ADME-guided design and synthesis of aryloxanyl pyrazolone derivatives to block mutant superoxide dismutase 1 (SOD1) cytotoxicity and protein aggregation: Potential application for the treatment of amyotrophic lateral sclerosis

Article abstract of DOI:10.1021/jm2014277

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. The arylsulfanyl pyrazolone (ASP) scaffold was one of the active scaffolds identified in a cell-based high throughput screening assay targeting mutant Cu/

Full text of DOI:10.1021/jm2014277

Article
pubs.acs.org/jmc
ADME-Guided Design and Synthesis of Aryloxanyl Pyrazolone
Derivatives To Block Mutant Superoxide Dismutase 1 (SOD1)
Cytotoxicity and Protein Aggregation: Potential Application for the
Treatment of Amyotrophic Lateral Sclerosis
Tian Chen,† Radhia Benmohamed,‡ Jinho Kim,§,∥ Karen Smith,⊥ Daniel Amante,§,∥
Richard I. Morimoto,# Donald R. Kirsch,‡ Robert J. Ferrante,§,∥ and Richard B. Silverman*,†,∞
†Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, United States
‡Cambria Pharmaceuticals, Cambridge, Massachusetts, 02142, United States
§Departments of Neurological Surgery, Neurology, and Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213,
United States
∥Geriatric Research Educational and Clinical Center (00-GR-H), V.A. Pittsburgh Healthcare System, 7180 Highland Drive,
Pittsburgh, Pennsylvania 15206, United States
⊥Bedford VA Medical Center, 200 Springs Road, Bedford, Massachusetts 01730, United States
#Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois
60208-3500, United States
∞Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery,
Northwestern University, Evanston, Illinois 60208-3113, United States
S
* Supporting Information
ABSTRACT: Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease
currently without a cure. The arylsulfanyl pyrazolone (ASP) scaffold was one of the active
scaffolds identified in a cell-based high throughput screening assay targeting mutant Cu/
Zn superoxide dismutase 1 (SOD1) induced toxicity and aggregation as a marker for ALS.
The initial ASP hit compounds were potent and had favorable ADME properties but had
poor microsomal and plasma stability. Here, we identify the microsomal metabolite and
describe synthesized analogues of these ASP compounds to address the rapid metabolism.
Both in vitro potency and pharmacological properties of the ASP scaffold have been
dramatically improved via chemical modification to the corresponding sulfone and ether
derivatives. One of the ether analogues (13), with superior potency and in vitro
pharmacokinetic properties, was tested in vivo for its pharmacokinetic profile, brain penetration, and efficacy in an ALS mouse
model. The analogue showed sustained blood and brain levels in vivo and significant activity in the mouse model of ALS, thus
validating the new aryloxanyl pyrazolone scaffold as an important novel therapeutic lead for the treatment of this
neurodegenerative disorder.
have advanced the understanding of the molecular mechanisms
and underlying pathogenesis of this disease.10 Mutations in
SOD1 are believed to be responsible for about 20% of all FALS
cases.11,12 The latter findings resulted in the development of
transgenic mouse models, which have further elucidated
pathophysiological mechanisms as well as provided a model
for preclinical drug trials.13 The clinical and pathological
features of FALS and sporadic ALS are similar, which supports
the strategy of using laboratory models of FALS mutations for
elucidating disease pathogenesis and for identifying potential
therapeutic targets for both forms of the disease.14,15 Protein
aggregation is a pathological hallmark of ALS;13,16 the
INTRODUCTION
■
Amyotrophic lateral sclerosis (ALS), also known as Lou
Gehrig’s disease, is a fatal neurodegenerative disease resulting
in progressive muscle loss and paralysis.1 The incidence of ALS
is 1−2 per 100 000 per year,2,3 with approximately 2−5 years
from diagnosis to death. There is a disproportionate incidence
of ALS among military personnel, particularly among Gulf War
veterans.4−6 No effective treatment for the disease is currently
known.7 Riluzole, the only FDA approved drug for treating
ALS, extends patient lives by 2−3 months.8
The sporadic disease (SALS) accounts for approximately
90% of all cases and familial disease (FALS) the remaining 10%.
Over 100 mutations in Cu/Zn superoxide dismutase 1 (SOD1)
have been identified since the pioneering work of Brown and
colleagues in identifying this FALS gene,9 and studies on SOD1
Received: October 21, 2011
Published: December 22, 2011
© 2011 American Chemical Society
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appearance of intracellular inclusions containing detergent-
insoluble SOD1 protein aggregates represents a common early
event associated with SOD1 cellular toxicity.17,1
In previous studies, PC12 cells expressing mutant G93A
SOD1 were utilized in a cell-based high throughput screening
(HTS) assay.18,19 The arylsulfanyl pyrazolone (ASP) scaffold
was identified as one of the active scaffolds showing protection
against cytotoxicity from protein aggregation from a 50 000-
compound library screened in the HTS assay (Figure 1; two
was obtained from 2-bromoacetyl bromide and N,O-dimethyl-
hydroxylamine hydrochloride.26 The corresponding β-hydrox-
yester intermediates were prepared by condensation of 42−47
with the enolate of ethyl acetate. The aryloxanyl pyrazolones
(13−19) were obtained by treatment of the β-hydroxyester
intermediates with hydrazine. Intermediates 39 and 41 were
obtained via Suzuki coupling of aryl bromides 38 and 40,
respectively, with phenylboronic acid using a palladium
catalyst.27
To avoid hydrolysis of Weinreb amide 36 under concen-
trated and strong basic conditions,28,29 3,5-dichlorophenol (34)
was allowed to react with ethyl 2-bromoacetate and then
treated with NaOH/H2O to afford 35. This intermediate was
transformed to 42 with oxalyl chloride and N,O-dimethylhy-
droxylamine hydrochloride, and the Weinreb amide was
converted to 13 as described above.
Microsomal Stability. ASP analogues, like 5-((2,4-
dichloro-5-methylphenylthio)methyl)-1H-pyrazol-3(2H)-one
(48, Figure 2) and 5-((4-chloro-2,5-dimethylphenylthio)-
methyl)-1H-pyrazol-3(2H)-one (49), were found to have low
mouse liver microsomal stability. One possible site of
metabolism by cytochrome P450 (CYP) isozymes30 is the
aromatic methyl group. Therefore, an analogue lacking an
aromatic methyl group (1) was tested for microsomal stability
in the presence of NADPH, and the results were compared to
minaprine (50), an antidepressant drug known to undergo
rapid microsomal metabolism, as a positive control.31 After
incubation for 20 min with rat liver microsomes, minaprine and
1 were metabolized by 41% and 88%, respectively (Figure 3).
Therefore, the aromatic methyl group is not responsible for the
metabolic instability of 1. On the basis of the above result, the
most likely site of CYP metabolism is the sulfide sulfur atom,
which is known to undergo oxidative metabolism.32
Metabolite Profiling of 1. Metabolic profiling was carried
out on 1 despite its relatively low in vitro potency and because
it contained a chlorine atom, which has a natural isotope
abundance (35Cl/37Cl = 3:1) that can simplify the mass spectral
analysis. The two likely metabolites are the corresponding
sulfoxide (4) and sulfone (6), which were synthesized as shown
in Scheme 1.
Figure 1. ASP HTS hits: R1, alkyl or substituted aromatic groups; R2,
proton or substituted aromatic groups; R, various alkyl and/or halogen
groups.
general structures were active, where type II was, in general,
more potent than type I).20 Structural optimization of this
scaffold led via 1 (EC50 = 1.93 μM) and 2 (EC50 = 0.71 μM) to
a more potent analogue (3) than the original screening hits,
with an EC50 of 170 nM. In vitro pharmacokinetic (PK) and in
vivo brain/plasma ratio studies were also performed at an early
stage of this drug development project. Other than having low
microsomal stability and plasma stability, the ASP scaffold
generally showed good PK properties and permeated the
blood−brain barrier.21 Identifying and modifying the metabolic
liability in the ASP scaffold, therefore, became a priority. The
results of the metabolism studies, SAR investigations of the new
scaffold, PK and toxicity properties, the ability to cross the
blood−brain barrier, and effects on an ALS mouse model are
described herein.
Compound 1 (15 μM) was incubated with rat liver
microsomes at 37 °C for 0, 5, 10, 20, and 60 min in the
presence of NADPH. HPLC analysis showed a new peak (5.90
min) that was not present prior to the addition of microsomes
and that was identified as the corresponding sulfoxide by
comparison with those of synthetic standards of sulfoxide 4
(5.95 min) and sulfone 6 (10.8 min) (Figure 4).
Because of the limit of HPLC detection, the metabolite
profiling of 1 was also carried out by LC/MS/MS. To assist in
this analysis, an analogue of 1 with 15N substituted for both
pyrazole nitrogens was synthesized by the route in Scheme 1
using 15NH2−15NH2 in place of hydrazine. Both 1 and [15N2]1
(5 μM) were incubated with rat liver microsomes in the
presence and absence of NADPH. After 0, 10, 20, 40, and 60
min, the samples were processed and analyzed by LC/MS/MS.
Both compounds, in the presence of NADPH but not in its
absence, produced one new peak not detected in the controls
without microsomes or compound. The mass spectrum of the
new peak corresponded to the sulfoxide (4). No sulfone was
detected. The rate of formation of sulfoxide corresponded well
with the loss of the sulfide (Figure 5).
RESULTS AND DISCUSSION
■
Chemistry. ASP analogues were synthesized as described
earlier.21 The syntheses of type II ASP analogues with sulfoxide
and sulfone linkers in place of the sulfide are shown in Scheme
1. The sulfide linker β-ketoester intermediates (20−22) were
treated with VO(acac)2-TBHP22,23 to give the sulfoxide and
24
sulfone intermediates (23−26) or with H2O2 to give sulfone
27, which were treated with hydrazine to give the
corresponding sulfoxide (4, 5) and sulfone (6−8) products.
The corresponding type II aryloxanyl pyrazolone derivatives
(9−14) were prepared from phenol and ethyl 4-chloroacetoa-
cetate to generate the β-hydroxyester intermediates (28−33),25
followed by treatment with hydrazine (Scheme 2). Although
this two-step synthetic method is simple and direct, it is not
efficient mainly because of the low stability of the enolate
derivatives from both the ethyl 4-chloroacetoacetate and the β-
hydroxyester intermediates. Improved strategies to prepare the
β-hydroxyester intermediates are described in Scheme 3.
Compounds 42−47 were synthesized from the corresponding
phenols and 2-bromo-N-methoxy-N-methylacetamide, which
Metabolism Guided Design and Synthesis of ASP
Analogues. Because of the rapid metabolism of 1, the sulfide
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a
Scheme 1
a
Reagents and conditions: (a) TBHP, VO(acac)2, DCM, room temperature, overnight; (b) H2O2, AcOH, EtOAc, room temperature; (c) NH2NH2,
EtOH, room temperature, overnight.
a
Scheme 2
In Vitro ADME Assays. Microsomal Stability. Human
and mouse microsomal stabilities of sulfone 7 and ether
analogues 9 and 13 were tested at 5 μM at 37 °C for 1 h in the
presence and absence of NADPH (Table 2). All three
compounds showed good stability.
Aqueous Solubility. The aqueous solubility of 7, 9, and 13
was evaluated by diluting them from a stock solution in DMSO
to a final concentration of 1% DMSO in PBS (Table 3). The
maximum solubility was considered to be the highest
concentration that showed no precipitation. All three
compounds showed good aqueous solubility.
a
Reagents and conditions: (a) ethyl 4-chloroacetoacetate, NaH, THF,
Caco-2 Permeability. Compounds 7, 9, and 13 all had
high33 permeability in the Caco-2 permeability assay (Table 4).
Protection of Primary Cortical Neurons. It is not
uncommon to observe that compounds that are active in tissue
culture cells will not be active in differentiated neurons.
Therefore, sulfide 2, sulfone 7, and ether linker analogue 13
were tested in a primary cortical neuron cell protection assay
that showed that all of these compounds were active. This issue
was of particular concern because compounds from another
scaffold, cyclohexane 1,3-diones, identified in the original high-
throughput screen had poor activity in this assay, potentially
due to poor cell permeability.34
PK Profiling of 13. Sulfone 7 and ether linker analogues 9
and 13 all showed good or adequate in vitro ADME properties.
Compound 13 was selected based on its potency and in vitro
pharmacological profile for in vivo PK profiling and efficacy in a
mouse model of ALS.
Mouse in Vivo Steady-State Level and Blood−Brain
Barrier Penetration for 13. The in vivo steady-state level of
ether analogue 13 from plasma was determined in mice. The
animals were administered 300 mg/kg 13 and sacrificed at
progressive time points (0, 3, 6, 12, and 24 h, Table 5). Blood
and brain samples were harvested and analyzed by mass
spectrometry using ESI ionization in the MRM mode.
In Vivo Rat PK Profiling of 13. Compound 13 was
administered to Sprague−Dawley rats both intravenously and
DMF, −20 to 70 °C; (b) NH2NH2, EtOH, room temperature,
overnight.
was replaced by sulfoxide, sulfone, and ether functional groups,
synthesized as shown in Schemes 1−3. These compounds were
screened in the toxicity protection assay, and compounds that
retained activity in this assay were tested for in vitro
microsomal stability.
Mutant SOD1-Induced Cytotoxicity Protection Assay
of Modified Compounds. All of the ASP analogues exhibited
100% viability in the cytotoxicity protection assay except for 4
(maximum viability of ∼35%). The EC50 values of these
analogues in the protection assay are summarized in Table 1. In
general, the potencies of the analogues were greater with the
ether linkage when compared with the sulfide, sulfone, and
sulfoxide. The low sulfoxide potency indicated that metabolism
of the ASPs to the corresponding sulfoxide results in
deactivation of the compounds and that pursuing a prodrug
strategy with the thioether linked compounds would not be
possible. Therefore, a series of ether analogues, aryloxanyl
pyrazolones, was synthesized (Table 1). Ether 13 (CMB-
087229) was the most potent (67 nM) of these compounds.
Analogues of 13, having F, CF3, Br, and Ph in place of the Cl
atoms, indicated that size and electronics were important
features at the meta-positions; the potency decreased in the
following order: Cl > CF3 > F > Br > Ph.
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a
Scheme 3
a
Reagents and conditions: (a) ethyl 2-bromoacetate, NaOEt, EtOH, reflux, overnight; (b) NaOH, H2O, 80 °C, overnight; (c) oxalyl chloride, DCM,
DMF, room temperature, 4 h; (d) N,O-dimethylhydroxylamine hydrochloride, DIEA, DCM, room temperature, 1 h; (e) N,O-dimethylhydroxyl-
amine hydrochloride, K2CO3, Et2O, H2O, room temperature, 30 min; (f) phenylboronic acid, PdCl2(PPh3)2, K2CO3, dioxane, H2O, 100 °C, 16 h;
(g) NaOEt, EtOH, 70 °C, overnight; (h) EtOAc, LiHMDS, THF, −78 °C, overnight; (i) NH2NH2, EtOH, room temperature, overnight.
Figure 2. Compounds tested in metabolic studies.
expressed in human embryonic kidney cells (HEK293) was
evaluated using a patch-clamp technique, the most definitive in
vitro hERG inhibition assay.35 A positive control (E-4031) was
used to confirm the sensitivity of this test system, and the
TurboSol evaluation system was performed on 13 to confirm
that its insolubility was not a reason for low hERG inhibition
observed in this test system (see Supporting Information for
results). The mean percent hERG antagonism (two experi-
ments) was 0.6 1.5%. These results indicate that 13 does not
affect the hERG channel.
Effect of 13 on Cytochrome P450 Isozymes. Compound 13
was tested for its inhibition of CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4, present in human liver microsomes.
IC50 values of 13 to CYP isozymes inhibition were evaluated
using single drug substrates except for CYP3A4 (testosterone
and midazolam tested). IC50 values were >50 μM except for
CYP2C19, which was 20 μM.
Effect of 13 on Enzymes and Receptors. Compound 13 was
screened at 10 μM in duplicate by MDS Pharma Services
(Taipei, Taiwan) with LeadProfilingScreen, a suite of 68 in
vitro enzyme and receptor assays of the most commonly
Figure 3. Microsomal stability of minaprine (50) and 1. Rat liver
microsomes were incubated at 37 °C for 20 min in the presence of 15
μM compound.
orally at 1 mg/kg in a single bolus dose. The results are
summarized in Table 6.
Effect of 13 on the Potassium Channel. The in vitro effect
of 13 (10 μM) on the hERG potassium channel current
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ALS mice in a dose dependent manner in comparison to
untreated G93A mice (Figure 6). The most efficacious dose (20
mg/kg) resulted in a life extension of 13.3%.
DISCUSSION
■
We had previously found that ASP compounds are
metabolically unstable.29 In vitro metabolic studies were carried
out to determine the basis of microsomal instability. The early
analogues had aromatic methyl groups (48 and 49) and a
sulfide sulfur atom. Metabolic degradation of an ASP without
an aromatic methyl (1) was more rapid than that of minaprine
(50), a CNS drug known to undergo rapid metabolism,
indicating that the instability was not caused by the aromatic
methyl (Figure 3). Following microsome incubation, a new
metabolite of 1 was identified by HPLC (Figure 4), which
comigrated with the corresponding sulfoxide (4); no sulfone
(6) was detected. The formation of sulfoxide 4 quantitatively
followed the degradation of 1 (Figure 5). The structure of the
NADPH-dependent metabolite was further confirmed by LC/
MS/MS; the NADPH dependence suggests that metabolism is
catalyzed by a cytochrome P450.
Identification of the metabolite of 1 as the corresponding
sulfoxide, which had poor activity in the cytotoxicity protection
assay, guided our redesign of the arylsulfanyl pyrazolones to the
corresponding sulfones and ethers (aryloxanyl pyrazolones). In
general, the potency of compounds with the various linker
groups increases (EC50 decreases) in the order ether > sulfide >
sulfone ≫ sulfoxide.
Figure 4. HPLC traces of the incubation of 1 (15 μM) with rat liver
microsomes at 37 °C.
A series of ether analogues was synthesized (Table 1), and all
of the compounds were assayed in the cytotoxicity protection
assay, which uses PC12 cells that express mutant G93A SOD1
as a YFP fusion protein.20 In general, the most potent
analogues were the 3,5-disubstituted phenyl analogues, where
dichloro > dibromo > ditrifluoromethyl > difluoro > diphenyl.
The dichloro analogue (13) has an EC50 of 67 nM.
Sulfone 7, ether 9, and ether 13 were much more
metabolically stable than arylsulfanyl pyrazolone 1,29 with the
ether being considerably more stable than the sulfone (Table
2). Because of the stability of the ethers, further ADME testing
was carried out with the most potent analogue (13).
observed adverse CNS, cardiovascular, pulmonary, and
genotoxic activities. No significant activity was detected in
any of the 68 assays. See Supporting Information for results.
Effect of 13 Administration on SOD1 G93A ALS Mice.
Control and transgenic mice of the same age ( 3 days) and
from the same “f” generation were selected from multiple litters
to form experimental cohorts. The tolerable dose range for 13
was determined in wild type mice by increasing the dose b.i.d.
one-fold each ip injection; the maximum tolerated dose was 75
mg/kg. On the basis of the studies performed to determine
tolerance and blood brain levels, the dose levels of 1.0, 10, and
20 mg/kg once a day were administered, starting at 6 weeks of
age, throughout the lives of the G93A mice. Administration of
13 resulted in a significant extension in survival in the G93A
The aqueous solubility of ether 13 was good (250 μM)
compared with that of 48 (56 μM21), although the solubility of
sulfone 7 was greater than twice that of 13. Permeability
through Caco-2 monolayer cells correlates with in vivo
Figure 5. Rate of formation of sulfoxide 4 and rate of metabolism of 1.
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a
Table 1. SAR of ASP Analogues with -S-, -SO-, -SO2-, and -O- Linkers
X
compd
R
EC50 (μM)
S
1
4-Cl
1.93
0.71
0.17
>32
2.88
4.7
2
2.6-di-Cl
3,5-di-Cl
4-Cl
3
SO
4
5
4-Cl-2,5-di-Me
4-Cl
SO2
6
7
3,5-di-Cl
4-Cl-2,5-di-Me
4-Cl
1.41
1.47
0.79
1.96
0.72
0.87
0.067
0.58
1.07
0.51
1.02
2.84
3.70
8
O
9
10
11
12
13
14
15
16
17
18
19
4-Et
3-Et
3-t-Bu
3,5-di-Cl
3,5-di-CF3
3,5-di-F
3,5-di-Br
3-Br
3-Ph
3,5-di-Ph
a
Average Z′ factor is 0.5.
c
Table 2. In Vitro Microsomal Stability of 7, 9, and 13
human
mouse
b
b
T1/2
T1/2
a
compd CL′int (mL min−1 kg−1)
a
−1
(min)
CL′int NADPH-free (mL min−1 kg−1) CL′int (mL min−1 kg−1)
(min)
CL′int NADPH-free (mL min−1 kg )
7
4.5
15.3
25
>60
139
93
1.3
0
15.3
24.5
64
>60
143
36
0
9
6.1
21
13
13
a
b
c
Microsomal intrinsic clearance. Half-life. Experimental procedures and data analysis are described in ref 29. Data were obtained from Apredica
and Biogen Idec.
a
Table 3. In Vitro Aqueous Solubility of Compounds 7, 9, and
13
Table 5. In Vivo Plasma Concentration of Compound 13
a
time (h)
plasma concn (μg/mL [μM])
maximum solubility (μM)
0
0
3
88.8 [342]
90 [347]
64.4 [248]
1.7 [6.5]
compd
45 min
16 h
6
7
≥500
250
≥500
250
12
24
9
13
250
250
a
a
Brain and plasma samples were analyzed at Apredica, Inc.
Data were obtained at Apredica Inc. Experimental procedures and
data analysis are described in ref 21.
13 suggest that these compounds have high membrane
permeability36 and are likely poor substrates for efflux transport
proteins, such as P-glycoprotein.37
To determine if these compounds are active in other types of
cells, including neurons, a screen of selected compounds was
carried out with four other cell types: SHSY-5Y, HeLa,
HEK293, and primary cortical neuronal cells. Ether 13 was
active in all four cell types, which is a positive indication of
utility for the treatment of ALS, a non cell autonomous
disease.38
a
Table 4. In Vitro Caco-2 Permeability of 7, 9, and 13
b
b
Papp(A→B)
Papp(B→A)
compd
(10−6 cm/s)
(10−6 cm/s)
ratio (B→A)/(A→B)
7
27.0
43.0
36.7
4.4
0.2
0.6
0.4
9
28.0
14.1
13
a
Data were obtained from Apredica Inc. Experimental procedures and
b
data analysis are described in ref 21. Apparent permeability.
Compound 13 was evaluated for its metabolic stability and
blood−brain barrier penetration in mice. Mouse plasma
stability was good, peaking at 3−6 h; after 4 h 13 was detected
at 194 μM in brain tissue. The loss of 13 does not follow first-
order kinetics following intraperitoneal administration. Phar-
intestinal permeability. The efflux of compounds in the
opposite direction also can be estimated by this method. The
high permeability values (Papp = 27−43 × 10−6 cm/s) and low
efflux ratios (Papp(B→A)/Papp(A→B) of 0.2−0.6) of 7, 9, and
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a
Table 6. Rat Plasma PK Profile of Compound 13
AUC last
(ng h/mL)
AUC/dose
T1/2
(h)
CL
Vss
(L/kg)
Cmax
(ng/mL)
Tmax
(h)
F
(%)
(ng h mL−1 dose−1)
(mL min−1 kg−1)
iv
179
42
184
50
2.1
3.6
92
5.7
po
39
0.25
27
a
Single bolus administration of 1 mg/kg iv and po to SD rats. Data were obtained from Biogen Idec.
Figure 6. Kaplan−Meier plot of 13-treated SOD1 G93A ALS mice: untreated group, 125.7 4.9 days; group 1 (1 mg/kg), 129.2 5.3 days; group
2 (10 mg/kg), 135 5.5 days; group 3 (20 mg/kg), 142.5 8.2 days; p < 0.05.
macokinetics could be influenced by the high plasma protein
binding by 13 (98% in rat and human), which can limit
compound bioavailability in the plasma compartment, thereby
prolonging the half-life.39,40 However, high plasma protein
binding alone does not determine the effect on the
pharmacokinetic properties; the binding affinity (on/off rate)
also is a major factor. Pharmacokinetics in Sprague−Dawley
rats was investigated both by iv and po administration. The
compound appears to be reasonably stable and is orally active;
half-lives were 2.1 and 3.6 h, respectively, for these two routes
of administration with oral bioavailability (F) of 27%, which is
above the typical cutoff for continued development of drug
candidates41 (Table 6). The difference in plasma stability from
the mouse and rat studies could be the result of a combination
of factors, including different species (mouse vs rat), different
amount of compound used (5 mg/kg vs 1 mg/kg), and route of
administration (ip vs iv).
Toxicity is an important cause for attrition of drug candidates
at later stages of drug development. To reduce the time and
effort of drug discovery, various toxicological criteria need to be
met early in the development of drug candidates. One is the
effect of the compound on the human ether-a-go-go-related
gene (hERG), a gene that encodes a cardiac potassium ion
channel to regulate cardiac repolarization, which determines
cardiac rhythmicity. When a compound inhibits the hERG ion
channel, it can produce a disorder called long QT syndrome,
resulting in torsade de pointes, heart arrhythmias and
potentially sudden death. Therefore, it is essential that drug
candidates do not affect hERG. Compound 13 did not
antagonize hERG at 10 μM.
Cytochrome P450 isozymes are also associated with drug
toxicity.42 Molecules that either inhibit or up-regulate these
enzymes can cause drug−drug interactions. If they inhibit the
isozymes, metabolism of other drugs is blocked. If they up-
regulate the isozymes, it leads to rapid metabolism of drugs.
The IC50 of compound 13 was >50 μM for human liver
CYP1A2, CYP2C19, CYP2D6, and CYP3A4 and was 20 μM
for CYP2C9, indicating that 13 should not cause drug−drug
interactions.
To determine if there are other receptors or enzymes that
might be potential off-targets, 13 was screened by MDS
Pharma Services (Taipei, Taiwan) using its LeadProfi-
lingScreen, a suite of 68 in vitro enzyme and receptor assays
of the most commonly observed adverse CNS, cardiovascular,
pulmonary, and genotoxic activities. Compound 13 showed no
significant binding to any of these targets at 10 μM, suggesting
that it is highly target selective.
Expression of genes with ALS-associated G93A SOD1
mutations produces a striking ALS phenotype motor neuron
degeneration and paralysis in rats.43,44 Transgenic mice that
express human G93A mutant SOD1 develop hind limb
weakness, muscle wasting, and neuropathological sequelae
similar to those observed in both familial and sporadic ALS
patients.45 Candidate compounds are commonly tested in the
ALS mouse model to evaluate their potential for the treatment
of ALS.13 The in vivo efficacy of riluzole, the only FDA-
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Beckman System Gold chromatograph (model 125P solvent module
and model 166 detector). All tested compounds had a purity of at least
95% as demonstrated by HPLC (see Supporting Information).
5-(4-Chlorophenylthio)-1H-[15N2]pyrazol-3(2H)-one (3). 4-
Chlorothiophenol (1.1 g, 7.61 mmol) was mixed with ethyl 4-
chloroacetoacetate (0.95 mL, 7.00 mmol) in CH2Cl2 (100 mL) at 0
°C. Triethylamine (1.5 mL, 10.8 mmol) was then added dropwise.
After the resulting suspension was stirred at 0 °C for another 30 min,
the reaction mixture was poured into water, and the aqueous layer was
extracted with EtOAc. The combined organic layer was washed with
saturated NaHCO3, HCl (0.25 N), brine, concentrated in vacuo, and
purified by flash column chromatography (ethyl acetate/hexanes = 1/
9) to afford 20 (1.82 g) as a light yellow oil, which was not very stable.
Therefore, 20 was used directly in the next step immediately after flash
column chromatography purification. A proton NMR spectrum was
approved drug for ALS, was tested in G93A SOD1 mice and
showed a lifespan extension of 7.5% when tested at 16 mg/kg46
and of 11% at 22 mg/kg.47 The in vivo efficacy of 13 in the
mutant SOD1 G93A ALS mouse model was assessed to
validate the aryloxanyl pyrazolone scaffold as a potential
therapy for ALS,. Compound 13 produced a life extension of
13.3% at 20 mg/kg (Figure 6), comparable to or superior to
riluzole.
CONCLUSIONS
■
We had previously identified arylsulfanyl pyrazolones (ASP) as
a class of compounds exhibiting good potency against toxicity
from protein aggregation by mutant SOD1.21 These com-
pounds could not be developed further, however, because of
poor metabolic stability. Here we identify the cause for
metabolic instability as the sulfide sulfur atom, which is
oxygenated to the corresponding sulfoxide and show that the
resulting sulfoxide compounds have significantly lower activity
than the sulfides. Conversion to the corresponding sulfones and
ethers, the aryloxanyl pyrazolones, resulted in much greater
metabolic stability. The most potent analogue was aryloxanyl
pyrazolone 13, with an IC50 of 67 nM, having good aqueous
solubility, excellent Caco-2 permeability with low efflux
potential, a rat plasma half-life of 3.6 h, rat oral bioavailability
of 27%, neuron permeability, good mouse blood−brain barrier
penetration (194 μM after 4 h), no effect on the hERG channel
or 68 off-target proteins, and a life extension of G93A ALS mice
of 13.3% at 20 mg/kg, as good or better than that previously
reported for riluzole, the only FDA-approved therapeutic for
ALS. These results support 13 as a novel drug candidate for the
treatment of ALS.
1
taken immediately after the flash column purification. H NMR (400
MHz, CDCl3, δ): 7.37 (dd, J = 18.4, 8.4 Hz, 4H), 4.12 (d, J = 14.0, 7.2
Hz, 2H), 4.06 (s, 2H), 3.72 (s, 2H), 1.21 (t, J = 7.2 Hz, 3H).
Compound 20 (0.52 g, 1.89 mmol) was stirred in EtOH (15 mL) and
H2O (6 mL), then 15NH215NH2·H2SO4 (0.25 g, 1.89 mmol) and
NaHCO3 (0.32, 3.79 mmol) were added. The resulting solution was
stirred overnight at room temperature, during which time a precipitate
formed. The precipitate was filtered, washed with cold EtOH, and
dried in vacuo to afford 3 (0.21 g, 44%, two steps) as a white solid. 1H
NMR (500 MHz, DMSO-d6, δ): 11.48 (br s, 1H), 9.48 (br s, 1H),
7.36 (s, 4H), 5.30 (s, 1H), 4.08 (s, 2H). 13C NMR (125 MHz, DMSO-
d6, δ): 161.3, 139.3, 135.0, 130.5, 129.8, 128.8, 89.6, 27.7. HRMS (m/
z): [M + H]+ calcd for C10H9Cl15N2OS, 243.0142; found 243.0192.
5-(4-Chlorophenylsulfinyl)-1H-pyrazol-3(2H)-one (4). Com-
pound 20 was obtained from 4-chlorothiophenol and ethyl 4-
chloroacetoacetate and used directly after flash column chromatog-
raphy as described above. Compound 20 (1.27 g, 4.66 mmol) was
mixed with tert-butyl hydrogen peroxide (70 wt % in water, 1.1 mL,
7.69 mmol) in CH2Cl2 (50 mL) at room temperature, and vanadyl
acetylacetonate (0.1% mol) was added slowly. Additional tert-butyl
hydrogen peroxide (0.5 mL, 3.50 mmol) was added to the reaction
mixture after 2 h. The resulting suspension was stirred overnight at
room temperature. The reaction mixture was then concentrated under
vacuum and purified by flash column chromatography (ethyl acetate/
hexanes = 1/4) to afford compound 23 (0.98 g) as a light yellow solid,
which was not very stable. A proton NMR spectrum was taken
EXPERIMENTAL SECTION
■
Chemistry. General Methods, Reagents, and Materials. All
reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI)
or Alfa Aesar (Ward Hill, MA) and were used without further
purification, unless stated otherwise. Tetrahydrofuran was distilled
under nitrogen from sodium/benzophenone. Dichloromethane was
redistilled from CaH2 under nitrogen. Other dry solvents were directly
purchased. Thin-layer chromatography was carried out on E. Merck
precoated silica gel 60 F254 plates. Compounds were visualized with
ferric chloride reagent or a UV lamp. Column chromatography was
performed with E. Merck silica gel 60 (230−400 mesh). Proton
nuclear magnetic resonances (1H NMR) were recorded in deuterated
solvents on a Varian Inova 400 (400 MHz), a Varian Inova 500 (500
MHz), or a Bruker 500 (500 MHz) spectrometer. Chemical shifts are
reported in parts per million (ppm, δ) using various solvents as
1
immediately after the flash column purification. H NMR (500 MHz,
CDCl3, δ): 7.46−7.34 (m, 4H), 4.20 (q, J = 7.5 Hz, 2H), 4.13 (s, 2H),
3.80 (s, 2H), 1.28 (t, J = 7.5 Hz, 3H). Therefore, 23 was used directly
in the next step immediately after the flash column chromatography
purification. Compound 23 (0.50 g, 1.73 mmol) was stirred in EtOH
(6 mL), and an ethanolic solution of NH2NH2 (2 N, 0.87 mL, 1.74
mmol) was added. The resulting solution was stirred overnight at
room temperature, during which time a precipitate formed. The
precipitate was filtered, washed with cold EtOH, and dried in vacuo to
1
afford 4 (0.10 g, 17%, three steps) as a white solid. H NMR (500
1
MHz, DMSO-d6, δ): 11.49 (br s, 1H), 9.75 (br s, 1H), 7.62 (d, J = 8.0
Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 5.17 (s, 1H), 4.13−3.99 (m, 2H).
13C NMR (125 MHz, DMSO-d6, δ): 160.9, 142.5, 135.7, 132.1, 129.1,
internal standards (CDCl3, δ 7.26 ppm; DMSO-d6, δ 2.50 ppm). H
NMR splitting patterns are designated as singlet (s), doublet (d),
triplet (t), or quartet (q). Splitting patterns that could not be
interpreted or easily visualized were recorded as multiplet (m) or
broad (br). Coupling constants are reported in hertz (Hz). Proton-
decoupled carbon (13C NMR) spectra were recorded on a Varian
Inova 500, a Varian Inova 400, or a Bruker 500 (125.7, 100.6, and
125.7 MHz, respectively) spectrometer and are reported in ppm using
various solvents as internal standards (CDCl3, δ 77.23 ppm; DMSO-
d6, δ 39.52 ppm). Electrospray mass spectra were obtained using an
LCQ-Advantage spectrometer with methanol as the solvent in the
positive ion mode. High-resolution mass spectrometry was carried out
using a VG70-250SE mass spectrometer. Chemical ionization (CI) or
electron impact (EI) was used as the ion source. Elemental analyses
were performed by Atlantic Microlab Inc., Norcross, GA. All final
compounds were analyzed for purity by HPLC using a Luna C18 (2)
column (4.6 mm × 150 mm, 5 μm; Phenomenex, Torrance, CA) at a
flow rate of 1 mL/min with multiple HPLC conditions. Sample elution
was detected by absorbance at 254 nm. HPLC was performed on a
126.3, 90.2, 53.3. HRMS (m/z): [M + H]+ calcd for C10H9ClN2O2S,
256.007 33; found 256.007 32.
5-(4-Chloro-2,5-dimethylphenylsulfinyl)-1H-pyrazol-3(2H)-
one (5). Analogous to 4, compound 22 was prepared from 4-chloro-
2,5-dimethylbenzenethiol (9 g, 52.1 mmol) to afford 22 (13.11 g).
Immediately after flash chromatography, 22 (4.69 g, 14.2 mmol) was
converted to 24 (3.20 g). Immediately after flash column
chromatography, 24 (1.39 g, 4.00 mmol) was converted to 5 as a
white solid (0.42 g, 19%, three steps). 1H NMR (500 MHz, DMSO-d6,
δ): 11.52 (br s, 1H), 9.50 (br s, 1H), 7.58 (s, 1H), 7.37 (s, 1H), 5.22
(s, 1H), 4.03−3.93 (m, 2H), 2.35 (s, 3H), 2.15 (s, 3H). 13C NMR
(125 MHz, DMSO-d6, δ): 161.1, 140.8, 135.7, 134.7, 134.1, 132.5,
130.3, 126.1, 90.9, 52.3, 19.3, 16.8.
5-(4-Chlorophenylsulfonyl)-1H-pyrazol-3(2H)-one (6). Com-
pound 20 was obtained from 4-chlorothiophenol and ethyl 4-
chloroacetoacetate and used directly after flash column as described
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Article
above. Compound 20 (3.0 g, 11.0 mmol) was mixed with tert-butyl
hydrogen peroxide (70 wt % in water, 1.5 mL, 10.5 mmol) in CH2Cl2
(50 mL) at room temperature, and vanadyl acetylacetonate (0.1%
mol) was added slowly. Additional tert-butyl hydrogen peroxide (6
mL, 41. 94 mmol) was added to the reaction mixture gradually until all
starting material was consumed, as determined by TLC. The resulting
suspension was stirred overnight at room temperature. The reaction
mixture was then concentrated under vacuum and purified by flash
column chromatography (ethyl acetate/hexanes = 1/4) to afford 25
(1.61 g) as a light yellow oil, which was not very stable. Therefore, 25
was used directly in the next step immediately after flash column
chromatography purification. Compound 25 (1.60 g, 5.25 mmol) was
stirred in EtOH (6 mL), and an ethanolic solution of NH2NH2 (2 N,
2.64 mL, 5.28 mmol) was added. The resulting solution was stirred
overnight at room temperature, during which time a precipitate
formed. The precipitate was filtered, washed with cold EtOH, and
dried under vacuum to afford 6 (0.46 g, 12%, three steps) as a white
solid. 1H NMR (500 MHz, DMSO-d6, δ): 11.24 (br s, 1H), 7.75 (d, J
= 9.0 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 5.21 (s, 1H), 4.56 (s, 2H). 13C
NMR (125 MHz, DMSO-d6, δ): 159.1, 139.0, 137.2, 130.1, 129.4,
89.9, 54.1. HRMS (m/z): [M + H]+ calcd for C10H9ClN2O3S,
272.002 24; found 272.001 64.
yellowish oil. The obtained mixture was used directly in the next step.
To the mixture of 28 and 4-chlorophenol (10.7 g, assumed 41.7
mmol) was added an ethanolic solution of NH2NH2 (2 N, 14.5 mL,
29.0 mmol). The resulting solution was stirred overnight at room
temperature, during which time a precipitate formed. The precipitate
was filtered, washed with cold EtOH, and dried under vacuum to
afford 9 (1.38 g, 13%, two steps) as a white solid. 1H NMR (500 MHz,
DMSO-d6, δ): 11.79 (br s, 1H), 9.56 (br s, 1H), 7.33 (d, J = 9.0 Hz,
2H), 7.02 (d, J = 9.0 Hz, 2H), 5.52 (s, 1H), 4.92 (s, 2H). 13C NMR
(125 MHz, DMSO-d6, δ): 160.2, 157.0, 139.2, 129.3, 124.6, 116.6,89.5,
62.1.
5-((4-Ethylphenoxy)methyl)-1H-pyrazol-3(2H)-one (10).
Analogous to 9, compound 29 was prepared from 4-ethylphenol
(3.0 g, 24.6 mmol). The mixture of 29 and 4-ethylphenol (4.05 g,
assumed 16.2 mmol) was converted to 10 (1.62 g, 30%, two steps) as a
white solid. 1H NMR (500 MHz, DMSO-d6, δ): 11.75 (br s, 1H), 9.50
(br s, 1H), 7.10 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 5.50 (s,
1H), 4.87 (s, 2H), 2.54−2.51 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C
NMR (125 MHz, DMSO-d6, δ): 160.0, 156.2, 140.3, 136.1, 128.7,
114.6, 89.4, 61.6, 27.4, 16.0.
5-((3-Ethylphenoxy)methyl)-1H-pyrazol-3(2H)-one (11).
Analogous to 9, compound 30 was prepared via method A from 3-
ethylphenol (3.0 g, 24.6 mmol). The mixture of 30 and 3-ethylphenol
(1.81 g, assumed 7.23 mmol) was converted to 10 (0.30 g, 6%, two
5-(3,5-Dichlorophenylsulfonyl)-1H-pyrazol-3(2H)-one (7).
Analogous to 6, compound 21 (4.20 g) was prepared from 3,5-
dichlorobenzenethiol (2.5 g, 14.0 mmol). Immediately after flash
chromatography, 21 (4.10 g, 13.3 mmol) was converted to 26 (2.24
g). Immediately after flash chromatography, 26 (2.14 g, 6.31 mmol)
1
steps) as a white solid. H NMR (500 MHz, DMSO-d6, δ): 11.79 (br
s, 1H), 9.55 (br s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.83−6.79 (m, 3H),
5.52 (s, 1H), 4.89 (s, 2H), 2.56 (q, J = 7.5 Hz, 2H), 1.16 (t, J = 7.5 Hz,
3H). 13C NMR (125 MHz, DMSO-d6, δ): 160.7, 158.2, 145.4, 139.3,
129.3,120.4, 114.3, 111.8, 89.8, 61.0, 28.3, 15.6.
5-((3-tert-Butylphenoxy)methyl)-1H-pyrazol-3(2H)-one (12).
Analogous to 9, compound 31 was prepared via method A from 3-tert-
butylphenol (3.0 g, 20.0 mmol). The mixture of 31 and 3-tert-
butylphenol (2.47 g, assumed 8.86 mmol) was treated with NH2NH2
(2 N, 4.40 mL, 8.80 mmol) to give 10 (1.52 g, 10%, two steps) as a
white solid. 1H NMR (500 MHz, DMSO-d6, δ): 11.72 (br s, 1H), 9.50
(br s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.97−6.80 (m, 3H), 5.52 (s, 1H),
4.91 (s, 2H). 13C NMR (125 MHz, DMSO-d6, δ): 160.7, 157.9, 152.4,
138.7, 129.0, 117.8, 112.3, 111.1, 89.7, 61.0, 34.5, 31.3.
5-((3,5-Dichlorophenoxy)methyl)-1H-pyrazol-3(2H)-one
(13). Analogous to 9, compound 32 was prepared via method A from
3,5-dichlorophenol (2.0 g, 12.3 mmol). The mixture of 32 and 3,5-
dichlorophenol (1.10 g, assumed 3.78 mmol) was treated with
NH2NH2 (2 N, 1.90 mL, 3.80 mmol) to give 13 (66.1 mg, 2%, two
steps) as a white solid. See below (method B) for characterization of
13.
1
was converted to 7 as a white solid (0.38 g, 10%, three steps). H
NMR (500 MHz, DMSO-d6, δ): 11.62 (br s, 1H), 9.55 (br s, 1H),
7.77−7.36 (m, 3H), 5.26 (s, 1H), 4.70 (s, 2H). 13C NMR (125 MHz,
DMSO-d6, δ): 161.3, 141.2, 135.0, 133.8, 129.5, 126.7, 92.1, 52.5.
HRMS (m/z): [M + H]+ calcd for C10H8Cl2N2O3S, 301.0408; found
301.0415.
5-(4-Chloro-2,5-dimethylphenylsulfonyl)-1H-pyrazol-3(2H)-
one (8). Compound 22 was obtained from 4-chloro-2,5-dimethyl-
benzenethiol and ethyl 4-chloroacetoacetate and used directly after
flash column chromatography as described above. Compound 22
(4.68 g, 14.1 mmol) was mixed with AcOH (5 mL) in EtOAc (10
mL), and H2O2 (30% in water, 10 mL, 84.6 mmol) was added. The
resulting solution was left stirring at room temperature overnight after
which additional H2O2 (30% in water, 5 mL, 42.3 mmol) was added.
The reaction mixture was then evaporated under vacuum and purified
by flash column chromatography (ethyl acetate/hexanes = 1/3) to
afford 27 (4.34 g) as a yellowish oil, which was not very stable.
Therefore, 27 was used directly in the next step immediately after flash
column chromatography purification. Compound 27 (4.33 g, 11.9
mmol) was stirred in EtOH (20 mL), and an ethanolic solution of
NH2NH2 (2 N, 5.98 mL, 11.9 mmol) was added. The resulting
solution was stirred overnight at room temperature, during which time
a precipitate formed. The precipitate was filtered, washed with cold
EtOH, and dried under vacuum to afford 8 (0.71 g, 17%, three steps)
as a white solid. 1H NMR (500 MHz, DMSO-d6, δ): 11.62 (br s, 1H),
9.59 (br s, 1H), 7.67 (s, 1H), 7.54 (s, 1H), 5.24 (s, 1H), 4.49 (s, 2H),
2.47 (s, 3H), 2.33 (s, 3H). 13C NMR (125 MHz, DMSO-d6, δ): 160.3,
138.8, 137.7, 135.2, 133.9, 132.5,132.4, 131.0, 91.8, 53.1, 43.3, 19.1.
HRMS (m/z): [M + H]+ calcd for C12H13ClN2O3S, 306.9705; found
306.9713.
Synthesis of Ether Analogues (9−13) via Method A. 5-((4-
Chlorophenoxy)methyl)-1H-pyrazol-3(2H)-one (9). A solution of
4-chlorophenol (6.4 g, 50 mmol) in THF (25 mL) was treated with
NaH (60% in mineral oil, 2 g, 50 mmol) at 0 °C. In another flask, a
solution of ethyl 4-chloroacetoacetate (10.21 mL, 75 mmol) in THF
(25 mL) was treated with NaH (60% in mineral oil, 3.5 g, 75 mmol) at
−20 °C. The resulting yellowish suspension was slowly added to the
solution of sodium 4-chlorophenoxide, which was kept at 0 °C. After
the addition of DMF (10 mL), the reaction temperature was slowly
raised to 70 °C. After the reaction mixture was stirred at 70 °C
overnight, it was cooled and evaporated to dryness. The residue was
purified by flash column chromatography (ethyl acetate/hexanes = 1/
9) to afford 28 (10.7 g, contains 30% 4-chloroacetonacetate) as a
5-((3,5-Bis(trifluoromethyl)phenoxy)methyl)-1H-pyrazol-
3(2H)-one (14). Analogous to 9, compound 33 was prepared via
method A from 3,5-bis(trifluoromethyl)phenol (1.00 mL, 5.93 mmol).
The mixture of 33 and 3,5-bis(trifluoromethyl)phenol (0.72 g,
assumed 2.00 mmol) was treated with NH2NH2 (2 N, 1.00 mL,
1
2.00 mmol) to give 14 (0.40 g, 20%, two steps) as a white solid. H
NMR (500 MHz, DMSO-d6, δ): 11.62 (br s, 1H), 9.50 (br s, 1H),
7.99 (s, 2H), 7.86 (s, 1H), 5.35 (s, 1H), 4.31 (s, 2H). 13C NMR (125
MHz, DMSO-d6, δ): 159.8, 140.9, 131.2, 130.9, 130.5, 130.2, 127.6,
127.2, 124.5, 121.8, 119.1, 118.8, 89.0, 27.6.
Synthesis of Ether Analogues (13, 15−19) via Method B.
Detailed experimental procedures and characterization of 35, 36, 39,
41, and 42−47 can be found in the Supporting Information.
5-((3,5-Dichlorophenoxy)methyl)-1H-pyrazol-3(2H)-one
(13). EtOAc (3.20 mL. 32.7 mmol) was added to a solution of
LiHMDS (1 N in THF, 75.0 mL, 75.0 mmol) at 0 °C and stirred. After
60 min, a THF solution of 42 (8.55 g, 32.4 mmol) was added dropwise
at −78 °C. After the resulting solution was stirred at −78 °C for
another 8 h, the reaction mixture was quenched with diluted HCl
(0.25 N), the pH was adjusted to 3−5, and the aqueous layer was
extracted with Et2O. The combined organic layer was dried over
Na2SO4 and concentrated under vacuum. The residue was purified by
flash column chromatography (ethyl acetate/hexanes = 1/9) to give
ethyl 4-(3,5-dichlorophenoxy)-3-oxobutanoate (3.28 g) as a white
solid. The proton NMR spectrum was taken immediately after
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recrystallization (ethyl acetate/hexanes). 1H NMR (500 MHz, CDCl3,
δ): 7.02 (t, J = 1.7 Hz, 1H), 6.81 (d, J = 1.7 Hz, 2H), 4.68 (s, 2H), 4.21
(q, J = 7 Hz, 2H), 3.61 (s, 2H), 1.28 (t, J = 7 Hz). Ethyl 4-(3,5-
dichlorophenoxy)-3-oxobutanoate was used directly in the next step
immediately after the flash column chromatography purification.
Ethanolic hydrazine (2 N, 5.60 mL, 11.2 mmol) was added to a
solution of ethyl 4-(3,5-dichlorophenoxy)-3-oxobutanoate (3.28 g,
11.3 mmol) in EtOH (25 mL). The resulting solution was stirred at
room temperature overnight. The reaction mixture was then
evaporated under vacuum, purified by flash column chromatography
(ethyl acetate/hexanes = 1/2), and recrystallized in ethyl acetate/
hexanes to give 13 (0.88 g, 11%, two steps) as white crystals. 1H NMR
(500 MHz, DMSO-d6, δ): 11.82 (br s, 1H), 9.54 (br s, 1H), 7.16 (s,
1H), 7.12 (d, J = 1.5 Hz, 2H), 5.53 (s, 1H), 4.99 (s, 2H). 13C NMR
(125 MHz, DMSO-d6, δ): 161.0, 159.4, 137.8, 134.6, 120.6, 114.2,
90.7, 61.5. Anal. Calcd for C10H8Cl2N2O2: C, 46.36; H, 3.11; Cl,
27.37; N, 10.81. Found: C, 46.40; H, 3.08; Cl, 27.24; N, 10.65. All
values are given as percentages.
5-((3,5-Difluorophenoxy)methyl)-1H-pyrazol-3(2H)-one (15).
Analogous to 13, compound 15 was prepared via method B from 43
(1.24 g, 5.36 mmol) to afford initially ethyl 4-(3,5-difluorophenoxy)-3-
oxobutanoate (0.89 g, 3.44 mmol). Because of instability, immediately
after flash column chromatography, ethyl 4-(3,5-difluorophenoxy)-3-
oxobutanoate (0.89 g, 3.44 mmol) was treated with NH2NH2 (2 N in
EtOH, 1.70 mL, 3.40 mmol) to give 15 as a white solid (50.9 mg, 5%,
two steps). 1H NMR (500 MHz, DMSO-d6, δ): 11.82 (br s, 1H), 9.53
(br s, 1H), 6.80−6.79 (m, 3H), 5.56 (s, 1H), 4.95 (s, 2H). 13C NMR
(125 MHz, DMSO-d6, δ): 0.1, 164.0, 162.1, 162.0, 160.0, 146.4, 137.8,
99.1, 98.9, 96.4, 90.7, 61.8.
(125 MHz, DMSO-d6, δ): 161.3, 159.1, 142.3, 140.0, 139.0, 129.0,
127.8, 127.0, 118.0, 112.3, 90.4, 61.2.
In Vitro Microsomal Stability of Compound 1 and
Minaprine. General Methods, Reagents, and Materials. The
microsome assay was measured on a Beckman HPLC system using
Luna C18 (2) column (4.6 mm × 150 mm, 5 μm; Phenomenex,
Torrance, CA) at a flow rate of 1 mL/min at 254 nm for minaprine
and 260 nm for 1. Solid phase extraction was carried out using Waters
Sep-PakVac C18 1 cc cartridges (Waters Chromatography, Milford,
MA). NADPH regeneration solutions and rat liver microsomes were
purchased from Bectin-Dickinson.
To PBS (10 mL) was added NADPH regeneration buffer A (500
μL) and NADPH regeneration buffer B (100 μL) to make the PBS+
solution. To the PBS+ solution (255 μL) was added the compound of
interest (30 μL of 150 μM stock in PBS+) in an Eppendorf tube. Each
reaction mixture was carried out in PBS (pH 7.4), which contained 1
mg/mL microsomal protein with 1.3 mM NADP+, 3.3 mM glucose 6-
phosphate, 0.4 U/mL glucose 6-phosphate dehydrogenase, and 3.3
mM magnesium chloride. The tubes were vortexed and equilibrated at
37 °C. The samples were incubated at 37 °C for 0 and 20 min after the
rat liver microsomes (15 μM) were added. Acetonitrile (40 μL) and
haloperidol (internal standard, 100 μL) were added as a quench
solution. The reaction mixture was vortexed and left in a −78 °C ice
bath immediately after it was quenched. The samples were centrifuged
(13.4 krpm, 12 min), and the supernatant was loaded onto a Waters
Sep-PakVac C18 1 cc SPE cartridge. The loaded SPE column was
washed with H2O (2 × 1 mL), and the compounds were eluted with
acetonitrile/H2O = 1/1 (2 × 1 mL). The solvent was removed under
vacuum. The samples were reconstituted in DMSO/H2O = 1/9 (25
μL), and an aliquot (20 μL) was analyzed by HPLC (isocratic,
acetonitrile/H2O = 25/75, 60 min, 0.1% TFA). The response ratio
(RR) was calculated by dividing the peak area of the analyte by the
peak area of the internal standard. Peak integrals were normalized by
dividing by the area for the peak at t = 0 min. Sample HPLC spectrum
and RR data can be found in the Supporting Information.
Metabolite Profiling of Compound 1. Compound 1 was
incubated with rat liver microsomes and NADPH regeneration buffers
as described above for the in vitro microsomal stability study of 1. To
the PBS+ solution (85 μL) was added 1 (10 μL of 150 μM stock in
PBS+). The sample was incubated at 37 °C for 0, 5, 10, 20, 40, and 60
min after the rat liver microsomes (15 μM) were added. Acetonitrile
(50 μL) was added as a stop solution. The mixture was vortexed and
left on a −78 °C ice bath immediately after it was quenched. The
samples were centrifuged (13.4 krpm, 12 min), and the supernatant
(20 μL) was directly analyzed by HPLC (isocratic, acetonitrile/H2O =
25/75, 30 min, 0.1% TFA). Compounds 4 (20 μL, 10 μM) and 6 (20
μL, 10 μM) were analyzed by the same HPLC program. Spectra of the
microsomal incubation residue of 1, standard solution of 4, and
standard solution of 6 were compared in parallel.
Further Metabolite Profiling of 1 and 3. Metabolite studies
were also performed at Apredica, Inc. (Watertown, MA). Samples
were analyzed by LC/MS/MS using either an Agilent 6410 mass
spectrometer coupled with an Agilent 1200 HPLC instrument and a
CTC PAL chilled autosampler or an ABI2000 mass spectrometer
coupled with an Agilent 1100 HPLC instrument and a CTC PAL
chilled autosampler. After separation of compounds on a C18 reverse
phase HPLC column using an acetonitrile−water gradient system,
peaks were analyzed by mass spectrometry using ESI ionization in
MRM mode.
Each test agent (1 and 3) was incubated in duplicate at 5 μM with
or without rat liver microsomes in the presence and absence of
NADPH. After 0, 10, 20, 40, and 60 min of incubation, an aliquot was
removed from each reaction and the protein was precipitated with
acetonitrile containing internal standard. The supernatant was
analyzed by LC/MS/MS. Detailed experimental data and HPLC and
MS spectra can be found in the Supporting Information.
5-((3,5-Dibromophenoxy)methyl)-1H-pyrazol-3(2H)-one
(16). Analogous to 13, compound 16 was prepared via method B from
44 (1.07 g, 3.03 mmol) to afford initially ethyl 4-(3,5-dibromophe-
noxy)-3-oxobutanoate (0.35 g). Immediately after flash column, ethyl
4-(3,5-dibromophenoxy)-3-oxobutanoate (0.35 g, 0.92 mmol) was
treated with NH2NH2 (2 N in EtOH, 0.46 mL, 0.92 mmol) to give 16
as a white solid (23.7 mg, 2%, two steps). 1H NMR (500 MHz,
DMSO-d6, δ): 11.80 (br s, 1H), 9.52 (br s, 1H), 7.39 (s, 1H), 7.28 (m,
2H), 5.55 (s, 1H), 4.99 (s, 1H). 13C NMR (125 MHz, DMSO-d6, δ):
161.0, 159.5, 137.7, 125.9, 122.8, 117.4, 90.6, 61.5.
5-((3-Bromophenoxy)methyl)-1H-pyrazol-3(2H)-one (17).
Analogous to 13, compound 17 was prepared via method B from
45 (2.43 g, 8.87 mmol) to afford initially ethyl 4-(3-bromophenoxy)-3-
oxobutanoate (1.07 g). Immediately after flash column, ethyl 4-(3-
bromophenoxy)-3-oxobutanoate (1.07 g, 3.55 mmol) was treated with
NH2NH2 (2 N in EtOH, 1.70 mL, 3.40 mmol) to give 17 as a white
1
solid (0.42 g, 18%, two steps). H NMR (500 MHz, DMSO-d6, δ):
11.78 (br s, 1H), 9.50 (br s, 1H), 7.26−7.23 (m, 1H), 7.14−7.13 (m,
1H), 7.02−7.00 (m, 2H), 5.58 (s, 1H), 4.96 (s, 2H). 13C NMR (125
MHz, DMSO-d6, δ): 161.6, 159.1, 138.4, 131.2, 123.8, 122.1, 117.6,
114.3, 90.5, 61.3.
5-((Biphenyl-3-yloxy)methyl)-1H-pyrazol-3(2H)-one (18).
Analogous to 13, compound 18 was prepared via method B from
46 (0-.74 g, 2.74 mmol) to afford initially ethyl 4-(biphenyl-3-yloxy)-3-
oxobutanoate (0.50 g). Immediately after flash column, ethyl 4-
(biphenyl-3-yloxy)-3-oxobutanoate (0.50 g, 1.68 mmol) was treated
with NH2NH2 (2 N in EtOH, 0.84 mL, 1.68 mmol) to give 18 as a
1
white solid (0.19 g, 27%, two steps). H NMR (500 MHz, DMSO-d6,
δ): 11.80 (br s, 1H), 9.50 (br s, 1H), 7.67−6.99 (m, 9H), 5.55 (s, 1H),
5.02 (s, 2H). 13C NMR (125 MHz, DMSO-d6, δ): 161.3, 158.6, 138.7,
141.7, 140.0, 130.0, 128.9, 127.7, 126.8, 119.4, 113.9, 113.0, 90.3, 60.8.
5-((5-Phenylbiphenyl-3-yloxy)methyl)-1H-pyrazol-3(2H)-one
(19). Analogous to 13, compound 19 was prepared via method B from
47 (0.61 g, 1.75 mmol) to afford initially ethyl 4-(5-phenylbiphenyl-3-
yloxy)-3-oxobutanoate (0.45 g). Immediately after flash column, ethyl
4-(5-phenylbiphenyl-3-yloxy)-3-oxobutanoate (0.45 g, 1.21 mmol) was
treated with NH2NH2 (2 N in EtOH, 0.60 mL, 1.20 mmol) to give 19
as a white solid (0.19 g, 27%, two steps). 1H NMR (500 MHz, DMSO-
d6, δ): 11.82 (br s, 1H), 9.50 (br s, 1H), 7.76 (d, J = 8.0 Hz, 4H),
7.50−7.38 (m, 7H), 7.27 (s, 2H), 5.60 (s, 1H), 5.13 (s, 2H). 13C NMR
Mutant SOD1-Induced Cytotoxicity Protection Assay.
Viability and EC50 values of 1−19 were determined according to the
previously reported assay procedure.20 PC12 cells expressing mutant
G93A SOD1 were seeded at 15 000 cells/well in 96-well plates and
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Journal of Medicinal Chemistry
Article
incubated for 24 h prior to compound addition. Compounds were
assayed in 12-point dose−response experiments to determine potency
and efficacy. The highest compound concentration tested was 32 μM,
which was decreased by one-half with each subsequent dose. After 24
h of incubation with the compounds, MG132 was added at a final
concentration of 100 nM. MG132 is a well-characterized proteasome
inhibitor, which enhances the appearance of protein aggregation by
blocking the proteosomal clearance of aggregated proteins. Cell
viability was measured 48 h later using a fluorescent viability probe,
Calcein-AM (Molecular Probes). Briefly, cells were washed twice with
PBS, Calcein-AM was added at a final concentration of 1 μM for 20
min at room temperature, and fluorescence intensity was read in a
POLARstar fluorescence plate reader (BMG). Fluorescence data were
coupled with compound structural data, then stored and analyzed
using the CambridgeSoft Chemoffice Enterprise Ultra software
package.
In Vitro ADME Assays. In vitro microsomal stability of 7 and 9,
aqueous solubility, and Caco-2 permeability of 7, 9, and 13 were
determined according to the previously reported procedure.21 In vitro
microsomal stability of 13 was obtained from Biogen Idec.
Preliminary Cortical Neuron Permeability. Primary rat cortical
tissue was purchased from Neuromics Inc., Edina, MN, and used to
initiate primary cortical neuron cultures. The tissue was isolated from
microsurgically dissected E18 embryonic Sprague−Dawley or Fischer
344 rat brain and shipped in a nutrient rich medium under
refrigeration.
To isolate neurons, the tissue was incubated with papain at 2 mg/
mL in Hibernate without calcium for 30 min at 37 °C. The enzymatic
solution was then removed and 1 mL of culture medium (Neurobasal,
B27, 0.5 mM glutamine) was added. A sterile Pasteur pipet was used to
gently disperse the cells, which were then washed, resuspended, and
counted. The cells were plated on poly-D-lysine coated 96-well plates
at a density of 20 000 cells/well and incubated at 37 °C in a 5% CO2-
humidified atmosphere for 5 days prior to use in compound testing. By
microscopic inspection, the resulting culture consisted of ∼90%
neurons.
Test compounds were assayed in six-point dose response
experiments. The highest compound concentration tested was 100
μM, which was then diluted by approximately one-third with each
subsequent dose. After 24 h of incubation with the compounds,
MG132 was added at a final concentration of 100 nM, a dose that
produces an approximately 70% loss of viability. Cell viability was
measured 48 h later using the fluorescent viability probe, Calcein-AM
(Molecular Probes, Invitrogen, Carlesbad, CA). Briefly, cells were
washed twice with PBS, Calcein-AM was added at a final concentration
of 1 μM for 20 min at room temperature, and fluorescence intensity
was then read in a POLARstar fluorescence plate reader (BMG).
Compounds that restored viability at any dose to a level equal to or
higher than 5 standard deviations from MG132 controls were
considered active.
weight, and parentage were used for placing individual mice into
experimental groups/cohorts. Wild type mice were used for initial
toxicity, tolerability, and pharmacokinetics studies. The tolerable dose
range and LD50 for 13 was determined in the wild type mice by
increasing the dose b.i.d. 1-fold each injection and observed at 75 mg/
kg. Drug steady-state level was determined in animals that had been
dosed for 1 week prior to sacrifice. The dosing levels of 1.0, 10, and 20
mg/kg once a day were administered throughout the levels of the
G93A mice.
Efficacy was measured using end points that clearly indicate
neuroprotective function. These include amelioration of degenerative
changes in the spinal cord, improved motor function, and prolonged
survival. The mice cohorts were sacrificed at end stage disease using
criteria for euthanasia, followed temporally for survival analyses. Mice
were observed three times daily (morning, noon, and later afternoon)
throughout the experiment. Mice were euthanized when disease
progression was sufficiently severe that they were unable to initiate
movement and right themselves after gentle prodding for 30 s.
Data sets were generated and analyzed for each clinical and
neuropathological measure. Effects on behavior and neuropathology
were compared in treatment and control groups. Dose-dependent
effects were assessed in each treatment group using multiple two-sided
ANOVA tests. Multiple comparisons in the same subject groups were
dealt with post hoc. Kaplan−Meier analysis was used for survival
function. All other statistical analyses were performed using Student’s t
test. Data are expressed as the mean SEM. Statistical comparisons of
histological data were made by ANOVA.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and data for 35, 36, 39, 41, and 42−47;
HPLC data and spectra for 1−19; HPLC spectra and data of
microsomal stability of minaprine and 1; data and spectra of
metabolite profiling of 1 and 3; bioanalysis data of in vivo
mouse steady-state level study and in vivo blood−brain barrier
study of 13; data for the effect of 13 on hERG potassium
channel; data for the effect of 13 on enzymes and receptors.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Address: Department of Chemistry, Northwestern University,
2145 Sheridan Road, Evanston, IL 60208-3113. Phone: 1-847-
491-5653. Fax: 1-847-491-7713. E-mail: Agman@chem.
northwestern.edu.
ACKNOWLEDGMENTS
■
In Vivo Mouse Drug Steady-State Level Determination and
in Vivo Brain Penetration. See Supporting Information.
We thank the National Institutes of Health (Grant
1R43NS057849), the ALS Association (TREAT program),
the Department of Defense (Grant AL093052), and the Center
for ALS Research at the Univesity of Pittsburgh, PA, for their
generous support of the research project. The authors are
grateful to Biogen Idec Inc. for carrying out the in vivo rat PK
profiling tests, in vitro P450 reversible inhibition study, the
plasma binding affinity assay, and the hERG inhibition assays;
and MDS Pharma Service (now Ricerca Biosciences, LLC) for
carrying out the LeadProfilingScreen of 13.
In Vivo PK Profiling. In vivo PK profiling was performed by
administering a 300 mg/kg bolus intraperitoneal injection of 13 into
B6SJL mice. Blood and brain samples were collected at 0, 1, 3, 6, 12,
and 24 h time points and flash frozen in liquid nitrogen, stored at −80
°C, and shipped to Apredica, Inc. for analysis. The analysis was carried
out as for in vivo mouse drug steady-state level determination and in
vivo brain penetration described in Supporting Information.
Effect of Compound 13 on Potassium Channels. FASTPatch
hERG screen assay, using cloned hERG potassium channels expressed
in human embryonic kidney cells (HEK293), was carried out by
ChanTest Corporation (Cleveland, OH).
In Vivo SOD1 G93A ALS Mice Study of 13. G93A SOD1 mice
and littermate SOD1 mice were mated with B6SJL females, and the
offspring were genotyped by PCR using tails. A 12 h light−dark cycle
was maintained, and animals were given free access to food and water.
Control and transgenic mice of the same age ( 3 days) and from the
same “f” generation were selected from multiple litters to form
experimental cohorts (n = 20 per group). Standardized criteria for age,
ABBREVIATIONS USED
■
ADME, absorption, distribution, metabolism, and excretion;
ALS, amyotrophic lateral sclerosis; ASP, arylsulfanyl pyrazo-
lone; CYP, cytochrome P450; FALS, familial amyotrophic
lateral sclerosis; HEK, human embryonic kidney; hERG, human
ether-a-go-go-related gene; HTS, high-throughput screening;
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amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase
1 aggregates. J. Cell Biol. 2005, 171, 75−85.
MRM, multiple reaction monitoring; PK, pharmacokinetics;
SOD1, Cu/Zn superoxide dismutase 1; SALS, sporadic
amyotrophic lateral sclerosis
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protective against G93A SOD1 toxicity for the treatment of
amyotrophic lateral sclerosis. Amyotrophic Lateral Scler. Other Mot.
Neuron Disord. 2011, 12, 87−96.
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