Pyrimidine-2,4,6-trione derivatives and their inhibition of mutant SOD1-dependent protein aggregation. Toward a treatment for amyotrophic lateral sclerosis

Article abstract of DOI:10.1021/jm101549k

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, most often from respiratory failure. The only FDA-approved drug for the treatment of ALS, riluzole, only extends the median survival in patients by 2-3 months. There is an urgent need for novel therapeutic strategies for this devastating disease. Using a high-throughput screening assay targeting an ALS cultured cell model (PC12-G93A-YFP cell line), we previously identified three chemotypes that were neuroprotective. We present a further detailed analysis of one promising scaffold from that group, pyrimidine-2,4,6-triones (PYTs), characterizing a number of PYT analogues using SAR and ADME. The PYT compounds show good potency, superior ADME data, low toxicity, brain penetration, and excellent oral bioavailability. Compounds from this series show 100% efficacy in the protection assay with a good correlation in activity between the protection and protein aggregation assays. The modifications of the PYT scaffold presented here suggest that this chemical structure may be a novel drug candidate scaffold for use in clinical trials in ALS.

Full text of DOI:10.1021/jm101549k

ARTICLE
pubs.acs.org/jmc
Pyrimidine-2,4,6-trione Derivatives and Their Inhibition of Mutant
SOD1-Dependent Protein Aggregation. Toward a Treatment for
Amyotrophic Lateral Sclerosis
Guoyao Xia,†,# Radhia Benmohamed,‡ Jinho Kim,§ Anthony C. Arvanites,‡,4 Richard I. Morimoto,||
Robert J. Ferrante,§ Donald R. Kirsch,‡ and Richard B. Silverman*,†,^
†Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, United States
‡Cambria Pharmaceuticals, Cambridge, Massachusetts 02142, United States
§Geriatric Research Education and Clinical Center, Bedford Veterans Administration Medical Center, Bedford, Massachusetts 01730,
United States, and Department of Neurology, Laboratory Medicine and Pathology, and Psychiatry, Boston University School of
Medicine, Boston, Massachusetts 02118, 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
b
ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative
disease characterized by the progressive loss of motor neurons, leading to muscle
weakness, paralysis, and death, most often from respiratory failure. The only FDA-
approved drug for the treatment of ALS, riluzole, only extends the median survival
in patients by 2-3 months. There is an urgent need for novel therapeutic strategies
for this devastating disease. Using a high-throughput screening assay targeting an
ALS cultured cell model (PC12-G93A-YFP cell line), we previously identified three
chemotypes that were neuroprotective. We present a further detailed analysis of one promising scaffold from that group, pyrimidine-
2,4,6-triones (PYTs), characterizing a number of PYT analogues using SAR and ADME. The PYT compounds show good potency,
superior ADME data, low toxicity, brain penetration, and excellent oral bioavailability. Compounds from this series show 100%
efficacy in the protection assay with a good correlation in activity between the protection and protein aggregation assays. The
modifications of the PYT scaffold presented here suggest that this chemical structure may be a novel drug candidate scaffold for use
in clinical trials in ALS.
’ INTRODUCTION
exceed $200000 per year,6 placing immense emotional and
economic burdens on patients and their families. There is no
effective treatment for ALS; riluzole, a presumptive antiglutama-
tergic agent,7 is the only FDA-approved therapeutic drug for
ALS, extending the median survival by only 2-3 months.8,9
While the vast majority of ALS cases occur sporadically, about
10% of ALS cases are familial. ALS pathology is caused by the
degeneration of large pyramidal neurons in the motor cortex and
associated corticospinal tracts, lower motor neurons originating
in the brainstem nuclei, and the spinal cord anterior horn. There
has been accumulating evidence that ALS is a noncell-autono-
mous disease in which glia play a prominent active role in disease
progression.10 Major neuropathological findings include (1)
neurofilamentous swelling of proximal axons and accumulation
of neurofilament and peripherin in axons and cell bodies; (2)
perikaryal inclusion bodies containing phosphorylated
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenera-
tive disease characterized by the progressive loss of motor
neurons, leading to muscle atrophy, paralysis, and death within
3-5 years.1 While sporadic (∼90% of cases) and familial forms
of the disease display similar clinical features, a direct causative
pathway to neuronal dysfunction and death has not yet been
established. The similarities in the clinical and pathological
features of familial ALS and sporadic ALS have led investigators
to use the models of genetic mutations associated with familial
ALS as a strategy for elucidating disease pathogenesis and
defining novel treatments for both forms of the disease. The
incidence of ALS is ∼2 cases per 100000 individuals, which
corresponds to a 1 in 2000 lifetime risk to develop the disease,
with death occurring within 2-5 years of diagnosis.2 An in-
creased prevalence of ALS has been observed,3 with the world-
wide affected population estimated to reach ∼108000 patients by
2012,4 with one-third residing in the United States.5 Disability is
significant, and patient care costs in the late stages of ALS can
Received: December 4, 2010
Published: March 04, 2011
r
2011 American Chemical Society
2409
dx.doi.org/10.1021/jm101549k J. Med. Chem. 2011, 54, 2409–2421
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Parallel Synthesis of PYTs
Scheme 2. Synthesis of PYT Analogues with Different R1 and
R2 Groups
neurofilament proteins, ubiquitin, and mutant Cu/Zn super-
oxide dismutase (SOD1) in some cases of familial ALS; (3)
fragmentation of the Golgi apparatus and formation of cytoplas-
mic neuronal inclusion bodies (Bunina bodies); (4) reduced
diameter of distal neurons; and (5) axonal Wallerian degenera-
tion and attenuation of dendrites. The genetic linkage of several
mutations in the gene for Cu/Zn superoxide dismutase (SOD1),
which promote protein aggregation and seen in about 20% of the
cases of familial ALS,11 provided the first indication of a potential
causal factor in the disease process.
Scheme 3. Synthesis of PYT Analogues with Different Types
of R3 Groups
Imbalances in protein homeostasis, which can be caused by
cell stress or expression of certain mutant proteins, can result in
the appearance of alternative conformational states that are able
to self-associate to form protein aggregates and inclusion bodies.
Aberrant protein aggregation has been reported as a common
feature in familial as well as sporadic forms of ALS and mutant
SOD1 transgenic mouse models.12,13 A cultured cell model,
PC12-G93A-YFP cell line, developed by Morimoto and co-
workers,14 was utilized in a high throughput assay to identify
compounds that protect against mutant SOD1-induced cyto-
toxicity. Two assays were established: a cytotoxicity protection
assay, in which compounds were screened for their ability to
protect cells from the cytotoxic effects of aggregated mutant
SOD1, and a protein aggregation assay, in which compounds that
are active in the cytotoxicity protection screen were tested for
their ability to reduce the aggregation of mutant SOD1.15
A library of approximately 50000 small molecules was as-
sembled for screening. The library was constructed to address
two goals: maximization of chemical structural diversity and
inclusion of biologically well-characterized compounds. Pyrimi-
dine-2,4,6-triones (PYT) were identified among the active
compounds and selected as one of the scaffolds for chemistry
optimization. To the best of our knowledge, PYT compounds
have not been reported in any publication or patent for the
treatment of ALS and, therefore, represent a novel chemical
scaffold for this disease. Compounds from this series show 100%
efficacy in the protection assay with a good correlation in activity
between the protection and protein aggregation assays. Thus, it is
reasonable to deduce that these compounds protect PC12 cells
against mutant SOD1-induced cytotoxicity, at least in part, by
reducing SOD1 protein aggregation and that these compounds
are not themselves cytotoxic.
acid/acetic anhydride at 60-90 ꢀC to provide PYT com-
pounds in moderate to high yields.17 Most of the final products
were easily recrystallized from ether/hexanes. The reactions
were carried out in 20 mL vials over a period of 2 days, and
often 8-12 reactions were set up at the same time on pie
blocks with an IKA stir plate. This method readily resulted in
high yields and excellent purity of diverse PYT analogues for
SAR studies.
A new synthetic route was designed for the synthesis of PYT
analogues with different R1 and R2 groups as shown in Scheme 2.
By changing the reaction solvent, temperature, and the amount
of starting material, a precursor of the urea compound could be
obtained with very high yield after purification, and this reaction
could be carried out on a large scale. The precursor reacted with
another amine to provide the desired urea intermediate with
different R1 and R2 groups. Without any further purification, PYT
compounds with different R1 and R2 groups could be achieved
using parallel synthesis procedures.
Further modifications at the R3 position of PYT were executed
as shown in Scheme 3. Following the same parallel synthesis
technology, reactions between crude urea intermediates and
substituted malonic acid provided the PYT analogues with sp3
hybridized groups, such as methyl and dimethyl groups. Intro-
duction of an sp2 hybridized group on the PYT ring at the R3
position was accomplished by a Knoevenagel condensation
between PYT analogues with aldehydes catalyzed by a mixture
of benzoic acid and piperidine.18
’ CHEMISTRY
Parallel synthesis technology was applied to the synthesis of
PYT compounds. As shown in Scheme 1, starting from commer-
cially available S,S-dimethyl carbonodithioate and amines, urea
intermediates were synthesized in high yields.16 After the solvent
was removed under vacuum, without any further purification, the
crude urea compounds were treated with malonic acid in acetic
Further attempts to modify the PYT ring to enol forms were
successfully performed, as shown in Scheme 4.
Reactions between urea intermediates, such as 48, with
oxalyl dichloride or succinyl dichloride provided the desired
2410
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Scheme 4. Synthesis of Enol Forms of PYT Analogues
Scheme 5. Synthesis of Other PYT Analogues
When one of the R groups on the PYT ring is H, the
compounds (1 and 2) exhibited no activity (Figure 2).
When both R groups are alkyl groups, the potency increased
with the size of the alkyl chain (compare 3, 4, and 5). The
introduction of Ph rings to the side chain of PYTs generally
increased the potency. Comparing 6-8 and 10, it is obvious
that the greater the number of carbon atoms in the chain,
the higher the potency of the analogues. However, bulkiness in
the side chain significantly reduces potency, such as in 11. Chan-
ging the phenyl ring to a heterocycle, such as a thiophene ring, did
not improve the potency (7 vs 12). Further introduction of other
atoms, such as O atoms, to provide additional possible binding sites
also did not improve the potency (8 vs 13). However, a phenyl ring
resulted in compounds that were generally more potent than those
with a much smaller ethyl group (13 vs 14).
The introduction of Ph rings resulted in PYT analogues with
higher potency and with further opportunities for SAR studies. PYT
analogues with different substituted groups on the Ph rings were
synthesized by the same parallel synthesis technology (Figure 3).
PYT analogues with electron-withdrawing groups on the Ph
rings showed greater potency than those with electron-donating
groups. The position of substitution on the Ph ring did not show
obvious effects on the potency (compare 15, 16, and 17). Diffe-
rent sized electron-withdrawing groups, such as F and Cl, showed
similar small effects on the potency (16 vs 18 and 17 vs 19).
Figure 1. PYT compounds.
five-membered ring products (49-51) or seven-membered ring
product, such as 52 (Scheme 5).
’ SCREENING RESULTS
Compound activity was determined using the previously
described cytotoxicity protection assay.15 As shown in Figure 1,
PYT compounds contain three carbonyl groups and two nitro-
gen atoms. From the initial high throughput screen, active
compounds had R3 as hydrogen or the carbon between the
carbonyls was sp2 hybridized (alkenyl groups). When R3 was
alkyl, the compounds were usually inactive. When both R1 and R2
were H, the PYT compounds showed very weak activity. When
R1 and R2 were alkyl chains with two or more carbons, the
compounds showed good potency. Some compounds with R1 as
hydrogen and R2 as alkyl (>2 carbons) also showed moderate to
good potency, depending on the R3 group.
2411
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Figure 2. PYT analogues with simple R groups.
Additional electron-withdrawing groups on the Ph ring slightly
increased the potency of the PYTs (compare 18 and 19 to 20).
Extra electron-donating groups on the Ph ring decreased the
potency of PYTs (21 vs 22).
one side chain increased the potency slightly (29 vs 7; 28 vs 31),
and an electron-donating group decreased the potency (30 vs 7).
The introduction of a basic nitrogen into the side chain of the
PYT led to an inactive analogue (34).
PYT analogues with different R1 and R2 groups did not show
any obvious improvement in potency despite several combina-
tions of different side chain groups (Figure 4). The range of
potency was approximately 1-4 μM. If one side chain was held
constant and the other side chain modified, the change in the
variable side chain would affect the potency of the compound as
discussed above; however, the trends were not followed when
both chains were modified. Comparing 23 to 27, the change in
potency was very small although the size of the right side chain
was modified very differently. The introduction of heterocycles
or O atoms on one side chain affected the potency only very
slightly (28, 31, 32, and 33). An electron-withdrawing group on
As shown in Figure 5, 37 and 38, with sp3 hybridized groups,
had no activity, and even 39, with an sp2 hybridized group but no
aromatic ring at the end of the double bond, was devoid of
activity. An sp2 hybridized group with an aromatic ring was key to
good potency. The aromatic ring could be a phenyl ring or other
heterocycle. The electron density and substitution pattern on the
phenyl ring at R3 did not have much effect on the potency (40-
43). A substituted pyrrole ring at R3 enhanced potency, at least in
comparison of 44 and 45.
The conformation of the six-membered ring of the PYT core
was elucidated by X-ray crystallography of 3 and 43. As shown in
Figure 6, the six-membered ring of the PYT core of 3 and 43 is
2412
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Figure 3. PYT analogues with different substituted groups on the Ph rings.
flat, similar to a benzene ring, with both substituents cis. This
unusual structure may be responsible for the activity.
Compounds 46 and 47 (Scheme 4), the vinyl chloride and
enol ether, respectively, derived from 7, are still planar but are
inactive. Therefore, planarity alone is not sufficient for activity.
To test the effect of core ring size, 49-51, having a five-
membered ring, and 52, the corresponding seven-membered
ring analogue, were synthesized (Scheme 5); none was active.
Possibly the distance or angle between the carbonyls is critical for
activity.
two carbons to give 10 was beneficial for enhancement of the
potency and solubility but detrimental for microsomal and
plasma stability. The plasma protein binding for 7 (at 2 μM
concentration) was 95% for rat and 90% for human plasma.
Caco-2 permeability assays were carried out on 7, 10, and 16
(Table 2). Permeability for 7 and 16 is very good, with favorable
asymmetry ratios. Although permeability for 10 is good, there
appears to be a potential efflux problem. The ability of 7 to
penetrate the blood-brain barrier was confirmed, showing a 3:1
blood:brain ratio (150 μM:50 μM) (Figure 9). Although blood is
measured in microliters and brain in milligrams, the density of
blood plasma is 1.025 mg/μL.19 Therefore, the weight of blood is
roughly equivalent to its volume, and a direct comparison between
the amount of compound in blood and brain can be made. This
indicates that sufficient compound can be delivered to the brain to
attain maximum activity, based on the in vitro cell assay.
’ MUTANT SOD1-INDUCED CYTOTOXICITY PROTEC-
TION ASSAY
PC12 cells expressing G93A SOD1-YFP were employed to
develop a high throughput assay to identify compounds that
block mutant SOD1 toxicity. Figure 7 is a representative dose-
response curve for 7 in this assay.
The IC50 values for 7 with the major CYP isozymes present in
human liver microsomes, CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4, were all >50 μM, indicating its lack of interference
with major metabolism enzymes in liver.
’ INHIBITION OF HIGH CONTENT AGGREGATION OF
G85R SOD1 BY 7
The human ether-a-go-go related gene (hERG) encodes for a
protein that is the inner pore-forming portion of a potassium
channel in heart muscle. This potassium ion channel controls the
flow of Kþ ions across the cell membrane. It has been reported
that compounds that inhibit this channel produce heart arrhyth-
mias (acquired long QT syndrome),20 which can lead to death in
some cases, and unfortunately in some instances this toxic
liability was identified only after clinical introduction of the drug.
Therefore, it is imperative that compounds do not block the
hERG channel. The in vitro effect of 7 on hERG potassium
channel current expressed in human embryonic kidney cells
(HEK293) was evaluated at room temperature using a patch-
clamp technique, the most definitive in vitro hERG inhibition
assay. A positive control (E-4031) was used to confirm the
sensitivity of the test system, and the TurboSol evaluation system
The reduction in the number of aggregates of G85R SOD1 by
7 was found to be concentration dependent (Figure 8). Fluo-
rescence images of cells treated with MG132 and with MG132
plus 10 μM compound 7 showed a significant reduction in
aggregates in the 7-treated cells (see Supporting Information).
’ IN VITRO AND IN VIVO ADME STUDIES
In vitro ADME studies were conducted on 7, 10, and 16. As
shown in Table 1, compound 7 showed the best overall
combination of potency, PBS solubility, and microsomal and
plasma stabilities. Fluorine substitution on the phenyl ring of 7
(compound 16) worsened the ADME properties except for
plasma stability. Extending the two-carbon side chains of 7 by
2413
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Figure 4. PYT analogues with different R1 and R2 groups.
was performed to confirm that poor compound solubility was not
a reason for low hERG inhibition in the test system. As shown in
Table 3, there is little hERG channel blockage by 7.
In vivo PK profiling of 7 at 1 mg/kg bolus intravenous and oral
administration doses in Sprague-Dawley rats was determined
(Table 4). Although a moderate plasma clearance was observed
for both iv and po administration, the oral bioavailability was
98%, indicating extremely good oral absorption.
A profiling screen was conducted for 7 with a broad package of
68 in vitro radioligand binding assays covering proteins involved
in the most commonly observed adverse CNS, cardiovascular,
pulmonary, and genotoxic events in order to detect potential
adverse activity, additional unexpected activity, relative selec-
tivity, and specificity (MDS Pharma Services). Compound 7 was
tested at a 10 μM concentration in duplicate. No significant
inhibitory effect was noted [the four receptors showing the
greatest effects (and % inhibition) were dopamine D3 (41%),
sodium channel site 2 (21%), estrogen ERR (20%), and GABA
transporter (19%); see Supporting Information for detailed
results], suggesting that 7 would be unlikely to introduce adverse
effects related to the enzymes and receptors tested.
’ CONCLUSIONS
PYT analogues were identified as potential drug candidates
with good potency, ADME properties, and protein profiling
properties. Some generalities about the SAR of PYT analogues
can be made: (1) The size of the R1/R2 group is critical for good
potency. (2) Electron density changes on the Ph ring of the R1/
R2 groups do not affect the potency. (3) Other potential
pharmacophores on the R1/R2 groups (e.g., oxygen atoms) do
not affect potency. (4) R3 groups with sp2 hybridization at the
core ring increase potency but increase the molecular weight as
well. (5) A six-membered flat ring of PYT is essential for good
potency. One PYT compound, 7, has exhibited a desirable
combination of potency, ADME properties, low toxicity, brain
penetration, oral bioavailability, and protein profiling and has
been selected for further animal testing; this may be a novel drug
candidate scaffold for use in ALS clinical trials.
’ EXPERIMENTAL SECTION
General Procedures. All reactions were carried out in oven- or
flame-dried glassware under an atmosphere of air unless otherwise
2414
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Figure 5. PYT analogues with different R3 groups.
noted. Except as otherwise indicated, all reactions were magnetically
stirred and monitored by analytical thin-layer chromatography using
Whatman precoated silica gel flexible plates (0.25 mm) with F254
indicator or Merck precoated silica gel plates with F254 indicator.
Visualization was accomplished by UV light (256 nm) or by potassium
permanganate and/or phosphomolybdic acid solution as an indicator.
Flash column chromatography was performed using silica gel 60 (mesh
230-400) supplied by E. Merck. Yields refer to chromatographically
and spectrographically pure compounds, unless otherwise noted. Com-
mercial grade reagents and solvents were used without further purifica-
tion except as indicated below. Tetrahydrofuran (THF) was distilled
from sodium benzophenone ketyl under an atmosphere of dry nitrogen.
1H NMR and 13C NMR spectra were recorded on a Bruker Avance III
(500 MHz 1H, 125 MHz 13C) with DCH Cryo-Probe. Chemical shift
values (δ) are reported in parts per million (ppm) relative to CDCl3 [δ
7.26 ppm (1H), 77.16 ppm (13C)]. The proton spectra are reported as
follows δ (multiplicity, number of protons). Multiplicities are indicated
by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (heptet),
m (multiplet), and br (broad). Elemental analyses were performed by
Atlantic Microlab Inc., Norcross, GA. The C, H, and N analyses were
performed by combustion using automatic analyzers, and all of the
compounds analyzed showed >95% purity.
vacuo, the crude urea residue and malonic acid (0.5 mmol) were
dissolved in 3 mL of glacial acetic acid. The mixture was heated to
60 ꢀC, and 2 mL of acetic anhydride was added over 30 min. The
reaction mixture was heated with stirring at 90 ꢀC for 4 h. The solvent
was removed under vacuum at 60 ꢀC, and the residue was recrystallized
from ether/hexanes or purified by silica gel chromatography to provide
the desired PYT analogue.
1,3-Diphenethylurea (48). 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 4
H), 7.21 (m, 2 H), 7.10 (m, 4 H), 4.46 (br, 2 H), 3.38 (m, 4 H), 2.76 (t, 4
H); 13C NMR (125 MHz, CDCl3) δ 158.1, 139.2, 128.9, 128.6, 126.5,
41.7, 36.5.
1-Butylpyrimidine-2,4,6(1H,3H,5H)-trione (1). 1H NMR (500 MHz,
CDCl3) δ 8.25 (s, 1 H), 3.90 (t, 2 H), 2.17 (s, 2 H), 1.60 (m, 2 H), 1.35
(m, 2 H), 0.94 (t, 3 H); 13C NMR (125 MHz, CDCl3) δ 170.6, 161.0,
149.0, 126.8, 40.8, 24.6, 20.1, 13.8. EC50 > 32 μM.
1
1-Cyclohexylpyrimidine-2,4,6(1H,3H,5H)-trione (2). H NMR (500
MHz, CDCl3) δ 7.92 (s, 1 H), 4.66 (m, 1 H), 2.70 (s, 2 H), 2.33 (m, 2
H), 1.84 (m, 2 H), 1.65 (m, 2 H), 1.36 (m, 2 H), 1.25 (m, 2 H); 13C
NMR (125 MHz, CDCl3) δ 196.4, 171.2, 160.9, 148.9, 96.0, 54.7, 29.1,
26.4, 25.2, 24.7. Elemental analysis: Comb. Anal. (C10H14N2O3),
Calculated: C, 57.13; H, 6.71; N, 13.33; O, 22.83. Found: C, 56.92;
H, 6.31; N, 11.21. Purity: >95%. EC50 > 32 μM.
1
Typical Procedure for Parallel Synthesis of PYT Analogues. S,S-
Dimethyl carbonodithioate (0.5 mmol) was added dropwise to a
solution of amine (1.05 mmol) in MeOH (5 mL) with stirring, and
the mixture was heated at 60 ꢀC for 24 h. After removal of solvent in
1,3-Dipropylpyrimidine-2,4,6(1H,3H,5H)-trione (3). H NMR (500
MHz, CDCl3) δ 3.81 (m, 4 H), 3.64 (s, 2 H), 1.61 (m, 4 H), 0.92 (t, 6
H); 13C NMR (125 MHz, CDCl3) δ 164.7, 151.5, 137.8, 43.6, 39.7, 21.3,
11.3. Elemental analysis: Anal. (C10H16N2O3), Calculated: C, 56.59; H,
2415
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
13C NMR (125 MHz, CDCl3) δ 164.5, 136.0, 129.3, 128.7, 128.2, 45.2, 39.8.
EC50 4.58 μM.
1,3-Diphenethylpyrimidine-2,4,6(1H,3H,5H)-trione (7). 1H NMR-
(500 MHz, CDCl3) δ 7.29 (m, 4 H), 7.22 (m, 6 H), 4.08
(m, 4 H), 3.56 (s, 2 H), 2.86 (m, 4 H); 13C NMR (125 MHz, CDCl3)
δ 164.4, 151.1, 137.8, 129.0 128.7, 126.8, 43.1, 39.6, 34.1. Elemental
analysis: Comb. Anal. (C20H20N2O3), Calculated: C, 71.41; H, 5.99;
N, 8.33; O, 14.27. Found: C, 71.29; H, 5.91; N, 8.27. Purity: >95%. EC50
1.68 μM.
1,3-Bis(3-phenylpropyl)pyrimidine-2,4,6(1H,3H,5H)-trione (8). 1H
NMR (500 MHz, CDCl3) δ 7.25 (m, 4 H), 7.18 (m, 6 H), 3.89 (m, 4
H) 3.34 (s, 2 H), 2.68 (m, 4 H) 1.97 (m, 4 H); 13C NMR (125 MHz,
CDCl3) δ 164.5, 151.2, 140.9, 128.4, 128.2, 126.0, 41.8, 39.3, 33.0, 28.4.
EC50 1.32 μM.
1,3-Bis(3,3-diphenylpropyl)pyrimidine-2,4,6(1H,3H,5H)-trione (9).
1H NMR (500 MHz, CDCl3) δ 7.29-7.27 (m, 16 H), 7.17 (m, 4 H),
4.03 (d, 2 H), 3.90 (m, 4 H), 3.06 (s, 2 H), 2.42 (m, 4 H); 13C NMR (125
MHz, CDCl3) δ 164.6, 151.2, 144.2, 128.7, 127.5, 126.5, 49.7, 41.4, 39.1,
32.7. EC50 1.39 μM.
1,3-Bis(4-phenylbutyl)pyrimidine-2,4,6(1H,3H,5H)-trione (10). 1H
NMR (500 MHz, CDCl3) δ 7.28 (m, 4 H), 7.19 (m, 6 H), 3.89 (t, 4
H), 3.61 (s, 2 H), 2.65 (t, 4 H), 1.65 (m, 8 H); 13C NMR (125 MHz,
CDCl3) δ 164.6, 151.4, 142.0, 128.5, 128.4, 125.9, 41.9, 39.6, 35.5, 28.6,
27.6. Elemental analysis: Comb. Anal. (C24H28N2O3), Calculated: C,
73.44; H, 7.19; N, 7.14; O, 12.23. Found: C, 73.29; H, 7.26; N, 7.20.
Purity: >95%. EC50 0.68 μM.
1,3-Bis(2,3-dihydro-1H-inden-1-yl)pyrimidine-2,4,6(1H,3H,5H)-trione (11).
1H NMR (500 MHz, CDCl3) δ 7.25 (m, 8 H), 5.46 (m, 2 H), 2.98 (m, 2 H),
2.86 (m, 2 H), 2.59 (m 2 H), 2.02 (m, 2 H), 1.80 (m, 2 H); 13C NMR (125
MHz, CDCl3) δ170.0, 143.5, 143.2, 128.0, 126.8, 124.9, 124.1, 54.8, 34.1, 30.3,
23.6. EC50 > 32 μM.
Figure 6. X-ray structure of the PYT core from different angles.
1,3-Bis(2-(thiophen-2-yl)ethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (12).
1H NMR (500 MHz, CDCl3) δ 7.16 (m, 2 H), 6.94 (m, 2 H), 6.86 (m 2
H), 4.15 (m, 4 H), 3.61 (s, 2 H), 3.11 (m, 4 H); 13C NMR (125 MHz,
CDCl3) δ164.4, 151.1, 139.8, 127.2 125.9, 124.3, 43.1, 39.7, 27.9. Elemental
analysis: Comb. Anal. (C16H16N2O3S2), Calculated: C, 55.15; H, 4.63; N,
8.04; O, 13.78; S, 18.40. Found: C, 55.15; H, 4.54; N, 8.04. Purity: >95%.
EC50 2.16 μM.
1,3-Bis(2-phenoxyethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (13). 1H
NMR (500 MHz, CDCl3) δ 7.24 (m, 4 H), 6.94 (m, 2 H), 6.86 (m, 4 H),
4.32 (t, 4 H), 4.19 (m, 4 H), 3.63 (s, 2 H); 13C NMR (125 MHz, CDCl3)
δ 164.5, 158.3, 151.4, 129.6, 121.3, 114.6, 64.2, 41.0, 39.7. Elemental
analysis: Comb. Anal. (C20H20N2O5), Calculated: C, 65.21; H, 5.47; N,
7.60; O, 21.72. Found: C, 65.36; H, 5.53; N, 7.59. Purity: >95%. EC50
3.26 μM.
Figure 7. Dose-response curve for 7 in G93A SOD1 protection assay.
1,3-Bis(2-ethoxyethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (14). 1H
NMR (500 MHz, CDCl3) δ 4.16 (t, 4 H), 3.68 (m, 4 H), 3.56 (s, 2
H), 3.45 (m, 4 H), 1.15 (t, 6 H); 13C NMR (125 MHz, CDCl3) δ 167.7,
149.8, 66.8, 66.2, 47.3, 41.5, 39.7, 15.0. EC50 7.75 μM.
7.60; N, 13.20; O, 22.61. Found: C, 56.30; H, 7.51; N, 13.25;. Purity:
>95%. EC50 7.68 μM.
1,3-Dicyclohexylpyrimidine-2,4,6(1H,3H,5H)-trione (4). 1H NMR
(500 MHz, CDCl3) δ 4.58 (m, 2 H), 3.59 (s, 2 H), 2.25 (m, 4 H),
2.25 (m, 4 H), 1.84 (m, 4 H), 1.63 (m, 6 H), 1.35-1.19 (m, 6 H); 13C
NMR (125 MHz, CDCl3) δ 165.1, 151.4, 55.4, 41.1, 29.2, 26.4, 25.2.
Elemental analysis: Comb. Anal. (C16H24N2O3), Calculated: C, 65.73;
H, 8.27; N, 9.58; O, 16.42. Found: C, 65.86; H, 8.32; N, 9.53. Purity:
>95%. EC50 2.97 μM.
1,3-Bis(2-fluorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (15).
1H NMR (500 MHz, CDCl3) δ 7.20 (m, 4 H), 7.05 (m, 4 H), 4.12
(t, 4 H), 3.55 (s, 2 H), 2.91 (t, 4 H); 13C NMR (125 MHz, CDCl3) δ
164.4, 151.1, 131.3, 128.8, 128.7, 125.0, 124.3, 115.5, 41.7, 39.5, 27.6.
EC50 3.29 μM.
1,3-Bis(3-fluorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (16).
1H NMR (500 MHz, CDCl3) δ 7.30 (m, 2 H), 7.04-6.96 (m, 6 H),
4.10 (m, 4 H), 3.64 (s, 2 H), 2.89 (m, 4 H); 13C NMR (125 MHz,
CDCl3) δ 164.3, 151.1, 140.3, 130.2, 124.7, 116.0, 115.9, 113.9,
42.9,39.6, 33.8. EC50 3.23 μM.
1,3-Bis(4-fluorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (17).
1H NMR (500 MHz, CDCl3) δ 7.20 (m, 4 H), 7.00 (m, 4 H), 4.06
(m, 4 H), 3.60 (s, 2 H), 2.84 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ
164.3, 151.2, 130.5, 115.9, 115.6, 115.4, 43.1, 39.6, 33.3. Elemental
analysis: Comb. Anal. (C20H18F2N2O3), Calculated: C, 64.51; H, 4.87;
1,3-Bis(3,3-dimethylbutyl)pyrimidine-2,4,6(1H,3H,5H)-trione (5).
1H NMR (500 MHz, CDCl3) δ 3.88 (m, 4 H), 3.63 (s, 2 H), 1.46 (m, 4
H), 0.97 (S, 9 H); 13C NMR (125 MHz, CDCl3) δ 164.5, 151.5, 41.1,
39.8, 39.1, 30.0, 29.2. Elemental analysis: Comb. Anal. (C16H28N2O3),
Calculated: C, 64.83; H, 9.52; N, 9.45; O, 16.19. Found: C, 64.93; H,
9.62; N, 9.42. Purity: >95%. EC50 2.03 μM.
1,3-Dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione (6). 1H NMR (500 MHz,
CDCl3) δ 7.41 (m, 4 H), 7.30 (m, 6 H), 5.03 (s, 4 H), 3.69 (s, 2 H);
2416
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Figure 8. Concentration-dependent reduction of aggregates by 7 in the high content G85R-SOD1 aggregation assay.
Table 1. ADME Properties for Selected PYT Compounds
Table 2. Caco-2 Permeability Assays
compd
concn (μM)
assay duration (h)
mean A f B Papp (10-6 cm s-1
)
mean B f A Papp (10-6 cm s-1
)
asymmetry ratioa
7
10
16
50
50
50
2
2
2
73.4
9.2
26.0
30.2
17.1
0.4
3.3
0.3
66.3
a Papp(B f A)/Papp(A f B).
1,3-Bis(3-chlorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (18).
1H NMR (500 MHz, CDCl3) δ 7.25 (m, 6 H), 7.15 (m, 2 H), 4.09
(m, 4 H), 3.65 (s, 2 H), 2.86 (m, 4 H); 13C NMR (125 MHz, CDCl3)
δ 164.3, 151.1, 139.8, 134.4, 130.0, 129.2, 127.1, 42.9, 39.6, 33.7. EC50
2.97 μM.
1,3-Bis(4-chlorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (19).
1H NMR (500 MHz, CDCl3) δ 7.28 (m, 4 H), 7.17 (m, 4 H), 4.05
(m, 4 H), 3.61 (s, 2 H), 2.83 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ
164.3, 151.1, 136.2, 132.7, 130.4, 128.8, 42.9, 39.6, 33.4. EC50 3.26 μM.
1,3-Bis(2,4-dichlorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(20). 1H NMR (500 MHz, CDCl3) δ 7.37 (m, 2 H), 7.18 (m, 4 H), 4.11
(t, 4 H), 3.60 (s, 2 H), 2.98 (t, 4 H); 13C NMR (125 MHz, CDCl3) δ
164.3, 151.1, 135.0, 134.5, 133.5, 131.9, 129.5, 127.4, 41.4, 39.6, 31.3.
EC50 1.87 μM.
Figure 9. Blood brain penetration of 7.
F, 10.20; N, 7.52; O, 12.89. Found: C, 63.23; H, 4.56; N, 6.85. Purity:
>95%. EC50 3.45 μM.
1,3-Bis(4-methoxyphenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(21). 1H NMR (500 MHz, CDCl3) δ 7.15 (m, 4 H), 6.84 (m, 4 H), 4.05
2417
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
Table 3. Effect of 7 on the hERG Channel
compd
n
concn (μM)
mean hERG inhibition (%)
std deviation
std error
7
2
2
10
4.9
1.8
0.7
1.3
0.5
positive control
0.5
98.7
Table 4. Plasma PK Profile of 7 (1 mg/kg iv and 1 mg/kg po) in Sprague-Dawley Rats Administered in PBS
route
AUC last (ng h/mL)
AUC/dose (ng h/mL/dose)
t1/2 (h)
Cl (mL/min/kg)
133
Vss (L/kg)
9.0
Cmax (ng/mL)
Tmax (h)
F (%)
3
3
iv
121
124
126
124
0.95
1.4
po
55
0.75
98
(m, 4 H), 3.77 (s, 6 H), 3.57 (s, 2 H), 2.81 (m, 4 H); 13C NMR (125
MHz, CDCl3) δ 164.4, 158.5, 151.2, 130.6, 129.8 128.7, 114.0, 55.3,
43.3, 39.6, 33.2. Elemental analysis: Comb. Anal. (C22H24N2O5),
Calculated: C, 66.65; H, 6.10; N, 7.07; O, 20.18. Found: C, 66.50; H,
5.98; N, 7.06. Purity: >95%. EC50 3.36 μM.
1,3-Bis(3,4-dimethoxyphenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(22). 1H NMR (500 MHz, CDCl3) δ 6.78 (m, 3 H), 4.07 (m, 4 H), 3.89
(s, 6 H), 3.84 (s, 6 H), 3.61 (s, 2 H), 2.81 (m, 4 H); 13C NMR (125 MHz,
CDCl3) δ 164.4, 151.2, 149.0, 147.9, 130.2, 121.0, 112.0, 111.2, 56.0,
43.3, 39.7, 33.8. EC50 11.59 μM.
5-Methyl-1,3-diphenethylpyrimidine-2,4,6(1H,3H,5H)-trione (37).
1H NMR (500 MHz, CDCl3) δ 7.29 (m, 4 H), 7.22 (m, 6 H), 4.11 (m, 4
H), 3.34 (q, 1 H), 2.90 (m, 4 H), 1.45 (d, 3 H); 13C NMR (125 MHz,
CDCl3) δ 168.7, 150.9, 137.8, 129.1 128.6, 126.8, 44.1, 43.1, 34.0, 14.7.
Elemental analysis: Comb. Anal. (C21H22N2O3), Calculated: C, 71.98;
H, 6.33; N, 7.99; O, 13.70. Found: C, 69.98; H, 6.17; N, 7.84. Purity:
>95%. EC50 > 32 μM.
5,5-Dimethyl-1,3-diphenethylpyrimidine-2,4,6(1H,3H,5H)-trione
(38). 1H NMR (500 MHz, CDCl3) δ 7.29-7.21 (m, 10 H), 4.10 (m, 4
H), 2.29 (t, 4 H), 1.33 (s, 6 H); 13C NMR (125 MHz, CDCl3) δ 172.4,
150.5, 137.8, 129.2, 128.6, 126.8, 47.3, 42.9, 34.0, 24.9. EC50 > 32 μM.
Typical Procedure for Parallel Synthesis of PYT Analogues with
Different R1 and R2 Groups. S,S-Dimethyl carbonodithioate (0.5 mmol)
was added dropwise to a solution of R1NH2 (0.75 mmol) in H2O (5 mL)
with stirring at room temperature, and the mixture was stirred for 24 h.
After removal of solvent in vacuo, the residue was recrystallized from
ether/hexanes to afford R1NHCOSMe.
129.1 128.7, 126.8, 43.1, 42.2, 39.7, 34.1, 31.9, 29.3, 28.0, 26.9, 22.7,
14.2. EC50 0.71 μM.
1-(2-Cyclohexenylethyl)-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-
trione (25). 1H NMR (500 MHz, CDCl3) δ 7.32-7.25 (m, 5 H), 5.39
(s, 1 H), 4.13 (m, H), 3.97 (t, 2 H), 3.61 (s, 2 H), 2.91 (m, 2 H), 2.19
(m, 2 H), 1.96-1.86 (m, 4 H), 1.64-1.54 (m, 4 H); 13C NMR
(125 MHz, CDCl3) δ 164.5, 164.4, 151.2, 137.9, 134.6, 129.0 128.7,
126.8, 123.9, 43.1, 40.6, 39.6, 36.2, 34.1, 28.1, 25.4, 22.9. 22.3. EC50
1.74 μM.
1-Benzyl-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-trione (26). 1H
NMR (500 MHz, CDCl3) δ 7.44-7.22 (m, 10 H), 5.05 (s, 2 H), 4.12
(m, 2 H), 3.63 (s, 2 H), 2.92 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ
164.6, 164.3, 151.3, 137.7, 136.1, 129.2, 129.0 128.7, 128.6, 128.1, 126.8,
45.0, 43.2, 39.6, 34.0. EC50 1.90 μM.
1-Phenethyl-3-(3-phenylpropyl)pyrimidine-2,4,6(1H,3H,5H)-trione
(27). 1H NMR (500 MHz, CDCl3) δ 7.30-7.20 (m, 10 H), 4.07 (m, 2
H), 3.96 (t, 2 H), 3.49 (s, 2 H), 2.90 (m, 2 H), 2.70 (t, 2 H), 1.99 (m, 2
H); 13C NMR (125 MHz, CDCl3) δ 164.6, 164.4, 151.2, 141.1, 137.8,
129.0, 128.7, 128.5, 128.2, 126.8, 126.1, 43.1, 42.0, 39.5, 34.1, 33.1, 28.6.
EC50 1.88 μM.
1-Phenethyl-3-(2-(thiophen-2-yl)ethyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (28). 1H NMR (500 MHz, CDCl3) δ 7.30-7.14 (m, 6 H), 6.94
(m, 2 H), 4.17 (m, 2 H), 3.90 (m, 2 H), 3.64 (s, 2 H), 3.17 (m, 2 H),
2.73-2.67 (m, 4 H), 1.67 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ
196.5, 169.4, 160.8, 151.2, 142.0, 139.8, 128.5, 127.1, 125.9, 125.7, 124.3,
96.0, 43.0, 42.7, 42.5, 41.4, 29.6, 28.8, 27.9, 24.9. EC50 3.39 μM.
1-(4-Chlorophenethyl)-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-
trione (29). 1H NMR (500 MHz, CDCl3) δ 7.26 (m, 7 H), 7.20 (m, 2
H), 4.09 (m, 4 H), 3.61 (s, 2 H), 2.87 (m, 4 H); 13C NMR (125 MHz,
CDCl3) δ 164.4, 164.3, 151.1, 137.8, 136.2, 132.7, 130.4, 129.0,
128.8, 128.7, 126.9, 43.2, 42.9, 39.6, 34.1, 33.4. Elemental analysis:
Comb. Anal. (C20H19ClN2O3), Calculated: C, 64.78; H, 5.16; Cl,
9.56; N, 7.55; O, 12.94. Found: C, 64.96; H, 5.19; N, 7.55. Purity:
>95%. EC50 1.19 μM.
1-(4-Methoxyphenethyl)-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-
trione (30). 1H NMR (500 MHz, CDCl3) δ 7.31 (m, 2 H), 7.26 (m, 3
H), 7.17 (m, 2 H), 6.86 (m, 2 H), 4.08 (m, 4 H), 3.81 (s, 3 H), 3.60 (s, 2
H), 2.87 (t, 2 H), 2.83 (t, 2 H); 13C NMR (125 MHz, CDCl3) δ 164.4,
158.5, 151.2, 137.8, 130.0, 129.8, 129.0 128.7, 126.8, 114.0, 55.3, 43.3,
43.1, 39.6, 34.1, 33.2. Elemental analysis: Comb. Anal. (C21H22N2O4),
Calculated: C, 68.84; H, 6.05; N, 7.65; O, 17.47. Found: C, 68.65; H,
6.00; N, 7.59. Purity: >95%. EC50 2.94 μM.
A solution of R1NHCOSMe (0.5 mmol) and R2NH2 (0.5 mmol) in
MeOH (5 mL) was heated at 60 ꢀC with stirring for 24 h. After removal
of solvent in vacuo, the crude urea residue and malonic acid (0.5 mmol)
were dissolved in 3 mL of glacial acetic acid. The mixture was heated to
60 ꢀC, and 2 mL of acetic anhydride was added over 30 min. The
reaction mixture was heated with stirring at 90 ꢀC for 4 h. The solvent
was removed under vacuum at 60 ꢀC, and the residue was recrystallized
from ether/hexanes or purified by silica gel chromatography to provide
the desired PYT analogue.
S-Methyl 4-Fluorophenethylcarbamothioate. 1H NMR (500 MHz,
CDCl3) δ 7.13 (m, 2 H), 6.99 (m, 2 H), 5.41 (br, 1 H), 3.51 (m, 2 H),
2.80 (m, 2 H), 2.34 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 167.9,
162.7, 160.8, 134.2, 130.3, 130.2, 115.6, 115.5, 42.7, 35.2, 12.4.
1-Cyclohexyl-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-trione (23).
1H NMR (500 MHz, CDCl3) δ 7.29 (m, 2 H), 7.24 (m, 3 H), 4.59 (m, 1
H), 4.08 (m, 2 H), 3.58 (s, 2 H), 2.89 (m, 2 H), 2.22 (m, 2 H), 1.84 (m, 2
H), 1.67-1.57 (m, 3 H), 1.34-1.22 (m, 3 H); 13C NMR (125 MHz,
CDCl3) δ 164.9, 164.8, 151.2, 137.9, 129.0 128.6, 126.8, 55.4, 43.1, 40.3,
34.1, 29.1, 26.4, 25.2. EC50 1.67 μM.
1-(4-Fluorophenethyl)-3-(2-(thiophen-2-yl)ethyl)pyrimidine-2,4,6-
(1H,3H,5H)-trione (31). 1H NMR (500 MHz, CDCl3) δ 7.20-7.15 (m,
3 H), 7.00-6.94 (m, 3 H), 6.86 (d, 1 H), 4.16 (t, 2 H), 4.05 (m, 2 H),
3.60 (s, 2 H), 3.12 (t, 2 H), 2.84 (t, 4 H); 13C NMR (125 MHz, CDCl3)
δ 164.4, 162.8, 160.9, 151.1, 139.6, 133.5, 130.5, 127.2, 125.9, 124.3,
115.6, 115.4, 43.2, 43.0, 39.6, 33.3, 27.9. EC50 2.16 μM.
1
1-Octyl-3-phenethylpyrimidine-2,4,6(1H,3H,5H)-trione (24). H
NMR (500 MHz, CDCl3) δ 7.30-7.23 (m, 5 H), 4.12 (m, 2 H), 3.83
(t, 2 H), 3.61 (s, 2 H), 2.90 (t, 2 H), 1.54 (m, 2 H), 1.28 (m, 10 H),
0.88 (t, 3 H); 13C NMR (125 MHz, CDCl3) δ 164.5, 151.3, 137.9,
1-(4-Fluorophenethyl)-3-(2-phenoxyethyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (32). 1H NMR (500 MHz, CDCl3) δ 7.30 (m, 2 H), 7.21 (m, 2
2418
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
H), 6.97 (m, 3 H), 6.89 (m, 2 H), 4.35 (t, 2 H), 4.19 (t, 2 H), 4.09 (m, 2
H), 3.64 (s, 2 H), 2.89 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ 164.4,
164.3, 162.8, 160.9, 158.3, 151.3, 133.5, 130.5, 129.6, 121.3, 115.6, 115.4,
114.5, 64.2, 43.2, 41.0, 39.6, 33.2. EC50 1.32 μM.
1-(4-Fluorophenethyl)-3-(2-isopropoxyethyl)pyrimidine-2,4,6(1H,
3H,5H)-trione (33). 1H NMR (500 MHz, CDCl3) δ 7.21 (m, 2 H), 6.99
(m, 2 H), 4.07 (m, 4 H), 3.64 (s, 2 H), 3.62-3.56 (m, 3 H), 3.28 (t, 2 H),
1.12 (d, 6 H); 13C NMR (125 MHz, CDCl3) δ 164.6, 162.8, 160.9,
151.3, 133.5, 130.5, 115.6, 71.6, 64.1, 43.2, 41.4, 39.7, 33.3, 22.1.
Elemental analysis: Comb. Anal. (C17H21FN2O4), Calculated: C,
60.70; H, 6.29; F, 5.65; N, 8.33; O, 19.03. Found: C, 60.56; H, 6.23;
N, 8.24. Purity: >95%. EC50 2.97 μM.
1-(4-Fluorophenethyl)-3-(2-morpholinoethyl)pyrimidine-2,4,6(1H,
3H,5H)-trione (34). 1H NMR (500 MHz, CDCl3) δ 7.19 (m, 2 H), 6.96
(m, 2 H), 4.08 (m, 4 H), 3.75-3.66 (m, 4 H), 2.87 (m, 4 H), 2.78-2.61
(m, 6 H); 13C NMR (125 MHz, CDCl3) δ 196.4, 162.7, 160.8, 134.8,
130.5, 130.4, 115.5, 115.3, 96.0, 53.7, 42.4, 33.5. EC50 > 32 μM.
1-(4-Fluorophenethyl)-3-(4-phenylbutyl)pyrimidine-2,4,6(1H,3H,
5H)-trione (35). 1H NMR (500 MHz, CDCl3) δ 7.29 (m, 3 H), 7.20 (m,
4 H), 6.90 (m, 2 H), 4.01 (m, 2 H), 3.90 (m, 2 H), 3.54 (s, 2 H), 2.80 (m,
2 H), 2.56 (m, 2 H), 1.55 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
164.5, 162.8, 160.9, 151.3, 142.0, 133.5, 130.5, 128.5, 126.0, 115.5, 115.4,
43.1, 42.0, 39.6, 35.5, 33.3, 28.7, 27.6. Elemental analysis: Comb.
Anal. (C22H23FN2O3), Calculated: C, 69.09; H, 6.06; F, 4.97;
N, 7.33; O, 12.55. Found: C, 68.92; H, 6.55; N, 7.19. Purity: >95%.
EC50 3.55 μM.
1-(2,2-Diphenylethyl)-3-(4-phenylbutyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (36). 1H NMR (500 MHz, CDCl3) δ 7.31-7.19 (m, 15 H), 4.60
(m, 2 H), 4.55 (t, 1 H), 3.81 (m, 2 H), 3.43 (s, 2 H), 2.64 (m, 2 H),
1.63-1.54 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ 196.3, 164.6,
164.4, 151.3, 142.0, 141.4, 141.0, 128.8, 128.6, 128.5, 128.4, 128.1, 127.1,
126.8, 126.0, 48.6, 45.6, 41.8, 39.5, 35.5, 28.5, 27.5. EC50 3.61 μM.
Typical Procedure for Synthesis of PYT Analogues with R3 Groups by
Knoevenagel Condensation. Into a mixture of 7 (0.5 mmol), piperidine
(0.05 mmol), benzoic acid (0.05 mmol), and benzene (10 mL) was
added slowly the aldehyde (0.5 mmol). The reaction apparatus was
equipped with a Dean-Stark trap, and the reaction mixture was refluxed
for 8 h. After cooling the mixture to room temperature and washing it
with water, the solvent was removed in vacuo, and the residue was
purified by silica gel chromatography to provide the desired PYT
analogue.
127.6, 126.7, 126.6, 125.8, 114.9, 114.6, 70.4, 43.8, 43.1, 34.3. EC50 2.84
μM.
1,3-Dicyclohexyl-5-(2,4-dimethoxybenzylidene)pyrimidine-2,4,6-
(1H,3H,5H)-trione (43). 1H NMR (500 MHz, CDCl3) δ 8.51 (s, 1 H),
8.39 (m, 1 H), 6.56 (m, 1 H), 6.42 (m, 1 H), 4.75 (m, 2 H), 3.88 (s, 6 H),
2.34 (m, 4 H), 1.82 (m, 4 H), 1.66 (m, 6 H), 1.32 (m, 6 H); 13C NMR
(125 MHz, CDCl3) δ 165.9, 163.4, 162.4, 161.3, 153.0, 151.1, 135.6,
115.7, 104.9, 97.5, 55.9, 55.7, 54.8, 29.3, 26.6, 25.4. Elemental analysis:
Comb. Anal. (C25H32N2O5), Calculated: C, 68.16; H, 7.32; N, 6.36; O,
18.16. Found: C, 67.98; H, 7.36; N, 6.36. Purity: >95%. EC50 2.58 μM.
5-((1H-Pyrrol-2-yl)methylene)-1,3-diphenethylpyrimidine-2,4,6-
(1H,3H,5H)-trione (44). 1H NMR (500 MHz, CDCl3) δ 8.34 (s, 1 H),
7.56 (m, 1 H), 7.29 (m, 8 H), 7.22 (m, 2 H), 7.15 (m, 1 H), 6.56 (m, 1
H), 4.20 (m, 4 H), 2.92 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ 163.4,
163.0, 150.8, 143.1, 138.5, 131.5, 129.7, 129.1, 128.6, 126.7, 126.6, 114.8,
105.8, 43.6, 43.2, 34.3. EC50 3.55 μM.
5-((2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene)-1,3-diphe-
1
nethylpyrimidine-2,4,6(1H,3H,5H)-trione (45). H NMR (500 MHz,
CDCl3) δ 8.62 (s, 1 H), 7.55 (m, 4 H), 7.31 (m, 7 H), 7.21 (m, 4 H), 4.18
(m, 4 H), 2.94 (m, 4 H), 2.32 (s, 3 H), 2.05 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 164.0, 161.4, 151.3, 149.5, 138.9, 138.8, 137.1, 129.9,
129.1, 128.6, 128.5, 127.7, 126.5, 118.8, 111.2, 107.6, 43.6, 42.8, 34.4,
12.9, 11.9. EC50 0.87 μM.
Synthesis of 6-Chloro-1,3-diphenethylpyrimidine-2,4(1H,3H)-dione
(46). A mixture of 7 (0.5 mmol) and POCl3 (2 mL) was heated at 100 ꢀC
with stirring for 8 h. After cooling the mixture to room temperature, the
mixture was poured into 20 mL of EtOAc. After being washed with water
and dried over Na2SO4, the solvent was removed in vacuo, and the
residue was purified by silica gel chromatography to provide 46 in 85%
yield. 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 6 H), 7.21 (m, 4 H), 5.90
(s, 1 H), 4.24 (t, 2 H), 4.13 (t, 2 H), 2.95 (t, 2 H), 2.89 (t, 2 H); 13C
NMR (125 MHz, CDCl3) δ 160.5, 150.6, 145.6, 138.3, 137.1, 129.1,
129.0, 128.9, 128.6, 127.1, 126.7, 102.3, 48.3, 43.1, 34.9, 33.6. Elemental
analysis: Comb. Anal. (C20H19ClN2O3), Calculated: C, 67.70; H, 5.40;
Cl, 9.99; N, 7.89; O, 9.02. Found: C, 66.95; H, 5.55; N, 7.82. Purity:
>95%. EC50 > 32 μM.
Synthesis of 6-Methoxy-1,3-diphenethylpyrimidine-2,4(1H,3H)-
dione (47). A mixture of 46 (0.5 mmol) and NaOEt (0.05 mmol) was
refluxed in MeOH (5 mL) with stirring for 6 h. After cooling the mixture
to room temperature, the mixture was poured into 20 mL of EtOAc.
After being washed with water and dried over Na2SO4, the solvent was
removed in vacuo, and the residue was purified by silica gel chromatog-
raphy to provide compound 47 in about 50% yield. 1H NMR (500 MHz,
CDCl3) δ 7.24-7.08 (m, 10 H), 4.96 (s, 1 H), 4.05 (m, 4 H), 3.62 (s, 3
H), 2.82 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ 162.9, 161.2, 150.8,
138.8, 138.2, 129.2, 129.0 128.6, 128.5 126.7, 126.5, 56.8, 43.6, 42.6,
34.8, 34.0. EC50 > 32 μM.
5-(Cyclohexylmethylene)-1,3-diphenethylpyrimidine-2,4,6(1H,3H,
5H)-trione (39). 1H NMR (500 MHz, CDCl3) δ 7.27 (m, 4 H), 7.22 (m,
6 H), 4.16 (m, 2 H), 4.03 (m, 2 H), 3.37 (m, 1 H), 2.87 (m, 4 H), 1.66
(m, 5 H), 1.16-0.48 (m, 6 H); 13C NMR (125 MHz, CDCl3) δ 168.8,
151.1, 137.8, 129.1 128.6, 126.8, 47.3, 42.9, 39.3, 34.7, 34.0, 32.8, 26.3,
26.0. EC50 > 32 μM.
Typical Procedure for the Synthesis of PYT Analogues with 5- or
7-Membered Ring Cores. Into a mixture of 48 (0.5 mmol), Et3N
(1.1 mmol), and THF (5 mL) was added slowly oxalyl dichloride
(0.5 mmol for 5-membered core) or succinyl dichloride (0.5 mmol for
7-membered core). After being stirred at room temperature for 10 h, the
reaction was quenched with water and poured into 20 mL of EtOAc. The
mixture was washed with water and brine and dried over Na2SO4. The
solvent was removed in vacuo, and the residue was purified by silica gel
chromatography to provide the desired PYT analogue.
5-(2,6-Dichlorobenzylidene)-1,3-diphenethylpyrimidine-2,4,6(1H,
3H,5H)-trione (40). 1H NMR (500 MHz, CDCl3) δ 8.28 (s, 1 H), 7.30-
7.14 (m, 13 H), 4.11 (m, 2 H), 4.00 (s, 2 H), 2.86 (m, 2 H), 2.76 (m, 2
H); 13C NMR (125 MHz, CDCl3) δ 160.3, 158.9, 151.7, 150.3, 138.0,
137.9, 133.1, 132.9, 129.1, 129.0, 128.7, 128.6, 127.9, 126.8, 126.7, 123.4,
43.7, 43.1, 34.1, 34.0. EC50 3.10 μM.
5-(2-Methylbenzylidene)-1,3-diphenethylpyrimidine-2,4,6(1H,3H,
5H)-trione (41). 1H NMR (500 MHz, CDCl3) δ 8.66 (s, 1 H), 7.43-
7.14 (m, 14 H), 4.12 (m, 2 H), 4.04 (m, 2 H), 2.87 (m, 2 H), 2.80 (m, 2
H), 2.29 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 161.7, 159.6, 158.8,
150.6, 138.5, 138.2, 132.8, 131.6, 130.2, 130.0, 129.1, 128.7, 128.6, 126.8,
126.7, 125.3, 118.8, 43.7, 43.0, 34.2, 20.4. EC50 2.23 μM.
5-(4-(Benzyloxy)benzylidene)-1,3-diphenethylpyrimidine-2,4,6(1H,
3H,5H)-trione (42). 1H NMR (500 MHz, CDCl3) δ 8.28 (s, 1 H), 8.02
(d, 2 H), 7.22 (m, 5 H), 7.09 (m, 10 H), 6.84 (d, 2 H), 4.97 (s, 2 H), 3.97
(m, 4 H), 2.70 (m, 4 H); 13C NMR (125 MHz, CDCl3) δ 163.5, 162.8,
160.6, 159.0, 150.7, 138.4, 137.9, 135.9, 129.2, 129.1, 128.9, 128.6, 128.5,
1,3-Diphenethylimidazolidine-2,4,5-trione (49). 1H NMR (500
MHz, CDCl3) δ 7.24-7.09 (m, 10 H), 3.79 (m, 4 H), 2.86 (m, 4 H);
13C NMR (125 MHz, CDCl3) δ 156.4, 153.5, 136.8, 128.9, 128.8, 127.2,
40.5, 33.9. Elemental analysis: Comb. Anal. (C19H18N2O3), Calculated:
C, 70.79; H, 5.63; N, 8.69; O, 14.69. Found: C, 71.03; H, 5.75; N, 8.73.
Purity: >95%. EC50 > 32 μM.
1
1-Cyclohexylimidazolidine-2,4,5-trione (50). H NMR (500 MHz,
CDCl3) δ 8.08 (s, 1 H), 4.04 (m, 1 H), 2.07 (m, 2 H), 1.88 (m, 2 H), 1.70
2419
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
(m, 3 H), 1.31 (m, 3 H); 13C NMR (125 MHz, CDCl3) δ 156.6, 156.1,
152.2, 52.7, 29.6, 25.7, 24.8. EC50 > 32 μM.
’ ACKNOWLEDGMENT
We thank the National Institutes of Health (1R43NS057849),
the ALS Association (TREAT program), the Department of
Defense (AL093052), the ALS Therapy Alliance, and the
Veterans Administration at the Edith Nourse Rogers Memorial
Veterans Hospital, Bedford, MA, for their generous support of
the research project. We are grateful to Biogen Idec (Cambridge,
MA) for financial assistance related to the DMPK assays and to
Dr. Robert Scannevin (Biogen Idec) for coordinating that effort.
1,3-Dicyclohexylimidazolidine-2,4,5-trione (51). 1H NMR (500
MHz, CDCl3) δ 4.01 (m, 2 H), 2.07 (m, 4 H), 1.86 (m, 4 H), 1.66
(m, 6 H), 1.30 (m, 6 H); 13C NMR (125 MHz, CDCl3) δ 156.6, 153.6,
52.5, 29.6, 25.7, 24.9. Elemental analysis: Comb. Anal. (C15H22N2O3),
Calculated: C, 64.73; H, 7.97; N, 10.06; O, 17.24. Found: C, 64.80; H,
7.99; N, 9.94. Purity: >95%. EC50 > 32 μM.
1,3-Diphenethyl-1,3-diazepane-2,4,7-trione (52). 1H NMR (500
MHz, CDCl3) δ 7.24-7.09 (m, 10 H), 3.59 (m, 4 H), 2.82 (m, 4 H),
2.55 (s, 4H); 13C NMR (125 MHz, CDCl3) δ 176.2, 159.3, 139.3, 128.7,
128.3, 127.1, 44.8, 39.9, 26.4. EC50 > 32 μM.
’ ABBREVIATIONS
Mutant SOD1-Induced Cytotoxicity Protection Assay. Cells
were seeded at 15000 cells/well in 96-well plates and incubated
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 incubation with the compounds, MG132 was added at a final
concentration of 100 nM. MG132 is a well-characterized proteasome
inhibitor, which would be expected to enhance the appearance of
protein aggregation by blocking the proteosomal clearance of
aggregated proteins. Cell viability was measured 48 h later using
the 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.
ADME, absorption, distribution, metabolism, excretion; ALS,
amyotrophic lateral sclerosis; PYT, pyrimidine-2,4,6-trione;
SAR, structure-activity relationship; SOD1, Cu/Zn superoxide
dismutase.
’ REFERENCES
(1) Brown, R. W. J. The Motor Neuron Diseases. In Harrison’s
Principles of Internal Medicine; Fauchi, A. S., Braunwald, E., Isselbacher,
K. J., WIlson, J. D., Martin, J. B., Kasper, D. L., Hauser, S. L., Longo, D. L.,
Eds.; McGraw-Hill: New York, 1998; pp 2368-2371.
(2) Rothstein, J. D. Current hypotheses for the underlying biology of
amyotrophic lateral sclerosis. Ann. Neurol. 2009, S3–S9.
(3) Worms, P. M. The epidemiology of motor neuron diseases: a
review of recent studies. J. Neurol. Sci. 2001, 191, 3–9.
(4) Waldmeier, P. C.; Tatton, W. G. Interrupting apoptosis in
neurodegenerative disease: potential for effective therapy? Drug Discov.
Today 2004, 9, 210–218.
In Vitro and in Vivo ADME Studies. In vitro ADME
properties of PYT compounds were tested at Apredica, Inc. (Watertown,
MA), a contract research organization. To assess levels of drug
present in blood and brain tissue specimens, a 50 mg/kg dose was
peritoneally administered to wild-type B6SJL mice (n = 6). An
untreated group (n = 6) was used as a negative control. Blood and
brain were removed 1 h after injection and rapidly quenched at -
80 ꢀC. Specimens were subsequently mixed with extraction buffer
(100% ice-cold methanol), sonicated on ice for 20 s, and then spun at
15000 rpm at 4 ꢀC for 30 min. Supernatant (100 μL) was then
analyzed using a Ceas 16 channel EC-HPLC. Experiments were
performed in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals.
(5) Gitkin, A.; Harris, G.; Birchenough, J.; Boylan, B. C.; Whitesell,
C. Nervous Breakdown: A Detailed Analysis of the Neurology Market; UBS
Warburg LLC: New York, 2001.
(6) Mitsumoto, H.; Rabkin, J. G. Palliative care for patients with
amyotrophic lateral sclerosis: “prepare for the worst and hope for the
best”. J. Am. Med. Assoc. 2007, 298, 207–216.
(7) Jimonet, P.; Audiau, F.; Barreau, M.; Blanchard, J.-C.; Boireau,
A.; Bour, Y.; Colꢀeno, M.-A.; Doble, A.; Doerflinger, G.; Do Huu, C.;
Donat, M.-H.; Duchesne, J. M.; Ganil, P.; Guꢀerꢀemy, C; Honorꢀe, E.; Just,
B.; Kerphirique, R.; Gontier, S.; Hubert, P.; Laduron, P. M.; Le Blevec, J.;
Meunier, M.; Miquet, J.-M.; Nemecek, C.; Pasquet, M.; Piot, O.; Pratt, J.;
Rataud, J.; Reibaud, M.; Stutzmann, J.-M.; Mignani, S. Riluzole series.
Synthesis and in vivo “antiglutamate” activity of 6-substituted-2-ben-
zothiazolamines and 3-substituted-2-imino-benzothiazolines. J. Med.
Chem. 1999, 42, 2828–2843.
(8) Bensimon, G.; Lacomblez, L.; Meininger, V. A controlled trial of
riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group. N.
Engl. J. Med. 1994, 330, 585–591.
(9) Francis, K.; Bach, J. R.; DeLisa, J. A. Evaluation and rehabilitation
of patients with adult motor neuron disease. Arch. Phys. Med. Rehabil.
1999, 80, 951–963.
(10) Ilieva, H.; Polymenidou, M.; Cleveland, D. W. Non-cell auton-
omous toxicity in neurodegenerative disorders: ALS and beyond. J. Cell
Biol. 2009, 187 (6), 761–772.
Radioligand Binding Assays for Activity Profiling. These
studies were performed at MDS Pharma Services (Taipei, Taiwan).
’ ASSOCIATED CONTENT
S
Supporting Information. X-ray analysis of 3 and 43, in
b
vitro pharmacokinetic studies, experimentals, and radioligand
assay data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(11) Rosen, D. R.; Siddique, T.; Patterson, D.; Figlewicz, D. A.; Sapp,
P.; Hentati, A.; Donaldson, D.; Goto, J.; O’Regan, J. P.; Deng, H.-X.;
Rahmani, Z.; Krizus, A.; McKenna-Yasek, D.; Cayabyab, A.; Gaston,
S. M.; Berger, R.; Tanzi, R. E.; Halperin., J. J.; Herzfeldt, B.; Van den
Bergh, R.; Hung, W.-Y.; Bird, T.; Deng, G.; Mulder, D. W.; Smyth, C.;
Laing, N. G.; Soriano, E.; Pericak-Vance, M. A.; Haines, J.; Rouleau,
G. A.; Gusela, J. S.; Horvitz, H. R.; Brown, R. H., Jr. Mutations in Cu/Zn
superoxide dismutase gene are associated with familial amyotrophic
lateral sclerosis. Nature 1993, 362, 59–62.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 847-491-5663. Fax: 847-491-7713. E-mail: Agman@chem.
northwestern.edu.
Present Addresses
#Chicago Discovery Solutions, 23561 W. Main St., Plainfield,
IL 60544.
(12) Gurney, M. E.; Pu, H.; Chiu, A. Y.; Dal Canto, M. C.; Polchow,
C. Y.; Alexander, D. D.; Caliendo, J.; Hentati, A.; Kwon, Y. W.; Deng,
H. X.; Chen, W.; Zhai, P.; Sufit, R. L.; Siddique, T. Motor neuron
4Harvard Stem Cell Institute, Harvard University, Cambridge,
MA 02138.
2420
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421

Journal of Medicinal Chemistry
ARTICLE
degeneration in mice that express a human Cu,Zn superoxide dismutase
mutation. Science 1994, 264, 1772–1775.
(13) Bruijn, L. I.; Becher, M. W.; Lee, M. K.; Anderson, K. L.;
Jenkins, N. A.; Copeland, N. G.; Sisodia, S. S.; Rothstein, J. D.; Borchelt,
D. R.; Price, D. L.; Cleveland, D. W. ALS-linked SOD1 mutant G85R
mediates damage to astrocytes and promotes rapidly progressive disease
with SOD1-containing inclusions. Neuron 1997, 18, 327–338.
(14) Morimoto, R. I. Proteotoxic stress and inducible chaperone
networks in neurodegenerative disease and aging. Genes. Dev. 2008, 22
(11), 1427–1438.
(15) Benmohamed, R.; Arvanites, A. C.; Kim, J.; Ferrante, R. J.;
Silverman, R. B.; Morimoto, R. I.; Kirsch, D. R. Identification of
compounds protective against G93A SOD1 toxicity for the treatment
of amyotrophic lateral sclerosis. Amyotroph. Lat. Scler. 2011, 12(2),
87-96.
(16) Artuso, E.; Carvoli, G.; Degani, L.; Fochi, R.; Magistris, C. A
general, facile, and safe procedure for the preparation of S-methyl N-
alkylthiocarbamates by methylthiocarbonylation of primary aliphatic
amines with S,S-dimethyl dithiocarbonate. Synthesis 2007, 1096–1102.
(17) Ingle, V. N.; Gaidhane, P. K.; Dutta, S. S.; Naha, P. P.; Sengupta,
M. S. Synthesis of novel galactopyranosyl-derived spiro barbiturates. J.
Carbohydr. Chem. 2006, 661–671.
(18) Ragoussis, N. Modified Knoevenagel condensations: synthesis
of (E)-3-alkenoic acids. Tetrahedron Lett. 1987, 93–96.
(19) Robertson, J. S. Blood pressure maxima in humans. J. Clin. Basic
Cardiol. 2006, 9, 37–40.
(20) (a) Towart, R.; Linders, J. T. M.; Hermans, A. N.; Rohrbacher,
J.; van der Linde, H. J.; Ercken, M.; Cik, M.; Roevens, P.; Teisman, A.;
Gallacher, D. J. Blockade of the IKs potassium channel: an overlooked
cardiovascular liability in drug safety screening? J. Pharmacol. Toxicol.
Methods 2009, 60 (1), 1–10. (b) Du, L.; Li, M.; You, Q. The interactions
between hERG potassium channel and blockers. Curr. Top. Med. Chem.
2009, 9 (4), 330–338. (c) Mitcheson, J. S. hERG potassium channels
and the structural basis of drug-induced arrhythmias. Chem. Res. Toxicol.
2008, 21 (5), 1005–1010.
2421
dx.doi.org/10.1021/jm101549k |J. Med. Chem. 2011, 54, 2409–2421