Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency and Pharmacokinetics while Mitigating Metabolic Liabilities

Article abstract of DOI:10.1021/acs.jmedchem.6b01851

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as Na_V1.7 inhibitors that demonstrate high levels of selectivity over other Na_V isoforms. The optimization of a series of internal Na_V1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.

Full text of DOI:10.1021/acs.jmedchem.6b01851

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Article
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Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency
and Pharmacokinetics While Mitigating Metabolic Liabilities
Matthew M. Weiss, Thomas A. Dineen, Isaac E. Marx, Steven Altmann, Alessandro A Boezio,
Howard Bregman, Margaret Y. Chu-Moyer, Erin F. DiMauro, Elma Feric Bojic, Robert S. Foti,
Hua Gao, Russell F. Graceffa, Hakan Gunaydin, Angel Guzman-Perez, Hongbing Huang, Liyue
Huang, Michael Jarosh, Thomas Kornecook, Charles R. Kreiman, Joseph Ligutti, Daniel S.
La, Min-Hwa Jasmine Lin, Dong Liu, Bryan D. Moyer, Hanh Nho Nguyen, Emily A Peterson,
Paul E. Rose, Kristin Taborn, Beth D. Youngblood, Violeta L. Yu, and Robert T. Fremeau, Jr.
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01851 • Publication Date (Web): 13 Mar 2017
Downloaded from http://pubs.acs.org on March 14, 2017
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Sulfonamides as Selective Na 1.7 Inhibitors:
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Optimizing Potency and Pharmacokinetics While
Mitigating Metabolic Liabilities
*
,†
†,#
†,#
§,#
Matthew M. Weiss, Thomas A. Dineen, Isaac E. Marx, Steven Altmann, Alessandro
†
†
†
†
§,#
Boezio, Howard Bregman, Margaret ChuꢀMoyer, Erin F. DiMauro, Elma Feric Bojic,
‡
ǁ
†
ǁ,#
†
Robert S. Foti, Hua Gao, Russell Graceffa, Hakan Gunaydin, Angel GuzmanꢀPerez,
†
,#
‡
§
α
†
Hongbing Huang, Liyue Huang, Michael Jarosh, Thomas Kornecook, Charles R. Kreiman,
α
†
‡
α
α
Joseph Ligutti, Daniel S. La, MinꢀHwa Jasmine Lin, Dong Liu, Bryan D. Moyer, Hanh N.
†
,#
†,#
±
§
§
Nguyen, Emily A. Peterson, Paul E. Rose, Kristin Taborn, Beth D. Youngblood, Violeta
§
§,#
Yu, and Robert T. Fremeau, Jr.
†
ǁ
‡
Departments of Therapeutic Discovery, Department of Molecular Engineering, Department of
§
±
Pharmacokinetics and Drug Metabolism, Department of Neuroscience and Department of
Biologics, Amgen, Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States
α
Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks,
California 91320, United States
KEYWORDS. Sodium channel, Na 1.7, Na 1.5, pain, pregnane X receptor, CYP3A4 induction,
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sulfonamide, cLogD
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ABSTRACT
Several reports have recently emerged regarding the identification of heteroarylsulfonamides as
Na 1.7 inhibitors that demonstrate high levels of selectivity over other Na isoforms. The
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optimization of a series of internal Na 1.7 leads that address a number of metabolic liabilities
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including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the
identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic
profiles and were devoid of the aforementioned liabilities. Key to achieving this within a series
prone to transporterꢀmediated clearance was the identification of a small range of optimal cLogD
values and the discovery of subtle PXR SAR that was not lipophilicityꢀdependent. This enabled
the identification of compound 20 which was advanced into a target engagement
pharmacodynamic model where it exhibited robust reversal of histamineꢀinduced scratching
bouts in mice.
INTRODUCTION
The transmembrane voltageꢀgated sodium channel Na 1.7 has received considerable attention
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as a target for the treatment of pain. There is a wealth of genetic evidence linking Na 1.7 and
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the pain processing pathway. This evidence comes mostly in the form of lossꢀofꢀfunction and
gainꢀofꢀfunction mutations in SCN9A, the gene encoding for Na 1.7. The former leads to a rare
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condition known as congenital indifference to pain (CIP), which manifests in the inability of
individuals to sense painful stimuli while not significantly impacting motor or cognitive
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function.
The latter leads to numerous painful conditions including primary inherited
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erythromelalgia, paroxysmal extreme pain disorder, and small fiber neuropathy.
These
observations have spurred considerable efforts towards the development of potent and selective
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inhibitors of Na 1.7. One of the primary challenges associated with these efforts has been the
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identification of isoformꢀselective inhibitors, in particular those that demonstrate suitable levels
of selectivity over Na 1.5, which is expressed in cardiac myocytes and plays a key role in
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cardiovascular function.
Over the past several years, novel leads within a class of highly isoformꢀselective
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heteroarylsulfonamideꢀcontaining Na 1.7 inhibitors were initially reported by Pfizer/Icagen and
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later advanced by Genentech/Xenon.
We recently published findings detailing efforts
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associated with a novel series of isoformꢀselective heteroarylsulfonamides. While this work
culminated in the identification of a moderately potent, isoformꢀselective Na 1.7 inhibitor that
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demonstrated excellent rodent pharmacokinetics and robust activity in a Na 1.7ꢀdependent
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pharmacodynamic model, this compound and generally those in this class suffered from several
metabolic liabilities, including the activation of the nuclear hormone receptor pregnane X
receptor (PXR) which can manifest in the induction of multiple drug metabolizing enzymes,
including CYP3A4. Additionally, lead compounds contained a 2ꢀaminothiazole, a motif prone
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to bioactivation, and as such represented a toxicity liability. Herein we report efforts to obviate
the metabolic liabilities associated with this class of compounds while improving potency and
maintaining isoform selectivity and favorable pharmacokinetics.
RESULTS AND DISCUSSION
The series of Na 1.7 inhibitors that we previously disclosed are represented by quinazoline 1
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(
Table 1). This class of compounds suffered from metabolic liabilities that included activation
of the nuclear hormone receptor PXR, a ligandꢀdependent transcription factor that serves a
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critical role in regulating detoxifying genes such as CYP3A4. Consistent with PXR activation,
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incubation of 1 with human hepatocytes led to the induction of CYP3A4. Given the wealth of
therapeutics metabolized by this CYP isoform and the potential for induction to lead to drugꢀ
1
2
drug interactions (DDIs), we focused on mitigating this liability. Previous work found that the
incorporation of a 2ꢀaminothiazole warhead afforded inhibitors with a unique balance of potency
and favorable pharmacokinetics, however this moiety presented a potential bioactivation risk. It
has been well established that 2ꢀaminothiazoles are prone to epoxidation and subsequent scission
to yield a reactive thiourea metabolite that is capable of oxidizing glutathione (GSH) and hence
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has the potential for idiosyncratic adverse drug reactions (IADRs).
It was demonstrated that
upon incubation of 1 with human liver microsomes (HLMs), GSH, and nicotinamide adenine
dinucleotide phosphate (NADPH), both the corresponding thiourea and the GSHꢀadduct were
observed. The adducts observed were presumably the result of oxidation of the C4ꢀC5 double
bond followed by subsequent nucleophillic attach of GSH at the carbon adjacent to the thiazole
nitrogen (C4). While the amount of these potentially reactive intermediates could be reduced
upon the introduction of substituents onto the thiazole, the steep SAR limited what was tolerated
(
e.g., CH , F, Cl, CN) and in none of these cases could the formation of the thiourea and the
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GSHꢀadduct be completely obviated. With the goal of identifying a clinically useful Na 1.7
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inhibitor, efforts focused on addressing these liabilities while improving potency and achieving a
preclinical pharmacokinetic profile that would portend favorable human pharmacokinetics.
To address the issue of bioactivation, we considered the utility of a compound previously
reported by Amgen wherein the 2ꢀaminothiazole had been replaced with a 4ꢀaminopyrimidine
(
2). While this ring mitigated the potential for the formation of reactive metabolites, and
maintained a favorable pharmacokinetic profile, the 4ꢀaminopyrimidine brought with it a loss in
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potency, a modest level of CYP3A4 inhibition (IC50 ~ 7 ꢀM) and significant induction of
CYP3A4.
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Table 1. Representative Amgen Na 1.7 Inhibitors
V
Compound
1
0.16
2
0.73
3
0.15
Na 1.7 IC (ꢀM)
V
50
Na 1.5 IC (ꢀM)
>30
>30
>30
V
50
a
Rat iv CL [CLu] (L/hr/kg)
0.54 [27]
0.013 [12]
128
0.26 [11]
0.055 [1.6]
177
1.7 [390]
0.049 [9.1]
12
b
Dog iv CL [CLu] (L/hr/kg)
c
CYP3A4 induction (%)
CYP3A4 inhibition IC (ꢀM)
>50
7.2
1.3
50
cLogD
2.1
1.7
2.1
a
b
0.5 mg/kg in DMSO to male rats. CLu = unbound clearance = total clearance/fup. 0.25 mg/kg
in DMSO to male dogs. CLu = unbound clearance = total clearance/f where f is the unbound
up
up
c
fraction in plasma. Expressed as a percentage of the induction seen with rifampicin when both
compounds are incubated with human hepatocytes at a concentration of 10 ꢁM.
Utilizing 2 as a starting point, our efforts were focused on improving potency, mitigating
CYP3A4 inhibition and reducing PXR activation and/or CYP3A4 induction. One strategy
1
4
frequently used to help mitigate PXR activation is the incorporation of polarity. Our previous
efforts had demonstrated that while potency could be retained upon introduction of polarity
within the core of the heterocyclic sulfonamide the increased polar surface area (PSA) brought
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with it a significant increase in the rate of in vivo clearance.
This latter trend was attributed to
the observation that this series was prone to transporterꢀmediated clearance. LogD is one of the
most important molecular properties correlating with biliary clearance, wherein reduced LogD
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often trends with an increase in biliary clearance. Thus, it was anticipated that an approach
wherein polarity would be incorporated to attenuate PXR activation would be challenging due to
the anticipated detrimental impact this would have on the pharmacokinetic profile. An
alternative strategy that was also pursued was one focused on the strategic introduction of
substituents to induce unfavorable steric interactions between the inhibitor and the nuclear
receptor. These approaches would be undertaken with the goal of identifying an inhibitor with a
cLogD between 1.5 – 3.0, a range that we believed could give rise to an inhibitor with a
favorable pharmacokinetic profile and also fall within the generally acceptable range for drugꢀ
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like molecules.
Earlier work within this series had revealed a significant amount of latitude when additional
lipophilicity was incorporated at the para position of the aryl Cꢀring. While we were mindful of
the already elevated cLogD of compound 2, we were optimistic that the acidic nature of these
molecules would allow for the incorporation of additional lipophilicity and added molecular
weight without a detrimental effect on physicochemical properties (e.g., solubility) and key in
vitro assays (e.g., microsomal clearance assays). As an initial foray into this area, the paraꢀCF3
moiety of 2 was replaced with a metaꢀfluoro substituted aryl ring to provide 3. This compound
demonstrated a 5ꢀfold increase in Na 1.7 potency, maintained a high level of selectivity over
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Na 1.5 and interestingly, did not lead to CYP3A4 induction. Compound 3 demonstrated low
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turnover in human, rat and dog liver microsomes (CLint <14 ꢀL/min/mg), excellent passive
permeability (27 ꢀcm/sec) and high aqueous solubility (344 ꢀM in PBS). Furthermore 3
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exhibited low to moderate iv clearance (CL), both total and unbound (CLu), in rat and dog. The
disparity in the rates of clearance across species (~40x) was partially attributed to the
involvement of hepatobiliary transporters. A series of hepatocyte experiments were undertaken
where it was found that compound 3 was rapidly removed from the medium when incubated
with attached rat hepatocytes. This process could then be inhibited by the organic anionꢀ
transporting polypeptide 1B1 (OATP1B1) and multidrug resistanceꢀassociated protein 2 (MRP2)
inhibitor MKꢀ571 ((E)ꢀ3ꢀ(((3ꢀ(2ꢀ(7ꢀchloroquinolinꢀ2ꢀyl)vinyl)phenyl)((3ꢀ(dimethylamino)ꢀ3ꢀ
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oxopropyl)thio)methyl)thio)propanoic acid) demonstrating that this compound was a substrate
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for uptake transporters.
It has been reported that for biliaryꢀexcreted drugs there can be a
significant species differences in clearance. For these reasons, we chose to drive this program on
both rat and dog iv CL given the uncertainties associated with the translation of preclinical
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pharmacokinetics to humans for drugs cleared via a biliary mechanism.
A brief exploration of SAR around the C and D rings found that the ideal substitution pattern
was one wherein both were substituted at the meta position and the Cꢀring was further
substituted with a methoxy group at the carbon ortho to the Bꢀring core (vide infra). Early in
these explorations it was found that a ~3ꢀfold boost in potency could be obtained by
incorporation of chlorine on the Cꢀring (4; Table 2). This modification did not lead to an
increase in turnover in liver microsomes, hence this functionality was maintained in subsequent
SAR efforts.
Table 2 illustrates efforts undertaken to identify a suitable alternative to the 4ꢀaminopyrimidine
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that would retain the potency, but lack the CYP3A4 inhibition, associated with this ring.
A
number of heteroaromatic Aꢀrings were evaluated and generally it was found that 5ꢀmembered
heterocycles were more potent than their 6ꢀmembered congeners (7 – 10 vs. 4 – 6). All of the
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compounds, with the exception of 2ꢀaminoxazole 9, exhibited low turnover in both human and
rat liver microsomes. Additionally, apart from 7, which was a potent inhibitor of CYP3A4 (0.80
ꢀ
M) and was only modestly selective over Na 1.5 (1.5 ꢀM), all of the compounds in Table 2
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were devoid of CYP3A4 inhibition (>27 ꢀM) and demonstrated high levels of selectivity over
Na 1.5 (IC s > 10 ꢀM). The Na 1.7 potencies are noteworthy as we had previously observed
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that compounds lacking an aryl Dꢀring were not tolerant of a wide range of Aꢀrings and exhibited
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steep SAR in this region of the molecule. Specifically, without a Dꢀring, compounds containing
a 1,2,4ꢀaminothiadiazole were considerably more potent than their 2ꢀaminothiazole and 4ꢀ
aminopyrimidine counterparts which were in turn significantly more potent than a variety of
other Aꢀrings evaluated. However, the 1,2,4ꢀaminothiadiazole generally conferred poor passive
permeability (<5 ꢀcm/sec) and high in vivo CL, hence the decision to initially pursue compounds
containing a 2ꢀaminothiazole. Despite the aforementioned bioactivation risk associated with this
moiety, it was somewhat unique within compounds without a Dꢀring in that it could provide
compounds with an overall good balance of potency and favorable pharmacokinetics.
Interestingly, within the context of aryl Dꢀringꢀcontaining compounds, the previously observed
steep SAR associated with the Aꢀring was no longer apparent. A wide range of heterocycles
were tolerated in this region and the 1,2,4ꢀaminothiadiazole (7) did not stand out with respect to
Na 1.7 potency. In light of their potent inhibition of Na 1.7 and lack of CYP3A4 inhibition,
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compounds 8 and 10 were further profiled to evaluate their in vivo pharmacokinetics.
Upon measurement of their rates of iv clearance in both rat and dog, it was found that 8 stood
out in this regard, exhibiting a significantly better pharmacokinetic profile in both species. The
elevated clearance associated with 10 was attributed to the slightly reduced cLogD (1.9) and
hence a likely increased susceptibility to transporterꢀmediated clearance. While 8 was found to
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activate PXR and lead to CYP3A4 induction (58% POC @ 10 ꢀM), it was unique in that it
merged potent inhibition of Na 1.7 (<50 nM) and favorable pharmacokinetics, a previously
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elusive profile within this series of inhibitors. It should be noted that the need to identify very
low clearance molecules was particularly important within this class of compounds. It is well
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known that most acidic molecules demonstrate volume of distribution (V ) at or below
dss
extracellular fluid volume, hence the identification of compounds that will demonstrate halfꢀlives
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1
suitable for onceꢀdaily dosing requires very low clearance values. We hypothesized that the
addition of polarity and/or modifications elsewhere in the molecule could address the PXR
activation observed with this compound. Ultimately the potency and pharmacokinetics of 8
outweighed the induction and we chose to conduct subsequent SAR efforts utilizing the 3ꢀ
aminoisoxazole Aꢀring.
Table 2. Selected AꢀRing SAR
b
c
HLM / RLM
Rat iv CL
[CLu]
Dog iv CL
[CLu]
(L/hr/kg)
Na 1.7
PXR
activation
V
Cpd
4
A
CLint
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cLogD
2.6
IC (µM)
50
(
ꢀL/min/mg)
(L/hr/kg)
0.065
0.12
<14 / <14
24
31
ꢀꢀ
ꢀꢀ
ꢀꢀ
5
<14 / 32
2.6
ꢀꢀ
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
6
7
0.14
<14 / 29
52
3.2
1.9
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
0.023
<14 / <14
5.9
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
8
9
0.049
0.22
<14 / 18
46 / 98
68
38
10
2.6
2.7
1.9
0.047 [42]
ꢀꢀ
0.003 [1.6]
ꢀꢀ
1
0
0.047
<14 / 20
1.6 [940]
0.17 [154]
a
Expressed as a percentage of the induction seen with rifampicin when both compounds are used
b
at a concentration of 10 ꢁM. 0.5 mg/kg in DMSO to male rats. CLu = unbound clearance = total
c
clearance/f where f is the unbound fraction in plasma. 0.25 mg/kg in DMSO to male dogs.
CLu = unbound clearance = total clearance/fup.
up
up
Attempting to alleviate PXR via a judicious reduction in LogD, we turned our attention to the
incorporation of polarity into the central core of this class of compounds and prepared a number
of different [6,6]ꢀheterocyclic sulfonamides, representatives of which are illustrated in Table 3.
As previously observed, it was found that an increase in PSA was generally well tolerated, with
compounds showing potency values comparable to 8. This was true for compounds that spanned
a range of cLogDs (1.3 – 2.8). Unfortunately, in most cases the addition of polarity did not help
mitigate PXR activation (11 – 14). Compounds 15 and 16 both demonstrated reduced levels of
PXR activation and were evaluated in vivo to understand the implications that increased polarity
would have on the pharmacokinetics. In both cases, the added polarity manifested in a
significant increase in both the total and unbound clearance, further highlighting the challenges
associated with designing a potent compound without PXR activation from within this series of
inhibitors without adversely impacting pharmacokinetics. Interestingly, while compounds 11 –
1
3 and 15 demonstrated high levels of selectivity over Na 1.5 (>10 ꢀM), 14 and 16 were
V
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Page 12 of 78
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2
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
somewhat unique in that they both showed modestꢀtoꢀpotent inhibition of this isoform (0.14 and
0.82 ꢀM, respectively).
Table 3. Selected modifications to the central Bꢀring core
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
b
c
HLM/RLM
CLint
(ꢀL/min/mg)
Rat iv CL
[CLu]
Dog iv CL
[CLu]
(L/hr/kg)
Na 1.7
PXR
activation
v
Cpd
X1
a
cLogD
2.6
IC (µM)
50
(L/hr/kg)
8
0.051
0.037
0.12
<14 / 18
21 / 58
16 / 25
68
59
54
0.047 [42] 0.003 [1.9]
1
1
1
2
1.9
1.1
ꢀꢀ
0.12 [25]
0.11 [31]
N
N
2.1 [620]
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Page 13 of 78
Journal of Medicinal Chemistry
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4
5
6
7
8
9
1
3
0.039
<14 / 21
69
2.8
ꢀꢀ
ꢀꢀ
N
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
O
1
1
4
5
0.028
<14 / <14
95
1.7
ꢀꢀ
ꢀꢀ
ꢀꢀ
0.085
0.041
15 / 28
28
27
1.8
1.3
0.13 [140]
0.82 [510]
2
.1
16
<14 / 18
[
1,500]
a
Expressed as a percentage of the activation response seen with rifampicin when both
compounds are used at a concentration of 10 ꢁM. 0.5 mg/kg in DMSO to male rats. CLu =
unbound clearance = total clearance/f where f is the unbound fraction in plasma. 0.25 mg/kg
in DMSO to male dogs. CLu = unbound clearance = total clearance/fup
b
c
up
up
Focusing on Dꢀring SAR, it was found that substituting the fluorine and chlorine atoms on 8 to
provide 17 did not significantly impact potency or PXR activation, hence subsequent Dꢀring
SAR was explored within the context of a metaꢀfluoro substituted Cꢀring (Table 4). A wide
range of substituents and substitution patterns were tolerated although substitution at the meta
and para positions generally led to more potent inhibitors. All compounds were highly selective
over Na 1.5 (>10 ꢀM) and demonstrated low turnover in rat and human liver microsomes. After
V
a significant amount of SAR, a trend emerged wherein potency could be maintained and PXR
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Journal of Medicinal Chemistry
Page 14 of 78
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2
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activation could reliably be avoided with Dꢀrings that contained relatively bulky substituents at
the meta and/or para positions. This can be seen by comparing compounds 17 – 18 and 22 with
19 – 21 wherein the latter, despite being more lipophilic, do not lead to PXR activation. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
decreased PXR activation observed with compounds like 20 may be the result of a steric clash
between the inhibitor and the nuclear hormone receptor. The strategy was routinely employed as
it allowed for balancing the lipophilicity needed for potency and good pharmacokinetics with
low PXR activation. The incorporation of polarity, while tolerated from a potency perspective,
did not reduce PXR activation (23).
Table 4. Exploration of DꢀRing SAR
HLM/RLM
Na 1.7
IC (µM)
50
PXR
activation
v
Cpd
R1
R2
R3
X
CLint
a
cLogD
(
ꢀL/min/mg)
1
1
7
8
Cl
F
H
H
H
H
C(H)
C(F)
C(H)
C(H)
C(H)
C(H)
N
0.037
0.040
0.060
0.036
0.24
36 / 34
30 / <14
35 / 21
34
47
1
2.7
2.3
3.1
2.9
2.9
2.5
1.2
19
20
CH
3
Cl
H
H
CF
H
H
<14 / <14
<14 / <14
36 / 34
1
3
2
2
2
1
2
3
CF
3
H
0
CH
F
H
H
0.047
0.068
54
151
3
H
OCH
<14 / 21
3
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Page 15 of 78
Journal of Medicinal Chemistry
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2
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4
5
6
7
8
9
a
Expressed as a percentage of the activation response seen with rifampicin when both
compounds are used at a concentration of 10 ꢁM.
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
To further support the trend outlined in Table 4, a number of analogs were prepared to probe the
impact of a bulky meta substituent on PXR activation. Illustrated in Table 5 are additional
representative examples, within the context of differentially substituted Cꢀrings, wherein the
incorporation of a trifluoromethyl group on the Dꢀring obviated the PXR activation associated
with analogs that lacked this bulky substituent (24 vs. 25; 26 vs 27). These examples provide
another illustration of PXR activation being presumably being mitigated by the introduction of
substituents which lead to steric interactions between the inhibitor and the nuclear receptor.
Table 5. Obviating PXR activation with the incorporation of a bulky substituent on the Dꢀring
Na 1.7 IC
PXR
v
50
Cpd
R1
R2
R3
Z
a
cLogD
(
µM)
activation
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Journal of Medicinal Chemistry
Page 16 of 78
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
4
5
6
F
CF
F
H
H
H
H
F
H
F
H
H
0.17
0.061
0.14
38
7
2.1
2.7
1.9
2.4
3
CN
CN
54
5
27
CF
3
H
0.028
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
Expressed as a percentage of the activation response seen with rifampicin when both
compounds are used at a concentration of 10 ꢁM.
In light of the favorable balance of potency and lack of both PXR activation and
2
1
CYP3A4 induction (7% POC @ 10 ꢀM) demonstrated by 20, this compound was further
profiled. The pharmacokinetic profile across rat and dog is illustrated in Table 6. The total and
unbound clearance in both rat and dog were acceptable and consistent with the previously
observed trend of higher clearance in rat vs. dog, resulting in a shorter t in rat vs dog (6.2 vs.
1
/2
2
0 hr). In accord with the high aqueous solubility (432 ꢀM in simulated intestinal fluid, pH 6.8),
the compound demonstrated good oral absorption and acceptable bioavailability in both rat and
dog (%F = 54 – 60).
Table 6. Pharmacokinetic Profiles and Plasma Protein Binding of 20
a
b
iv
po
c
CL [CLu]
L/h/kg)
AUC
(ꢀM*hr)
plasma protein
binding (fup)
species
rat
Vdss (L/kg)
t1/2 (h)
tmax (h)
%F
(
0.23 [37]
0.023 [3.6]
2.1
6.2
20
44.2
101
6.0
2.0
54
63
0.0087
dog
0.56
0.0066
a
rat: 0.5 mg/kg in DMSO; dog: 0.25 mg/kg in DMSO. CLu = unbound clearance = total
clearance/f . rat: 10 mg/kg oral dose as a solution in 1%Tween 80/2% HPMC/97%water/KOH
at pH = 10; dog: 2 mg/kg (dog) oral dose as a solution in 30% HPBCD/70%water/NaOH at pH =
10. CLu = unbound clearance = total clearance/f , where f is the unbound fraction in plasma.
b
up
c
up
up
The selectivity of 20 across the Na isoforms was determined by evaluation using the
V
IonWorks Quattro (IWQ) electrophysiology platform. As illustrated in Table 7, 20 demonstrated
very high levels of selectivity (>400ꢀfold) against the Na isoforms that were evaluated. No
V
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23
inhibition of hERG was observed (hERG PatchXpress IC >10 ꢀM) and while the compound
5
0
was devoid of activity on CYP3A4, 2D6 and 1A2 (IC >15 ꢀM), it did show potent inhibition of
5
0
CYP2C9 (IC = 0.43 ꢀM). Addressing this latter liability will be the subject of a subsequent
5
0
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
manuscript.
Table 7. Selectivity profile of 20 against Na 1.1 – Na 1.6
V
V
Na 1.1
Na 1.3
Na 1.4
Na 1.5
Na 1.6
IC (ꢀM)
50
Na 1.7
IC (ꢀM)
50
V
V
V
V
V
V
IC (ꢀM)
IC (ꢀM)
50
IC (ꢀM)
50
IC (ꢀM)
50
5
0
1
7
6.9
12
12
16
0.017
The stateꢀdependent block of Na 1.7 observed with our previously reported
V
heteroarylsulfonamides was also evident with the inhibitors reported herein and was confirmed
8
with manual electrophysiology measurements. When 20 was evaluated at a holding potential
wherein the channels were in a closed, fullyꢀinactivated state (ꢀ140 mV), the IC was rightꢀ
5
0
shifted ~200ꢀfold [4.0 ꢀM], clearly demonstrating the stateꢀdependent block of Na 1.7.
V
The observation that 20 was potent against mouse, but not rat, Na 1.7 (mouse / rat Na 1.7
V
V
PatchꢀXpress IC : 0.036 / 4.0 ꢀM) necessitated the evaluation of in vivo pharmacology in mice.
5
0
The aforementioned lack of potency on rat Na 1.7 was not particulalry surprising as this has
V
8
previously been observed with compounds from this general class of inhibitors. As a means to
measure Na 1.7 target engagement in vivo, 20 was evaluated in a mouse histamineꢀinduced
V
scratching model. There is a significant amount of data implicating Na 1.7 as an important
V
mediator in the itch pathway, including human genetic evidence that SCN9A gainꢀofꢀfunction
2
2
mutations can lead to paroxysmal itch, as well as the near complete lack of scratching behavior
2
3
in Na 1.7 knockout mice in response to an intradermal histamine challenge. It has also been
V
demonstrated that the transmission of both pruriceptive and nociceptive sensory information into
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Journal of Medicinal Chemistry
Page 18 of 78
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the spinal cord occurs via axons of Cꢀfibers and hence supports the utility of a histamineꢀinduced
scratching model in preclinical and clinical settings. Oral administration of 20 to C57Bl/6 mice
demonstrated a robust reduction in the number of scratching bouts induced by the intradermal
injection of histamine (Figure 1). It should be noted that this dose, and a similar exposure, did
not lead to a significant reduction in activity in a separate openꢀfield activity study in naïve mice.
The high level of target coverage required to elicit a robust response (45ꢀfold over the mouse
Na 1.7 IC ; mouse plasma protein binding (f ): 0.0116) and the steep doseꢀresponse are in line
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
V
50
up
with the pharmacokinetic/pharmacodynamic (PK/PD) relationship that has been generated with a
range of internal compounds that span a number of different chemotypes and will be the subject
8
, 26
of a future publication.
(A)
Dose (mg/kg) [plasma]total (ꢀM) [plasma]unbound (ꢀM)
3
0
21.2 ± 9.55
106 ± 22.3
137 ± 25.7
0.246
1.23
1.59
1
00
00
3
(B)
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Page 19 of 78
Journal of Medicinal Chemistry
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5
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7
8
9
Figure 1. (A) Reduction of scratch bouts in a mouse histamineꢀinduced scratch model with
vehicle, 20, and DPH (diphenhydramine, 30 mg/kg po dosing). *, p < 0.05, ***, p < 0.0001
versus vehicle group (oneꢀway ANOVA followed by Dunnett’s tests). (B) Total and unbound
plasma exposure levels of 20.
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
CHEMISTRY
The synthesis of pyrimidine 3 is outlined in Scheme 1. Benzyl mercaptan was coupled with 6ꢀ
bromoꢀ1ꢀchloroisoquinoline (28) to provide an intermediate that was converted to
pentafluorophenyl (PFP) ester 29 following oxidation of the thioether to the sulfonyl chloride
25
and treatment with pentafluorophenol.
Pyrimidine 30 was obtained following the
deprotonation of 4ꢀaminopyrimidine with lithium bis(trimethylsilyl)amide (LHMDS) and the
addition of this anion into the activated PFP ester. These conditions proved to be applicable for
the incorporation of a wideꢀrange of heterocyclic amines. Coupling of 30 with 4ꢀchloroꢀ2ꢀ
methoxyphenyl)boronic acid proceeded smoothly to deliver an intermediate that could
subsequently be elaborated to 3 using standard Suzuki coupling conditions.
a
Scheme 1. Synthesis of pyrimidine analog 3 .
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Page 20 of 78
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 120
2
3
ºC, 50%; (b) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione, MeCN, AcOH, H O, 0 ºC, ii)
pentafluorophenol, TEA, DCM, 68%; (c) pyrimidinꢀ4ꢀamine, LHMDS, THF, 60%; (d) (4ꢀ
chloroꢀ2ꢀmethoxyphenyl)boronic acid, K CO , Pd(PPh ) , dioxane, H O, ꢀW, 100 °C, 71%; (e)
3ꢀfluorophenyl)boronic acid, dicyclohexyl(2',6'ꢀdimethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀyl)phosphine,
chloro(2ꢀdicyclohexylphosphinoꢀ2',6'ꢀdimethoxyꢀ1,1'ꢀbiphenyl)[2ꢀ(2ꢀaminoethylphenyl)]Pd(II)
DCM, K PO , dioxane, H O, ꢀW, 120 °C, 52%.
2
2
3
3 4
2
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
2
Scheme 2 illustrates the routes that were used to access isoquinoline analogs 4 – 10 wherein
modifications were made to the Aꢀring region of the molecule. Synthesis of these compounds
necessitated the preparation of (2ꢀchloroꢀ3'ꢀfluoroꢀ5ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid
(33), which was prepared in an efficient three step sequence starting with 2ꢀbromoꢀ1ꢀchloroꢀ4ꢀ
methoxybenzene (31) and proceeding through iodide 32. The derived boronic acid was coupled
to chloroisoquinoline 29 to provide a PFPꢀester that enabled diversification to compounds 4 – 6
and 8 – 9 using the aforementioned LHMDSꢀmediated conditions. The coupling of 29 and 1,2,4ꢀ
aminothiadiazole to furnish 7 was carried under slightly modified conditions that utilized cesium
carbonate. The synthesis of 10 necessitated the preparation of tertꢀbutyl 1,2,4ꢀoxadiazolꢀ3ꢀ
2
6
ylcarbamate to enable smooth addition of this aminoheterocycle into PFPꢀester 29 which was
subsequently coupled with 33 to provide the fully elaborated compound (10) in acceptable yield.
a
Scheme 2. Synthesis of Aꢀring analogs 4 – 10
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Journal of Medicinal Chemistry
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7
8
9
10
11
12
13
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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
a
Reagents and conditions: (a) 28, K CO , Pd(PPh ) , dioxane, H O, 50 °C, 80%; (b) R ꢀNH ,
2
3
3 4
2
1
2
LHMDS, THF, 0 °C, 34 – 91%; (c) 1,2,4ꢀthiadiazoleꢀ5ꢀamine, Cs CO , CH CN, 64%; (d) tertꢀ
butyl 1,2,4ꢀoxadiazolꢀ3ꢀylcarbamate, Cs CO , DMF, 50%; (e) (3ꢀfluorophenyl)boronic acid,
K CO , Pd(PPh ) , dioxane, H O, 90 °C, 74%; (f) NIS, H SO , AcOH, DCM, 85%; (g) nꢀBuLi,
B(iOPr) , THF, ꢀ78 °C; NaOH, 66%.
2
3
3
2
3
2
3
3 4
2
2
4
3
The synthesis of quinazoline 11 is detailed in Scheme 3. The sequence involves the twoꢀ
step conversion of 7ꢀbromoquinazolinꢀ4ꢀol (34) to PFP ester 35 using conditions similar to those
described above. Installation of protected amine 38, which was derived from 3ꢀaminoisoxazole
(
37), delivered a hydroxyquinazoline that was chlorinated with POCl to furnish an intermediate
3
that was subsequently converted to 36 using standard Suzuki conditions. Coupling of this
bromide with the appropriate boronic acid followed by acidꢀmediated removal of the PMB
protecting group furnished 11 in good overall yield.
a
Scheme 3. Synthesis of quinazoline 11
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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3
4
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5
5
6
0
1
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0
1
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9
0
1
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0
1
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7
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9
0
1
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3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 50 ºC,
2
3
9
7%; (b) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione, MeCN, AcOH, H O, 0 ºC, ii)
2
pentafluorophenol, TEA, DCM, 15%; (c) 38, LHMDS, THF, ꢀ78 ºC, 90%; (d) POCl , DIPEA,
DCE, 70 ºC, 52%; (e) Pd(PPh ) , K CO , 4ꢀbromoꢀ5ꢀchloroꢀ(2ꢀmethoxyphenyl)boronic acid,
dioxane, H O, 60 ºC, 65%; (f) i) Pd(PPh ) , K CO , (3ꢀfluorophenyl)boronic acid, H O, 100 ºC,
ii) TFA, 80 ºC, 67%; (g) 4ꢀmethoxybenzaldehyde, acetic acid, molybdenum dichloride dioxide,
3
3
4
2
3
2
3 4
2
3
2
phenylsilane, CH ΟΗ, water, 74%.
3
The preparation of phthalazinone 12 followed the seven step, sixꢀpot sequence outlined in
Scheme 4. This sequence was initiated with the hydrolysis of 6ꢀbromoꢀ1,4ꢀdichlorophthalazine
(
39) which gave rise to an inseparable mixture of isomeric chlorophthalazinones that were
carried forward through the benzyl thioether installation and oxidation sequence highlighted in
Scheme 1. Separation of the isomers by silica gel chromatography provided PFP ester 40 in 38%
overall yield. Installation of PMBꢀprotected 2ꢀaminoisoxazole furnished 41 in modest yield.
Completion of the synthesis involved coupling of 41 with boronic acid 33, methylation of the
phthalazinone and removal of the PMB protecting group to deliver 12 in good yield.
a
Scheme 4. Synthesis of phthalazinone 12
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33
34
35
36
37
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
60
a
Reagents and conditions: (a) DMSO, H O, 100 ºC, 99%; (b) Xantphos, Pd (dba) , benzyl
2
2
3
mercaptan, DIPEA, dioxane, 50 ºC, 84%; (c) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀ
dione, MeCN, AcOH, H O, 0 ºC, ii) pentafluorophenol, TEA, DCM, 38%; (d) Nꢀ(4ꢀ
methoxybenzyl)isoxazolꢀ3ꢀamine, LHMDS, THF, ꢀ78 ºC, 26%; (e) (2ꢀchloroꢀ3'ꢀfluoroꢀ5ꢀ
methoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid, chloro(2ꢀdicyclohexylphosphinoꢀ2′,6′ꢀdimethoxyꢀ
1
2
,1′ꢀbiphenyl)[2ꢀ(2′ꢀaminoꢀ1,1′ꢀbiphenyl)]palladium(II), K PO , dioxane, H O, ꢀW, 100 ºC,
3
4
2
5
1%; (f) i) CH I, K CO , DMF, ii) TFA, triflic acid, DCM, 74%.
3
2
3
Isoquinolinone 13 was prepared utilizing the Scheme illustrated in Scheme 5 and mirrors that
used for the preparation of 11. The sequence started with 7ꢀbromoꢀ3ꢀmethoxyisoquinoline (42)
and involved installation of the benzyl mercaptan which upon oxidation and trapping with
pentafluorophenol led to concomitant chlorination at the 4ꢀposition to provide PFP ester 43 in
excellent yield. Advancement to elaborated biaryl 44 proceeded uneventfully as previously
described. Exposure of 44 to methyl iodide unveiled the methylated dihydroisoquinolinone core
which following removal of the protecting group provided 13.
a
Scheme 5. Synthesis of isoquinolinone 13
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4
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5
5
5
5
6
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 60 ºC,
2
3
1
00%; (b) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione, MeCN, AcOH, H O, 0 ºC, ii)
2
pentafluorophenol, TEA, DCM, 94%; (c) 38, LHMDS, THF, ꢀ78 ºC, 52%; (d) 2ꢀchloroꢀ3'ꢀ
fluoroꢀ5ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid, Cl Pd(AmPhos) , K PO , dioxane, H O,
1
2
2
3
4
2
00 °C, 49%; (e) NaI, CH I, CH CN, 80 °C, 38%; (f) TFA, 80 ºC, 91%.
3 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
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0
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0
1
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0
1
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4
5
6
7
8
9
0
The preparation of aminophthalazine 14 commences with conversion of 7ꢀbromocinnolinꢀ
(1H)ꢀone (45) to the corresponding benzyl thioether which upon sequential treatment with Nꢀ
4
bromosuccinimide (NBS) and POBr gave dibromide 461 in 58% overall yield (Scheme 6). An
3
efficient three step sequence similar to the one described above afforded sulfonamide 47, which
upon Pdꢀmediated installation of tertꢀbutylsulfinamide and deprotection furnished 14 in modest
overall yield.
a
Scheme 6. Synthesis of aminophthalazine 14
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 110 ºC,
2
3
9
8%; (b) NBS, DMSO, 90 ºC, 73%; (c) POBr , CH3CN, DIPEA, 90 ºC, 82%; (d) (2ꢀchloroꢀ3'ꢀ
3
fluoroꢀ5ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid, Cl Pd(dppf) DCM, K CO , dioxane, H O,
5
pentafluorophenol, TEA, DCM, 88%; (f) isoxazolꢀ3ꢀamine, LHMDS, THF, ꢀ78 ºC, 100%; (g) i)
tertꢀbutylsulfinamide, Pd(OAc) , Xantphos, Cs CO , dioxane, 100 °C; ii) HCl, dioxane, 28%.
2
2
3
2
3%; (e) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione, MeCN, AcOH, H O, 0 ºC, ii)
2
2
2
3
Scheme 7 outlines the protocol used to access hydroxyisoquinoline 15. The sequence was
initiated with the conversion of 48 to the benzyl thioether which upon chlorination with POCl3
and deprotection of the methoxy group with BBr provided 49 in excellent yield. Completion of
3
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the synthesis involved preparation of the sulfonyl chloride, direct addition of aminoisoxazole 38
into this intermediate (50) followed by Suzuki coupling and deprotection.
a
Scheme 7. Synthesis of hydroxyisoquinoline 15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
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42
43
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 110 ºC,
2
3
100%; (b) POCl , DCE, 90 ºC, 99%; (c) BBr , DCM, 0 ºC, 98%; (d) AcOH, sulfuryl chloride,
CH CN, H O, 65%; (e) 33, LHMDS, THF, ꢀ78 ºC, 59%; (f) i) (2ꢀchloroꢀ3'ꢀfluoroꢀ5ꢀmethoxyꢀ
[
2
3
3
3
2
1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid, Pd(PPh ) , K CO , dioxane, H O, ꢀW, 100 ºC, ii) triflic acid,
3
4
2
3
2
1%.
The synthesis of isoquinolinone 16 was initiated with the conversion of 6ꢀbromoꢀ1,3ꢀ
dichloroisoquinoline (51) to protected sulfonamide 52 using a three step sequence similar to
those already described (Scheme 8). Coupling of this intermediate with boronic acid 33 gave
rise exclusively to 53 in excellent yield.
Palladiumꢀmediated conversion of the
chloroisoquinoline to the dihydroisoquinolinone followed by removal of the PMB protecting
group furnished 16 in good to excellent yield.
a
Scheme 8. Synthesis of Isoquinolinone 16
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9
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
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3
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0
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0
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5
6
7
8
9
0
a
Reagents and conditions: (a) Xantphos, Pd (dba) , benzyl mercaptan, DIPEA, dioxane, 60 ºC,
2
3
1
00%; (b) i) 1,3ꢀdichloroꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione, MeCN, AcOH, H O, 0 ºC, ii)
2
pentafluorophenol, TEA, DCM, 94%; (c) 38, LHMDS, THF, ꢀ78 ºC, 100%; (d) Cl Pd(dppf)
DCM, (2ꢀchloroꢀ3'ꢀfluoroꢀ5ꢀmethoxyꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)boronic acid 33, Na CO , dioxane, 50
°
2
2
3
C, 84%; (e) KOH, Pd (dba) , 2ꢀdiꢀtertꢀbutylphosphinoꢀ2',4',6'ꢀtriisopropylbiphenyl, 100 °C,
2 3
100%; (f) TFA, 80 °C, 98%.
Preparation of compounds 17 – 23 followed chemistry similar to that described above
and is outlined in Scheme 9. Conversion of 29 to biaryl sulfonamide 54a-c was followed by
coupling to the appropriate boronic acid to deliver final compounds 17 – 27.
a
Scheme 9. Synthesis Cꢀ and Dꢀring isoquinoline analogs 17 – 27
a
Reagents and conditions: (a) boronic acid, K CO , Pd(PPh ) , dioxane, H O, 50 °C, 72 – 85%,
2
3
3 4
2
(
b) isoxazolꢀ3ꢀamine, LHMDS, THF, ꢀ78 ºC, 69 – 97%; (c) boronic acid, Pd catalyst, ligand,
base, dioxane, water, ꢀW, 120 °C, 11 – 56%.
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CONCLUSIONS
In conclusion, optimization efforts successfully addressed a number of metabolic liabilities
associated with a previously reported series of bicyclic sulfonamide Na 1.7 inhibitors. These
V
10
11
12
13
14
15
16
17
18
19
20
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23
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32
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35
36
37
38
39
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41
42
43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
efforts focused on improving potency, removing a bioactivation risk and mitigating CYP
induction and inhibition liabilities. Critically important to overcoming these liabilities while
retaining favorable pharmacokinetics within a series prone to transporterꢀmediated clearance was
maintaining the cLogD within a tight range and the discovery of subtle PXR SAR that was not
dependent solely on lipophilicity but leveraged a molecular recognition approach. While this
approach required an increase in molecular weight, rigorous attention to key in vitro metabolic
properties enabled the idnetification of inhibitors devoid of the liabilities associated with the
strating point from which this series was derived. Ultimately, this was achieved with the
strategic introduction and substitution of an aromatic ring and led to 20 which demonstrated a
robust response in a Na 1.7 target engagement pharmacodynamic model. Additional efforts to
V
further improve the potency and pharmacokinetics while addressing remaining metabolic
liabilities (i.e., CYP2C9 inhibition) of the bicyclic sulfonamide series will be reported in due
course.
EXPERIMENTAL SECTION
Chemistry. All reagents were purchased from commercial suppliers and used as is unless
otherwise noted. Anhydrous solvents were obtained from Aldrich or EM Science. An inert
atmosphere of nitrogen or argon was used for reactions involving airꢀ or moistureꢀsensitive
reagents. A Biotage Initiator reactor from Biotage AB, Uppsala, Sweden was used for all
1
microwave assisted reactions. H NMR spectra were recorded on a Bruker AVꢀ400 (400 MHz)
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9
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
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5
5
6
spectrometer at ambient temperature or on a Varian 400 MHz spectrometer. Chemical shifts are
reported in parts per million (ppm, δ units) downfield from tetramethylsilane. Data are reported
as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br =
broad, m = multiplet), coupling constants, and number of protons. Purity for final compounds
was greater than 95% and was measured using Agilent 1100 series high performance liquid
chromatography (HPLC) systems with UV detection at 254 nm (system A, Agilent Zorbax
Eclipse XDBꢀC8 4.6 mm × 150 mm, 5 ꢁm, 5−100% CH CN in H O with 0.1% TFA for 15 min.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
at 1.5 mL/min.; system B, Waters Xterra 4.6 mm × 150 mm, 3.5 ꢁm, 5−95% CH CN in H2O
3
with 0.1% TFA for 15 min. at 1.0 mL/min.). Exact mass confirmation was performed on an
Agilent 1100 series high performance liquid chromatography (HPLC) system (Santa Clara, CA,
U.S.) by flow injection analysis, eluting with a binary solvent system A and B (A, water with
0.1% FA; B, ACN with 0.1% FA) under isocratic conditions (50% A/50% B) at 0.2 mL/min.
with MS detection by an Agilent G1969A timeꢀofꢀflight (TOF) mass spectrometer (Santa Clara,
CA, U.S.).
1-(3'-fluoro-3-methoxy-[1,1'-biphenyl]-4-yl)-N-(pyrimidin-4-yl)isoquinoline-6-
sulfonamide (3). Step 1: A 2ꢀneck roundꢀbottom flask was charged with 6ꢀbromoꢀ1ꢀchloroꢀ
isoquinoline 28 (5.43 mL, 37.5 mmol), Xantphos (1.08 g, 1.87 mmol), and Pd (dba) (0.858 g,
2
3
0.937 mmol). The flask was flushed with Ar (g), then dioxane (74.9 mL) and N,Nꢀ
diisopropylethylamine (13.1 mL, 74.9 mmol) were added in sequence. The flask was fitted with
a reflux condenser and placed in a 120 °C heating bath for 10 min., at which point the mixture
was at a gentle reflux. Benzyl thioether (4.65 mL, 39.3 mmol) was added dropwise via syringe
over 3 min. The reaction was maintained at 120 °C for 1 hr at which point the mixture was
cooled to rt. After 2 h, a solid had crystallized out. The mixture was filtered, the filtrate was
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concentrated, and the crude product was purified by chromatography on silica gel (120ꢀg Rediꢀ
Sep Gold column, 0ꢀ30% EtOAc/Heptane) to give 6ꢀ(benzylthio)ꢀ1ꢀchloroisoquinoline (5.34 g,
18.7 mmol, 50% yield) as an offꢀwhite solid that was used directly in the next step.
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
Step 2: A roundꢀbottom flask was charged with 6ꢀ(benzylthio)ꢀ1ꢀchloroisoquinoline (601 mg,
2.10 mmol), acetonitrile (19.8 mL), acetic acid (742 µl), and water (495 µl) to give a thin
suspension. The flask was cooling in an iceꢀbath for 10 min., then 1,3ꢀdichloroꢀ5,5ꢀ
dimethylimidazolidineꢀ2,4ꢀdione (829 mg, 4.21 mmol) was added in one portion. After stirring
for 30 min., 2,3,4,5,6ꢀpentafluorophenol (774 mg, 4.21 mmol) was added followed by
triethylamine (730 µl, 5.26 mmol). The cooling bath was removed and the reaction was stirred
for 1 hour. The mixture was diluted with EtOAc (50 mL), washed with water (2 × 50 mL),
washed with brine, and dried over sodium sulfate. The mixture was concentrated and purified by
chromatography on silica gel (40ꢀg RediꢀSep Gold column, 0ꢀ20% EtOAc/Heptane) to give
perfluorophenyl 1ꢀchloroisoquinolineꢀ6ꢀsulfonate 29 (0.590 g, 1.44 mmol, 68% yield) as white
1
powder. H NMR (400 MHz ,CDCl ) δ = 8.63 (d, J = 8.9 Hz, 1 H), 8.57 (d, J = 1.8 Hz, 1 H),
3
8.52 (d, J = 5.7 Hz, 1 H), 8.18 (dd, J = 1.9, 8.9 Hz, 1 H), 7.85 ꢀ 7.76 (m, 1 H). Mass Spectrum
+
(ESI) m/z 410.0 [M+H] .
Step 3: A roundꢀbottom flask was charged with perfluorophenyl 1ꢀchloroisoquinolineꢀ6ꢀ
sulfonate 29 (1.035 g, 2.53 mmol), pyrimidinꢀ4ꢀamine (0.264 g, 2.78 mmol), and THF (12.6 mL)
to give a cloudy, yellow suspension. The flask was cooled in an iceꢀbath for 10 min., then
lithium bis(trimethylsilyl)amide (1M in THF) (5.30 mL, 5.30 mmol) was added dropwise over 1
min. to give an orange mixture. After 20 min. trifluoroacetic acid (0.584 mL, 7.58 mmol) was
added dropwise, then the mixture was diluted with DCM (ca. 30 mL). The mixture was stirred
for 10 min. while warming to room temperature and was then filtered. The collected solid was
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