The discovery and evaluation of diaryl ether heterocyclic sulfonamides as URAT1 inhibitors for the treatment of gout

Article abstract of DOI:10.1039/c6md00190d

Elevated serum uric acid levels can lead to gout which remains an area of unmet medical need. Following an unexpected clinical uricosuric effect observed with a sulfonamide compound, PF-05089771 (9), subsequently attributed to weak URAT1 inhibition, the o

Full text of DOI:10.1039/c6md00190d

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Owen, A. Pike, C. Benn, E. Armstrong, D. C. Blakemore, M. Bictash, K. Costelloe, E. Impey, P. Milliken, E.
Mortimer-Cassen, H. Pearce, B. Pibworth and G. Toschi, Med. Chem. Commun., 2016, DOI:
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The discovery and evaluation of diaryl ether heterocyclic
sulfonamides as URAT1 inhibitors for the treatment of gout†
R. Ian Storer,a* Robert M. Owen,a Andy Pike,b* Caroline L. Benn,c Emma Armstrong,c David C.
Blakemore,a Magda Bictash,c Kathryn Costelloe,c Emma Impey,c Philip H. Milliken,d Elisabeth
Mortimer-Cassen,d,‡ Hannah J. Pearce,e Benjamin Pibworth,f Gianna Toschif
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Elevated serum uric acid levels can lead to gout which remains an area of unmet medical need. Following an unexpected
clinical uricosuric effect observed with a sulfonamide compound, PF-05089771 (9), subsequently attributed to weak
URAT1 inhibition, the optimization of a series of acidic sulfonamides as selective URAT1 inhibitors was undertaken.
Compounds 10f and 10i were identified as suitable leads for more extensive profiling after exhibiting high URAT1
inhibitory potency and subsequently demonstrated promising preclinical ADME and safety profiles.
CN
Introduction
O
O
N
O
H
N
HN
N
Uric acid (1) is an endogenous metabolite resulting from the
O
S
O
NH
N
H
N
O
N
H
metabolism of purines and is also present in the diet. In most
mammals, uric acid is principally cleared via metabolism to
allantoin by the enzyme uricase. However, humans do not
express functional uricase and the major route of uric acid
clearance is via renal excretion. Hyperuricemia describes
elevations in serum levels of uric acid (>6.0 mg/dL) which,
despite often being asymptomatic, can lead to an increased
risk of urate crystal formation due to the limited solubility of
uric acid at these concentrations. At higher concentrations
(>6.8 mg/dL), crystals of uric acid have the propensity to
collect around the peripheral joints of the limbs leading to
gout, a disease characterized by painful inflammatory joint
flares. If untreated, crystal deposits called tophi can form and
lead to irreversible damage to the joints.1-6
H
N
OH
uric acid (1)
allopurinol (2)
1966-present
febuxostat (3)
2009-present
Figure 1. Uric acid (1) and XO inhibitor drugs.
An alternative approach to the reduction of serum uric acid
levels is to increase renal clearance using uricosuric agents.
Typically ≥90% of filtered urate is actively reabsorbed in the
renal proximal tubule via organic anion transporters,
principally URAT1 (SLC22A12).7-8 Therefore, inhibition of
URAT1 results in reduction of serum uric acid levels by
increasing renal clearance. This has been demonstrated
genetically, with non-functional missense mutations leading to
hypouricemia, and pharmacologically by URAT1 inhibitors such
as benzbromarone (
4
) and probenecid (
5
) (Figure 2).9
Hyperuricemia is commonly treated using xanthine oxidase
(XO) inhibitors, allopurinol (2) and febuxostat (3), which
reduce the rate of uric acid synthesis (Figure 1). However, an
appreciable proportion of patients do not respond effectively
to XO inhibitors.1
Br
O
O
OH
Br
O
Cl
O
)
R
O
OH
N
S
O
probenecid (
IC50 = 15 ꢀM
O
O
O
5
)
6
4
( )
fenofibrate (
R=iPr, IC50 = >75 ꢀM
benzbromarone
IC50 = 22 nM
ꢀ
R =H, IC50 = 11
M
N
N
N
OH
OH
O
S
Br
N
S
O
8
)
7
verinurad (RDEA3170) (
IC50 = 23 nM
lesinurad (RDEA594) ( )
CN
IC50 = 6.7 ꢀM
Figure 2. Clinical URAT1 inhibitors.
Benzbromarone (4) was first marketed for gout treatment in
1976, but withdrawn in 2003 due to compound-related
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hepatotoxicity (Figure 2).10-11 However, prior to withdrawal, 1) The relevant effect is urate renal clearance, Emax and
benzbromarone ( ) had been shown to be an effective baseline are GFR and 5% of GFR respectively (normal urate
4
treatment and proven successful in XO non-responding renal clearance, although it should be noted that this number
patients. Recently there has been renewed interest in may not hold for individuals with highly elevated urate).
uricosuric agents with a number of companies pursuing URAT1 2) Unbound plasma concentration is a valid surrogate of the
inhibitors either by repurposing secondary uricosuric drugs ultrafiltrate drug concentration in the proximal tubule.
such as fenofibrate (
Notable examples include lesinurad (RDEA594,
verinurad (RDEA3170, ), developed by Ardea Biosciences and
6
) or by developing novel clinical agents.12 3) Renal clearance is the only significant route of urate
and clearance in humans.
7)
8
later acquired by AstraZeneca with lesinurad gaining approval
for treating gout in late 2015.1
Results and Discussion
Whilst developing a NaV1.7 sodium channel inhibitor, PF-
05089771 (9) (Figure 3), unexpected secondary effects on uric
Although acidic sulfonamides are interesting acid isosteres due
to potential to span a range of pKa values they have not
previously been described as URAT1 inhibitors. In order to
capitalize on this finding, and in the absence of detailed
structural biology information on the target binding site, a
screening campaign of the company file was undertaken
focusing on lower molecular weight (MW <450) and logD (<3)
acids. In particular, an extensive compound legacy of diaryl
ether sulfonamides existed from the NaV1.7 program, which
acid were observed in clinical trials: A decrease in serum urate
was accompanied by an increase in urinary excretion. At doses
of >450 mg b.i.d., where PF-05089771 (9) plasma Cave unbound
was ~60 nM, serum urate reduction approached ~50%, similar
to that achieved by benzbromarone.13 This effect was
subsequently attributed to inhibition of URAT1 (IC50 = 3.5 ꢀM).
S
F
O
O
had originally delivered compound 9. A bespoke set of 20k
Cl
S
N
N
acidic compounds, rich in sulfonamides, was tested in a
functional radiolabel proximity assay measuring uric acid
uptake into URAT1 transfected cells (see supporting
information). Pleasingly, this campaign delivered a high hit rate
H
PF-05089771 (9)
URAT1 IC50 = 3.5 ꢀM
logD 2.6, clogP 4.2
MW 500
pKa (acid) 6.2
O
Cl
H2N
HN
N
Figure 3. Clinical compound: secondary uricosuric effect.
with 6% of compounds showing >30% inhibition at 10 ꢀM.
Within these hits, a number of reasonably potent and
lipophilic efficient acidic diaryl ether sulfonamides were
identified as shown graphically in Figure 4.18-19
Despite this interesting clinical result, a specific gout clinical
trial was not undertaken with PF-05089771 ( ). This was
9
largely due to the high doses of the compound (>450 mg bid)
required to deliver robust levels of efficacy, in combination
with a complex 10-step synthesis, leading to a prohibitively
high projected cost of goods for the gout marketplace. We
therefore undertook the simplification and optimization of this
acidic sulfonamide template towards improved URAT1 potency
and herein describe a novel chemotype for URAT1 inhibition.
However, the availability of the clinical data for PF-05089771
(9) proved useful in defining the desired target profile.
Determining the requisite potency level for advancing a
compound was hampered by the lack of a valid preclinical
model, due to the effect of uricase in non-human mammals.
This dictated the use of existing clinical data as the only guide
to the required potency. However, direct clinical PK/PD
comparison between known uricosuric compounds is
challenging, due to both the lack of consistent data sets and
complicating factors such as active metabolites e.g.
benzbromarone,14 active renal excretion which may imply that
ultrafiltrate concentrations in the proximal tubule significantly
differ from unbound plasma e.g. lesinurad,15-16 or potential
Figure 4. Screening campaign of diaryl ethers from company compound collection.
organic anion transporter polypharmacology e.g. probenecid.9,
Interestingly, despite a diverse range of compounds being
tested, derived from a range of different amino-heterocycles,
sulfonamide core ring substitutions and diaryl ether
substituents, the most lipophilic efficient hits fell broadly into
two structural subseries. As highlighted in Figure 4, the first
subseries was derived from 1,2,4- or 1,3,4-aminothiadiazoles,
with a nitrile substituted core ring and diarylether motif
comprising an ortho-heterocycle substitution.18 This sub-series
17
However, for our clinical compound, PF-05089771 (
9), and
simple literature examples e.g. fenofibrate (
6) (albeit this
compound is a prodrug of fenofibric acid with the parent ester
being essentially inactive in our hands) we tentatively
rationalized the low fraction of IC50 required to drive efficacy
using an Emax+baseline type response with the following
assumptions:
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of compounds generally exhibited high molecular weight (MW
>450) and high TPSA (>150 Å2) which resulted in low passive
permeability (RRCK <5x10-6 cm/sec). Previous experience with
compounds possessing similar physicochemical properties on
the NaV1.7 project suggested that this subseries would likely
suffer from poor and unpredictable preclinical PK due to low
absorption and high unbound clearance driven by organic
anion-transporting polypeptide (OATP) mediated hepatic
uptake.20 As a result, this sub-series was not actively pursued.
The other efficient subseries from the initial compound screen
Table 2. Selected compound optimisation.
URAT1 IC50 LE
(nM)
HLMc
logDb clogP
(LipE)a
Clint
No.
10f
Structure
12
0.38
(4.2)
1.8
1.9
2.0
3.7
3.6
3.7
3.4
<13
<8
(n=27)
was based on
a 2-amino-5-fluoropyridine sulfonamide
NC
Cl
O
183
0.33
(3.1)
headgroup with a nitrile-substituted phenyl core (10).19 These
compounds were characterised by lower molecular weight
(MW <450) and lower TPSA (<130 Å2) which resulted in higher
levels of passive permeability (RRCK >10x10-6 cm/sec). In
addition, several of these hits already possessed good URAT1
potency (URAT1 IC50 = <100 nM) and promising levels of ligand
efficiency (LE >0.35) and ligand lipophilic efficiency (lipE = ~4).
As a result, this subseries became the main focus of further
optimisation and characterisation work.
10g
10h
10i
(n=8)
92
0.34
(3.3)
134
<10
(n=2)
1
0.43
(5.6)
2.0
(n=9)
aLipE = -log(IC50)-clogP; blogD shake flask octanol/H2O; HLM (ꢀL/min/mg)
c
It was decided to optimise compounds from this subseries,
maintaining the p-CN group for potency and oxidative
metabolism stability characteristics. As fluorinated compound
10e had shown moderate potency (URAT1 IC50 = 57 nM) and
Table 1. Selected hits from initial screening campaign.
good HLM stability (<8 ꢀL/min/mg), it was anticipated that the
analogous Cl analogue 10f should be approximately half a log
unit more lipophilic and thereby ~5x more potent if lipE were
maintained whilst retaining good stability to microsomal
oxidation. As a result, compound 10f was synthesised along
URAT1 IC50 LE
(nM)
HLMc
Clint
No.
10a
Structure
logDb clogP
(LipE)a
with isomers 10g
exhibited lead-like levels of both URAT1 inhibition potency
(IC50 = 12 nM) and microsomal stability (HLM <13 L/min/mg).
The direct isomers 10g 10h were less promising, both
& 10h (Table 2). Pleasingly, compound 10f
19
0.37
(3.2)
2.3
-
4.5
3.2
3.2
3.3
3.3
163
<8
(n=7)
ꢀ
74
0.36
(3.9)
10b
10c
10d
10e
&
(n=5)
exhibiting lower URAT1 inhibition potency. Also, as previously
206
0.33
(3.5)
observed, compound 10h with m-CN showed high microsomal
-
65
83
<8
(n=2)
turnover (HLM 134 ꢀL/min/mg).
57
0.35
(3.9)
The diaryl ether ring system of 10 was further investigated for
additional potential lipophilic efficiency gains. Surveying the
broader SAR from the original screening campaign, an
unoptimised example was noted where a para-hydroxymethyl
substituent appeared to provide an improvement in lipophilic
efficiency. As a result, this group was introduced into the
preferred template to provide compound 10i. Pleasingly, this
hydroxymethyl group provided a significant improvement for
compound 10i in terms of both potency (URAT1 IC50 = 1 nM)
and lipophilic efficiency (lipE = 5.6) as compared to compound
10f (URAT1 IC50 = 12 nM, lipE = 4.2). Compound 10i also
1.5
1.4
(n=12)
53
0.35
(4.0)
(n=12)
aLipE = -log(IC50)-clogP; blogD shake flask octanol/H2O; cHLM (ꢀL/min/mg)
Example pyridine sulfonamides hits (10) are illustrated by
selected compounds 10a-e (Table 1). These all exhibited
moderate to good lipophilic efficiencies (lipE = 3.2-4.0) and
demonstrated promising potency in the IC50 = <100 nM range.
However, the SAR for stability to oxidation in human liver
microsomes proved interesting. As expected, more lipophilic
maintained good microsomal stability (HLM <10
ꢀL/min/mg),
making this a suitable lead alongside compound 10f
.
and
π-electron rich diaryl ether systems generally exhibited
high rates of oxidation eg 10a (HLM 163 L/min/mg).
Once initial lead compound (10f) had been identified, a
systematic optimisation of the template was carried out (Table
3, compounds 11-26). This included exploration of alternative
5- and 6-membered heterocycle sulfonamide headgroups and
core ring substitutions.18-19 However, although a number of
derivatives were synthesised and tested, no improvements to
either potency or lipophilic efficiency were observed
suggesting the original core template (10) obtained from the
original file mining and screening to be optimal.
ꢀ
Conversely, more polar and electron poor systems were
generally more stable. However, a p-CN substituent appeared
optimal for microsomal stability (comparing iso-lipophilic p-CN
10b vs m-CN 10c: HLM <8 vs 65 ꢀL/min/mg respectively).
Interestingly, this trend for superior HLM stability for p-CN vs
m-CN also held for the corresponding fluorinated derivatives
10d vs 10e (HLM 83 vs <8 ꢀL/min/mg).
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Accordingly, compounds 10f and 10i were selected for further
profiling. Both compounds were taken through in vitro ADME,
safety and polypharmacology studies (Table 4).
Table 3. Selected sulfonamide heterocycle and core substitution SAR.
URAT1 IC50 LE
(nM)
No.
11
Structure
clogP
(LipE)a
Table 4. Broader profiles of lead compounds 10f & 10i
115
0.35
(3.3)
3.6
(n=4)
Compound
10f
Compound
10i
Property
Physicochemical properties
Molecular weight (Da)
clogP / logD
395
0.32
(2.8)
12
13
14
15
16
3.6
2.7
2.8
2.7
2.4
429
3.7 / 1.8
5.8
434
3.4 / 2.1
5.7
(n=3)
pKa
1823
(n=3)
0.29
(3.0)
TPSA (Å)
124
121
Solubility at pH6.5 (
ꢀ
M)
7.7
15.3
ADME in vitro profiles
RRCK (x10-6 cm/sec)
P-gp efflux ratio
1954
(n=2)
0.28
(2.9)
16
3.5
12
1.9
BCRP efflux ratio
122
34
5147
(n=3)
0.26
(2.6)
HLM, Clint
RLM, Clint
DLM, Clint
(
ꢀ
L/min/mg)
<13
<10
(
ꢀ
L/min/mg)
<14
<14
(
ꢀ
L/min/mg)
L/min/106 cells)
<18
<18
HHEP, Clint
RHEP, Clint
DHEP, Clint
(
ꢀ
23
23
3868
(n=1)
0.27
(3.0)
(
ꢀ
L/min/106 cells)
L/min/106 cells)
10
82
(
ꢀ
<6
<6
human plasma fu
1.2 x 10-3
2.1 x 10-3
4.1 x 10-3
8.3 x 10-3
4.3 x 10-3
1.4 x 10-2
2982
(n=2)
0.28
(2.8)
17
18
19
2.7
3.4
3.4
rat plasma fu
dog plasma fu
OATP cell uptake
58
0.38
(3.8)
OATP1B1 (transfected/WT)
OATP1B3 (transfected/WT)
OATP2B1 (transfected/WT)
DDI CYP inhibition
1.003
1.067
1.232
1.181
1.124
1.480
(n=3)
110
0.36
(3.6)
(n=2)
CYP1A2, inh. IC50
CYP2C8, inh. IC50
CYP2C9, inh. IC50
(
ꢀ
M)
M)
M)
>30
11.9
4.2
>30
9.9
(ꢀ
(ꢀ
2.0
426
0.33
(4.0)
20
21
2.4
2.7
(n=5)
CYP2C19, inh. IC50
(
ꢀ
M)
17.2
>30
17.5
17.5
>30
>30
CYP2D6, inh. IC50
CYP3A4, inh. IC50
(
ꢀ
M)
(
ꢀ
Anion transporter inhibition
M)
1123
(n=4)
0.31
(3.2)
OAT1 IC50
OAT3 IC50
OAT4 IC50
Safety
(
(
(
ꢀ
ꢀ
ꢀ
M)
M)
M)
14.0
0.72
14.0
14.3
0.5
15
3358
(n=2)
0.27
(2.4)
22
23
3.1
3.6
THLE cytotoxicity (
IVMN (+/- S4)
ꢀ
M)
103
105
88
0.37
(3.5)
negative
negative
negative
negative
(n=1)
Ames (+/- S4)
Ion channel pharmacologya
382
0.32
(2.8)
24
25
3.6
hERG ephys. IC50 M)
(
ꢀ
67
>100
>30
>30
>10
(n=3)
NaV1.5 ephys. IC50
NaV1.7 ephys. IC50
(
ꢀ
ꢀ
M)
M)
>30
>30
4.8
(
244
0.33
(2.4)
GABAA Ki (ꢀM)
4.2
3.8
(n=1)
aFull Cerep polypharmacology profiles included in SI section.
37
0.37
(3.6)
Pleasingly, neither compound appeared to present any serious
liabilities in these assays. In terms of in vitro ADME, the
compounds showed good levels of passive permeability in the
RRCK cell line and metabolic stability in both human
26
(n=3)
aLipE = -log(IC50)-clogP
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microsomes and hepatocytes.21 Although some inhibition of Evidence for formation of a potentially reactive species
CYP2C9 was observed (10f, CYP2C9 IC50 = 4.2 M; 10i, CYP2C9 following loss of the chlorobenzonitrile moiety in combination
IC50 = 2.0 M) this was still relatively weak in comparison to with addition of glutathione was observed in human
ꢀ
ꢀ
the primary pharmacology, so deemed to present low DDI risk. hepatocytes with the presence of a glutathione conjugate and
Both compounds also looked promising for safety with low subsequent breakdown products. However, it was present
cytotoxicity potential in a THLE cell line and negative results in only in trace amounts in dog and was not detectable in rats
both Ames mutagenicity and in vitro micronucleus genetic (see SI for metabolite scheme).
toxicity studies. Off-target risks were assessed through in vitro Compound 10i, while having excellent i.v. PK in dogs with low
polypharmacology panels. Interestingly, compound 10f did Clu and Vss, showed highly variable i.v. PK in the rat (which
show some weak ꢀM-level off-target activity in a few instances precluded the calculation of accurate PK parameters) and
whereas compound 10i showed no off-target pharmacology in variable oral PK in both species. Again, a limited contribution
the test panel (see supporting information for full of biliary clearance was noted in bile duct cannulated rats with
polypharmacology panel profiles across both compounds). ~10% of the dose of compound 10i excreted in bile as
Both compounds were progressed into low dose i.v. and oral unchanged parent. In human microsomes and hepatocytes,
(p.o.) in vivo PK in rat and dog (Table 5).
the major metabolite was an oxidation of the hydroxymethyl
group to the carboxylic acid. In rat hepatocytes, significant
glucuronidation, believed to be of the hydroxymethyl group,
was also noted.
Table 5. Preclinical PK for compounds 10f & 10i
PF-05089771 Compound Compound
Property
The ratio of uptake rates between cell lines expressing human
OATP1, OATP3, or OATP4, and wild-type was close to 1 for
both compounds, suggesting a low contribution of OATP
driven active hepatic uptake to the clearance.
9
10f
10i
Rat PK
Dose i.v./p.o. (mg/kg)
Clp (mL/min/kg)
Clu (mL/min/kg)
T1/2 (h)
1.0/3.0
6.0
0.76/2.3
0.48
229
1.0/3.0
Both compounds showed significant efflux by BCRP in bi-
447
4.4
See text &
supporting
information*
directional transport experiments and, in the case of 10f
a
7.1
modest additional effect of P-gp. While at relatively high
preclinical doses the compounds showed good oral absorption,
this may in part reflect saturation of the efflux transporters
and the possibility of efflux affecting absorption at low clinical
dose levels cannot be ruled out.
Vss (L/kg)
1.3
0.25
100
F (%)
27
Dog PK
Dose i.v./p.o. (mg/kg)
Clp (mL/min/kg)
Clu (mL/min/kg)
T1/2 (h)
0.1/3.0
4.8
0.5/1.0
0.39
95
0.5/1.0
1.3
Compounds 10f and 10i were synthesized on sufficient scale to
advance to preclinical in vivo toxicology studies. The synthesis
of all compounds was initially carried out in 2-steps starting
from commercially available sulfonyl chloride 27 (Scheme 1).
Amide formation with 5-fluoropyridin-2-amine provided
common intermediate 28 that was subsequently converted to
the final compounds 10a-i via SNAr displacement with the
corresponding phenols 29. For compound 10f, this method
proved high yielding and was directly scaled to provide 113 g
of material in 50% yield over 2-steps and in >99% purity after
recrystallization from ethyl acetate. However, although
compound 10i was initially prepared in an analogous manner
641
1.4
93
6.7
3.9
Vss (L/kg)
0.31
35
0.18
100
0.27
50
F (%)
*Rat i.v. data for compound 10i were highly variable and not suitable for the
calculation of PK parameters.
The preclinical pharmacokinetics of the parent series of the
NaV1.7 blockers (typically high MW >450 and lipophilicity),
were typically characterized by moderate to high Clp in
rodents, driven by OATP transporter-mediated liver uptake
and biliary clearance22 of parent compound, and
uncharacteristically high Vss for acidic molecules, which may
using the primary alcohol containing phenol 29i
, this
displacement proved low yielding (29% yield).
also partly reflect liver uptake.23 While PF-05089771 (
9)
showed exceptional preclinical PK within the series, typically in
vitro to in vivo extrapolation was challenging.
In contrast, 10f demonstrated excellent PK in both rats and
dogs with low Clu, low Vss, typical of an acidic molecule, and
very high oral bioavailability (Table 5). In contrast to the parent
series, the biliary excretion in bile duct cannulated rats was
low, with <0.5% of the dose excreted in bile as unchanged
parent. Renal clearance was <<GFR x fu in rats and dogs
(~0.0001 mL/min/kg in both species), indicating net re-
absorption and a low contribution to total clearance. The
major oxidative metabolite in human microsomes and
hepatocytes was an oxidation of the chlorophenyl ring, which
was seen in trace quantities in rat and not present in dog.
Scheme 1. (a) Pyridine, 23 ˚C, 2 h, (b) K2CO3, DMSO, 80 ˚C, recrystallization EtOAc.
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In order to support larger scale synthesis, several alternatives Cardiovascular assessment of compound 10f in the rat at 10,
were investigated for compound 10i. It was anticipated that 30 and 100 mg/kg identified a decrease in heart rate and body
the main issue in the reaction was the unprotected benzyl temperature at 10 mg/kg, with a small biphasic change in
alcohol. Disappointingly, protection of the benzyl alcohol with blood pressure (increase followed by decrease) at 30 and 100
a silyl group, acetate group or dimethoxybenzyl group did not mg/kg. The single dose unbound Cmax and Cave concentrations
improve yields to any significant extent. However, adjustment at the lowest dose of 10 mg/kg were 59 and 41 nM,
of the oxidation level to the aldehyde or ester both proved respectively.
effective in increasing the yield of the reaction. A screen of a Similarly, compound 10i was administered orally to rats at 30,
range of bases identified K3PO4 and K2HPO4 to provide the 100 and 300 mg/kg/day and to dogs at 10, 30 and 100
cleanest reactions with K2HPO4 giving the highest overall yield. mg/kg/day. In rats the compound was well tolerated up to 300
While K2HPO4 proved effective for the SNAr with either the mg/kg/day and there were no test article-related clinical signs
aldehyde or ester present, it was established that on scale-up, or changes in body weight, food consumption, hematology,
the cleanest and highest yielding reaction occurred in the clinical chemistry, organ weights, necropsy, or microscopic
presence of the aldehyde. As a result, aldehyde-containing observations. At the 300 mg/kg dose unbound Cmax and Cave
phenol 29j was applied to give SNAr adduct 10j in 77% yield. concentrations were 537 and 240 nM respectively. Similarly, in
This was followed by subsequent reduction of the aldehyde to dogs compound 10i was tolerated up to 100 mg/kg/day, and
primary alcohol in 96% yield to provide the >250g of did not result in direct clinical pathology or histologic test
compound 10i in a 68% yield over 3 steps.
article-related findings. Treatment related clinical signs of
emesis and salivation were mainly present in high dose
animals (100 mg/kg/day). Findings indirectly-related to
treatment with 10i were slightly decreased hepatic glycogen
content and a decrease in serum glucose at 100 mg/kg,
secondary to emesis. At the 100 mg/kg dose unbound Cmax and
Cave concentrations were 3905 and 1019 nM respectively.
Cardiovascular assessment of compound 10i was conducted in
the rat at 3, 30, and 100 mg/kg, with no adverse effects
observed up to 30 mg/kg but an increase in blood pressure
was observed at 100 mg/kg. The single dose unbound Cmax and
Cave concentrations at the 30 mg/kg no cardiovascular effect
dose were 98 and 43 nM, respectively.
Scheme 2. (a) K2HPO4, DMSO, 100 ˚C, 2.5 h (b) NaBH4, MeOH, rt, 45 min.
Both compounds 10f and 10i were taken into 14-day repeat-
dose exploratory in vivo toxicity studies in rat and dog, with
single-dose cardiovascular assessment in the rat. Compound
10f was administered to both rats and dogs at 30, 100 and 300
mg/kg/day. In rat, compound related mortality was observed
at 300 mg/kg/day and microscopic changes were noted in the
kidney at 300 mg/kg/day (hypertrophy/hyperplasia of the
urothelium and increased mitoses in the collecting ducts), and
Conclusions
In summary, the first example of a series of potent acidic
sulfonamide URAT1 inhibitors is described. Following
elucidation that clinically efficacious diaryl ether sulfonamide
compound
9 likely derived its uricosuric activity via inhibition
of URAT1, this template was subsequently optimized for lower
molecular weight, lipophilicity and synthetic complexity. This
led to the design of potent sulfonamide compounds 10f and
10i. Profiling through detailed in vitro and in vivo preclinical
ADME and safety studies highlighted that both compounds 10f
and 10i exhibited promising preclinical PK and safety profiles
making them interesting candidates for possible further
development with the principal issues being the potential
formation of reactive species for 10f and low confidence in
human PK prediction for 10i. In the event, further refinement
of the series produced more favourable compounds which are
the subject of a separate manuscript.24
liver at
≥100 mg/kg/day (hepatocellular hypertrophy, with
focal necrosis at 300 mg/kg/day). The maximum tolerated
dose was therefore 100 mg/kg. On day 14 at the 30 mg/kg
dose, at which there were no test article-related observations,
unbound Cmax and Cave concentrations were 179 and 123 nM
respectively. In dogs, compound 10f was tolerated up to 300
mg/kg/day. Alanine aminotransferase (ALT) levels were
decreased in males at doses ≥30 mg/kg/day and females at
≥100 mg/kg/day, without any pathologic correlation in the
liver.
A histologic change in testes was noted at 300
mg/kg/day, correlating with immaturity. However, evidence of
testicular immaturity was also observed in the vehicle animal.
On day 14 at the 30 mg/kg dose, unbound Cmax and Cave
concentrations were 7419 and 3980 nM respectively. While
some ALT observations were made at this dose, without
pathological correlates, the exposure reached suggests an
adequate therapeutic margin could be achieved.
Acknowledgements
We thank Joe Warmus for assistance with compound
characterization, Peakdale Molecular Ltd for the synthesis of
compound analogues and York Bioanalytical Solutions for the
generation of ADME data. We also thank Laura Kammonen
6 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
and Paula Cawkill for cell culture and thank Katrina Gore and w/w, 1.5 mol of citric acid for 1 mol of product) and water (1
Phil Stanley for statistical data analysis support.
L). The organic phase was concentrated in vacuo to give crude
material (136 g). The compound was crystallised from EtOAc
(1.6 L, 12 mL/g) to give material (110 g). This material was
combined with 35 g of previously obtained material then
partially dissolved in boiling ethyl acetate (~1.5 mL, 10 mL/g
ca.). The mixture was left to cool overnight to room
temperature and filtered to afford the title compound as a
Abbreviations
ADME, absorption, distribution, metabolism and excretion;
BCRP, breast cancer resistance protein; Cave, average plasma
concentration; Cmax, maximum plasma concentration; Clp,
plasma clearance; Clu, unbound clearance; CYP, cytochrome
P450; DDI, drug-drug interaction; DHEP, dog hepatocytes;
DLM, dog liver microsomes; F, bioavailability; fu, fraction
unbound; GFR, glomerular filtration rate; HHEP, human
hepatocytes; HLM, human liver microsomes, IC50, half-
maximum inhibitory concentration; IVMN, in vitro
micronucleus; LE, ligand efficiency; LipE, lipophilic efficiency;
OAT, organic anion transporter; OATP, organic anion-
transporting polypeptide; PD, pharmacodynamics; P-gp, P-
glycoprotein; PK, pharmacokinetics; ppb, plasma protein
binding; RHEP, rat hepatocytes; RLM, rat liver microsomes;
RRCK, Ralph Russ canine kidney cell line; SLC, solute carrier;
SNAr, nucleophilic aromatic substitution; sUA, serum uric acid;
T1/2, half life; TPSA, topological polar surface area; Vss,
apparent volume of distribution at steady state
1
white solid (10f, 113 g, 54%). H NMR (400 MHz, d6-DMSO) δ
11.34 (br. s., 1H), 8.44 (d, J= 2.34 Hz, 1H), 8.22 (d, J= 2.73 Hz,
1H), 8.16 (dd, J= 2.30, 9.00 Hz, 1H), 8.13 (d, J= 8.59 Hz, 1H),
7.83 (d, J= 2.34 Hz, 1H), 7.71 (ddd, J= 3.10, 8.60, 8.60 Hz, 1H),
7.47 (dd, J= 2.34, 8.98 Hz, 1H), 7.32 (d, J= 8.98 Hz, 1H), 7.13
(dd, J= 3.90, 8.98 Hz, 1H); 13C NMR (101 MHz, d6-DMSO) δ
160.6, 158.6, 155.2 (d, J= 248.0 Hz), 148.0, 138.0, 137.2, 137.0,
135.6 (m), 134.6, 134.0, 126.8 (d, J= 22.7 Hz), 122.4, 120.4,
119.1, 116.1, 114.8, 114.6 (d, J= 4.40 Hz), 109.7, 104.2; 19F
NMR (376 MHz, d6-DMSO) δ -134.44: HPLC (system 1, 4.5 min,
acid) Rt 3.03 min, ELSD >95% purity; LRMS m/z 428.95 [MH]+;
HRMS (ESI) m/z: [MH]+ Calcd for C19H10ClFN4O3S 429.0219,
found 429.0221.
4-(3-Chloro-4-(hydroxymethyl)phenoxy)-3-cyano-N-(5-
fluoropyridin-2-yl)benzene sulfonamide (10i)
To a suspension of 4-(3-chloro-4-formylphenoxy)-3-cyano-N-
(5-fluoropyridin-2-yl) benzenesulfonamide (10j, 276.9 g, 0.64
mol) in methanol (5.5 L) at 0 °C was added NaBH4 portion-wise
over 25 min. The reaction was stirred at rt for 1.5 h, cooled to
0 °C then water (4.8 L) slowly added. The reaction mixture was
warmed to rt and 1M HCl (2.5 L) was slowly added resulting in
a suspension of the product. The mixture was left to stir at rt
for 1 h and then the suspension was filtered and dried for 16 h
at 40 °C under vacuum to give crude product. The crude
product was dissolved in acetone (2.5 L) and then silica (380 g)
was added to the vessel. The mixture was stirred at rt for 20
min and then filtered through a silica pad and washed with
acetone (1.5 L). Water (10 L) was slowly added resulting in the
precipitation of the product. The mixture was left to stir at rt
for 1 h and then the suspension was filtered and the residue
dried overnight at 40 °C to yield 4-(3-chloro-4-
(hydroxymethyl)phenoxy)-3-cyano-N-(5-fluoropyridin-2-
Experimental Section
In vitro assays
Full assay protocol for the URAT1 assay is provided in the
supporting information.
In vivo studies
All experiments involving animals were conducted in our
AAALAC-accredited facilities and were reviewed and approved
by the Pfizer Institutional Animal Care and Use Committee. The
acute rat toleration study was conducted by TCG Life Sciences
(TCGLS). TCGLS adheres to the ethical guidelines of Committee
for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Govt. of India under the supervision of a
fully compliant Institutional Animal Ethics Committee (IAEC).
General chemistry experimental
yl)benzenesulfonamide as a cream powder (10i, 268.2 g, 96%).
1H NMR (400 MHz, d6-DMSO) δ 11.31 (br. s., 1H), 8.39 (d, J=
2.34 Hz, 1H), 8.22 (d, J= 2.73 Hz, 1H), 8.12 (dd, J= 2.34, 8.98 Hz,
1H), 7.70 (dd, J= 8.60, 3.10 Hz, 1H), 7.66 (d, J= 8.59 Hz, 1H),
7.48 (d, J= 2.73 Hz, 1H), 7.30 (dd, J= 2.34, 8.59 Hz, 1H), 7.12
(dd, J= 3.51, 8.98 Hz, 1H), 7.05 (d, J= 8.90 Hz, 1H), 5.50 (t, J=
5.46 Hz, 1H), 4.59 (d, J= 5.07 Hz, 2H); 13C NMR (101 MHz, d6-
DMSO) δ 162.48, 156.42 (d, J= 247 Hz), 152.96, 147.99, 138.19,
135.63 (d), 135.54, 134.66, 133.88, 132.52, 130.17, 126.88 (d,
J= 19.1 Hz), 121.82, 120.02, 117.05, 115.11, 114.54 (d, J=
5.14Hz), 102.89, 60.38; 19F NMR (376 MHz, CDCl3) δ -134.5:
HPLC (system 1, 25 min, acid) Rt 14.09 min, ELSD 98.9% purity;
LRMS m/z 434.02 [MH]+; HRMS (ESI) m/z: [MH]+ Calcd for
C19H13ClFN3O4S 434.0372, found 434.0369.
Details of general methods, experimental details for
compounds, HPLC conditions and spectra for lead compounds
10f and 10i can be found in the supporting information.
Selected experimental details for the synthesis of compounds
10f and 10i only are outlined below.
4-(3-Chloro-4-cyanophenoxy)-3-cyano-N-(5-fluoro-pyridin-2-
yl)benzenesulfonamide (10f)
A
mixture
of
3-cyano-4-fluoro-N-(5-fluoropyridin-2-
yl)benzenesulfonamide (28, 110 g, 0.38 mol), 2-chloro-4-
hydroxybenzonitrile (29f, 85.8 g, 0.56 mol) and K2CO3 (154 g,
1.12 mol) in DMSO (1.1 L) was heated to 80 °C for 32 h, then
stirred for an additional 16 h at rt. The reaction mixture was
poured in to water (3 L). The combined organic layers were
washed with water (1 L), citric acid solution (117 g, in 3.5 L, 3%
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DOI: 10.1039/C6MD00190D
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Journal Name
7. Y. Lu; T. Nakanishi; I. Tamai Drug Metab. Pharmacokinet.,
2013, 28, 153.
8. T. Nakanishi; K. Ohya; S. Shimada; N. Anzai; I. Tamai Nephrol.,
Dial., Transplant., 2013, 28, 603.
4-(3-Chloro-4-formylphenoxy)-3-cyano-N-(5-fluoropyridin-2-
yl)benzenesulfonamide (10j)
A
suspension of 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)
9. K. Maeda; Y. Tian; T. Fujita; Y. Ikeda; Y. Kumagai; T. Kondo; K.
Tanabe; H. Nakayama; S. Horita; H. Kusuhara; Y. Sugiyama Eur. J.
Pharm. Sci., 2014, 59, 94.
10. A. Felser; P. W. Lindinger; D. Schnell; D. V. Kratschmar; A.
Odermatt; S. Mies; P. Jeno; S. Krahenbuhl Toxicology, 2014, 324,
136.
11. M.-H. H. Lee; G. G. Graham; K. M. Williams; R. O. Day Drug
Saf., 2008, 31, 643.
12. D. Uetake; I. Ohno; K. Ichida; Y. Yamaguchi; H. Saikawa; H.
Endou; T. Hosoya Intern. Med., 2010, 49, 89.
13. Z. Ali; K. J. Butcher; R. P. Butt; S. J. Felstead; S. Glatt; R. M.
McKernan; M. Panesar, 2013, WO2013057722.
14. S. Uchida; K. Shimada; S. Misaka; H. Imai; Y. Katoh; N. Inui; K.
Takeuchi; T. Ishizaki; S. Yamada; K. Ohashi; N. Namiki; H.
Watanabe Drug Metab. Pharmacokinet., 2010, 25, 605.
15. Z. Shen; C. Rowlings; B. Kerr; V. Hingorani; K. Manhard; B.
Quart; L.-T. Yeh; C. Storgard Drug. Des. Devel. Ther., 2015, 9,
3423.
16.
http://www.accessdata.fda.gov/drugsatfda_docs/label/2015/20
7988lbl.pdf.
17. Y. Hagos; D. Stein; B. Ugele; G. Burckhardt; A. Bahn J. Am.
Soc. Nephrol., 2007, 18, 430.
18. R. M. Owen; R. I. Storer, 2014, WO2014170793.
19. R. I. Storer; N. A. Swain; R. M. Owen, 2014, WO2014170792.
20. H. M. Jones; R. P. Butt; R. W. Webster; I. Gurrell; P. Dzygiel;
N. Flanagan; D. Fraier; T. Hay; L. E. Iavarone; J. Luckwell; H.
Pearce; A. Phipps; J. Segelbacher; B. Speed; K. Beaumont Clin.
Pharmacokinet., 2016, Ahead of Print.
21. L. Di; C. Whitney-Pickett; J. P. Umland; H. Zhang; X. Zhang; D.
F. Gebhard; Y. Lai; J. J. Federico; R. E. Davidson; R. Smith; E. L.
Reyner; C. Lee; B. Feng; C. Rotter; M. V. Varma; S. Kempshall; K.
Fenner; A. F. El-kattan; T. E. Liston; M. D. Troutman J. Pharm.
Sci., 2011, 100, 4974.
benzene sulfonamide (28, 108.2 g, 0.37 mol), K2HPO4 (191.3 g,
1.10 mol) and 2-chloro-4-hydroxybenzaldehyde (29j, 63.1 g,
0.40 mmol) in DMSO (760 mL) was heated to 100 °C and left to
stir for 2.5 h then cooled to rt. The reaction mixture was
poured into water (3.0 L) resulting in some precipitation of
product. EtOAc (3.0 L) was added and the aq. layer acidified to
pH 3 using conc. HCl. The aq. layer was removed and the
resulting suspension filtered and solid washed with EtOAc (200
mL) and 1M HCl (20 mL) to give crude product. The organic
filtrates were retained and washed with 1M HCl (700 mL) and
sat. brine (2 × 700 mL) and dried over MgSO4, filtered and
concentrated to yield more crude product. Both batches of
crude product were combined and slurried in EtOAc (750 mL)
at reflux for 45 min. The mixture was cooled to rt, filtered and
the solid washed with EtOAc (2× 100 mL). The product was
died overnight under vacuum at 40 °C to yield 4-(3-chloro-4-
formylphenoxy)-3-cyano-N-(5-fluoropyridin-2-yl)benzene
1
sulfonamide (10j, 121.1 g, 77%) as a cream powder. H NMR
(400 MHz, d6-DMSO) δ 11.34 (br. s, 1H), 10.28 (s, 1H), 8.42 (d,
J= 2.3 Hz, 1H), 8.20 (d, J= 3.1 Hz, 1H), 8.14 (dd, J= 9.0 Hz, 2.4
Hz, 1H), 7.95 (d, J= 8.6 Hz, 1H), 7.69 (td, J= 8.6 Hz, 3 Hz, 1H),
7.63 (d, J= 2.4 Hz, 1H), 7.38 (dd, J= 8.2, 2.5 Hz, 1H), 7.28 (d, J=
8.9 Hz, 1H), 7.09 (dd, J= 9.2, 3.7 Hz, 1H); 19F NMR (376 MHz,
d6-DMSO) δ -134.4; HPLC (system 1, 4.5 min, acid) Rt 3.58 min,
ELSD >95% purity; LRMS m/z 432.09 [MH]+.
3-Cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzene sulfonamide
(28)
22. K. M. Giacomini; S.-M. Huang; D. J. Tweedie; L. Z. Benet; K. L.
R. Brouwer; X. Chu; A. Dahlin; R. Evers; V. Fischer; K. M. Hillgren;
K. A. Hoffmaster; T. Ishikawa; D. Keppler; R. B. Kim; C. A. Lee; M.
Niemi; J. W. Polli; Y. Sugiyama; P. W. Swaan; J. A. Ware; S. H.
Wright; S. Wah Yee; M. J. Zamek-Gliszczynski; L. Zhang Nat. Rev.
Drug Disc., 2010, 9, 215.
23. A.-K. Sohlenius-Sternbeck; U. Fagerholm; J. Bylund
Xenobiotica, 2013, 43, 671.
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Bictash; K. Cheung; K. Costelloe; E. Dardennes; E. Impey; P. H.
Milliken; E. Mortimer-Cassen; H. J. Pearce MedChemComm,
2016, Submitted.
3-Cyano-4-fluorobenzene-1-sulfonyl chloride (27, 60.3 g, 274
mmol) was added to a solution of 5-fluoropyridin-2-amine
(40.0 g, 357 mmol) and pyridine (67 mL, 823 mmol) in DCM (1
L) at rt then stirred for 3 h. The solvent was removed under
vacuum and the residue stirred in dilute HCl (2N, 850 mL) for
16 h. The precipitate was removed by filtration and the residue
washed with water (200 mL) and dried under high vacuum
overnight. The crude material was triturated with TBME (500
mL) to give the title compound (28, 70.3 g, 87%) as an orange
1
solid. H NMR (400 MHz, d6-DMSO) δ 11.45 (bs, 1H), 8.44 (dd,
J= 6.0, 2.4 Hz, 1H), 8.26-8.24 (m, 1H), 8.18 (d, J= 3.2 Hz, 1H),
7.72-7.66 (m, 2H), 7.11-7.08 (m, 1H); 19F-NMR (376 MHz, d6-
DMSO) δ -101.50, -134.20: HPLC (system 2, 4.5 min, acid) Rt
2.55 min, ELSD >95% purity; LRMS m/z 296.06 [MH]+.
References
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8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6MD00190D
The discovery and optimization of a series of acidic heterocyclic sulfonamides that are potent and selective URAT1 inhibitors is described.
1