestingly, when 48 was tested 3380



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Figure 2. Effects of 7, 27, and 48 in MIA-OA pain model. (A) Oral acute dosing of 7 in the rat monoiodoacetate-induced osteoarthritis model (MIAOA) with a grip force end point. The vehicle control group was assigned a value of 0%, whereas a naive group was assigned a value of 100%. The percent effect for each dose-group was expressed as % return to normal grip force as found in the naive group and calculated using the formula % return to normalcy = [(treatment CFmax − vehicle CFmax)/(naive CFmax − vehicle CFmax)] × 100, where CFmax is maximum compressive force (see Supporting Information for complete procedure). (B) Oral acute doing of 27 in the MIA-OA model as described above. (C) Oral acute and subchronic dosing of 48 in the MIA-OA model with solid line representing acute dosing and dashed line subchronic dosing as described in the Experimental Section. Dicolfenac (30 mg/kg po) was administered as a positive control in all acute experiments.

doses from the subchronic study with 48 were 0.96 μg/mL and 0.19 μg/mL for 1 mg/kg and 0.3 mg/kg, respectively.

with subchronic dosing twice a day for 7 days, we observed a shift in efficacious plasma levels. On day 1, results were consistent with acute administration but on day 7 (shown in dashed line in Figure 2C) there was a significant shift in efficacy. In this subchronic dosing study, significant effects were observed at 0.3 mg/kg and the EC50 shifted to 800 ng/mL. We hypothesized that in this subchronic dosing paradigm, drug levels achieved a steady state at the site of action (neurons) resulting in improved efficacy. Importantly, none of the compounds tested exhibited any CNS side effects as assessed by high-dose locomotor activity and rotorod performance assays in rodents (data not shown). To better understand the efficacy of these analogs in the OAGF model, plasma levels were obtained and are shown in Table 4. For analog 7, robust efficacy was observed at 1.05 μg/mL or 2.9 μM which was sufficient to cover the FRET IC50 of 1.9 μM. Higher drug levels were obtained with both 27 and 48, consistent with their improved rat PK profiles relative to 7 and also exceed their FRET IC50 values. Plasma levels for the lower

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CONCLUSION In summary, we discovered two series of indazole analogs that exhibited activity at Nav1.7. The 3-hydroxyindazole series was observed to have reasonable Nav1.7 activity and moderate selectivity versus Nav1.5. Analog 7 in this series exhibited robust activity in the monoiodoacetate induced osteoarthritis model of pain with a comparatively low efficacious plasma concentration (500 ng/mL). Unfortunately, despite good activity in an acute pain model, the suboptimal pharmacokinetic profile of 7 and related analogs precluded further development. A related 4-substituted indazole series was also investigated. Analogs in this series exhibited good to moderate activity at Nav1.7 as measured by the FRET-membrane potential assay and electrophysiology and showed moderate to good selectivity versus the cardiac sodium channel Nav1.5. Furthermore, this series demonstrated improved pharmacokinetic profiles with good rat bioavailabilities and half-lives. In this series, we noted that the 4-indazole side chain influenced the P-gp efflux activity and thus determined the extent of CNS penetration. Amides such as 27 or 48 were P-gp efflux substrates and, as such, were minimally present in the CNS, while a ketone substituent (32) or aryl group (33−35) yielded analogs that were centrally active. Two analogs (27 and 48) in this series were tested in the monoiodoacetate induced osteoarthritis pain model. Both analogs demonstrated robust activity in acute testing with

Table 4. Plasma Concentrations in the OA-GF Pain Model OA-GF plasma concn (μg/mL) at 7 27 48

3 mg/kg

10 mg/kg

30 mg/kg

0.05 2.7 0.69

0.38 6.8 3.3

1.05 10.5 6.3 3381



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J = 7.6, 1.6 Hz, 1H), 7.44−7.40 (m, 1H), 7.35 (dddq, J = 9.5, 4.9, 2.4, 1.8 Hz, 1H), 7.31−7.24 (m, 2H), 7.22−7.17 (m, 2H), 5.10 (s, 2H), 3.76−3.67 (m, 6H), 3.60−3.53 (m, 2H); MS (APCI) m/z 374 [M + H]+. 2-((5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yl)oxy)-1morpholinopropan-1-one (5). Step 1. A mixture of 5-fluoro-1-(2fluorophenyl)-1H-indazol-3-ol (3, from synthesis of 4, step 2), methyl 2-bromopropanoate (0.713 g, 4.27 mmol), and Cs2CO3 (1.45 g, 4.46 mmol) in acetonitrile (12 mL) was stirred for 16 h. The mixture was then diluted with ethyl acetate (10 mL) and washed with 1 N NaOH (10 mL) and brine (10 mL). The organics were dried over Na2SO4 and purified via column chromatography (SiO2, 100% hexanes to 50% ethyl acetate/hexanes) to give methyl 2-((5-fluoro-1-(2-fluorophenyl)1H-indazol-3-yl)oxy)propanoate (methyl ester of 6) (1.03 g, 3.1 mmol, 76% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.60−7.50 (m, 4H), 7.46−7.30 (m, 3H), 5.30 (q, J = 6.8 Hz, 1H), 3.69 (s, 3H), 1.64 (d, J = 6.9 Hz, 3H); MS (APCI) m/z 333 [M + H]+. Step 2. A mix of methyl 2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol3-yloxy)propanoate (methyl ester of 6 from step 1, 1.03 g, 3.10 mmol) and 1 N NaOH (3.8 mL, 3.80 mmol) in methanol (3 mL) and THF (9 mL) was allowed to stir for 1 h and then was diluted with ethyl acetate (5 mL). The mixture was acidified with 1 N HCl (4 mL), and the layers were separated. The organic layer was washed with water (5 mL) and brine (5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give 2-(5-fluoro-1-(2fluorophenyl)-1H-indazol-3-yloxy)propanoic acid, 6 (926 mg, 2.91 mmol, 94% yield). 1H NMR (300 MHz, DMSO-d6) δ 13.00 (s, 1H), 7.62−7.45 (m, 4H), 7.44−7.30 (m, 3H), 5.21 (q, J = 7.0 Hz, 1H), 1.62 (d, J = 7.0 Hz, 3H); MS (APCI) m/z 319 [M + H]+. Step 3. A mixture of 2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol-3yloxy)propanoic acid (step 2, 0.926 g, 2.91 mmol) and oxalyl chloride (0.33 mL, 3.78 mmol) in CH2Cl2 (10 mL) with catalytic DMF (0.1 mL) was stirred at ambient temperature for 90 min and then was concentrated under reduced pressure. The material was dissolved in CH2Cl2 (10 mL), and then morpholine (0.28 mL, 3.21 mmol) and pyridine (0.35 mL, 4.33 mmol) were added and the mixture was allowed to stir for 16 h. The reaction mixture was then diluted with ethyl acetate (10 mL), washed with saturated aqueous NaHCO3 (5 mL) and brine (mL), and dried over anhydrous Na2SO4. The material was then filtered, concentrated under reduced pressure, and purified via column chromatography (SiO2, 18% ethyl acetate/CH2Cl2) to give 2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)-1-morpholinopropan-1-one, 5 (1.06 g, 2.75 mmol, 94% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.62−7.46 (m, 4H), 7.44−7.30 (m, 3H), 5.70 (q, J = 6.6 Hz, 1H), 3.68−3.46 (m, 7H), 3.47−3.35 (m, 1H), 1.54 (d, J = 6.6 Hz, 3H); MS (APCI) m/z 388 [M + H]+. 1-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one (7). To a solution of 5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-ol (3, from synthesis of 4, step 2, 2.5 g, 10.15 mmol), 1-(2-hydroxyethyl)-2-imidazolidinone (Alfa Aesar, 2.11 g, 16.3 mmol), and tri-N-butylphosphine (4.01 mL, 16.3 mmol) in toluene (60 mL) was added 1,1′-(azodicarbonyl)dipiperidine (4.10 g, 16.3 mmol) portionwise over 5 min. The mixture was warmed to 80 °C and was allowed to stir for 5 h. The mixture was then allowed to cool to ambient temperature and was stirred for 16 h. The reaction mixture was diluted with toluene (10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate was concentrated under reduced pressure and purified via column chromatography (SiO2, 5% ethyl acetate/hexanes to 75% ethyl acetate/hexanes) to give 1-(2-(5-fluoro1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one, 7 (2.9 g, 8.09 mmol, 80% yield). 1H NMR (300 MHz, methanol-d4) δ 7.60 (td, J = 7.7, 1.7 Hz, 1H), 7.50−7.31 (m, 4H), 7.28−7.21 (m, 2H), 4.57 (t, J = 5.2 Hz, 2H), 3.71−3.62 (m, 4H), 3.43−3.35 (m, 2H); MS (DCI) m/z 359 [M + H]+. 1-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)1H-imidazol-2(3H)-one (8). To a solution of 5-fluoro-1-(2fluorophenyl)-1H-indazol-3-ol (3, from synthesis of 4, step 2, 4 g, 16.25 mmol) in DMF (40 mL) at ambient temperature was added NaH (60 wt %, 2.60 g, 65.0 mmol). This mixture was stirred at ambient temperature for 30 min, and then 1-(2-chloroethyl)-1,3-

higher plasma levels required for activity relative to analog 7. However, upon subchronic dosing of 48 for 7 days, a favorable shift toward higher efficacy was observed at the same plasma levels. Both 27 and 48 remain compounds of interest for potential clinical development.

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EXPERIMENTAL SECTION

Biological Assays. Procedures for all biological assays are included in the Supporting Information. Chemistry. Proton NMR spectra were obtained on a Varian Inova 500 (500 MHz), Varian Inova 400 (400 MHz), Varian 400-MR (400 MHz), Varian UNITY plus 300 (300 MHz) with chemical shifts (δ) reported relative to tetramethylsilane as an internal standard. Column chromatography was carried out on silica gel 60 (230−400 mesh). Dry organic solvents (CH2Cl2, CH3CN, DMF, etc.) were purchased from Aldrich packaged under nitrogen Sure-Seal bottles. Preparative reversed phase HPLC (Gilson 333/334 pump system and Gilson 215 liquid handler, custom packed YMC TriArt C18 20 μm column, 50 mm × 150 mm, flow rate 80 mL/min with solvents as described in individual experiments. Thin-layer chromatography was performed using 250 mm silica gel 60 glass-backed plates with F254 as indicator. The purity of all final compounds was assessed to be ≥95% as determined by HPLC (data are included in the Supporting Information). All starting materials were commercially available and were obtained from Aldrich unless otherwise specified. 2-((5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yl)oxy)-1morpholinoethanone (4). Step 1. To a solution of 2-amino-5fluorobenzoic acid (Matrix Scientific, 21.7 g, 140 mmol) in THF (500 mL) at 0 °C was added carbonyldiimidazole (23.8 g, 147 mmol) using a mechanical stirrer. The ice bath was removed after the addition was complete, and the mixture was stirred at ambient temperature for 1.5 h. Hünig’s base (58.6 mL, 336 mmol) was added and the solids dissolved completely. The 2-fluorophenylhydrazine hydrochloride (25.0 g, 154 mmol) was added portionwise over 15 min, and then the mixture was allowed to stir at ambient temperature for 18 h. The mixture was quenched with aqueous HCl (1 N) to pH 1, and then aqueous NaOH (50%) was added until pH 7. The layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organics were washed with water (100 mL), saturated aqueous NaHCO3 (100 mL), and brine (100 mL), and then were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the resulting 2-amino-5-fluoro-N′-(2fluorophenyl)benzohydrazide (40 g, 129 mmol, 92% yield) was carried on without further purification to step 2. MS (DCI) m/z 264 [M + H]+. Step 2. A mixture of 2-amino-5-fluoro-N′-(2-fluorophenyl)benzohydrazide (from step 1) (37 g, 141 mmol) in ethanol (450 mL) and aqueous HCl (1 N, 422 mL, 422 mmol) was warmed to 76 °C and was allowed to stir until all solids were dissolved. Then sodium nitrite (29.1 g, 422 mmol) in water (50 mL) was added dropwise via addition funnel over 1 h. The mixture was allowed to stir at 76 °C for 1 h and then was allowed to cool to ambient temperature and was stirred for 16 h. The solids were isolated via filtration and washed with water (2 × 20 mL) to give 5-fluoro-1-(2-fluorophenyl)-1H-indazol-3ol, 3 (20.5 g, 83 mmol, 59% yield). 1H NMR (300 MHz, DMSO-d6) δ 11.39−10.89 (m, 1H), 7.60 (td, J = 7.8, 1.6 Hz, 1H), 7.55−7.41 (m, 3H), 7.41−7.23 (m, 3H); MS (DCI) m/z 247 [M + H]+. Step 3. To a solution of 5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-ol (3, from step 2) (2.25 g, 9.14 mmol) in DMF (35 mL), NaH (60 wt %, 1.1 g, 27.4 mmol) was added portionwise at ambient temperature, and the mixture was stirred for 15 min. To this dark brown suspension was added 2-bromo-1-morpholinoethanone (2.38 g, 11.42 mmol) in DMF (1 mL), and the reaction mixture was stirred for another 1 h. Water was added and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified via column chromatography (SiO2, 30% ethyl acetate/hexanes to 100% ethyl acetate) to give 2-((5-fluoro-1-(2fluorophenyl)-1H-indazol-3-yl)oxy)-1-morpholinoethanone, 4 (2.58 g, 6.9 mmol, 76% yield). 1H NMR (400 MHz, chloroform-d) δ 7.54 (td, 3382



Article

Step 2. To a solution of 2-(2-(5-fluoro-1-(2-fluorophenyl)-1Hindazol-3-yloxy)ethylamino)ethanol (from step 1, 0.21 g, 0.630 mmol) in THF (5 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 9.40 μL, 0.063 mmol) followed by N,N′-carbonyldimidazole (CDI, 0.102 g, 0.630 mmol). The mixture was allowed to stir at ambient temperature for 4 h. The mixture was quenched with saturated aqueous NaHCO3 (5 mL) and diluted with ethyl acetate (5 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organics were dried over anhydrous Na2SO4, filtered, concentrated, and purified via column chromatography (SiO2, 5% ethyl acetate/hexanes to 75% ethyl acetate/hexanes) to give 3-(2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)oxazolidin-2-one, 12 (0.215 g, 0.598 mmol, 95% yield). 1H NMR (300 MHz, methanol-d4) δ 7.61 (td, J = 7.7, 1.7 Hz, 1H), 7.51−7.31 (m, 4H), 7.29−7.22 (m, 2H), 4.62 (t, J = 5.1 Hz, 2H), 4.34 (dd, J = 9.1, 7.1 Hz, 2H), 3.85−3.73 (m, 4H); MS (DCI) m/z 360 [M + H]+. 1-(2-(1-(2,4-Difluorophenyl)-5-fluoro-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one (15). Step 1. Sodium nitrite (6.67 g, 97 mmol) in water (25 mL) was added dropwise to a solution of 2amino-5-fluorobenzoic acid (15 g, 97 mmol) in 2 N HCl (100 mL) at 0 °C. The mixture was stirred for 30 min, and then sodium sulfite (30.5 g, 242 mmol) in water (120 mL) was added in one portion. The cooling bath was removed, and the reaction was stirred for 2 h. Concentrated HCl (31.8 mL, 387 mmol) was added slowly, and the mixture was stirred at ambient temperature for 18 h. The mixture was then heated to 80 °C for 4 h. After cooling to ambient temperature, the pH was adjusted to 5−5.5 with 2 N NaOH (125−130 mL). The mixture was filtered to obtain the precipitate, 5-fluoro-1H-indazol-3-ol (10 g, 65.7 mmol, 68% yield). 1H NMR (300 MHz, DMSO-d6) δ 11.55 (s, 1H), 10.52 (s, 1H), 7.39−7.27 (m, 2H), 7.18 (td, J = 9.1, 2.7 Hz, 1H); MS (DCI) m/z 170 [M + NH4]+. Step 2. To a solution of 5-fluoro-1H-indazol-3-ol (from step 1, 10 g, 65.7 mmol) and Boc2O (30.5 mL, 131 mmol) in acetonitrile (200 mL) was added 4-dimethylaminopyridine (DMAP, 0.803 g, 6.57 mmol). This mixture was allowed to stir at ambient temperature for 18 h. The mixture was concentrated under reduced pressure, and the residue was stirred with 7 N NH3/methanol (10 mL) for 2 h. The material was again concentrated under reduced pressure and dried under vacuum. The crude tert-butyl 5-fluoro-3-hydroxy-1H-indazole-1-carboxylate, 13 (18 g, 71.4 mmol, >100% yield) was carried forward without further purification. 1H NMR (300 MHz, DMSO-d6) δ 8.00 (dd, J = 9.0, 4.3 Hz, 1H), 7.57−7.39 (m, 2H), 3.32 (s, 1H), 1.60 (s, 9H); MS (DCI) m/z 170 [M + NH4]+. Step 3. To a solution of tert-butyl 5-fluoro-3-hydroxy-1H-indazole-1carboxylate (13, from step 2, 16.6 g, 65.7 mmol), 1-(2-hydroxyethyl)2-imidazolidinone (Alfa Aesar, 13.7 g, 105 mmol), and tri-Nbutylphosphine (25.9 mL, 105 mmol) in toluene (200 mL) was added 1,1′-(azodicarbonyl)dipiperidine (26.5 g, 105 mmol) portionwise over 5 min. The mixture was warmed to 80 °C and was allowed to stir for 5 h. The mixture was allowed to cool to ambient temperature and then was allowed to cool to ambient temperature and was stirred for 16 h. The mixture was diluted with toluene (10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate was concentrated under reduced pressure and the material was purified via column chromatography (SiO2, 5% ethyl acetate/hexanes to 75% ethyl acetate/hexanes) to give tert-butyl 5-fluoro-3-(2-(2oxoimidazolidin-1-yl)ethoxy)-1H-indazole-1-carboxylate (6 g, 16.5 mmol, 25% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.05−7.95 (m, 1H), 7.59−7.45 (m, 2H), 6.34 (s, 1H), 4.50 (t, J = 5.3 Hz, 2H), 3.58−3.44 (m, 4H), 3.26−3.17 (m, 2H), 1.63 (s, 9H); MS (DCI) m/z 382 [M + NH4]+. Step 4. To a solution of tert-butyl 5-fluoro-3-(2-(2-oxoimidazolidin1-yl)ethoxy)-1H-indazole-1-carboxylate (from step 3, 1.5 g, 4.12 mmol) in CH2Cl2 (25 mL) at 0 °C was added TFA (12.7 mL, 165 mmol). The mixture was allowed to warm to ambient temperature and was stirred for 2 h. The mixture was concentrated under reduced pressure and purified via column chromatography (SiO2, 10% ethyl acetate/hexanes to 100% ethyl acetate to 9:1:0.1 ethyl acetate/ methanol/triethylamine) to give 1-(2-(5-fluoro-1H-indazol-3-yloxy)-

dihydro-2H-imidazol-2-one (Chembridge, 4.76 g, 32.5 mmol) in DMF (10 mL) was added. The mixture was warmed to 50 °C and was allowed to stir for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO3 (5 mL) and diluted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organics were dried over anhydrous Na2SO4, filtered, concentrated, and purified via column chromatography (SiO2, 1% ethyl acetate/heptanes to 30% ethyl acetate/heptanes) to give 1-(2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)-1H-imidazol-2(3H)-one, 8 (0.95 g, 2.67 mmol, 16% yield). 1H NMR (300 MHz, methanol-d4) δ 7.59 (td, J = 7.7, 1.6 Hz, 1H), 7.49−7.30 (m, 4H), 7.28−7.20 (m, 2H), 6.57 (d, J = 3.0 Hz, 1H), 6.38 (d, J = 3.0 Hz, 1H), 4.64 (dd, J = 5.8, 4.6 Hz, 2H), 4.17− 4.09 (m, 2H); MS (DCI) m/z 357 [M + H]+. 3-(2-(1H-Imidazol-1-yl)ethoxy)-5-fluoro-1-(2-fluorophenyl)1H-indazole (9). To a solution of 5-fluoro-1-(2-fluorophenyl)-1Hindazol-3-ol (3, from synthesis of 4, step 2, 2.3 g, 9.34 mmol), 1-(2hydroxyethyl)imidazole (Oakwood, 1.05 g, 9.34 mmol), and tri-Nbuylphosphine (2.305 mL, 9.34 mmol) in toluene (60 mL) was added 1,1′-(azodicarbonyl)dipiperidine (2.357 g, 9.34 mmol) portionwise over 5 min. The mixture was warmed to 80 °C and was allowed to stir for 5 h. The mixture was allowed to cool to ambient temperature, then was stirred for 16 h. The reaction mixture was diluted with toluene (10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate was concentrated under reduced pressure and the material was purified via column chromatography (SiO2, 5% ethyl acetate/hexanes to 75% ethyl acetate/hexanes) to give 3-(2-(1H-imidazol-1-yl)ethoxy)5-fluoro-1-(2-fluorophenyl)-1H-indazole, 9 (2.4 g, 7.05 mmol, 75% yield). 1H NMR (300 MHz, methanol-d4) δ 7.76 (d, J = 1.2 Hz, 1H), 7.58 (td, J = 7.7, 1.5 Hz, 1H), 7.51−7.30 (m, 4H), 7.27−7.22 (m, 3H), 6.98 (t, J = 1.1 Hz, 1H), 4.71 (dd, J = 5.6, 4.3 Hz, 2H), 4.55 (dd, J = 5.5, 4.5 Hz, 2H); MS (DCI) m/z 341 [M + H]+. 1-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)3-methylimidazolidin-2-one (10). To a solution of 1-(2-(5-fluoro1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one 7 (0.20 g, 0.56 mmol) in DMF (5 mL) at ambient temperature was added NaH (60 wt %, 0.089 g, 2.23 mmol). This mixture was stirred at ambient temperature for 1 h, and then iodomethane (0.042 mL, 0.670 mmol) was added. The mixture was allowed to stir at ambient temperature for 3 h, and then the mixture was quenched with saturated aqueous NaHCO3 (5 mL) and diluted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organics were dried over anhydrous Na2SO4, filtered, concentrated, and purified via column chromatography (SiO2, 5% ethyl acetate/heptanes to 100% ethyl acetate to 10% methanol in ethyl acetate) to give 1-(2-(5-fluoro-1-(2fluorophenyl)-1H-indazol-3-yloxy)ethyl)-3-methylimidazolidin-2-one, 10 (0.17 g, 0.46 mmol, 82% yield). 1H NMR (300 MHz, methanol-d4) δ 7.60 (td, J = 7.6, 1.7 Hz, 1H), 7.50−7.31 (m, 4H), 7.28−7.22 (m, 2H), 4.61−4.53 (m, 2H), 3.72−3.64 (m, 2H), 3.59−3.50 (m, 2H), 3.37−3.31 (m, 2H), 2.74 (s, 3H); MS (DCI) m/z 373 [M + H]+. 3-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)oxazolidin-2-one (12). Step 1. To a solution of 5-fluoro-1-(2fluorophenyl)-1H-indazol-3-ol (3, from synthesis of 4, step 2, 1.34 g, 5.43 mmol), 2-(3oxazolidine)ethanol (Frinton Labs, 0.633 mL, 5.98 mmol), and tri-N-buylphosphine (1.47 mL, 5.98 mmol) in toluene (20 mL) was added 1,1′-(azodicarbonyl)dipiperidine (1.51 g, 5.98 mmol) portionwise over 5 min. The mixture was warmed to 80 °C and was allowed to stir for 5 h. The mixture was allowed to cool to ambient temperature and was stirred for 16 h. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure and purified via column chromatography (SiO2, 10% ethyl acetate/hexanes to 100% ethyl acetate to 9:1:0.1 ethyl acetate/methanol/triethylamine) to give 2-(2-(5-fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethylamino)ethanol, 11 (0.762 g, 2.286 mmol, 42% yield). 1H NMR (300 MHz, methanol-d4) δ 7.60 (td, J = 7.6, 1.6 Hz, 1H), 7.50−7.31 (m, 4H), 7.28−7.23 (m, 2H), 4.60−4.54 (m, 2H), 3.77−3.68 (m, 2H), 3.20−3.12 (m, 2H), 2.89−2.81 (m, 2H); MS (DCI) m/z 334 [M + H]+. 3383



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ethyl)imidazolidin-2-one, 14 (0.71 g, 2.69 mmol, 65% yield). 1H NMR (300 MHz, DMSO-d6) δ 12.03 (s, 1H), 7.39 (dd, J = 9.0, 4.2 Hz, 1H), 7.31 (dd, J = 8.8, 2.4 Hz, 1H), 7.23 (td, J = 9.1, 2.4 Hz, 1H), 6.33 (s, 1H), 4.39 (t, J = 5.5 Hz, 2H), 3.52−3.43 (m, 4H), 3.22 (tt, J = 7.5, 1.1 Hz, 2H); MS (DCI) m/z 265 [M + H]+. Step 5. To a pressure tube were added 1-(2-(5-fluoro-1H-indazol-3yloxy)ethyl)imidazolidin-2-one (14 from step 4, 0.3 g, 1.135 mmol), copper(I) iodide (10.8 mg, 0.057 mmol), and potassium phosphate tribasic (0.506 g, 2.38 mmol). This mixture was degassed three times with a nitrogen backflush each time. The 2,4-difluoro-1-iodobenzene (0.163 mL, 1.362 mmol) was added followed by trans-N,N′dimethylcyclohexane-1,2-diamine (0.036 mL, 0.227 mmol) and toluene (5 mL). The pressure tube was sealed, and the mixture was warmed to 110 °C and was allowed to stir for 18 h. The material was allowed to cool to ambient temperature and then was filtered through Celite with ethyl acetate. The filtrate was concentrated under reduced pressure and purified via column chromatography (SiO2, 1% ethyl acetate/hexanes to 50% ethyl acetate/hexanes) to give 1-(2-(1-(2,4difluorophenyl)-5-fluoro-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one, 15 (0.19 g, 0.505 mmol, 44.5% yield). 1H NMR (300 MHz, methanold4) δ 7.63 (td, J = 8.7, 5.8 Hz, 1H), 7.43−7.35 (m, 1H), 7.31−7.20 (m, 3H), 7.19−7.11 (m, 1H), 4.58−4.53 (m, 2H), 3.70−3.62 (m, 4H), 3.43−3.35 (m, 2H); MS (DCI) m/z 377 [M + H]+. 1-(2-(5-Fluoro-1-(4-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one (16). Copper(I) iodide (3.60 mg, 0.019 mmol), potassium phosphate tribasic (0.169 g, 0.795 mmol), trans-N,N′dimethylcyclohexane-1,2-diamine (0.012 mL, 0.076 mmol), 4fluoroiodobenzene (0.044 mL, 0.378 mmol), and 1-(2-(5-fluoro-1Hindazol-3-yloxy)ethyl)imidazolidin-2-one (14 from synthesis of 15, step 4, 0.12 g, 0.454 mmol) were combined in dioxane (1.0 mL) in a pressure tube. The tube was purged with nitrogen, sealed, and heated at 110 °C for 4 h. The reaction was allowed to cool to ambient temperature, then was diluted with ethyl acetate (5 mL), filtered through Celite, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 100% EtAOc and then 1% methanol in ethyl acetate) to give 1-(2-(5-fluoro-1-(4-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one, 16 (105 mg, 0.293 mmol, 77% yield). 1H NMR (300 MHz, methanol-d4) δ 7.72−7.62 (m, 3H), 7.38 (dd, J = 8.3, 2.4 Hz, 1H), 7.31−7.22 (m, 3H), 4.62− 4.55 (m, 2H), 3.70−3.62 (m, 4H), 3.44−3.34 (m, 2H); MS (ESI+) m/ z 359 [M + H]+. tert-Butyl 2-(1-(2-Fluorophenyl)-1H-indazol-4-yloxy)ethylcarbamate (19). Step 1. A mixture of 4-(benzyloxy)-1Hindazole (enamine, 0.2 g, 0.892 mmol), potassium phosphate tribasic (0.398 g, 1.873 mmol), 2-fluoroidodobenzene (0.104 mL, 0.892 mmol), copper(I) iodide (8.49 mg, 0.045 mmol), and trans-N,N′dimethylcyclohexane-1,2-diamine (0.028 mL, 0.178 mmol) in dioxane (1.78 mL) in a pressure tube was flushed with nitrogen. The tube was sealed and was heated at 110 °C for 20 h. The reaction mixture was allowed to cool to ambient temperature and then diluted with ethyl acetate (10 mL), filtered through Celite, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 10% ethyl acetate in hexanes) to give 4-(benzyloxy)-1-(2fluorophenyl)-1H-indazole (0.25 g, 0.785 mmol, 88% yield). 1H NMR (300 MHz, methanol-d4) δ 8.31 (d, J = 0.9 Hz, 1H), 7.61 (td, J = 7.6, 1.7 Hz, 1H), 7.53 (dt, J = 8.0, 2.6 Hz, 3H), 7.46−7.30 (m, 6H), 6.92 (ddd, J = 8.2, 2.8, 0.8 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H), 5.30 (s, 2H); MS (DCI) m/z 319 [M + H]+. Step 2. A mixture of 4-(benzyloxy)-1-(2-fluorophenyl)-1H-indazole (from step 1, 0.25 g, 0.785 mmol), 10 wt % Pd/C (0.084 g, 0.079 mmol), and ammonium formate (0.248 g, 3.93 mmol) in methanol (4.0 mL) was warmed to 40 °C and was allowed to stir for 18 h. The reaction mixture was then allowed to cool to ambient temperature and was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 25% ethyl acetate in hexanes) to give 1-(2-fluorophenyl)1H-indazol-4-ol, 18 (0.14 g, 0.613 mmol, 78% yield). 1H NMR (300 MHz, methanol-d4) δ 8.28 (d, J = 1.1 Hz, 1H), 7.60 (td, J = 7.6, 1.6 Hz, 1H), 7.56−7.49 (m, 1H), 7.44−7.35 (m, 3H), 7.25 (dd, J = 8.4,

7.7 Hz, 1H), 6.79 (dd, J = 8.4, 2.9 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H); MS (DCI) m/z 229 [M + H]+. Step 3. To 1-(2-fluorophenyl)-1H-indazol-4-ol (18 from step 2, 0.7 g, 3.07 mmol) in dimethylacetamide (DMA, 5.0 mL) was added sodium hydride (60 wt %, 0.123 g, 3.07 mmol), and the mixture was stirred for 15 min at ambient temperature. Then tert-butyl N-(2bromoethyl)carbamate (1.38 g, 6.13 mmol) was added, and the reaction mixture was stirred at ambient temperature for 20 h. The reaction was quenched with saturated aqueous NH4Cl (5 mL), and the mixture was partitioned between ethyl acetate (10 mL) and aqueous 10% Na2CO3. The organic phase was washed with water (1 × 5 mL) and brine (1 × 5 mL) and then was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 25% ethyl acetate in hexanes) to give tert-butyl 2-(1-(2-fluorophenyl)-1H-indazol-4-yloxy)ethylcarbamate, 19 (1.0 g, 2.69 mmol, 88% yield). 1H NMR (300 MHz, methanol-d4) δ 8.33 (d, J = 1.0 Hz, 1H), 7.66−7.48 (m, 2H), 7.47−7.31 (m, 3H), 6.91 (ddd, J = 8.5, 3.0, 0.9 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H), 4.22 (t, J = 5.6 Hz, 2H), 3.55 (t, J = 5.5 Hz, 2H), 1.45 (s, 9H); MS (DCI) m/z 372 [M + H]+. tert-Butyl 3-(1-(2-Fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidine-1-carboxylate (23). Step 1. To a mixture of 2-bromo6-fluorobenzaldehyde (Combi-Blocks, 10 g, 49 mmol) and (2fluorophenyl)hydrazine hydrochloride (8.01 g, 49.3 mmol) in Nmethyl-2-pyrrolidinone (100 mL) at ambient temperature was added cesium carbonate (33.7 g, 103 mmol). The resulting slurry was heated to 140 °C. After 1 h, the reaction was allowed to cool to ambient temperature and water was added (300 mL). The slurry was stirred for 1 h; then the solids were collected by filtration, washed with water, and dried in a vacuum oven at 50 °C to give 4-bromo-1-(2-fluorophenyl)1H-indazole, 21 (13.0 g, 44.7 mmol, 91% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 0.5 Hz, 1H), 7.72 (td, J = 7.8, 1.6 Hz, 1H), 7.66−7.38 (m, 6H); MS (DCI) m/z 291, 293 [M + H]+. Step 2. To a solution of 4-bromo-1-(2-fluorophenyl)-1H-indazole (21 from step 1, 5.00 g, 17.2 mmol) and imidazolidin-2-one hydrate (16.3 g, 86.0 mmol) in dimethoxyethane (DME), (100 mL) at ambient temperature were added cesium carbonate (8.39 g, 25.8 mmol), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (0.80 g, 1.37 mmol), and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 0.63 g, 0.69 mmol). This mixture was heated to 80 °C and was allowed to stir for 16 h. The mixture was allowed to cool to ambient temperature and was partitioned between water (250 mL) and ethyl acetate (200 mL). The organic phase was washed with water (200 mL) and brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2 100% CH2Cl2 to 90% ethyl acetate/ CH2Cl2, then 10% methanol/CH2Cl2). The resulting material was dissolved in 10:1 methyl tert-butyl ether (MTBE)/CH2Cl2 (5 volumes), and the resultant mixture was heated to reflux. The mixture was allowed to cool to ambient temperature with stirring. The resulting solids were isolated via filtration, washed with methyl tertbutyl ether, and dried to provide 1-(1-(2-fluorophenyl)-1H-indazol-4yl)imidazolidin-2-one, 22 (2.58 g, 8.7 mmol, 51% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.43 (d, J = 0.6 Hz, 1H), 7.67 (td, J = 7.8, 1.5 Hz, 1H), 7.62−7.50 (m, 2H), 7.43 (ddd, J = 18.7, 11.7, 4.7 Hz, 2H), 7.14 (d, J = 7.4 Hz, 2H), 7.06 (dd, J = 8.4, 2.9 Hz, 1H), 4.15−4.05 (m, 2H), 3.51 (t, J = 7.8 Hz, 2H); MS (ESI+) m/z 297 [M + H]+. Step 3. To a solution of the product of 1-(1-(2-fluorophenyl)-1Hindazol-4-yl)imidazolidin-2-one (22 from step 2, 1.54 g, 5.20 mmol) and di-tert-butyl dicarbonate (2.41 mL, 10.4 mmol) in acetonitrile (20 mL) was added 4-dimethylaminopyridine (0.063 g, 0.520 mmol). This mixture was allowed to stir at ambient temperature for 18 h. The mixture was concentrated under reduced pressure, and the residue was purified via column chromatography (SiO2, 5% ethyl acetate/heptanes to 100% ethyl acetate) to give the tert-butyl 3-(1-(2-fluorophenyl)-1Hindazol-4-yl)-2-oxoimidazolidine-1-carboxylate, 23 (1.6 g, 4.04 mmol, 78% yield). 1H NMR (300 MHz, methanol-d4) δ 8.38 (d, J = 1.1 Hz, 1H), 7.62 (td, J = 7.6, 1.7 Hz, 1H), 7.59−7.52 (m, 1H), 7.51−7.44 (m, 1H), 7.44−7.37 (m, 2H), 7.25−7.19 (m, 1H), 7.18 (d, J = 7.5 Hz, 1H), 4.14−3.96 (m, 4H), 1.58 (s, 9H); MS (ESI+) m/z 397 [M + H]+. 3384



Article

2-{3-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-2-oxoimidazolidin-1-yl}acetamide (24). To a solution of 1-(1-(2-fluorophenyl)-1Hindazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 0.69 g, 2.33 mmol) in N,N-dimethylformamide (10 mL) at ambient temperature was added NaH (60% dispersion in mineral oil, 0.47 g, 11.6 mmol). This mixture was stirred at ambient temperature for 30 min, and then 2-iodoacetamide (1.29 g, 6.99 mmol) was added. The mixture was warmed to 45 °C, was stirred for 3 h, was allowed to cool to ambient temperature, and was quenched with saturated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 1% ethyl acetate/heptanes to 30% ethyl acetate/heptanes) to give 2-{3-[1-(2fluorophenyl)-1H-indazol-4-yl]-2-oxoimidazolidin-1-yl}acetamide, 24 (0.45 g, 1.27 mmol, 55% yield). 1H NMR (300 MHz, methanol-d4) δ 8.43 (d, J = 1.3 Hz, 1H), 7.62 (td, J = 7.5, 1.6 Hz, 1H), 7.58−7.50 (m, 1H), 7.48−7.35 (m, 3H), 7.14 (dd, J = 8.1, 3.7 Hz, 2H), 4.18− 4.09 (m, 2H), 4.02 (s, 2H), 3.77−3.68 (m, 2H); MS (ESI+) m/z 354 [M + H]+. N-Cyclopropyl-2-{3-[1-(2-fluorophenyl)-1H-indazol-4-yl]-2oxoimidazolidin-1-yl}acetamide (25). Step 1. To a solution of the product of 1-(1-(2-fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 3.0 g, 10.1 mmol) in THF (20 mL) at ambient temperature was added sodium hydride (60% dispersion in mineral oil, 0.607 g, 15.2 mmol). After 5 min, tert-butyl 2bromoacetate (1.9 mL, 13 mmol) was added, and the mixture was stirred for 1 h. The mixture was quenched with water (150 mL), the layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 200 mL). The combined organic layers were concentrated under reduced pressure, and the residue was purified by column chromatography (SiO2, 50% ethyl acetate/heptane) to provide tertbutyl 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidin-1yl)acetate (3.37 g, 8.2 mmol, 81%). 1H NMR (400 MHz, DMSOd6) δ 8.40 (s, 1H), 7.68 (td, J = 7.9, 1.5 Hz, 1H), 7.63−7.50 (m, 2H), 7.48−7.38 (m, 2H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.4, 2.9 Hz, 1H), 4.16−4.05 (m, 2H), 3.99 (s, 2H), 3.69−3.57 (m, 2H), 1.47 (s, 9H); MS (ESI+) m/z 411 [M + H]+. Step 2. To a solution of tert-butyl 2-(3-(1-(2-fluorophenyl)-1Hindazol-4-yl)-2-oxoimidazolidin-1-yl)acetate (from step 1, 19.9 g, 48.5 mmol) in CH2Cl2 (65 mL) at ambient temperature was added TFA (65.0 mL) dropwise via addition funnel over 20 min. This mixture was allowed to stir at ambient temperature for 6 h and then was concentrated under reduced pressure and diluted with toluene. The material was again concentrated under reduced pressure. The dilution with toluene and concentration was repeated two additional times to give 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidin-1yl)acetic acid, 30 (17.0 g, 47.8 mmol, 99% yield). 1H NMR (300 MHz, methanol-d4) δ 8.39 (s, 1H), 7.62 (td, J = 7.6, 1.6 Hz, 1H), 7.57−7.50 (m, 1H), 7.50−7.35 (m, 3H), 7.18−7.09 (m, 2H), 4.18− 4.05 (m, 4H), 3.76 (dd, J = 9.1, 6.8 Hz, 2H); MS (ESI+) m/z 355 [M + H]+. Step 3. To a solution of 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)2-oxoimidazolidin-1-yl)acetic acid (30 from step 2, 0.25 g, 0.706 mmol), cyclopropylamine (0.056 mL, 0.776 mmol), and N-ethyl-Nisopropylpropan-2-amine (0.493 mL, 2.82 mmol) in THF (5 mL) was added N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 0.295 g, 0.776 mmol). This mixture was allowed to stir at ambient temperature for 16 h and then was quenched with water (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 20% hexanes/ ethyl acetate to 100% ethyl acetate to 9:1:0.1 ethyl acetate/methanol/ triethylamine) to provide N-cyclopropyl-2-{3-[1-(2-fluorophenyl)-1Hindazol-4-yl]-2-oxoimidazolidin-1-yl}acetamide, 25 (0.15 g, 0.381 mmol, 54% yield). 1H NMR (400 MHz, methanol-d4) δ 8.43 (d, J =

0.9 Hz, 1H), 7.62 (td, J = 7.7, 1.7 Hz, 1H), 7.59−7.51 (m, 1H), 7.48− 7.37 (m, 3H), 7.16−7.10 (m, 2H), 4.12 (dd, J = 8.9, 6.8 Hz, 2H), 3.97 (s, 2H), 3.70 (dd, J = 8.9, 6.9 Hz, 2H), 2.76−2.68 (m, 1H), 0.75 (td, J = 7.1, 5.1 Hz, 2H), 0.59−0.52 (m, 2H); MS (ESI+) m/z 394 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-[2-oxo-2-(pyrrolidin-1-yl)ethyl]imidazolidin-2-one (26). To a solution of 1-(1-(2fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 1.2 g, 4.05 mmol) in dimethylformamide (DMF) (20 mL) at ambient temperature was added NaH (60% dispersion in mineral oil, 0.81 g, 20.3 mmol). This mixture was stirred at ambient temperature for 30 min, and then 2-bromo-1-(pyrrolidin-1-yl)ethanone (ChemDiv, 2.33 g, 12.15 mmol) was added. The mixture was warmed to 45 °C and was allowed to stir for 3 h. The mixture was quenched with saturated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 1% ethyl acetate/heptanes to 30% ethyl acetate/heptanes) to give the 1-[1-(2-fluorophenyl)-1H-indazol4-yl]-3-[2-oxo-2-(pyrrolidin-1-yl)ethyl]imidazolidin-2-one, 26 (1.25 g, 3.07 mmol, 76% yield). 1H NMR (400 MHz, methanol-d4) δ 8.43 (s, 1H), 7.62 (td, J = 7.6, 1.6 Hz, 1H), 7.59−7.49 (m, 1H), 7.49−7.37 (m, 3H), 7.18−7.08 (m, 2H), 4.17 (s, 2H), 4.13 (dd, J = 9.0, 7.0 Hz, 2H), 3.75 (dd, J = 8.9, 6.9 Hz, 2H), 3.52 (dt, J = 19.5, 6.8 Hz, 4H), 2.04 (q, J = 6.8 Hz, 2H), 1.92 (q, J = 6.8 Hz, 2H); MS (ESI+) m/z 408 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-{2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethyl}imidazolidin-2-one (27). To a solution of 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidin1-yl)acetic acid (30 from step 2 of procedure for 25, 1.12 g, 3.16 mmol), (S)-(+)-3- fluoropyrrolidine hydrochloride (0.437 g, 3.48 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.814 mL, 4.66 mmol) in THF (15 mL) was added N-[(dimethylamino)-1H-1,2,3triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 1.32 g, 3.48 mmol). This mixture was allowed to stir at ambient temperature for 16 h and then was quenched with water (5 mL) and diluted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organics were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified via column chromatography (SiO2, 20% hexanes/ethyl acetate to 100% ethyl acetate to 9:1:0.1 ethyl acetate/methanol/triethylamine) to provide 1-[1-(2-fluorophenyl)-1H-indazol-4-yl]-3-{2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethyl}imidazolidin-2-one, 27 (1.25 g, 2.94 mmol, 93% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 0.8 Hz, 1H), 7.68 (td, J = 7.8, 1.6 Hz, 1H), 7.63−7.49 (m, 2H), 7.49− 7.34 (m, 2H), 7.18 (d, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.4, 3.0 Hz, 1H), 5.59−5.14 (m, 1H), 4.26−3.95 (m, 4H), 3.92−3.47 (m, 6H), 2.36− 1.85 (m, 2H); MS (ESI+) m/z 426 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-{2-[(3R)-3-fluoropyrrolidin-1-yl]-2-oxoethyl}imidazolidin-2-one (28). To a solution of 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidin1-yl)acetic acid (30 from step 2 of procedure for 25, step 2, 0.2 g, 0.564 mmol), (R)-(−)-3-fluoropyrrolidine hydrochloride (CombiBlocks, 0.078 g, 0.621 mmol), and N-ethyl-N-isopropylpropan-2amine (0.814 mL, 4.66 mmol) in THF (3 mL) was added N[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HATU, 0.236 g, 0.621 mmol). This mixture was allowed to stir at ambient temperature for 16 h and then was quenched with water (5 mL) and diluted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organics were dried over anhydrous Na2SO4, filtered, concentrated, and purified via column chromatography (SiO2, 20% hexanes/ethyl acetate to 100% ethyl acetate to 9:1:0.1 ethyl acetate/ methanol/triethylamine) to provide (R)-1-(1-(2-fluorophenyl)-1Hindazol-4-yl)-3-(2-(3-fluoropyrrolidin-1-yl)-2-oxoethyl)imidazolidin-2one, 28 (0.23 g, 0.541 mmol, 96% yield). 1H NMR (500 MHz, 3385



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DMSO-d6) δ 8.40 (d, J = 0.9 Hz, 1H), 7.67 (td, J = 7.8, 1.7 Hz, 1H), 7.63−7.51 (m, 2H), 7.50−7.42 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.4, 2.9 Hz, 1H), 5.51−5.27 (m, 1H), 4.22−4.01 (m, 4H), 3.70−3.61 (m, 4H), 3.60−3.45 (m, 1H), 3.36 (td, J = 11.4, 6.9 Hz, 1H), 2.35−1.95 (m, 2H); MS (ESI+) m/z 426 [M + H]+ 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-{2-[(3S)-3-hydroxypyrrolidin-1-yl]-2-oxoethyl}imidazolidin-2-one (29). To a solution of the product of 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2oxoimidazolidin-1-yl)acetic acid (30 from step 2 of procedure for 25, step 2, 0.2 g, 0.564 mmol), (S)-3-hydroxypyrrolindine (0.052 mL, 0.62 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.81 mL, 4.66 mmol) in THF (5 mL) was added N-[(dimethylamino)-1H-1,2,3triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 0.24 g, 0.62 mmol). This mixture was allowed to stir at ambient temperature for 16 h and then was quenched with water (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 20% hexanes/ethyl acetate to 100% ethyl acetate to 9:1:0.1 ethyl acetate/methanol/triethylamine) to provide 29 (0.20 g, 0.47 mmol, 84% yield). 1H NMR (300 MHz, methanol-d4) δ 8.42 (d, J = 0.9 Hz, 1H), 7.62 (td, J = 7.6, 1.6 Hz, 1H), 7.58−7.51 (m, 1H), 7.49−7.35 (m, 3H), 7.18−7.07 (m, 2H), 4.59−4.37 (m, 1H), 4.18−4.09 (m, 4H), 3.80−3.43 (m, 6H), 2.21−1.85 (m, 2H); MS (ESI+) m/z 424 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-{2-[(3S)-3-fluoropiperidin-1-yl]-2-oxoethyl}imidazolidin-2-one (31). To a mixture of 2-(3-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxoimidazolidin-1-yl)acetic acid (30 from step 2 of procedure for 25, step 2, 1.10 g, 3.10 mmol), (S)-3-fluoropiperidine hydrochloride (Synthonix, 442 mg, 3.17 mmol), and trimethylamine (1.09 mL, 7.76 mmol) in N,Ndimethylformamide (5 mL) was added N-[(dimethylamino)-1H1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 1.42 g, 3.73 mmol). The reaction was stirred at ambient temperature for 18 h. The resulting solution was filtered through a glass microfiber frit and purified by preparative HPLC [custom packed YMC TriArt C18 20 μm column, 50 mm × 150 mm, flow rate 80 mL/min, 10−100% gradient of acetonitrile in buffer (0.025 M aqueous ammonium bicarbonate, adjusted to pH 10 with ammonium hydroxide)] to give 31 (1.05 g, 2.39 mmol, 77% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.40 (s, 1H), 7.68 (td, J = 7.8, 1.6 Hz, 1H), 7.63−7.51 (m, 2H), 7.48−7.37 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.08 (dd, J = 8.4, 2.9 Hz, 1H), 4.96− 4.64 (m, 1H), 4.28−3.76 (m, 6H), 3.66−3.54 (m, 3H), 3.35−3.26 and 3.08−3.00 (two m, 1H; amide rotamers), 1.96−1.41 (m, 4H); MS (ESI+) m/z 440 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-(4-methyl-2oxopentyl)imidazolidin-2-one (32). To a solution of 1-(1-(2fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 2.0 g, 6.75 mmol) in N,N-dimethylformamide (15 mL) at ambient temperature was added NaH (60% dispersion in mineral oil, 0.810 g, 20.3 mmol). This mixture was stirred at ambient temperature for 30 min, and then 1-bromo-4-methylpentane-2-one (1.57 g, 8.77 mmol) was added. The mixture was allowed to stir at ambient temperature for 24 h, and then the mixture was quenched with saturated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 1% ethyl acetate/heptanes to 30% ethyl acetate/heptanes to 10% methanol/ethyl acetate) to give 1-[1-(2fluorophenyl)-1H-indazol-4-yl]-3-(4-methyl-2-oxopentyl)imidazolidin2-one, 32 (0.65 g, 1.65 mmol, 24% yield). 1H NMR (300 MHz, methanol-d4) δ 8.37 (d, J = 1.1 Hz, 1H), 7.66−7.58 (m, 1H), 7.58− 7.50 (m, 1H), 7.50−7.36 (m, 3H), 7.19−7.09 (m, 2H), 4.21 (s, 2H), 4.17−4.07 (m, 2H), 3.73−3.62 (m, 2H), 2.41 (d, J = 6.9 Hz, 2H), 2.19

(dp, J = 13.5, 6.7 Hz, 1H), 0.99 (d, J = 6.6 Hz, 6H); MS (ESI+) m/z 395 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-(1,3-oxazol-2ylmethyl)imidazolidin-2-one (33). To a solution of 1-(1-(2fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 0.26 g, 0.877 mmol) in N,N-dimethylformamide (5 mL) at 0 °C was added NaH (60% dispersion in mineral oil, 0.175 g, 4.39 mmol). This mixture was allowed to warm to ambient temperature and was stirred for 30 min, and then 2-chloromethyloxazole (JWPharmlab, 0.113 g, 0.965 mmol) was added. The mixture was warmed to 40 °C and was allowed to stir for 3 h, and then the mixture was quenched with saturated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 1% ethyl acetate/heptanes to 30% ethyl acetate/heptanes) to give 1-[1-(2-fluorophenyl)-1H-indazol-4yl]-3-(1,3-oxazol-2-ylmethyl)imidazolidin-2-one, 33 (0.18 g, 0.477 mmol, 54% yield). 1H NMR (300 MHz, methanol-d4) δ 8.41 (d, J = 0.9 Hz, 1H), 7.95 (d, J = 0.9 Hz, 1H), 7.62 (td, J = 7.6, 1.6 Hz, 1H), 7.58−7.51 (m, 1H), 7.49−7.37 (m, 3H), 7.22−7.11 (m, 3H), 4.70 (s, 2H), 4.14 (dd, J = 9.0, 6.8 Hz, 2H), 3.77−3.68 (m, 2H); MS (ESI+) m/z 378 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-[(6-methylpyrazin2-yl)methyl]imidazolidin-2-one (34). To a solution of 1-(1-(2fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 190 mg, 0.64 mmol) in THF (4 mL) at 0 °C was added NaH (60% dispersion in mineral oil, 103 mg, 2.56 mmol). The mixture was allowed to warm to ambient temperature over a period of 30 min. 2-(Chloromethyl)-6-methylpyrazine (Small Molecules Inc., 155 mg, 1.09 mmol) was added. The resulting mixture was stirred at ambient temperature for 18 h. The reaction was quenched by the slow addition of methanol (3 mL), and the resulting mixture was concentrated in vacuo. The residue was dissolved in N,Ndimethylformamide (3 mL), filtered through a glass microfiber frit, and purified by preparative HPLC [custom packed YMC TriArt C18 20 μm column, 50 mm × 150 mm, flow rate 80 mL/min, 20−100% gradient of acetonitrile in buffer (0.025 M aqueous ammonium bicarbonate, adjusted to pH 10 with ammonium hydroxide)] to give 1[1-(2-fluorophenyl)-1H-indazol-4-yl]-3-[(6-methylpyrazin-2-yl)methyl]imidazolidin-2-one, 34 (138 mg, 0.34 mmol, 54% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.50 (d, J = 5.1 Hz, 1H), 8.44−8.44 (m, 2H), 7.68 (td, J = 7.9, 1.7 Hz, 1H), 7.62−7.51 (m, 2H), 7.48−7.39 (m, 2H), 7.20 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.3, 2.9 Hz, 1H), 4.60 (s, 2H), 4.12 (dd, J = 8.8, 6.7 Hz, 2H), 3.66−3.56 (m, 2H), 2.53 (s, 3H); MS (ESI+) m/z 403 [M + H]+. 1-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-3-(pyridin-4-yl)imidazolidin-2-one (35). To a pressure tube were added 1-(1-(2fluorophenyl)-1H-indazol-4-yl)imidazolidin-2-one (22 from procedure for 23, step 2, 0.25 g, 0.844 mmol) in dioxane (3 mL), CuI (Strem, 8 mg, 0.042 mmol), and potassium phosphate tribasic (Strem, 0.38, 1.772 mmol). This mixture was degassed three times with a nitrogen backflush each time. 4-Iodopyridine (TCI-US, 0.114 mL, 1.097 mmol) was added followed by trans-N,N′-dimethylcyclohexane-1,2-diamine (0.027 mL, 0.169 mmol). The mixture was warmed to 110 °C and was allowed to stir for 48 h. The material was allowed to cool to ambient temperature and then was filtered through diatomaceous earth, rinsing with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 1% ethyl acetate/hexanes to 50% ethyl acetate/hexanes) to give 1-[1(2-fluorophenyl)-1H-indazol-4-yl]-3-(pyridin-4-yl)imidazolidin-2-one, 35 (0.26 g, 0.696 mmol, 83% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.47−8.40 (m, 3H), 7.82−7.64 (m, 3H), 7.63−7.54 (m, 2H), 7.54− 7.42 (m, 2H), 7.27 (d, J = 7.5 Hz, 1H), 7.21 (dd, J = 8.5, 3.0 Hz, 1H), 4.30−4.05 (m, 4H); MS (ESI+) m/z 374 [M + H]+. (S)-4-(1-(2-Fluorophenyl)-1H-indazol-4-yl)-1-(2-(3-fluoropyrrolidin-1-yl)-2-oxoethyl)piperazin-2-one (38). Step 1. Nitrogen was bubbled through a mixture of 4-bromo-1-(2-fluorophenyl)-1Hindazole (21 from procedure for 23, step 1, 1.07 g, 3.68 mmol), 3386



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1H), 3.63−3.54 (m, 2H), 3.44−3.38 (m, 1H), 3.09 (ddd, J = 9.3, 7.4, 1.5 Hz, 1H), 2.97 (dd, J = 9.2, 1.5 Hz, 1H), 2.83−2.73 (m, 2H), 2.40 (ddd, J = 21.1, 9.3, 7.5 Hz, 2H), 1.50 (s, 9H). MS (ESI+) m/z 317 (M + H)+. Step 2. To a solution of tert-butyl 5-benzyl-1-oxohexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (step 1, 2.44 g, 7.71 mmol) in CH2Cl2 (40.0 mL) was added TFA (10.0 mL, 130 mmol). The reaction was stirred at ambient temperature for 1 h and then was concentrated under reduced pressure and dissolved in CH2Cl2 (25 mL). This solution was washed with 1 M NaOH (2 × 10 mL) and brine (1 × 5 mL), and the organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give 5benzylhexahydropyrrolo[3,4-c]pyrrol-1(2H)-one, 41 (1.21 g, 5.61 mmol, 73% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.48 (s, 1H), 7.35−7.19 (m, 5H), 3.53 (s, 2H), 3.47−3.38 (m, 1H), 2.92 (dd, J = 9.7, 3.0 Hz, 1H), 2.89−2.78 (m, 2H), 2.76−2.67 (m, 1H), 2.62 (dd, J = 9.3, 2.3 Hz, 1H), 2.39 (dd, J = 9.2, 7.0 Hz, 1H), 2.28 (dd, J = 14.0, 5.7 Hz, 1H). MS (DCI+) m/z 217 (M + H)+. Step 3. Nitrogen was vigorously bubbled through a mixture of 4bromo-1-(2-fluorophenyl)-1H-indazole (21 from procedure for 23, step 1, 1.39 g, 4.76 mmol), 5-benzylhexahydropyrrolo[3,4-c]pyrrol1(2H)-one (41 from step 2, 1.21 g, 5.61 mmol), copper(I) iodide (0.0531 g, 0.279 mmol), and potassium phosphate tribasic (2.16 g, 10.2 mmol) in dioxane (20.0 mL) for 20 min. The trans-N,N′dimethylcyclohexane-1,2-diamine (0.15 mL, 0.951 mmol) was added, and the reaction was warmed to 105 °C for 48 h. The mixture was allowed to cool to ambient temperature and was filtered through Celite. The filtrate was concentrated under reduced pressure and purified via column chromatography (SiO2, 50% ethyl acetate/ heptanes to 100% ethyl acetate) to give 5-benzyl-2-(1-(2-fluorophenyl)-1H-indazol-4-yl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one, 42 (1.69 g, 3.97 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.23 (d, J = 0.8 Hz, 1H), 7.70 (td, J = 7.8, 1.6 Hz, 1H), 7.65−7.53 (m, 2H), 7.52−7.42 (m, 2H), 7.36−7.29 (m, 4H), 7.29−7.21 (m, 3H), 4.29 (t, J = 9.2 Hz, 1H), 3.70 (dd, J = 9.7, 2.9 Hz, 1H), 3.64 (dd, J = 30.4, 13.1 Hz, 2H), 3.26−3.18 (m, 1H), 3.10 (d, J = 9.0 Hz, 1H), 3.07−2.98 (m, 1H), 2.88 (dd, J = 9.4, 1.7 Hz, 1H), 2.54 (dd, J = 9.3, 7.4 Hz, 1H), 2.45 (dd, J = 8.9, 7.6 Hz, 1H). MS (ESI+) m/z 427 (M + H)+. (3aS,6aR)-2-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-5-(3-hydroxy-3-methylbutanoyl)hexahydropyrrolo[3,4-c]pyrrol1(2H)-one (48). Step 1. To a solution of 2-oxo-2,4-dihydropyrrole-1carboxylic acid tert-butyl ester, 39 (Chem-Impex, 17 g, 90.5 mmol) in CH2Cl2 (200 mL) at 0 °C was added TFA (TFA, 2.53 mL, 32.8 mmol) followed by the (R)-(+)-N-methoxymethyl-N-(trimethylsilyl)methyl-1-phenylethylamine, 43 (Small Molecules, 21.5 g, 82 mmol) in CH2Cl2 (15 mL) dropwise via syringe pump over 2 h. The mixture was allowed to warm slowly to ambient temperature and was stirred for 16 h. The mixture was quenched with saturated aqueous NaHCO3 (30 mL), and the layers were separated. The aqueous layer was extracted with CH2Cl2 (3 × 25 mL), and the combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 100% CH2Cl2 to 20% ethyl acetate/CH2Cl2 to 40% ethyl acetate/CH2Cl2) to give the first eluting isomer, (3aR,6aS)tert-butyl 1-oxo-5-((R)-1-phenylethyl)hexahydropyrrolo[3,4-c]pyrrole2(1H)-carboxylate, 44 (11.8 g, 26.7 mmol, 33% yield), and the second eluting isomer (3aS,6aR)-tert-butyl 1-oxo-5-((R)-1-phenylethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate, 45 (10.8 g, 32.6 mmol, 40% yield) (stereochemistry of second eluting isomer (45) confirmed by X-ray crystal structure, data not shown). Data for first eluting isomer, 44: 1H NMR (400 MHz, chloroform-d) δ 7.35−7.21 (m, 5H), 3.88 (dd, J = 10.9, 9.5 Hz, 1H), 3.46−3.28 (m, 2H), 3.24− 3.12 (m, 1H), 3.06 (t, J = 8.7 Hz, 1H), 2.78−2.62 (m, 1H), 2.50 (dd, J = 9.6, 1.5 Hz, 1H), 2.49−2.40 (m, 1H), 2.36−2.21 (m, 1H), 1.55 (d, J = 4.4 Hz, 9H), 1.37 (dd, J = 14.3, 5.4 Hz, 3H); MS (ESI+) m/z 331 [M + H]+. Data for second eluting isomer, 45: 1H NMR (400 MHz, chloroform-d) δ 7.34−7.17 (m, 5H), 3.90 (dt, J = 19.1, 9.6 Hz, 1H), 3.54 (dd, J = 11.1, 3.1 Hz, 1H), 3.31−3.16 (m, 1H), 3.10−2.98 (m, 1H), 2.93 (d, J = 9.3 Hz, 1H), 2.84−2.65 (m, 2H), 2.59−2.44 (m,

piperazin-2-one (0.384 g, 3.83 mmol), bis(dibenzylideneacetone)palladium(0) (Strem, 0.137 g, 0.238 mmol), 1,3-bis(2,6-diisopropylphenyl)-1H-imidazol-3-ium chloride (Strem, 0.299 g, 0.704 mmol), and cesium carbonate (3.75 g, 11.5 mmol) in dioxane (18.4 mL) for 20 min. The reaction was then warmed to 90 °C and was allowed to stir for 2 days. The reaction was allowed to cool, filtered through a plug of Celite with ethyl acetate and the filtrate was concentrated under reduced pressure and purified via column chromatography (SiO2, 10% methanol in CH2Cl2) to give 4-(1-(2-fluorophenyl)-1Hindazol-4-yl)piperazin-2-one, 36 (0.247 g, 0.797 mmol, 22% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.50 (d, J = 0.9 Hz, 1H), 8.07 (s, 1H), 7.66 (td, J = 7.8, 1.6 Hz, 1H), 7.62−7.49 (m, 2H), 7.48−7.39 (m, 1H), 7.31 (dd, J = 8.3, 7.8 Hz, 1H), 6.87 (dd, J = 8.4, 3.0 Hz, 1H), 6.57 (dd, J = 7.4, 3.4 Hz, 1H), 3.86 (s, 2H), 3.67−3.58 (m, 2H), 3.48−3.38 (m, 2H). MS (ESI+) m/z 311 (M + H)+. Step 2. Sodium hydride (60% in mineral oil, 0.0695 g, 1.74 mmol) was added portionwise to a solution of 4-(1-(2-fluorophenyl)-1Hindazol-4-yl)piperazin-2-one (from step 1, 0.116 g, 0.375 mmol) in DMF (5.0 mL) at 0 °C. The mixture was allowed to stir at 0 °C for 30 min, and then ethyl iodoacetate (0.080 mL, 0.677 mmol) was added. The reaction mixture was allowed to warm ambient temperature and was allowed to stir for 16 h. Sodium hydroxide (1 M, 1 mL) was added, and the reaction mixture was stirred for an additional 4 h. The mixture was neutralized to pH 7 with 1 M HCl and partitioned between CH2Cl2 and water. The aqueous fraction was extracted with CH2Cl2 (2 × 5 mL). The combined organic fractions were washed with brine (1 × 5 mL), dried over anhydrous MgSO4, and concentrated under reduced pressure to give 2-(4-(1-(2-fluorophenyl)-1H-indazol-4-yl)-2-oxopiperazin-1-yl)acetic acid, 37 (0.138 g. 0.375 mmol, 100% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 0.8 Hz, 1H), 7.66 (td, J = 7.8, 1.6 Hz, 1H), 7.62−7.50 (m, 2H), 7.48−7.39 (m, 1H), 7.36−7.28 (m, 1H), 6.89 (dd, J = 8.4, 2.9 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 4.13 (s, 2H), 3.97 (s, 2H), 3.76−3.69 (m, 2H), 3.67−3.58 (m, 2H). MS (ESI+) m/z 369 (M + H)+. Step 3. To a mixture of 2-(4-(1-(2-fluorophenyl)-1H-indazol-4-yl)2-oxopiperazin-1-yl)acetic acid (from step 2, 0.138 g, 0.375 mmol), (S)-(+)-3-fluoropyrrolidine hydrochloride (0.0727 g, 0.579 mmol), and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (HATU, 0.1560 g, 0.410 mmol) in THF (1.5 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.3 mL, 1.718 mmol). The mixture was stirred at ambient temperature for 1 h. The mixture was partitioned between ethyl acetate (5 mL) and water (5 mL). The organic fraction was washed with water (1 × 5 mL) and brine (1 × 5 mL), concentrated under reduced pressure, and purified via column chromatography (SiO2, 10% methanol in CH2Cl2) to give (S)-4-(1-(2-fluorophenyl)-1H-indazol-4-yl)-1-(2-(3-fluoropyrrolidin-1-yl)-2-oxoethyl)piperazin-2-one, 38 (34.2 mg, 0.078 mmol, 21% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.52 (d, J = 0.7 Hz, 1H), 7.67 (td, J = 7.9, 1.6 Hz, 1H), 7.63−7.51 (m, 2H), 7.44 (td, J = 7.7, 1.5 Hz, 1H), 7.36−7.28 (m, 1H), 6.88 (dd, J = 8.4, 2.8 Hz, 1H), 6.61 (d, J = 7.6 Hz, 1H), 5.51−5.25 (m, 1H), 4.36−4.17 (m, 2H), 3.98 (s, 2H), 3.87−3.38 (m, 8H), 2.31−2.03 (m, 2H). MS (ESI+) m/z 440 (M + H)+. 5-Benzyl-2-(1-(2-fluorophenyl)-1H-indazol-4-yl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one (42). Step 1. A solution of tert-butyl 2-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate, 39 (Chem-Impex, 1.64 g, 8.95 mmol) in CH2Cl2 (20 mL) was cooled to 0 °C, and TFA (0.15 mL, 1.947 mmol) was added followed by the slow addition of Nmethoxymethyl-N-(trimethylsilyl)benzylamine, 40 (2.8 mL, 10.94 mmol) in CH2Cl2 (4.00 mL) via an addition funnel over 90 min. Once the addition was complete, the reaction was allowed to warm to ambient temperature and was allowed to stir for 16 h. The reaction mixture was then diluted with CH2Cl2 (5 mL) and washed with saturated aqueous NaHCO3 (2 × 5 mL) and brine (1 × 5 mL). The combined organics were concentrated under reduced pressure and purified via column chromatography (SiO2, 50% ethyl acetate/ heptanes to 100% ethyl acetate) to give tert-butyl 5-benzyl-1oxohexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (2.44 g, 7.71 mmol, 86% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.36 (dd, J = 8.1, 6.8 Hz, 2H), 7.30 (d, J = 7.3 Hz, 3H), 3.90 (dd, J = 10.9, 9.1 Hz, 3387



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2H), 1.54 (s, 9H), 1.36 (d, J = 6.2 Hz, 3H); MS (ESI+) m/z 331 [M + H]+. Step 2. To a solution of (3aS,6aR)-tert-butyl 1-oxo-5-((R)-1phenylethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (45, second eluting isomer from step 1, 21.5 g, 65.1 mmol) in CH2Cl2 (200 mL) at 0 °C was added TFA (90 mL, 1171 mmol). This mixture was allowed to stir at ambient temperature for 2 h and then was concentrated under reduced pressure to give (3aR,6aR)-5-((R)-1phenylethyl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one (15 g, 65.1 mmol, 100% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.47−7.21 (m, 5H), 3.50 (t, J = 8.8 Hz, 1H), 3.30 (q, J = 6.6 Hz, 1H), 3.14 (dd, J = 9.1, 1.7 Hz, 1H), 2.99−2.78 (m, 3H), 2.50−2.35 (m, 3H), 1.39 (d, J = 6.6 Hz, 3H); MS (ESI+) m/z 231 [M + H]+. Step 3. A mixture of 4-bromo-1-(2-fluorophenyl)-1H-indazole (21 from procedure for 23, 19 g, 65.3 mmol), (3aR,6aR)-5-((R)-1phenylethyl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one (from step 2, 15.03 g, 65.3 mmol), copper(I) iodide (0.62 g, 3.26 mmol), potassium phosphate tribasic (29.1 g, 137 mmol), and trans-N,N′-dimethylcyclohexane-1,2-diamine (1.9 mL, 13.05 mmol) in dioxane (200 mL) was flushed with nitrogen. The mixture was then warmed to 110 °C and was allowed to stir for 16 h. The reaction mixture was allowed to cool to ambient temperature and then diluted with ethyl acetate (100 mL), filtered through Celite, and concentrated under reduced pressure. The residue was purified via column chromatography (SiO2, 5% ethyl acetate/hexanes to 65% ethyl acetate/hexanes) to give (3aS,6aR)-2-[1-(2-fluorophenyl)-1H-indazol-4-yl]-5-[(1R)-1phenylethyl]hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one, 46 (25.1 g, 57 mmol, 87% yield). 1H NMR (500 MHz, methanol-d4) δ 8.28 (s, 1H), 7.65 (td, J = 7.8, 1.6 Hz, 1H), 7.57 (tdd, J = 7.2, 4.9, 1.7 Hz, 1H), 7.51 (dd, J = 8.5, 7.4 Hz, 1H), 7.43 (q, J = 8.9, 8.1 Hz, 2H), 7.39−7.35 (m, 2H), 7.33−7.27 (m, 3H), 7.25−7.18 (m, 2H), 4.25 (dd, J = 9.8, 8.7 Hz, 1H), 3.64 (dd, J = 9.9, 3.1 Hz, 1H), 3.48 (dd, J = 9.5, 1.7 Hz, 1H), 3.35−3.31 (m, 2H), 3.07−3.00 (m, 1H), 2.65 (dd, J = 9.9, 2.3 Hz, 1H), 2.61−2.55 (m, 1H), 2.50 (dd, J = 9.8, 7.2 Hz, 1H), 1.44 (d, J = 6.6 Hz, 3H); MS (ESI+) m/z 441 [M + H]+. Step 4. To a solution of (3aS,6aR)-2-[1-(2-fluorophenyl)-1Hindazol-4-yl]-5-[(1R)-1-phenylethyl]hexahydropyrrolo[3,4-c]pyrrol1(2H)-one (46 from step 3, 24.1 g, 54.7 mmol) in CH2Cl2 (200 mL) at 0 °C was added 1-chloroethyl chloroformate (23.9 mL, 219 mmol). The mixture was stirred at 0 °C for 1 h and then was allowed to warm to ambient temperature and was stirred for 2 h. The mixture was warmed to 50 °C, was stirred for an additional 1 h, and then was allowed to cool to ambient temperature and was concentrated under reduced pressure. The residue was dissolved in methanol (100 mL), and the mixture was warmed to reflux. The solution was stirred at reflux for 2 h and then was allowed to stir overnight at ambient temperature. The mixture was concentrated under reduced pressure, and the residue was purified via column chromatography (SiO2, 5% ethyl acetate/heptanes to 100% ethyl acetate to 9:1:0.1 ethyl acetate/ methanol/triethylamine) to give (3aS,6aR)-2-(1-(2-fluorophenyl)-1Hindazol-4-yl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one, 47 (13.5 g, 40.1 mmol, 73.4% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.44 (s, 1H), 7.69 (td, J = 7.9, 1.7 Hz, 1H), 7.64−7.52 (m, 2H), 7.52−7.42 (m, 2H), 7.31−7.25 (m, 2H), 4.33 (dd, J = 10.1, 7.1 Hz, 1H), 3.87 (dd, J = 10.3, 1.8 Hz, 1H), 3.57 (dtt, J = 20.4, 9.4, 4.9 Hz, 2H), 3.31−3.26 (m, 4H); MS (ESI+) m/z 337 [M + H]+. Step 5. To a solution of (3aS,6aR)-2-(1-(2-fluorophenyl)-1Hindazol-4-yl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one (47 from step 4, 1.36 g, 4.04 mmol), β-hydroxyisovaleric acid (0.48 mL, 4.45 mmol), and N-ethyl-N-isopropylpropan-2-amine (2.8 mL, 16.2 mmol) in THF (30 mL) was added (dimethylamino)-N,N-dimethyl(3-oxido-1H[1,2,3]triazolo[4,5-b]pyridin-1-yl)methaniminium hexafluorophosphate (HATU, 1.69 g, 4.45 mmol) portionwise over 15 min. This mixture was allowed to stir at ambient temperature for 3 h and then was quenched with water (10 mL) and diluted with ethyl acetate (10 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified via column chromatography (SiO2, 20% hexanes/ethyl acetate to 100% ethyl acetate to 15% methanol in ethyl acetate) to

provide (3aS,6aR)-2-[1-(2-fluorophenyl)-1H-indazol-4-yl]-5-(3-hydroxy-3-methylbutanoyl)hexahydropyrrolo[3,4-c]pyrrol-1(2H)-one, 48 (1.2 g, 2.75 mmol, 68% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.27 (dd, J = 2.9, 0.7 Hz, 1H), 7.68 (td, J = 7.9, 1.5 Hz, 1H), 7.64−7.52 (m, 2H), 7.52−7.42 (m, 2H), 7.31−7.22 (m, 2H), 4.84 (d, J = 11.3 Hz, 1H), 4.33 (ddd, J = 9.9, 6.2, 3.8 Hz, 1H), 4.04−3.96 (m, 1H), 3.93−3.85 (m, 1H), 3.82−3.78 (m, 2H), 3.61−3.37 (m, 2H), 3.31− 3.11 (m, 1H), 2.48−2.36 (m, 2H), 1.19 (t, J = 5.7 Hz, 6H); MS (ESI−) m/z 435 (M − H)−. (3aS,6aS)-5-[1-(2-Fluorophenyl)-1H-indazol-4-yl]-1-(3-hydroxy-3-methylbutanoyl)hexahydropyrrolo[3,4-b]pyrrol6(1H)-one (55). Step 1. To a 2 L round-bottom flask were added 3buten-1-ol, 49 (59.5 mL, 693 mmol) in CH2Cl2 (800 mL), and the mixture was cooled to 4 °C with an ice bath. Triethylamine (97 mL, 690 mmol) was added slowly over 5 min followed by dropwise addition of methanesulfonyl chloride (53.8 mL, 693 mmol). An 8−9 °C exothermic reaction was observed. The reaction mixture was stirred for 60 min with an internal temperature maintained between 4 and 8 °C. The reaction mixture was combined with 1 N HCl (200 mL), and the layers were separated. The organic layer was washed with saturated aqueous NaHCO3 (200 mL) and brine (200 mL), then was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give but-3en-1-yl methanesulfonate (96 g, 639 mmol, 92% yield) which was used without further purification. 1H NMR (500 MHz, DMSO-d6) δ 5.80 (ddt, J = 16.9, 10.2, 6.6 Hz, 1H), 5.17 (dq, J = 17.3, 1.6 Hz, 1H), 5.11 (ddt, J = 10.3, 2.2, 1.3 Hz, 1H), 4.24 (t, J = 6.5 Hz, 2H), 3.16 (s, 3H), 2.44 (qt, J = 6.5, 1.4 Hz, 2H); MS (DCI) m/z 151 [M + H]+. Step 2. To a 500 mL round-bottom flask containing but-3-en-1-yl methanesulfonate (from step 1, 50.0 g, 333 mmol) were added acetonitrile (100 mL) and (S)-1-phenylethanamine (64.4 mL, 499 mmol). The reaction was heated to 85 °C and was allowed to stir for 16 h. The mixture was allowed to cool to ambient temperature and was diluted with tert-butyl methyl ether (300 mL). The layers were separated, and the organic layer was washed with 2 N NaOH (50 mL) and dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, eluted with heptane/ethyl acetate 0−50% over 50 min) to provide N-[(1S)-1phenylethyl]but-3-en-1-amine (11.94 g, 68.1 mmol, 20% yield). 1H NMR (400 MHz, CDCl3) δ 7.35−7.27 (m, 3H), 7.26−7.19 (m, 2H), 5.74 (ddt, J = 17.1, 10.2, 6.8 Hz, 1H), 5.06 (dq, J = 17.2, 1.6 Hz, 1H), 5.01 (ddt, J = 10.2, 2.1, 1.2 Hz, 1H), 3.76 (q, J = 6.6 Hz, 1H), 2.62− 2.45 (m, 2H), 2.22 (qt, J = 7.2, 1.3 Hz, 2H), 1.48−1.39 (m, 1H), 1.34 (d, J = 6.6 Hz, 3H); MS (DCI) m/z 176 [M + H]+. Step 3. To a 1 L round-bottom flask were added N-[(1S)-1phenylethyl]but-3-en-1-amine (from step 2, 27.4 g, 156 mmol), dimethyl sulfoxide (100 mL), and N,N-diisopropylethylamine (54.1 mL, 312 mmol). Methyl bromoacetate (17.3 mL, 187 mmol) was then added dropwise, and the reaction mixture was allowed to stir for 2 h. The mixture was poured into ethyl acetate (100 mL) and washed with saturated aqueous NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, eluted with heptane/ethyl acetate 0−5% 60 min gradient) to give methyl N-but-3-en-1-yl-N-[(1S)-1-phenylethyl]glycinate, 50 (32.7 g, 132 mmol, 85% yield). 1H NMR (400 MHz, CDCl3) δ 7.46−7.13 (m, 5H), 5.75 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 5.10−4.87 (m, 2H), 4.03 (q, J = 6.8 Hz, 1H), 3.66 (s, 3H), 3.55−3.17 (m, 2H), 2.68 (ddd, J = 8.2, 6.9, 1.8 Hz, 2H), 2.19 (dddq, J = 8.3, 7.0, 5.5, 1.7 Hz, 2H), 1.35 (d, J = 6.7 Hz, 3H); MS (ESI) m/z 248 [M + H]+. Step 4. To a 500 mL round-bottom flask under nitrogen were added methyl N-but-3-en-1-yl-N-[(1S)-1-phenylethyl]glycinate (50 from step 3, 15 g, 61 mmol) and diethyl ether (300 mL), and the solution was cooled to −74 °C (dry ice/acetone). Lithium bis(trimethylsilyl)amide (1 M in THF, 66.7 mL, 66.7 mmol) was then added dropwise with the internal temperature being maintained below −65 °C. After the addition was complete, the reaction was allowed to warm to 0 °C over 25 min and then was stirred for an additional 20 min at 0 °C (ice/ water). The reaction was again cooled to −74 °C, and zinc bromide (28.7 g, 127 mmol in diethyl ether 300 mL) was added dropwise over 20−30 min, with the internal temperature kept below −60 °C during 3388



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Hz, 1H), 3.54 (dd, J = 11.2, 2.5 Hz, 1H), 3.44 (d, J = 8.9 Hz, 1H), 2.88 (ddd, J = 8.8, 7.2, 3.5 Hz, 1H), 2.61 (dtd, J = 20.7, 8.6, 6.1 Hz, 1H), 2.08 (dddd, J = 15.6, 9.6, 6.6, 3.4 Hz, 1H), 1.76−1.58 (m, 2H), 1.57− 1.41 (m, 12H); MS (DCI) m/z 331 [M + H]+. Step 8. To a solution of tert-butyl (3aS,6aS)-6-oxo-1-[(1S)-1phenylethyl]hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (from step 7, 9.69 g, 29.3 mmol) in CH2Cl2 (200 mL) at 0 °C was added TFA (40.7 mL, 528 mmol) dropwise over 30 min. After the addition as complete, the ice bath was removed and the mixture was allowed to stir at ambient temperature for 2 h. The mixture was concentrated under reduced pressure and dissolved in CH2Cl2 (200 mL). Saturated aqueous NaHCO3 (50 mL) was added dropwise via addition funnel, and then the layers were separated. The aqueous layer was extracted with CH2Cl2 (3 × 100 mL), and the combined organic fractions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give (3aS,6aS)-1-[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one, 52 (6.71 g, 29.1 mmol, 99% yield), which was used without further purification. 1H NMR (500 MHz, DMSO-d6) δ 7.48 (s, 1H), 7.37−7.13 (m, 5H), 4.03 (q, J = 6.8 Hz, 1H), 3.31 (dd, J = 9.8, 7.7 Hz, 1H), 3.17 (d, J = 9.0 Hz, 1H), 2.92 (ddd, J = 9.8, 2.1, 1.0 Hz, 1H), 2.80−2.59 (m, 2H), 2.37 (td, J = 9.0, 6.2 Hz, 1H), 1.94 (dddd, J = 12.1, 9.2, 6.2, 3.3 Hz, 1H), 1.47 (ddt, J = 12.1, 8.8, 7.2 Hz, 1H), 1.37 (d, J = 6.8 Hz, 3H); MS (DCI) m/z 231 [M + H]+. Step 9. To a 200 mL flask were added (3aS,6aS)-1-[(1S)-1phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one (52 from step 8, 9.58 g, 32.9 mmol), 4-bromo-1-(2-fluorophenyl)-1H-indazole (21 from procedure for 23, step 1, 6.89 g, 29.9 mmol), copper(I) iodide (0.285 g, 1.496 mmol), and potassium phosphate tribasic (13.34 g, 62.8 mmol). The contents were purged with nitrogen for 5 min, and then trans-N,N′-dimethylcyclohexane-1,2-diamine (0.944 mL, 5.98 mmol) and dioxane (80 mL) were added. Nitrogen was blown through the system for 5 min. The mixture was warmed to 100 °C and allowed to stir for 90 min. The material was allowed to cool to ambient temperature and then was filtered through diatomaceous earth with ethyl acetate (500 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatograph (SiO2, eluting with 5% ethyl acetate/hexanes to 65% ethyl acetate/ hexanes) to give (3aS,6aS)-5-[1-(2-fluorophenyl)-1H-indazol-4-yl]-1[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one, 53 (10.20 g, 23.2 mmol, 77% yield). 1H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 1.0 Hz, 1H), 7.60 (td, J = 7.9, 1.9 Hz, 1H), 7.51−7.46 (m, 2H), 7.46−7.21 (m, 8H), 7.12 (d, J = 7.4 Hz, 1H), 4.40 (q, J = 6.8 Hz, 1H), 4.14 (dd, J = 9.7, 7.3 Hz, 1H), 3.70−3.64 (m, 2H), 2.96 (ddd, J = 8.9, 7.1, 4.5 Hz, 1H), 2.94−2.87 (m, 1H), 2.80 (dt, J = 9.2, 7.2 Hz, 1H), 2.24 (dddd, J = 12.6, 9.1, 6.9, 4.7 Hz, 1H), 1.86−1.76 (m, 1H), 1.55 (d, J = 6.8 Hz, 3H); MS (DCI) m/z 441 [M + H]+. Step 10. To a 500 mL stainless steel pressure vessel were added (3aS,6aS)-5-[1-(2-fluorophenyl)-1H-indazol-4-yl]-1-[(1S)-1phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one (53 from step 9, 23.78 g, 54 mmol), trifluoroethanol (170 mL), and 20% Pd(OH)2 on carbon, wet (3.3 g, 37 mmol), and the mixture was shaken at ambient temperature under 30 psi of hydrogen gas for 30 min. The mixture was filtered and washed with trifluoroethanol (20 mL) and the filtrate was concentrated in vacuo to give (3aS,6aS)-5-[1-(2fluorophenyl)-1H-indazol-4-yl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)one, 54 (19.06 g, 56.7 mmol, >100% yield), which was used without purification. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 1.0 Hz, 1H), 7.64−7.04 (m, 8H), 4.33 (dd, J = 10.0, 7.5 Hz, 1H), 4.17 (d, J = 8.1 Hz, 1H), 3.91 (q, J = 8.8 Hz, 1H), 3.78−3.69 (m, 1H), 3.26−2.93 (m, 2H), 2.28 (dtd, J = 12.9, 9.0, 7.4 Hz, 1H), 1.83 (ddt, J = 13.1, 7.1, 4.0 Hz, 1H); MS (DCI) m/z 337 [M + H]+. Step 11. To a 1 L round-bottom flask with (3aS,6aS)-5-[1-(2fluorophenyl)-1H-indazol-4-yl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)one (54 from step 10, 21.0 g, 62.6 mmol) in N,N-dimethylformamide (100 mL), 3-hydroxy-3-methylbutanoic acid (9.46 mL, 75 mmol), and triethylamine (9.60 mL, 68.9 mmol) was added a N,N-dimethylformamide (25 mL) solution of (dimethylamino)-N,N-dimethyl(3-oxido1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)methaniminium hexafluorophosphate (HATU, 26.2 g, 68.9 mmol). The reaction mixture was allowed

the addition. The reaction was then allowed to warm to ambient temperature over 30 min and was stirred for 10 min. The reaction was then cooled to 0 °C, and iodine (16.2 g, 63.7 mmol, in 320 mL diethyl ether) was added dropwise with the internal temperature being maintained below 10 °C (25−30 min for addition). After the addition was complete, the mixture was allowed to warm to ambient temperature and was stirred for 1 h. The mixture was diluted with diethyl ether (250 mL) and washed with saturated aqueous Na2S2O3 (50 mL) and saturated aqueous NH4Cl (50 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to provide methyl (3R)-3-(iodomethyl)-1-[(1S)-1-phenylethyl]-Lprolinate (21.95 g, 58.8 mmol, 96% yield) which was carried on without further purification. 1H NMR (500 MHz, DMSO-d6) δ 7.38− 7.12 (m, 5H), 3.67−3.54 (m, 4H), 3.37 (d, J = 7.6 Hz, 1H), 3.15−2.98 (m, 2H), 2.96−2.86 (m, 2H), 2.65 (dq, J = 10.5, 7.8 Hz, 1H), 2.19− 2.01 (m, 1H), 1.66−1.47 (m, 1H), 1.23 (d, J = 6.6 Hz, 3H); MS (DCI) m/z 374 [M + H]+. Step 5. To a 500 mL round-bottom flask containing the methyl (3R)-3-(iodomethyl)-1-[(1S)-1-phenylethyl]-L-prolinate (from step 4, 12.0 g, 32.2 mmol) were added N,N-dimethylformamide (120 mL) and sodium azide (4.18 g, 64.3 mmol). The reaction mixture was warmed to 50 °C and was stirred for 2 h. The mixture was diluted with ethyl acetate (500 mL) and washed with brine (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give methyl (3S)-3-(azidomethyl)-1-[(1S)-1-phenylethyl]-Lprolinate, 51 (9.81 g, 34 mmol, >100% yield) which was used without purification. 1H NMR (500 MHz, CDCl3) δ 7.42−7.07 (m, 5H), 3.71 (q, J = 6.6 Hz, 1H), 3.65 (s, 3H), 3.43 (d, J = 8.1 Hz, 1H), 3.33−3.13 (m, 2H), 3.07 (td, J = 9.1, 3.3 Hz, 1H), 2.91 (q, J = 7.9 Hz, 1H), 2.68− 2.48 (m, 1H), 2.03 (dtd, J = 11.4, 7.8, 3.2 Hz, 1H), 1.72 (dtd, J = 12.4, 9.7, 7.5 Hz, 1H), 1.36 (d, J = 6.6 Hz, 3H); MS (DCI) m/z 275 [M + H]+. Step 6. To a 500 mL round-bottom flask were added methyl (3S)-3(azidomethyl)-1-[(1S)-1-phenylethyl]-L-prolinate (51 from step 5, 16.7 g, 58.0 mmol), 2-methyl THF (160 mL), and water (160 mL). Triphenylphosphine (23 g, 87 mmol) was added, and the reaction mixture was warmed to 74 °C and was allowed to stir for 90 min. The reaction was allowed to cool to ambient temperature and was transferred to a separatory funnel. The mixture was extracted with ethyl acetate (2 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, eluting with 0−100% ethyl acetate/heptane over 30 min with a 20 min hold and then ramped to 100% ethyl acetate with 10% methanol) to give (3aS,6aS)-1-[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one (9.46 g, 41.1 mmol, 71% yield). 1H NMR (400 MHz, CDCl3) δ 7.43−7.17 (m, 5H), 6.10 (s, 1H), 4.15 (dq, J = 21.4, 7.0 Hz, 1H), 3.46 (dd, J = 9.8, 7.6 Hz, 1H), 3.36 (d, J = 8.9 Hz, 1H), 3.12 (ddd, J = 9.9, 2.1, 1.1 Hz, 1H), 2.92− 2.71 (m, 1H), 2.55 (td, J = 9.0, 6.3 Hz, 1H), 2.16−1.98 (m,1H), 1.65 (ddt, J = 12.5, 8.9, 7.2 Hz, 1H), 1.51 (d, J = 6.8 Hz, 4H); MS (DCI) m/z 231 [M + H]+. Step 7. To a 500 mL round-bottom flask were added (3aS,6aS)-1[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one (from step 6, 9.46 g, 41.1 mmol), N,N-dimethylformamide (100 mL), di-tertbutyl dicarbonate (10.76 g, 49.3 mmol), and N,N-diisopropylethylamine (14.21 mL, 82 mmol), and the reaction mixture was stirred at ambient temperature. The reaction was proceeding slowly so a catalytic amount of 4-(dimethylamino)pyridine (100 mg) was added, and the reaction was allowed to stir for 16 h. The mixture was poured into CH2Cl2 (200 mL) and was washed with 1 N HCl (20 mL), saturated NaHCO3 (20 mL), and brine (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, eluted with cyclohexane/0−20% THF over 60 min with 60 min hold) to afford tert-butyl (3aS,6aS)-6-oxo-1-[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (9.69 g, 29.3 mmol, 71% yield) and a small amount of the isomer, tert-butyl (3aR,6aR)-6-oxo-1[(1S)-1-phenylethyl]hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.314 g, 1.0 mmol, 2% yield). 1H NMR (400 MHz, CDCl3) δ 7.51−6.91 (m, 5H), 4.21 (q, J = 6.8 Hz, 1H), 3.71 (dd, J = 11.1, 7.8 3389



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to stir for 30 min and then was poured into ethyl acetate (500 mL) and transferred to a separatory funnel. The material was washed with water (100 mL) and brine (100 mL), and the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, eluted with 0−100% EtOAc/heptanes) to provide a 98:2 mixture of enantiomers as assessed by chiral supercritical fluid chromatography analysis using a Chiralcel OD-H column. The material was further purified on a preparative Chiralcel OD-H column eluting with 30% methanol/CO2 at 80 mL/min to give (3aS,6aS)-5-[1-(2-fluorophenyl)-1H-indazol-4-yl]-1-(3-hydroxy-3-methylbutanoyl)hexahydropyrrolo[3,4-b]pyrrol-6(1H)-one, 55 (17.01 g, 39 mmol, 62% yield). [α]D20 −119.20° (c 0.25, methanol); 1H NMR (500 MHz, DMSO-d6) δ 8.35 (d, J = 1.0 Hz, 1H), 7.69 (td, J = 7.9, 1.8 Hz, 1H), 7.65−7.41 (m, 4H), 7.37−7.22 (m, 2H), 5.12 (d, J = 8.0 Hz, 1H), 4.96 (s, 1H), 4.30 (ddd, J = 9.9, 6.4, 3.6 Hz, 1H), 3.78−3.45 (m, 3H), 3.27−3.14 (m, 1H), 2.82 (d, J = 15.1 Hz, 1H), 2.72 (d, J = 15.0 Hz, 1H), 2.40− 2.19 (m, 1H), 1.87 (dq, J = 13.0, 9.0 Hz, 1H), 1.22 (d, J = 11.8 Hz, 6H); MS (DCI) m/z 437 [M + H]+.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00063. Experimental procedures for all biological experiments and compound purity data (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 847-937-0721. Notes

The authors declare the following competing financial interest(s): All authors are employees, former employees, or retirees of AbbVie. This study was sponsored by AbbVie. AbbVie contributed to the study design, research, interpretation of data, writing, reviewing, and approving the publication.

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ACKNOWLEDGMENTS The authors acknowledge Xiangdong Xu, Kelly Desino, Stella Z. Doktor, Anthony Lee, Hong Liu, AbbVie HT-ADME group. ABBREVIATIONS USED CIP, congenital insensitivity to pain; Nav1.7, voltage-gated sodium channel type 7; SCN9A, sodium channel, voltage-gated, type IX, R subunit; SAR, structure−activity relationship; FRET, fluorescence resonance energy transfer; MIA-OA, monoiodoacetate-induced osteoarthritis model; HEK, human embryonic kidney; MDCK, Madin−Darby canine kidney; CNS, central nervous system; PK, pharmacokinetic; Clint,u, in vitro intrinsic clearance, unbound; F, oral bioavailability; CDI, carbonyldiimidazole; TFA, trifluoroacetic acid; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; Hunig’s base, N,Ndiisopropylethylamine; MsCl, methanesulfonyl chloride; DMAP, 4-dimethylaminopyridine; LDA, lithium diisopropylamide; DMSO, dimethyl sulfoxide

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REFERENCES

(1) Cummins, T. R.; Rush, A. M. Voltage-gated sodium channel blockers for the treatment of neuropathic pain. Expert Rev. Neurother. 2007, 7, 1597−1612. 3390



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