Substituted Indazoles as Nav1. 7 Blockers for the Treatment of Pain

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Article V

Substituted Indazoles as Na 1.7 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 R Neelands, Mark A Matulenko, Jerome F Daanen, Madhavi V Pai, Nayereh Ghoreishi-Haack, Cenchen Zhan, Xu-Feng Zhang, and Michael E. Kort J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00063 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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Journal of Medicinal Chemistry

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 †

† AbbVie,

Research and Development, 1 North Waukegan Road, North Chicago, IL 60064, USA

††Aptinyx,

1801 Maple Ave. #4300 Evanston, IL 60201

ACS Paragon Plus Environment


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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 drug-like 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 mono-iodoacetate-induced osteoarthritis model of pain and sub-chronic dosing of 48 showed a shift to a lower EC50 over seven days.

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-of-function 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-of-function SCN9A mutations can result in painful conditions such as inherited erythromelalgia and paroxysmal extreme pain disorder.7,8 Furthermore, knock-out 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, non-selective sodium channel blockers approved


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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 towards 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. 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 drug-like 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). We then focused on 4substituted indazole analogs which exhibited improved selectivity versus Nav1.5 and improved pharmacokinetic properties. Select compounds were interrogated in the rat mono-iodoacetate-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 osteoarthritis pain is supported by the reported knockout studies and additional literature.9,19,20 Figure 1. General Structures of 3 and 4-substituted indazoles

Chemistry



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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 carbonyl dimidazole (CDI) and Hünig’s base to give the intermediate hydrazide which when treated with NaNO2 and HCl at 76 °C gave 3-hydroxy-indazole 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-imidazol2-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. Scheme 1. Preparation of 3-Hydroxyindazoles


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The 3-hydroxy-indazole 3 was also subjected to Mitsunobu coupling conditions21 in the presence of commercially available 1-(2-hydroxyethyl)-2-imidazolidinone or 1-(2-hydroxyethyl)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

1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU) to generate oxazolidinone 12. Scheme 2. Preparation of 3-Hydroxyindazoles 15 and 16

Analogs 15 and 16 were obtained as shown in Scheme 2. The 3-hydroxy-indazole 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)-2imidazolidinone and removal of the Boc group resulted in intermediate 14. Buchwald coupling22,23 of 14 with 2,4-difluoro-1-iodobenzene 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 palladium on carbon to give 18. Alkylation of 18 with t-butyl (2-bromoethyl)carbamate and NaH resulted in Boc-amine 19. Scheme 3. Preparation of 4-Substituted Indazole 19



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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-2-pyrrolidinone at 140 °C to give intermediate 21. Aryl bromide 21 then underwent Pd-catalyzed coupling with imidazolidin-2-one 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 Boc-protected giving 23 and was N-arylated with 4-iodopyridine in the presence of CuI to give analog 35. Intermediate 22 was also treated with t-butylbromoacetate and NaH and the resulting t-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 Indazoles


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F

N

F O

+

F

f N

2

N

O

30

22

21

O N

NH

Br 20

N

b

N

H2N NH

Br

F

N

a

F

N

N

O OH

c

g e

d NH2

24: R1 =

N

25: R =

N O

N

O

F

N

NR1

F

N N

N N

O

O

O N

N

N

F

O

28: R =

N

F

29: R =

N

OH

31: R =

N

N

NR2

O 23

N

O

O

N 34: R1 =

N

27: R =

N N

O

F

N

32: R1 =

33: R1 =

H N

N

O 26: R1 =

F

N 35

F

N (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, CuI, trans-N1,N2-dimethylcyclohexane-1,2-diamine, K3PO4, 1,4-dioxane, 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

Piperazinone analog 38 was prepared as described in Scheme 5. Coupling of piperazin-2-one with bromo-indazole intermediate 21 using Pd2(dba)3 and Nolan’s catalyst gave 36. Alkylation with ethyl 2iodoacetate 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. Scheme 5. Preparation of 4-Substituted Indazole 38



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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. Scheme 6. Preparation of 4-Substituted Indazole 42

H N O

N O

+

N

O

N

O

a

b N

Si

O

O

N Ph

39

F

N

40

N

41 Ph 42

(a) (i) trifluoroacetic acid (cat), CH2Cl2, 86% yield; (ii) trifluoroacetic acid (8 eq), CH2Cl2, 73% yield (b) 21, CuI, trans-N1,N2-dimethylcyclohexane-1,2-diamine,K3PO4, 1,4-dioxane, 83% yield

The chiral bicycle 48 was also prepared starting with 39 and using commercially available αmethylbenzyl 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


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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. Scheme 7. Preparation of 4-Substituted Indazole 48

N O

(R)

N O

N

H

O H b

N

N

Si

39

H

a

+ O

N

O

H

O

O

O

O

O

43

44

45

F

N

F

N

N

F

N

N

N N H

O

N

H N

H

O

H

O H

H N H

46

N

d

c

N

OH

O

47

48

(a) trifluoroacetic acid, CH2Cl2, 40% yield 45 separated from ~1:1 mixture of 44 and 45; (b) (i) TFA, CH2Cl2, quantitative yield; (ii) 21, CuI, 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

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 αmethylbenzyl group shown. But-3-en-1-ol (49) was treated with MsCl to give the intermediate mesylate which then underwent displacement with (S)-1-phenylethanamine followed by alkylation with methyl bromoacetate to give 50. Sequential treatment of 50 with lithium diisopropyl amide and zinc bromide followed by iodine gave the cyclized iodo-intermediate which was reacted with sodium azide to give 51.



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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 which underwent hydrogenation to provide amine 54. Amide coupling with 3-hydroxy-3-methylbutanoic acid resulted in formation of desired analog 55. Scheme 8. Preparation of 4-Substituted Indazole 55 N3

H

b

a

c

N

OH

CO2Me

H Ph

Ph

Ph 49

50

51

N

e

H O Ph

N

N f N

N F

N O

H H NH

O

H H N O

53

F

N

F

N N

O

52

H N

N

N

(S)

d NH

CO2Me

54

OH

55

(a) (i) MsCl, Et3N, CH2Cl2, 92% yield; (ii) (S)-1-phenylethanamine, CH3CN, 85 °C, 20% yield; (iii) methyl bromoacetate, iPr2EtN, DMSO, 85% yield; (b) (i) LDA, Et2O, -78 °C; ZnBr2, Et2O, -74 °C to r.t.; iodine, Et2O, 0 °C, 96% yield; (ii) NaN3, DMF, 50 °C, quantitative yield; (c) (i) Ph3P, 1:1 MeTHF/H 2O, 74 °C; (ii) Boc2O, DMAP (cat), i-Pr2EtN, DMF; minor isomer separated out; 71% yield; (iii) TFA, CH2Cl2, 99% yield; (d) 21, CuI, 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-3methylbutanoic acid, HATU, Et3N, DMF, 62% yield.


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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.18 Virtually no activity was observed at the resting state (-100 mV) for all analogs and, as such, all were determined to be statedependent. 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 mono-iodoacetate-induced osteoarthritis model (MIA-OA, grip force endpoint) wherein an intraarticular injection of mono-iodoacetate causes osteoarthritis-like knee joint lesions. Analogs were then administered orally and the rats were tested for grip force deficit of hind limbs.26 Results and Discussion Our work began with the 3-hydroxy indazole 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



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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, 4h 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, 4h in plasma) but poor metabolic stability (hClint,u = 19 L/h/Kg). 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/Kg). Rat PK studies demonstrated that 7 had similar bioavailability (F = 17%) to 4 (F = 22%) but was stable in plasma (>80% remaining, 4h in plasma) although 7 did exhibit a short half-life 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 (vida 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 = 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.


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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). Table 1: In Vitro Activity of 3-Hydroxy Indazoles EP hNav1.7 hNav1.5 hCl,int,ud rCl,int,u IC50, -65 IC50c (µM) (L/h/Kg) (L/h/Kg) IC50a (µM) mVb (µM) FRET hNav1.7

R1

%F

t1/2 (h)

R2 tetracaine

0.056 ± 0.002

0.32 0.65 ± 0.3 (manual)

4

2-F

0.76 ± 0.05

1.4

5

2-F

0.11 ± 0.009

0.62

7

2-F

1.9 ± 0.16

2.7

8

2-F

0.96 ± 0.32

9

2-F

1.1 ± 0.14

7

10

2-F

0.32 ± 0.002

1.2

12

2-F

2.6 ± 0.09

15

2,4-F

2 ± 0.30

12

1 ± 0.24

7

11

22

1.6

19

43

27

2

2.8 ± 1.0

11

83

17

0.4

5.1 ± 1.2

12

47

35

0.4

2 ± 0.04

9

69

28

1.1

60

432

41

149

9

37

71

0.94

3.4 ± 0.92

a

Nav1.7 IC50 values were determined by least-squares fitting of a logistic equation to data from full eightpoint, half-log concentration–response curves using a FRET-membrane potential assay as described in the Experimental Section. Data is 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.



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With consistently poor metabolic stability for the 3-substituted indazole series and narrow SAR, adequate progress was not made towards 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/Kg) and rat liver microsomes (hClint,u = 513 L/h/Kg). 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 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 (vida infra).

It was also noted that the corresponding (R)-3-

fluoropyrrolidine 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)-3-fluoropiperidine 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.


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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 knock-out 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 P-gp mediated efflux. We further noted that the P-gp efflux ratio (ratio ≥ 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 P-gp 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, methyl pyrazine 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,



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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 (vida 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.


Page 17 of 59

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


Table 2: In Vitro Activity of 4-Hydroxy Indazoles

F

N N

EP FRET hNav1.7 hNav1.5 hCl,int,ud hNav1.7 IC50, -65 IC50c (µM) (L/h/Kg) a IC50 (µM) mVb (µM)

rCl,int,u (L/h/Kg)

%F

t1/2 (h)

P-gp efflux ratio

([plasma]free/[ brain]free)

0.98

1.6

BI

R

tetracaine

0.056 ± 0.002

0.32 0.65 ± 0.3 (manual)

19

0.58 ± 0.010

23

0.73 ± 0.090

2.3

24

0.84 ± 0.06

25

43

513

5 ± 1.6

5.9

7.2

100

2

22

13.6

2.5

19*

8.2

11.8

55

0.6

11.4

10.9

0.92 ± 0.054

1.1

>33

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,


Page 53 of 59

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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-(2-fluorophenyl)-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-oxido-1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)methaniminium

hexafluorophosphate (HATU, 26.2 g, 68.9 mmol). The reaction mixture was allowed 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]+. Abbreviations: 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: mono-iodoacetate-induced osteoarthritis model; HEK: human embryonic kidney; MDCK: Madin-Darby Canine Kidney; CNS: central nervous



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system; PK, pharmacokinetic; Clint,u: in vitro intrinsic clearance, unbound; F: oral bioavailability; CDI: carbonyl diimidazole; TFA: trifluoroacetic acid; HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate); Hunig’s Base: N,N-diisopropylethylamine; MsCl: methanesulfonyl chloride; DMAP: 4-dimethylaminopyridine; LDA: lithium diisopropylamide; DMSO: dimethyl sulfoxide. Acknowledgements:

Xiangdong Xu, Kelly Desino, Stella Z. Doktor, Anthony Lee, Hong Liu,

AbbVie HT-ADME group. Disclosures: All authors are employees, former employees, or retirees of AbbVie. This study was sponsored by AbbVie. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the publication. Supporting Information Available: Experimental procedures for all biological experiments and compound purity data are available in the Supporting Information.

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. 2 Rush, A. M.; Cummins, T. R.; Waxman, S. G. Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons. J. Physiol. 2007, 579, 1-14. 3 Dabby, R. Pain disorders and erythromelalgia caused by voltage-gated sodium channel mutations. Curr. Neurol. Neurosci. Rep. 2012, 12, 76-83. 4 Dib-Hajj, S. D., Cummins, T. R.; Black, J. A.; Waxman, S. G. From genes to pain: Nav1.7 and human pain disorders. Trends Neurosci. 2007, 30, 555-563.


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5 Waxman, S. G. Neuroscience: Channelopathies have many faces. Nature 2011, 472, 173-174. 6 Zufall, F.; Pyrski, M.; Weiss, J.; Leinders-Zufall, T. Link between pain and olfaction in an inherited sodium channelopathy. Arch. Neurol. 2012, 1-5. 7 Dib-Hajj, S. D.; Yang, Y.; Black, J. A.; Waxman, S. G. The Na(V)1.7 sodium channel: from molecule to man. Nat. Rev. Neurosci. 2013, 14, 49-62. 8 Waxman, S. G. “Neuroscience: Channelopathies have many faces. Nature 2011, 472, 173-174. 9 Minett, M. S.; Nassar, M. A.; Clark, A. K.; Passmore, G.; Dickenson, A. H.; Wang, F.; Malcangio, M.; Wood, J. N. Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons. Nat Commun. 2012, 3, 791. 10 Dworkin, R. H.; O’Connor, A. B.; Audette, J.; Baron, R.; Gourlay, G. K.; Haanpaa, M. L.; Kent, J. L.; Krane, E. J.; Lebel, A. A.; Levy, R. M.; Mackey, S. C.; Mayer, J.; Miaskowski, C.; Raja. S. N.; Rice, A. S.; Schmader, K. E.; Stacey, B.; Stanos, S.; Treede, R. D.; Turk, D. C.; Walco, G. A.; Wells, C. D. Recommendations for the pharmacological management of neuropathic pain: an overview and literature update. Mayo Clin. Proc. 2010, 85, S3-14. 11 Goldberg, Y. P.; Price, N.; Namdari, R.; Cohen, C. J.; Lamers, M. H.; Winters, C.; Price, J.; Yound, C. E.; Verschoof, H.; Sherrington, R.; Pimstone, S. N.; Hayden, M. R. Treatment of Na(v)1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker. Pain 2012, 153, 80-85. 12 Focken, T.; Liu, S.; Chahal, N.; Dauphinais, M.; Grimwood, M. E.; Chowdhury, S.; Hemeon, I.; Bichler, P.; Bogucki, D.; Waldbrook, M.; Bankar, G.; Sojo, L. E.; Young, C.; Lin, S.; Shuart, N.; Kwan, R.; Pang, J.; Chang, J. H.; Safina, B. S.; Sutherlin, D. P.; Johnson, J. P. Jr.; Dehnhardt, C. M.; Mansour,



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T. S.; Oballa, R. M.; Cohen, C. J.; Robinette, C. L. Discovery of aryl sulfonamides as isoform-selective inhibitors of Nav1.7 with efficacy in rodent pain models. Med. Chem. Lett. 2016, 7, 277-282. 13 de Lera Ruiz, M.; Kraus, R. L. Voltage-gated sodium channels: structure, function, pharmacology, and clinical indications. J. Med. Chem. 2015, 58, 7093−7118. 14 Beaudoin, S.; Laufersweiler, M. C.; Markworth, C. J.; Marron, B. E.; Millan, D. S.; Rawson, D. J.; Reister, S. M.; Sasaki, K.; Storer, R. I.; Stupple, P. A.; Swain, N. A.; West, C. W.; Zhou, S. Sulfonamide derivatives. WO2010079443A1, US8907101B2, 2010. 15 Convergence Pharmaceuticals, CNV1014802: http://www.convergencepharma.com/ (accessed March, 8, 2016); http://clinicaltrials.gov/ct2/results?term=CNV1014802 (accessed March, 8, 2016). 16 Bagal, S. K.; Chapman, M. L.; Marron, B. E.; Prime, R.; Storer, R. I.; Swain, N. A. Recent progress in sodium channel modulators for pain. Bioorg. Med. Chem. Lett. 2014, 24, 3690-3699. 17 Gonzales, J. E.; Termin, A. P.; Wilson, D. M. Small molecule blockers of voltage-gated sodium channels. Methods and principles in medicinal chemistry. In Voltage-gated Ion Channels as Drug Targets; Triggle, D. J., Gopalakrishnan, M.; Rampe, D.; Zheng, W. Eds.; Wiley-VCH: Weinheim, 2006, pp 168-192. 18 Nardi, A.; Damann, N.; Hertrampf, T.; Kless, A. Advances in targeting voltage-gated sodium channels with small molecules. ChemMedChem 2012, 7, 1712-1740. 19 Thakur, M.; Dawes, J. M.; McMahon, S. B. Genomics of pain in osteoarthritis. Osteoarthritis Cartilage 2013, 21, 1374-1382.


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20 Staunton, C. A.; Lewis, R.; Barrett-Jolley, R. Ion channels and osteoarthritic pain: potential for novel analgesics. Curr. Pain Headache Rep. 2013, 17, article 398, 1-9. 21 Tsunoda, T.; Yamamiya, Y.; Ito, S. 1,1′-(azodicarbonyl)dipiperidine-tributylphosphine, a new reagent system for mitsunobu reaction. Tetrahedron Lett. 1993, 34, 1639–1642. 22 Kwong, F. Y.; Klapars, A.; Buchwald, S. L. Copper-catalyzed coupling of alkylamines and aryl iodides: an efficient system even in an air atmosphere. Org. Lett. 2002, 4, 581-584. 23 Jiang, L., Job, G. E, Klapars, A., Buchwald, S. L. Copper-catalyzed coupling of amides and carbamates with vinyl halides. Org. Lett. 2003, 5, 3667-3669. 24 Chang, L. L.; Yang, G. X.; McCauley, E.; Mumford, R. A.; Schmidt, J. A.; Hagmann, W. K. Constraining the amide bond in N-sulfonylated dipeptide VLA-4 antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 1688-1691. 25 Harmer, A. R.; Abi-Gerges, N.; Easter, A.; Woods, A.; Lawrence, C. L.; Small, B. G.; Valentin, J.-P.; Pollard, C. E. Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks™. J. Pharmacol. Toxicol. Methods 2008, 57, 30-41. 26 Chandran, P.; Pai, M.; Blomme E. A.; Hsieh, G. C.; Decker, M. W.; Honore, P. Pharmacological modulation of movement-evoked pain in a rat model of osteoarthritis. Eur. J. Pharmacol. 2009, 613, 39-45. 27 Magano, J.; Waldo, M.; Greene, D.; Nord, E. The synthesis of (S)-5-fluoro-1-(2-fluorophenyl)-3(piperidin-3-ylmethoxy)-1H-indazole, a norephinephrine/serotonin reuptake inhibitor for the treatment of fibromyalgia. Org. Process Res. Dev. 2008, 12, 877-883.



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28 Lynch, S. M.; Tafesse, L.; Carlin, K.; Ghatak, P.; Shao, B.; Abdelhamin, H.; Kyle, D. J.; N-Aryl azacycles as novel sodium channel blockers. Biorg. Med. Chem. Lett. 2015, 25, 48-52.


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Table of Contents Graphics

Oral efficay in rat MIA-OA pain model with acute and subchronic dosingl N N

F

***

10 0

H

O H

N

OH

O

***

***

60 40

***

***

80 N

% E ffec t

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


***

20 0

Na v1.7 IC50 (manual) Inactivated: 0.34 µM

0.3

1

3

10

3 0 Diclo 30

48 (mg/kg, p.o.)


Substituted Indazoles as Nav1. 7 Blockers for the Treatment of Pain

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