Identification of selective acyl sulfonamide-cycloalkylether inhibitors of

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Identification of selective acyl sulfonamide-cycloalkylether inhibitors of the voltage gated sodium channel (NaV) 1.7 with potent analgesic activity Shaoyi Sun, Qi Jia, Alla Y Zenova, Michael S Wilson, Sultan Chowdhury, Thilo Focken, Jun Li, Shannon Decker, Michael E Grimwood, Jean-Christophe Andrez, Ivan Hemeon, Tao Sheng, Chien-An Chen, Andy White, David H. Hackos, Lunbin Deng, Girish Bankar, Kuldip Khakh, Elaine Chang, Rainbow Kwan, Sophia Lin, Karen Nelkenbrecher, Benjamin D. Sellers, Antonio G DiPasquale, Jae Chang, Jodie Pang, Luis Sojo, Andrea Lindgren, Matthew Waldbrook, Zhiwei Xie, Clint Young, James P Johnson, C. Lee Robinette, Charles J Cohen, Brian Salvatore Safina, Daniel P. Sutherlin, Daniel Fred Ortwine, and Christoph M Dehnhardt J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01621 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Identification of selective acyl sulfonamidecycloalkylether inhibitors of the voltage gated sodium channel (NaV) 1.7 with potent analgesic activity Shaoyi Sun,┼ Qi Jia,┼ Alla Y. Zenova,┼ Michael S. Wilson,┼ Sultan Chowdhury, ┼,  Thilo Focken,┼ Jun Li,╪ Shannon Decker,┼ Michael E. Grimwood,┼ Jean-Christophe Andrez, ┼ Ivan Hemeon,┼ Tao Sheng, ┼, Chien-An Chen, #, Andy White, #,○ David H. Hackos,╪ Lunbin Deng, ╪ Girish Bankar,┼ Kuldip Khakh,┼ Elaine Chang,┼ Rainbow Kwan, ┼ Sophia Lin,┼ Karen Nelkenbrecher,┼ Benjamin D. Sellers,╪ Antonio G. DiPasquale,╪ Jae Chang,╪ Jodie Pang,╪ Luis Sojo,┼ Andrea Lindgren,┼ Matthew Waldbrook,┼ Zhiwei Xie,┼ Clint Young,┼ James P. Johnson, ┼

C. Lee Robinette,┼ Charles J. Cohen,┼ Brian S. Safina,╪ Daniel P. Sutherlin,╪ Daniel F.

Ortwine *,╪ and Christoph M. Dehnhardt *,┼ ┼Xenon

Pharmaceuticals Inc, 200-3650 Gilmore Way, Burnaby, BC, V5G 4W8 Canada;

╪Genentech

Inc, 1 DNA Way, South San Francisco, California 94080-4990, USA; #Chempartner,

Building No. 5, 998 Halei Rd., Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai, P. R. China.


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KEYWORDS Voltage-gated sodium channel, SCN9A, NaV1.7, NaV1.5, pain, inherited erythromelalgia, formalin test.

ABSTRACT

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes (HLM). Further design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability utilizing induced fit docking in our previously disclosed X-ray co-crystal of the NaV 1.7 voltage sensing domain (VSD). These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.

INTRODUCTION Drugs that block voltage-gated sodium channels (NaVs) are widely used therapeutically as antiarrhythmics, anticonvulsants, anesthetics, and analgesics. Non-subtype selective sodium channel blockers such as mexiletine and carbamazepine have been used for treatment of neuropathic pain


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conditions. Unfortunately, this class of drugs is known to exhibit dose-limiting side effects in patients, such as somnolence, dizziness, and nausea, that limit their utility as analgesic agents.1, 2 These adverse effects are thought to be derived in large part from a lack of selectivity on the NaV family of channels. Nine NaV isoforms are known (NaV1.1–NaV1.9), and there are indications3 that isoform selective blockers may have an improved therapeutic index compared to the nonselective compounds presently in use. In particular, NaV1.7 is a highly validated target for the treatment of pain conditions based upon human genetic studies.4 Congenital insensitivity to pain (CIP), caused by a genetic deficiency of the SCN9A gene encoding NaV1.7, is a rare autosomal recessive disorder characterized by the complete absence of pain sensation typically associated with noxious stimuli.5–10 Individuals with CIP show no deficits in motor or cognitive function.11 Conversely, gain of function mutations in SCN9A result in painful conditions, including inherited erythromelalgia (IEM),12–15 paroxysmal extreme pain disorder (PEPD)16 and small fiber neuropathies.17 These findings indicate that NaV1.7 has an essential role in mediating pain signaling in humans and suggests that selective NaV1.7 antagonism may be a highly effective strategy to treat painful conditions.4 NaV1.5 is the predominant sodium channel in cardiac myocytes and has been identified as an important off-target to avoid in NaV1.7 drug discovery.

Inhibition of NaV1.5 has been

demonstrated to lead to cardiac arrhythmias.18 As a consequence, our optimization campaign focused on establishing selectivity for NaV1.7 over NaV1.5. Due to a high sequence homology among NaV isoforms, compounds that are highly specific for NaV1.7 have been difficult to identify despite considerable efforts by the pharmaceutical industry. Recently, subtype selective small molecule NaV1.7 inhibitors have been reported.19–25 An example is aryl sulfonamide 1 (PF05089771) (Figure 1), which selectively (>600 fold) blocks NaV1.7 over NaV1.5, and was


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advanced into clinical trials.20a,b,d Our earlier work described the discovery of two structurally distinct series of aryl sulfonamide NaV1.7 inhibitors exemplified by 221a and 3,21b as well as the X-ray structure of the binding site on hNaV1.7.26 We also recently disclosed a novel class of triazolyl sulfonamide NaV1.7 inhibitors, exemplified by 4.21c F F

H N

O

Cl

S

O

Cl

N

F

H N

O S

O

S

O

N N

H 2N

S N

O F H 2N

N H

1 (PF-05089771)

2 O

N N

N

O N S H O

O N H

N

S

N Cl

N O

3

F

O

O S O

N

N

OMe

O F 5 (PF-05241328)

O

N H

F

O 4

N

O

F

HN N

O

S

F

O Cl

H N

O

Cl

Cl 6

Figure 1. Structures of selected subtype-selective NaV1.7 inhibitors. Although potent on NaV1.7 and highly selective against NaV1.5, aryl sulfonamides such as 321b displayed poor ADME properties. Therefore, we sought to identify alternative chemical series to mitigate these liabilities. A literature survey on selective NaV1.7 inhibitors revealed a primary focus on aryl sulfonamides, with only a single paper on a companion series of acyl sulfonamides.22a This report did not contain a characterization of the analogues in in vivo models of pain. We and others21b,22a have found that for many series, large multiples of the NaV1.7 IC50 values are needed to achieve acceptable efficacy in in vivo models. We recently reported in vivo studies on a class of acyl sulfonamides37 that showed efficacy at low NaV1.7 IC50 multiples. However, the design and optimization of compounds that are highly active in in vivo assays has so far been elusive, which prompted the current study on acyl sulfonamide optimization that focused on improving in vivo efficacy.


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An analysis of the patent landscape through 2013 for aryl and acyl sulfonamide NaV1.7 inhibitors27 guided our start of the program. We noticed that acyl sulfonamides occupy a more favorable physicochemical property space such as lower molecular weight (MW) and clogP (Figure 2). For example, aryl sulfonamides displayed an average clogP of 4.1 and average MW of 490, while acyl sulfonamides showed an average clogP of 3.0 and an average MW of 400. Given that optimization very often involves increases in molecular weight and clogP, acyl sulfonamides represented an attractive series for progression within Lipinski property space which was an important criterion for us at the start of the program.28 Additionally, acyl sulfonamides represented a less explored chemical series compared to aryl sulfonamides,20a,22a providing an opportunity to deepen our understanding of this class of compounds. Known acyl sulfonamides such as 5 (PF-05241328)20a or 622a have a highly planar architecture due to the presence of multiple aromatic rings. The number of aromatic rings in a molecule has been shown to negatively affect aqueous solubility, and has been associated with an increased attrition risk in drug development,29 suggesting a need for the identification of acyl sulfonamides with improved drug-like properties. Our strategy to increase favorable properties focused partly on increasing fsp3 (fraction of sp3 carbons relative to the total carbon count).30 These efforts culminated in 33, a potent, molecularly selective and metabolically stable NaV1.7 inhibitor that demonstrated efficacy in various models of pain.


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Figure 2. Acyl sulfonamides generally occupy a more favorable physiochemical property space (average cLogP of 3.0 versus 4.1 and MW of 400 versus 490 for acyl versus aryl sulfonamides) for achieving oral bioavailability. RESULTS AND DISCUSSION Compounds were evaluated in vitro in a radioligand binding assay (LBA) that measured the displacement of aryl sulfonamide [3H]GX–545 from HEK membranes that expressed hNaV1.7 including β1.26 Effects in cells were assessed by whole cell patch voltage clamp electrophysiology measurements (EP) of sodium currents mediated by human hNaV1.7 and hNaV1.5 heterologously expressed in HEK293 cells.26 The LBA assay was used as a high throughput assay to guide design and to establish binding to a preferred site in voltage sensing domain 4 (VSD4) of NaV1.7.26 The functional potency data obtained by EP is biologically more relevant as it tracks the movement of the channel in a cell but is associated with a larger measurement error. 31 Given these assay behaviors, we were most interested in compounds that showed high potencies in both assays.


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Acyl sulfonamide 7,21c,32 possessing a pendant adamantyl group, was discovered in early efforts to explore ether linked saturated rings. This compound inhibited hNaV1.7 with an IC50 of 0.07 M in the LBA (Table 1). A voltage–clamp assay confirmed the ligand binding result (IC50 = 0.006 M) and indicated that 7 was highly selective against hNaV1.5 (IC50 = 1.9 M, NaV1.5/NaV1.7 = 340-fold). Further profiling showed that 7 had good permeability, moderate solubility (31 M), no significant CYP inhibition for isoforms tested (> 10 M for CYPs 3A4, 1A2, 2C19, 2C9 and 2D6), and no hERG liability (IC50> 10 M). However, 7 showed moderate and poor stabilities in mouse and human liver microsomes (MLM = 32ml/min/Kg, HLM = 19ml/min/kg), respectively. Initial in vivo pharmacokinetic (PK) evaluation in mouse showed good oral bioavailability and exposure. The observed exposure, despite moderate mouse liver microsomal stability, was most likely a result of high plasma protein binding (> 99.9% bound) that restricted the intrinsic clearance of the molecule. We were cognizant of the fact that high plasma protein binding would require exquisite potency to achieve efficacy in vivo without requiring very high exposure. In addition, optimization of metabolic stability is particularly important for these highly bound analogues as their low volume of distribution requires good metabolic stability to achieve a half-life suitable for BID dosing. Hence, our optimization focused particularly on increasing potency and HLM stability. Given its potency, selectivity, and biopharmaceutical properties, 7 represented a good starting point for a lead optimization campaign.


7

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Table 1. In vitro activity and pharmacokinetic properties of compound 7.

[3H]GX-545 LBA IC50 (M)

0.07

hNaV1.7 EP IC50 (M)

0.006

hNaV1.5 EP IC50 (M)/ sel. h1.5/1.7

1.9/340

LM CLhep (ml/min/kg) 19 / 28 human/mouse MDCKIPapp (A to B)

13 x 10-6 cm/s

PPB (%)h / m

99.9 / 99.9

Mouse IV PK (1 mg/kg)

CL: 9.2 ml/min/kg, Vss: 1.2 l/kg, t½: 1.7 h AUC: 240 µMh, t½: 3.5 h, Cmax: 45 µM, F: 100%,

Mouse PO PK (30 mg/kg)

To better understand how the present acyl sulfonamides might interact with VSD4 of the NaV1.7 receptor, we modeled representative analogs into the crystal structure of VSD4 via induced fit docking. A proposed fit of 7 in VSD4 is shown in Figure 3, supported in part by mutagenesis and small molecule X-ray data. For the aryl sulfonamide GX-936 a large loss of potency was seen for the R1608A and R1608K mutations (3000x and 400x, respectively) while the R1602A mutation caused little change in affinity.26 In contrast, mutagenesis studies with acyl sulfonamide 7 showed that both R1608 and R1602 were required for binding, with 300x and 50x


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decreases in potency observed for the R1608A and R1602A mutations, respectively.

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We

therefore postulated that the acyl sulfonamide warhead anion is delocalized between the carbonyl and sulfonyl moieties, making hydrogen bonding and ionic interactions with both R1608 and R1602. Sulfonyl oxygens are known to form hydrogen bonds with arginines, although the interaction is often weak. The combination of the SO2-to-R1608 and the amide carbonyl oxygen to R1602 interactions proposed for acyl sulfonamides rationalizes the roughly equipotent affinity between the acyl and aryl class of inhibitors. A small molecule X-ray structure of the sodium salt of 7 grown in aqueous media (Figure 4) shows a sodium ion complexed to an ionized amide carbonyl oxygen, which may partially exist as its imino tautomer, consistent with our hypothesis and binding to R1602 in VSD4. This proposed fit results in the linker phenyl ring being offset from the analogous aryl sulfonamide phenyl ring in the X-ray crystal structure of GX-936,26 and is consistent with the lack of corresponding SAR between aryl and acyl sulfonamides. Specifically, the linker phenyl in 7 is predicted to be translated approximately 1.5-1.7 angstroms out of the active site, providing room for the adamantyl group and substituted cyclohexyl rings of the present series to fit into a mostly hydrophobic region flanked by W1538, V1541, F1583, M1582, and the sidechain carbon atoms of D1586. This fit would drive the chlorine of 7 into a different region than that occupied by the cyano in GX-936, essentially occupying the center of the channel. The X-ray structure of NaV1.7 VSD4 represents one conformation of a flexible system designed to enable outward movement of S4 during a depolarization. This flexibility and induced fit of ligands introduces uncertainty in the modeling calculations. Other states of this protein can no doubt exist. Nonetheless, the modeling experiments using induced fit docking


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have served to rationalize parts of the SAR such as phenyl ring and sulfonamide substitution and provided design guidance throughout the project.

Figure 3. Induced fit docking of 7 into the VSD4 of the hNaV1.7 receptor, shown in cyan. The active site cavity and helices are from the induced fit version. Key residues and distances are shown.

The co-crystal structure of GX-936 and VSD4 of NaV1.726 (PDB code 5EK0) is

superimposed and shown in orange.


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Figure 4. Small molecule crystal structure of the sodium salt of 7 (crystals grown in 50% MeOH solution), with sodium ions complexed to the charged oxygen atom shown as orange spheres. Two molecules of methanol are also shown. For details, see the Supporting Information.

With this data in hand, we focused our attention on improving the potency and metabolic stability of compound 7. We were cognizant of the fact that large lipophilic groups like adamantane are main contributors to high metabolic clearance, so our initial SAR exploration focused on a diverse set of replacements aimed at understanding the impact on potency and metabolic stability (Table 2). Since metabolic stability is very often linked to lipophilicity,33 we also tracked measured LogD (mLogD) and cLogP as well as ligand lipophilic efficiency (LLE) (calculated using hNaV1.7 EP IC50 and cLogP) in the initial set of compounds to understand if there is a correlation. In addition, lowering lipophilicity is an established medicinal chemistry strategy to decrease the risk for drug promiscuity and unwanted pharmacology.34 The differences


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between cLogP and mLogD reflects the compound ability to exist as deprotonated species at pH=7.4. Cyclohexane 8 demonstrated a 7-fold loss in hNaV1.7 potency, but a slight increase in HLM stability compared to 7. Incorporation of an oxygen atom into the cyclohexyl ring (9) was not tolerated. Extending the linker by one carbon (10) only slightly increased the potency while negatively effecting metabolic stability. Shortening the linker (11) or reducing the ring size by one carbon (12) resulted in a further decrease in potency but enhanced metabolic stability. As expected, metabolic stability and lipophilicity of the left-hand portion of the molecule were generally correlated, with cyclopentyl ether 12 and ethylcyclopropyl ether 13 being the most stable in this subset. However, both displayed unacceptably low potency in the LBA. In this initial library, we found a relatively good correlation between mLogD and HLM stability. For example, 13 showed one of the lowest mLogD values (0.4) and was also the most stable in HLMs (HLM Cl hep = 7ml/min/kg). On the other hand, the homologated cyclohexane 10, which showed one of the highest LogD’s compared to other analogues in this subset, had low stability in HLMs (HLM Clhep = 19ml/min/kg). However, in a larger data set this tendency could not be confirmed. We suspect structural features are overriding the HLM-lipophilicity trend. Considering that 7 displayed the highest potency in a functional assay and the highest LLE, we used this building block for further optimization to explore the SAR of substituents on the phenyl core (Table 3). A 2-fluoro substitution was essential for hNaV1.7 potency, as removal (14) or replacement with chlorine (15) resulted in significant drops in potency. Replacement of the 5chlorine in 7 with a fluorine (16) also significantly reduced potency. Likewise, replacement of the 5-chlorine with methyl (17), ethyl (18) or isopropyl (19) were not beneficial to potency.


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However, introducing a cyclopropyl group as a chloro replacement (20) turned out to be highly favorable, providing a greater than 10–fold potency enhancement with minimal increases in molecular weight or lipophilicity (LogP = 3.5 and 3.6 for 7 and 20, respectively) resulting in a significant gain in LLE. Unfortunately, modification of the phenyl ring had no impact on HLM stability. The marked increase in potency for 20 over 7 may be a result of the cyclopropyl making additional hydrophobic contacts with the protein, or simply being more compatible with the adjacent hydrophobic membrane environment.

Table 2. Effects of adamantane replacements on NaV1.7 potency and HLM stability.

F

O

N H

1

R

O S O

O Cl

Compd

hNaV1.7 IC50 (M)

R1



7 8 9

 O 



10 11 12





LBAa

EPb

HLM CLhep mLogDd cLogP (ml/min/kg)c

0.07

0.006

19

2.4

3.5

4.8

0.18

0.043

15

1.4

3.2

4.2

>10

7.4

nd

0.21

1.7

3.4

0.50

0.017

19

1.5

3.6

4.2

1.2

0.26

10

1.3

3

3.6

0.52

0.068

7

1.1

2.7

4.5

LLEe


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13

2.7



0.44

7

0.4

2.4

4

a

Ligand binding assay using [3H]GX-545. IC50s are an average of at least two independent determinations. b

IC50s are an average of two independent determinations.

c

Predicted hepatic Clearance CLhep extrapolated from in vitro experiments in human liver microsomes (CLHep = 19 ml/min/kg correlates to a 100% extraction ratio);35 nd: not determined. d Measured e LLE

[email protected]

was calculated using hNaV1.7 EP IC50 and cLogP

Table 3. Effect of substitutions on the phenyl core on NaV1.7 potency and HLM stability. Y

O

O S N H O

O X

hNaV1.7 IC50 (M)

HLM CLhep

LBAa

EPb

(ml/min/kg)c

H

0.33

0.059

19

3.8

Cl

Cl

2.3

0.57

nd

2.2

16

F

F

0.64

0.041

19

2.9

17

Me

F

0.037

0.006

19

4.9

18

Et

F

0.015

0.006

19

4.7

19

i-Pr

F

0.028

0.010

19

3.8

F

0.006

0.0003

19

5.9

Compd

X

Y

14

Cl

15

20



LLEd

a

Ligand binding assay using [3H]GX-545 IC50s are an average of at least two independent determinations.


14


b

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c

Predicted hepatic Clearance CLhep extrapolated from in vitro experiments in human liver microsomes (CLHep =19 mL/min/kg correlates to a 100% extraction ratio);35 nd: not determined. d LLE


Given the high potency and LLE for 20, we now explored ways of improving the metabolic stability of the adamantane itself. Metabolite identification (MetID) studies on 7 indicated that the adamantal skeleton was subject to oxidative metabolism. We therefore decided to investigate blocking potential sites of metabolism and reducing lipophilicity by incorporating polar substituents on this ring system. As shown in Table 4, introduction of a polar substituent (21) significantly enhanced HLM stability, but compromised potency on hNaV1.7 Other attempts to block the potential sites of oxidative metabolism via introduction of fluorine (22 and 23) resulted in negligible effect on HLM stability for 22 and markedly decreased HLM for 23. Both 22 and 23 displayed significant loss of hNaV1.7 potency compared to 7. Given 23 had relatively good potency and stability, we wanted to probe if replacing Cl with cyclopropyl would increase potency and retain HLM stability as seen for 20. While cyclopropyl analogue 24 did indeed show potency increases in ligand binding and EP, its HLM stability decreased significantly. Finally, substitution of each tertiary carbon on the adamantane with fluorine (25) substantially improved stability but again at a marked cost in hNaV1.7 potency and LLE relative to 7 or 20.


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Table 4. Effects of adamantane modification on NaV1.7 potency and HLM stability. F R1

O

O S

N H

O

O X

Compd

LBAa

EPb

Cl

>10

2.2

9

3.1



Cl

0.357

0.031

18

4.3



Cl

0.290

0.011

12

4.4

0.008

0.0031

16

4.8

2.398

0.752

6.7

3.5

X

21

HO

22

F

23

F

hNaV1.7 IC50 (M)

HLM CLhep LLEd (ml/min/kg)c

R1



F

24



F



F

F

25



F

Cl

F

a

Ligand binding assay using [3H]GX-545 IC50s are an average of at least two independent determinations. b


c

Predicted hepatic Clearance CLhep extrapolated from in vitro experiments in human liver microsomes (CLHep =19 ml/min/kg correlates to a 100% extraction ratio);35 nd: not determined. d LLE



16


F

O

F

O N H

S

Page 18 of 74

O

O N H

O

S O

O

O O 26 m/z 368

M1 (62%), M2 (15%), M3 (19%) m/z 384

Figure 5. Summary of the major metabolic pathway of 26.

Lessons taken from the SAR developed in Tables 2-4 influenced the design of the next round of analogs (Table 5). Specifically, our goal was to incorporate strategies used successfully to block metabolism of the adamantyl group on the synthetically less complex cyclohexyl ring while also taking advantage of potency improvements realized in the core. We also wanted to maintain the relatively high fsp3 count of the overall molecule. Replacing the 5-chloro in 8 with a cyclopropane (26) led to a significant boost in NaV1.7 potency, particularly in the functional assay, but unfortunately retained the HLM instability seen with previous analogs. MetID studies revealed three major metabolites resulting from oxidation of the cyclohexyl ring (Figure 5). Further improvements in stability were obtained through gem-difluorination at the 4-position of the cyclohexyl ring (27). This compound also retained some of the LLE from 26. However, it showed only moderate LBA potency compared to others in the series. We next turned to examining the effect of varying the acyl sulfonamide substitution on potency and stability. Interestingly, replacement of the N-methylsulfonyl substituent in 27 with N-cyclopropysulfonyl (28) or N-((2-methoxyethyl)sulfonyl (29) enhanced stability. Potency was retained in 28, while 29 was much less potent, presumably due to size limitations in this region of the protein.


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From this small SAR study of the warhead, we learned that our ability to modulate potency and stability via changes in acyl sulfonamide substitution was limited. We therefore turned our attention back to the cyclohexyl-ether appendage. We examined the effect of adding methyl groups36 by incorporation of a substituent on the tertiary carbon at the 1-position of the cyclohexyl ring. Compound 30 provided a moderate improvement in both potency and stability over 29. Increasing the size of the substituent at the tertiary position had a detrimental effect on stability (31). Therefore, the methyl group was incorporated as a preferred substituent for further cyclohexane explorations. Replacing the 4-difluoro group in 30 with a trifluoromethyl (32) retained potency relative to 30, but decreased stability.

As an alternative to blocking

metabolically labile sites by fluorine atoms, we also explored the incorporation of strained systems. An example is 33, which showed the highest functional activity against hNaV1.7 (IC50 = 0.6 nM) and retained moderate stability in human microsomes and hepatocytes (Table 6.) Compounds 30 and 33 were selected for further characterization against a panel of human and rodent NaV isoforms as they showed distinct advantages over other analogs. Compound 33 showed the highest potency in EP, high LLE, and moderate stability, while 30 showed acceptable potency, HLM stability, and displayed the highest LLE in the cyclohexane series. Although 33 showed good selectivity over hNaV1.5, it was only about 10- and 2-fold selective against hNaV1.1 and hNaV1.2. Compounds 30 and 33 also did not inhibit (IC50 >10uM) mNaV1.8 in TTX-resistant channels in mouse dorsal root ganglion neurons. Compound 30 showed high selectivity over hNaV 1.5 and greater selectivity against hNaV 1.1 than acyl sulfonamide 33.


18


Page 20 of 74

Table 5. Effects of cyclohexane modification on NaV1.7 potency and HLM stability. F

O

N H

1

R

O S

R2 O

O

hNaV1.7 IC50 (M) Compd

R1

26



27

F F

28

F F

29

F F

30

31

32

33

R2





F F

EPb

Me

0.022

0.0007

19

5.9

Me

0.093

0.015

4.9

4.5



0.033

0.008

3.7

3.9

0.304

0.075

2.7

3.7



0.015

0.003

2.2

4.0



0.017

0.005

9.5

3.3



0.010

0.006

13

3.2



0.015

0.0006

12

4.6



F



Et

LLEd

LBAa



F

HLM CLhep (ml/min/ kg)c

O



F3C





a

Ligand binding assay using [3H]GX-545. IC50s are an average of at least two independent determinations.


19

Page 21 of 74 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


b


c

Predicted hepatic Clearance CLhep extrapolated from in vitro experiments in human liver microsomes (CLHep =19 ml/min/kg correlates to a 100% extraction ratio);35 nd: not determined. d LLE


Table 6. In vitro profiles of compounds 30 and 33 30

33

3 / 2.5 / 9

0.6 / 2.2 / 3.7

260 / 48 / 6 / 49

50 / 6 / 2 / 38

26

38

2 / 38 / 10 / 7

12 / 65 / 22 / nd

2.8 / 17 / 4.3 / 3.5

13 / 6.0 / 5.2 / 7.6

> 10 / > 10 / > 10 / > 10 / > 10

> 10 / 6.1 / > 10 / > 10 / > 10

22

9

Compound NaV1.7 IC50 (nM)a human / mouse / rat hNaV 1.5, 1.1, 1.2, 1.6 IC50 (nM)a % inh. TTX-R @ (10µM) Liver Microsomes CLhep (ml/min/kg)b human / mouse / rat / dog Hepatocytes CLhep (mL/min/kg)c human / mouse / rat / dog CYP 3A4 / 2C9 / 2C19 / 2D6 / 1A2 IC50 (M) MDCK1 Papp (A to B) x 10-6 cm/s Ratio (BA / AB)


20


Page 22 of 74

0.55

1.0

Papp (A to B) x 10-6 cm/s

11

3.7

Ratio (BA / AB)

1.4

1.2

Papp (A to B) x 10-6 cm/s

0.4

5.1

Ratio (BA / AB)

56

2.1

human / mouse / rat

99.6 / 99.8 / 99.8

99.9 / 99.9 / 99.9

Kinetic Solubility (M )

32

10

mLogD/LLE(EP/mLogD)d,e

1.4/6.8

2.1/7.1

MDCK1-MDR1

MDCK2-mBCRP

PPB (%)

aIC

values are an average of two independent determinations using PatchXpress electrophysiology technique. 50

bPredicted

hepatic clearance (CLhep) extrapolated from in vitro human, mouse and rat microsome experiments.35 cPredicted

hepatic clearance (CLhep) extrapolated from in vitro human, mouse, rat and dog hepatocyte experiments.35 d Measured e LLE

[email protected]

was calculated using hNaV1.7 EP IC50 and mLogD

Compounds 30 and 33 were also tested against mouse and rat NaV1.7 where they showed potencies similar to that of the human isoform. The invariant human, rat, and mouse NaV1.7 potencies are a distinct characteristic the present acyl sulfonamide series relative to other


21

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reported acyl and aryl sulfonamides (e.g., 4) that bind to the VSD4 site. We believe that the increased conformational flexibility between cycloalkyether and phenyl core allow our compounds to adopt a binding mode in rat NaV1.7 that is similar the one shown in for compound 7 in Figure 3, and adapt that binding mode to residue changes in the VSD between human, mouse, and rat. The less flexible, more deeply binding aryl sulfonamides such as 4 cannot readily adjust to the residue differences in the rNaV1.7 binding pocket, and are less potent as a result. Gratifyingly, both 30 and 33 were selective for hNaV1.7 over hNaV1.5. No significant DDI risk was observed, as 30 and 33 did not show notable CYP inhibition (Table 6). We tracked permeability in a mouse MDCK2-BCRP cell line, which contains the main transporter found at the blood brain barrier, to better understand the brain exposure of our compounds. Compound 33 had good permeability and low efflux, while 30 proved to be a mBCRP substrate. Both compounds showed high protein binding in human, mouse and rat (Table 6). We also evaluated 33 in a CEREP panel of 33 receptors at 10uM concentration, where we saw >75% inhibition of only two targets: NaV ion channels (80%), and PPAR (91%). The measured LogD’s at pH 7.4 were acceptable for both 30 (mlogD=1.4) and 33 (mLogD=2.1). Noticeably, LLE when calculated based on mLogD was high for both 30 and 33, and in line with low off target activity described for 33.

Table 7. Pharmacokinetic profiles for compounds 30 and 33

Compd

Species

30

Mousea

30

Ratb

t½ (h)

IV CL (ml/min/kg)

PO Vss (l/kg)

t½(h)

Cmax (µM )

AUC (µMh)

F(%)

2.1

1.6

0.28

15.6

69

350

49

9.0

0.4

0.29

16.9

25

160

66


22


33

Mousea

33

Ratb

33

Dogc

Page 24 of 74

1.5

3.2

0.41

9.2

22

113

31

8.1

1.2

0.88

nc

13

78

49

5.4

0.3

0.14

6.0

9

83

68

aAverage

of 3 male FVB mice, dosed IV with 1 mg/kg of compound 30 or 33 in 5% DMSO/35% PEG400/60% PBS or dosed PO with 30 mg/kg of compound 30 or 33 as MCT suspension. bAverage

of 3 male Sprague-Dawley rats, dosed IV with 1 mg/kg of compound 30 or 33 10% DMSO/50% PEG400/40% PBS or dosed PO with 5 mg/kg of compound 30 or 33 MCT suspension. nc: not calculated.

in as

cAverage

of 3 male beagle dogs, dosed IV with 33 in 0.5 mg/kg in 15% EtOH, 60% PEG400 in water, and orally dosed with 31 at 1 mg/kg as MCT suspension.

Based on their overall in vitro potencies and DMPK profiles, 30 and 33 were selected for further in vivo evaluation. Their PK profiles were determined in mouse and rat, 33 was also evaluated in dog. Results are summarized in Table 7. Consistent with the predicted CLhep and high plasma protein binding, both compounds exhibited very low plasma clearance (CL). Both 30 and 33 displayed moderate half-lives (t½) in mice. Both molecules showed increased halflives in the rat IV experiments, with 33 showing acceptable half lives in dog IV and PO studies. They also displayed low volume of distributions (Vss) values in these animal species, presumably because of high plasma protein binding.

In agreement with their high permeability and

solubility, both compounds displayed good to moderate oral plasma exposures (AUC) and bioavailabilities (F). The oral bioavailabilities of 33 in mice, rats, and dogs were 31%, 49% and 68%, respectively.


23

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To determine if the PK and potencies of our compounds were sufficient to demonstrate analgesia in vivo, 30 and 33 were evaluated in transgenic mice21c,37 that express a human variant of NaV1.7 that results in IEM (mutation I848T).38 Aconitine-induced pain in these mice provides a novel way to quantify engagement of hNaV1.7 in vivo and facilitates the characterization of molecularly selective inhibitors that, most often, show species specificity for rodent orthologs. This assay reports the number of nociceptor responses (shaking and licking) in response to the sodium channel activator aconitine. As illustrated in Figures 6 and 7, 30 and 33 demonstrated dose-related inhibition of aconitine-induced nociceptive behaviors, matched by dose-dependent increases in plasma concentrations. Consistent with its significantly higher functional potency against NaV1.7, 33 was much more potent in this study demonstrating >75% analgesic efficacy starting at a plasma concentration of approximately 3.1 M (which corresponds to a unbound plasma concentration of Cpu = 3nM, approximately 5-fold the hNaV1.7 IC50) (0.3 mg/kg, oral dose). At these very low efficacious free plasma concentrations, we did not expect to see significant inhibition of other NaV isoforms. Plasma concentrations of 33 for the 3 and 10 mg/kg groups were measured as 25 and 79 M, respectively. The brain concentrations were 1.9 and 6.2 M, respectively, which provided a low brain to plasma ratio of approximately 0.08. We were gratified to see this remarkable efficacy for 30 and 33 despite their high plasma protein binding. The significantly higher efficacy of 33 over 30 was rationalized by its 5-fold higher potency in the functional assay for inhibition of hNaV1.7. In vivo effects of 33 were further assessed in a formalin-induced pain model in mice,39 an assay that has been used to evaluate analgesic effects for many sodium channel blockers.23, 24, 25b,40 This pain model utilizes formalin as a pain-inducing agent and generates a biphasic pain response. The initial response (phase I), occurring within 5– 10 minutes post-injection of formalin, indicates an acute localized irritation (partially due to


24


Page 26 of 74

activation of TRPA1 channels41), while a delayed response (Phase II) that begins 10–15 minutes post-injection indicates neurogenic inflammation.42 As illustrated in Figure 8, 33 produced significant response reduction in phase II of the formalin assay at a PO dose of 10 mg/kg. The plasma concentration of 33 was measured at the end of the study (approximately 3 hours after oral dosing) to be 50 µM. Despite the modest selectivity for the inhibition of hNaV1.7 over hNaV1.1 and hNaV1.2, 33 was well tolerated in mice, as there were no clinical observations over the course of this study. We also evaluated 30 and 33 for inhibition of mNaV1.8 in TTXresistant channels in mouse dorsal root ganglion neurons, and found minimal inhibition (IC50 >10 µM), indicating that their efficacy was not caused by inhibition of this isoform.

Figure 6. Effects of 30 on aconitine-induced pain in humanized IEM transgenic mice. Each bar represents the mean of at least 4 animals. Error bars represent standard errors of the mean. ****: p < 0.0001 versus vehicle group. Statistical significance of results was calculated by two-way ANOVA using Prism version 7 (GraphPad software)


25

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Figure 7. Effects of 33 on aconitine-induced pain in humanized IEM transgenic mice. Each bar represents the mean of at least 4 animals, except the 0.3 mg/kg and 1mg/kg groups (3 animals for each group at these doses) Error bars represent standard errors of the mean. ****: p < 0.0001 and ***: p < 0.005 versus vehicle group for nociceptive events. Statistical significance of results was calculated by two-way ANOVA using Prism version 7 (GraphPad software)


26


Page 28 of 74

Figure 8. Dose response effect of 33 in the mouse formalin model. (A) Time course of inhibition of formalin-evoked pain behavior. Each point represents the mean number of times that a mouse (n = 4) shook or licked its injected paw over a 5 min period. (B) Quantification of the response to formalin binned into two different phases: phase I (0–10 min after formalin injection) and phase II (11–40 min after formalin injection). Each bar represents the mean of 4 animals. Error bars represent standard errors of the mean. ***: p = 0.0021 versus vehicle group. Statistical


27

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significance of results was calculated by two-way ANOVA using prism version 7 software (Graphpad software)

CHEMISTRY The general synthetic routes to prepare acyl sulfonamides 7–33 are outlined in Schemes 1–8. For syntheses of analogues 7–14, 16, 21–23 and 25 (Scheme 1), a simple and efficient nucleophilic aromatic substitution of intermediates 37, 38 and 39 with an appropriate alcohol was applied. This method was general, and a wide range of alcohols could be used in a library fashion to prepare the desired molecules 7–14, 16, 21–23 and 25.

Scheme 1. Syntheses of compounds 7–14, 16, 21–23 and 25 a Y

O

Y

a

Z

O

O N H

F

O

34: X = Cl, Y = F, Z = OH 35: X = Cl, Y = H, Z = OH 36: X, Y = F, Z = Cl

O

R

X 37: X = Cl, Y = F 38: X = Cl, Y = H 39: X, Y = F

O N H

1

F

X

Y

b

S

S O

O

X 7-13, 21-23, 25: X = Cl, Y = F 14: X = Cl, Y = H 16: X, Y = F

a

Reagents and conditions: (a) for 37 and 38: MeSO2NH2, EDCI, DMAP, CH2Cl2, rt, 58–96%; for 39: MeSO2NH2, Et3N, CH3CN, rt, 54%; (b) R1OH, t-BuOK, DMSO, rt, 9–58%.

Scheme 2. Synthesis of compound 15a

Br

Br

a Cl

Cl 40

b

O O S N H O

O

O

HO

Cl

Cl

Cl

41

Cl 15


28


Page 30 of 74

Reagents and conditions: (a) adamantan-1-ylmethyl methanesulfonate, K2CO3, DMF, 65 C, 71%; (b) MeSO2NH2, Mo(CO)6, Pd(OAc)2, Xantphos, Et3N, 1,4-dioxane, 100 C, microwave irradiation, 45 min, 10%. a

Analogue 15 was synthesized in a two-step sequence from phenol 40 which was alkylated to give intermediate 41. Compound 15 was prepared from 41 via introduction of the acyl sulfonamide group by a palladium-catalyzed carbonylation.43 Compound 17 was prepared from 42 as detailed in Scheme 3, involving a nucleophilic aromatic substitution and an acyl sulfonamide formation in the presence of EDCI and DMAP. The synthesis of analogue 18 involved the esterification of 34, nucleophilic aromatic substitution of 44 with (adamantan-1yl)methanol yielding the predominantly para-regioisomer in 62% yield, Suzuki-Miyaura crosscoupling reaction44 of 45 with vinylboronic acid pinacol ester, hydrolysis of 46 with TFA, hydrogenation

of

47,

and

finally

acyl

sulfonamide

formation

of

acid

48

with

methanesulfonamide (Scheme 4). For analogue 19, a method analogous to that used for 18 was applied. In this preparation, vinylboronic acid pinacol ester was replaced by 2-isopropenyl4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Scheme 5).

Scheme 3. Synthesis of compound 17a

F

F

O OH

OH

a O

F Me 42

F

O b

O N H

S O

O Me

Me 43

O

17

Reagents and conditions: (a) 1-adamantanemethanol, t-BuOK, DMSO, rt to 50 C, 17%; (b) MeSO2NH2, EDCI, DMAP, CH2Cl2, rt, 27%. a


29

Page 31 of 74 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


Scheme 4. Synthesis of compound 18 a F

F

O t

a

O Bu

OtBu O

O

F

O

c

t

b

O Bu

34

F

O

Cl

Cl

45

44 F

O

F OH

d

46 O

F OH

e

O

O N H

f

O

S O

O Et

47

O

Et 18

48

Reagents and conditions: a) di-tert-butyl dicarbonate, DMAP, tert-Butanol, 50 C, 99%; (b) (adamantan-1-yl)methanol, Cs2CO3, DMSO, 70 C, 62%; (c) vinylboronic acid pinacol ester, Pd2(dba)3, t-Bu3P∙HBF4, Na2CO3, 1, 4-dioxane, H2O, 100 C, 92%; (d) TFA, CH2Cl2, rt, 97%; (e) Pd-C, H2, EtOAc, rt, 74%; (f) MeSO2NH2, EDCI, DMAP, CH2Cl2, rt, 69%. a

Scheme 5. Synthesis of compound 19 a F

O

F t

45

O Bu

a

O

F OR

b

O

49

c

50: R = tBu 51: R = H

O N H

d

O

O

S O

O

19

a

Reagents and conditions: (a) 2-isopropenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd2(dba)3, t-Bu3P∙HBF4, Na2CO3, 1, 4-dioxane, H2O, 100 C, 64%; (b) Pd-C, H2, EtOAc, rt, 94%; (c) TFA, CH2Cl2, rt, 84%; (d) MeSO2NH2, EDCI, DMAP, CH2Cl2, rt, 33 %.

The syntheses of 20, 26-32 started from an aromatic nucleophilic substitution of 44 with an appropriate alcohol in the same manner as described in preparation of 45. Compounds 45, 52-56 were then subjected to the Suzuki-Miyaura cross-coupling reaction44 with cyclopropylboronic acid to provide the corresponding cyclopropyl intermediates 57-62. After treatment with TFA, the resulting acids 63–68 were coupled with alkylsulfonamide to afford the desired acyl


30


Page 32 of 74

sulfonamides 20 and 26–32 (Scheme 6), respectively. Analogue 24 was directly prepared from 23 in a one-step transformation using the Suzuki-Miyaura cross-coupling reaction44 (Scheme 7). Compound 33 was synthesized in multiple steps from intermediate 44 (Scheme 8). In this preparation, the dioxolane protecting group in 70 was selectively removed with TFA in a mixture of THF and H2O without affecting the tert-butyl ester group. Simmons-Smith cyclopropanation of 72 proceeded smoothly with the use of chloroiodomethane and diethylzinc45 to afford the key intermediate 73.

Scheme 6. Syntheses of compounds 20, 26–32a

F 44

F

O OtBu

a R1

b

O

F OR

R1

O

d

R1

O

O

O 2 S R N H O

O

Cl 57-62: R = tBu

45, 52-56

20, 26-32

c 63-68: R = H

Reagents and conditions: (a) R1OH, Cs2CO3, DMSO, 70 C, 43–95%.; (b) cyclopropylboronic acid, Pd(OAc)2, Cy3P∙HBF4, K3PO4, toluene, H2O, 100 C, 86–99%.; (c) TFA, CH2Cl2, rt, 50–86%.; (d) R2SO2NH2, EDCI, DMAP, CH2Cl2, rt, 40–70%.. a

Scheme 7. Syntheses of compound 24a F

O

N H F F

O

F

O S O

a

23

O N H

F Cl

O

S O

O

F 24

a

Reagents and conditions: (a) cyclopropylboronic acid, Pd(OAc)2, Cy3P∙HBF4, toluene, H2O, 100 C, 86%.

K3PO4,


31

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Scheme 8. Syntheses of compounds 33 a F

F

O t

O Bu

a 44

O

O

OtBu

b

O

69

71

70 F

F

O OtBu

d

O

F OR

e

g

O

O

72

OtBu O

O

O

O

c

O

O

Cl

F

O

f

O

O N H

S O

O

73: R = tBu 74: R = H

33

a

Reagents and conditions: (a) (8-methyl-1,4-dioxaspiro[4.5]decan-8-yl)methanol, Cs2CO3, DMSO, 70 C, 54%; (b) cyclopropylboronic acid, Pd(OAc)2, Cy3P∙HBF4, K3PO4, toluene, H2O, 100 C, 83%; (c) TFA, H2O, THF, rt, 90%; (e) MePPh3Br, LiHMDS, THF, -20 C to rt, 82%; (e) ClCH2I, Et2Zn, ClCH2CH2Cl, 0 C, 93%; (f) TFA, CH2Cl2, rt, 90%; (g) cyclopropanesulfonamide, EDCI, DMAP, CH2Cl2, rt, 41%.

CONCLUSION This paper details the discovery of 33 a highly potent and selective NaV1.7 inhibitor that displayed favorable PK in rodents, acceptable subtype selectivity over other NaV isoforms, and efficacy in in vivo models of pain as well as hNaV1.7. target engagement models. Starting with an analysis of the physicochemical property profiles of previously reported aryl- and acylsulfonamides, followed by library syntheses, we identified lead molecule (7). Guided by induced fit docking into our recently published X-ray co-crystal structure of VSD4 of NaV1.7, we simplified the adamantane to a cyclohexane in compound 26 and conducted metabolite ID studies to guide further designs. Significant improvements in metabolic stability were achieved by identifying the primary metabolites of 26 and utilizing these data to systematically modify the


32


Page 34 of 74

structures to address proposed liabilities. These efforts culminated in the identification of 33 a highly potent and efficacious tool molecule. Finally, we also disclosed CEREP™ screening data and inhibition data against TTX resistant currents in mouse DRG’s that provided clear evidence that the in vivo activity of compound 33 was primarily caused by its inhibition of NaV1.7 channels.

EXPERIMENTAL SECTION General. Chemicals, reagents and solvents were purchased from commercial sources and were either used as supplied or purified using reported methods. Final compounds reported herein exhibited spectral data consistent with their proposed structure by nuclear magnetic resonance spectra (1H NMR and 13C NMR) and mass spectra data. NMR spectra were recorded on a Bruker Avance 300 spectrometer with chemical shifts () reported in parts-per-million (ppm) relative to the residual signal of the deuterated solvent. 1H NMR data are tabulated in the following order: multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constants in Hertz and number of protons. Mass spectra were obtained using a Waters 2795/ZQ LC/MS system (Waters Corporation, Milford, MA). Final compounds were greater than 95% pure as determined by analytical HPLC on Agilent 1200 systems (Agilent Technologies, Santa Clara, CA) using an EMD Chromolith SpeedROD RP-18e column (4.6 mm i.d. x 50 mm length) (Merck KGaA, Darmstadt, Germany). The mobile phase consisted of a gradient of component “A” (0.1% v/v aqueous trifluoroacetic acid) and component “B” (acetonitrile) at a flow rate of 1 mL/min. The gradient program used was as follows: initial conditions 5% B, hold at 5% B for 1 min., linear ramp from 5% to 95% B over 5 min., 100% B for 3 min., return to initial conditions for 1 min. Peaks were detected at a wavelength of 254 nm with an Agilent photodiode array


33

Page 35 of 74 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


detector. Melting points were determined on a Fisher-Johns melting point apparatus and were uncorrected.

Chemical

names

were

generated

using

ChemBioDraw

version

12.0

(CambridgeSoft, Cambridge, MA.). 4-(Adamantan-1-ylmethoxy)-5-chloro-2-fluoro-N-(methylsulfonyl)benzamide (7). To a mixture of adamantan-1-ylmethanol (1.00 g, 6.0 mmol) in anhydrous DMSO (40 mL) was added potassium t-butoxide (1.68 g, 15.0 mmol) at room temperature. The resulting mixture was stirred at room temperature for 30 min followed by the addition of 37 (1.62 g, 6.0 mmol). The reaction mixture was stirred at room temperature for 16 h. The mixture was cooled to 0 ºC and quenched with hydrochloride acid (1N, 30 mL) followed by extraction with ethyl acetate (200 mL). The organic layer was washed with water (2 x 40 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo, the residue was purified by silica gel column chromatography eluted with a gradient of 10-60% ethyl acetate (containing 2% acetic acid) in hexanes to afford 7 as a colorless solid (1.17 g, 46%). mp 194 –196 °C (EtOAc/hexanes) 1H NMR (300 MHz, DMSO–d6)  12.10 (s br, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.23 (d, J = 12.5 Hz, 1H), 3.72 (s, 2H), 3.35 (s, 3H), 1.99 (s br, 3H), 1.75–1.64 (m, 12H).

13C

NMR (75 MHz,

DMSO–d6)  162.3 (d, J = 2.2 Hz), 159.8 (d, JC-F = 255 Hz), 158.3 (d, JC-F = 11 Hz ), 130.6 (d, JC-F = 4 Hz), 116.8 (d, JC-F = 3 Hz ), 113.6 (d, JC-F = 14 Hz), 102.7 (d, JC-F = 28 Hz), 79.1, 41.2, 38.5, 36.4, 33 .4, 27.3. MS (ES-) m/z: 414.1, 416.1 (M - 1).

5-Chloro-4-(cyclohexylmethoxy)-2-fluoro-N-(methylsulfonyl)benzamide

(8).

Using

a

method analogous to the preparation of compound 7, 8 was prepared from 37 and cyclohexylmethanol as colorless solid (0.11 g, 30%). 1H NMR (300 MHz, DMSO–d6)  12.04 (s


34


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br, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 12.6 Hz, 1H), 3.93 (d, J = 5.9 Hz, 2H), 3.31 (s, 3H), 1.79–1.60 (m, 6H), 1.29–0.97 (m, 5H). MS (ES-) m/z: 362.1, 364.1 (M - 1).

5-Chloro-2-fluoro-N-(methylsulfonyl)-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzamide (9). Using a method analogous to the preparation of compound 7, 9 was prepared from 37 and (tetrahydro-2H-pyran-4-yl)methanol as a colorless solid (0.160 g, 15%). 1H NMR (300 MHz, DMSO-d6)  12.10 (s br, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 12.5 Hz, 1H), 4.03 (d, J = 6.4 Hz, 2H), 3.91–3.86 (m, 2H), 3.38–3.30 (m, 5H), 2.10–2.00 (m, 1H), 1.70–1.65 (m, 2H), 1.43– 1.29 (m, 2H). MS(ES-) m/z: 364.1, 366.1 (M - 1).

5-chloro-4-(2-cyclohexylethoxy)-2-fluoro-N-(methylsulfonyl)benzamide

(10).

Using

a

method analogous to the preparation of compound 7, 10 was prepared from 37 and 2cyclohexylethanol as a colorless solid (0.05 g, 14%). 1H NMR (300 MHz, DMSO-d6)  12.05 (s, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 12.4Hz, 1H), 4.17 (t, J = 6.6 Hz, 2H), 3.29 (s, 3H), 1.76-1.41 (m, 8H), 1.27-0.90 (m, 5H); MS(ES-) m/z: 376.1, 378.1 (M - 1). 5-Chloro-4-(cyclohexyloxy)-2-fluoro-N-(methylsulfonyl)benzamide (11). Using a method analogous to the preparation of compound 7, 11 was prepared from 37 and cyclohexanol as a colorless solid (0.03 g, 9%). 1H NMR (300 MHz, DMSO–d6)  12.03 (s br, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 12.7 Hz, 1H), 4.64-4.57 (m, 1H), 3.31 (s, 3H), 1.88-1.82 (m, 2H), 1.69– 1.64 (m, 2H), 1.54–1.27 (m, 6H). MS (ES-) m/z: 348.1, 350.1 (M - 1).

5-Chloro-4-(cyclopentylmethoxy)-2-fluoro-N-(methylsulfonyl)benzamide (12). Using a method analogous to the preparation of compound 7, 12 was prepared from 37 and


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cyclopentylmethanol as a colorless solid (0.14 g, 40%). 1H NMR (300 MHz, DMSO-d6)  12.09 (s br, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.25 (d, J = 12.5 Hz, 1H), 4.04 (d, J = 6.8 Hz, 2H), 3.35 (s, 3H), 2.39–2.29 (m, 1H), 1.82–1.73 (m, 2H), 1.65–1.52 (m, 4H), 1.40–1.30 (m, 2H). MS (ES-) m/z: 348.1, 350.1 (M - 1).

5-Chloro-4-(2-cyclopropylethoxy)-2-fluoro-N-(methylsulfonyl)benzamide (13). Using a method analogous to the preparation of compound 7, 13 was prepared from 37 and 2cyclopropylethan-1-ol as a colorless solid (0.26 g, 52%). 1H NMR (300 MHz, DMSO–d6)  12.13 (s br, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 12.5 Hz, 1H), 4.20 (t, J = 6.3 Hz, 2H), 3.35 (s, 3H), 1.70–1.63 (m, 2H), 0.87–0.79 (m, 1H), 0.47–0.41 (m, 2H), 0.17–0.12 (m, 2H). MS (ES-) m/z: 33 4.1, 33 6.1 (M - 1).

4-(Adamantan-1-ylmethoxy)-3-chloro-N-(methylsulfonyl)benzamide (14). Using a method analogous to the preparation of compound 7, 14 was prepared from 38 and (adamantan-1yl)methanol as a colorless solid (0.34 g, 52%). 1H NMR (300 MHz, DMSO–d6)  12.07 (s br, 1H), 8.04–7.94 (m, 2H), 7.25 (d, J = 8.5 Hz, 1H), 3.72 (s, 2H), 3.36 (s, 3H), 1.99 (s br, 3H), 1.71–1.66 (m, 12H). MS (ES-) m/z: 396.1, 398.1 (M - 1).

4-(adamantan-1-ylmethoxy)-2,5-dichloro-N-(methylsulfonyl)benzamide (15). A mixture of 41 (0.16 g, 0.41 mmol), methanesulfonamide (0.12 g, 1.23 mmol), Mo(CO)6, (0.11 g, 0.41 mmol) and Et3N (0.23 mL, 1.64 mmol) in 1, 4-dioxane was purged with nitrogen for 5 min, Xantphos (0.35 g, 0.082 mmol) and Pd(OAc)2 (0.01 g, 0.041 mmol) were added. The reaction mixture was heated at 100 °C for 45 min under microwave irradiation, and then cooled to room


36


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temperature. The reaction was repeated twice on 0.82 mmol scale. The reaction mixtures were combined and concentrated in vacuo.

The residue was diluted with EtOAc (40 mL) and

saturated aqueous NH4Cl solution (10 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 25 mL). The combined organic extract was washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography eluted with a gradient of 10% to 40% EtOAc (containing 0.2% acetic acid) in hexanes to afford 15 as a colorless solid (0.09 g, 10%). 1H

NMR (300 MHz, CDCl3)  8.96 (s br, 1H), 7.99 (s, 1H), 6.91 (s, 1H), 3.60 (s, 2H), 3.42 (s,

3H), 2.09–2.01 (m, 3H), 1.83–1.64 (m, 12H). MS (ES -) m/z: 430.2 (M -1), 432.2 (M -1).

4-((adamantan-1-ylmethoxy)-2,5-difluoro-N-(methylsulfonyl)benzamide (16). Using a method analogous to the preparation of compound 7, 16 was prepared from 39 and adamantan-1ylmethanol as a colorless solid (0.145 g, 36%): 1H NMR (300 MHz, DMSO-d6)  12.01 (s, 1H), 7.53 (dd, J = 6.8, 11.3 Hz, 1H), 7.23 (dd, J = 6.8, 12.3 Hz, 1H), 3.68 (s, 2H), 3.31 (s, 3H), 1.95 (s, 3H), 1.71-1.58 (m, 12H); MS(ES-) m/z: 398.1 (M - 1).

4-(Adamantan-1-ylmethoxy)-2-fluoro-5-methyl-N-(methylsulfonyl)benzamide (17). To a mixture of 43 (0.16 g, 0.50 mmol) in anhydrous CH2Cl2 (25 mL) was added EDCI (0.22 g, 1.15 mmol), DMAP (0.14 g, 1.15 mmol), and MeSO2NH2 (11.44 g, 119.0 mmol) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was quenched by addition of 1M HCl solution (5 mL) at 0 C. The organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 25 mL). The combined organic extract was washed with water and brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in


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vacuo, and the residue was crystallized from EtOAc and hexanes to afford 17 as a colorless solid (0.06 g, 27%). 1H NMR (300 MHz, DMSO–d6)  11.85 (s br, 1H), 7.48 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 13.0 Hz, 1H), 3.62 (s, 2H), 3.33 (s, 3H), 2.15 (s, 3H), 1.99 (s br, 3H), 1.75–1.66 (m, 12H). MS (ES+) m/z: 396.2 (M + 1); MS (ES-) m/z: 394.2 (M - 1).

4-(Adamantan-1-ylmethoxy)-5-ethyl-2-fluoro-N-(methylsulfonyl)benzamide (18). Using a method analogous to the preparation of 17, compound 18 was prepared from 48 and MeSO2NH2 as a colorless solid (0.17 g, 69%). 1H NMR (300 MHz, DMSO–d6)  11.87 (s br, 1H), 7.46 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 13.1 Hz, 1H), 3.62 (s, 2H), 3.34 (s, 3H), 2.58 (q, J = 7.5 Hz, 2H), 1.99 (s br, 3H), 1.75–1.64 (m, 12H), 1.15 (t, J = 7.5 Hz, 3H). MS (ES+) m/z: 410.2 (M + 1); MS (ES-) m/z: 408.3 (M - 1).

4-(Adamantan-1-ylmethoxy)-2-fluoro-5-isopropyl-N-(methylsulfonyl)benzamide

(19).

Using a method analogous to the preparation of 17, compound 19 was prepared from 51 and MeSO2NH2 as a colorless solid (0.16 g, 33 %). 1H NMR (300 MHz, CDCl3)  8.73 (d, J = 16.5 Hz, 1H), 7.93 (d, J = 9.6 Hz, 1H), 6.57 (d, J = 14.7 Hz, 1H), 3.53 (s, 2H), 3.42 (s, 3H), 3.33 – 3.19 (m, 1H), 2.05 (s, 3H), 1.84–1.62 (m, 12H), 1.23 (d, J = 6.9 Hz). MS (ES -) m/z: 422.3 (M 1). 4-(Adamantan-1-ylmethoxy)-5-cyclopropyl-2-fluoro-N-(methylsulfonyl)benzamide

(20).

Using a method analogous to the preparation of 17, compound 20 was prepared from 63 and MeSO2NH2 as a colorless solid (0.45 g, 70%). mp 158-159 °C (EtOAc/hexanes). 1H NMR (300 MHz, CDCl3)  8.72 (d, J = 16.2Hz, 1H), 7.58 (d, J = 9.3Hz, 1H), 6.56 (d, J = 14.7 Hz, 1H), 3.56 (s, 2H), 3.41 (s, 3H), 2.09–2.00 (m, 4H), 1.81–1.58 (m, 12H), 0.99–0.91 (m, 2H), 0.70-0.62


38


(m, 2H);

13C

Page 40 of 74

NMR (75 MHz, CDCl3)  163.9 (d, JC-F = 11 Hz), 160.5 (d, JC-F = 246 Hz), 161.5

(d, JC-F = 3 Hz ), 129.9 (d, JC-F = 2 Hz), 128.2 (d, JC-F = 3 Hz ), 108.6 (d, JC-F = 10 Hz), 98.7 (d, JC-F = 29 Hz), 78.9, 41.8, 39.3, 36.9, 33 .8, 27.9, 9.3, 7.2. MS (ES+) m/z: 422.2 (M - 1); MS (ES) m/z: 420.2 (M - 1).

5-Chloro-2-fluoro-4-((3-hydroxyadamantan-1-yl)methoxy)-N-(methylsulfonyl)benzamide (21). Using a method analogous to the preparation of compound 7, 21 was prepared from 37 and 3-(hydroxymethyl)adamantan-1-ol as a colorless solid (0.25 g, 29%). 1H NMR (300 MHz, DMSO-d6)  12.11 (s br, 1H), 7.76 (d, J = 6.6 Hz, 1H), 7.23 (d, J = 12.3 Hz, 1H), 4.36 (s br, 1H), 3.78 (s, 2H), 3.35 (s, 3H), 2.14 (s br, 2H), 1.68–1.39 (m, 12H). MS (ES-) m/z: 430.1, 432.1 (M 1).

5-Chloro-2-fluoro-4-((3-fluoroadamantan-1-yl)methoxy)-N-(methylsulfonyl)benzamide (22). Using a method analogous to the preparation of compound 7, 22 was prepared from 37 and (3-fluoroadamantan-1-yl)methanol as a colorless solid (0.37 g, 58%). 1H NMR (300 MHz, DMSO–d6)  12.12 (s br, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.24 (d, J = 12.4 Hz, 1H), 3.85 (s, 2H), 3.35 (s, 3H), 2.30 (s, 2H), 1.82–1.76 (m, 6H), 1.61–1.52 (m, 6H). MS (ES-) m/z: 432.1, 434.1 (M - 1).

5-Chloro-4-((4,4-difluoroadamantan-1-yl)methoxy)-2-fluoro-N(methylsulfonyl)benzamide (23). Using a method analogous to the preparation of compound 7, 23 was prepared from 37 and (4,4-difluoroadamantan-1-yl)methanol as a colorless solid (0.47 g, 42%). 1H NMR (300 MHz, CDCl3)  8.69 (s br, 1H), 8.06 (d, J = 8.4Hz, 1H), 6. 65 (d, J =


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13.8Hz, 1H), 3.61 (s, 2H), 3.39 (s, 3H), 2.28 (s br, 2H), 2.05–1.87 (m, 5H), 1.78–1.66 (m, 6H). MS (ES+) m/z: 452.1, 454.1 (M + 1); MS (ES-) m/z: 450.2, 452.2 (M - 1). 5-cyclopropyl-4-((4,4-difluoroadamantan-1-yl)methoxy)-2-fluoro-N(methylsulfonyl)benzamide (24). To a mixture of 23 (0.37 g, 0.81 mmol), cyclopropylboronic acid (0.21 g, 2.44 mmol) and K3PO4 (1.72 g, 8.10 mmol) in toluene (30 mL) and water (1.5 mL) under a nitrogen atmosphere was added Cy3P∙HBF4 (0.06 g, 0.16 mmol) and Pd(OAc)2 (0.02 g, 0.9 mmol). The reaction mixture was heated to 100 °C for 18 hours and then cooled to ambient temperature. 5% aqueous hydrochloric acid (20 mL) was added and the mixture was extracted with EtOAc (100 mL x 3), the combined organics were washed with brine; dried over anhydrous sodium sulphate and concentrated in vacuo. Purification of the residue by column chromatography (10% to 30% gradient ethyl acetate in hexanes) afforded the title compound as a colorless solid (0.32 g, 86%): 1H NMR (300 MHz, CDCl3)  8.69 (br s,1H), 7.59 (d, J = 9.0Hz, 1H), 6.53 (d, J = 14.1Hz, 1H), 3.59 (s, 2H), 3.39 (s, 3H), 2.28 (br s, 2H), 2.08-1.92 (m, 6H), 1.53-1.51 (m, 6H), 0.97-0.89 (m, 2H), 0.66-0.60 (m, 2H); MS(ES-) m/z: 456.2 (M - 1).

5-chloro-2-fluoro-N-(methylsulfonyl)-4-((3,5,7-trifluoroadamantan-1yl)methoxy)benzamide (25). Using a method analogous to the preparation of compound 7, 25 was prepared from 37 and (3,5,7-trifluoroadamantan-1-yl)methanol as a colorless solid (0.04 g, 28%): 1H NMR (300 MHz, DMSO-d6) δ 12.10 (br s, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 12.28 Hz, 1H), 4.00 (s, 2H), 3.22 (s, 3H), 2.26-2.15 (m, 3H), 2.07-1.97 (m, 3H), 1.82-1.74 (m, 6H); MS (ES-) m/z 468.21, 470.20 (M - 1).


40


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4-(Cyclohexylmethoxy)-5-cyclopropyl-2-fluoro-N-(methylsulfonyl)benzamide (26). Using a method analogous to the preparation of 17, compound 26 was prepared from 64 and MeSO2NH2 as a colorless solid (0.19 g, 40%). 1H NMR (300 MHz, CDCl3)  8.71 (d, J = 16.5 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 6.56 (d, J = 14.7 Hz, 1H), 3.82 (d, J = 5.7 Hz, 2H), 3.41 (s, 3H), 2.11–2.01 (m, 1H), 1.96–1.68 (m, 6H), 1.39–1.04 (m, 5H), 0.98–0.90 (m, 2H), 0.69–0.63 (m, 2H). MS (ES -) m/z: 368.3 (M -1).

5-Cyclopropyl-4-((4,4-difluorocyclohexyl)methoxy)-2-fluoro-N(methylsulfonyl)benzamide (27). Using a method analogous to the preparation of 17, compound 27 was prepared from 65 and MeSO2NH2 as a colorless solid (0.04 g, 13%). 1H NMR (300 MHz, CDCl3)  8.69 (d, J = 15.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 6.55 (d, J = 14.4 Hz, 1H), 3.88 (d, J = 9.0 Hz, 2H), 3.39 (s, 3H), 2.25–2.11 (m, 2H), 2.04–1.66 (m, 6H), 1.54–1.41 (m, 2H), 0.97–0.89 (m, 2H), 0.67–0.61 (m, 2H). MS (ES-) m/z: 404.1 (M - 1).

5-cyclopropyl-N-(cyclopropylsulfonyl)-4-((4,4-difluorocyclohexyl)-methoxy)-2fluorobenzamide (28). Using a method analogous to the preparation of 17, compound 28 was prepared from 65 and cyclopropanesulfonamide as a colorless solid (0.15 g, 46%). 1H NMR (300 MHz, CDCl3)  8.68 (d, J = 16.2 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 6.56 (d, J = 14.4 Hz, 1H), 3.89 (d, J = 6.0 Hz, 2H), 3.15–3.04 (m, 1H), 2.24–1.66 (m, 12H), 1.19–1.10 (m, 2H), 0.98–0.89 (m, 2H), 0.69–0.63 (m, 2H). MS (ES +) m/z: 432.0 (M +1).


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5-Cyclopropyl-4-((4,4-difluorocyclohexyl)-methoxy)-2-fluoro-N-((2methoxyethyl)sulfonyl)benzamide (29). Using a method analogous to the preparation of 17, compound 29 was prepared from 65 and 2-methoxyethane-1-sulfonamide as a colorless solid (0.07 g, 26%). 1H NMR (300 MHz, CDCl3)  8.62 (d, J = 15.9 Hz, 1H), 1H), 7.56 (d, J = 9.0 Hz, 1H), 6.55 (d, J = 14.4 Hz, 1H), 3.91–3.75 (m, 6H), 3.30 (s, 3H), 2.24–1.42 (m, 10H), 0.97–0.88 (m, 2H), 0.71–0.60 (m, 2H). MS (ES +) m/z: 450.0 (M +1).

5-Cyclopropyl-N-(cyclopropylsulfonyl)-4-((4,4-difluoro-1-methylcyclohexyl)-methoxy)-2fluorobenzamide (30). Using a method analogous to the preparation of 17, compound 30 was prepared from 66 and cyclopropanesulfonamide as a colorless solid (0.17 g, 61%). mp 134 -135 °C. 1H NMR (300 MHz, CDCl3)  8.6 6 (d, J = 16.2 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 6.55 (d, J = 14.4 Hz, 1H), 3.74 (s, 2H), 3.14–3.03 (m, 1H), 2.05–1.73 (m, 7H), 1.66–1.59 (m, 2H), 1.48– 1.41 (m, 2H), 1.20–1.10 (m, 5H), 0.97–0.89 (m, 2H), 0.68–0.59 (m, 2H);

13C

NMR (75 MHz,

CDCl3)  163.3 (d, JC-F = 11 Hz), 161.1 (d, JC-F = 3 Hz), 160.4 (d, JC-F = 247 Hz), 129.9 (d, JC-F = 2 Hz), 128.8 (d, JC-F = 3 Hz), 123.3 (t, JC-F = 241 Hz), 109.4 (d, JC-F = 10 Hz), 98.9, 98.5, 33.7 , 31.5, 30.7 (dd, JC-F = 6.93, 2.82 Hz), 29.6 (t, JC-F = 24.3 Hz), 21.2, 9.4, 7.1, 6.4. MS (ES +) m/z: 446.1 (M +1). Anal. Calcd. for C21H26F3NO4S: C, 56.62; H, 5.88; N, 3.14; Found: C, 56.80; H, 5.89; N, 3.15.

5-Cyclopropyl-N-(cyclopropylsulfonyl)-4-((1-ethyl-4,4-difluorocyclohexyl)methoxy)-2fluorobenzamide (31). Using a method analogous to the preparation of 17, compound 31 was prepared from 67 and cyclopropanesulfonamide as a colorless solid (0.17 g, 37 %). 1H NMR (300 MHz, CDCl3)  8.68 (d, J = 16.2 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 6.59 (d, J = 14.4 Hz,


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1H), 3.79 (s, 2H), 3.15-3.05 (m, 1H), 2.04–1.86 (m, 5H), 1.81–1.57 (m, 6H), 1.50–1.41 (m, 2H), 1.19–1.10 (m, 2H), 0.98–0.84 (m, 5H), 0.69–0.61 (m, 2H). MS (ES -) m/z: 458.3 (M -1).

5-Cyclopropyl-N-(cyclopropylsulfonyl)-2-fluoro-4-((1-methyl-4(trifluoromethyl)cyclohexyl)methoxy)benzamide (32). Using a method analogous to the preparation of 17, compound 32 was prepared from 68 and cyclopropanesulfonamide as a colorless solid (0.11 g, 61%). 1H NMR (300 MHz, DMSO–d6)  11.82 (s br, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 13.1 Hz, 1H), 3.93 (s, 2H), 3.12-3.03 (m, 1H), 2.30–2.20 (m, 1H), 2.05– 1.96 (m, 1H), 1.87–1.83 (m, 2H), 1.73–1.68 (m, 2H), 1.54–1.41 (m, 2H), 1.34–1.24 (m, 2H), 1.13–1.08 (m, 4H), 1.05 (s, 3H), 0.92–0.85 (m, 2H), 0.69–0.63 (m, 2H). MS (ES+) m/z: 478.1 (M + 1); MS (ES-) m/z: 476.2 (M - 1).

5-Cyclopropyl-N-(cyclopropylsulfonyl)-2-fluoro-4-((6-methylspiro[2.5]octan-6yl)methoxy)benzamide (33). Using a method analogous to the preparation of 17, compound 33 was prepared from 74 and cyclopropanesulfonamide as a colorless solid (0.05 g, 41%). mp 134– 135 C . 1H NMR (300 MHz, CDCl3) δ 8.70 (d, J = 16.3 Hz, 1H), 7.62 (d, J = 9.1 Hz, 1H), 6.59 (d, J = 14.5 Hz, 1H), 3.76 (s, 2H), 3.16–3.06 (m, 1H), 2.09–1.95 (m, 1H), 1.69–1.40 (m, 7H), 1.28–1.02 (m, 7H), 0.98–0.79 (m, 3H), 0.69–0.62 (m, 2H), 0.32–0.18 (m, 4H);

13C

NMR (75

MHz, CDCl3) δ 163.9 (d, J C-F = 11 Hz), 162.2, 161.2 (d, J C-F = 3 Hz), 158.9, 130.0 (d, J C-F = 3 Hz), 128.6 (d, J C-F = 3 Hz), 108.9 (d, J C-F = 10 Hz), 98.7 (d, J C-F = 29 Hz), 34.4, 33 .5, 31.5, 30.9, 22.2, 18.7, 11.9, 11.9, 9.4, 7.1, 6.4. MS (ES+) m/z: 436.2 (M + 1); MS (ES-) m/z: 434.2 (M - 1). Anal. Calcd. for C23H30FNO4S·0.2H2O: C, 62.91; H, 6.98; N, 3.19; Found: C, 62.73; H, 7.11; N, 3.20.


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5-Chloro-2,4-difluoro-N-(methylsulfonyl)benzamide (37). To a solution of 5-chloro-2,4difluorobenzoic acid (34) (15.0 g, 77.9 mmol) in anhydrous CH2Cl2 (250 mL) was added EDCI (22.6 g, 117.8 mmol), DMAP (21.6 g, 176.8 mmol), and MeSO2NH2 (11.4 g, 119.0 mmol) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was quenched by addition of 1M HCl solution (50 mL). The organic layer was separated and washed with brine; dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo, the residue was crystallized from EtOAc and hexanes (1:4, by volume) to give 37 as a colorless solid (12.3 g, 58%). 1H NMR (300 MHz, DMSO–d6)  12.40 (s br, 1H), 8.02–7.97 (m, 1H), 7.76–7.70 (m, 1H), 3.37 (s, 3H). MS (ES-) m/z: 268.1, 270.1 (M - 1).

3-Chloro-4-fluoro-N-(methylsulfonyl)benzamide (38). Using a method analogous to the preparation of compound 37, 38 was prepared from 5-chloro-4-difluorobenzoic acid (35) and MeSO2NH2 as a colorless solid (13.8 g, 96%). 1H NMR (300 MHz, DMSO–d6)  12.27 (s br, 1H), 8.21–8.18 (m, 1H), 8.00–7.95 (m, 1H), 7.62–7.56 (m, 1H), 3.38 (s, 3H).

2,4,5-Trifluoro-N-(methylsulfonyl)benzamide (39). To a solution of MeSO2NH2 (0.49 g, 5.14 mmol) and Et3N (4.3 mL, 30.8 mmol) in anhydrous CH3CN (15 mL) was added 2,4,5trifluorobenzoyl chloride (36) (1.00 g, 5.14 mmol) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was quenched by addition of 1M HCl (20 mL), and extracted with EtOAc (2 x 50 mL). The combined organic extracts was washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo, and the residue was crystallized from EtOAc and hexanes to give 39 as a light-brown solid (0.7 g, 54%). 1H NMR


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(300 MHz, CDCl3)  8.76 (d, J = 14.2 Hz, 1H), 8.00–7.92 (m, 1H), 7.15–7.06 (m, 1H), 3.43 (s, 3H). MS (ES-) m/z: 252.0 (M –1).

1-((4-Bromo-2,5-dichlorophenoxy)methyl)adamantine (41). To a mixture of 4-bromo-2,5dichlorophenol (40) (1.35 g, 5.58 mmol) and K2CO3 (0.85 g, 6.14 mmol) in DMF (10 mL) was added adamantan-1-ylmethyl methanesulfonate (1.50 g, 6.14 mmol). After stirring at 65 °C for 16 h and at 125 °C for 48 h, the reaction mixture was cooled to room temperature, diluted with water (40 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extract was washed with brine (50 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography eluted with a gradient of 5% to 10% EtOAc in hexanes to afford 41 as a colorless solid (1.70 g, 71%). 1H

NMR (300 MHz, CDCl3)  7.57 (s, 1H), 6. 96 (s, 1H), 3.51 (s, 2H), 2.05–1.97 (m, 3H), 1.81–

1.61 (m, 12H). 4-(Adamantan-1-ylmethoxy)-2-fluoro-5-methylbenzoic acid (43). To a solution of 1adamantane methanol (2.40 g, 14.4 mmol) in anhydrous DMSO (20 ml) was added t-BuOK (4.86 g, 43.3 mmol). After stirring at room temperature for 30 min, 5-methyl-2,4-difluorobenzoic acid (42) (2.50 g, 14.40 mmol) was added to the reaction mixture and stirred at 50 ºC for 72 h. The reaction mixture was cold to room temperature and acidified to pH = 1 with ice-cold 1M HCl solution, followed by addition of saturated NH4Cl solution (100 mL). The solid was collected by filtration and washed with H2O. The crude product was crystallized from EtOAc and hexanes to afford 43 as a colorless solid (0.77 g, 17%). 1H NMR (300 MHz, DMSO-d6)  12.70 (s br, 1H), 7.64 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 13.2 Hz, 1H), 3.60 (s, 2H), 2.14 (s, 3H), 1.99 (s br, 3H), 1.74–1.63 (m, 12H). MS (ES+) m/z: 437.2 (M +1).


45

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tert-Butyl 5-chloro-2,4-difluorobenzoate (44). To a solution of 34 (10.0 g, 51.93 mmol) and DMAP (0.02 g, 0.16 mmol) in t-BuOH (60 mL) was added di-tert-butyl dicarbonate (23.35 g, 106.98 mmol). The reaction mixture was heated at 50 °C for 16 h, and concentrated in vacuo. The residue was diluted with EtOAc (200 mL), washed with 1M HCl (50 mL), water and brine. Dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give 44 as a pale yellow oil (12.9 g, 99%). 1H NMR (300 MHz, CDCl3)  7.94 (t, J = 7.8 Hz, 1H), 6.95 (t, J = 9.4 Hz, 1H), 1.59 (s, 9H). tert-Butyl 4-(adamantan-1-ylmethoxy)-5-chloro-2-fluorobenzoate (45). To a mixture of 1adamantanemethanol (25.8 g, 0.155 mol) and 44 (50.0 g, 0.201 mol) in DMSO (250 mL) was added Cs2CO3 (109 g, 0.309 mol). The reaction mixture was heated at 70 °C for 20 h, then cooled to room temperature and filtered through a pad of celite®, and washed with EtOAc (600 mL). The filtrate was washed with water and brine; dried over anhydrous Na2SO4, filtered. The filtrate was concentrated in vacuo. The residue was triturated in methanol (300 mL) and filtered to afford 45 as a pale yellow solid (37.8 g, 62%). 1H NMR (300 MHz, CDCl3)  7.84 (d, J = 7.8 Hz, 1H), 6.59 (d, J = 12.3 Hz, 1H), 3.53 (s, 2H), 2.01 (s, 3H), 1.78–1.61 (m, 12H), 1.55 (s, 9H). tert-Butyl 4-(adamantan-1-ylmethoxy)-2-fluoro-5-vinylbenzoate (46). To a mixture of 45 (2.00 g, 5.06 mmol), vinylboronic acid pinacol ester (1.56 g, 10.1 mmol) and Na2CO3 (1.61 g, 15.2 mmol) in 1, 4-dioxane (20 mL) and water (5 mL) was added , t-Bu3P∙HBF4, (0.15 g, 0.51 mmol) and Pd2(dba)3 (0.023 g, 0.025 mmol) under a nitrogen atmosphere. The reaction mixture was heated to 100 C for 24 h, and then cooled to room temperature. Water (50 mL) was added and the mixture was extracted with EtOAc (3 x 100 mL). The combined organic extract was washed with brine; dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified over column chromatography eluted with 30% CH2Cl2 in


46


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hexanes to give 46 as a pale yellow solid (1.80 g, 92%). 1H NMR (300 MHz, CDCl3)  7.95 (d, J = 9.0 Hz, 1H), 6.98–6.86 (m, 1H), 6.52 (d, J = 12.0 Hz, 1H), 5.76 (d, J = 18.0 Hz, 1H), 5.26 (d, J = 9.0 Hz, 1H), 3.51 (s, 2H), 1.78–1.62 (m, 12H), 1.58–1.52 (m, 3H), 1.22 (s, 9H). MS (ES+) m/z: 387.2 (M + 1).

4-(Adamantan-1-ylmethoxy)-2-fluoro-5-vinylbenzoic acid (47). To a solution of 46 (1.80 g, 4.65 mmol) in CH2Cl2 (10 mL) was added TFA (10 ml). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. The residue was triturated in hexanes (50 mL), and the solid was collected by filtration to give 47 as a pale yellow solid (1.50 g, 97%). 1H

NMR (300 MHz, DMSO–d6)  12.94 (s br, 1H), 7.92 (d, J = 8.6 Hz, 1H), 6.96–6.81 (m, 2H),

5.84–5.78 (m, 1H), 5.32–5.28 (m, 1H), 3.62 (s, 2H), 1.95 (s, 3H), 1.70–1.60 (m, 12H). MS (ES+) m/z: 33 1.2 (M + 1); MS (ES-) m/z: 329.2 (M - 1).

4-(Adamantan-1-ylmethoxy)-5-ethyl-2-fluorobenzoic acid (48). A mixture of 47 (1.00 g, 3.03 mmol) in EtOAc (50 mL) was hydrogenated over 10% palladium on activated carbon (0.10 g) with atmosphere hydrogen at room temperature for 42 h. The mixture was filtered through a pad of celite®, and washed with EtOAc (50 mL). The filtrate was concentrated in vacuo, and the residue was recrystallized from EtOAc and hexanes to afford 48 as a colorless solid (0.75 g, 74%). 1H NMR (300 MHz, DMSO–d6)  12.80 (s br, 1H), 7.63 (d, J = 8.6 Hz, 1H), 6.87 (d, J = 13.2 Hz, 1H), 3.61 (s, 2H), 5.57 (d, J = 7.5 Hz, 2H), 1.99 (s, 3H), 1.75–1.63 (m, 12H), 1.14 (t, J = 7.5 Hz, 3H). MS (ES+) m/z: 33 3.2 (M + 1).


47

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tert-Butyl 4-(adamantan-1-ylmethoxy)-2-fluoro-5-(prop-1-en-2-yl)benzoate (49). Using a method analogous to the preparation of 46, compound 49 was prepared from 45 and 2isopropenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a colorless solid (1.94 g, 64%). 1H NMR (300 MHz, CDCl3)  7.69 (d, J = 8.7 Hz, 1H), 6.54 (d, J = 13.2 Hz, 1H), 5.17-5.13 (m, 1H), 5.10–5.07 (m, 1H), 3.50 (s, 2H), 2.15 (s, br, 3H), 2.02 (s br, 3H), 1.81–1.62 (m, 12H), 1.58 (s, 9H). MS (ES +) m/z: 401.2 (M + 1).

tert-Butyl 4-(adamantan-1-ylmethoxy)-2-fluoro-5-isopropylbenzoate (50). A mixture of 49 (1.94 g, 4.84 mmol) in EtOAc (50 mL) was hydrogenated over 10% palladium on activated carbon (0.19 g) with atmosphere hydrogen at room temperature for 16 h, filtered through a pad of celite®, and concentrated in vacuo to afford 50 as a pale yellow oil (1.84 g, 94%). 1H NMR (300 MHz, CDCl3)  7.69 (d, J = 8.7 Hz, 1H), 6.50 (d, J = 12.9 Hz, 1H), 3.49 (s, 2H), 3.30– 3.18 (m, 1H), 2.04 (s br, 3H), 1.83–1.64 (m, 12H), 1.58 (s, 9H), 1.22 (d, J = 6.9 Hz, 1H). MS (ES +) m/z: 403.3 (M + 1).

4-(Adamantan-1-ylmethoxy)-2-fluoro-5-isopropylbenzoic acid (51). Using a method analogous to the preparation of 47, compound 51 was prepared from 50 as a colorless solid (1.31 g, 84 %). 1H NMR (300 MHz, DMSO–d6)  12.82 (s br, 1H), 7.61 (d, J = 8.7 Hz, 1H), 6.84 (d, J = 13.2 Hz, 1H), 3.58 (s, 2H), 3.22–3.10 (m, 1H), 1.96 (s, 3H), 1.75–1.59 (m, 12H), 1.15 (d, J = 6.9 Hz, 6H). MS (ES +) m/z: 347.2 (M +1).

tert-Butyl

5-chloro-4-(cyclohexylmethoxy)-2-fluorobenzoate

(52).

Using

a

method

analogous to the preparation of 45, compound 52 was prepared from 44 and cyclohexylmethanol


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as a colorless oil (15.37 g, 43 %). 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 7.5 Hz, 1H), 6.61 (d, J = 12.0 Hz, 1H), 3.81 (d, J = 6.0 Hz, 2H), 1.90–1.68 (m, 6H), 1.57 (s, 9H), 1.39–1.04 (m, 5H).

tert-Butyl

5-cyclopropyl-4-((4,4-difluoro-cyclohexyl)methoxy)-2-fluorobenzoate

(53).

Using a method analogous to the preparation of 45, compound 53 was prepared from 44 and (4,4-difluorocyclohexyl)methanol as a pale yellow oil (5.9 g, 80%). 1H NMR (300 MHz, CDCl3)  7.88 (d, J = 7.8 Hz, 1H), 6.62 (d, J = 12.0 Hz, 1H), 3.88 (d, J = 6.0 Hz, 2H), 2.23–1.92 (m, 7H), 1.57 (m, 9H), 1.50–1.42 (m, 2H). tert-Butyl 5-chloro-4-((4,4-difluoro-1-methylcyclohexyl)methoxy)-2-fluorobenzoate (54). Using a method analogous to the preparation of 45, compound 54 was prepared from 44 and (4,4-difluoro-1-methylcyclohexyl)methanol as a colorless oil (3.69 g, 51%). 1H NMR (300 MHz, CDCl3)  7.88 (d, J = 7.5 Hz, 1H), 6.62 (d, J = 12.0 Hz, 1H), 3.74 (s, 2H), 2.06–1.55 (m, 17H), 1.16 (s, 3H). MS (ES +) m/z: 393.1 (M +1).

tert-Butyl

5-chloro-4-((1-ethyl-4,4-difluorocyclohexyl)methoxy)-2-fluorobenzoate

(55).

Using a method analogous to the preparation of 45, compound 55 was prepared from 44 and (4,4-difluoro-1-ethylcyclohexyl)methanol as colorless oil (5.97 g, 77 %). 1H NMR (300 MHz, CDCl3)  7.88 (d, J = 7.8 Hz, 1H), 6.63 (d, J = 11.7 Hz, 1H), 3.78 (s, 2H), 2.02–1.86 (m, 4H), 1.74–1.67 (m, 4H), 1.59–1.57 (m, 11H), 0.87 (t, J = 7.51 Hz, 3H). MS (ES +) m/z: 351.2 (M 56).


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tert-Butyl-5-chloro-2-fluoro-4-((1-methyl-4-(trifluoromethyl)cyclohexyl)methoxy)benzoate (56). Using a method analogous to the preparation of 45, compound 56 was prepared from 44 and (1-methyl-4-(trifluoromethyl)cyclohexyl)methanol as a colorless oil (6.20 g, 95 %). 1H NMR (300 MHz, CDCl3)  7.88 (d, J = 7.7 Hz, 1H), 6.65 (d, J = 12.0 Hz, 1H), 3.85 (s, 2H), 1.96–1.93 (m, 2H), 1.85–1.79 (m, 2H), 1.58 (s, 9H), 1.53–1.39 (m, 3H), 1.34–1.23 (m, 2H), 1.11 (s, 3H).

tert-Butyl 4-(adamantan-1-ylmethoxy)-5-cyclopropyl-2-fluorobenzoate (57). To a mixture of 45 (15.8 g, 40.0 mmol), cyclopropylboronic acid (5.16 g, 60.0 mmol), K3PO4 (38.2 g, 180.0 mmol) in toluene (160 mL) and H2O (8 mL) under a nitrogen atmosphere was added Cy3P∙HBF4 (1.47 g, 3.99 mmol) and Pd(OAc)2 (0.45 g, 2.00 mmol). The reaction mixture was heated to 100 °C for 18 h and then cooled to room temperature. H2O (100 mL) was added, and the mixture was extracted with EtOAc (3 x 100 mL), the combined organic extract was washed with brine; dried over anhydrous Na2SO4 and filtrated. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography eluted with 5% ethyl acetate in hexanes afforded 57 as a colorless solid (13.8 g, 86%). 1H NMR (300 MHz, CDCl3)  7.37 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 12.9 Hz, 1H), 3.49 (s, 2H), 2.05–1.95 (m, 4H), 1.78–1.61 (m, 12H), 1.55 (s, 9H), 0.91–0.84 (m, 2H), 0.64–0.58 (m, 2H).

tert-Butyl 4-(cyclohexylmethoxy)-5-cyclopropyl-2-fluorobenzoate (58). Using a method analogous to the preparation of 57, compound 58 was prepared from 52 as a colorless oil (13.73 g, 88 %). 1H NMR (300 MHz, CDCl3) δ 7.36 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 12.9 Hz, 1H), 3.78


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(d, J = 5.7 Hz, 2H), 2.07–1.98 (m, 1H), 1.92–1.69 (m, 6H), 1.56 (s, 9H), 1.39–1.02 (m, 5H), 0.93–0.85 (m, 2H), 0.67–0.61 (m, 2H). MS (ES+) m/z: 387.2 (M + 39).

tert-Butyl

5-cyclopropyl-4-((4,4-difluorocyclohexyl)methoxy)-2-fluorobenzoate

(59).

Using a method analogous to the preparation of 57, compound 59 was prepared from 53 and cyclopropylboronic acid as a pale yellow oil (5.37 g, 86%). 1H NMR (300 MHz, CDCl3)  7.39 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 12.6 Hz, 1H), 3.85 (d, J = 5.7 Hz, 2H), 2.24–1.91 (m, 8H), 1.59– 1.47 (m, 11H), 0.94–0.84 (m, 2H), 0.70–0.58 (m, 2H). MS (ES-) m/z: 383.0 (M - 1).

tert-Butyl 5-cyclopropyl-4-((4,4-difluoro-1-methylcyclohexyl)-methoxy)-2-fluorobenzoate (60). Using a method analogous to the preparation of 57, compound 60 was prepared from 54 and cyclopropylboronic acid as a colorless oil (3.69 g, 99 %). 1H NMR (300 MHz,CDCl3)  7.42 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 12.6 Hz, 1H), 3.72 (s, 2H), 2.06–1.73 (m, 8H), 1.65–1.62 (m, 1H), 1.57 (s, 9H), 1.15 (s, 3H), 0.94–0.85 (m, 2H), 0.66–0.59 (m, 2H). MS (ES +) m/z: 399.2 (M + 1).

tert-Butyl-5-cyclopropyl-4-((1-ethyl-4,4-difluorocyclohexyl)methoxy)-2-fluorobenzoate (61). Using a method analogous to the preparation of 57, compound 61 was prepared from 55 and cyclopropylboronic acid as colorless oil (5.66 g, 94 %). 1H NMR (300 MHz, CDCl3)  7.41 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 12.6 Hz, 1H), 3.74 (s, 2H), 2.02–1.86 (m, 5H), 1.82–1.60 (m, 6H), 1.55 (s, 9H), 0.92–0.82 (m, 5H), 0.65–0.56 (m, 2H). MS (ES +) m/z: 413.1 (M + 1).


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tert-butyl

5-cyclopropyl-2-fluoro-4-((1-methyl-4-

(trifluoromethyl)cyclohexyl)methoxy)benzoate (62). Using a method analogous to the preparation of 45, compound 62 was prepared from 56 and cyclopropylboronic acid as a pale yellow oil (1.66 g, 96 %). 1H NMR (300 MHz, CDCl3)  7.42 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 12.6 Hz, 1H), 3.81 (s, 2H), 2.00–1.95 (m, 3H), 1.84–1.79 (m, 2H), 1.57 (s, 9H), 1.55–1.41 (m, 3H), 1.33 –1.22 (m, 2H), 1.11 (s, 3H), 0.91–0.85 (m, 2H), 0.64–0.59 (m, 2H).

4-(Adamantan-1-ylmethoxy)-5-cyclopropyl-2-fluorobenzoic acid (63). Using a method analogous to the preparation of 47, compound 63 was prepared from 57 as a colorless solid (10.1 g, 85%). 1H NMR (300 MHz, DMSO–d6)  12.77 (s br, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 13.2 Hz, 1H), 3.59 (s, 2H), 2.04–1.92 (m, 4H), 1.71–1.58 (m, 12H), 0.91–0.83 (m, 2H), 0.59– 0.52 (m, 2H). 4-(Cyclohexylmethoxy)-5-cyclopropyl-2-fluorobenzoic acid (64). Using a method analogous to the preparation of 47, compound 64 was prepared from 58 as a colorless solid (6.64 g, 58 %). 1H

NMR (300 MHz, DMSO–d6)  12.79 (s br, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 13.2

Hz, 1H), 3.88 (d, J = 5.7 Hz, 2H), 2.06–1.96 (m, 1H), 1.86–1.62 (m, 6H), 1.34–1.02 (m, 5H), 0.93–0.85 (m, 2H), 0.62–0.55 (m, 2H). MS (ES-) m/z: 291.3 (M - 1). 5-Cyclopropyl-4-((4,4-difluorocyclohexyl)-methoxy)-2-fluorobenzoic acid (65). Using a method analogous to the preparation of 47, compound 65 was prepared from 59 as a colorless solid (2.29 g, 50 %). 1H NMR (300 MHz, DMSO-d6)  12.83 (s br, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 13.2 Hz, 1H), 3.96 (d, J = 6.0 Hz, 2H), 2.10–1.74 (m, 8H), 1.47–1.33 (m, 2H), 0.92– 0.84 (m, 2H), 0.61–0.54 (m, 2H). MS (ES+) m/z: 329.1 (M + 1).


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5-Cyclopropyl-4-((4,4-difluoro-1-methylcyclohexyl)methoxy)-2-fluorobenzoic acid (66). Using a method analogous to the preparation of 47, compound 66 was prepared from 60 as a colorless solid (2.74 g, 86 %). 1H NMR (300 MHz, CDCl3)  7.56 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 12.9 Hz, 1H), 3.75 (s, 2H), 2.08–1.75 (m, 7H), 1.66–1.55 (m, 2H), 1.16 (s, 3H), 0.97–0.88 (m, 2H), 0.68–0.61 (m, 2H). MS (ES+) m/z: 343.2 (M + 1).

5-Cyclopropyl-4-((1-ethyl-4,4-difluorocyclohexyl)methoxy)-2-fluorobenzoic

acid

(67).

Using a method analogous to the preparation of 47, compound 67 was prepared from 61 as a colorless solid (3.497 g, 72 %). 1H NMR (300 MHz, CDCl3)  9.31 (s br, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 12.6 Hz, 1H), 3.77 (s, 2H), 2.02-1.85 (m, 5H), 1.82–1.57 (m, 6H), 0.96– 0.84 (m, 5H), 0.67–0.59 (m, 2H). MS (ES +) m/z: 357.2 (M + 1).

5-Cyclopropyl-2-fluoro-4-((1-methyl-4-(trifluoromethyl)cyclohexyl)methoxy)benzoic acid (68). Using a method analogous to the preparation of 47, compound 68 was prepared from 62 as a colorless solid (1.30 g, 81 %). 1H NMR (300 MHz, DMSO–d6)  12.84 (s br, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 13.2 Hz, 1H), 3.92 (s, 2H), 2.26–2.20 (m, 1H), 2.04–1.95 (m, 1H), 1.87–1.83 (m, 2H), 1.72–1.67 (m, 2H), 1.54–1.41 (m, 2H), 1.34–1.23 (m, 2H), 1.05 (s, 3H), 0.91–0.85 (m, 2H), 0.60–0.55 (m, 2H). MS (ES-) m/z: 373.2 (M - 1).

tert-Butyl 5-chloro-2-fluoro-4-((8-methyl-1,4-dioxaspiro[4.5]decan-8-yl)methoxy)benzoate (69). Using a method analogous to the preparation of 45, compound 69 was prepared from 44


53

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and (8-methyl-1,4-dioxaspiro[4.5]decan-8-yl)methanol as a colorless oil (5.13 g, 54%). 1H NMR (300 MHz, CDCl3)  7.87 (d, J = 7.9 Hz, 1H), 6.62 (d, J = 12.3 Hz, 1H), 3.95 (s, 4H), 3.74 (s, 2H), 1.76–1.60 (m, 8H), 1.58 (s, 9H), 1.15 (s, 3H). MS (ES+) m/z: 359.1 (M - 55).

tert-Butyl-5-cyclopropyl-2-fluoro-4-((8-methyl-1,4-dioxaspiro[4.5]decan-8yl)methoxy)benzoate (70). Using a method analogous to the preparation of 57, compound 70 was prepared from 69 and cyclopropylboronic acid as a colorless oil (2.59 g, 83%). 1H NMR (300 MHz, CDCl3)  7.41 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 12.7 Hz, 1H), 3.95 (s, 4H), 3.71 (s, 2H), 2.05–1.94 (m, 1H), 1.77–1.60 (m, 8H), 1.57 (s, 9H), 1.14 (s, 3H), 0.93–0.85 (m, 2H), 0.65– 0.59 (m, 2H). MS (ES+) m/z: 365.2 (M - 55).

tert-Butyl-5-cyclopropyl-2-fluoro-4-((1-methyl-4-oxocyclohexyl)methoxy)benzoate

(71).

To a solution of 70 (2.59 g, 6.16 mmol) in THF (6 mL) and H2O (4.3 mL) was added TFA (2.2 mL). After stirring at room temperature for 18 h, the reaction was quenched by addition of 2 M aqueous NaOH solution (15 mL), and then extracted with EtOAc (3 x 50 mL). The combined organic extract was washed with H2O and brine; dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography eluted with a gradient of 0% to 15% EtOAc in hexanes to afford 71 as a colorless oil (2.09 g, 90%). 1H NMR (300 MHz, CDCl3)  7.42 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 12.5 Hz, 1H), 3.82 (s, 2H), 2.56–2.36 (m, 4H), 2.05–1.78 (m, 5H), 1.57 (s, 9H), 1.30 (s, 3H), 0.92–0.86 (m, 2H), 0.65–0.60 (m, 2H). MS (ES+) m/z: 321.2 (M - 55).


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tert -Butyl 5-cyclopropyl-2-fluoro-4-((1-methyl-4-methylenecyclohexyl)methoxy)benzoate (72). To a cooled (-20 ˚C) suspension of methyltriphenylphosphonium bromide (1.44 g, 4.02 mmol) in THF (12 mL) was added LiHMDS (1.0M solution in THF, 4.0 mL, 4.00 mmol). After stirring at -20 C for 90 min, a solution of 71 (1.00 g, 2.68 mmol) in THF (6 mL) was added. The reaction mixture was slowly warmed to room temperature and stirred for 12 h. The reaction was quenched by addition of saturated aqueous NH4Cl solution (20 mL) and diluted with ethyl acetate (100 mL). The organic layer was separated and the aqueous was extracted with EtOAc (3 x 30 mL). The combined organic extract was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography eluted with a gradient of 0% to 5% EtOAc in hexanes to afford 72 as a colorless oil (0.83 g, 82%). 1H NMR (300 MHz, CDCl3)  7.41 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 12.7 Hz, 1H), 4.65 (s, 2H), 3.71 (s, 2H), 2.30–2.15 (m, 4H), 2.05–1.93 (m, 1H), 1.68–1.50 (m, 13H), 1.15 (s, 3H), 0.93–0.86 (m, 2H), 0.66–0.60 (m, 2H). MS (ES+) m/z: 319.1 (M - 55).

tert-Butyl-5-cyclopropyl-2-fluoro-4-((6-methylspiro[2.5]octan-6-yl)methoxy)benzoate (73). To a cooled (0 ˚C) solution of 72 (0.30 g, 0.80 mmol) and chloroiodomethane (0.21 mL, 2.65 mmol) in 1,2-dichloroethane (2 mL) was added by diethylzinc (1.0 M solution in hexanes, 1.33 mL, 1.33 mmol). After stirring at 0 ˚C for 2 h, the reaction was quenched by addition of 1N HCl solution (5 mL), and extracted with CH2Cl2 (3 x 30 mL). The combined organic extract was dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography eluted with a gradient of 0% to 5% EtOAc in hexanes to afford 73 as a colorless oil (0.29 g, 93%). 1H NMR (300 MHz, CDCl3)  7.41 (d, J =


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8.5 Hz, 1H), 6.52 (d, J = 12.8 Hz, 1H), 3.72 (s, 2H), 2.09–1.94 (m, 1H), 1.73–1.42 (m, 14H), 1.19–1.03 (m, 5H), 0.97–0.84 (m, 3H), 0.70–0.59 (m, 2H), 0.33 –0.16 (m, 4H). MS (ES+) m/z: 33 3.2 (M - 55).

5-Cyclopropyl-2-fluoro-4-((6-methylspiro[2.5]octan-6-yl)methoxy)benzoic acid (74). To a cooled (0 C) solution of 73 (0.28 g, 0.71 mmol) and anisole (0.11 mL, 1.07 mmol) in CH2Cl2 (3 mL) was added TFA (0.75 mL, 9.61 mmol). After stirring for 4 h at 0 C, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with H2O (5 x 10mL) until the last wash was neutral as monitored by pH paper. The organic layer was dried over anhydrous Na2SO4 sulfate and filtered. The filtrate was concentrated in vacuo to afford 74 as a colorless solid (0.21 g, 90%). 1H NMR (300 MHz, CDCl3)  7.48 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 13.0 Hz, 1H), 3.71 (s, 2H), 2.08–1.90 (m, 1H), 1.78–1.38 (m, 6H), 1.17–1.02 (m, 4H), 0.95–0.80 (m, 3H), 0.68–0.56 (m, 2H), 0.33 –0.13 (m, 4H). MS (ES-) m/z: 33 1.3 (M - 1).

Molecular Modeling. Induced fit docking of compound 7 to the hNaV1.7 VSD4 active site. Using the X-ray structure of NaV1.726 as the source, chain B of the protein was first isolated and prepped using the Protein Preparation Wizard.46 Compound 7 was docked into the VSD4 active site via induced fit docking46b,47 using default settings which optimized side chains within 5Å of the ligand in addition to docking. The centroid of the co-crystal structure ligand GX-936 was used as the center of the grid box. A single receptor-ligand model was chosen from the resulting set through visual inspection. Compound 7 was then minimized in the active site.


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Use of Animal Subjects. All studies involving animals were either approved by the Animal Care Committee of Xenon Pharmaceuticals (Burnaby, BC, Canada) and performed in compliance with the Canadian Council on Animal Care guidelines (mouse efficacy and mouse PK studies) or approved by and conformed to the guidelines and principles set by the Institutional Animal Care and Use Committee of Genentech.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Description of in vitro biological assays, in vivo rodent pain assays, acute dissociation of mouse DRG neuron and patch clamp recording, CEREP screening data for compound 33, 1HNMR

spectra of compounds 7–33 , HPLC purity data for compounds 7, 20, 26, 30, and

33, experimental procedure and small molecules X-Ray coordinates for compound 7 (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone 604-484-3336 *E-mail: [email protected]. Phone 650-225-2749


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Present Addresses For

S.C.: Syrachem Sciences Inc, 7600 Glover Road, Langley, BC Canada.

ForT.S.:

Inception Sciences Canada, 887 Great Northern Way, Suite 210, Vancouver, BC V5T

4T5, Canada. For ○For

C.C.: WuXi AppTec Co., Ltd., 288 FuTe Zhong Road, Shanghai 200131, P. R. China. A.W.: College of Pharmacy, University of Michigan, 428 Church St, Ann Arbor, MI

48109-1065, United States. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors thank Eric Kam for assistant in preparation of compounds 9, 12 and 13; Noah Stuart, Sam Goodchild for additional EP data and Rhena Yoo for assistance with ligand binding assay test. We also thank Bruce Roth, Tarek S. Mansour, Renata M. Oballa, and the XenonGenentech JRC for their leadership and support. In addition, we want to thank James Empfield and Steven Wesolowski for helpful revisions of this manuscript. ABBREVIATIONS AUC, area under curve; BCRP, breast cancer resistance protein; BID, twice a day; CIP, congenital insensitivity to pain; Cmax, maximal concentration; CL, plasma clearance; CLhep hepatic clearance; cLogP, calculated logarithm of partition coefficient; CNS, central nervous system; CYP, Cytochromes P450; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide;


58


DMSO,

dimethyl

sulfoxide;

DRG,

dorsal

root

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ganglion;

EDCI,

1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide; EP, electro-physiology; F, oral bioavailability; HH, human hepatocyte; HEK, human embryonic kidney; hERG, human Ether-a-go-go Related Gene; HLM, human liver microsome; IEM, inherited erythromelalgia; LBA, ligand binding assay; LLE, lipophilic ligand efficiency; MCT, 0.5% w/w methylcellulose and 0.2% v/v Tween-80 in water; MDCKI, Madin-Darby canine kidney cell line I; MDRI, multi-drug resistant protein I, also refer to P-glycoprotein; MetID, metabolite identification; mLogD, measured distribution coefficient at pH7.2; NaV1.7, voltage-gated sodium channel type 7; NaV1.5, voltage-gated sodium channel type 5; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0); PCy3, tricyclohexylphosphine; PEPD, paroxysmal extreme pain disorder; PK, pharmacokinetic; PNS, peripheral nervous system; PX, PatchXpress; PPB, plasma protein binding; SCN9Agene encoding the sodium channel NaV1.7; SAR, structure-activity relationship; TFA, trifluoroacetic acid; t½, terminal half-life; VSD4, voltage sensing domain 4; Vss, volume of distribution.

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Table of Contents Graphic 254x190mm (96 x 96 DPI)


Identification of selective acyl sulfonamide-cycloalkylether inhibitors of

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