Discovery of Pyrrolidine Sulfonamides as Selective and Orally Bioavailable Antagonists of Transient Receptor Potential Vanilloid-4 (TRPV4)

Article abstract of DOI:10.1021/acs.jmedchem.8b01317

A novel series of pyrrolidine sulfonamide transient receptor potential vanilloid-4 (TRPV4) antagonists was developed by modification of a previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon binding to the target protein. The new template was initially discovered as a minor regio-isomeric side product formed during routine structure-activity relationship (SAR) studies, and further optimization resulted in highly potent compounds with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for identifying new heart failure medicines.

Full text of DOI:10.1021/acs.jmedchem.8b01317

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Article
Discovery of Pyrrolidine Sulfonamides as Selective and Orally Bioavailable
Antagonists of Transient Receptor Potential Vanilloid-4 (TRPV4)
Edward Brnardic, Guosen Ye, Carl A Brooks, Carla Donatelli, Linda Barton, John McAtee, Robert
Sanchez, Arthur Shu, Karl F Erhard, Lamont Terrell, Grazyna Graczyk-Millbrandt, Yanan He, Melissa
Costell, David J Behm, Theresa J Roethke, Patrick Stoy, Dennis A. Holt, and Brian G. Lawhorn
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01317 • Publication Date (Web): 10 Oct 2018
Downloaded from http://pubs.acs.org on October 11, 2018
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Discovery of Pyrrolidine Sulfonamides as Selective and
Orally Bioavailable Antagonists of Transient Receptor
Potential Vanilloid-4 (TRPV4)
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Edward J. Brnardic* , Guosen Ye , Carl Brooks , Carla Donatelli , Linda Barton , Jeff McAtee ,
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Robert M. Sanchez , Arthur Shu , Karl Erhard , Lamont Terrell , Grazyna Graczyk-Millbrandt ,
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Yanan He , Melissa H. Costell , David J. Behm , Theresa Roethke , Patrick Stoy , Dennis A. Holt ,
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Brian G. Lawhorn
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‡et
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Heart Failure Discovery Performance Unit, Flexible Discovery Unit and Platform Technology and
Sciences.
GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, United States
ABSTRACT:
A novel series of pyrrolidine sulfonamide TRPV4 antagonists was developed by modification of a
previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that
improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon
binding to the target protein. The new template was initially discovered as a minor regio-isomeric side
product formed during routine SAR studies, and further optimization resulted in highly potent compounds
with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were
achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound
GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in
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vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for
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identifying new heart failure medicines.
KEYWORDS: Transient Receptor Potential Vanilloid-4, TRPV4, pyrrolidine sulfonamide, lead
optimization
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■
INTRODUCTION:
Heart failure (HF) is an incurable, progressive disease that affects over 6 million people in America and
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significantly erodes quality of life. A common symptom of heart failure is pulmonary congestion, which
leads to shortness of breath, a decrease in exercise capacity, and an increase in heart failure rates and
hospital visits.2-3 In heart failure the decreased ability of the left ventricle to pump blood into the
peripheral circulatory system (indicated by a reduced ejection fraction and/or left ventricular dilation)
results in an accumulation of blood in the pulmonary veins, producing an increase in blood pressure within
these veins. Sustained high pressure in the pulmonary veins causes fluid to flow from the pulmonary
circulation into the surrounding alveoli which are responsible for transfer of oxygen to the bloodstream.
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Current therapies such as beta blockers, ACE inhibitors, angiotensin receptor blockers and diuretics are
unable to adequately treat HF-induced pulmonary edema and thus heart failure remains a significant area
of unmet medical need.
Transient Receptor Potential Vanilloid-4 (TRPV4), a member of the transient receptor potential (TRP)
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ligand-gated ion channel family that regulates Ca /Na influx into cells, is expressed at high levels in
the pulmonary vascular endothelial cells 7-8 which form a critical part of the alveolar septal barrier.
TRPV4 is activated by a variety of stimuli including warm temperatures, hypotonicity, physical cell
stress/pressure and pharmacological activators.8-13 Activation of TRPV4 in response to increased
pulmonary venous pressure causes endothelial cell contraction and detachment. This reduces the integrity
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of the alveolar septal barrier and enhances permeability, resulting in pulmonary edema and impairment
of gas exchange.14-15 Recently, TRPV4 antagonists such as GSK2193874,16 and a Piperidine-
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Benzimidazole GSK TRPV4 antagonist (Figure 1) among others,
have demonstrated the prevention
and resolution of pulmonary edema in in vivo models, suggesting a potential clinical benefit of inhibiting
TRPV4 function in the treatment of acute and/or chronic heart failure associated lung congestion.20
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Figure 1. TRPV4 Antagonists that have demonstrated efficacy in the prevention of lung edema.
Herein we describe the discovery and optimization of a novel series of pyrrolidine sulfonamides as
TRPV4 antagonists.
■
RESULTS AND DISCUSSION:
21
Piperidine 1 and azetidine 2 (Figure 2) were previously reported to be potent antagonists of TRPV4
and their activity was confirmed in our own TRPV4 assay (IC50 = 130 nM and 50 nM respectively).22
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F
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hTRPV4 IC50 = 130 nM
hTRPV4 IC50 = 50 nM
Figure 2. Azetidine and Piperidine TRPV4 Leads
The literature reported SAR study was limited and the few analogs that were reported varied only in the
substitution pattern on both aromatic rings.21 Therefore, we initiated an investigation to expand the SAR
of this promising template by focusing on modifications of the central core including changes in the
central ring size substituents. To assess the value of the hydroxy substituent, the des-hydroxy analog of
1
was synthesized (Scheme 1). Alkylation of 3-fluoro-4-cyano-phenol (4) with N-Boc-4-bromomethyl
piperidine (3) gave aryl ether (5) which was deprotected under acidic conditions and then treated with
2,4-dicholorobenzenesulfonyl chloride (6) to give sulfonamide 7 (IC50 = 6300 nM). The removal of the
hydroxy substituent resulted in a 50-fold loss in potency highlighting its critical importance for TRPV4
binding.
Scheme 1.
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Modification of the central ring size from the 6-membered piperidine (1) and the 4-membered azetidine
(2) to the unreported pyrrolidine was accomplished via a separate route (Scheme 2). Wittig reaction of
ketone 8 with methyl triphenyl phosphonium bromide in the presence of potassium tert-butoxide afforded
olefin 9. Bromination with NBS in the presence of water gave an equal mix of bromo-hydroxy regio
isomers that were subsequently cyclized using sodium hydride to give epoxide 10. The epoxide was then
opened with 3-fluoro-4-cyano-phenol (4) to give intermediate 11 which was deprotected under acidic
conditions and sulfonylated with 2,4-dichlorobenzenesulfonyl chloride (6) to give hydroxy pyrrolidine
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Scheme 2.
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The novel pyrrolidine core (12, IC50 = 150 nM) maintained potency relative to the previously reported
piperidine (1, IC50 = 130 nM) and azetidine (2, IC50 = 50 nM) while offering potential SAR advantages
due to its asymmetry and synthetic accessibility. Additionally, the pyrrolidine core has reduced
conformational flexibility compared to the piperidine while allowing for more substitution patterns as
compared to the azetidine core. We were eager to explore the SAR around the pyrrolidine core and
initially we sought to re-evaluate the position of the hydroxyl substituent to determine its optimal
placement for a possible interaction with TRPV4 (Scheme 3).
The methylene hydroxy pyrrolidine intermediate 13, which was initially synthesized according to
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literature precedent, was protected as the TBS-ether before hydroborating the olefin using 9-BBN and
sodium perborate to give cis- and trans-hydroxymethyl isomers 14 and 15. The cis- and trans-isomers
were separated by silica gel column chromatography and the stereochemistry of 14 and 15 was later
assigned based on inference from analytical data on final products. Etherification of hydroxymethyl
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isomers 14 and 15 was accomplished by treatment with mesylate 16 under basic conditions to give 17
and 20 respectively. The synthesis was completed via Boc deprotection using TFA, followed by
sulfonylation with 2,4-dichlorobenzene sulfonyl chloride to give sulfonamides 18 and 21, and finally silyl
deprotection using TBAF to afford racemic mixtures of cis-isomer 19, and trans-isomer 22. The
stereochemistry of 19 and 22 was assigned by the observation of nOe between the pyrrolidine C3 proton
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and the pyrrolidine C4 proton in compound 19, and the observation of nOe between the pyrrolidine C3
proton and the aryl ether methylene protons in 22.
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Scheme 3.
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Shifting the hydroxyl group of 12 to the neighboring position to give cis- and trans-diastereomers (19
IC50 = 1500 nM and 22 IC50 = 1000 nM) resulted in a moderate loss in potency (6-10 fold). Compounds
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indicating a significant benefit of the 4-hydroxy substituent. We hypothesized there may be two separate
hydroxy binding interactions with TRPV4 that are engaged as suggested by compounds 12, and 19 or 22,
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and thus we explored these possible interactions by simultaneously incorporating two hydroxyl
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substituents on the central ring (26, Scheme 4).
Synthesis of the cis-diol 26 (Scheme 4) began with epoxidation of allylic alcohol 13 which resulted in
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the isolation of a single epoxide diastereomer (23) which was assigned the cis- orientation. Presumably,
the hydroxyl group coordinates with m-CPBA and directs the epoxidation to the same face of the
pyrrolidine ring. Epoxide 23 was ring opened with 3-fluoro-4-cyanophenol (4) under basic conditions
and isolation of the major reaction product 24, followed by deprotection and sulfonylation with 6, gave
sulfonamide 26. Interestingly, an unknown minor side product 25 with identical molecular weight as 24
was also isolated from the epoxide ring opening in 8% yield. The minor isomer was elaborated to the
final product 27 to facilitate its structural assignment and confirm the assignment of 26. Following
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deprotection and sulfonylation, 25 was converted into 27 which by MS and H NMR appeared to be an
isomer of 26.
Scheme 4.
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The structure of 26 was determined using H and C GASPE spectra, and 2-D NMR data (gCOSY,
gHMQC, gHMBC). The atom connectivity of 26 was assigned based on the observation of HMBC
correlations from C1 of the aryl ether to the exocylclic methylene protons, C3 of the pyrrolidine to the
exocyclic methylene protons, and C4 of the pyrrolidine to the exocyclic methylene protons. COSY
correlations observed between the C4 proton and the secondary hydroxy, the C4 proton and a C5 proton,
and between the two C2 protons confirmed the assignment. The stereochemistry of 26 was assigned based
on nOe observations showing both the secondary hydroxy and tertiary hydroxy protons are near the
pyrrolidine C2-Ha and C5-Hb protons, indicating the two hydroxy groups occupy the same face of the
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ring. Likewise, the exocyclic methylene and the C4 methine share nOe to both the C2-Hb and C5-Ha
protons.
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The structure of 27 was determined using 2-D NMR studies (gCOSY45, gHMQC, gHMBC and 13C
gaspe spectra). The atom connectivity of 27 was assigned based on the observation of an HMBC
correlation from C1 of the aryl ether to the pyrrolidine C3 proton which indicated that 27 was a
regioisomer of 26 with connection of the benzonitrile occuring through the secondary center of the
pyrrolidine rather than through the primary hydroxyl. A COSY correlation between the exocyclic
methylene protons and the primary OH proton was observed that confirmed this assignment. The
stereochemistry of 27 was assigned by the observation of nOe between the methine proton at the C3
position of the pyrrolidine and the exocyclic methylene protons, and the absence of nOe from the C3
pyrrolidine methine proton to both the OH protons, providing evidence that 27 was the cis-diastereomer
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Figure 3). Based on the structures of starting material and the product obtained we propose that 25 is
formed from 24 under the reaction conditions via a Smiles rearrangment26-29 (Figure 3) where an
intramolecular displacement at the aromatic ring is initiated by the nucleophilic secondary hydroxide and
the reaction proceeds through a 6-membered transition state.
Figure 3. Mechanism of Formation of 25
When evaluated for activity against TRPV4, compound 26 (IC50 = 150 nM) showed equivalent activity
to its monohydroxy analog 12 (IC50 = 150 nM), indicating the secondary hydroxyl offers no additional
benefit in this template. Thus, the role of the secondary hydroxyl in 19 and 22 may be similar to that of
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the tertiary hydroxyl of 12, with the C3 placement being preferred for TRPV4 recognition. Remarkably,
when compound 27 was tested against TRPV4 it displayed similar activity (IC50 = 100 nM) as its
regioisomer 26 despite lacking the preferred C3-OH. We suspected that the potency of 27 may arise from
its reduced conformational flexibility resulting from the removal of the methylene linker through direct
linkage of the phenol to the pyrrolidine ring.
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Given its unique activity and novel structure, 27 was selected as a lead for further SAR studies. We
hypothesized that the substituents of 27 may produce conformational bias on the pyrrolidine template30-
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and also suspected the hydroxy substituents could be involved in specific interactions with TRPV4. To
understand the critical features of 27 required for TRPV4 binding, we aimed to deconstruct the pyrrolidine
diol core of 27 with particular intent on understanding the role of each hydroxyl group. Recognizing that
the stereochemical array of substituents found serendipitously in 27 would likely be important for TRPV4
recogntion but may not be optimal, another key element of our strategy was the systematic evaluation of
the stereochemical presentation of each substituent. Thus, we designed synthetic routes that would allow
us to evaluate all of the possible stereoisomers of a complete series of mono-hydroxy and des-hydroxy
pyrrolidine analogs of 27.
Synthesis of the des-hydroxy pyrrolidine analog (Scheme 5) was accomplished by first treating the
commercially available hydroxy pyrrolidine 28 with 2,4-dichlorobenzenesulfonyl chloride (6) under basic
conditions. The resulting sulfonamide 29 was condensed with 3-fluoro-4-cyanophenol (4) under
Mitsunobu conditions to form 30.
Scheme 5.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Surprisingly, the des-hydroxy pyrrolidine 30 (IC50 = 320 nM) showed only a 3-fold loss in potency vs.
7 (IC50 = 100 nM) compared to the 50-fold loss observed upon removal of the hydroxy group from the
2
piperidine series (cf 1 and 7). Not only did this result highlight the inherent entropic advantage of the
ether-linked pyrrolidine series, but it also indicated that 27 may not present an optimal arrangement of
hydroxy groups for TRPV4 binding.
To evaluate the effects of removing the tertiary hydroxyl of 27, two hydroxymethyl diastereomers
(
Scheme 6) were synthesized. Condensation of hydroxy pyrrolidine 13 with 3-fluoro-4-cyanophenol (4)
under Mitsunobu conditions, followed by hydroboration/oxidation of olefin 31 using 9-BBN and sodium
32
perborate gave both the minor trans- and major cis-hydroxymethyl pyrrolidines 32 and 33. Boc removal
followed by sulfonylation with 2,4-dichlorobenzenesulfonyl chloride (6) afforded compounds 34 and
35.33
Scheme 6.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
While the trans-hydroxymethyl isomer 34 (IC50 = 200 nM) showed little improvement in potency
compared to the des-hydroxy compound 30 (IC50 = 320 nM), the cis-hydroxymethyl diastereomer 35 (IC50
= 50 nM) showed a 6-fold improvement, confirming our hypothesis that the hydroxyl groups in 27 may
not be optimally placed for an interaction with TRPV4. Compound 34 was however, 2-fold less potent
than 27 indicating the potential importance of the tertiary C3-pyrrolidine hydroxyl group.
Next, we assessed the individual C3-hydroxy diastereomers (Scheme 7) of 27 lacking only the
3
4
hydroxymethyl group, which were synthesized by ring-opening of epoxide 36 with 3-fluoro-4-cyano
phenol (4) under basic conditions to give the trans-isomer 37. Intermediate 37 was either directly
deprotected and sulfonylated to give the trans-hydroxypyrrolidine sulfonamide 38, or it was inverted via
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Page 14 of 56
esterification under Mitsunobu conditions with para-nitrobenzoic acid followed by ester hydrolysis with
LiOH to give the cis-hydroxypyrrolidine 39. Compound 39 was then deprotected under acidic conditions
and sulfonylated to give the cis-hydroxypyrrolidine sulfonamide 40.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 7.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Similar to the C4-hydroxymethyl substituent, we found that the C4-hydroxy substituent was not
optimally oriented in 27 as the cis-hydroxypyrrolidine analog 40 (IC50 = 100 nM) showed only a 3-fold
improvement in potency compared to the des-hydroxy pyrrolidine 30 (IC50 = 320 nM), while the trans-
hydroxypyrrolidine diastereomer 38 (40 nM) showed an 8-fold improvement. Thus, deconstructing diol
2
7 (IC50 = 100 nM) into its mono hydroxy substituents revealed there was no advantage in combining the
cis-hydroxy of 40 (IC50 = 100 nM) and trans-hydroxymethyl of 34 (IC50 = 200 nM) suggesting that neither
of the pyrrolidine C4-substituents of 27 were presented in an optimal configuration. The substantial
potency enhancements (> 6-fold) offered by both 35 and 38 over 30 prompted synthesis of 42, an analog
that combines each of these substituents.
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Journal of Medicinal Chemistry
Compound 42, the diastereomer of 27, was synthesized (Scheme 8) from intermediate 31. The Boc
1
2
protecting group was removed with trifluoroacetic acid, and treatment of the unprotected pyrrolidine TFA
salt with 2,4-dichlorobenzenesulfonyl chloride (6) and aqueous K CO in DCM gave sulfonamide 41.
Dihydroxylation of the exocyclic olefin with catalytic OsO , using NMO as a co-oxidant, provided trans-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
4
pyrrolidine diol 42. The stereochemical configuration of 42 was assigned based on an nOe between the
C3 pyrrolidine methine proton and the tertiary hydroxy proton, and the lack of an nOe between the C3
pyrrolidine methine proton and the exocyclic methylene protons. The dihydroxylation proceeds in
excellent yield and with high diastereoselectivity (dr 99:1) with the trans-isomer presumably arising
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
through steric bias wherein OsO preferentially approaches the olefin on the ring face opposite the
4
phenoxy substituent. Notably, the stereochemical outcome is the inverse of that observed when
epoxidizing the hydroxy olefin 13 (Scheme 4). Thus, both cis- and trans-pyrrolidine isomers can be
accessed in highly stereoselective fashion by selection of appropriate intermediate and reagent
combinations.
Scheme 8.
Gratifyingly, the SAR was almost completely additive when combining the trans-hydroxy C4-
substituent (8-fold potency enhancement over des-hydroxy pyrrolidine 30) with the cis-hydroxymethyl
C4-substituent (6-fold) to give diol 42 which exhibited exceptional potency (IC50 = 4 nM) representing a
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8
0-fold improvement over the unsubstituted pyrrolidine 30 . The additive SAR demonstrates that each
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydroxyl group of 42 is independently responsible for potency enhancements and plays a unique role in
TRPV4 recognition. Notably, the trans-configured tertiary hydroxyl group elicits a strong bias on the
pucker of the pyrrolidine ring, with the trans-diaxial orientation of the C3-phenoxy and C4-hydroxyl
being the favored low energy conformation based on ab initio QM calculations (Figure 4).35 This
conformation maximizes gauche effects in the pyrrolidine ring and minimizes dipole repulsion of the
neighboring C-O bonds.30-31 Thus, we speculate the potency gains arising from the tertiary-hydroxyl may
be due to its impact on preorganizing the pyrrolidine template into the TRPV4 binding orientation. The
primary hydroxyl group may reinforce this conformation through an intramolecular H-bond with the
tertiary hydroxyl or may engage in a direct interaction with the TRPV4 protein.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Representation of the low energy conformation of 42.
The exceptional TRPV4 antagonist activity of 42 prompted further profiling of the trans-oriented
pyrrolidine diol template. Thus, 42, which had been prepared as a racemic mixture, was separated into
its constituent enantiomers using chiral chromatography and the configuration of each enantiomer was
assigned by ab initio vibrational circular dichroism (VCD) analysis. The TRPV4 activity was found to
predominately reside in the (3S,4R)-enantiomer 51 (IC50 = 2.5 nM), with the (3R,4S)-enantiomer showing
100-fold less potency (IC50 = 250 nM).
Scheme 9.
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Journal of Medicinal Chemistry
Br
1
2
3
4
5
6
7
8
9
N
O
N
Br
HO
Br
O
O
n
2N NaOH
Me S+ I-, BuLi
3
N
o
N
o
o
MeCN/H O, 10 C
THF, 23 C
N
THF, -20 C
2
Boc
Boc
Boc
96% over 2 steps
85%
43
(+/-) 44
36
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
Chiral
Resolution
PhCOCl, Et N
KOH
3
O
O
O
O
N
o
o
DCM, 23 C
MeOH, 23 C
Boc
N
N
80%
Boc
Boc
99%
(+/-) 13
(+/-) 45
46
F
F
HO
O
O
4
, PS-PPh , DIAD
TFA
NC
6, K CO
3
3
2
NC
N
o
o
o
THF, 23 C
DCM, 23 C
N
DCM/H O, 23 C
2
N
H
Boc
.
Boc
TFA
45% over 3 steps
49
4
7
48
F
F
O
O
OH
O
OH
NC
NC
OsO , NMO
N
S
4
N
S
O
O
O
o
THF, 23 C
Cl
Cl
5
0
60%
51
Cl
Cl
A chiral synthesis of 51 was developed and optimized to access large scale quantities for biological
evaluation (Scheme 9). Beginning with commercially available N-Boc-pyrroline (43), bromination with
1
,3-dibromo-5,5-dimethylhydantoin in acetonitrile/water afforded the trans-bromo-hydroxypyrrolidine
4 which was cyclized to form epoxide 36 in an aqueous solution of sodium hydroxide. Exposure of 36
4
to an excess of dimethylsulfonium methylide (generated from the reaction of trimethylsulfonium iodide
with n-butyl lithium) produced allylic alcohol 13 which was then treated with benzoyl chloride and
triethylamine to give the benzoyl ester intermediate 45. Separation of the enantiomers of 45 was
accomplished using Chiral HPLC and the absolute configuration of the enantiomers was assigned by ab
initio VCD analysis.35 Ester hydrolysis of the (S)-enantiomer 46 using KOH in MeOH produced the (S)-
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enantiomer of the Boc protected hydroxyl pyrrolidine 47. Compound 47 was converted to the aryl ether
8 by reaction with 3-fluoro-4-cyanophenol (4) using Mitsunobu conditions (PS-PPh /DIAD) which
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
3
occurred with clean inversion of stereochemistry to provide the desired (R)-phenyl ether. The Boc
protecting group was removed with trifluoroacetic acid to give the TFA salt of pyrrolidine 49. Treatment
of the pyrrolidine 49 with 2,4-dichlorobenzenesulfonyl chloride (6) and K
synthesis was completed via dihydroxylation of the exocyclic olefin of the pyrrolidine ring with catalytic
OsO using NMO as a co-oxidant, which provided 51.
2
CO
3
gave sulfonamide 50. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Table 1. Profile of Trans-pyrrolidine-diol 51 vs Piperidine 1
Compound
hTRPV4 IC50 a
ChromlogD b
Aq. Solubility
Rat CL c
1
51
2.5 nM
4.6
130 nM
6.3
7.5 µM
360 µM
58
1
9
ml/min/kg ml/min/kg
Rat MRT c
1.5 h
56%
0.9 h
35%
Rat Oral F c
Rat PPB, f d
Rat CL d
1.3%
1461
5.9%
966
u
u
ml/min/kg ml/min/kg
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Journal of Medicinal Chemistry
a.hTRPV4 potency was determined in a FLIPR assay.22 b.ChromlogD was used to measure lipophilicity. c.Rat
pharmacokinetic properties of clearance (CL) and mean resonance time (MRT) measurements were derived from iv leg and
oral bioavailability (Oral F) measurements were derived from oral leg.39-40 d.Unbound clearance (CLu) was determined
based on invitro plasma protein binding (PPB).39-40
38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Trans-pyrrolidine diol 51 is a novel, advanced lead compound for identifying TRPV4 antagonist drug-
candidates. A side-by-side assessment of the biological, physicochemical, and pharmacokinetic
properties of 51 and the reported lead 1 was conducted (Table 1).37 Compound 51 exhibits a 50-fold
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8
improvement in TRPV4 activity, significantly reduced lipophilicity as measured by ChromLogD, and
higher aqueous solubility. The pharmacokinetic properites of 1 and 51 are similar, with 51 showing a
moderate clearance, MRT and oral biovailability in rats.39-40 Importantly, 51 demonstrates excellent
selectivity for TRPV4, exhibiting little or no activity against a broad panel of TRP channels (IC50 > 10
µM for TRPA1, TRPV1, TRPM2, TRPM4, TRPM8, TRPC3, TRPC4, TRPC5, TRPC6). We sought to
further improve the potency and modulate the PK properties of 51 by optimizing both the aryl ether and
the sulfonamide substituents.
Evaluation of a small set of aryl ether substituent patterns revealed that the SAR for the pyrrolidine diol
was analogous to that reported for piperidine 1 and the 3-fluoro, 4-nitrile substitution pattern of 51 was
found to be optimal for potency and pharmacokinetics on the trans-pyrrolidine diol template (data not
shown).21 The similarity in aryl ether and sulfonamide SAR between 1, 2 and 51 suggests an overlap in
binding modes between the two series and confirms that the conformational rigidity and introduction of
the diol are the primary drivers of the enhanced potency in the pyrrolidine diol template. We then focused
our attention on the optimization of the sulfonamide moiety to further build on the SAR previously
established for the piperidine and azetidine cores.21 We discovered that substituting in the 3- position
consistently led to a loss in potency regardless of the electronic nature of the substituents, suggesting a
possible steric component. In contrast, 2-substituents were found to enhance activity compared to the
unsubstituted benzene, suggesting that the ortho-substitution may favorably bias the analogs into the
TRPV4 binding conformation. Notably, 2-substituted aryl sulfonamides are expected to adopt a bisected
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conformation about the S-Ar bond whereas unsubstituted aryl sulfonamides prefer a perpindicular
orientation.41 Small, electron withdrawing groups offered the most potency enhancement in both the 2-
and 4-position indicating an apparent preference for an electron-deficient aryl sulfonamide. The SAR
was additive when combining substitution in the 2- and 4-positions and of particular interest was the 2-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CN,4-CF -sulfonamide 52 which balanced potency (1 nM) and lipophilicity (ChromlogD = 4.6) with
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
moderate CL and good oral bioavailability (72%).
Table 2. Profile of Trans-pyrrolidine-diol 52
Compound
hTRPV4 IC50 a
ChromlogD b
Aq. Solubility
Rat CL c
52
1 nM
4.6
90 µM
8
2
ml/min/kg
Rat MRT c
0.8 h
Rat Oral F c
72%
Rat PPB, f d
Rat CL d
4.9%
u
1400
ml/min/kg
u
22
38
a.hTRPV4 potency was determined in a FLIPR assay. b.ChromlogD was used to measure lipophilicity. c.Rat
pharmacokinetic properties of clearance (CL) and mean resonance time (MRT) measurements were derived from iv leg and
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Journal of Medicinal Chemistry
oral bioavailability (Oral F) measurements were derived from oral leg.39-40 d.Unbound clearance (CLu) was determined
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
based on invitro plasma protein binding (PPB).39-40
An established in vivo pharmacokinetic/pharmacodynamic (PK/PD) model of TRPV4-mediated
pulmonary edema was used to evaluate the effects of Pyrrolidine 52 on mean arterial pressure (MAP) and
lung wet weight (normalized to body weight; LW/BW) in Sprauge Dawley rats following 10 min i.v.
infusion with the TRPV4 agonist GSK1016790A at 10 µg/kg/min.12,20,42 Prior to agonist administration,
rats were pre-treated with vehicle or a dose-range of Pyrrolidine 52 via a regimen consisting of an i.v.
bolus loading dose (0.03, 0.08, and 0.23 mg/kg) followed by a continuous 60 min i.v. infusion (0.6, 1.7
and 5.1 µg/kg/min) to provide steady-state plasma exposure. Steady-state plasma concentrations were 30
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
±
9.8, 40 ± 7.1, and 90 ± 1.3 nM, respectively.
In vehicle treated rats, GSK1016790A significantly decreased MAP and increased the LW/BW ratio,
while treatment with Pyrrolidine 52 inhibited these responses in a dose-dependent manner (Figure 5).
Full inhibition of these responses was achieved using the 5.1 µg/kg/min dose, demonstrating protection
against TRPV4-mediated blood pressure and pulmonary edema effects in the rat. Notably, the observed
effects in the agonist-driven PK/PD experiment correlate with prevention of lung edema in heart failure
disease models including acute pressure overload and chronic myocardial infarction studies in rodents. 20
2
5
0
*** ****
*
**9
0
1.3 nM
-
-
-
25
50
75
40 7.1 nM
30 9.8 nM
Vehicle (n=3)
52 (5.1 ug/kg/min i.v., n=3)
2 (1.7 ug/kg/min i.v., n=3)
2 (0.6 ug/kg/min i.v., n=3)
#
##
8
5
5
-100
0
2
4
6
10
GSK1016790A Infusion Time (min)
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FIGURE 5. Effect of TRPV4 antagonist TRPV4 agonist-induced reduction of mean arterial pressure
MAP; A) and elevation of lung weight normalized to body weight (LW/BW; B).43-44
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
*
*p<0.01, ***p<0.001, ****p<0.0001 vs. vehicle
#
##
p<0.05, p<0.001 vs. baseline
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
■ CONCLUSION:
In summary, SAR studies on a reported piperidine sulfonamide TRPV4 antagonist led to a novel series
of pyrrolidine sulfonamide TRPV4 antagonists. The key pyrrolidine diol core arose serendipitously as an
unexpected side-product from a routine synthetic reaction that proved to have potent TRPV4 activity.
The template was optimized by identifying the preferred stereochemical presentation of the diol moiety.
The enhanced potency of the pyrrolidine diol template was primarily attributed to an increase in structural
rigidity (reduction of entropy) via the deletion of the aryl ether methylene linker, truncation of the
piperidine ring to the pyrrolidine, and conformationl bias imparted by the diol substituents. The TRPV4
activity and oral bioavailability were further enhanced through modifications of the sulfonamide aryl
substituent pattern. Robust efficacy in the in vivo PK/PD model was observed with the optimal exemplar
from the series (52) and therefore, pyrrolidine sulfonamide 52 (GSK3395879) represents an optimized
lead with drug like properties from a chemical series that has potential therapeutic value for the treatment
of pulmonary edema associated with heart failure.
■
EXPERIMENTAL SECTION:
45
46
General Experimental. Biological assays and Rat Pharmacokintetics were conducted as described.
The purity of each antagonist 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
22
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Journal of Medicinal Chemistry
were conducted using a Waters Acquity SQD with pos/neg switching in ESI mode. Preparative HPLC
was conducted on a Waters 2525 system at a flow rate of 50 ml/min with 254 nm detection using a Sunfire
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C18 OBD 5 μm column (30 × 150 mm) with a gradient of MeCN/H
2
O/0.1% TFA. Flash column
chromatography was conducted on silica gel eluting with EtOAc-hexanes, EtOAc-heptane, or
MeOH−CH
-((1-((2,4-dichlorophenyl)sulfonyl)-4-hydroxypiperidin-4-yl)methoxy)-2-fluorobenzonitrile (1),21 4-
(1-((2,4-dichlorophenyl)sulfonyl)-3-hydroxyazetidin-3-yl)methoxy)-2-fluorobenzonitrile (2),23 (+/-)
2
Cl mixtures. The following intermediates were synthesized following literature procedures:
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
(
tert-butyl
3-hydroxy-4-methylenepyrrolidine-1-carboxylate
(13),23
4-cyano-3-fluorophenyl
2
4
34
methanesulfonate (16), tert-butyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (36). Other required
reagents and building blocks were either purchased and used as is or synthesized as described below.
Tert-butyl 4-((4-cyano-3-fluorophenoxy)methyl)piperidine-1-carboxylate (5). A solution of tert-
butyl 4-(bromomethyl)piperidine-1-carboxylate 3 (500 mg, 1.797 mmol) and cesium carbonate (878 mg,
2
.70 mmol) in DMSO (2 ml) and water (2 ml) was treated with 2-fluoro-4-hydroxybenzonitrile 4 (296
mg, 2.157 mmol) and the resulting mixture was was subjected to microwave irradiation for 30 minutes at
00 °C. The mixture was diluted with water and extracted three times with EtOAc. The combined organic
1
extracts were washed with brine, dried over Na
2
SO , filtered, and concentrated to give 5 (500 mg, 83%)
4
as a colorless oil: MS (m/z) 357.2 (M + Na+).
4
-((1-((2,4-dichlorophenyl)sulfonyl)piperidin-4-yl)methoxy)-2-fluorobenzonitrile (7). A solution
of 5 (500 mg, 1.5 mmol) in DCM (6 ml) was treated with a solution of 4M HCl in dioxane (3 ml) and the
reaction solution was stirred for 1 h at 23 °C and then concentrated to give a white solid. The solid
material was re-dissolved in DCM (6 ml) and subsequently treated with triethylamine (1.04 ml, 7.48
mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride 6 (551 mg, 2.24 mmol) and the resulting mixture was
stirred for 3 hours at 23 °C. The mixture was diluted with dicholoromethane, washed with water, washed
with brine, dried over Na
2
SO
4
, filtered, concentrated, and subjected first to flash chromatography (0-10%
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Page 24 of 56
EtOAc/Hexanes) and then to reverse phase HPLC (MeCN/Water/0.1% TFA) to give 7 (22 mg, 3%) as a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
white solid: H NMR (400 MHz, DMSO-d
6
) δ ppm 7.97 (d, J=8.53 Hz, 1H), 7.93 (d, J=2.01 Hz, 1H),
7.82 (t, J=8.41 Hz, 1H), 7.66 (dd, J=2.01, 8.53 Hz, 1H), 7.16 (dd, J=2.38, 11.92 Hz, 1H), 6.96 (dd, J=2.26,
8
1
.78 Hz, 1H), 3.97 (d, J=6.27 Hz, 2H), 3.75 (d, J=12.55 Hz, 2H), 2.73-2.84 (m, 2H), 1.87-1.99 (m, 1H),
.76-1.85 (m, 2H), 1.29 (dq, J=4.02, 12.30 Hz, 2H). MS (m/z) 443.1 (M + H+).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Tert-butyl
3-methylenepyrrolidine-1-carboxylate
(9).
A
solution
of
bromo(methyl)triphenylphosphorane (19.3 g, 54.0 mmol) in diethyl ether (150 ml) was treated with KOt-
Bu (6.06 g, 54.0 mmol) and the mixture was stirred at 25 °C for 1 hour before being treated with a solution
of tert-butyl 3-oxopyrrolidine-1-carboxylate 8 (10.0 g, 54.0 mmol) in dry diethyl ether (100 ml) and the
resulting mixture was stirred overnight at 25 °C. The mixture was diluted with water and extracted three
times with EtOAc. The combined organic extracts were washed three times with brine, dried over
Na
2
SO
4
, filtered, concentrated and subjected to flash chromatography (0-40% EtOAc/hexanes) to give 9
1
(
5.2 g, 58 %): H NMR (400 MHz, CHLOROFORM-d) δ 4.98 (td, J=1.94, 7.15 Hz, 2H), 3.94 (s, 2H),
3.48 (t, J=7.40 Hz, 2H), 2.57 (t, J=6.78 Hz, 2H), 1.49 (s, 9H). MS (m/z) 205.9 (M + Na+).
(
+/-) Tert-butyl 1-oxa-5-azaspiro[2.4]heptane-5-carboxylate (10). A solution of 9 (1.00 g, 5.46
mmol) in DMSO (10 ml) was cooled to 0 °C and treated with 1-bromopyrrolidine-2,5-dione (1.94 g, 10.9
mmol) and water (0.197 ml, 10.9 mmol). The mixture was warmed to 25 °C and stirred overnight. The
reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic
extracts were washed three times with water, dried over Na
2
SO , filtered, concentrated and subjected to
4
flash chromatography (0-40% EtOAc/hexanes) to give a mixture of tert-butyl 3-(bromomethyl)-3-
hydroxypyrrolidine-1-carboxylate and tert-butyl 3-bromo-3-(hydroxymethyl)pyrrolidine-1-carboxylate
+
regioisomers (800 mg, 58%): MS (m/z) 223.9 (M + H – t-Bu).
The hydroxy bromo regioisomers (800 mg, 2.86 mmol) were dissolved in THF (20 ml) and cooled to 0
°
C before being treated with sodium hydride (156 mg of a 60% dispersion in mineral oil, 3.89 mmol).
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The mixture was warmed to 25 °C and stirred overnight and then quenched with the addition of aqueous
saturated ammonium chloride solution. The mixture was diluted with water and extracted three times
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with EtOAc. The combined organic extracts were washed three times with brine, dried over Na
2
SO ,
4
filtered, concentrated and subjected to flash chromatography (0-60% EtOAc/hexanes) to give 10 (380 mg,
1
7
2
4 %): H NMR (400 MHz, CHLOROFORM-d) δ 3.63 (m, 3H), 3.30 (d, J=11.80 Hz, 1H), 2.96 (s, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.16-2.40 (m, 1H), 1.77-1.93 (m, 1H), 1.49 (s, 9H).
(+/-) Tert-butyl 3-((4-cyano-3-fluorophenoxy)methyl)-3-hydroxypyrrolidine-1-carboxylate (11).
A solution of 10 (5.10 g, 25.6 mmol) and 2-fluoro-4-hydroxybenzonitrile (4) (4.21 g, 30.7 mmol) in
isopropanol (150 ml), DMSO (30 ml) and water (30 ml) was treated with cesium carbonate (10.0 g, 30.7
mmol) and the mixture was heated overnight at 60 °C. The volatile solvents were removed under reduced
pressure and the mixture was diluted with water and extracted three times with EtOAc. The combined
organic extracts were washed three times with brine, dried over Na
SO
2 4
, filtered, concentrated and
1
subjected to flash chromatography (0-70% EtOAc/hexanes) to give 11 (4.3 g, 50%): H NMR (400 MHz,
DMSO-d
6
) δ 7.78-7.93 (m, 1H), 7.20 (dd, J=2.38, 11.92 Hz, 1H), 7.01 (dd, J=2.26, 8.78 Hz, 1H), 5.27 (s,
1
H), 4.09 (d, J=2.76 Hz, 2H), 3.15-3.53 (m, 4H), 1.89-2.09 (m, 1H), 1.74-1.87 (m, 1H), 1.40 (d, J=3.01
+
Hz, 9H). MS (m/z) 280.9 (M + H – t-Bu).
(
+/-)
fluorobenzonitrile (12). A solution of 11 (280 mg, 0.832 mmol) in DCM (20 ml) was treated with TFA
1.5 ml, 19.5 mmol) and the solution was stirred for 2 hours at 25 °C. The solvent was removed under
4-((1-((2,4-Dichlorophenyl)sulfonyl)-3-hydroxypyrrolidin-3-yl)methoxy)-2-
(
reduced pressure and the resulting residue was redissolved and concentrated from DCM/hexanes several
times to give the trifluoroacetic acid salt of 2-fluoro-4-((3-hydroxypyrrolidin-3-yl)methoxy)benzonitrile
1
(
290 mg, 100%): H NMR (400 MHz, DMSO-d
6
) δ 8.87-9.34 (m, 2H), 7.87 (t, J=8.28 Hz, 1H), 7.21 (dd,
J=2.01, 11.80 Hz, 1H), 7.02 (dd, J=1.76, 8.78 Hz, 1H), 5.77 (s, 1H), 4.10-4.28 (m, 2H), 3.36 (dd, J=5.40,
8.66 Hz, 2H), 3.09-3.26 (m, 2H), 1.89-2.15 (m, 2H). MS (m/z) 237.0 (M + H+).
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A
solution of the trifluoroacetic acid salt of 2-fluoro-4-((3-hydroxypyrrolidin-3-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yl)methoxy)benzonitrile (100 mg, 0.286 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (70.3 mg,
0.286 mmol) in DCM (10 ml) was treated with triethylamine (0.100 ml, 0.716 mmol) and the reaction
mixture was stirred overnight at 25 °C. The mixture was diluted with water and extracted three times
with DCM. The combined organic extracts were passed through a phase separator, concentrated and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
subjected to flash chromatography (0-70% EtOAc/hexanes) to give 12 (120 mg, 89 %): H NMR (400
MHz, DMSO-d
6
) δ 8.00 (d, J=8.53 Hz, 1H), 7.89 (d, J=2.01 Hz, 1H), 7.84 (t, J=8.28 Hz, 1H), 7.64 (dd,
J=1.76, 8.53 Hz, 1H), 7.09-7.19 (m, 1H), 6.96 (dd, J=1.88, 8.66 Hz, 1H), 5.45 (s, 1H), 3.99-4.16 (m, 2H),
3
.54 (dd, J=5.14, 8.66 Hz, 2H), 3.46 (d, J=10.29 Hz, 1H), 3.28-3.37 (m, 3H), 1.98-2.10 (m, 1H), 1.87-
1.96 (m, 1H). MS (m/z) 444.8 (M + H+).
(
+/-) Tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (13).
A suspension of
trimethylsulfonium iodide (762 g, 3740 mmol) in THF (2500 ml) at −10 °C was treated with nBuLi (1380
ml, 3460 mmol) over 45 min while maintaining an internal temperature between −5 and −10 °C. After
stirring for an additional 30 minutes the mixture was treated with a solution of tert-butyl 6-oxa-3-
azabicyclo[3.1.0]hexane-3-carboxylate 36 (173 g, 934 mmol) in THF (500 ml) over 20 min before being
warmed to 23 °C and stirred overnight. The mixture was diluted with TBME (2L) and washed with water
(
2L). The aqueous was extracted once more with 1L TBME and the combined orgainc layers were washed
with brine, dried over magnesium sulfate, filtered and concentrated to give 13 (158 g, 85%) as a yellow
1
oil: H NMR (400 MHz, DMSO-d
6
) δ 5.36 (d, J=5.27 Hz, 1H), 5.13 (br. s., 1H), 5.06 (br. s., 1H), 4.44
(
d, J=4.77 Hz, 1H), 3.76-3.99 (m, 2H), 3.49-3.59 (m, 1H), 2.94-3.07 (m, 1H), 1.40 (s, 9H).
(+/-) Cis-tert-butyl 3-((tertbutyldimethylsilyl)oxy)-4-(hydroxymethyl)pyrrolidine-1-carboxylate
(
14) and (+/-) Trans-tert-butyl 3-((tertbutyldimethylsilyl)oxy)-4-(hydroxymethyl)pyrrolidine-1-
carboxylate (15). A solution of tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate 13 (6.8 g,
3
4.1 mmol) in DMF (100 ml) was treated with TBSCl (6.17 g, 41.0 mmol) followed by imidazole (4.65
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g, 68.3 mmol) and the mixture was stirred overnight at 23 °C. The reaction mixture was diluted with
water and the product was extracted three times with EtOAc. The combined organic layers were washed
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
three times with brine, dried over Na
2
SO
4
, filtered, concentrated and subjected to flash chromatography
(10-60% EtOAc/hexanes) to
give
(+/-)
tert-butyl
3-((tert-butyldimethylsilyl)oxy)-4-
1
methylenepyrrolidine-1-carboxylate (9.4 g, 88 %) as a light yellow oil: H NMR (400 MHz, DMSO-d
6
)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
δ 5.09 (br. s., 2H), 4.53-4.74 (m, 1H), 3.77-3.96 (m, 2H), 3.51-3.69 (m, 1H), 2.87-3.11 (m, 1H), 1.40 (s,
9
H), 1.24 (br. s., 1H), 0.73-0.96 (m, 9H), 0.09 (s, 6H).
A 0.5 M solution of 9-borabicyclo[3.3.1]nonane in THF (15 ml, 7.5 mmol) was treated with a solution
of (+/-) tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-methylenepyrrolidine-1-carboxylate (2 g, 6.4 mmol)
in THF (2 ml) and the mixture was stirred for 1 hour before being diluted with water (5 ml). The reaction
mixture was then treated with sodium perborate tetrahydrate (9.8 g, 64 mmol) and the mixture was
vigorously stirred for 1 hr. The suspension was diluted with saturated aq. NaHCO
aqueous phase was extracted three times with EtOAc. The combined organic layers dried over with
Na SO , filtered, concentrated and subjected to flash chromatography (0-50% EtOAc/hexanes) to give 14
3
solution and the
2
4
(
520 mg, 23%) and 15 (170 mg, 8%) as colorless oils.
1
1
4: H NMR (400 MHz, CHLOROFORM-d) δ 4.45 (d, J=3.26 Hz, 1H), 3.82-3.95 (m, 1H), 3.69-3.81
(
(
m, 1H), 3.40-3.61 (m, 2H), 3.33 (d, J=8.78 Hz, 2H), 2.17-2.42 (m, 2H), 1.48 (s, 9H), 0.92 (s, 9H), 0.13
+
d, J=4.52 Hz, 6H). MS (m/z) 276.1 (M + H – t-Bu).
1
1
5: H NMR (400 MHz, CHLOROFORM-d) δ 4.10-4.29 (m, 1H), 3.51-3.75 (m, 4H), 3.08-3.25 (m,
2
3
H), 2.20-2.41 (m, 1H), 1.63-1.68 (m, 1H), 1.48 (s, 9H), 0.84-1.03 (m, 9H), 0.03-0.20 (m, 6H). MS (m/z)
32.2 (M + H+).
(+/-)
Cis-tert-butyl
3-((tert-butyldimethylsilyl)oxy)-4-((4-cyano-3-
fluorophenoxy)methyl)pyrrolidine-1-carboxylate (17). A solution of 14 (360 mg, 1.1 mmol) and 4-
cyano-3-fluorophenyl methanesulfonate 16 (230 mg, 1.1 mmol) in DMF (4.9 ml) was treated with Cs
2
CO
3
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(
420 mg, 1.3 mmol) and the mixture was heated overnight at 100 °C. The reaction mixture was cooled to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
room temp, diluted with water (5 ml) and extracted three times with EtOAc. The combined organic layers
were washed several times with brine, dried over Na
2
SO
4
, filtered, concentrated and subjected to flash
1
chromatography (0-40% EtOAc/hexanes) to give 17 (180 mg, 35%) as a white solid: H NMR (400 MHz,
CHLOROFORM-d) δ 7.55 (t, J=7.65 Hz, 1H), 6.65-6.85 (m, 2H), 4.36-4.56 (m, 1H), 4.19 (dt, J=2.64,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.34 Hz, 1H), 3.99 (td, J=6.15, 8.53 Hz, 1H), 3.37-3.69 (m, 3H), 3.26 (td, J=10.13, 17.38 Hz, 1H), 2.63
(
d, J=4.27 Hz, 1H), 1.49 (s, 9H), 0.86 (s, 9H), 0.09 (s, 3H), -0.01 (d, J=5.02 Hz, 3H). MS (m/z) 451.1 (M
H+).
+
(
+/-)
Cis-4-((4-((tert-butyldimethylsilyl)oxy)-1-((2,4-dichlorophenyl)sulfonyl)pyrrolidin-3-
yl)methoxy)-2-fluorobenzonitrile (18). A solution of 17 (180 mg, 0.40 mmol) in DCM (1 ml) was
treated with trifluoroacetic acid (0.40 ml) for 30 minutes at 23 °C. The reaction mixture was concentrated,
diluted with water (5 ml) and adjusted to pH of 8-9 using 2N aq. NaOH solution. The reaction mixture
was extracted four times with DCM and the combined organic layers were dried over Na
2
SO , filtered
4
and concentrated to give (+/-) cis-4-((4-((tert-butyldimethylsilyl)oxy)pyrrolidin-3-yl)methoxy)-2-
1
fluorobenzonitrile (120 mg, 84%) as a red oil. H NMR (400 MHz, CHLOROFORM-d) δ 7.54 (dd,
J=7.53, 8.78 Hz, 1H), 6.60-6.83 (m, 2H), 4.35-4.50 (m, 1H), 4.19 (t, J=8.53 Hz, 1H), 3.97 (dd, J=6.02,
8.78 Hz, 1H), 3.13 (dd, J=8.53, 11.04 Hz, 1H), 2.97-3.07 (m, 1H), 2.80-2.96 (m, 2H), 2.45-2.62 (m, 1H),
.79-0.92 (m, 9H), 0.02-0.16 (m, 3H), 0.00 (s, 3H). MS (m/z) 351.1 (M + H+).
0
A
mixture
of
(+/-)
cis-4-((4-((tert-butyldimethylsilyl)oxy)pyrrolidin-3-yl)methoxy)-2-
fluorobenzonitrile (120 mg, 0.35 mmol) and potassium carbonate (120 mg, 0.88 mmol) in chloroform (1
ml) and water (1 ml) was treated with 2,4-dichlorobenzene-1-sulfonyl chloride 6 (130 mg, 0.54 mmol)
and the reaction mixture was stirred overnight at 23 °C. The reaction mixture was diluted with saturated
aqueous Na
2
CO (5 ml) and extracted three times with DCM. The combined organic layers were washed
3
with brine, filtered through a phase separator, concentrated, and subjected to flash chromatography (0-
28
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Page 29 of 56
Journal of Medicinal Chemistry
1
4
0% EtOAc/hexanes) to give 18 (140 mg, 70%) as a colorless oil. H NMR (400 MHz, CHLOROFORM-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
d) δ 8.03 (d, J=8.53 Hz, 1H), 7.49-7.63 (m, 2H), 7.39 (dd, J=2.01, 8.53 Hz, 1H), 6.58-6.83 (m, 2H), 4.47-
4.60 (m, 1H), 4.15 (t, J=8.41 Hz, 1H), 3.97 (dd, J=6.27, 8.78 Hz, 1H), 3.76 (dd, J=7.78, 9.03 Hz, 1H),
3
.66 (dd, J=3.89, 10.67 Hz, 1H), 3.33-3.48 (m, 2H), 2.60-2.81 (m, 1H), 0.67-0.89 (m, 9H), 0.03 (s, 3H),
-
0.02 (s, 3H). MS (m/z) 559.1 (M + H+).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
+/-)
fluorobenzonitrile (19). A solution of 18 (140 mg, 0.26 mmol) in THF (5.5 ml) was treated with TBAF
1M in THF, 0.78 ml, 0.78 mmol) and stirred for 1 hour at 23 °C. The reaction mixture was quenched
with addition of saturated aq. NH Cl solution, the volatile solvents were removed under reduced pressure
and the mixture was extracted three times with EtOAc. The combined organic layers were dried over
Cis-4-((1-((2,4-dichlorophenyl)sulfonyl)-4-hydroxypyrrolidin-3-yl)methoxy)-2-
(
4
Na
2
SO
4
, filtered, concentrated and subjected to flash chromatography (0-50% EtOAc/hexanes) to give 19
1
(
56 mg, 46%) as a white solid. H NMR (400 MHz, CHLOROFORM-d) δ 8.06 (d, J=8.53 Hz, 1H,
benzenesulfonamide C6-H), 7.50-7.64 (m, 2H, benzenesulfonamide C3-H, arylether C5-H), 7.41 (dd,
J=2.01, 8.53 Hz, 1H, benzenesulfonamide C5-H), 6.65-6.85 (m, 2H, arylether C2-H, arylether C6-H),
4
.59 (d, J=3.76 Hz, 1H, pyrrolidine C3-H), 4.28 (dd, J=7.40, 9.16 Hz, 1H, ArOCHH), 4.08 (dd, J=6.90,
.16 Hz, 1H, ArOCHH), 3.78 (dd, J=8.16, 9.41 Hz, 1H, pyrrolidine C5-H), 3.66-3.73 (m, 1H, pyrrolidine
9
C2-H), 3.58-3.64 (m, 1H, pyrrolidine C2-H), 3.42 (t, J=9.79 Hz, 1H, pyrrolidine C5-H), 2.71-2.85 (m,
H, pyrrolidine C4-H), 1.89 (d, J=4.52 Hz, 1H, OH). 13C NMR (126 MHz, CHLOROFORM-d) δ 164.5
1
(
arylether C3), 163.5 (arylether C1), 139.6 (benzenesulfonamide C4), 135.4 (benzenesulfonamide C1),
134.3 (arylether C5), 133.3 (benzenesulfonamide C2), 132.8 (benzenesulfonamide C6), 131.9
benzenesulfonamide C3), 127.4 (benzenesulfonamide C5), 114.1 (nitrile CN), 111.5 (arylether C6),
(
1
4
02.8 (arylether C2), 93.7 (arylether C4), 70.5 (pyrrolidine C3), 65.9 (CH
2
OAr), 56.4 (pyrrolidine C2),
1
13
8.0 (pyrrolidine C5), 43.7 (pyrrolidine C4). For compound 19, the H NMR and C NMR resonances
1
3
were assigned based on gCOSY45, gHMQC, gHMBC, and C GASPE spectra, and all 2D correlations
agree with the assigned structure. MS (m/z) 445.0 (M + H+).
29
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