Design and Optimization of Sulfone Pyrrolidine ... - ACS Publications

Article Cite This: J. Med. Chem. 2018, 61, 11209−11220

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Design and Optimization of Sulfone Pyrrolidine Sulfonamide Antagonists of Transient Receptor Potential Vanilloid‑4 with in Vivo Activity in a Pulmonary Edema Model Joseph E. Pero,*,† Jay M. Matthews,‡ David J. Behm,‡ Edward J. Brnardic,‡ Carl Brooks,‡ Brian W. Budzik,† Melissa H. Costell,‡ Carla A. Donatelli,‡ Stephen H. Eisennagel,‡ Karl Erhard,† Michael C. Fischer,§ Dennis A. Holt,‡ Larry J. Jolivette,‡ Huijie Li,† Peng Li,† John J. McAtee,‡ Brent W. McCleland,† Israil Pendrak,† Lorraine M. Posobiec,§ Katrina L.K. Rivera,‡ Ralph A. Rivero,† Theresa J. Roethke,‡ Matthew R. Sender,† Arthur Shu,† Lamont R. Terrell,‡ Kalindi Vaidya,§ Xiaoping Xu,‡ and Brian G. Lawhorn‡ †

Flexible Discovery Unit, ‡Heart Failure Discovery Performance Unit, and §Platform Technology and Sciences, GlaxoSmithKline, 1250 Collegeville Road, Collegeville, Pennsylvania 19426, United States S Supporting Information *

ABSTRACT: Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternativeand potentially superiortherapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

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INTRODUCTION Despite numerous medical advancements and increased awareness of risk factors, heart disease remains one of the leading causes of death.1 Specifically, approximately 2% of adults in developed countries have experienced congestive heart failure (HF), with this number rising to 6−10% for those exceeding 65 years of age.2 Over a 12-month period postdiagnosis, the risk of death is approximately 35%, which is comparable to that of severe cancers.3 HF and its related side effects also pose a significant economic burden on society, with total associated costs in the United States alone projected to increase from $31 billion in 2012 to $70 billion in 2030.4 HF is caused by the inability of an impaired left ventricle to pump sufficient blood into peripheral circulation. This results in elevated pulmonary venous pressure (PVP) and disrupts the septal barrier, which separates the circulatory aqueous environment from the alveolar airspaces.5,6 With an increased flow of fluid from pulmonary circulation into the alveolar space, HF patients frequently suffer from pulmonary edema, © 2018 American Chemical Society

which significantly compromises their quality of life and can prove fatal if left untreated.7 Transient receptor potential vanilloid-4 (TRPV4) receptors are members of the TRP superfamily of cation channels.8 These tetrameric voltage-gated channels are highly expressed in lung endothelial cells9,10 and are upregulated in HF patients.11 Their direct association with pulmonary edema has been implicated by studying the effects of selective TRPV4 antagonist GSK2193874 (compound 1, Figure 1a) in rodents.11 In both acute and chronic preclinical models, pretreatment with GSK2193874 inhibited HF-induced pulmonary edema and increased arterial oxygenation.11 In addition, GSK2193874 was effective at reversing HF-induced pulmonary edema in mice subjected to myocardial infarction for one week prior to treatment.11 Received: August 29, 2018 Published: November 30, 2018 11209

DOI: 10.1021/acs.jmedchem.8b01344 J. Med. Chem. 2018, 61, 11209−11220

Journal of Medicinal Chemistry

Article

Figure 1. Previously-reported TRPV4 antagonists from GSK (a,b) and Pfizer (c).

Figure 2. Scaffold modification of 3 to give pyrrolidines 4 and 5.

Figure 3. (a) Replacement of phenoxy ether functionality with sulfone, leading to 7. (b) Additional stereoisomers of sulfone pyrrolidine diol 7.

line (GSK) include 1-(4-piperidinyl)-benzimidazoles14 and spirocarbamates (2, Figure 1b),15,16 while Pfizer has disclosed azetidine sulfonamides (3, Figure 1c) derived from a highthroughput screen.17 With its promising ligand efficiency (LE)18 and lipophilic ligand efficiency (LLE),19 3 was previously utilized as a starting point in efforts directed toward discovering novel TRPV4 antagonists.20 This led to the discovery of pyrrolidine sulfonamide lead 4, which displayed encouraging in vitro activity (IC50 = 32 nM) at recombinant hTRPV4 channels21 with comparable LE (0.40) and LLE (3.9) relative to 3 (Figure 2). Subsequent scaffold optimization20 led to diol 5, which featured excellent LLE (5.3) due to enhanced TRPV4 activity (IC50 = 5.0 nM) in conjunction with reduced lipophilicity (clogP22 = 3.0).

Importantly, strong evidence indicates that these effects may be relevant to humans. GSK2193874 was found to be potent in recombinant hTRVP4 channels (IC50 = 50 nM) and prevented agonist-driven cellular contraction in human umbilical vein endothelial cells.11 These experimental findings suggest that TRPV4 blockade has the potential to lead to a novel treatment for HF-induced pulmonary edema that could prove more effective than current standards of care (i.e., diuretics, nitroglycerin, angiotensin antagonists), which have been associated with unfortunate side effects including hypotension and deteriorating renal function.12,13 As such, a number of selective TRPV4 antagonists have been disclosed in the recent literature with superior druglike properties (i.e., reduced molecular weight, lower lipophilicity) to GSK2193874. Examples from GlaxoSmithK11210



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despite elevated total clearance (25 mL/min/kg), the mean residence time28 of 7 (MRT = 1.7 h) was nearly equivalent to that of 6 because of its higher volume of distribution (Vdss = 2.3 L/kg). A comparison of the PK properties of 7 with corresponding ether 5 provided additional validation of the sulfone structural class. Ether 5 featured higher total clearance (74 mL/min/kg) and clearance adjusted for unbound fraction (CL/f u = 15) as well as a shorter MRT (0.6 h), potentially due to oxidative metabolism of the phenoxy ether moiety. Furthermore, ether 5 exhibited reduced oral bioavailability (46%) in relation to sulfone 7. In order to facilitate analog preparation, a generalized29 synthetic route toward the sulfone-functionalized pyrrolidine diols was established (Scheme 1). Chiral resolution of known tert-butyl-3-hydroxy-4-methylenepyrrolidine-1-carboxylate30 provided the requisite (S)-isomer 11. Formation of the corresponding mesylate followed by SN2 displacement with an aryl thiol under basic conditions afforded thioether 12. Oxidation with m-CPBA generated sulfone 13 which then underwent dihydroxylation to give diol 14 in high diastereomeric fidelity. Establishing the sulfone moiety prior to treatment with osmium tetroxide proved critical, as dihydroxylation of thioether 12 proceeded with significantly lower diastereoselectivity.31 With advanced intermediate 14 in hand, acid-mediated cleavage of the Boc-carbamate followed by sulfonamide formation under standard conditions generated targeted compounds of generic structure 15. With a superior balance of potency and PK properties being the ultimate goal, the versatility of this synthetic sequence allowed structure−activity relationships of both the aryl sulfonamide and sulfone moieties to be explored (Table 2). Compound 16 features the installation of a fluoro substituent at R2 that proved to be a potency enhancer in the aforementioned azetidine17 and phenoxy ether pyrrolidine20 structural classes. However, while compound 16 indeed demonstrated slight improvements in TRPV4 activity (IC50 = 2.0 nM) and LLE (5.6) relative to its des-fluoro counterpart 7, these benefits were counterbalanced by an erosion in PK properties as exemplified by higher clearance adjusted for unbound fraction (CL/f u = 14), shorter MRT (0.8 h), and reduced oral bioavailability (51%). One possible rationalization of the higher unbound clearance associated with 16 is that its more electrophilic aryl sulfone functionality was subjected to

The present work describes an analogous structural class exemplified by sulfone 6, which was viewed as a lead compound superior to ether 4 because of reduced lipophilicity (cLogP = 2.7) and improved LLE (4.9) (Figure 3a). Design elements toward an optimal balance of TRPV4 activity, selectivity, and pharmacokinetic (PK) properties are detailed. Evaluation of leading compounds in an in vivo rat pulmonary edema assay indicative of therapeutic potential is also provided.

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RESULTS AND DISCUSSION At the outset of our optimization efforts, the potencyenhancing diol of the ether structural class20 was incorporated to afford compound 7, which displayed improvements in both TRPV4 activity (IC50 = 7.9 nM) and LLE (5.2) with only a slight increase in lipophilicity (cLogP = 2.9). Notably, the stereoisomers of 7 were also explored (Figure 3b, compounds 8−10), with each exhibiting considerably weaker activity as measured by a fluorometric imaging plate reader (FLIPR).21 An investigation of the PK properties of 7 further substantiated the diol chemotype (Table 1). Following Table 1. PK Properties (Rat) of Compounds 5, 6, and 7a Cmpd

PSA

Perm (nm/s)

5 6 7

111 116 136

370 780 450

Vdss CL (mL/min/kg) CL/f u (L/kg) 74 9 25

15 30 9

2.4 1.1 2.3

MRT (h)

F (%)

0.6 2.0 1.7

46 100 100

a PSA = total polar surface area; Perm = artificial membrane permeability; CL = total clearance; f u = fraction unbound (rat); Vdss = volume of distribution; MRT = mean resonance time; F = oral bioavailability.

intravenous (i.v.) dosing in rats (1 mpk), 7 exhibited reduced clearance adjusted for unbound fraction23 (CL/f u = 9) relative to parent compound 6 (CL/f u = 30), suggesting that the potency-enhancing diol functionality did not pose an additional metabolic liability. Furthermore, high permeability24 (450 nm/s) and 100% oral bioavailability (F, 2 mpk p.o.) were maintained despite an elevated total polar surface area (PSA = 136). This could be rationalized by a reduction in solventaccessible PSA due to conformational restriction imparted by intramolecular hydrogen bonds, which were confirmed by vibrational circular dichroism (VCD) studies.25−27 In addition, Scheme 1. General Synthesis of Sulfone Pyrrolidine diolsa

a Reagents and conditions: (a) MsCl, triethylamine (TEA), dichloromethane (DCM); (b) arylthiol, K2CO3, N,N-dimethylformamide (DMF) (74− 81% over 2 steps); (c) m-CPBA, DCM (59−89%); (d) OsO4, 4-methylmorpholine N-oxide (NMO), tetrahydrofuran (THF) (81−98%, >95:5 dr); (e) HCl/1,4-dioxane or trifluoroacetic acid (TFA), DCM; (f) arylsulfonyl chloride, NaHCO3, THF (26−85% over 2 steps).

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Table 2. Structure−Activity Relationships of Sulfone Pyrrolidine Diolsa

Cmpd

R1

R2

R3

R4

IC50 (nM)

LLE

PSA

CL (mL/min/kg)

CL/f u

Vdss (L/kg)

MRT (h)

F (%)

7 16 17 18 19

CN CN CN CN Cl

H F H H H

Cl Cl CN Cl Cl

Cl Cl Cl CN CN

7.9 2.0 2.0 3.2 3.2

5.2 5.6 6.8 6.9 5.8

136 136 160 160 136

25 25 82 35 49

9 14 7 4 12

2.3 1.2 2.6 2.2 4.5

1.7 0.8 0.5 1.0 1.6

100 51 25 15 100

Human TRPV4 IC50 values measured by a calcium flux FLIPR assay (see Supporting Information for details); LLE = lipophilic ligand efficiency; PSA = total polar surface area; CL = total clearance; f u = fraction unbound (rat); Vdss = volume of distribution; MRT = mean resonance time; F = oral bioavailability. a

Table 3. Incorporation of Heteroatoms into the Pyrrolidine Diol Scaffolda

Cmpd

X

Y

Z

Z′

R

IC50 (nM)

LLE

PSA

CL (mL/min/kg)

CL/f u

Vdss (L/kg)

MRT (h)

F (%)

19 20 21 22 23 24

CH CH CH N CH CH

CH N N CH CH CH

O O O O NH O

O O O O O NH

Cl H CF3 Cl Cl Cl

3.2 631 20 5.0 20 40

5.8 4.9 6.0 5.7 5.0 4.2

136 149 149 149 143 143

49 22 310 23 52 48

12 1.2 66 11 4.8 8.9

4.5 4.1 3.6 2.0 3.2 2.7

1.6 3.0 0.2 1.4 1.0 0.9

100 62 8 62 38 80


(1.0 h), oral bioavailability remained poor (F = 15%). Given this result, subsequent targets were aimed at reducing PSA in an effort to improve cell permeability and enhance gastrointestinal absorption. To this end, compound 19 featured a single-point modification involving replacement of the cyano substituent on the aryl sulfone with a chloride (R1 = Cl). Relative to parent compound 18, this maintained potency (IC50 = 3.2 nM) while reducing PSA to 136. Despite an increase in both total CL (49 mL/min/kg) and CL/f u (12), MRT was elongated because of an elevated volume of distribution (Vdss = 4.5 L/kg). Gratifyingly, the oral bioavailability improved to 100%, likely driven by enhanced cell permeability (350 nm/s) and reduced oxidative (first-pass) metabolism of the aryl sulfone functionality.32 Our interest in compound 19 was further buoyed by its clean off-target profile. It exhibited high TRP selectivity (TRPA1, TRPV1, TRPM2, TRPM8, TRPC3-C6 IC50 > 30 μM) and weak activity (IC50 > 10 μM) against a panel of receptors including CYPs, hERG, and CaV1.2. Furthermore, it was inactive up to 100 μM in an assay measuring timedependent inhibition (TDI) of CYP3A4.

increased nucleophilic addition by glutathione (GSH), which was suggested by trapping experiments in rat liver microsomes in which only 46% of the parent remained after incubation (30 min). In contrast, analogous studies with compound 7 showed 89% of the parent remaining after incubation. Replacing the ortho-chloro substituent of the aryl sulfonamide with a cyano group (R3 = CN, compound 17) resulted in a fourfold increase in potency (IC50 = 2.0 nM) relative to parent compound 7. While the LLE improved to 6.8, the PK properties in rats diminished. In particular, the MRT decreased to 0.5 h due to a rise in total clearance (CL = 82 mL/min/kg), and the oral bioavailability was only 25%. Metabolite ID studies in rat hepatocytes suggested that this increased metabolism was largely driven by GSH-mediated sulfonamide cleavage. In relation to 17, compound 18 featured a transposition of the chloro and cyano substituents on the aryl sulfonamide. This modification preserved the excellent LLE of 17 as well as reduced total clearance (CL = 35 mL/min/kg) and clearance adjusted for unbound fraction (CL/f u = 4). However, while the enhanced metabolic stability was reflected in a longer MRT 11212



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Table 4. Profiles of Amino Alcohols 25, 26, and 27a

Cmpd

X

stereochem.

IC50 (nM)

LLE

PSA

CL (mL/min/kg)

CL/f u

Vdss (L/kg)

MRT (h)

F (%)

25 26 27

CH N N

3S,4S 3S,4S 3R,4S

0.3 0.5 0.5

7.6 7.4 7.4

142 154 154

12 12 1.7

2.7 3.8 1.7

4.0 1.3 0.2

5.7 1.9 2.2

22 64 56


Scheme 2. Synthesis of Amino Alcohol 25a

Reagents and conditions: (a) 1. TFA, DCM; 2,2-chloro-4-cyanobenzene-1-sulfonyl chloride, TEA, DCM (63%); (b) MsCl, TEA, DCM, 0 °C (88%); (c) 4-chlorobenzenethiol, K2CO3, DMF, (59%); (d) OsO4, NMO, THF (37%); (e) MsCl, TEA, K2CO3, DCM, MeOH (52%); (f) NH3, EtOH, 70 °C, 99%; (g) Boc2O, TEA, THF (82%); (h) m-CPBA, DCM (99%); (i) TFA, DCM (66%).

a

Scheme 3. Synthesis of Amino Alcohols 26 and 27a

a

Reagents and conditions: (a) 1. MsCl, TEA, DCM; 2,5-chloropyridine-2-thiol, K2CO3, DMF (91%); (b) OsO4, NMO, THF [78% (diastereomeric mixture)]; (c) propane-2,2-diol, pyridinium p-toluenesulfonate (PPTS), DCM [38% (26b), 38% (26c)]; (d) 1. TFA, DCM; 2. NaHCO3, THF, H2O, 2-chloro-4-cyanobenzene-1-sulfonyl chloride [82% (26d), 85% (26dd)]; (e) 1. MsCl, TEA, DCM; 2. NaN3, DMF [39% (26e), 83% (26ee)]; (f) 1. PMe3, THF; 2. Boc2O, TEA, THF [67% (26f), 77% (26ff)]; (g) m-CPBA, DCM [98% (26g), 77% (26gg)]; (h) TFA, DCM [54% (26), 50% (27)].

f u = 1.2), and nearly a twofold improvement in MRT (3 h). Unfortunately, however, the TRPV4 potency of 20 (IC50 = 631 nM) was approximately 200-fold weaker than that of parent compound 19. A significant percentage of this activity could be recovered by the reintroduction of a lipophilic ortho substituent on the aryl sulfonamide moiety. In a representative example, compound 21 (R = CF3) demonstrated an in vitro

Additional structural modifications were investigated to reduce lipophilicity in hopes of further enhancing PK properties. This involved the insertion of heteroatoms into the various subunits of the scaffold (Table 3). Compound 20, featuring a pyridyl sulfonamide functionality (Y = N), demonstrated reduced total clearance (CL = 22 mL/min/ kg), outstanding clearance adjusted for unbound fraction (CL/ 11213



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activity (IC50 = 20 nM) and LLE (6.0) comparable to 19. However, 21 also featured a startling erosion of PK properties (CL = 310 mL/min/kg, CL/f u = 66, MRT = 0.2 h, F = 8%), prompting us to shift our attention to other subunits. Installation of a heteroaromatic sulfone proved to be better tolerated in regard to both TRPV4 activity and PK properties. The most encouraging results were attained with a 2-pyridyl sulfone, in which compound 22 displayed nearly identical TRPV4 potency (IC50 = 5 nM), LLE (5.7), and MRT (1.4 h) relative to direct comparator 19. Notably, oral bioavailability was reduced (F = 62%), possibly due to limited membrane permeability caused by an increase in total PSA (PSA = 149). Structural diversity was further broadened by the exploration of sulfoximine (compound 2333 ) and sulfonimidamide (compound 2434) analogs. While the latter featured excellent oral bioavailability in rats (F = 80%), neither exemplars offered an advantage over parent compound 19 in terms of TRPV4 activity, LLE, or PK properties (CL, MRT, and F). With the inability of compounds 20−24 to improve on the overall profile of compound 19, focus was subsequently shifted toward modifications of the diol subunit (Table 4). Specifically, amino alcohol 25 (Scheme 2), a direct comparator of 19, emerged as a benchmark compound with exquisite TRPV4 activity (IC50 = 0.3 nM) and exceptional LLE (7.6). Its rat PK properties were also promising, highlighted by reduced total clearance (CL = 12 mL/min/kg) and CL/f u (2.7) in addition to an MRT of nearly 6 h. Compound 26 (Scheme 3), the 2-pyridyl sulfone analog of 25, featured improved oral bioavailability (F = 64%), while maintaining subnanomolar potency (IC50 = 0.5 nM) and excellent clearance adjusted for unbound fraction (CL/f u = 3.8). The MRT of compound 26 (1.9 h), while diminished relative to 25, remained comparable to that of leading diol 19. Importantly, compound 27, the (3R,4S)-diastereomer of 26, was equipotent in the FLIPR assay (IC50 = 0.5 nM), thereby constituting divergent structure−activity relationships relative to the diol chemotype (Figure 3). Furthermore, it exhibited reduced CL/f u (1.7) with similar MRT (2.2 h) and oral bioavailability (56%) in comparison to 26. The encouraging oral bioavailability of 26 and 27 could be due to facilitated gastrointestinal absorption driven by their enhanced aqueous solubility35 (500 μM), which exceeded that of 25 (280 μM). Along with a potential reduction in first-pass metabolism, this could also explain the improvement relative to diols with similar PSA such as 17 and 18, each of which exhibited lower aqueous solubility (380 and 400 μM, respectively). In contrast to the diol exemplars, the off-target profiles of these amino alcohols featured several areas of concern. For one, compound 25 displayed activity in a phenotypic assay measuring phospholipidosis36 risk (pMEC = 4.7 and response = 425%),37 which is not uncommon to lipophilic amines containing aromatic residues (Table 5). Cationic amphiphilic drugs (CADs) have been reported to cause phospholipidosis in humans, and activity in the phenotypic screen has correlated with a CAD “likeness” score exceeding 50.38 With a CAD score defined as the sum of indicators of lipophilicity [chromatographic hydrophobicity index (CHI) immobilized artificial membrane (IAM)] 39 and basicity [ΔCHI (pH10.5− pH7.4)],39 design elements to address phospholipidosis risk have been well-established. Indeed, amino alcohols 26 and 27, each featuring lower lipophilicity (CHI IAM = 42 and 36, respectively) and basicity [ΔCHI (pH10.5−pH7.4) = −2.2 and 3.7, respectively] relative to 25 [CHI IAM = 49, ΔCHI

Table 5. Off-Target Profiles of Amino Alcohols 25, 26, and 27a phospholipidosis

Cmpd

hERG IC50 (μM)

CAD likeness

pMEC

25 26 27

5.3 8.0 8.0

58 40 40

4.7

Design and Optimization of Sulfone Pyrrolidine ... - ACS Publications

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