Design and Optimization of Sulfone Pyrrolidine Sulfonamide Antagonists of Transient Receptor Potential Vanilloid-4 with in Vivo Activity in a Pulmonary Edema Model

Article abstract of DOI:10.1021/acs.jmedchem.8b01344

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.

Full text of DOI:10.1021/acs.jmedchem.8b01344

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Article
Design and Optimization of Sulfone Pyrrolidine Sulfonamide
Antagonists of Transient Receptor Potential Vanilloid-4
(TRPV4) with In Vivo Activity in a Pulmonary Edema Model
Joseph E. Pero, Jay Matthews, David J Behm, Edward Brnardic, Carl A Brooks, Brian W
Budzik, Melissa Costell, Carla Donatelli, Stephen Eisennagel, Karl F Erhard, Michael Fischer,
Dennis A. Holt, Larry Jolivette, Huijie Li, Peng Li, John McAtee, Brent W McCleland, Israil
Pendrak, Lorraine Posobiec, Katrina Rivera, Ralph A. Rivero, Theresa J Roethke, Matthew
Sender, Arthur Shu, Lamont Terrell, Kalindi Vaidya, Xiaoping Xu, and Brian G. Lawhorn
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01344 • Publication Date (Web): 30 Nov 2018
Downloaded from http://pubs.acs.org on December 1, 2018
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of their duties.

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Journal of Medicinal Chemistry
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Design and Optimization of Sulfone Pyrrolidine
Sulfonamide Antagonists of Transient Receptor
Potential Vanilloid-4 (TRPV4) with In Vivo Activity in
a Pulmonary Edema Model
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Joseph E. Pero*,ɸ, Jay M. Matthews†, David J. Behm†, Edward J. Brnardic†, Carl Brooks†, Brian W.
Budzikɸ, Melissa H. Costell†, Carla A. Donatelli†, Stephen H. Eisennagel†, Karl Erhardɸ, Michael C.
Fischerǁ, Dennis A. Holt†, Larry J. Jolivette†, Huijie Liɸ, Peng Liɸ, John J. McAtee†, Brent W.
McClelandɸ, Israil Pendrakɸ, Lorraine M. Posobiecǁ, Katrina L.K. Rivera†, Ralph A. Riveroɸ, Theresa J.
Roethke†, Matthew R. Senderɸ, Arthur Shuɸ, Lamont R. Terrell†, Kalindi Vaidyaǁ, Xiaoping Xu †, Brian
G. Lawhorn†
ɸFlexible Discovery Unit, †Heart Failure Discovery Performance Unit and ǁPlatform Technology and
Sciences
GlaxoSmithKline, 1250 Collegeville Road, Collegeville, Pennsylvania, 19426, United States
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 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
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described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential
1
2
3
is highlighted herein.
4
5
6
■ INTRODUCTION:
7
8
9
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 post-diagnosis, 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
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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, which significantly compromises their quality of life and can prove fatal if left
untreated.7
TRPV4 receptors are members of the Transient Receptor Potential (TRP) superfamily of cation
channels.8 These tetrameric voltage-gated channels are highly expressed in lung endothelial cells9,10 and
are up-regulated 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, pre-treatment 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 (MI) for one week
prior to treatment.11
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Journal of Medicinal Chemistry
Importantly, strong evidence indicates that these effects may be relevant to humans. GSK2193874
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3
4
5
was found to be potent in recombinant hTRVP4 channels (IC50 = 50 nM), and prevented agonist-driven
cellular contraction in human umbilical vein endothelial cells (HUVEC’s).11
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8
9
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 drug-like properties (i.e.
reduced molecular weight, lower lipophilicity) to GSK2193874. Examples from GlaxoSmithKline
include 1-(4-piperidinyl)-benzimidazoles14 and spirocarbamates (2, Figure 1b),15,16 while Pfizer has
disclosed azetidine sulfonamides (3, Figure 1c) derived from a high-throughput screen.17
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a)
b)
c)
Me
Me
NC
O
N
O
NH
Me
O
O
N
OH
O
CN
N
N
N
S
Br
N
N
O
Me
O
N
CF3
Cl
Cl
Compound 3 (Pfizer)
hTRPV4 IC50 = 21 nM
MW = 413, cLogP = 3.9
LE = 0.41, LLE = 3.8
Compound 1 (GSK2193874)
hTRPV4 IC50 = 50 nM
MW = 692, cLogP = 8.1
LE = 0.22, LLE = -0.8
Compound 2 (GSK)
hTRPV4 IC50 = 7.5 nM
MW = 448, cLogP = 3.6
LE = 0.34, LLE = 4.5
Figure 1. Previously-reported TRPV4 antagonists from GSK (a, b) and Pfizer (c).
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
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3 (Figure 2). Subsequent scaffold optimization20 led to diol 5, which featured excellent LLE (5.3) due to
1
2
3
enhanced TRPV4 activity (IC50 = 5.0 nM) in conjunction with reduced lipophilicity (cLogP22 = 3.0).
4
5
6
7
NC
8
9
O
OH
O
OH
scaffold
modification
diol
incorporation
OH
NC
NC
O
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OH
N
O S
O
N
N
O S
O
O S
O
Cl
Cl
Cl
Cl
Cl
Cl
Compound 3 (Pfizer)
hTRPV4 IC50 = 21 nM
cLogP = 3.9
Compound 4 (GSK)
hTRPV4 IC50 = 32 nM
cLogP = 3.6
Compound 5 (GSK)
hTRPV4 IC50 = 5.0 nM
cLogP = 3.0
LE = 0.41, LLE = 3.8
LE = 0.40, LLE = 3.9
LE = 0.41, LLE = 5.3
Figure 2. Scaffold modification of 3 to give pyrrolidines 4 and 5.
The present work describes an analogous structural class exemplified by sulfone 6, which was
viewed as a lead compound superior to ether 4 due to 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.
■ RESULTS AND DISCUSSION:
At the outset of our optimization efforts, the potency-enhancing 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 in the primary FLIPR21 assay.
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Journal of Medicinal Chemistry
O
O
O
a)
O
S
OH
S
OH
1
2
3
4
5
6
Sulfone
incorporation
Diol
incorporation
O
O
OH
NC
NC
NC
OH
3
4
3
4
3
4
N
N
S
N
O
S
O
S
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
7
8
9
Compound 6 (3S, 4S)
hTRPV4 IC50 = 25 nM
cLogP = 2.7
Compound 7 (3S, 4R)
hTRPV4 IC50 = 7.9 nM
cLogP = 2.9
Compound 4 (3S,4S)
hTRPV4 IC50 = 32 nM
cLogP = 3.6
10
11
12
13
14
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16
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19
20
21
22
23
LE = 0.37, LLE = 4.9
LE = 0.37, LLE = 5.2
LE = 0.40, LLE = 3.9
b)
NC
O
O
O
O
O
O
S
OH
S
OH
S
OH
NC
NC
OH
OH
OH
3
4
3
4
3 4
N
N
N
O
S
O
S
O
S
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
Compound 8 (3R, 4R)
hTRPV4 IC50 = 1585 nM
cLogP = 2.9, LLE = 2.9
Compound 9 (3S, 4S)
hTRPV4 IC50 = 50 nM
cLogP = 2.9, LLE = 4.4
Compound 10 (3R, 4S)
hTRPV4 IC50 = 159 nM
cLogP = 2.9, LLE = 3.9
Figure 3. (a) Replacement of phenoxy ether functionality with sulfone, leading to 7. (b) Additional stereoisomers of sulfone
pyrrolidine diol 7.
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An investigation of the PK properties of 7 further substantiated the diol chemotype (Table 1).
Following intravenous (i.v.) dosing in rat (1 mpk), 7 exhibited reduced clearance adjusted for unbound
fraction23 (CL/fu = 9) relative to parent compound 6 (CL/fu = 30), suggesting that the potency-enhancing
diol functionality did not pose an additional metabolic liability. Furthermore, high permeability24 (450
nm/sec) 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 solvent-accessible PSA due to
conformational restriction imparted by intramolecular hydrogen bonds, which were confirmed by
vibrational circular dichroism (VCD) studies.25,26,27. In addition, 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 due to its
higher volume of distribution (Vdss = 2.3 L/kg).
Table 1. PK properties (rat) of Compounds 5, 6 and 7a
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Cmpd PSA
Perm
CL
CL/fu Vdss MRT
F
1
2
3
(nm/sec) (mL/min/kg)
(L/kg) (h) (%)
4
5
6
7
8
5
6
7
111
116
136
370
780
450
74
9
15
30
9
2.4
1.1
2.3
0.6
46
2.0 100
1.7 100
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25
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aPSA = total polar surface area; Perm = artificial membrane permeability; CL = total clearance; fu = fraction unbound (rat);
Vdss = volume of distribution; MRT = mean resonance time; F = oral bioavailability
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/fu = 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 a 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.
Scheme 1. General synthesis of sulfone pyrrolidine diolsa
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Journal of Medicinal Chemistry
O
R
R
HO
O
S
S
1
2
3
4
a, b
d
c
N
N
N
Boc
Boc
Boc
11
12
13
5
6
7
8
9
O
R
O
S
R
O
N
O
N
S
OH
OH
OH
e, f
OH
10
11
12
13
O S
O
Boc
14
15
R'
aReagents and conditions: (a) MsCl, TEA, DCM; (b) arylthiol, K2CO3, DMF (74-81% over 2 steps); (c) m-CPBA, DCM (59-
89%); (d) OsO4, NMO, THF (81-98%, >95:5 dr); (e) HCl/1,4-dioxane or TFA, DCM; (f) arylsulfonyl chloride, NaHCO3, THF
(26-85% over 2 steps).
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With a superior balance of potency and PK properties being the ultimate goal, the versatility of
this synthetic sequence allowed the 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. 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/fu = 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 increased nucleophilic addition by glutathione (GSH), which was
suggested by trapping experiments in rat liver microsomes in which only 46% of parent remained after
incubation (30 min). Analogous studies with compound 7 showed 89% of parent remaining after
incubation.
Table 2. Structure-activity relationships of sulfone pyrrolidine diolsa
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R2
O
1
2
O
S
OH
R1
OH
3
4
5
6
N
S
O
O
R3
R4
7
8
9
10
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12
13
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19
20
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Cmpd R1 R2 R3 R4
IC50
(nM)
7.9
LLE PSA
CL
CL/fu Vdss MRT
F
(mL/min/kg)
(L/kg) (h) (%)
7
CN
CN
CN
CN
Cl
H
F
Cl Cl
Cl Cl
CN Cl
Cl CN
Cl CN
5.2
5.6
6.8
6.9
5.8
136
136
160
160
136
25
25
82
35
49
9
14
7
2.3
1.2
2.6
2.2
4.5
1.7 100
16
17
18
19
2.0
0.8
0.5
1.0
51
25
15
H
H
H
2.0
3.2
4
3.2
12
1.6 100
ahuman 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; fu = fraction unbound (rat); Vdss = volume of
distribution; MRT = mean resonance time; F = oral bioavailability
Replacing the ortho-chloro substituent of the aryl sulfonamide with a cyano group (R3 = CN,
Compound 17) resulted in a 4-fold increase in potency (IC50 = 2.0 nM) relative to parent compound 7.
While the LLE improved to 6.8, the PK properties in rat 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
glutathione-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/fu = 4). While the
enhanced metabolic stability was reflected in a longer MRT (1.0 h), oral bioavailability remained poor (F
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Journal of Medicinal Chemistry
= 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/fu (12), MRT was elongated due to 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/sec) and reduced oxidative (first-pass) metabolism
of the aryl sulfone functionality.32
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5
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7
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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 CYP’s, hERG and CaV1.2. Furthermore, it was inactive
up to 100 µM in an assay measuring time-dependent inhibition (TDI) of CYP3A4.
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/fu
= 1.2), and nearly a 2-fold 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 re-introduction of a lipophilic ortho-substituent on
the aryl sulfonamide moiety. In a representative example, Compound 21 (R = CF3) demonstrated an in
vitro 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/fu = 66, MRT = 0.2 h, F = 8%), prompting us to shift
our attention to other subunits.
Table 3. Incorporation of heteroatoms into the pyrrolidine diol scaffolda
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Z
X
O
1
2
S
OH
Cl
OH
3
4
N
S
Z'
5
O
6
R
Y
CN
7
8
9
10
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12
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16
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Cmpd
X
Y
Z
Z'
R
IC50 LLE PSA
(nM)
CL
CL/fu Vdss MRT
F
(mL/min/kg)
(L/kg) (h) (%)
19
20
21
22
23
24
CH CH
O
O
O
O
O
O
O
O
O
Cl
H
3.2
5.8 136
49
22
12
1.2
66
4.5
4.1
3.6
2.0
3.2
2.7
1.6 100
CH
CH
N
N
N
631 4.9 149
3.0
0.2
1.4
1.0
0.9
62
8
CF3 20
6.0 149
5.7 149
5.0 143
4.2 143
310
23
CH
Cl
Cl
5.0
20
40
11
62
38
80
CH CH NH
CH CH
52
4.8
8.9
O
NH Cl
48
ahuman 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; fu = fraction unbound (rat); Vdss = volume of
distribution; MRT = mean resonance time; F = oral bioavailability
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 polar surface area (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 rat (F = 80%), neither exemplar offered an advantage over parent compound 19 in terms of TRPV4
activity, LLE, or PK properties (CL, MRT, F).
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Journal of Medicinal Chemistry
With the inability of Compounds 20-24 to improve on the overall profile of Compound 19, focus
1
2
was subsequently shifted toward modifications of the diol subunit (Table 4). Specifically, amino alcohol
25, a direct comparator of 19, emerged as a benchmark compound with exquisite TRPV4 activity (IC50 =
0.3 nM) and exceptional LLE (7.6). Its PK properties in rat were also promising, highlighted by reduced
total clearance (CL = 12 mL/min/kg) and CL/fu (2.7) in addition to an MRT of nearly 6 hours. Compound
26, the 2-pyridyl sulfone analog of 25, featured improved oral bioavailability (F = 64%) while maintaining
sub-nanomolar potency (IC50 = 0.5 nM) and excellent clearance adjusted for unbound fraction (CL/fu =
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 SAR relative to the diol chemotype (Figure 3).
Furthermore, it exhibited reduced CL/fu (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, which each exhibited lower aqueous
solubility (380, 400 µM, respectively).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Profiles of amino alcohols 25, 26 and 27a
O
X
O
S
OH
Cl
NH2
4
3
N
O
S
O
Cl
CN
Cmpd
X
stereochem. IC50 LLE PSA
(nM)
CL
CL/fu Vdss MRT
(L/kg) (h) (%)
4.0 5.7 22
F
(mL/min/kg)
12
25
CH
3S, 4S
0.3
7.6 142
2.7
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26
27
N
N
3S, 4S
3R, 4S
0.5
0.5
7.4 154
7.4 154
12
3.8
1.7
1.3
0.2
1.9
2.2
64
56
1
2
3
1.7
4
5
6
7
8
9
ahuman 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; fu = fraction unbound (rat); Vdss = volume of
distribution; MRT = mean resonance time; F = oral bioavailability
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
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, response = 425%)37, which is not uncommon to lipophilic amines
containing aromatic residues (Table 5). Cationic amphiphilic drugs (CAD’s) 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 (CHI 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, 36,
respectively) and basicity (ΔCHI (pH10.5 – pH7.4) = -2.2, 3.7, respectively) relative to 25 (CHI IAM =
49, ΔCHI (pH10.5 – pH7.4) = 9), were gratifyingly inactive in the phenotypic assay (pMEC < 4).
Table 5. Off-target profiles of amino alcohols 25, 26 and 27a
Phospholipidosis
Cmpd hERG IC50
(µM)
CAD
likeness
58
pMEC
Response37
3A4 TDI (100
3A4 RI IC50
(%)
425
--
µM)
yes
no
(µM)
0.1
25
26
27
5.3
8.0
8.0
4.7
< 4
< 4
40
0.3
40
--
no
4.0
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Journal of Medicinal Chemistry
aDetails for hERG, phospholipidosis, 3A4 TDI and 3A4 RI assays described in Supporting Information; pMEC = -
1
2
3
log(minimum effective concentration)
4
5
6
7
8
9
Compounds 26 and 27 also held additional important advantages over 25. Whereas the latter
exhibited time-dependent inhibition of human CYP3A4 at 100 µM, exemplars 26 and 27 were inactive at
the same concentration, potentially a result of their reduced basicity. This may also have contributed to
their lower activity (IC50 = 8 µM) against the cardiac hERG channel. Ultimately, compound 27 emerged
as the most promising amino alcohol within this structural class, with reduced DDI (drug-drug interaction)
risk relative to 25 and 26 as demonstrated by its weaker activity in an assay measuring reversible inhibition
of CYP3A4 (IC50 = 4 µM).
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
Leading diol 19 and amino alcohol 27 were each investigated in a rodent in vivo assay suggestive
of therapeutic potential for pulmonary edema (Figure 4). In this model, rats were treated with vehicle or
TRPV4 antagonist prior to administration of known TRPV4 agonist, GSK1016790A (100 µg/kg total i.v.
dose).11,40,41 Importantly, previous studies have shown that the effects of GSK1016790A correlate with
those induced by myocardial infarction in rodents.11 In the vehicle-treated group, GSK1016790A
demonstrated robust pulmonary edema as quantified by an increase in wet lung weight/body weight ratio
relative to naïve animals. Gratifyingly, significant attenuation of agonist effects was observed with diol
19 (2.3 µg/kg/min i.v., p < 0.01 vs. vehicle) and amino alcohol 27 (0.2 µg/kg/min i.v., p < 0.05 vs.
vehicle).42
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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
Figure 4. Effects of TRPV4 agonist GSK1016790A (10 µg/kg/min i.v. for 10 min) on lung wet weight/body weight ratio
(LW/BW) in vehicle-treated rats and rats pre-treated with TRPV4 antagonists 19 (a) and 27 (b). Full inhibition of agonist-
mediated LW/BW response was achieved with the 2.3 µg/kg/min dose of 19 (99% inhibition) and the 0.2 µg/kg/min dose of
27 (88% inhibition). Corresponding steady-state plasma concentrations are indicated above and data are presented as mean ±
SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001 vs. vehicle.43
Further details regarding the in vivo activity of Compounds 19 and 27 are provided in Table 6.
Notably, despite slightly weaker in vitro potency in rat, Compound 19 featured superior activity (IC50 =
33 nM)44 in the in vivo pulmonary edema model in comparison to Compound 27 (IC50 = 611 nM).
Correcting for rat unbound fraction (rfu) generated a “free” in vivo IC50 of 0.3 nM for diol 19, a significant
improvement over that associated with amino alcohol 27 (8.5 nM).
Table 6. In vivo activity of Compounds 19 and 27 in the rat pulmonary edema modela
Cmpd rTRPV4 in vivo
rfu
“Free” in vivo
IC50
(nM)
4.0
IC50
(nM)
33
(%)
IC50 (nM)
19
4.2
0.3
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Journal of Medicinal Chemistry
27
1.0
611
1.4
8.5
1
2
3
4
5
arat TRPV4 IC50 values measured by a calcium flux FLIPR assay (see Supporting Information for details); rfu = fraction
unbound (rat); conc = concentration
6
7
8
9
Ultimately, with enhanced in vivo activity, less off-target promiscuity and dose-proportional oral
exposures in both rat and dog (Table 7), diol 19 was selected over amino alcohol 27 for further
progression. To our delight, a 7-day rat safety assessment (100/300 mpk) proceeded with no compound-
related macro- or microscopic findings. Based on scaling of preclinical PK data, the exposures at 300 mpk
provided minimum rat safety margins of 68-fold and 42-fold over the predicted human Cmax and AUC,
respectively, with Cmin set at the concentration required for full inhibition in the rat pulmonary edema
assay (Figure 4a).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 7. PK studies with Compound 19 in rat and doga
Species
rat
p.o. dose (mpk)
AUC0-t (µg·h/mL)
DNAUC0-t (h·kg/L)
2.4
98
1.1
40
0.5
0.4
2.9
3.0
rat
dog
dog
0.9
30
2.5
90
a n = 3 animals; AUC0-t = area under the plasma concentration time curve to last time point with quantifiable drug; DNAUC0-t
= dose-normalized AUC0-t.
■ CONCLUSIONS:
A novel class of sulfone pyrrolidine TRPV4 antagonists45 has been discovered, with exemplars
featuring excellent potency and tractability with respect to physicochemical and PK properties. Structure-
activity relationships within a diol chemotype were investigated in order to balance potency with desirable
PK properties, whereas efforts within an amino alcohol sub-series leveraged exquisite in vitro activity
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while addressing off-target concerns. Leading exemplars from both chemotypes (19 and 27) demonstrated
blockade of TRPV4 agonist effects in an in vivo model indicative of therapeutic potential. Of these
compounds, 19 emerged as a viable candidate for HF studies which can prove instrumental in assessing
the utility of TRPV4 antagonists in the treatment of HF-induced pulmonary edema.
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
■ EXPERIMENTAL SECTION:
Chemistry. The syntheses of diol 19 and amino alcohols 25-27 are described below. Detailed synthetic
protocols and spectroscopic characterizations for Compounds 4, 6-7, 16-18 and 20-24 are provided in the
Supporting Information (along with procedures for biological assays and pharmacokinetic studies). The
purity of each compound was determined to be >95% on a Waters Acquity LCMS equipped with a
Acquity™ UPLC BEH C18 1.7mm, 2.1 x 50 mm column using a gradient of 3% to 100% MeCN, 0.1%
formic acid at 1 ml/min flow rate with an Acquity PDA detector at 210 and 350 nm. Mass determinations
were conducted using a Waters Acquity SQD with positive/negative switching in ESI mode.
Scheme 2. Synthesis of amino alcohol 25a
HO
HO
MsO
S
S
OH
Cl
Cl
a
b
c
d
OH
N
N
S
N
S
N
S
N
S
Boc
O
O
O
O
O
O
O
11
O
Cl
CN
Cl
CN
Cl
CN
Cl
CN
16bb
16dd
16aa
16cc
O
S
OH
S
OH
Cl
O
O
Cl
S
S
OH
NHBoc
CN
NH2
Cl
Cl
NH2
f
h, i
e
g
N
S
N
S
N
S
N
S
O
O
O
O
O
O
O
O
Cl
Cl
CN
Cl
CN
Cl
CN
25a
25b
25c
25
aReagents 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%).
Scheme 3. Synthesis of amino alcohols 26 and 27a
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Journal of Medicinal Chemistry
N
Me
O
5
Me
S
OH
N
N
N
HO
Cl
OH
1
2
3
4
S
S
OH
S
4 3
d
a
b
c
O
Cl
Cl
Cl
OH
9
N
N
N
O
S
N
N
Boc
O
Boc
Boc
Boc
26b: (5R, 9S)
26c: (5S, 9S)
26d: (3R, 4S)
26dd: (3S, 4S)
Cl
CN
11
18aa
26a
5
6
7
8
9
10
11
12
13
14
O
N
O
N
N
N
O
O
S
OH
S
OH
S
OH
S
OH
Cl
Cl
Cl
Cl
NHBoc
CN
NHBoc
CN
NH2
N3
4
3
4 3
4
3
4
3
f
e
g
h
N
N
N
S
N
O
S
O
S
O
O
S
O
O
O
O
Cl
Cl
Cl
CN
Cl
CN
26e: (3S, 4S)
26ee: (3R, 4S)
26f: (3S, 4S)
26ff: (3R, 4S)
26g: (3S, 4S)
26gg: (3R, 4S)
26: (3S, 4S)
27: (3R, 4S)
aReagents 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, 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)).
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
(R)-tert-Butyl 3-((4-chlorophenyl)thio)-4-methylenepyrrolidine-1-carboxylate (12, R = para-
chloro): To a stirred mixture of (S)-tert-butyl 3-hydroxy-4 methylenepyrrolidine-1-carboxylate (11, 12.33
g, 61.9 mmol, 1 equiv) in DCM (120 mL) was added TEA (12.94 mL, 93 mmol, 1.5 equiv) in one portion.
The mixture was subsequently cooled in an ice bath to give an internal temperature of 5 °C.
Methanesulfonyl chloride (8.51 g, 74.3 mmol, 1.2 equiv) was added dropwise via a syringe over 40 min
such that the internal temperature did not exceed 2 °C. Following the addition, the reaction vessel was
removed from the bath and the mixture was allowed to stir at ambient temperature for 45 min. Following
this duration, the mixture was diluted with 50 mL of DCM and washed with 40 mL of water. The layers
were separated and the organic layer was dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to provide the crude mesylate as a light brown oil (19.9 g), which was immediately
carried forward without further purification. To a stirred mixture of the crude mesylate and 4-
chlorobenzenethiol (9.40 g, 65.0 mmol) in DMF (125 mL) in an ice bath was added K2CO3 (powder form,
12.83 g, 93 mmol) portion-wise. Following the addition, the reaction vessel was removed from the bath
and the mixture was allowed to stir at ambient temperature for 1 h. Following this duration, the stirring
was stopped and the mixture was allowed to age at RT for 40 min. To this mixture was added 1:1
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EtOAc/hexane (400 mL), and ice water (200 mL) sequentially such that the internal temp remained below
20 °C. The resulting mixture was filtered and the filtrate was phase-separated. The organic extract was
washed with water (200 mL, then 2 x 150 mL) dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to give a yellow oil. The crude product was subsequently dissolved in DCM (50
mL), absorbed onto Isolute and concentrated to remove the residual DCM. Purification by silica gel
chromatography (0-30% EtOAc:Hexanes) afforded 12 as a clear oil (14.9 g, 74%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.40 (br. s., 9 H) 3.39 (dd, J=11.80, 2.51 Hz, 1 H) 3.69 (dd, J=11.80, 6.27 Hz, 1 H)
3.89 (d, J=12.05 Hz, 1 H) 4.00 - 4.11 (m, 1 H) 4.47 (br. s., 1 H) 5.01 (br. s., 1 H) 5.11 (s, 1 H) 7.35 - 7.51
(m, 4 H); m/z 269.9 [MH-t-Bu]+.
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
(R)-tert-Butyl 3-((4-chlorophenyl)sulfonyl)-4-methylenepyrrolidine-1-carboxylate (13, R = para-
chloro): To a cooled solution of (R)-tert-butyl 3-((4-chlorophenyl)thio)-4-methylenepyrrolidine-1-
carboxylate (14.57 g, 44.7 mmol, 1 equiv) in 250 mL of DCM was added m-CPBA (25 g, 112 mmol, 2.5
equiv) portion-wise over 7 min such that the internal temperature stayed ≤ -5 °C. After 1 h, to the cold
mixture was added 10% wt aqueous Na2S2O3 solution (140 mL) portion-wise over 40 min such that the
internal temperature remained ≤ -2 °C. To the cooled mixture was added 325 mL of saturated aqueous
NaHCO3 over 30 min such that the internal temperature remained ≤ 1 °C. The pH of the resulting mixture
was 9. The cold mixture was phase-separated. The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure to approximately 50 mL total volume. The solution was
adsorbed onto Isolute and concentrated to remove residual DCM. Purification by silica gel
chromatography (0-70% EtOAc:Hexanes) afforded 13 (R = para-chloro) as a white foam (15.9 g, 89%
yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.41 (s, 4 H) 1.36 (s, 5 H) 3.53 - 3.72 (m, 2 H) 3.72 - 3.85
(m, 1 H) 3.86 - 3.99 (m, 1 H) 4.67 (d, J=7.28 Hz, 1 H) 5.18 (br.s., 1 H) 5.39 (br. s., 1 H) 7.76 (d, J=8.28
Hz, 2 H) 7.85 (d, J=7.53 Hz, 2 H); m/z 380.0 [M+Na]+.
(3R,4S)-tert-Butyl-4-((4-chlorophenyl)sulfonyl)-3-hydroxy-3-(hydroxymethyl)pyrrolidine-1-
carboxylate (14, R = para-chloro): To a stirred, cooled mixture of (R)-tert-butyl 3-((4-
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chlorophenyl)sulfonyl)-4-methylenepyrrolidine-1-carboxylate (15.9 g, 44.4 mmol) and NMO (10.41 g,
89 mmol, 2 equiv) in 300 mL of THF was added OsO4 solution in t-butanol (25 mL, 2.5% wt, 1.99 mmol,
0.045 equiv) via syringe over 4 min such that the internal temperature did not exceed 15 °C. After
completion of addition, the internal temperature was allowed to gradually rise to 22-23 °C within 45 min
and remained at 23 °C. After 3 h, an additional 2.8 mL of OsO4 solution (total 0.05 equiv added) was
added. After 5 h, stirring was stopped and the mixture was aged at RT over 14 h. The yellow solution was
subsequently chilled in an ice bath and saturated aqueous Na2SO3 (100 mL) was added over 10 min
(keeping internal temperature at approximately 9 °C), bringing the pH of the reaction mixture to 9-10.
The mixture was stirred in the ice bath for 1 h. Following this duration, the ice bath was removed. To the
cold mixture was added 100 mL of water and the resulting suspension was filtered through Celite. The
cake was washed with 300 mL of EtOAc. The filtrate was phase-separated. The organic layer was washed
with brine (30 mL), dried over anhydrous Na2SO4 and DARCO (activated charcoal), filtered, and
concentrated under reduced pressure. The wet residue was taken up in 200 mL of DCM and washed with
20 mL of brine. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced
pressure to afford an off-white foamy residue. The residue was re-dissolved in DCM and purified by
silica gel chromatography (5-70% (3:1 EtOAc:EtOH):Hexanes) to provide 14 (R = para-chloro) as an
off-white solid (14.1 g, 81% yield). 1H NMR (400MHz, DMSO-d6) δ ppm 7.91 (d, J=8.5 Hz, 2H), 7.77
(d, J=8.3 Hz, 2H), 5.53 (d, J=9.0 Hz, 1H), 4.85 (d, J=4.3 Hz, 1H), 3.98 (d, J=4.5 Hz, 1H), 3.82 - 3.42 (m,
5H), 3.28 - 3.16 (m, 1H), 1.40 (s, 9H); m/z 414.1 [M+Na]+.
1
2
3
4
5
6
7
8
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(3R,4S)-4-((4-Chlorophenyl)sulfonyl)-3-(hydroxymethyl)pyrrolidin-3-ol, hydrochloride: (3R,4S)-
tert-Butyl-4-((4-chlorophenyl)sulfonyl)-3-hydroxy-3-(hydroxymethyl)pyrrolidine-1-carboxylate (14, R
= para-chloro, 14.0 g, 35.7 mmol) was dissolved in 4N HCl/1,4-dioxane solution (200 mL, 800 mmol)
and the resulting reaction mixture was stirred vigorously for 1 h. The slurry was diluted with TBME (50
mL), filtered, and the solid was washed with TBME (50 mL). The solid was subsequently dried under
vacuum to afford 10.8
g
(92% yield) of (3R,4S)-4-((4-chlorophenyl)sulfonyl)-3-
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(hydroxymethyl)pyrrolidin-3-ol, hydrochloride as a white solid. 1H NMR (400MHz, Methanol-d4) δ ppm
8.00 (d, J=8.5 Hz, 2H), 7.75 (d, J=8.5 Hz, 2H), 4.18 - 4.11 (m, 1H), 4.10 - 3.96 (m, 2H), 3.81 (dd, J=8.0,
13.6 Hz, 1H), 3.70 - 3.55 (m, 3H), 3.44 (d, J=11.8 Hz, 1H); m/z 292.0 [MH]+.
1
2
3
4
5
6
7
8
9
(S)-3-Chloro-4-((3-hydroxy-4-methylenepyrrolidin-1-yl)sulfonyl)benzonitrile (16aa): To
a
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25
26
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60
solution of (S)-tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (11, 10 g, 50.2 mmol) in DCM
(40 mL) was added TFA (58.0 mL, 753 mmol) and the resulting reaction mixture was stirred at RT for 1
h. Following this duration, the reaction contents were concentrated under reduced pressure to give a tan
oil, which was suspended in DCM (80 mL) and cooled in an ice bath. To this reaction mixture was added
TEA (35.0 mL, 251 mmol) cautiously, followed by 2-chloro-4-cyanobenzene-1-sulfonyl chloride (11.85
g, 50.2 mmol). The reaction mixture was subsequently warmed to RT and stirred at 23 °C for 1 h. The
mixture was then diluted with water (80 mL) and extracted with DCM (80 mL). The combined organics
were washed with brine and concentrated. Purification by silica gel chromatography (0-60%
EtOAc:Hexane) provided 16aa as a pale yellow solid (9.5 g, 63% yield). 1H NMR (400 MHz, DMSO-d6)
δ ppm 3.12 (dd, J=9.91, 5.65 Hz, 1 H) 3.64 (dd, J=10.04, 6.27 Hz, 1 H) 4.04 - 4.10 (m, 1 H) 4.49 (d,
J=5.27 Hz, 1 H) 5.11 (d, J=1.51 Hz, 1 H) 5.18 (d, J=1.76 Hz, 1 H) 5.56 (d, J=5.02 Hz, 1 H) 8.03 - 8.07
(m, 1 H) 8.11 - 8.14 (m, 1 H) 8.36 (d, J=1.26 Hz, 1 H); m/z 280.8 [MH-H2O+].
(S)-1-((2-Chloro-4-cyanophenyl)sulfonyl)-4-methylenepyrrolidin-3-yl methanesulfonate (16bb):
To a solution of (S)-3-chloro-4-((3-hydroxy-4-methylenepyrrolidin-1-yl)sulfonyl)benzonitrile (16aa,
9.50 g, 31.8 mmol) in DCM (80 mL) in an ice bath was added TEA (7.80 mL, 56.0 mmol) followed by
methanesulfonyl chloride (3.10 mL, 39.7 mmol). The resulting reaction mixture was stirred at RT for 1
h. Following this duration, water (80 mL) was added and the resulting layers were separated. The aqueous
layer was extracted with DCM (1 x 80 mL). The combined organics were washed with water (2 x), dried
over anhydrous MgSO4 and filtered. The filtrate was concentrated under reduced pressure to give an off-
white solid. Trituration with 15% EtOAc in hexane, filtration and concentration under reduced pressure
afforded 16bb as a white solid (10.6 g, 88% yield). 1H NMR (400 MHz, DMSO-d6)  ppm 3.24 (s, 3 H)
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Journal of Medicinal Chemistry
3.66 - 3.81 (m, 2 H) 4.02 (d, J=14.56 Hz, 1 H) 4.20 (d, J=14.31 Hz, 1 H) 5.45 (s, 1 H) 5.51 (d, J=1.76 Hz,
2 H) 7.99 - 8.09 (m, 1 H) 8.11 - 8.20 (m, 1 H) 8.37 (d, J=1.51 Hz, 1 H); m/z 280.8 [MH-H2O]+.
1
2
3
4
5
6
7
8
(R)-3-Chloro-4-((3-chlorophenyl)thio)-4-methylenepyrrolidin-1-yl)sulfonyl)benzonitrile (16cc):
To a solution of (S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-methylenepyrrolidin-3-yl methanesulfonate
(16bb, 2.5 g, 6.63 mmol) in chloroform (100 mL) at RT was added 4-chlorobenzenethiol (1.151 g, 7.96
mmol). Triethylamine (2.312 mL, 16.59 mmol) was then added and the resulting mixture was allowed to
stir at RT for 18 h. Following this duration, the reaction mixture was concentrated under reduced pressure
and purified by silica gel chromatography to give 16cc as a white solid (1.7 g, 59%). 1H NMR (400MHz,
CHLOROFORM-d) δ ppm 8.21 (d, J=8.0 Hz, 1H), 7.85 (s, 1H), 7.71 (d, J=8.3 Hz, 1H), 7.36 - 7.26 (m,
4H), 5.11 (d, J=7.3 Hz, 2H), 4.30 - 4.21 (m, 1H), 4.12 (t, J=14.2 Hz, 2H), 3.89 (dd, J=6.3, 11.0 Hz, 1H),
3.56 (dd, J=4.0, 11.0 Hz, 1H); m/z 425.0 [MH]+.
9
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3-Chloro-4-(((3R,4S)-4-((4-chlorophenyl)thio)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-3-
yl)sulfonyl)benzonitrile (16dd): To a solution of (R)-3-chloro-4-((3-((4-chlorophenyl)thio)-4-
methylenepyrrolidin-1-yl)sulfonyl)benzonitrile (16cc, 1.66 g, 3.90 mmol) in tetrahydrofuran (13.0 mL)
was added NMO (0.503 g, 4.29 mmol) and osmium tetroxide (2.5% in t-BuOH, 2.45 mL, 0.195 mmol)
sequentially to give a bright yellow solution. After 30 min, an additional 270 mg (0.6 equiv) of NMO was
added. After 30 min, an additional 150 mg (0.3 equiv) of NMO was added. After 10 min, saturated
aqueous NaHSO3 was added (40 mL) followed by DCM (100 mL). The layers were separated and the
organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
Purification by silica gel chromatography (0-50% EtOAc:Hexanes) afforded 16dd as a white solid (660
mg, 37% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.63 (br. s., 1 H) 2.03 (br. s., 1 H) 3.09
(s, 1 H) 3.50 (d, J=10.79 Hz, 1 H) 3.59 (dd, J=10.54, 2.76 Hz, 1 H) 3.70 - 3.77 (m, 2 H) 3.97 (d, J=11.29
Hz, 1 H) 4.03 (dd, J=10.54, 6.02 Hz, 1 H) 7.27 - 7.39 (m, 4 H) 7.69 (d, J=8.03 Hz, 1 H) 7.84 (s, 1 H) 8.19
(d, J=8.28 Hz, 1 H); m/z 441.0 [M-OH]+.
(R)-tert-Butyl 3-((5-chloropyridin-2-yl)thio)-4-methylenepyrrolidine-1-carboxylate (18aa):
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To a solution of (S)-tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (11, 2.0 g, 10.04 mmol)
in DCM (30 mL) was added TEA (2.1 mL, 15.07 mmol), followed by mesyl chloride (0.94 mL, 12.06
mmol) dropwise. The resulting reaction mixture was stirred at RT for 30 min. Following this duration,
the reaction mixture was diluted with water and extracted with dichloromethane. The organic extract was
washed with water, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated
under reduced pressure to give the desired mesylate. The intermediate was immediately dissolved in N,N-
dimethylformamide (100 mL). 5-Chloropyridine-2-thiol (1.46 g, 10.04 mmol) was then added, followed
by potassium carbonate (2.08 g, 15.06 mmol). The reaction mixture was stirred for 1 h at RT, diluted with
water and extracted with ethyl acetate. The organic extract was washed with water, dried over anhydrous
magnesium sulfate and filtered. The filtrate was subsequently concentrated under reduced pressure and
purified by silica gel chromatography (0-15% EtOAc:Hexanes) to give 18aa as a clear, colorless oil (3.0
1
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1
g, 91% yield). H NMR (400 MHz, DMSO-d6) δ ppm 1.39 - 1.41 (m, 9 H) 3.53 - 3.59 (m, 1 H) 3.86 -
4.00 (m, 2 H) 4.13 - 4.32 (m, 2 H) 4.84 - 4.97 (m, 1 H) 5.01 - 5.13 (m, 1 H) 7.41 - 7.45 (m, 1 H) 7.71 -
7.76 (m, 1 H) 8.46 - 8.49 (m, 1 H); m/z 327.0 [MH]+.
3-Chloro-4-(((3R,4S)-4-((4-chlorophenyl)sulfonyl)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-1-
yl)sulfonyl)benzonitrile (19): To
a
solution of (3R,4S)-4-((4-chlorophenyl)sulfonyl)-3-
(hydroxymethyl)pyrrolidin-3-ol, hydrochloride (9.66 g, 29.4 mmol) in THF (100 mL) was added saturated
aqueous NaHCO3 (150 mL) and the resulting reaction mixture was stirred for 5 min, to which was added
a suspension of 2-chloro-4-cyanobenzene-1-sulfonyl chloride (7.02 g, 28.8 mmol) in THF (150 mL)
portion-wise at 10 °C. The resulting reaction mixture was stirred for 1 h, at which an additional 2-chloro-4-
cyanobenzene-1-sulfonyl chloride (0.7 g) was added and the reaction mixture was stirred for an additional
1 h. Following this duration, the reaction mixture was diluted with EtOAc and H2O, the layers separated,
and the aqueous phase extracted with EtOAc. The combined organics were washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude oil was purified in two batches,
using flash column chromatography eluting with a 5% EtOAc-hexanes to 100% EtOAc gradient. The pure
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Journal of Medicinal Chemistry
product-containing fractions were combined and the solvent evaporated under reduced pressure to afford
an off-white foam (12.7 g). The foam was triturated with heptane (50 mL) and DCM (5 mL) and the
solvent was evaporated under reduced pressure and dried under vacuum to afford 11.6 g (80% yield) of
1
2
3
4
5
6
1
7
8
19 as an off-white solid. H NMR (400 MHz, DMSO-d6) δ ppm 3.38 (d, J=10.04 Hz, 1 H) 3.59 (dd,
9
J=11.29, 3.01 Hz, 1 H) 3.63 - 3.72 (m, 2 H) 3.74 - 3.87 (m, 2 H) 4.14 (dd, J=7.28, 3.01 Hz, 1 H) 4.94 (t,
J=5.40 Hz, 1 H) 5.76 (s, 1 H) 7.68 - 7.79 (m, 2 H) 7.82 - 7.90 (m, 2 H) 8.01 - 8.08 (m, 1 H) 8.14 (s, 1 H)
10
11
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59
60
13
8.36 (s, 1 H); C NMR (101 MHz, DMSO-d6) δ ppm 48.3, 56.8, 63.4, 68.6, 81.8, 116.9, 117.1, 130.3,
130.4, 131.9, 132.3, 132.4, 136.2, 137.8, 139.9, 140.9; LRMS: m/z 491.1 [MH]+. HRMS: m/z 602.96736
(observed), 602.96717 (calculated) (TFA-adduct). Chiral purity was determined to be 100% by chiral
HPLC (CHIRALPAK IC, 5 µ, 30 x 250 mm, 30:70 Heptane:EtOH). [α]D20 = +53° (c = 0.2, CH3OH).
3-Chloro-4-(((3S,7S)-7-((4-chlorophenyl)thio)-1-oxa-5-azaspiro[2.4]heptan-5-yl)sulfonyl-
benzonitrile (25a): To a solution of 16dd (0.700 g, 1.52 mmol) in DCM (20.0 mL) was added TEA
(0.380 mL, 2.73 mmol) followed by mesyl chloride (0.140 mL, 1.79 mmol). The resulting reaction
mixture was stirred at RT for 0.5 h. Following this duration, the reaction mixture was concentrated under
reduced pressure to afford a white solid. The solid was dissolved in MeOH (20 mL) to which was added
K2CO3 (0.316 g, 2.29 mmol). The resulting reaction mixture was stirred at RT for 3 h, diluted with H2O
and extracted with EtOAc. The combined organics were dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-
35% EtOAc:Hexanes) to afford 25a as a white solid (350 mg, 52% yield); m/z 441.0 [MH]+.
4-(((3S,4S)-3-(Aminomethyl)-4-((4-chlorophenyl)thio)-3-hydroxypyrrolidin-1-yl)sulfonyl-3-
chloro benzonitrile (25b): A suspension of 25a (0.100 g, 0.227 mmol) in 2 M NH3 / EtOH (2.0 mL, 4.00
mmol) was irradiated under microwave irradiation at 70 °C for 0.5 h. The reaction mixture was cooled to
ambient temperature and the solvent was evaporated under reduced pressure. The residue was dissolved
in MeOH and loaded onto a SCX cartridge (2.0 g), eluting with MeOH, followed by 2 M ammonia in
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MeOH solution. The ammonia solution was collected and concentrated under reduced pressure to afford
1
2
3
25b as an oil (100 mg, 99% yield); m/z 458.0 [MH]+.
4
5
6
7
8
tert-Butyl(((3S,4S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-((4-chlorophenyl)thio)-3-
hydroxypyrrolidin-3-yl)methyl)carbamate (25c) To a solution of 25b (0.100 g, 0.218 mmol) in THF
(3.0 mL) was added TEA (0.061 mL, 0.436 mmol), followed by Boc2O (0.061 g, 0.262 mmol) and the
resulting reaction mixture was stirred at RT for 1 h. The reaction mixture was diluted with H2O and
extracted with EtOAc. The combined organics were dried over MgSO4, filtered and concentrated under
reduced pressure. The crude residue was purified by silica gel chromatography (0-35% EtOAc:Hexanes)
to afford 25b as a colorless semi-solid (105 mg, 82% yield); m/z 558.2 [MH]+.
9
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4-(((3S,4S)-3-(Aminomethyl)-4-((4-chlorophenyl)sulfonyl)-3-hydroxypyrrolidin-1-yl)sulfonyl)-3-
chlorobenzonitrile (25): To a solution of 25c (0.105 g, 0.188 mmol) in DCM (3.0 mL) was added m-
CPBA (0.065 mL, 0.376 mmol), and the reaction mixture was stirred at ambient temperature for 24 h.
The reaction mixture was diluted with H2O and extracted with DCM. The combined organics were dried
over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified
by silica gel chromatography (0-40% EtOAc:Hexanes) to afford 110 mg (99% yield) of the corresponding
sulfone as a semi-solid (m/z 490.0 [MH]+). To a solution of the sulfone (110 mg, 0.186 mmol) in DCM
(1 mL) was added TFA (0.2 mL, 2.60 mmol) and the reaction mixture was stirred at RT for 0.5 h.
Following this duration, the reaction mixture was concentrated under reduced pressure, diluted with H2O,
neutralized with saturated aqueous NaHCO3 and extracted with EtOAc. The combined organics were
dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was purified
by silica gel chromatography (0-5% MeOH:DCM) and further purified by semi-prep HPLC, eluting with
a 10% MeCN-H2O to 90% MeCN-H2O gradient. The pure product-containing fractions were combined
and concentrated under reduced pressure to afford 25 as a white solid (64 mg, 66% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm 3.42 - 3.48 (m, 1 H) 3.56 - 3.62 (m, 1 H) 3.66 - 3.74 (m, 1 H) 3.81 - 3.94 (m, 1
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Journal of Medicinal Chemistry
H) 4.21 - 4.30 (m, 1 H) 6.31 - 6.59 (m, 1 H) 7.17 - 7.36 (m, 1 H) 7.83 (s, 4 H) 8.12 (s, 2 H) 8.39 (s, 1 H);
1
2
3
m/z 489.9 [MH]+.
4
5
6
(3R,4S)
and
(3S,4S)-tert-Butyl
4-((5-chloropyridin-2-yl)thio)-3-hydroxy-3-
7
8
9
(hydroxymethyl)pyrrolidine-1-carboxylate (26a): To a solution of 18aa (3.0 g, 9.18 mmol) in THF (30
mL) was added 50% aqueous NMO (2.85 mL, 13.8 mmol) followed by 2.5% OsO4 in t-BuOH (4.61 mL,
0.367 mmol) and the reaction mixture was stirred at RT for 2 h. Following this duration, the reaction
mixture was quenched with saturated aqueous Na2SO3 and extracted with EtOAc. The organic extracts
were washed with brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (0-55% EtOAc:Hexanes) to afford 26a as a
colorless oil (2.58 g, 78% yield, diastereomeric mixture). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.36 -
1.42 (m, 9 H) 3.15 - 3.31 (m, 2 H) 3.85 - 3.98 (m, 1 H) 4.00 - 4.07 (m, 2 H) 4.13 - 4.32 (m, 1 H) 4.86 -
5.09 (m, 1 H) 5.33 - 5.49 (m, 1 H) 7.27 - 7.49 (m, 1 H) 7.66 - 7.86 (m, 1 H) 8.36 - 8.61 (m, 1 H); m/z
305.0 [MH-Boc]+.
10
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19
20
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22
23
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25
26
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(5R,9S)-tert-Butyl 9-((5-chloropyridin-2-yl)thio)-2,2-dimethyl-1,3-dioxa-7-azaspiro[4.4]nonane-7-
carboxylate (26b) and (5S,9S)-tert-butyl 9-((5-chloropyridin-2-yl)thio)-2,2-dimethyl-1,3-dioxa-7-
azaspiro[4.4]nonane-7-carboxylate (26c): To a solution of 26a (2.58 g, 7.15 mmol) in DCM (20 mL)
was added 2,2-dimethoxypropane (2.64 mL, 21.4 mmol) and PPTS (0.136 g, 0.715 mmol) and the
resulting reaction mixture was stirred at RT for 0.5 h. Following this duration, the reaction mixture was
diluted with H2O and extracted with DCM. The organics were washed with brine, dried over anhydrous
MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel
chromatography (0-25% EtOAc:Hexanes). Pure product-containing fractions of the trans-isomer (first
eluting peak) were combined and concentrated under reduced pressure to afford 26b as a clear oil (1.1 g,
38% yield). Pure product-containing fractions of the cis-isomer (second eluting peak) were combined and
concentrated under reduced pressure to afford 26c as a clear oil (1.1 g, 38% yield). 26b: 1H NMR (400
MHz, DMSO-d6) δ ppm 1.30 - 1.56 (m, 15 H) 3.39 (d, J=12.80 Hz, 1 H) 3.51 (d, J=11.04 Hz, 1 H) 3.70
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- 3.93 (m, 1 H) 4.00 - 4.12 (m, 3 H) 4.37 (d, J=15.31 Hz, 1 H) 7.46 (d, J=8.53 Hz, 1 H) 7.83 (d, J=8.53
Hz, 1 H) 8.55 (s, 1 H); m/z 345.2 [MH-t-Bu]+. 26c: 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 - 1.44
(m, 15 H) 3.23 (d, J=11.80 Hz, 1 H) 3.45 - 3.61 (m, 2 H) 3.93 - 4.04 (m, 3 H) 4.29 - 4.49 (m, 1 H) 7.44
(d, J=8.53 Hz, 1 H) 7.79 (d, J=6.53 Hz, 1 H) 8.51 (s, 1 H); m/z 345.2 [MH-t-Bu]+.
1
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3-Chloro-4-(((3R,4S)-4-((5-chloropyridin-2-yl)thio)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-1-
yl)sulfonyl)benzonitrile (26d): To a solution of 26b (0.800 g, 1.99 mmol) in DCM (3.0 mL) was added
TFA (7.69 mL, 100 mmol) and the resulting reaction mixture was stirred for 0.5 h. Following this duration,
the reaction mixture was concentrated and treated with saturated aqueous NaHCO3. The resulting
suspension was diluted with THF (10 mL). A mixture of 2-chloro-4-cyanobenzene-1-sulfonyl chloride
(0.921 g, 3.90 mmol) in THF (10 mL) was added dropwise and the reaction mixture was allowed to stir
at RT for 0.5 h. The reaction mixture was diluted with H2O and extracted with EtOAc. The organics were
washed with brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel chromatography (0-60% EtOAc:Hexanes) to afford 26d as a semi-solid
(853 mg, 82% yield); m/z 460.1 [MH]+.
3-Chloro-4-(((3S,4S)-4-((5-chloropyridin-2-yl)thio)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-1-
yl)sulfonyl)benzonitrile (26dd) was prepared from 26c analogously to the synthesis of 26d from 26b to
1
afford 26dd as a white solid (390 mg, 85% yield). H NMR (400MHz, DMSO-d6) δ ppm 8.50 (s, 1H),
8.37 (s, 1H), 8.15 (d, J=8.0 Hz, 1H), 8.10 - 8.03 (m, 1H), 7.76 (dd, J=2.3, 8.5 Hz, 1H), 7.39 (d, J=8.5 Hz,
1H), 5.63 (s, 1H), 5.18 - 5.04 (m, 1H), 4.27 (t, J=9.2 Hz, 1H), 4.21 - 4.13 (m, 1H), 3.67 (d, J=10.3 Hz,
1H), 3.54 - 3.40 (m, 2H), 3.36 (br. s., 2H); m/z 460.3 [MH]+.
4-(((3S,4S)-3-(Azidomethyl)-4-((5-chloropyridin-2-yl)thio)-3-hydroxypyrrolidin-1-yl)sulfonyl)-3-
chlorobenzonitrile (26e): To a solution of 26d (0.280 g, 0.608 mmol) in DCM (8.0 mL) was added TEA
(0.153 mL, 1.09 mmol) followed by MsCl (0.057 mL, 0.730 mmol) and the reaction mixture was stirred
at RT for 10 min. Following this duration, the reaction mixture was diluted with H2O and extracted with
DCM. The combined organics were dried over anhydrous MgSO4, filtered and concentrated under
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Journal of Medicinal Chemistry
reduced pressure. The residue was dissolved in DMF (5.0 mL), to which was added NaN3 (0.099 g, 1.52
mmol) and the reaction mixture was stirred at 80 C for 40 min. The reaction mixture was subsequently
cooled to ambient temperature, diluted with H2O and extracted with EtOAc. The combined organics were
washed with H2O, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The
crude residue was purified by silica gel chromatography (0-25% EtOAc:Hexanes) to afford 26e as a
colorless wax (115 mg, 39% yield); m/z 485.1 [MH]+.
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4-(((3R,4S)-3-(Azidomethyl)-4-((5-chloropyridin-2-yl)thio)-3-hydroxypyrrolidin-1-yl)sulfonyl)-3-
chlorobenzonitrile (26ee) was prepared from 26dd analogously to synthesis of 26e from 26d to afford
26ee as a colorless wax (343 mg, 83% yield). 1H NMR (400MHz, DMSO-d6) δ ppm 8.49 (d, J=1.8 Hz,
1H), 8.38 (s, 1H), 8.15 (d, J=8.3 Hz, 1H), 8.10 - 8.02 (m, 1H), 7.79 (dd, J=2.3, 8.5 Hz, 1H), 7.42 (d, J=8.5
Hz, 1H), 6.16 (s, 1H), 4.28 (t, J=8.8 Hz, 1H), 4.12 (t, J=8.5 Hz, 1H), 3.64 - 3.43 (m, 5H); m/z 485.3
[MH]+.
tert-Butyl
(((3S,4S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-((5-chloropyridin-2-yl)thio)-3-
hydroxypyrrolidin-3-yl)methyl)carbamate (26f): To a solution of 26e (0.115 g, 0.237 mmol) in THF
(4.0 mL) was added a 1.0 M solution of PMe3 in THF (0.474 mL, 0.474 mmol) and the resulting reaction
mixture was stirred at RT for 24 h. Following this duration, the reaction mixture was loaded onto a SCX
cartridge and flushed with MeOH followed by 2 M NH3 in MeOH. The filtrate was concentrated under
reduced pressure to afford 92 mg (85% yield) of the requisite amine as a colorless wax (m/z 459.1 [MH]+).
The amine (92 mg, 0.200 mmol) was dissolved in THF (4.0 mL) to which was added Boc2O (0.060 g,
0.260 mmol) followed by TEA (0.056 mL, 0.401 mmol) and the reaction mixture was stirred at RT for
0.5 h. Following this duration, the reaction mixture was diluted with H2O and extracted with EtOAc. The
combined organics were dried over anhydrous MgSO4, filtered and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (0-30% EtOAc:Hexanes) to afford 26f as a
colorless semi-solid (75 mg, 67% yield); m/z 559.2 [MH]+.
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Page 28 of 37
tert-Butyl(((3R,4S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-((5-chloropyridin-2-yl)thio)-3-
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hydroxy-pyrrolidin-3-yl)methyl)carbamate (26ff) was prepared from 26ee analogously to the synthesis
of 26f from 26e to afford 26ff as a colorless semi-solid (178 mg, 77% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 1.38 (d, J=13.30 Hz, 9 H) 3.18 (d, J=4.77 Hz, 2 H) 3.48 (s, 1 H) 3.65 (d, J=10.54 Hz, 1 H) 4.01
- 4.15 (m, 2 H) 4.31 (s, 1 H) 5.35 - 5.61 (m, 1 H) 7.39 (d, J=8.53 Hz, 1 H) 7.76 (dd, J=8.53, 2.51 Hz, 1
H) 8.01 - 8.09 (m, 1 H) 8.16 (d, J=8.03 Hz, 1 H) 8.36 (s, 1 H) 8.49 (s, 1 H); m/z 559.5 [MH]+.
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tert-Butyl
(((3S,4S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-((5-chloropyridin-2-yl)sulfonyl)-3-
hydroxypyrrolidin-3-yl)methyl)carbamate (26g): To a solution of 26f (0.075 g, 0.134 mmol) in DCM
(4.0 mL) was added m-CPBA (0.077 g, 0.335 mmol) and the reaction mixture was stirred at RT for 3 h.
Following this duration, the reaction mixture was quenched with 10% aqueous Na2S2SO3 and the layers
were separated. The organic layer was washed with saturated aqueous NaHCO3, dried over anhydrous
MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel
chromatography (0-35% EtOAc:Hexanes) to afford 26g as a colorless semi-solid (78 mg, 98% yield); m/z
491.1 [MH-Boc]+.
tert-Butyl(((3R,4S)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-((5-chloropyridin-2-yl)sulfonyl)-3-
hydroxypyrrolidin-3-yl)methyl)carbamate (26gg) was prepared from 26ff analogously to the synthesis
of 26g from 26f to afford 26gg as a white solid (150 mg, 77% yield). 1H NMR (400 MHz, DMSO-d6) δ
ppm 1.35 (s, 9 H) 3.38 (br. s., 4 H) 3.94 (d, J=9.29 Hz, 1 H) 4.11 (br. s., 1 H) 4.37 (br. s., 1 H) 6.94 (br.
s., 1 H) 8.01 (d, J=8.53 Hz, 1 H) 8.05 (d, J=8.28 Hz, 1 H) 8.14 (d, J=8.03 Hz, 1 H) 8.25 - 8.31 (m, 1 H)
8.36 (s, 1 H) 8.82 (s, 1 H); m/z 491.4 [MH-Boc]+.
4-(((3S,4S)-3-(Aminomethyl)-4-((5-chloropyridin-2-yl)sulfonyl)-3-hydroxypyrrolidin-1-
yl)sulfonyl)-3-chlorobenzonitrile (26): To a solution of 26g (0.078 g, 0.132 mmol) in DCM (1.0 mL)
was added TFA (0.305 mL, 3.96 mmol) and the resulting reaction mixture was stirred at RT for 0.5 h.
Following this duration, the reaction mixture was concentrated under reduced pressure. The residue was
diluted with H2O and the resulting solution was basified with saturated aqueous NaHCO3. The white
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Journal of Medicinal Chemistry
precipitate was filtered, washed with H2O and dried under vacuum to afford 26 as a white solid (37 mg,
54% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.82 - 3.00 (m, 2 H) 3.43 (d, J=10.29 Hz, 1 H) 3.58
(d, J=10.04 Hz, 1 H) 3.69 (d, J=11.54 Hz, 1 H) 3.91 (d, J=4.02 Hz, 1 H) 4.29 (d, J=6.27 Hz, 1 H) 5.62 -
5.98 (m, 1 H) 8.04 (dd, J=13.05, 8.53 Hz, 2 H) 8.10 - 8.17 (m, 1 H) 8.29 - 8.40 (m, 2 H) 8.90 (s, 1 H); m/z
491.3 [MH]+.
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4-(((3R,4S)-3-(Aminomethyl)-4-((5-chloropyridin-2-yl)sulfonyl)-3-hydroxypyrrolidin-1-
yl)sulfonyl)-3-chlorobenzonitrile (27) was prepared from 26gg analogously to the synthesis of 26 from
26g to afford 27 as a white solid (61 mg, 50% yield). 1H NMR (400MHz, DMSO-d6) δ ppm 8.84 (s, 1H),
8.36 (s, 1H), 8.28 (d, J=8.5 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 8.03 (dd, J=8.3, 19.8 Hz, 2H), 4.49 (t, J=7.5
Hz, 1H), 4.16 - 4.02 (m, 1H), 3.88 (d, J=9.3 Hz, 1H), 3.51 (d, J=10.0 Hz, 1H), 3.40 (br. s., 2H), 2.81 (d,
J=8.3 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.7, 57.6, 64.3, 73.2, 77.9, 116.8, 117.3, 124.3,
132.6, 136.0, 136.2, 139.1, 140.4, 142.7, 145.2, 148.8, 155.7; LRMS: m/z 491.3 [MH]+. HRMS: m/z
491.0018 (observed), 491.0017 (calculated).
■ ASSOCIATED CONTENT:
The Supporting Information is available free of charge on the ACS Publications website at DOI: ***
Included are experimental details regarding the calcium flux FLIPR, CLND solubility, hERG, CaV1.2,
phospholipidosis, 3A4 TDI, 3A4 RI assays; in vivo pharmacokinetics studies; rat pulmonary edema model
and in vivo IC50 calculations; complete experimental procedures and compound characterization data
(NMR, MS); VCD analyses of Compounds 7 and 10; crystal structure of Compound 19 (PDF)
Molecular formula strings and associated biological data (CSV)
■ AUTHOR INFORMATION:
Corresponding Author
*E-mail for J.E.P.: joseph.e.pero@gsk.com. Phone: (484)-923-3836
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