Discovery of a Potent (4 R,5 S)-4-Fluoro-5-methylproline Sulfonamide Transient Receptor Potential Ankyrin 1 Antagonist and Its Methylene Phosphate Prodrug Guided by Molecular Modeling

Article abstract of DOI:10.1021/acs.jmedchem.8b00117

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To s

Full text of DOI:10.1021/acs.jmedchem.8b00117

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Discovery of a Potent (4R,5S)-4-Fluoro-5-Methylproline Sulfonamide
Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist and
its Methylene Phosphate Prodrug Guided By Molecular Modeling
Huifen Chen, Matthew Volgraf, Steven Do, Aleksandr Kolesnikov, Daniel G Shore, Vishal A. Verma,
Elisia Villemure, Lan Wang, Yong Chen, Baihua Hu, Ai-Jun Lu, Guosheng Wu, Xiaofeng Xu, Po-
wai Yuen, Yamin Zhang, Shawn D Erickson, Martin Dahl, Christine Brotherton-Pleiss, Suzanne Tay,
Justin Q. Ly, Lesley J Murray, Jun Chen, Desiree Amm, Wienke Lange, David H. Hackos, Rebecca
M. Reese, Shannon D. Shields, Joseph P. Lyssikatos, Brian S. Safina, and Anthony A. Estrada
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00117 • Publication Date (Web): 29 Mar 2018
Downloaded from http://pubs.acs.org on March 29, 2018
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Discovery of a Potent (4R,5S)ꢀ4ꢀFluoroꢀ5ꢀ
Methylproline Sulfonamide Transient Receptor
Potential Ankyrin 1 (TRPA1) Antagonist and its
Methylene Phosphate Prodrug Guided By Molecular
Modeling
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,
†
,†
†
†
†
Huifen Chen,* Matthew Volgraf,* Steven Do, Aleksandr Kolesnikov, Daniel G. Shore,
†
†
†
ø
ø
ø
Vishal A. Verma, Elisia Villemure, Lan Wang, Yong Chen, Baihua Hu, AiꢀJun Lu,
ø
ø
ø
ø
ǁ
Guosheng Wu, Xiaofeng Xu, Poꢀwai Yuen, Yamin Zhang, Shawn D. Erickson, Martin Dahl,
ǁ
ǁ
∆
∆
∆
§
Christine BrothertonꢀPleiss, Suzanne Tay, Justin Q. Ly, Lesley J. Murray, Jun Chen,
#
#
∞
∞
∞
Desiree Amm, Wienke Lange, David H. Hackos, Rebecca M. Reese, Shannon D. Shields,
†
†
†
Joseph P. Lyssikatos, Brian S. Safina, Anthony A. Estrada,
†
∞
§
Department of Discovery Chemistry, Department of Neurosciences, Department of
∆
Biochemical and Cellular Pharmacology, Department of Drug Metabolism and
Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA
ø
PharmaronꢀBeijing Co. Ltd., 6 Taihe Road, BDA, Beijing 100176, PR China
ǁ
Small Molecule Research, Pharmaceutical Research and Early Drug Development, Hoffmannꢀ
La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, USA
#
Ion Channels Group, Evotec AG; Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg,
Germany
*
Correspondence: chen.huifen@gene.com; volgraf.matthew@gene.com
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ABSTRACT
TRPA1 is a nonꢀselective cation channel expressed in sensory neurons where it functions as an
irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and
respiratory disease. To study its function in various disease contexts, we sought to identify novel,
potent and selective small molecule TRPA1 antagonists. Herein we describe the evolution of an
Nꢀisopropyl glycine sulfonamide lead (1) to a novel and potent (4R,5S)ꢀ4ꢀfluoroꢀ5ꢀmethylproline
sulfonamide series of inhibitors. Molecular modeling was utilized to derive low energy threeꢀ
dimensional conformations to guide ligand design. This effort led to compound 20, which
possessed a balanced combination of potency and metabolic stability, but poor solubility that
ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed
methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo
target engagement in a rat model of AITCꢀinduced pain.
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INTRODUCTION
Transient receptor potential ankyrin 1 (TRPA1) is one of the 28 members of the transient
receptor potential (TRP) channel family and the sole member of the TRPA subfamily in
1
mammals. Like all TRP channels, TRPA1 possesses a tetrameric structure where each subunit is
comprised of six transmembrane helices and 14 Nꢀterminal ankyrin repeats. TRPA1 acts as a
polymodal sensor for noxious external stimuli and is activated by a wide variety of small
molecule agonists including allylisothiocyanate (AITC), cinnamaldehyde, and acrolein via
2
,3,4
covalent, cysteineꢀreactive mechanisms.
There has been strong interest in TRPA1 as a drug target following several rodent knockout
5
,6,7,8,9
studies that suggested TRPA1 may play a central role in pain.
This is further supported by
human genetic evidence demonstrating that a gainꢀofꢀfunction mutation in TRPA1 causes
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familial episodic pain syndrome. Based on these data, several groups have reported potent and
1
1,12,13,14,15
orally bioavailable small molecule antagonists that target TRPA1.
Unfortunately,
conflicting reports in the literature have called into question the efficacy of TRPA1 antagonists
1
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in rodent models of inflammatory and neuropathic pain.
Most notably, the antagonist
AMG0902 achieved limited analgesic efficacy in several rat in vivo pain models despite systemic
free concentrations reaching 4ꢀfold coverage of the in vitro rat IC , suggesting that TRPA1
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activation may not significantly contribute to neural signaling in the relevant underlying pain
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pathways.
More recently, a growing body of literature supports the investigation of TRPA1 in
1
7,18
neuroinflammatory pathways involved in asthma.
TRPA1 expression in the primary afferent
sensory neurons that innervate the airways suggests that TRPA1 may act as a sensor for inhaled
1
9,20
irritants.
In support of this hypothesis, both genetic and pharmacological studies in rodents
show that interfering with TRPA1 function results in a decrease in the asthmatic response in an
2
1,22
ovalbuminꢀsensitized asthma model.
Despite these data, the exact role of TRPA1 in the
pathophysiology of asthma remains unclear.
1
1,12,13,14,15,23
While many series of TRPA1 inhibitors have now been reported,
at the time we
began our own work very few in vivo tool compounds were available in the literature. In
addition, it is likely that many of the reported antagonists bind at different receptor sites and thus
it is possible that the reported efficacy of each inhibitor class could vary accordingly. Based on
the literature evidence supporting TRPA1 as a target for both pain and asthma indications, we
initiated a program centered on the identification of novel small molecule inhibitors with good
oral bioavailability and the ability to demonstrate onꢀtarget activity in a rat model of AITCꢀ
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induced pain. Herein we describe our efforts towards the identification, optimization and in vivo
characterization of a novel series of proline sulfonamideꢀbased TRPA1 antagonists.
RESULTS AND DISCUSSION
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Compound 1 is an Nꢀisopropyl glycine sulfonamideꢀbased reversible TRPA1 inhibitor (Figure
1
) with moderate liver microsome stability in human and rat (LM H/R), and potency (IC )
50
against the human and rat TRPA1 channels of 0.283 ꢀM and 0.960 ꢀM, respectively. Due to the
2
4,25
desirable drugꢀlike properties of 1 (LogD = 2.7, lipophilic ligand efficiency
(LLE) = 3.9)
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.4
and related compounds, we decided to further explore this series of TRPA1 inhibitors with the
objective of improving potency and metabolic stability to enable in vivo characterization of
TRPA1 antagonists in an AITCꢀevoked rodent pain model.
Figure 1. Profile of Nꢀisopropyl glycine sulfonamide TRPA1 inhibitor 1.
26
Recently a cryoꢀEM structure of the fullꢀlength human TRPA1 channel has been published,
however, the resolution was not sufficient to enable structureꢀbased design or prediction of
binding mode for novel ligands. In the absence of structureꢀbased drug design, we used ligandꢀ
based modeling techniques to derive low energy conformations to guide structureꢀactivity
relationship (SAR) analysis and the lead optimization process. To sufficiently sample the
conformational landscape, we employed the mixed torsional/largeꢀscale low mode sampling
27
method in MacroModel™ as implemented in Maestro for conformational search. A diverse set
of 125 conformations with pairwise heavy atom rootꢀmeanꢀsquare deviation (RMSD) ranging
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from 0.3 to 5.4 Å were generated, followed by quantum mechanics (QM) optimizations of each
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conformer using Jaguar™ with the PoissonꢀBoltzmann solvation model for water.
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A)
B)
C)
D)
Figure 2. Lowest energy quantum mechanical (QM) threeꢀdimensional models of
representative compounds. (A) QM model of compound 1 colored in yellow. (B) Overlay of two
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distinct small molecule Xꢀray conformations of compound 1 (purple and red) to its lowest energy
QM model (yellow). (C) Overlay of QM models of compounds 1 (yellow), 2 (magenta), 3
(green), and 4 (cyan). (D) Closeꢀup view of the compound 1 (yellow) overlayed with compound
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(green).
After comparing the solution phase energies of the optimized conformations for compound 1,
we identified a Uꢀshape conformation as the lowest energy (Figure 2A), limiting the total and
polar surface area of the ligand. Existing structureꢀactivity relationships in the Gly sulfonamide
series were consistent with the Uꢀshape QM model (data not shown), giving us confidence in
using the model to guide lead optimization.
A small molecule Xꢀray structure of compound 1 lent further support to the Uꢀshape QM
model of 1. There are two independent conformations in the asymmetric unit cell; one highly
similar to our global minimum QM model with a heavy atom RMSD of 0.7 Å, and a second one
with a heavy atom RMSD of 1.5 Å (Figure 2B). Based on the Uꢀshape model, it was
hypothesized that cyclic amino acids would restrict the conformational flexibility to reinforce our
proposed Uꢀshape conformation.
Comparison of the fourꢀ, fiveꢀ, and sixꢀmembered ring analogs 2ꢀ4 (Table 1) showed a
dramatic preference for proline 3, with a nearly twoꢀfold improvement in potency (0.154 ꢀM)
relative to 1 (0.283 ꢀM). Notably, the fourꢀ and sixꢀmembered ring analogs 2 and 4 showed a 5ꢀ
and 10ꢀfold loss in potency, respectively. An overlay of all four models of compounds 1-4
(Figure 2C) suggested the 5ꢀmembered proline most effectively mimics the Nꢀisopropyl group of
glycine sulfonamide 1. The moderate potency and LLE improvement of compound 3 encouraged
us to further explore this series.
Table 1. Representative examples of cyclized sulfonamide SAR
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a
Antagonist IC , FLIPR assay. All IC values represent geometric means of at least two
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determinations.
b
LLE was calculated using LogD (LLE = pIC – LogD ).
7.4
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c
Liver microsomeꢀpredicted hepatic clearance.
d
H/R = human/rat.
Metabolite identification studies suggested oxidation of the pyrrolidine moiety of the proline as
the primary metabolic pathway for this class of molecules (data not shown). To block
metabolism, we explored both (4R)ꢀ and (4S)ꢀstereoisomers of 4ꢀfluoroproline (compounds 5 and
6
) to introduce polarity as well as potentially provide steric shielding from CYPꢀmediated
oxidative metabolism. While both molecules possessed significant loss in human TRPA1
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potency relative to 3 (1.2 and 4.4 ꢀM for 5 and 6, respectively), liver microsome data suggested
that the fluorination strategy was effective in mitigating some of the metabolic instability of the
unsubstituted proline. The potency loss of compound 5 could be the result of an unfavorable
intramolecular electronic repulsion between the 4ꢀfluoro and the phenyl ring of the sulfonamide,
while the loss in potency of compound 6 could not be rationalized based on the conformation
alone. Despite the potency loss in 5, we were delighted to see very low clearance in vivo (rat iv
CLp = 15 mL/min/kg) in agreement with the in vitro liver microsome data (RLM = 10
mL/min/kg).
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SAR from the Nꢀisopropyl glycine sulfonamide series suggested that both methyl groups of the
isopropyl were critical to potency (data not shown). Based on the overlay of models for
compounds 1 and 3 (Figure 2D), we hypothesized that addition of a (5S)ꢀmethyl at the C5
position of the proline might effectively mimic the Nꢀisopropyl of compound 1 and could be a
source of additional TRPA1 potency. Gratifyingly, the (5S)ꢀ5ꢀmethylproline 7 brought a greater
than 10ꢀfold increase in human TRPA1 potency relative to unsubstituted proline 3 (0.013 ꢀM vs.
0.154 ꢀM); the (5R)ꢀ5ꢀmethylproline stereoisomer 8, however, did not provide a potency benefit
(
human TRPA1 IC = 0.239 ꢀM). This result suggests that the (5S)ꢀmethyl may effectively fill a
5
0
hydrophobic pocket within the TRPA1 binding site and that the resulting van der Waals contacts
improve overall ligand binding and potency. Despite this gain in potency, the added methyl of 7
did not improve liver microsome stability relative to proline 3.
We next hypothesized that the combination of the metabolic stability of (4R)ꢀfluoro 5 and the
potency of (5S)ꢀmethyl 7 might balance competing potency/stability trends if employed in the
same compound. Consistent with this hypothesis, (4R,5S)ꢀ4ꢀfluoroꢀ5ꢀmethylproline 9 achieved a
balance of potency and stability with a human TRPA1 IC of 0.179 ꢀM and moderate liver
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microsome stability (HLM = 10 mL/min/kg, Table 1). We were pleased that optimization of the
substituents around the proline ring of 9 maintained acceptable lipophilic ligand efficiency (LLE
= 3.8) relative to prototype proline 3 (LLE = 4.1), while also incorporating metabolic stability.
One of the drawbacks of the proline decoration was the addition of three stereogenic centers
relative to the original Nꢀisopropyl glycine sulfonamide 1. Despite this additional synthetic
complexity, we chose the more potent (4R,5S)ꢀ4ꢀfluoroꢀ5ꢀmethylproline sulfonamide core as the
basis of our continued lead discovery efforts en route to a compound that could achieve in vivo
target engagement.
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Optimization efforts were next focused on the rightꢀhand side (RHS) biaryl portion of the
molecule where we hoped to find additional potency while continuing to improve metabolic
stability, using the Uꢀshape conformational model as a guide. We also began to closely track
compound solubility during this phase of the discovery effort as compound 9 possessed poor
kinetic solubility (2 ꢀM) despite a reasonably low LogD7.4 (2.7) and acceptable solubility index
28
(
5.7). As we began to rapidly assess oral absorption profiles of the (4R,5S)ꢀ4ꢀfluoroꢀ5ꢀ
methylproline series, it became clear that the poor solubility of the series was contributing to
poor absorption when dosed orally. To address both metabolic stability and solubility concerns,
we first sought to lower the LogD7.4 of the molecule through the addition of nitrogen atoms to
the RHS. To our satisfaction, 2ꢀtrifluoromethylpyrimidine 10 (Table 2) possessed a human
TRPA1 IC50 of 0.064 ꢀM, a nearly 3ꢀfold improvement relative to the parent 9, while
maintaining good in vitro stability. In addition, 10 also showed improved kinetic solubility (53
ꢀM) and LLE (4.5 versus 3.8). Conversely, the regioisomeric 2ꢀtrifluoromethylpyrazine 11 did
not improve TRPA1 potency (0.184 ꢀM) and provided only a modest improvement in kinetic
solubility (18 ꢀM). We believe that QM modeling provides at least some explanation for the
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potency difference between the regioisomers, as pyrimidine 10 was predicted to have a roughly
twoꢀfold enrichment in the lowest energy conformation relative to pyridine 9 due to the added
symmetry of the pyrimidine ring, while pyrazine 11 was predicted to have no such advantage as
a result of the added asymmetric nitrogen. Due to the clear benefit of the 2ꢀ
trifluoromethylpyrimidine, this moiety was employed in all subsequent analogs within the series.
Table 2. Representative examples of biaryl SAR
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
a
Antagonist IC , FLIPR assay. All IC values represent geometric means of at least two
5
0
50
determinations.
b
LLE was calculated using LogD7.4 (LLE = pIC – LogD ).
50
7.4
c
Liver microsomeꢀpredicted hepatic clearance.
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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
H/R = human/rat.
We next explored different substitutions at the 2ꢀposition of the central pyrimidine ring and
found that a wide range of substitutions were tolerated (data not shown). Cyclopropyl 12 was
among the most potent (human TRPA1 IC50 = 0.005 ꢀM, Table 2) while maintaining an
equivalent LLE. Although this compound was metabolically stable in vitro, it was significantly
less soluble (kinetic solubility < 1 ꢀM) than the unsubstituted pyrimidine 10.
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
We then studied the contributions of the pyrimidine nitrogens through the synthesis of pyridine
regioisomers 13 and 14. Both matched pair pyridines 13 and 14 were less efficient (LLE = 3.6
and 4.1, respectively) relative to pyrimidine 10, without the benefit of improved liver microsome
stability. Several electronꢀwithdrawing substituents of different polarity and sizes (cyano, chloro
and fluoro) were then installed at the newly available carbon atom on the pyridine ring in both
regioisomeric series; in all cases the substituents increased both TRPA1 potency and LLE.
Within the regioisomeric series of pyridine 13, nitrile 15 had the highest kinetic solubility (123
ꢀ
M) although without a significant increase in TRPA1 potency (human TRPA1 IC = 0.096
50
ꢀM, Table 2). However, chloropyridine 16 and fluoropyridine 17 provided efficient increases in
TRPA1 activity with IC values of 0.013 and 0.033 ꢀM, respectively.
5
0
Regioisomeric pyridines 18-20 were then synthesized, each displaying even larger increases in
potency than the matched pairs 15ꢀ17, with IC values from 0.005 to 0.010 ꢀM, and LLE values
5
0
ranging from 4.7 to 5.4 (Table 2). Compounds 18-20 possessed equal or better liver microsome
stabilities, yet lower kinetic solubilities relative to the benchmark pyrimidine 10. Notably,
fluoropyridine 20 was the most efficient molecule within the series (LLE = 5.4) with moderate
predicted metabolic stability (HLM = 10 mL/min/kg, Table 2) and a 35ꢀfold increase in potency
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relative to Nꢀisopropyl glycine sulfonamide 1, without the addition of lipophilicity or metabolic
liabilities.
To validate our Uꢀshape QM model for ligand conformation, we synthesized difluoropyridine
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
2
1. We hypothesized that fluorine substitution towards the concave edge of the ligand might
destabilize the preferred ligand conformation and reduce TRPA1 potency. As expected, we
observed a 41ꢀfold loss in potency from 20 to 21. This result further strengthened our Uꢀshape
hypothesis for future lead optimization efforts.
Fluoropyridine 20 was chosen for additional potency and DMPK profiling (Table 3) to enable
rat in vivo characterization. In line with other sulfonamideꢀbased TRPA1 inhibitors within the
series, potency against the rat TRPA1 receptor (0.036 ꢀM) was weaker than the human isoform
(0.008 ꢀM), with no measurable inhibition of other human TRP channels such as TRPV1 (IC50
>20 ꢀM) and TRPM8 (IC >20 ꢀM). Similarly, 20 did not show significant activity against the
50
human EtherꢀàꢀgoꢀgoꢀRelated Gene (hERG) potassium channel (14.3% inhibition at 10 ꢀM) in
an automated patch clamp assay conducted at Chantest (Cleveland, OH). Despite high MDCK
ꢀ6
ꢀ1
permeability (A:B = 11.2 x10 cm s ), poor measured kinetic solubility (<1 ꢀM) suggested that
2
0 may require formulation optimization to achieve sufficient oral exposure. Fluoropyridine 20
demonstrated good hepatocyte stability and reasonable free fraction in plasma (Table 3).
Consistent with in vitro liver microsome and hepatocyte data, 20 displayed low hepatic clearance
ꢀ1
in rat (CL = 4.4 mL/min/kg) with a moderate volume of distribution (V = 7.5 L kg ),
p
ss
translating into an in vivo halfꢀlife of 7.5 hours.
Table 3. Rat potency, TRP channel selectivity, physicochemical properties and DMPK summary
of 20
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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
a
Antagonist IC , FLIPR assay. All IC values represent geometric means of at least two
5
0
50
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
determinations.
b
MadinꢀDarby Canine Kidney cells.
c
Apicalꢀtoꢀbasolateral.
d
e
Cryopreserved hepatocyteꢀpredicted hepatic clearance.
H/R = human/rat.
ꢀ1
Oral rat PK of 20 was first investigated using crystalline material in a lowꢀdose (1 mg kg )
MCT (0.5% methylcellulose with 0.2% Tween 80 in water) suspension (Table 4). Oral
bioavailability was low (12%), suggestive of solubilityꢀlimited drug absorption and poor fraction
absorbed, while observed CMax and AUC0ꢀ24 exposure provided poor coverage of the rat IC in
50
ꢀ1
vivo (CMax,unbound / rat TRPA1 IC = 0.02). Higher doses (30 and 100 mg kg ) were explored
50
using an amorphous suspension for oral dosing by dissolving the crystalline solid in DMSO, then
precipitating in an aqueous MCT vehicle to achieve a final composition of 15% DMSO and 85%
ꢀ1
MCT. Oral exposure in the 30 mg kg experiment (AUC
= 32.8 ꢀM*h) was greater than
0
ꢀ24
ꢀ1
doseꢀproportional compared to the 1 mg kg MCT dosing, achieving an improved free
concentration multiple relative to the rat TRPA1 IC50 (1.8ꢀfold coverage of rat IC50).
ꢀ1
Unfortunately, efforts to increase the exposure of 20 at 100 mg kg were less than dose
proportional, suggesting solubilityꢀlimiting absorption despite the use of an amorphous
formulation. It became clear that the poor kinetic solubility (<1 ꢀM) was hindering the in vivo
characterization of 20 and other sulfonamideꢀbased TRPA1 inhibitors and that a new approach
would be needed to assess the pharmacology of TRPA1 in vivo.
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Table 4. TRPA1 potency, physicochemical properties and rat oral PK summary of 20 and
prodrug 22
NaO
NaO P
O
F
O
F
Prodrug 22:
N
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
Human TRPA1 IC50a: >20 M
N
N
N
Kinetic Solubility: 133 M
S O O
O
N
CF3
F
In vivo Target
Multiple
AUC0-24
CMax,unbound
po Dose
(mg/kg)
CMax
Vehicle
MCTb
.
Rat IC50d
Ex.
( M h)
( M)
20
1
0.56
32.8
0.03
2.33
0.02
1.81
DMSO/MCT
15/85)c
DMSO/MCT
30
(
100
39.6
0.88
78.7
2.51
0.07
6.24
1.95
0.05
3.85
(
15/85)
22
1
MCT
50
MCT
a
Antagonist IC , FLIPR assay. All IC values represent geometric means of at least two
5
0
50
determinations.
b
Compound dissolved in 0.5% methylcellulose with 0.2% Tween 80 in water.
c
Compound was formulated as amorphous suspension for oral dosing by dissolving the solid in
DMSO then precipitating in aqueous MCT vehicle. The final composition is 15% DMSO and
85% MCT.
d
Rat TRPA1 IC of active compound 20 (0.036 ꢀM).
5
0
Prodrugs are often employed to enhance aqueous solubility and oral absorption of poorly
soluble small molecule drugs. Many diverse solubilizing groups have been developed, including
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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
phosphates and carboxylic acids, to modify a variety of common functional handles present in
2
9,30
small molecule drugs.
In the case of 20, a highly soluble methylenephosphate group was
appended to the secondary amide moiety to yield prodrug 22 as the disodium salt (Table 4). If
successful, we believed this could be a broadly applicable strategy to the proline sulfonamideꢀ
based inhibitor class to improve both solubility and oral exposure.
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
Prodrug 22 lacked measurable human TRPA1 potency (Table 4) as a result of capping the
secondary amide with a methylene phosphate moiety. Measured kinetic solubility was 133 ꢀM,
greater than 100ꢀfold more soluble than the unfunctionalized matched pair 20 (kinetic solubility
ꢀ1
<1 ꢀM). Low dose (1 mg kg ) oral rat PK in MCT with prodrug 22 provided only modest
increases in observed CMax and AUC0ꢀ24h exposure of 20 when compared to an equivalent dose of
the parent drug 20. Meanwhile, prodrug 22 was not detected in any of the plasma samples, as the
methylene phosphate is cleaved prior to absorption at the brush border of the enterocytes by
2
9
ꢀ1
alkaline phosphatases. Gratifyingly, a 50 mg kg dose of prodrug 22 demonstrated a greater
than doseꢀproportional increase in oral exposure of 20. This resulted in higher exposures (CMax
2.33 ꢀM and AUC0ꢀ24h = 78.7 ꢀM*h) and free drug target multiples (CMax,unbound / rat TRPA1
=
ꢀ1
IC = 3.9) than the parent drug 20 at 100 mg kg (Table 4).
5
0
Prodrug 22 was selected for further in vivo benchmarking in a rat AITC target engagement
ꢀ1
assay at doses of 10, 30, and 50 mg kg (Figure 3). Behavioral responses evoked by
administration of the TRPA1 agonist AITC to rodents has previously been shown to be inhibited
11,12,13,14,15
by prior dosing of a TRPA1 antagonist.
Following an oral dose of 22, a solution of
0
.1% AITC is injected into the hind paw at the projected TMax (3 h). The time spent engaging in
nocifensive behavior (i.e. lifting, licking, shaking, or guarding the injected paw) was then
measured over a 5 minute period. Rats dosed with prodrug 22 displayed a statistically significant
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doseꢀdependent reduction in the amount of AITCꢀinduced nocifensive behavior relative to
ꢀ1
control, with complete suppression at 50 mg kg (Figure 3A). The rat target multiples achieved
ꢀ1
(C3h,unbound / rat TRPA1 IC ) for the 10, 30, and 50 mg kg doses were 1.6, 3.3, and 4.5 fold,
50
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
respectively. Using the individual exposureꢀresponse relationships (Figure 3B) an unbound in
vivo EC of 0.060 ꢀM was calculated, which is within twoꢀfold of the rat TRPA1 FLIPR assay
5
0
(0.036 ꢀM). This work suggests that the prolyl sulfonamideꢀbased TRPA1 inhibitors are suitable
in vivo tools for the study of TRPA1.
70
60
50
A)
40
30
2
0
0
0
1
5
9%
75%
100
0
mg/kg
10 mg/kg
30 mg/kg
50 mg/kg
Cunbound
= 1.6x
3.3x
4.5x
Rat TRPA1 IC50
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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B)
150
0
1
3
5
mg/kg
0 mg/kg
0 mg/kg
0 mg/kg
100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
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0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
50
0
0
0.01
0.1
1
Free plasma concentration (uM)
Figure 3. Summary of rat AITC target engagement upon oral dosing of prodrug 22. (A) Doseꢀ
dependent reduction in time spent engaging in nocifensive behaviors. (B) Exposureꢀresponse
relationship by individual animals.
Chemistry
The sulfonamideꢀbased TRPA1 inhibitors presented in Table 1 and 2 share a final threeꢀstep
sequence, as depicted in Scheme 1, from three component parts: a cyclic amino acid, a benzylic
biaryl amine, and 4ꢀfluorobenzenesulfonyl chloride. The amide bond is first formed between the
NꢀBocꢀprotected cyclic amino acid (23ꢀ29) and the appropriate benzylic amine via HATU,
followed by TFAꢀdeprotection of the NꢀBoc protecting group and final sulfonamide formation
between the free secondary amine and the sulfonyl chloride. Cyanopyridines 15 and 18 were
further modified from the corresponding chloropyridines 16 and 19, respectively, via palladiumꢀ
catalyzed cyanation.
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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
Scheme 1. General scheme for the synthesis of sulfonamideꢀbased TRPA1 inhibitors from amino
acids, biaryl amines, and 4ꢀfluorobenzenesulfonyl chloride. (i) HATU, DIPEA, DMF, rt; (ii)
TFA, DCM; (iii) 4ꢀfluorobenzenesulfonyl chloride, Et N, DCM; (iv) Zn(CN) , Pd (dba) , dppf,
3
2
2
3
o
DMF, mwave, 100ꢀ150 C ,1 h.
The various cyclic NꢀBoc amino acids used in Scheme 1 were obtained commercially (23, 24,
3
1
25, 26), synthesized via literature methods (27), or synthesized as described below (28, 29)
32
(
Scheme 2). (5R)ꢀsubstituted methylproline 28 was accessed from methoxypyrrolidine 30 via
Lewis acidꢀcatalyzed iminium formation and stereoselective methyl cuprate addition to afford
the methyl ester 31, which was converted to the acid 28 upon saponification with lithium
hydroxide.
(4S,5R)ꢀSubstituted 4ꢀfluoroꢀ5ꢀmethylproline 29 could be synthesized from commercially
3
3
available (2S,4R)ꢀ4ꢀhydroxyproline methyl ester via the substituted pyroglutamate 32 (Scheme
2
). Tebbe olefination provided NꢀBoc enamine 33, which could be diastereoselectively reduced
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1
1
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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
to the (5S)ꢀmethyl 34. Deprotection (ꢀ35), fluorination (ꢀ36), and saponification provided key
proline 29 that forms the basis of this work.
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
.
Scheme 2. Synthesis of substituted prolines 28 and 29. (i) BF Et O, CuBr, MeMgBr, Et O, ꢀ40
3
2
2
o
to ꢀ78 C to rt; (ii) LiOH, MeOH/H O; (iii) Cp Ti(CH ) , pyridine, (η⁵ꢀC₅H₅)₂TiCl₂, toluene, 67
2
2
3 2
o
o
C, 2 h; (iv) H , Pd/C, MeOH; (v) TBAF, THF; (vi) BAST, DCM, 0 C to rt, 64 h; (vii) NaOH,
2
o
MeOH/THF, 0 C to rt, 16 h.
Synthesis of the benzylic amine 41 begins via Suzuki crossꢀcoupling of 4,6ꢀdichloropyrimidine
and boronic acid 37 (Scheme 3). Cyanation (ꢀ39), a oneꢀpot reduction/Bocꢀprotection sequence
(ꢀ40), followed by acid deprotection then afforded the key biaryl amine 41. Together with the
various NꢀBoc amino acids shown in Scheme 1 (23-29), amine 41 affords final compounds 2ꢀ6
and 8ꢀ9 via HATU coupling, Bocꢀdeprotection, and sulfonylation.
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9
0
Scheme 3. Synthesis of amine 41. (i) 4,6ꢀdichloropyrimidine , Pd(PPh ) Cl , Na CO , 1,4ꢀ
3
2
2
2
3
o
dioxane, H O, 50 C, 48 h; (ii) NaCN, DBU, DMSO, H O; (iii) (Boc) O, H , Pd/C, MeOH; (iv)
2
2
2
2
HCl (gas), EtOAc.
Compound 7 was synthesized by a reverse sulfonylation / amide bond coupling sequence
3
4
described in Scheme 4. (5S)ꢀMethylproline ester 42 was first sulfonylated to afford
sulfonamide 43, which was then treated with lithium hydroxide to provide the carboxylic acid
4
4. HATU coupling with amine 41, then provided the final target 7.
Scheme 4. Synthesis of proline sulfonamide 7. (i) 4ꢀfluorobenzenesulfonyl chloride, Et N,
3
DMAP, DCM; (ii) LiOH, MeOH/H O; (iii) 41, HATU, DIPEA, DMF, rt.
2
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6
Discovery of the trifluoromethylpyrimidine present in final compounds 10 and 12ꢀ21 was
critical to the potency and efficiency of this class of TRPA1 inhibitors. As such, boronic ester 46
was a critical synthetic intermediate that was prepared on large scale to support this work (>150
3
5
0
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7
8
9
0
g). Towards this end, previously described bromopyrimidine 45 could be treated with
palladium acetate and bis(pinacolato)diboron to provide boronate 46 in 50% yield (Scheme 5).
o
Scheme 5. Synthesis of boronate ester 46. (i) (PinB) , Pd(OAc) , DMF, 80 C, 3 h.
2
2
Synthesis of the various biaryl amines used in Table 2 began with the synthesis of bisꢀ
3
6
pyrimidine 50 (Scheme 6), en route to final compound 10. Alcohol 47 was converted to
phthalimide 48 via Mitsunobu reaction, followed by Suzuki crossꢀcoupling between the
chloropyrimidine moiety in 48 and boronate 46 to provide protected bisꢀpyrimidine 49. Final
deprotection with hydrazine hydrate afforded the desired biaryl amine 50. Chloropyrimidine 48
could also be converted to the trimethylstannane 51 under palladiumꢀcatalyzed conditions.
Stannane 51 could then be crossꢀcoupled with 2ꢀchloroꢀ5ꢀ(trifluoromethyl)pyrazine to provide
access to biaryl phthalimide 52, which was then also deprotected with hydrazine to yield biaryl
amine intermediate 53 used in the synthesis of final compound 11.
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Scheme 6. Synthesis of amines 50, 53, and 59. (i) Phthalimide, DIAD, PPh , THF; (ii) 46,
3
o
o
Pd(dppf)Cl , K CO , 1,4ꢀdioxane, H O, 70 C, 3 h; (iii) hydrazine hydrate, MeOH, 50 C; (iv)
2
2
3
2
o
(
Me Sn) , Pd(PPh ) , toluene, 110 C, 45 min; (v) 2ꢀchloroꢀ5ꢀ(trifluoromethyl)pyrazine,
3 2 3 4
o
o
Pd(PPh ) , toluene, 110 C; (vi) POCl , 100 C, 12 h; (vii) phthalimide, DMF; (viii) 46,
3
4
3
o
Pd(dppf)Cl , K CO , DMF, 70 C, 12 h; (ix) cyclopropylboronic acid, Pd(dppf)Cl , K CO , 1,4ꢀ
2
2
3
2
2
3
o
o
dioxane, 90 C, 12 h; (x) hydrazine hydrate, MeOH, 50 C, 12 h.
Synthesis of 2ꢀsubstituted pyrimidines required access to 2,4ꢀdichloropyrimidine intermediate
5
5, which could be synthesized via treatment of pyrimidinedione 54 with phosphoryl chloride
(Scheme 6). Phthalimide alkylation to afford 56 then set the stage for difunctionalization of the
dichloropyrimidine group. The chlorine at the 4ꢀposition reacted first under palladiumꢀcatalyzed
conditions with boronate 46 to yield 2ꢀchloropyrimidine 57, which was again subjected to Suzuki
crossꢀcoupling conditions, now with cyclopropylboronic acid, to yield fully functionalized
bispyrimidine 58. Phthalimide deprotection then afforded the final biaryl amine intermediate 59
to be used towards the synthesis of final compound 12.
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9
0
o
Scheme 7. Synthesis of amines 62, 66, and 71. (i) 46, Pd(dppf)Cl , K CO , 1,4ꢀdioxane, 110 C,
2
2
3
o
1
6
2 h; (ii) hydrazine hydrate, EtOH, 80 C, 12 h; (iii) 46, Pd(dppf)Cl , K CO , 1,4ꢀdioxane, H O,
2
2
3
2
o
o
0 C, 3 h; (iv)NꢀBocꢀaminomethyltrifluoroborate, Pd(PPh ) Cl , Na CO , EtOH, H O, 85 C;
3
2
2
2
3
2
o
o
(v) HCl (gas), DCM; (vi) MsCl, Et N, DCM, 0 C; (vii) Boc NH, NaH, DMF, 0 to 60 C; (viii)
3
2
o
o
4
6, Pd(dppf)Cl , K CO , 1,4ꢀdioxane, H O, 90 C, 1 h; (ix) HCl (gas), DCM, MeOH, 0 C.
2 2 3 2
Scheme 7 describes the synthesis of biaryl amines 62, 66, and 71 used in the synthesis of final
3
7
compounds 13 and 15ꢀ17. Chloropyridine 60 was crossꢀcoupled with boronate 46 and the
phthalimide was deprotected under standard hydrazine conditions to provide 62. Towards 66,
2
,5ꢀdichloroꢀ4ꢀiodopyridine 63 was selectively functionalized at the 4ꢀposition using Suzuki
crossꢀcoupling conditions, again with boronate 46 as the coupling partner, to yield biaryl 64. Nꢀ
Bocꢀaminomethyltrifluoroborate was then used in a siteꢀselective Suzuki crossꢀcoupling at the 2ꢀ
position of dichloropyridine 64 to provide NꢀBoc amine 65, which was deprotected under acidic
conditions to provide the final amine intermediate 66. Lastly, primary alcohol 67 was converted
to bisꢀBocꢀamine 69 via a twoꢀstep protocol via mesylate 68 using Boc NH as a nucleophile
2
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following deprotonation with sodium hydride. Following crossꢀcoupling with 46, NꢀBocꢀbiaryl
7
0 was then deprotected under standard conditions to provide amine 71.
1
1
1
1
1
1
1
1
1
1
2
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Scheme 8. Synthesis of amines 74, 80, 84, and 90. (i) 46, Pd(dppf)Cl , K CO , 1,4ꢀdioxane,
2
2
3
o
o
H O, 80 C, 12 h; (ii) hydrazine hydrate, MeOH; (iii) BH ꢀTHF, THF, 0 C to rt; (iv) MsCl,
2
3
o
Et N, DCM, 0 C to rt; (v) Boc NH, NaH, DMF; (vi) 46, Pd(dppf)Cl , Cs CO , 1,4ꢀdioxane,
3
2
2
2
3
o
o
H O, 110 C, 5 h; (vii) HCl (gas), MeOH; (viii) NH in MeOH, 40 C, 6 h; (ix) CO (gas),
2
3
o
o
Pd(dppf)Cl , Et N, MeOH, 50 C; (x) DIBALꢀH, hexane, ꢀ78 C; (xi) phthalimide, DIAD, PPh ,
2
3
3
THF.
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6
The synthesis of regioisomeric pyridine intermediates 74, 80, 84, and 90 used in the
preparation of 14, 19, 20, and 21, respectively, are described in Scheme 8. Following similar
protocols as previously described, 2ꢀchloropyridine 72 could be coupled with boronic ester 46
0
1
2
3
4
5
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7
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9
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(ꢀ73) and the phthalimide could then be deprotected to afford the final biaryl amine 74. Access
to bisꢀBoc amine 78 began with borane reduction of carboxylic acid 75 (ꢀ76), followed by
mesylation (ꢀ77) and nucleophilic displacement by Boc NH. Standard Suzuki crossꢀcoupling
2
with boronate ester 46 selectively functionalized the chlorine at the 2ꢀposition of the pyridine
(ꢀ79), which could then be deprotected under acidic conditions to yield the final amine
intermediate 80. A similar but reversed sequence was used in the preparation of fluoropyridine
intermediate 84. 2ꢀBromopyridine 81 was first coupled with pyrimidine 46 to yield biaryl alcohol
8
2 which could then be mesylated (ꢀ83) and converted directly to the primary amine 84 via
o
treatment with ammonia in methanol at 40 C. Difluoropyridine intermediate 90 required a
slightly longer sequence to install the additional fluorine substituent. The process began with the
selective esterification of the iodine in 2ꢀbromoꢀ3,5ꢀdifluoroꢀ4ꢀiodopyridine 85 to afford methyl
ester 86, which was subsequently crossꢀcoupled with pyrimidine 46 to provide biaryl 87.
Reduction (ꢀ88), installation of the phthalimide protecting group via Mitsunobu (ꢀ89), and
final hydrazine deprotection, provided the biaryl amine intermediate 90.
Prodrug 22 is a synthetically modified form of fluoropyridine antagonist 20, yet initial attempts
to synthesize 22 directly from 20 proved unsuccessful due to difficulties alkylating the sterically
congested secondary amide. To circumvent this issue, a synthetic plan was developed that
allowed installation of the RHS biaryl at a late stage and conversion of the thiomethyl to the final
phosphate (Scheme 9). Towards this end, substituted proline carboxylic acid 29 was converted to
primary amide 91 via the mixed anhydride. The NꢀBoc protecting group was removed under
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standard acidic deprotection conditions (ꢀ92), followed by installation of the 4ꢀ
fluorophenylsulfonamide (ꢀ93) by treatment with the corresponding sulfonyl chloride reagent.
(Chloromethyl)(methyl)sulfide alkylation provided the secondary amide 94 which was then
treated with biaryl mesylate 83 (see Scheme 8) with sodium hydride to afford penultimate
intermediate 95. Oxidation of the thiomethyl group with Nꢀiodosuccinimide followed by
displacement with phosphoric acid provided the desired prodrug 22. As the salt form of the
phosphate was found to be critical to characterization and stability, the diꢀsodium salt was
prepared by addition of sodium carbonate prior to reversedꢀphase column chromatography.
1
1
1
1
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0
Scheme 9. Synthesis of prodrug 22. (i) (NH CO , (tꢀBuO) CO, pyridine, 1,4ꢀdioxane; (ii)
4
)2
3
2
HCl in EtOAc; (iii) 4ꢀfluorobenzenesulfonyl chloride, Et N, DCM; (iv) ClCH SCH , TFA,
3
2
3
o
DCM; (v) 83, NaH, NaI, THF, ꢀ5 C to rt; (vi) H O P, NIS, 3 Å mol. sieves; THF.
3
4
CONCLUSION
NꢀIsopropyl glycine sulfonamide TRPA1 antagonist (1) with moderate potency against the
human and rat TRPA1 channels (IC values of 0.283 and 0.960 ꢀM, respectively), provided the
5
0
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6
starting point for a lead optimization campaign aimed at establishing in vivo target engagement
in a rat model for pain. QM modeling provided low energy conformations that supported a Uꢀ
shape binding model, which ultimately resulted in the discovery of a (4R,5S)ꢀ4ꢀfluoroꢀ5ꢀ
methylproline core with a balance of potency (IC = 0.008 ꢀM) and in vitro stability. Careful
0
1
2
3
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5
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7
8
9
0
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0
modifications to the RHS biaryl aimed at increasing potency without eroding metabolic stability,
resulted in fluoropyridine 20, however solubilityꢀlimiting absorption prevented the use of 20 at
doses necessary to achieve target coverage in vivo. Ultimately, the highly soluble prodrug 22, a
methylene phosphateꢀderived version of 20, achieved doseꢀproportional exposures up to 50 mg
ꢀ1
kg and excellent target coverage in rat. Prodrug 22 was used in a rat AITC target engagement
study to demonstrate that the sulfonamide class of TRPA1 inhibitors is capable of reversing
behavioral responses evoked by AITC in rodents at in vivo concentrations similar to measured rat
TRPA1 FLIPR potencies. Further investigation of TRPA1 pharmacology in vivo using efficacy
models of acute pain and asthma will be reported in due course.
EXPERIMENTAL SECTION
Chemistry
General Methods. Unless otherwise indicated, all commercial reagents and anhydrous solvents
were used without additional purification. Intermediates 23-26, 37, 54, 63, and 75 were obtained
1
via commercial sources. HꢀNMR spectra were measured on Bruker Avance III 300, 400, or 500
1
3
MHz spectrometers. CꢀNMR spectra were measured on a Bruker Avance III 125.80 MHz
spectrometer. Chemical shifts (in ppm) were referenced to internal standard tetramethylsilane (δ
=
0 ppm). The reported carbon multiplicities and coupling constants are from CꢀF coupling.
Highꢀresolution mass spectrometry of final compounds were performed on a Thermo
UHPLC/QE with a ThermoꢀQ Exactive mass spectrometry detector using ESI ionization, after
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elution on a Acquity BEH C18 (2.1 mm × 50 mm; 1.7 ꢁm particle size) stationary phase using a
gradient of water/acetonitrile (3–97% over 7 min; 0.1% formic acid in both phases). Reactions
were monitored by walkup Shimadzu LCMS/UV system with LCꢀ30AD solvent pump, 2020
MS, Silꢀ30AC autosampler, SPDꢀM30A UV detector, CTOꢀ20A column oven, using 2ꢀ98%
acetonitrile/0.1% formic acid (or 0.01% Ammonia) over 2.5 min OR Waters Acquity LCMS
system using 2ꢀ98% acetonitrile/0.1% formic acid (or 0.1% Ammonia) over 2 min. Flash column
chromatography purifications were done on a Teledyne Isco Combiflash Rf utilizing Silicycle
HP columns. Reverseꢀphase purification was carried out on a Phenomenex GeminiꢀNX C18 (30
x 100mm,5um) with a gradient of 5−95% acetonitrile/water (with 0.1% Formic Acid or 0.1%
NH4OH) over 10 min at 60 mL/min. Preparative SFC separations were performed on a PIC
Solutions instrument, with conditions indicated in the Experimental Section. Analytical purity
was greater than 95% as determined by LCMS using UV 254 nM detection unless stated
otherwise. The melting point was determined by Differential Scanning Calorimetry (DSC) (TA
InstrumentsꢀWaters L.L.C.) by using 5mg of solid sample and measuring the onset melting
temperature.
1
1
1
1
1
1
1
1
1
1
2
2
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2
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2
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0
(S)-1-((4-fluorophenyl)sulfonyl)-N-((6-(6-(trifluoromethyl)pyridin-3-yl)pyrimidin-4-
yl)methyl)azetidine-2-carboxamide (2). The title compound was prepared from carboxylic acid
+
1
2
3 and amine 41 in a manner analogous to 20. LCMS, m/z = 496.1 [M+H] . H NMR (400 MHz,
DMSOꢀd ) δ 9.49 (d, J = 2.1 Hz, 1H), 9.30 (d, J = 1.3 Hz, 1H), 8.96 (t, J = 5.9 Hz, 1H), 8.80 (dd,
6
J = 8.3, 2.2 Hz, 1H), 8.16 (d, J = 1.2 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.05 – 7.97 (m, 2H), 7.56
(t, J = 8.8 Hz, 2H), 4.61 (dd, J = 17.3, 6.2 Hz, 1H), 4.51 (dd, J = 17.3, 5.7 Hz, 1H), 4.38 (dd, J =
9
.1, 7.7 Hz, 1H), 3.77 (td, J = 8.5, 4.2 Hz, 1H), 3.59 (q, J = 8.4 Hz, 1H), 2.36 – 2.13 (m, 2H).
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