Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7

Article abstract of DOI:10.1021/acs.jmedchem.7b00598

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective Na_V1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective Na_V1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

Full text of DOI:10.1021/acs.jmedchem.7b00598

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Article
Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-
yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-
ylbenzenesulfonamide (PF-05089771): Design and Optimization of
Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7
Nigel Alan Swain, Dave Batchelor, Serge Beaudoin, Bruce M. Bechle, Paul A Bradley, Alan D
Brown, Bruce Brown, Ken J Butcher, Richard P Butt, Mark L Chapman, Stephen Denton, David
Ellis, Sebastien Galan, Stephen M Gaulier, Ben S Greener, Marcel J. de Groot, Mel S Glossop, Ian
K Gurrell, Jo Hannam, Matthew S Johnson, Zhixin Lin, Christopher J Markworth, Brian E Marron,
David S Millan, Shoko Nakagawa, Andy Pike, David Printzenhoff, David J Rawson, Sarah J Ransley,
Steven M Reister, Kosuke Sasaki, R Ian Storer, Paul Anthony Stupple, and Christopher W West
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00598 • Publication Date (Web): 06 Jul 2017
Downloaded from http://pubs.acs.org on July 6, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of Clinical Candidate 4ꢀ[2ꢀ(5ꢀAminoꢀ1Hꢀ
pyrazolꢀ4ꢀyl)ꢀ4ꢀchlorophenoxy]ꢀ5ꢀchloroꢀ2ꢀfluoroꢀ
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Nꢀ1,3ꢀthiazolꢀ4ꢀylbenzenesulfonamide
(PFꢀ
05089771): Design and Optimization of Diaryl Ether
Aryl Sulfonamides as Selective Inhibitors of NaV1.7
Nigel. A. Swain,*,a,† Dave Batchelor,e Serge Beaudoin,d Bruce M. Bechle,c Paul A. Bradley,e Alan
D. Brown,a Bruce Brown,e Ken J. Butcher,e Richard P. Butt,a Mark L Chapman,d Stephen
Denton,e David Ellis,e Sebastien Galan,e Steven M. Gaulier,e Ben S. Greener,e Marcel J. de
Groot,e Mel S. Glossop,e Ian K. Gurrell,f Jo Hannam,e Matthew S. Johnson,d Zhixin Lin,d
Christopher J. Markworth,d Brian E. Marron,d David S. Millan,e Shoko Nakagawa,e Andy
Pike,b,† David Printzenhoff,d David J. Rawson,e Sarah J. Ransley,e Steven M. Reister,d Kosuke
Sasaki,e R. Ian Storer,a,† Paul A. Stupple,e Christopher W. West,d.
aWorldwide Medicinal Chemistry, bPharmacokinetics, Dynamics and Metabolism, Pfizer Ltd, The Portway, Granta Park,
c
Cambridge, CB21 6GS, UK. Worldwide Medicinal Chemistry, Pfizer Inc, Eastern Point Road, Groton, Connecticut 06340,
d
e
f
USA. Icagen Inc, 4222 Emperor Blvd # 350, Durham, NC 27703, USA. Worldwide Medicinal Chemistry, Pharmacokinetics,
Dynamics and Metabolism, Pfizer Ltd, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK.
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KEYWORDS. NaV1.7, pain, sodium channel, NaV1.5, CYP2C9 inhibition, microdose, aryl
sulfonamide
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ABSTRACT
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A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subꢀtype selective
NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of
structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven
drugꢀdrug interaction concerns to deliver the desired balance of preclinical in vitro properties.
Concerns over nonꢀmetabolic routes of clearance, variable clearance in preꢀclinical species and
subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a
human microdose study to determine clinical pharmacokinetics. The design strategies and
results from preꢀclinical PK and clinical human microdose PK data are described leading to the
discovery of the first subꢀtype selective NaV1.7 inhibitor clinical candidate PFꢀ05089771 (34)
which binds to a site in the voltage sensing domain.
INTRODUCTION
Voltageꢀgated sodium channels control the flow of sodium ions across cellular membranes and
are critical to the initiation and propagation of electrical impulses in excitable cells. There are
nine different human isoforms of sodium channels (NaV1.1ꢀNaV1.9) with varying tissue
expression patterns in neurons, cardiac and skeletal muscle.1 Several neuronally expressed
sodium channels have been linked to aberrant action potential firing associated with pain and
sodium channel inhibition has served as a point of pharmacological intervention in pain
management.2 Indeed, nonꢀselective NaV inhibitors such as lidocaine (1), mexilitine (2) and
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carbamazepine (3) demonstrate clinical efficacy for chronic pain (Figure 1). However, these
agents are of limited clinical utility due to doseꢀlimiting side effects associated with modulation
of nonꢀpainꢀrelated NaV subꢀtypes.3
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Figure 1. Selected examples of sodium channel inhibitors in clinical use.
Whilst several NaV subꢀtypes have been implicated as potential targets in reduction of pain the
most compelling human genetic evidence indicates NaV1.7 as critical for pain perception.4 Lossꢀ
ofꢀfunction mutations in the SCN9a gene (which encodes NaV1.7) result in the rare genetic
condition, congenital insensitivity to pain (CIP).5 Conversely, gainꢀofꢀfunction mutations of
SCN9a can lead to extreme pain conditions such as paroxysmal extreme pain disorder (PEPD)
and peripheral erythromelalgia (PE).6
This human genetic evidence has prompted the search for subꢀtype selective inhibitors of the
NaV1.7 channel as superior analgesics with a significantly reduced sideꢀeffect burden compared
with panꢀNaV blockade.7
We pioneered work which identified novel lead matter containing an aryl sulfonamide acid
isostere with superior subꢀtype selectivity particularly over the cardiac channel NaV1.5
(inhibition of which may lead to arrhythmias).8 Elegant mutagenesis studies suggested
molecules such as 5 (PFꢀ04856264) bind to a unique site in the voltage sensor domain IV which
is distinct from the local anesthetic and tetrodotoxin (4) pore binding sites (Figure 2).9 The
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existence of this novel binding site has recently been confirmed by a Xꢀray crystal structure of a
chimeric human NaV1.7 voltage sensor domain IV in complex with an aryl sulfonamide.10
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Figure 2. Transmembrane view of sodium channel from the bacterium Arcobacter butzleri
(NaVAb PDB code 3RVY)11 Domains DIꢀDIV are represented in different colors (DI – red, DII –
magenta, DIII – blue, DIV – green). The postulated binding sites are highlighted for local
anesthetics (yellow), tetrodotoxin (red) and acidic aryl sulfonamides (blue).
Many groups have subsequently actively targeted this novel binding site which offers enhanced
subꢀtype selectivity over traditional nonꢀselective pore region binding ligands.12 However, this
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has presented many medicinal chemistry and development challenges associated with
progression of high molecular weight, lipophilic, acidic small molecules.
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Herein we report the work that resulted in the discovery and optimization of a series of acidic
diaryl ether aryl sulphonamides leading to the delivery of a clinical candidate with enhanced subꢀ
type selectivity vs known agents.
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RESULTS AND DISCUSSION
Initial work focused on replacement of the 2ꢀamino thiazole moiety from early leads such as
compound 5 to mitigate the risk of idiosyncratic drug toxicity. The risk associated with a 2ꢀ
amino thiazole group is exemplified by Sudoxicam (6) which was removed from Phase III
clinical trials due to hepatotoxicity. Its suspension has been attributed to cytochrome P450
mediated ring oxidation to an electrophilic epoxide 7 (Figure 3).13 Subsequent hydrolytic ring
scission yields acylthiourea metabolite 8 capable of oxidizing glutathione and proteins.
Therefore, we sought suitable replacements for this known structural alert whereby the 2ꢀ
sulfonamido linked thiazoles 9 had the potential to undergo a similar metabolic fate as 6 to reveal
both reactive epoxide intermediates 10 and potentially hepatotoxic sulfonylthiourea adducts 11.
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Figure 3. Metabolic pathway of hepatotoxin Sudoxicam (6) and implication for acidic 2ꢀamino
5
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thiazole containing aryl sulfonamide NaV1.7 series.
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Replacement of the 2ꢀaminothiazole with a number of other 5 and 6 membered heterocycles (12ꢀ
20) was successful in maintaining good to excellent levels of hNaV1.7 activity whilst maintaining
high levels of NaV1.5 selectivity (Table 1). Furthermore, these alternative hetrerocycles
eliminated the potential for thiourea adducts formation resulting from cytochrome P450 (CYP)
mediated ring oxidation. Incorporation of an additional nitrogen atom into the thiazole ring
provided more acidic 1,2,4ꢀthiadiazole analogues 12-14 (pka’s ~3.5) and 1,3,4ꢀthiadiazole
regioisomers 15-16 (pka’s ~4.5) which demonstrated high lipophilic efficiency (LipE) and
selectivity over hNaV1.5.
Table 1. Selected SAR of core substitution and heterocyclic RHSa
HLM
hNaV1.7
IC50
hNaV1.5
IC50
CLint,
app
(µL/min/
mg)
CYP3A4
IC50 (nM) IC50 (nM)
CYP2C9
R1, R2
HET
clogDb
LipEd
(nM)c
(nM)c
N
S
S
S
H, CN
H, F
0.9
1.2
1.2
21
31
5
>30000
15060
22210
92
556
N.D.
<30
517
N.D.
39
6.8
6.3
7.1
12
13
14
N
N
>262
>320
N
N
F, F
N
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H, CN
F, Cl
0.5
1.2
1.8
2.2
244
20
25448
>30000
>10000
25786
68
>300
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N.D.
11068
>30000
26588
N.D.
100
1368
37
6.1
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H, CN
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229
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>10000
>10000
>320
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20
1058
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6.0
aN.D. = not determined. clogD calculated logD based on shakeꢀflask method in octanol and water at pH7.4. Vhalf inactivation
b
c
protocol using PatchXpress platform (mean IC50 >2 replicates).14 dLipE=ꢀlogIC50ꢀclogD.
Interestingly the 4ꢀthiazole analogue in combination with a nitrile core 17 lost significant LipE
although this could be regained by core substituent replacement to the more lipophilic F/Cl
analogue 18. Pleasingly 6ꢀmembered heterocyclic replacements such as fluoroꢀpyridyl 19 and
pyrimidyl 20 were also tolerated in combination with F/Cl core substituents although were not as
efficient as their thiadiazole counterparts. However, all these molecules suffered from high
human liver microsome (HLM) clearance with Nꢀdemethylation of the LHS pyrazole group
confirmed as the major metabolite in metabolite identification studies. Furthermore, we were
concerned by significant inhibition of the CYP isoforms 3A4 and 2C9.
Table 2. Broader NaV pharmacology profile for compounds 12 and 13.a
12
13
hNaV1.1 IC50 (nM)
>3000
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hNaV1.2 IC50 (nM)
hNaV1.3 IC50 (nM)
hNaV1.5 IC50 (nM)
hNaV1.6 IC50 (nM)
hNaV1.7 IC50 (nM)
hNaV1.8 IC50 (nM)
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>3000
>30000
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149
292
15060
510
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>10000
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>3000
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rNaV1.7 IC50 (nM)
aVhalf inactivation protocols using PatchXpress platform.14
Intriguingly, core substitution changes at R2 imparted a dramatic effect on the subꢀtype and
orthologue selectivity as demonstrated by a broader NaV pharmacology profiling of matched
pairs 12 and 13 (Table 2). With the exception of hNaV1.6 the selectivity window for nitrile core
analogue 12 was superior to its fluoro partner 13, particularly hNaV1.1 (>140x vs 7x
respectively) and hNaV1.3 (>140x vs 9x respectively). Furthermore, a shift in the rat NaV1.7
activity was noted (39x vs 4x for compounds 12 and 13 respectively) which remained a key
consideration in selecting molecules for in vivo evaluation throughout the drug discovery
campaign. The excellent LipE and NaV subꢀtype selectivity profile of the 1,2,4ꢀthiadiazole in
combination with a nitrile R2 substituent (as represented by compound 12) became the focus and
we turned our attention to solving the HLM and CYP inhibition liabilities through modifications
to the pendant orthoꢀsubstituted heterocycle (Table 3).
Table 3. Selected SAR of orthoꢀsubstituted phenyl substitution
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HLM
CLint,
app
(µL/min/
mg)
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IC50
hNaV1.5
IC50
CYP3A4
IC50
(nM)
CYP2C9
IC50 (nM)
R
HET
logDa
LipEc
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Cl
Cl
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4
25231
17060
11102
>10000
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28
<8
400
505
737
827
205
83
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CF3
Cl
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894
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Cl
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9
>10000
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205
279
6.4
25
CF3
CF3
CF3
0.9
0.7
0.7
325
324
13
25578
43699
35118
39
19
14
910
616
180
321
5.6
5.8
7.2
26
27
28
1271
2200
alogD measured via shakeꢀflask method in octanol and water at pH7.4. bVhalf inactivation protocol using PatchXpress platform.14
cLipE=ꢀlogIC50ꢀlogD.
Desꢀmethyl analogue 21 significantly increased metabolic stability in HLM compared to parent
molecule 12 whilst symmetrical pyrazole derivative 22 further increased LipE in combination
with low HLM. However, both analogues continued to suffer from high CYP2C9 inhibition (7x
and 20x for compounds 21 and 22 respectively) giving the potential for a significant drugꢀdrug
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interaction (DDI). To aid design of molecules with reduced CYP2C9 inhibition liability we
extended our studies to explore coꢀcrystallization of our NaV1.7 aryl sulfonamides in CYP2C9.
Pleasingly, we were able to solve a crystal structure of a related 2ꢀaminothiazole analogue 29 in
complex with CYP2C9 (Figure 4). Critically, the RHS acidic moiety interacts with a key
Arg108 residue orientating the LHS of the ligand towards the heme region. In this case the
pyrazole forms Tꢀstacking and Nꢀdonor interactions with the iron complex. Given the RHS
acidic heterocyclic sulfonamide group was a crucial pharmacophore for NaV1.7 inhibition our
strategy to decrease CYP2C9 inhibition was to disrupt the hemo interaction through steric and/or
electronic modifications to the pendent LHS heterocycle.
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Figure 4. (a) Structure and profile of 2ꢀaminothiazole analogue 29. (b) Xꢀray crystal structure of
29 in complex with CYP2C9 (PDB code 5K7K). Iron atom shown as a yellow sphere. (c) Key
interactions between 29 and CYP2C9. Pink arrows indicate Hꢀbond interactions with the protein
(dotted lines indicate interactions with sidechains and filled lines indicate interactions with the
backbone). Green arrows indicate stacking interactions while brown arrows indicate other
contacts (in this case, the interaction with Fe2+). Residues are colored by type: red are acidic
residues; green are hydrophobic residues; cyan are polar residues and blue are basic residues.
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Based on this hypothesis, substitution of the pyrazole group with either a methyl or amino group
provided potent, selective and high LipE ligands 23 and 24 respectively. Both compounds also
demonstrated a significant improvement in CYP2C9 selectivity (186x and 81x for compounds 23
and 24 respectively) likely due to unfavorable interactions with the protein. Conversely, sixꢀ
membered heterocycles proved more challenging replacements to boost the CYP2C9 selectivity
window. Aminoꢀpyridine 25 showed no improvement in CYP selectivity and diazole analogues
such as pyrimidine 26 and pyridazine 27 eroded NaV1.7 activities with concomitant shift in LipE
independent from CYP2C9 activity resulting in no selectivity. However, symmetrical pyridazine
analogue 28 provided an excellent balance of primary activity, subꢀtype selectivity and
lipophilicity in combination with reduced CYP liability (169x for 2C9 and 98x for 3A4) in one
of the most efficient molecules synthesized on the project (LipE = 7.2). The significant
reduction in CYP2C9 inhibition for pyridazine 28 was not expected and is a clear outlier from
related unsubstituted 6ꢀmembered heterocyclic ring systems. We hypothesized that the unusual
adjacent dual lone pair system of the pyridazine nitrogens form unfavorable interactions with the
heme region of CYP2C9. Unfortunately, attempts to better understand these interactions via coꢀ
crystallization of pyridazine containing analogues in CYP2C9 failed.
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Several lead compounds possessing the 1,2,4ꢀthiadiazole RHS with high LipE (>6.5) and low
intrinsic clearance (<30 µL/min/mg) in preꢀclinical species were evaluated in pharmacokinetic
(PK) studies (Table 4). Following intravenous (IV) dosing of pyrazole derivatives 22 and 23 in
rats and dogs much higher plasma clearances were observed compared to their corresponding
scaled in vitro microsomal Clint measurements suggesting a significant contribution from nonꢀ
metabolic clearance pathway(s). Oral dosing in rat for compound 23 and dog for compound 22
also demonstrated low bioavailability consistent with a contribution from incomplete absorption
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which we attributed, based on data from the RRCK assay, to low passive permeability for these
high TPSA, acidic (pKa ~3.5) molecules. Despite its reduced lipophilicity, pyridazine analogue
28 also showed very high clearance in rat and moderate clearance in dog. Following IV
administration of 28 to bile duct cannulated rats ~73% of the dose was excreted in bile as
unchanged drug during the 6 hour study suggesting the high clearance was nonꢀmetabolic. Early
PK work with this chemotype also confirmed our hypothesis that molecules in this
physicochemical space (high MW, high TPSA, acids) demonstrate very low levels of CNS
exposure. Given NaV1.7 is highly expressed in peripheral sensory neurons we rationalized only
peripheral block of NaV1.7 was required for efficacy and that limiting CNS penetration would be
desirable to further reduce centrally mediated toxicity effects (e.g. via inhibition of NaV1.1 and
NaV1.2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Physicochemical, in vitro ADME and preꢀclinical pharmacokinetic profiles for lead
1,3,4ꢀthiadiazoles.a
Compound
22
23
28
Physicochemical properties
MW (Da)
458
2.8
506
3.2
504
2.2
clogP
logD7.4
1.7
2.0
0.7
TPSA (Å)2
134
259
134
>300
131
>300
Solubility at pH6.5 (ꢀM)
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5
ADME in vitro profile
6
7
8
RRCK (x10ꢀ6 cm/sec)
2.1
1.1
0.7
9
HLM/RLM/DLM, Clint
(µL/min/mg)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17 / 26 / <5
28 / 29 / 9
14 / 23 / <5
Hhep, Rheps, DHeps Clint
(µL/min/106 cells)
18 / 5 / N.D.
34 / N.D./N.D.
0.0015, 0.0043, 0.0039
7 / 11 / N.D.
hPPB, rPPB, dPPB (Fu)
in vivo PK
<0.001, 0.0022, 0.0022
0.00543, 0.015, 0.021
Rat
1
Dog
0.1
6.5
2955
0.5
0.9
0.13
18
Rat
1
Dog
0.1
Rat
1
Dog
0.1
IV dose (mg/kg)
Clp (mL/min/kg)
Clu (mL/min/kg)
Vss (L/kg)
43
40.1
9325
6.3
5.1
3
21.2
5435
1.8
58.0
3866
1.3
0.2
3
10.0
476
1.2
19545
2.3
T1/2 (h)
<1
1.2
3.1
Oral dose (mg/kg)
Cmax (ng/mL)
N.D.
N.D.
N.D.
4
1
AUC0→∞ (ng*h/mL)
40
26
5
F (%)
23
<5
<1
aN.D. = not determined.
Several LHS substituents had now been identified which were optimized for LipE, metabolic
stability and CYP2C9 selectivity. Subsequent designs to reꢀexplore the core substituents and
alternative heterocyclic RHS groups were initiated in an attempt to reduce the TPSA and acidity
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and explore the impact on preꢀclinical clearance and oral absorption. Furthermore, earlier
evidence (as observed with representatives in Table 1) also suggested alternative nonꢀ1,2,4ꢀ
thiadiazole heterocycles could offer improvements in the selectivity over CYP3A4 inhibition,
reducing any associated DDI risk.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Utilizing pyridazine 28 as an advanced lead the nitrile core was switched to the lower TPSA
expressing F/Cl substitution pattern and less acidic RHS heterocyclic alternatives were
synthesized (Table 5). 1,3,4ꢀThiadiazole derivative 30 (measured pKa 4.0) maintained high
LipE and improved HLM Clint and CYP3A4 selectivity (536x) over compound 28. The 4ꢀ
thiazole derivative 31 (estimated pKa 5.6) also showed reduced CYP3A4 liability although this
more lipophilic expression showed moderate turnover in HLM and reduced LipE.
Table 5. Heterocyclic changes with an optimized pyridazyl LHS
HLM
hNaV1.7
IC50
hNaV1.5
IC50
CLint, app
(µL/min/
mg)
CYP3A4
IC50 (nM) IC50 (nM)
CYP2C9
HET
logDa
TPSA
LipEc
(nM)b
(nM)b
0.8
1.6
107
94
21
>30000
<8
>11248
>30000
1101
1365
6.9
6.0
30
31
26
22451
17
alogD measured via shakeꢀflask method in octanol and water at pH7.4. bVhalf inactivation protocol using PatchXpress platform.14
cLipE=ꢀlogIC50ꢀlogD.
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The high LipE and improved CYP2C9 profile for aminoꢀpyrazole example 24 (Table 3) was
intriguing although the high TPSA and HBD/HBA expression of this LHS moiety led to
significant concerns over oral absorption. However, an exploration of alternative RHS
expressions with the lower TPSA expressing F/Cl core was undertaken providing 1,3,4ꢀ
thiadiazole and 4ꢀthiazole derivatives 32-34, all showing an improved CYP3A4 selectivity
window whilst maintaining good levels of NaV1.7 activity (Table 6). Unfortunately, following a
3 mg/kg oral administration of high LipE thiadiazole 32 in rat, oral bioavailability was calculated
to be <5 % (despite clearance approximately 25% liver blood flow) suggesting poor absorption
with this high TPSA, moderately acidic (measured acidic pKa 4.3) example. However, the lower
TPSA and weakly acidic 4ꢀthiazole derivatives 33 and 34 (measured acidic pKa 6.3) both
demonstrated good oral bioavailability in rat and a PK summary for compound 34 is provided in
table 8 and will be discussed in more detail later in this manuscript.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. Heterocyclic changes with an optimized aminoꢀpyrazole LHS
HLM CLint,
app
(µL/min/mg)
CYP3A
4 IC50
(nM)
hNaV1.7
hNaV1.5
CYP2C9
IC50 (nM)
R
HET
logDa TPSA
LipEc
IC50 (nM)b IC50 (nM)b
CF3
CF3
0.9
2.2
136
123
8
9
14413
10549
<9
<9
>30000
>30000
658
552
7.2
5.8
32
33
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Cl
2.3
123
11
>10000
10
>30000
299
5.5
34
5
alogD measured via shakeꢀflask method in octanol and water at pH7.4. bVhalf inactivation protocol using PatchXpress platform.14
6
7
cLipE=ꢀlogIC50ꢀlogD.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
During the course of our SAR studies we identified that zwitterionic ligands (via incorporation of
a basic center distal to the key acidic pharmacophore) were also well tolerated as hNaV1.7
inhibitors. Alkylated azetidine substituted pyrazoles 35-38 provided molecules with good levels
of NaV1.7 activity and excellent selectivity against NaV1.5 (Table 7). Unfortunately PK profiling
of 1,2,4ꢀthiadiazole analogue 35 resulted in liver blood flow clearance in rat thwarting any desire
to progress further. Pleasingly, 4ꢀthiazole analogues 36ꢀ38 provided higher selectivity windows
over CYP inhibition. Furthermore, increasing the size of the R3 alkyl group from a methyl in
example 37 to ethyl analogue 38 was not detrimental to NaV1.7 activity or metabolic stability in
HLM and continued to widen the CYP inhibition window. Interesting, despite its lower LipE,
nitrile core example 36 showed impressive levels of NaV subꢀtype selectivity (see table 8 for full
pharmacology profile) consistent with earlier observations for the 1,3,4ꢀthiadiazole, nitrile core
acidic molecules.
Table 7. Optimization of zwitterionic substrate
HLM
hNaV1.7
hNaV1.5
CLint, app
(µL/min/
mg)
CYP3A4
IC50 (nM)
CYP2C9
IC50 (nM)
R1, R2
R3
HET
logDa
TPSA
LipEc
IC50 (nM)b IC50 (nM)b
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F, F
H, CN
F, F
Me
Et
1.0
1.4
1.3
1.6
102
113
89
3
46
7
29291
>30000
>30000
>10000
N.D.
13
427
264
17992
1012
3234
7.5
35
36
37
38
12239
15124
>30000
5.9
6.8
6.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
Et
27
F, F
89
10
25
alogD measured via shakeꢀflask method in octanol and water at pH7.4. bVhalf inactivation protocol using PatchXpress platform.14
cLipE=ꢀlogIC50ꢀlogD.
More extensive analysis of three advanced molecules (examples 30, 34 and 36) with superior in
vitro ADME and primary pharmacology profiles was carried out (Table 8). Upon full NaV
pharmacology profiling excellent subꢀtype selectivities were confirmed together with a general
shift in the orthologue activity which was most pronounced in rat. Compounds 30, 34 and 36
were also tested for offꢀtarget pharmacology against a broad range of receptors, enzymes and ion
channels at CEREP (Bioprint) and within Pfizer (see SI). Encouragingly, a low perceived safety
risk was posed by the levels of activity observed in these selectivity panels.
Generally within the series the preclinical in vivo PK was not reliably predicted by in vitro
metabolic Cllint using appropriate liver microsomes or hepatocyte systems. It was also notable
that there were distinct trends in PK between species with rats typically displaying high
clearance and atypical PK profiles for acidic molecules, such as high Vss, while dogs typically
showed lower clearance and more typical PK.15 Various pieces of in vitro data, including
Sandwich Cultured Human Hepatocytes (data not shown) and OATP expressing cell lines,
suggested a potential contribution to clearance from active hepatic uptake. It was also noted in
bile duct cannulated rat studies that these compounds showed significant contributions from
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biliary clearance of unchanged drug, although the relevance to other species was not well
understood.
7
8
9
The failure of in vitro scaling to predict preclinical PK, inconsistent preclinical PK between
species, and data suggesting the potential for a significant contribution from nonꢀmetabolic
clearance routes, raised significant concerns of the likely accuracy of human PK predictions for
compounds in this series. Therefore, these three compounds from the diaryl ether aryl
sulfonamides series described, 30, 34 and 36, were chosen for inclusion in a human microdose
study, where the PK profiles were determined following intravenous and oral administration.16
A fourth compound from an alternative indazole series was also included in the microdose study
and will be the topic of future disclosures. It is noteworthy that whilst LipE proved a key
parameter in ligand design to improve overall physicochemical properties for the series,
compounds 34 and 36 display some of the lowest LipEs reported for our most advanced
inhibitors. This is a reflection of the major challenges faced with this chemotype to deliver
structurally differentiated molecules for a human microdose study balancing low DDI risk, high
oral absorption and acceptable preꢀclinical clearance.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 8. Physicochemical properties, in vitro ADME, NaV pharmacology and preꢀclinical
pharmacokinetic profiles for lead microdose molecules.a
Compound
30
34
36
Physicochemical properties
MW (Da)
clogP
532
3.1
0.9
4.3
500
4.2
2.6
6.3
541
4.4
logD7.4
1.4
pKa acid, [base]
6.5 [7.4]
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TPSA (Å)2
107
286
123
113
3
4
5
6
7
8
Solubility at pH6.5 (ꢀM)
ADME in vitro profile
<0.3
>262
RRCK (x10ꢀ6 cm/sec)
MDCKꢀMDR1 Papp AB/BA (x10ꢀ6
cm/sec)
0.7
3.8
3.2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
<0.5/1.1
0.7/6.3
<0.5/11
HLM/RLM/DLM, Clint
(µL/min/mg)
<8 / <17 / <5
<8 / 8 / N.D.
<10 / <18 / <8
15 / <5 / N. D.
<20 / 41 / 23
7 / <5 / N.D.
Hhep, Rheps, DHeps Clint
(µL/min/106 cells)
hPPB, rPPB, dPPB (Fu)
0.0016, 0.0221, 0.0055
0.0015, 0.0134, 0.0075
0.0648, 0.125, 0.231
OATP1B1 (transfected/wild type)
OATP1B3 (transfected/wild type)
OATP2B1 (transfected/wild type)
NaV pharmacologyb
hNaV1.1 IC50 (nM)
hNaV1.2 IC50 (nM)
hNaV1.3 IC50 (nM)
hNaV1.4 IC50 (nM)
hNaV1.5 IC50 (nM)
hNaV1.6 IC50 (nM)
hNaV1.7 IC50 (nM)
hNaV1.8 IC50 (nM)
rNaV1.7 IC50 (nM)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2.05
2.13
1.83
8750
398
677
119
>10000
2044
>10000
>30000
>30000
1109
>10000
>10000
>10000
173
>10000
>10000
>30000
1636
21
15
46
>30000
5450
>10000
153
>10000
3140
dNaV1.7 IC50 (nM)
in vivo PK
132
16
263
Rat
1
Dog
0.1
Rat
1
Dog
0.1
Rat
1
Dog
0.1
15
IV dose (mg/kg)
Clp (mL/min/kg)
40.3
1824
2.0
6.0
448
2.7
>70
>560
Clu (mL/min/kg)
364
360
65
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Vss (L/kg)
T1/2 (h)
11.0
6.6
3
0.5
4.6
3
1.3
4.4
3
0.2
4.0
3
29.7
2.2
3
0.9
1.1
Oral dose (mg/kg)
Tmax (h)
N.D.
1.5
73.2
16
1.5
2890
54
2.0
541
27
0.9
998
35
0.3
92.7
68
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cmax (ng/mL)
F (%)
aN.D. = not determined. bVhalf inactivation protocols using PatchXpress platform.14
The data from the human microdose study are summarized in Table 9 and Figure 5. Compound
36 was ruled out based on its short plasma halfꢀlife as the peak to trough ratios, based on twiceꢀ
daily administration, would have required a high therapeutic index. Compounds 30 and 34
exhibited halfꢀlife values that would support twiceꢀdaily oral administration. However, on the
basis of superior NaV1.7 potency and a PBPK modelling prediction suggesting the clinical target
concentration of 3x IC50 unbound plasma concentration at trough would be obtained at a lower
dose, it was decided to progress compound 34 to further clinical trials. It was subsequently
shown to be safe and wellꢀtolerated up to a single oral dose of 2400 mg, providing significant
coverage of the NaV1.7 IC50 (Cmax free plasma concentration of 226 nM providing ~20x NaV1.7
IC50 at the 2400 mg dose).
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. (a) Structures of 30 (PFꢀ051864628), 34 (PFꢀ050897718) and 36 (PFꢀ051501228). (b)
Human mean plasma concentrations following 100 µg intravenous (blue) and 100 µg oral
microdoses (red).
Table 9. Human pharmacokinetic microdose data for 30, 34 and 36.
Compound
IV dose (µg)
30
34
36
100
100
100
Clp (mL/min/kg)
Clu (mL/min/kg)
Vss (L/kg)
1.2
745
0.18
4.5
0.5
350
0.15
6.5
3.6
55
0.41
2.2
T1/2 (h)
Oral dose (µg)
Cmax (ng/mL)
F (%)
100
4.8
100
11
100
0.67
52
101
110
Significant in vitro and in vivo profiling of aryl sulfonamide 34 and close structural analogue 33
has been reported in an earlier manuscript.17 Detailed manual electrophysiology characterization
confirm 34 is a stateꢀdependent and selective inhibitor of NaV1.7. Inhibitory effects of
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compound 34 on action potentials in spinal slice electrophysiology recordings, mouse and human
DRG neurons, in addition to in vivo efficacy of 33 in a mouse capsaicinꢀinduced neurogenic
flare model, further demonstrate the multiple contributions of NaV1.7 in nociceptor signaling.17
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11
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16
17
18
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20
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27
28
29
30
31
32
33
34
35
36
37
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SYNTHETIC CHEMISTRY
The key compounds 30, 34, 36 were prepared by SNAr reaction, joining a heteroaryl phenol
left hand side (LHS) with a suitably protected heteroaryl sulfonamide right hand side (RHS) via
an ether linkage.
Scheme 1. Synthesis of Compound 34a
aReagents and conditions. (a) CH3CH2OCHO, sodium, reflux, 16 h (b) (CH3)3CHNHNH2.HCl,
EtOH, reflux 24 h (c) (CF3CO)2O, Et3N, CH2Cl2, 16 h (d) BBr3, CH2Cl2, 0 oC, 45 min, 66% over
4 steps (e) diphenylphosphoryl azide, tꢀBuOH, Et3N, reflux, 18 h, 77% (f) LiHMDS, THF, 0 C,
30 min, 50% (g) i) NaH, DMF, ii) K2CO3, 55 C, 3 days iii) Na2CO3 (aq), CH3OH, 55 C 16 h,
iv) HCl, CH3OH, 50 oC, 16 h 12% over 4 steps.
o
o
o
For the synthesis of compound 34 (Scheme 1) the LHS was constructed in one pot by standard
aminopyrazole ring formation utilizing 2ꢀ(5ꢀchloroꢀ2ꢀmethoxyphenyl)acetonitrile 39, ethyl
formate and tꢀbutyl hydrazine hydrochloride followed by trifluoroacetate amine protection.
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Finally boron tribromide demethylation yielded the desired phenol 40. The protected heteroaryl
sulfonamide RHS was prepared by reaction of tertꢀbutyl thiazolꢀ4ꢀylcarbamate 42 (made via
Curtuis rearrangement of the corresponding thiazole carboxylic acid 41) with aryl sulfonyl
chloride 43 to generate the protected RHS 44. Base promoted SNAr between the LHS and RHS,
and subsequent protecting group removal yielded the desired final product 34.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of Compound 30a
aReagents and conditions: (a) LiHMDS, 2ꢀMeꢀTHF, ꢀ45 oC, 45 min.; (b) Compound 46, ꢀ45 oC
o
to 0 C, 2 h, 78%; (c) Iodine, Na2CO3, THF/Water, 24 h, 41 %; (d) CH3CN, CuI, (PPh3)4, CsF,
50 oC, 2 h, 68%; (e) K2CO3, DMSO, 16 h, 67%; (f) 4M HCl / dioxane, 3 h, 82%
Synthesis of compound 30 (Scheme 2) was initiated by aryl sulfonyl chloride 46 addition to
2,4ꢀdimethoxybenzyl (DMB) protected aminothiadiazole 45 to build the RHS for this scaffold.
Construction of the LHS was achieved by iodination of 4ꢀtrifluoromethyl phenol 48 to yield
compound 49 followed by copper mediated addition of 4ꢀ(tributylstannyl) pyridazine 50 to
construct diaryl phenol 51. Finally base mediated SNAr and HCl cleavage of the DMB protecting
group gave compound 30.
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Scheme 3. Synthesis of Compound 36a
3
4
5
6
7
8
9
10
11
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aReagents and Conditions (a) Compounds 53 and 54 (see SI for synthesis), EtOH, 0 C, 2 h,
42%; (b) LiHMDS, ꢀ78 oC to room temp., 16 h, 54%; (c) K2CO3, DMSO, room temp. 4 h, 24%;
(d) i) Chlororethyl chloroformate, proton sponge, CH2Cl2, room temp. 3.5 h. ii) CH3OH, reflux,
4 h; (e) 4M HCl/dioxane, 2 h, 33% over 3 steps; (f) Sodium triacetoxyborohydride,
CH2Cl2/CH3OH, Et3N, 0 oC, acetaldehyde, 1.5 h, 60%.
In an analogous manner the LHS of compound 36 (Scheme 3) was prepared by standard
condensation of enamine 53 with diphenylmethyl (DPM) protected azetidinylꢀhydrazine
hydrochloride 54 to generate the required phenol 55. The RHS 57 was prepared analogously to
that in Scheme 1 and subsequent SNAr provided fully protected 58. DPM removal via treatment
with proton sponge and chloroethyl chloroformate followed by complete Boc removal with HCl
gave the desired fully deprotected 59. Finally, reductive amination with acetaldehyde gave the
desired compound 36.
CONCLUSIONS
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In summary, a series of diaryl ether aryl sulfonamides has been identified as potent and highly
subtype selective inhibitors of NaV1.7. This high molecular weight, acidic chemotype
represented a significant challenge in human pharmacokinetic predictions due to nonꢀmetabolic
clearance pathways predominating. A human microdose study was therefore carried out on 3
diverse ligands from the series (varying pKa, lipophilicity and chemical diversity) to determine
basic PK parameters leading to the nomination of 34 as a clinical candidate. Furthermore, this
represents the first potent and selective molecule which binds to the domain IV voltage sensor
region of the sodium channel to progress into the clinic. The development of selective agents
such as 34 offers new potential therapeutic modalities for acute and chronic pain, which remain a
high medical need.
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EXPERIMENTAL SECTION
The synthesis of the lead compounds 30, 34, 36 are described below. Detailed experimental
procedures for the synthesis of all other compounds, their intermediates, and characterization
data are provided in the supporting information.
Materials and Methods. Unless otherwise noted, all materials were obtained from
1
commercial suppliers and used without further purification. H Nuclear magnetic resonance
(NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical
shifts (δ) are given in partsꢀperꢀmillion (ppm) (δ relative to residual solvent peak) using
conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad. The mass spectra (m/z) were recorded electrospray ionization (ESI). The
following abbreviations have been used for common solvents: CDCl3, deuterochloroform; d6ꢀ
DMSO, deuterodimethylsulfoxide; CD3OD, deuteromethanol. All solvents were reagent grade
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and, when necessary, were purified and dried by standard methods. Concentration of solutions
after reactions and extractions involved the use of a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Anhydrous solvents were obtained from commercial sources and used as
supplied. Organic solvents were dried over anhydrous magnesium sulfate or anhydrous sodium
sulfate. All final compounds had a high performance liquid chromatography (HPLC) purity of
95% or greater using one of the methods described in the supporting information.
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5-Chloro-2-fluoro-4-[2-pyridazin-4-yl-4-(trifluoromethyl)phenoxy]-N-1,3,4-thiadiazol-2-
ylbenzenesulfonamide (30). To a stirred solution of 5ꢀchloroꢀNꢀ(2,4ꢀdimethoxybenzyl)ꢀ2ꢀ
fluoroꢀ4ꢀ[2ꢀpyridazinꢀ4ꢀylꢀ4ꢀ(trifluoromethyl)phenoxy]ꢀNꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
ylbenzenesulfonamide (52) (28.1 g, 41.2 mmol) in 1,4ꢀdioxane (250 mL) at room temperature
was added a 4M solution of HCl in 1,4ꢀdioxane (300 mL) dropwise over 30 minutes. The
resulting suspension was left to stir at room temperature for 3 hours before concentration in
vacuo. The residue was azeotroped with diethyl ether (3 x 300 mL) followed by a diethyl ether
trituration (200 mL) to provide crude material as a fawn colored solid. This material was
suspended in methanol (200 mL) and filtered through Celite, washed with methanol (400 mL)
and the resulting filtrate concentrated in vacuo to give a sand colored solid. This material was
suspended in water (100 mL) and treated with concentrated ammonium hydroxide (60 mL)
portion wise until pH 9ꢀ10 was achieved. The resulting solution was washed with diethyl ether
(3 x 75 mL) and the aqueous layer acidified to pH=5 with citric acid. The mixture was then
extracted with ethyl acetate (3 x 200 mL) and brine (100 mL) added to aid separation. The
combined organic layers were washed with water (200 mL), dried over MgSO4 and concentrated
in vacuo to approximately 100 mL whereby a precipitate was observed. This mixture was
allowed to stand for 18 hours and the resulting solid filtered and washed with cold ethyl acetate
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(10 mL) and dried in vacuo at 60 °C to provide the title compound as a sand colored crystalline
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solid (17.5 g, 80%). H NMR (400 MHz, DMSOꢀd6) δ ppm 7.31 (d, J=8.60 Hz, 1 H) 7.51 (d,
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J=10.55 Hz, 1 H) 7.87 (dd, J=8.99, 2.34 Hz, 1 H) 7.92 ꢀ 8.03 (m, 2 H) 8.12 (d, J=2.34 Hz, 1 H)
8.83 (s, 1 H) 9.33 (dd, J=5.27, 1.37 Hz, 1 H) 9.52 (dd, J=2.54, 1.37 Hz, 1 H). HPLC (12min) tR=
8.35 min, HPLC purity >98%. LCMS (APCI) m/z 532 [M+H]+ HRMS (EI) m/z: [M+H]+ Calcd
for C19H11ClF4N5O3S2 531.9928, found 531.9919.
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4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,
3-thiazol-4-
ylbenzenesulfonamide (34). To a suspension of sodium hydride (29 mg, 1.2 mmol) in
dimethylformamide (1 mL) was added Nꢀ(1ꢀtertꢀbutylꢀ4ꢀ(5ꢀchloroꢀ2ꢀhydroxyphenyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)ꢀ2,2,2ꢀtrifluoroacetamide (40) (239 mg, 0.661 mmol) and stirred for 30 minutes. To
this was added tertꢀbutyl 5ꢀchloroꢀ2, 4ꢀdifluorophenylsulfonyl (thiazolꢀ4ꢀyl) carbamate (44) (206
mg, 0.501 mmol) and the reaction stirred for 24 hours. Potassium carbonate (40 mg, 0.3 mmol)
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was added and the reaction heated at 55 C for 3 days. The reaction was cooled, diluted with
ethyl acetate (10 mL) and the organic extract washed with water (10 mL) and saturated aqueous
sodium chloride solution (10 mL), dried over magnesium sulfate, filtered and concentrated in
vacuo.
Purification by automated flash column chromatography eluting with ethyl
acetate:hexanes (gradient 0:1 to 1:0) afforded intermediate protected product. This residue was
dissolved in methanol (1 mL) and sodium carbonate solution (2 M aqueous, 0.08 mL, 0.2 mmol)
and water (0.2 mL) added. The reaction was stirred at room temperature for 6 hours and then
heated at 55 °C for 16 hours before concentrating in vacuo and purification by flash
chromatogray eluting with methanol:dichloromethane (gradient 0:1 to 1:9). The resulting
purified material was dissolved in methanol (saturated in gaseous hydrogen chloride) and heated
at 50 oC for 16 hours. Purification by preparative HPLC afforded the title compound HCl salt as
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a white solid (31 mg, 12%). H NMR ꢀ HCl salt (600 MHz, DMSOꢀd6) δ ppm 6.98 (d, J=11.15
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Hz, 1 H) 7.10 (d, J=1.17 Hz, 1 H) 7.22 (d, J=8.80 Hz, 1 H) 7.47 (dd, J=8.51, 2.05 Hz, 1 H) 7.73
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(br. s., 1 H) 7.90 (d, J=7.04 Hz, 1 H) 7.94 (s, 1 H) 8.92 (d, J=1.17 Hz, 1 H) H NMR ꢀ Free
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base: (600 MHz, DMSOꢀd6) δ ppm 4.85 (br. s., 2 H) 6.72 (d, J=10.56 Hz, 1 H) 7.07 (d, J=1.17
Hz, 1 H) 7.21 (d, J=8.80 Hz, 1 H) 7.28 ꢀ 7.34 (m, 1 H) 7.42 (br. s., 1 H) 7.68 (br. s., 1 H) 7.89 (d,
J=7.04 Hz, 1 H) 8.90 (d, J=1.76 Hz, 1 H) 11.37 (br. s., 1 H) 11.72 (br. s., 1 H), HPLC (12min)
tR= 7.66 min, HPLC purity >98%. LRMS (ESI) m/z 501 [M+H]+; HRMS (EI) m/z: [M+H]+
Calcd for C18H13Cl2FN5O3S2 499.9821, found 499.9816.
4-{4-Chloro-2-[1-(1-ethylazetidin-3-yl)-1H-pyrazol-5-yl]phenoxy}-3-cyano-N-1,3-thiazol-
4-ylbenzenesulfonamide (36). To a suspension of 4ꢀ[2ꢀ(1ꢀazetidinꢀ3ꢀylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ4ꢀ
chlorophenoxy]ꢀ3ꢀcyanoꢀNꢀ1,3ꢀthiazolꢀ4ꢀylbenzenesulfonamide, (59) (500 mg, 0.797 mmol) in
methanol (4 mL) and dichloromethane (4 mL) was added triethylamine (161 mg, 1.59 mmol) and
the reaction cooled to 0 °C in an ice/water bath. To the suspension was added sodium
triacetoxyborohydride (422 mg, 1.99 mmol) and the reaction was then stirred at 0 °C for 10
minutes. Acetaldehyde (105 mg, 2.39 mmol) was added dropwise and the reaction stirred at 0 °C
for 1.5 hours. The solvent was removed in vacuo to give an orange oil which was partitioned
between dichloromethane (25 mL) and water (25 mL). The organic layer was separated and the
aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organic layers
were washed with saturated aqueous sodium chloride solution (20 mL) and filtered through a
phase separation cartridge. The solvent was removed in vacuo and the resulting solid was
triturated in hot ethyl acetate (10 mL), allowed to cool to room temperature and then filtered to
provide the title compound as a white solid (260 mg, 60%). 1H NMR (400 MHz, DMSOꢀd6) δ
ppm 1.04 ꢀ 1.13 (m, 3 H) 3.24 (q, J=6.64 Hz, 2 H) 4.16 (br. s., 2 H) 4.42 (br. s., 2 H) 5.11 (br. s.,
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