Structure-activity relationship of N,N′-disubstituted pyrimidinetriones as CaV1.3 calcium channel-selective antagonists for Parkinson's disease

Article abstract of DOI:10.1021/jm4005048

Ca_V1.3 L-type calcium channels (LTCCs) have been a potential target for Parkinson's disease since calcium ion influx through the channel was implicated in the generation of mitochondrial oxidative stress, causing cell death in the dopaminergic neurons. Selective inhibition of Ca_V1.3 over other LTCC isoforms, especially Ca_V1.2, is critical to minimize potential side effects. We recently identified pyrimidinetriones (PYTs) as a Ca_V1.3-selective scaffold; here we report the structure-activity relationship of PYTs with both Ca_V1.3 and Ca_V1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies allowed characterization of the Ca_V1.3 and Ca _V1.2 LTCCs binding sites. The SAR also identified four important moieties that either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at Ca_V1.3 and Ca_V1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for drug development.

Full text of DOI:10.1021/jm4005048

Article
pubs.acs.org/jmc
Structure−Activity Relationship of N,N′-Disubstituted
Pyrimidinetriones as CaV1.3 Calcium Channel-Selective Antagonists
for Parkinson’s Disease
Soosung Kang,† Garry Cooper,†,‡ Sara Fernandez Dunne,§ Chi-Hao Luan,§ D. James Surmeier,‡
and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular
Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208, United States
‡Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States
§High-Throughput Analysis Laboratory, Northwestern University, Evanston, Illinois 60208, United States
S
* Supporting Information
ABSTRACT: CaV1.3 L-type calcium channels (LTCCs) have
been a potential target for Parkinson’s disease since calcium
ion influx through the channel was implicated in the
generation of mitochondrial oxidative stress, causing cell
death in the dopaminergic neurons. Selective inhibition of
CaV1.3 over other LTCC isoforms, especially CaV1.2, is critical
to minimize potential side effects. We recently identified
pyrimidinetriones (PYTs) as a CaV1.3-selective scaffold; here
we report the structure−activity relationship of PYTs with
both CaV1.3 and CaV1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies
allowed characterization of the CaV1.3 and CaV1.2 LTCCs binding sites. The SAR also identified four important moieties that
either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at CaV1.3
and CaV1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for
drug development.
demonstrated in a PD model.11 Antagonization of CaV1.3
LTCCs could provide a means to diminish cell loss in PD by
protecting SNc dopaminergic neurons against toxins, thereby
INTRODUCTION
■
L-Type calcium channels1 (LTCCs) are cell membrane
proteins activated upon membrane depolarization, selectively
modulating calcium ion influx into the cells to initiate diverse
making CaV1.3 LTCCs an important therapeutic target for
PD.12,13 However, drug candidates must selectively antagonize
intracellular events such as synaptic transmission, secretion, and
CaV1.3 over other ion channels, especially over CaV1.2 LTCCs,
gene expression. Although these channels are multimeric
in order to avoid potential cardiovascular side effects.14
proteins of five subunits (α1, α2, β, γ, δ),2,3 the α1 subunit is
We have been interested in developing CaV1.3 selective
the major pore-forming subunit, having four different subtypes
(CaV1.1−1.4). CaV1.2 LTCCs are the major isoform (∼90%),4
inhibitors for treating PD; a calcium influx assay was developed
using fluorometric imaging plate reader15 (FLIPR) with a Fluo-
expressed in cardiac myocites, smooth muscle, pancreas, and
8 calcium dye16 and stably expressed CaV1.3 and CaV1.2
neurons, while CaV1.1 and CaV1.4 LTCCs are minor isoforms,
restricted to skeletal muscle and retina, respectively.5 CaV1.3
LTCCs in HEK293 cells. We tested diverse known CaV1.2
LTCCs blockers,17,18 such as isradipine (1), verapamil (2), and
LTCCs6 are expressed similarly as CaV1.2 LTCCs but are more
neuron specific (cerebral cortex, hippocampus, basal ganglia,
diltiazem (3), and synthesized over 100 modified DHPs19 and
various hydropyrimidines20 (4) to obtain CaV1.3 selective
habenula, and thalamus); they are thought to serve
predominantly as modulators in the neuronal system.7
antagonists; however, the best of these compounds have no
Our recent studies8 showed that CaV1.3 LTCCs are used for
greater than 2- to 3-fold selectivity for CaV1.3 over CaV1.2
LTCCs. More recently, high-throughput screening of molecular
rhythmic pacemaking in adult dopaminergic neurons of the
substantia nigra pars compacta (SNc).9,10 The engagement of
libraries identified pyrimidine-2,4,6-trione (PYT) 5 as a
potential scaffold for the selective inhibition of CaV1.3
CaV1.3 LTCCs during autonomous pacemaking increases with
LTCCs.21 After SAR-based initial modification of the scaffold,
we arrived at N-(3-chlorophenethyl)-N′-cyclopentylpyrimidine-
age in animal models of Parkinson’s disease (PD),8 but CaV1.3
LTCCs seem to be unnecessary for normal functioning of SNc
dopaminergic neurons. This reliance of CaV1.3 LTCCs elevates
mitochondrial oxidant stress in SNc dopaminergic neurons,
incurring continuous loss of SNc dopaminergic neurons, as
Received: April 7, 2013
© XXXX American Chemical Society
A
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Figure 1. Example of CaV1.3 blockers. 1, 2, and 3 are known nonselective LTCC blockers used for the initial CaV1.3 study. 4 is a potent and slightly
selective molecule. 5 is our initial hit from HTS. 6 is the most selective compound, and 7 is most potent compound in the previous study.
2,4,6-trione (6) as a selective CaV1.3 LTCC antagonist and N-
(4-chlorophenethyl)-N′-( )-endo-norbornylpyrimidine-2,4,6-
(1H,3H,5H)-trione (7) as a potent CaV1.3 LTCC antagonist
among the series of compounds (Figure 1). PYT is a
completely new scaffold as applied to the ion channels, and
selective inhibition of CaV1.3 LTCCs with PYTs is the first
approach for the treatment of PD as a potential cure. Herein,
we report on our lead optimization efforts to obtain diverse
potent and selective compounds.
cyclopentyl ring to investigate the electronic demands of the
cyclopentyl ring region. To explore the steric demands on the
ethylene linker of the N-phenethyl side chain while maintaining
cyclopentyl or endo-norbornyl as the other side chain, diverse
methyl, gem-dimethyl, phenyl, and carbonate moieties were
employed on the α- or β-position of the ethylene linker.
Similarly, diverse aromatic rings were employed for lead
optimization. The moieties used for the construction of the
PYT library are shown in Figure 3.
DESIGN
■
RESULTS AND DISCUSSION
■
Our previous study showed that, starting from symmetric N,N′-
disubstituted pyrimidine 2,4,6-trione (PYT) 5, changing a
phenethyl group to a cyclopentyl ring led to the discovery of
our most selective molecule, 6, and using an endo-norbornyl
group led to most potent molecule (7) in this scaffold. We
synthesized ∼100 PYT molecules utilizing electronically and/or
sterically diverse arylalkyls, alkyls, and three- to seven-
membered cycloalkyl rings. All selective compounds in this
class share N-cycloalkyl and N-arylethyl moieties as the side
chains. An overlapped visualization of compounds 6 and 7 was
used as the starting point for further derivatization to obtain
more potent and selective compounds. As shown in Figure 2,
Chemistry. A majority of PYT analogues were synthesized
from commercially available amines and isocyanates using the
previously established one-pot synthesis of N,N-disubstituted
PYTs, which involved the Wohler22 urea synthesis and a Biltz
̈
and Wittek23 condensation with activated malonic acid
(Scheme 1). Urea formation was accomplished by coupling
isocyanates with amines in dichloromethane. If the starting
amine was an acid salt, 1 equiv of triethylamine was added to
the reaction mixture to initiate urea formation. The addition of
malonyl chloride to the urea, performed under dilute
conditions (0.02 M in dichloromethane) to avoid intermo-
lecular acylation, provided the condensation products in
moderate to high yields. The use of these synthetic approaches
permitted the construction of the major library members in
sufficient quantities and purity for assay against CaV1.3 and
CaV1.2 LTCCs.
Commercially unavailable isocyanates and amines were
prepared from related ketones, acetonitriles, carboxylic acids,
and other amines. Enantiomerically pure (1S,2R,4R)-(−)-endo-
2-norbornyl isocyanate (10) and (1R,2S,4S)-(+)-endo-2-nor-
bornyl isocyanate (13) were prepared via a combination of the
Poll24 and Fukaya25 procedures (Scheme 2); asymmetric
Diels−Alder reactions of cyclopentadiene with an acrylate
using (R)-pantolactone or (S)-2-hydroxy-N-methylsuccinimide
chiral auxiliary provided chiral carboxylate intermediates. The
resulting mixture underwent saponification and catalytic
hydrogenation to give enantiomerically pure endo-norbornyl
carboxylic acids 9 and 12. Carbonyl azide formation, followed
by Curtius rearrangement, provided enantiomerically pure
(1S,2R,4R)- or (1R,2S,4S)-endo-norbornyl isocyanates (10, 13),
which were stored in the refrigerator and used for several
months without further purification.
Figure 2. Overlay of two standard compounds, 6 (orange) and 7
(blue). The figure was generated using Sybyl-X and PyMOL.
molecules 6 and 7 are only different at the cycloalkyl region and
the substitution of the aromatic ring. The two extra carbons of
the norbornyl ring in 7 or para-chloro substitution on the
aromatic ring make 7 3-fold more potent than 6 toward CaV1.3
LTCC. It seems that a hydrophobic site is key for selective
inhibition of CaV1.3 over CaV1.2 LTCCs. On the basis of this
analysis, various 2- or 3-alkyl substituted cycloalkyl derivatives,
as well as diverse bicycloalkyl derivatives, were prepared to
probe the steric demands arising from this hydrophobic binding
site. m-Chloro and p-chlorophenethyl groups were initially
chosen as the other side chain to verify the SAR. Since racemic
norbornyl derivatives showed good potency for CaV1.3 LTCCs,
we synthesized each enantiomer of the endo-norbornyl
derivatives to explore the effect of chirality. Diverse ethers
and esters were incorporated at the 1, 2, and 3 positions of the
Commercially unavailable methyl-substituted phenethyl-
amines (16a−d) required for the syntheses were prepared as
shown in Scheme 3.26 Methylation of commercially available
phenylacetonitriles (14a,b) with iodomethane, using sodium
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Figure 3. Diverse N-alkyl moieties used for side chains on PYTs.
Scheme 1. General Synthetic Process for Assembling the
PYT Ring
Scheme 3. Synthesis of Methyl-Substituted Phenethylamines
16a−d
a
a
a
Reagents and conditions: (a) isocyanate, CH2Cl2 (0.1 M), room
temp, 1−5 h; (b) malonyl chloride, CH2Cl2 (0.02 M), room temp, 1 h.
Scheme 2. Asymmetric Syntheses of endo-Nornornyl
a
Isocyanates 10 and 13
a
Reagents and conditions: (a) NaH, MeI, DMF, room temp,
overnight; (b) LiAlH4, THF, 0 °C, 2 h.
(N-Boc-amino)cyclopentyl alcohols in a two-step process:
esterification of the acids/alcohols with appropriate alcohols/
acids, and Boc deprotection with 50% TFA solution in
dichloromethane. Alkyl-substituted cycloalkylamines were
synthesized from the corresponding cyclopentanone by
reductive amination with a benzylamine (Scheme 4). Especially,
cis-2-methyl or 2,2-gem-dimethylcyclopentyl27 PYT derivatives
were produced by asymmetric reductive amination of a
cyclopentanone with a nonracemic α-methylbenzylamine,28
which occurs specifically from the face opposite29 that occupied
by the adjacent alkyl substituent, to furnish the racemic cis-
diastereomer in a low yield. This product was then coupled to
the isocyanate as before, and the ethylbenzene was removed by
treatment with 75% trifluoroacetic acid in dichloromethane.
The urea was cyclized with malonyl chloride to furnish the final
PYT ring. When the same procedure was applied for the
synthesis of 3-alkyl substituted cyclopentyl derivatives,
inseparable racemic mixtures were obtained.
a
Reagents and conditions: See Poll’s22 and Fukaya’s23 procedure. (a)
cyclopentadiene, TiCl4; (b) LiOH; (c) 10% Pd/C, H2; (d) (i) ethyl
chloroformate, triethylamine, 0 °C; (ii) NaN3, acetone−water, 2 h;
(iii) hexane, reflux 2 h.
hydride as base, yielded a mixture of mono- and dimethylated
acetonitriles (15a−d). Reduction of the nitriles with LiAlH4
provided the target phenethylamines (16a−d) in high yields.
A majority of alkoxyl or carboxylate substituted cyclopentyl-
amines used in these syntheses were easily prepared from the
corresponding (N-Boc-amino)cyclopentanecarboxylic acids or
C
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a
Scheme 4. Synthesis of Alkyl Substituted Cycloalkylamine
a
Reagents and conditions: (a) (i) (S)-α-methylbenzylamine, cat. TsOH, benzene, reflux, overnight; (ii) NaBH3CN, EtOH; *(R)-1-
phenylethanamine was used under the same conditions; (b) 2-(3-Cl-phenyl)ethyl isocyanate, CH2Cl2, room temp; (c) 75% TFA in CH2Cl2; (d)
malonyl chloride, CH2Cl2.
Biological Activity. The CaV1.3 and CaV1.2 LTCCs assays
were carried out with the synthesized PYT molecules using the
previously described high-throughput screen with a FLIPR
system and Fluo-8 calcium assay kit.19 Each IC50 value, the
concentration of test compounds required to inhibit 50% of
calcium-dependent fluorescence response in the assay, was
determined in triplicate (Tables 1−4). IC50 values and
associated standard deviations were calculated by curve fitting
of the percent inhibition data from the FLIPR assay to a
sigmoidal model for a one-site compound target interaction
using XLfit. The selectivity of antagonism was defined as the
inverse of the ratio of the IC50 value with CaV1.2 LTCCs to that
with CaV1.3 LTCCs. The Z′ score of the HTS was >0.6 on the
basis of the values from at least a one-half of a plate of positive
and negative controls, and standard deviations associated with
the calcium channel FLIPR assay were usually <20%.
To better understand the steric effect of the original N-
cyclopentyl, N-3-chlorophenethyl PYT molecule (6) on
potency and selectivity, our initial analogue efforts were
focused on variations of steric bulkiness at the cyclopentyl
moiety by alkyl substitutions on that ring. We used 3-
chlorophenethyl and 4-chlorophenethyl moieties as the other
side chain to confirm the substitution effect on the aromatic
ring. As shown in Table 1, analogues with 1-cis-methyl (19, 20,
24, 25), 1,1-dimethyl (21, 22, 26, 27), 2-methyl (30, 31), and
2,2-dimethyl (32, 33) substitutions on the cyclopentyl ring had
generally 1- to 4-fold increased binding affinity for both CaV1.3
and CaV1.2 LTCCs; 1,1-dimethyl-cyclopentyl compound 22
displayed an IC50 of 1.7 μM for CaV1.3 LTCCs. It seems that
the binding site preferentially recognizes the stereochemistry of
1,2-cis substituted compounds. The favored 1,2-substitution
pattern is also shown when utilizing cyclohexyl as the cycloalkyl
chain (41). This steric preference was slightly increased with
the combination of 4-chlorophenethyl as the other side chain
(compounds 20 vs 19, 25 vs 24, and 22 vs 21). Alkyl
substituted cyclopentyl derivatives had ∼3-fold lower IC50 for
CaV1.3 LTCCs, but they also had increased CaV1.2 binding
affinity; their CaV1.3 selectivities, therefore, were moderate to
low. However, it is noteworthy that the 3-methyl- or 3,3-
dimethylcyclopentyl derivatives still retained >7-fold selectivity
for CaV1.3 LTCCs. Those compounds having ( )-trans-2-
methylcyclopentyl (37, 38), 3-ethylcyclopentyl (31, 35), or 4-
methylcyclohexyl (39−42) moieties were similar in potency to
compound 6 but far less selective. In the bicyclic ring series, a
significant potency drop was noted by increasing the bulkiness
of the bicyclic rings. It seems the active site that interacts with
the cyclopentyl moiety is not big enough to fit bornyl (53, 54),
cis-myrtanyl (55, 56), or isopinocamphyl (57−60) groups;
these bulky molecules have almost no binding to either CaV1.3
or CaV1.2 LTCCs. Racemic endo-norbornyl compound 7 was
the most potent molecule in the previous study.19 To confirm
the stereoactivity relationship, we asymmetrically synthesized
each pair of enantiomers (49 and 50, 51 and 52, and 107 and
108) to isolate the more potent and selective enantiomer, as
each enantiomer of known chiral LTCC blockers displays
different efficacies.30 Although chiral compounds 50 and 51
displayed excellent selectivity (>48-fold) with good potency
(∼2.0 μM) for CaV1.3 LTCCs, there was only a small
stereorelated preference for CaV1.3 LTCCs with the
(1S,2R,4R)-norbornyl moiety; (1S,2R,4R)-norbornyl analogues
are 1.1- to 1.8-fold more potent than (1R,2S,4S)-norbornyl
analogues for CaV1.3 LTCCs. Presumably, each endo-norbornyl
group can freely rotate in the binding site; therefore, they are
recognized similarly.
The other interesting molecule resulting from this study is
cyclohexylethyl drivative (S)-N-(3-chlorophenethyl)-N-(1-
cyclohexylethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (43). This
compound is slightly more potent (IC50 = 4.3 μM) than lead
compound 6 and is a moderately selective antagonist for CaV1.3
LTCCs. In addition to 43, all three N-(1-cyclohexylethyl)
derivatives (44−46) in this study displayed good selectivity
over CaV1.2, with IC50 of 4.3−9.7 μM for CaV1.3 LTCCs.
These compounds have additional carbons between the
cycloalkyl and PYT rings that will allow further structural
variations on the cyclopentyl side chain.
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Table 1. IC50 Values and Selectivities of Various Cycloalkyl Analogues
a
b
Ratio of the IC50 values (CaV1.2/CaV1.3). Selectivity was confirmed from the previous study.
Another trend that emerges from the SAR of various PYT
binding affinity for both calcium channels. As shown by
compounds 65 and 67 in Table 2, charged molecules or even
an alcohol derivative (69) has no binding affinity for either
CaV1.3 or CaV1.2 LTCCs, even at 100 μM. A hydrophobic
molecules is the unfavorable effect of polar functionality on the
cyclopentyl ring. Previous SAR studies showed that an electron
lone pair or charge (hydrophilicity) on the aryl ring limits its
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Table 2. IC50 Values and Selectivity of Various Cycloalkyl Analogues
cycloalkyl chain also is favored, and bulky esters or ethers (63,
66, 68, 74) do not seriously reduce (1- to 2-fold reduction)
binding affinity for both CaV1.2 and CaV1.3 LTCCs. It is
thought that the hydrophilicity arising from the heteroatoms
are counterbalanced by the nonpolar groups, and so the ether
bond on the cycloalkyl chain can be utilized as a linker for
further modification. The cis-1,2-substitution pattern is also
favored when acetoxyl is substituted on the cyclopentyl chain
(71 and 72), even though it results in 6-fold reduced binding
affinity. The favored 3-substitution pattern for selectivity was
not observed with the ether or ester substituted derivatives;
presumably the binding site in CaV1.2 LTCCs recognizes
hydrophobic functional groups of various inhibitors differently.
When the 3-chlorophenethyl substituent was changed to
various fused aromatic rings, such as 1-tetrahydronaphthyl
(77), 1-indanyl (78), and 2-indanyl (79), their binding affinity
for CaV1.3 decreased 2-fold while their potency for CaV1.2
increased (Table 3). Although different lengths of alkyl linkers
(81−84) were incorporated to adjust the space of this region, it
was found that the ethyl linker was best for potency and
selectivity. It is not clear whether this region disfavors
hydrophilic functionalities, but carboxylate analogues 93−95
did not provide better potency or selectivity for CaV1.3 LTCCs.
Another trend that emerged from the ethyl linker region is the
steric preference; methyl- or dimethyl-substituted members or
phenyl substituted members (89, 90, 96−99) have 1- to 3-fold
improved binding affinity for CaV1.3 LTCCs. However, this
modification also results in better interaction with CaV1.2
LTCCs; the overall selectivity for CaV1.3 LTCCs was 1- to 5-
fold. 3,4- or 3,5-Dichloro and 3,5-bis(trifluoromethyl) sub-
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chlorophenethyl (100), α-methyl-4-chlorophenethyl (101), β-
methyl-3-chlorophenethyl (102), β,β-dimethyl-3-chlorophe-
nethyl (103), 2-(bis-3-(trifluoromethyl)phenyl)ethyl (104), 2-
(3,5-dichlorophenyl)ethyl (105), or 2-(3-(trifluoromethyl)-
phenyl)ethyl (106−108) led to good CaV1.3 LTCCs inhibitory
activity, with IC50 values of 1.8−3.6 μM. Compounds 106−
108, as racemic mixtures or individual enantiomers, gave IC50
values of 2−3 μM for CaV1.3 and ∼40-fold selectivity over
CaV1.2 LTCCs. The (1S,2R,4R)-endo analogue (108) is slightly
more potent than the (1R,2S,4S)-endo analogue (107), but they
are almost the same in their CaV1.3 LTCCs inhibitory activity
and selectivity.
Table 3. IC50 Values and Selectivities of Various Arylalkyl
Analogues
CONCLUSION
■
Our SAR study of N-(3-chlorophenethyl)-N′-cyclopentylpyr-
imidine-2,4,6-(1H,3H,5H)-trione (6) highlights several ad-
vancements in lead optimization of the PYT scaffold. One of
the trends found from these analogues is that the endo-
norbornyl moiety provides high selectivity for CaV1.3 LTCCs
with good binding affinity if an appropriate arylalkyl moiety is
used as the other side chain. Second, both CaV1.3 and CaV1.2
LTCCs have limited space to interact with various steric
requirements at the cycloalkyl ring binding site; however,
insertion of a one methylene linker between the cycloalkyl and
PYT rings retains selectivity. Third, inactive compounds at both
CaV1.3 and CaV1.2 LTCCs have a charged ion or electron lone
pair near the cycloalkyl ring, implying that strong polarity
should be avoided for antagonism of CaV1.3 LTCCs. Fourth,
both CaV1.3 and CaV1.2 Ca2+ channels preferentially recognize
the regiochemistry of 1,2/1,3-substituted cycloalkyl derivatives.
The overall SAR of the PYT analogues identified four
important series of molecules that either retained selectivity
or enhanced binding affinity: The chiral N-endo-norbornyl
analogues 50 (CaV1.3 IC50 = 2.1 0.4 μM, selectivity ≥ 48),
51 (CaV1.3 IC50 = 2.0 0.1 μM, selectivity ≥ 50), and 108
(CaV1.3 IC50 = 2.1
0.1 μM, selectivity = 36) have low
micromolar activity against CaV1.3 LTCCs while retaining >36
selectivity. Dimethyl substitution on the cyclopentyl ring (22,
CaV1.3 IC50 = 1.7 0.1 μM) or on the arylethyl side chain (98,
CaV1.3 IC50 = 2.4 0.2 μM) leads to improved potency for
CaV1.3. Among the studied PYT molecules, 3,4- or 3,5-
disubstituted phenethyl derivatives (85, CaV1.3 IC50 = 1.4 0.1
μM; 86, CaV1.3 IC50 = 1.2
0.1 μM) are the most potent
inhibitors for CaV1.3 LTCC. N-1-Cyclohexylethyl analogue 43
(CaV1.3 IC50 = 4.3 0.5 μM, selectivity ≥ 29) is one of the
potent and selective molecules; this side chain could be further
modified. Our study highlights several advancements in the lead
optimization and diversification toward the drug development
of PYTs. The superimposition of the energy-minimized
structures of selective or potent molecules highlights the
overlapping and variable regions (Figure 4); these hotspots are
located at or near the cycloalkyl and aromatic rings and should
be important for future modifications.
EXPERIMENTAL SECTION
■
stitution on the aromatic ring (85−87) led to ∼5-fold
improved potency (IC50 = 1.2−1.4 μM) for CaV1.3 LTCCs,
but selectivities were moderate.
Previously, CaV1.3 inhibitory activity was improved with
substitution of the cyclopentyl group by the endo-norbornyl
group; thus, a small series of norbornyl analogues (100−106)
were prepared to determine optimal arylalkyl moiety (Table 4).
The combination of endo- norbornyl with β-methyl-4-
General Methods for Biology. Experimental procedures for the
construction and stable transfection of HEK293 cells with CaV1.3 and
CaV1.2 and procedure for high-throughput screening were previously
reported.17,19 Rat CaV1.3α1D (GenBank code AF370010) containing
all alternative splice sites, rat CaVβ3 (GenBank code M88751), rat
CaVα2δ-1 (GenBank code 286488), and rabbit CaV1.2α1C (GenBank
code P15381) cDNAs were used. Screen Quest Fluo-8 no wash
calcium assay kit (ABD Bioquest Inc., Sunnyvale, CA, U.S.) on a
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Table 4. IC50 Values and Selectivities of Various Norbornyl Analogues
Finnigan LCQ mass spectrometer. High resolution mass spectra were
obtained using an Agilent 6210 LC-TOF spectrometer. Melting points
were measured in open capillaries on a Buchi B-540 melting point
̈
analyzer. The purity of the compounds was evaluated on an Agilent
1260 reverse phase analytical HPLC system using an Agilent Zorbax
Eclipse XDB-C18 (4.6 mm × 50 mm, 5 μm) reverse phase column
with UV absorbance and evaporative light scattering detection. Optical
rotations were measured using an AA-100 polarimeter. The chiral
purity of the compounds was evaluated on a Beckman Gold HPLC
system using Daicel CHIRALPAK AD-RH or OD-RH (4.6 mm × 150
mm, 5 μm) reverse phase chiral columns.
Figure 4. Overlay of the most interesting molecules: (A) selective
compounds 43 (green), 50 (blue), and 51 (puple); (B) potent
compounds 22 (skyblue), 85 (pink), 86 (white), and 98 (blue). The
picture was generated using Sybyl-X and PyMOL.
General Procedure for PYT Synthesis Using Amine and
Isocyanate. Method A. To a solution of an amine (1 mmol) in dry
dichloromethane (10 mL), was added an isocyanate (1 mmol), and the
mixture was stirred at room temperature for 1−5 h. After dilution with
dry dichloromethane (40 mL), malonyl chloride (1.1 mmol) was
added dropwise under vigorous stirring at room temperature for 5
min. The resulting pale yellow solution was stirred for an additional 1
h and washed with brine (50 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated at reduced pressure to a small
volume. The resultant reaction mixture was purified using a silica gel
cartridge (24 g) with an Agilent 971-FP purification system to give
analytically pure compounds (20−90% yield).
(1S,2R,4R)-Bicyclo[2.2.1]heptane-2-isocyanate (10). Precursor
9 was prepared according to Poll24 and Fukaya.25 To a solution of
ethyl chloroformate (2.04 mL, 21.4 mmol) in hexane (20 mL) was
added dropwise a solution of 9 (3.0 g, 21.4 mmol) and triethylamine
(3.07 mL, 22 mmol) in hexane (20 mL) at 0 °C. After being stirred for
30 min at the same temperature, the mixture was quickly filtered. The
filtrate was dried under vacuum and redissolved in acetone (20 mL).
To this solution, a solution of sodium azide (2.2 g, 32 mmol) in water
FLIPR tetra (Molecular Devices LLC, Sunnyvale, CA, U.S.) were used
for the high-throughput assay. HEK293 cell density was 4 × 104 cells/
well and cultured in DMEM with 10% FBS for 4 days.
General Methods for Synthesis and Structure Character-
ization. Reagents were purchased from Sigma-Aldrich, Alfa-Aesar,
TCI America, and Chem-Impex and were used without further
purification. Solvents were purified by passage through a solvent
column composed of activated alumina and a supported copper redox
catalyst. Normal phase flash column chromatography was performed
using SuperFlash Si 50 prepacked silica cartridges with an Agilent 971-
FP flash purification system. Reaction progress was monitored by thin-
layer chromatography (TLC) carried out on SiliCycle silica gel plates
(2.5 cm × 7.5 cm, 250 μm thick, 60 F254), visualized by using UV
1
(254 nm) and ninhydrin stain. H NMR and 13C NMR spectra were
recorded in the indicated solvent on a Bruker Avance-III (500 and 126
1
MHz for H and 13C, respectively) spectrometer. Chemical shifts are
given in ppm (δ) relative to the internal standard, and coupling
constants (J) are in hertz (Hz). MS was performed on a system
consisting of an electrospray ionization (ESI) source in a Thermo
H
dx.doi.org/10.1021/jm4005048 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
(20 mL) was added dropwise at 0 °C, and stirring continued for 2 h at
the same temperature. The mixture was poured into ice−water (50
mL), and the organic materials were extracted with hexane (50 mL).
The organic layer was dried over anhydrous MgSO4, filtered, and then
heated to reflux for 5 h. After the mixture was cooled to room
temperature, the solvent was removed by vacuum evaporation to give a
crude product of 10 (2.26g, 75%). The product was used in the next
step without further purification. 1H NMR (500 MHz, CDCl3) δ 3.87
(m, 1H), 2.35 (t, J = 4.3 Hz, 1H), 2.25 (t, J = 4.6 Hz, 1H), 2.12−1.98
(m, 1H), 1.79 (m, 1H), 1.67−1.54 (m, 1H), 1.47 (m, 1H), 1.38−1.29
(m, 3H), 1.05 (m, 1H); 13C NMR (126 MHz, CDCl3) δ 121.20,
described for 16a. Pale yellow oil; H NMR (500 MHz, CDCl3) δ
7.66−7.45 (m, 4H), 3.62 (m, 1H), 2.81 (m, 2H), 1.50 (d, J = 6.6 Hz,
3H). MS (ESI): calculated for C10H12F3N [M + H]+, 204.09; found,
204.43.
2-Methyl-2-(3-(trifluoromethyl)phenyl)propan-1-amine
(16d). 16d was prepared (yield 68%) from 15d following a similar
reaction procedure described for 16a. 1H NMR (500 MHz, CDCl3) δ
7.71−7.30 (m, 4H), 2.90 (s, 2H), 1.40 (s, 6H); 13C NMR (126 MHz,
CDCl3) δ 147.04, 130.77 (q, J = 32.3 Hz), 129.59, 129.08, 128.85,
123.40 (d, J = 3.9 Hz), 123.15 (m), 122.80 (d, J = 3.7 Hz), 52.91,
38.96, 26.14. MS (ESI): calculated for C11H14F3N [M + H]+, 218.12;
found, 218.23.
(1S,2R)-2-Methyl-N-((S)-1-phenylethyl)cyclopentanamine
(18a). A mixture of 2-methylcyclopentanone (0.294 g, 3.0 mmol), (S)-
1-phenylethanamine (0.774 mL, 6.0 mmol), and a catalytic amount of
TsOH in benzene (25 mL) was refluxed overnight. After being cooled
to room temperature, the reaction mixture was diluted with 10 mL of
absolute EtOH, and sodium cyanoborohydride (569 mg, 9 mmol) was
then added to the reaction mixture in three portions over a ∼5 h
period. The reaction mixture was quenched with brine (50 mL),
partitioned with ethyl acetate (50 mL), dried over MgSO4, and
concentrated on reduced pressure. The resulting residue was subjected
to silica gel chromatography to purify the major component as 18a
55.10, 42.28, 39.32, 37.36, 36.79, 29.54, 21.88. [α]25 (c 1, CHCl3) =
D
−1.3.
(1R,2S,4S)-Bicyclo[2.2.1]heptane-2-isocyanate (13). 13 was
prepared from 12 following a similar reaction procedure described for
10, and spectral data were identical to those of 10. [α]25D (c 1, CHCl3)
= +1.5.
2-(3-Chlorophenyl)propanenitrile (15a) and 2-(3-Chloro-
phenyl)-2-methylpropanenitrile (15b). To a solution of (3-
chlorophenyl)acetonitrile (3.0 g, 20 mmol) in DMF (40 mL) was
added NaH (1.6 g, 40 mmol, 60% in oil) portionwise at room
temperature. After the mixture was stirred for 15 min, MeI (4.56 g, 32
mmol) was added. The mixture was stirred overnight. The reaction
mixture was quenched with saturated NH4Cl (80 mL), extracted with
ethyl acetate (200 mL), dried over MgSO4, and concentrated at
reduced pressure. The organic residue was purified by flash column
chromatography to give 15a (0.49 g, 15%) and 15b (1.61 g, 45%).
15a: 1H NMR (500 MHz, CDCl3) δ 7.46−7.13 (m, 4H), 3.91 (q, J =
7.3 Hz, 1H), 1.68 (d, J = 7.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
138.90, 135.03, 130.49, 128.40, 127.03, 124.98, 120.97, 30.97, 21.33.
MS (ESI): calculated for C9H8Cl [M + H]+, 166.04; found, 166.19.
15b: 1H NMR (500 MHz, CDCl3) δ 7.45−7.29 (m, 4H), 1.73 (s, 6H);
13C NMR (126 MHz, CDCl3) δ 143.41, 130.28, 128.12, 126.13,
1
(0.238 g, 36%). H NMR (500 MHz, CDCl3) δ 7.49−7.04 (m, 5H),
3.78 (q, J = 6.6 Hz, 1H), 2.82 (m, 1H), 2.10 (m, 1H), 1.74−1.56 (m,
3H), 1.46−1.35 (m, 1H), 1.33 (d, J = 6.5 Hz, 3H), 1.28 (m, 2H), 0.87
(d, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 146.29, 128.30,
126.72, 126.67, 59.51, 56.26, 34.02, 31.51, 30.18, 24.99, 20.25, 13.56.
MS (ESI): calculated for C14H21N [M + H]+, 204.18; found, 204.14.
(1R,2S)-2-Methyl-N-((R)-1-phenylethyl)cyclopentanamine
(23a). 23a was prepared (yield 33%) with (R)-1-phenylethanamine
following a similar reaction procedure described for 18a. Spectral data
were identical to those of 18a.
125.45, 123.95, 123.49, 37.05, 29.04. MS (ESI): calculated for
(S)-2,2-Dimethyl-N-((S)-1-phenylethyl)cyclopentanamine
(18b). 18b was prepared (yield 63%) from (S)-1-phenylethanamine
and 2,2-dimethylcyclopentanone following a similar reaction proce-
dure described for 18a. 1H NMR (500 MHz, CDCl3) δ 7.49−7.06 (m,
5H), 3.91 (q, J = 6.6 Hz, 1H), 2.56 (dd, J = 9.6, 7.7 Hz, 1H), 2.44−
2.30 (m, 1H), 1.82−1.70 (m, 1H), 1.61−1.54 (m, 1H), 1.48−1.36 (m,
3H), 1.35 (d, J = 6.6 Hz, 3H), 1.30−1.19 (m, 1H), 1.05 (s, 3H), 0.89
(s, 3H); 13C NMR (126 MHz, CDCl3) δ 146.72, 128.29, 126.76,
126.24, 65.58, 57.30, 40.90, 39.96, 31.87, 28.15, 24.36, 21.14, 19.71.
MS (ESI): calculated for C15H24ClN [M + H]+, 218.19; found, 218.18.
(R)-2,2-Dimethyl-N-((R)-1-phenylethyl)cyclopentanamine
(23b). 23b was prepared (yield 60%) from 2,2-dimethylcyclopenta-
none and (R)-1-phenylethanamine following a similar reaction
procedure described for 18a. Spectral data were identical to those of
18b.
C10H12F3N [M + H]+, 204.10; found, 204.24.
2-(3-(Trifluoromethyl)phenyl)propanenitrile (15c) and 2-
Methyl-2-(3-(trifluoromethyl)phenyl)propanenitrile (15d). 15c
and 15d were prepared (15c = 5%, 15d = 59% yield) from (3-
(trifluoromethyl)phenyl)acetonitrile following a similar reaction
1
procedure described for 15a,b. 15c: H NMR (500 MHz, CDCl3) δ
7.68−7.48 (m, 4H), 4.01 (q, J = 7.3 Hz, 1H), 1.71 (d, J = 7.3 Hz, 3H);
13C NMR (126 MHz, CDCl3) δ 138.01, 131.65 (q, J = 32.6 Hz),
130.20, 129.83, 125.12 (d, J = 4.0 Hz), 123.72 (q, J = 272.5 Hz),
123.64 (q, J = 3.9 Hz), 120.81, 31.17, 21.37. MS (ESI): calculated for
1
C10H8F3N [M + H]+, 200.07; found, 200.23. 15d: H NMR (500
MHz, CDCl3) δ 7.81−7.43 (m, 4H), 1.79 (s, 6H); 13C NMR (126
MHz, CDCl3) δ 142.48, 131.56 (q, J = 32.2 Hz), 129.62, 128.82,
124.87 (d, J = 4.3 Hz), 123.79, 123.69 (q, J = 277 Hz), 121.82 (d, J =
3.8 Hz), 37.20, 29.06. MS (ESI): calculated for C10H8F3N [M + H]+,
214.20; found, 214.23.
3-Methyl-N-((S)-1-phenylethyl)cyclopentanamine (29a). Dia-
stereomeric mixture 29a was prepared (yield 71%) from 3-
methylcyclopentanone and (S)-1-phenylethanamine following a
2-(3-Chlorophenyl)propan-1-amine (16a). To a solution of 15a
(0.40 g, 2.45 mmol) in THF (25 mL) at 0 °C was added 1.0 M
solution of LiAH4 (7.35 mL, 7.35 mmol). After being stirred for 2 h at
the same temperature, the mixture was warmed to room temperature,
quenched with saturated NH4Cl (30 mL), and partitioned with ethyl
acetate (30 mL). The organic layer was washed with brine, dried over
1
similar reaction procedure described for 18a. H NMR (500 MHz,
CDCl3) δ 7.48−7.31 (m, 12H), 7.26−7.12 (m, 4H), 4.07−3.94 (m,
1H), 3.14−2.89 (m, 1H), 2.21−2.05 (m, 1H), 1.91−1.75 (m, 2H),
1.74−1.64 (m, 1H), 1.55 (dd, J = 6.8, 2.1 Hz, 3H), 1.41−1.13 (m,
2H), 1.01−0.91 (m, 3H); 13C NMR (126 MHz, CDCl3) δ 141.56,
128.79, 127.86, 127.24, 56.97, 56.87, 56.63, 56.05, 41.09, 39.99, 33.34,
32.99, 32.50, 32.25, 32.13, 30.96, 29.62, 24.13, 22.64, 20.44. MS (ESI):
calculated for C14H21N [M + H]+, 204.18; found, 204.12.
3,3-Dimethyl-N-((S)-1-phenylethyl)cyclopentanamine (29b).
Diastereomeric mixture 29b was prepared (yield 75%) from 3,3-
dimethylcyclopentanone and (S)-1-phenylethanamine following a
similar reaction procedure described for 18a. Major isomer: 1H
NMR (500 MHz, CDCl3) δ 7.36−7.20 (m, 5H), 3.98−3.77 (m, 1H),
2.93−2.78 (m, 1H), 1.95−1.87 (m, 1H), 1.75−1.67 (m, 1H), 1.57−
1.54 (m, 3H), 1.47−1.39 (m, 4H), 0.96 (s, 3H), 0.93 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ 146.52, 128.31, 126.76,126.31, 57.28,
46.85, 38.86, 35.29, 29.36, 29.24, 20.72. MS (ESI): calculated for
C15H24ClN [M + H]+, 218.19; found, 218.13.
1
MgSO4, and concentrated to give 16a (0.253g, 61%). H NMR (500
MHz, CDCl3) δ 7.44−7.15 (m, 4H), 3.65 (m, 1H), 2.77 (m, 2H), 1.23
(d, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 147.09, 134.97,
130.50, 128.36, 127.00, 124.99, 49.12, 30.92, 21.28. MS (ESI):
calculated for C9H12ClN [M + H]+, 170.07; found, 169.85.
2-(3-Chlorophenyl)-2-methylpropan-1-amine (16b). 16b was
prepared (yield 73%) from 15b following a similar reaction procedure
1
described for 16a. Pale yellow oil; H NMR (500 MHz, CDCl3) δ
7.43−7.10 (m, 4H), 2.79 (s, 2H), 1.30 (s, 6H); 13C NMR (126 MHz,
CDCl3) δ 149.42, 134.34, 129.64, 126.63, 126.15, 124.43, 54.47, 39.97,
26.22. MS (ESI): calculated for C10H15ClN [M + H]+, 184.09; found,
183.96.
2-(3-(Trifluoromethyl)phenyl)propan-1-amine (16c). 16c was
prepared (yield 61%) from 15c following a similar reaction procedure
I
dx.doi.org/10.1021/jm4005048 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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3-Ethyl-N-((S)-1-phenylethyl)cyclopentanamine (29c). Dia-
stereomeric mixture 29c was prepared (yield 74%) from 3-ethyl-
cyclopentanone and (S)-1-phenylethanamine following a similar
reaction procedure described for 18a. Isomeric mixture: 1H NMR
(500 MHz, CDCl3) δ 7.40−7.09 (m, 5H), 3.81 (p, J = 6.8 Hz, 1H),
3.01−2.82 (m, 1H), 2.20−2.04 (m, 1H), 1.98−1.75 (m, 2H), 1.69−
1.57 (m, 1H), 1.52−1.35 (m, 3H), 1.34 (d, J = 6.8 Hz, 3H), 1.28−1.19
(m, 2H), 0.89−0.80 (m, 3H); 13C NMR (126 MHz, CDCl3) δ 146.03,
128.35, 126.77, 126.75, 126.63, 126.59, 56.98, 56.85, 56.57, 56.50,
56.22, 56.20, 56.14, 45.03, 41.09, 40.54, 40.35, 40.07, 39.87, 39.75,
39.65, 38.94, 38.67, 38.58, 34.24, 33.39, 32.79, 31.93, 31.28, 31.16,
30.08, 29.89, 29.36, 29.24, 29.23, 29.21, 29.16, 28.44, 24.70, 24.65,
24.60, 12.87. MS (ESI): calculated for C15H23N [M + H]+, 218.19;
found, 218.14.
CHCl3) = −6.3, chiral HPLC (Daicel AD-RH, 4.6−150 mm, 5 μm;
70% acetonitrile/30% 0.05 M pH 2 phosphate buffer; 0.4 mL/min;
268 nm) tR = 33.217 min; >99% ee. 46, HPLC purity: 99.3%, [α]25D (c
2, CHCl3) = +6.5, chiral HPLC (Daicel AD-RH, 4.6−150 mm, 5 μm;
70% acetonitrile/30% 0.05 M pH 2 phosphate buffer; 0.4 mL/min;
268 nm) tR = 37.199 min; >99% ee.
1 -( (1R , 2S , 4 S ) - B i c yc lo [ 2 . 2 . 1 ] h e p t an -2 - yl ) - 3 - ( 3 -
chlorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (49) and
1-((1S,2R,4R)-Bicyclo[2.2.1]heptan-2-yl)-3-(3-chlorophenethyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (51). White powder; mp 155−
1
157 °C; H NMR (500 MHz, CDCl3) δ 7.28−7.11 (m, 4H), 4.69−
4.56 (m, 1H), 4.18−4.03 (m, 2H), 3.64 (dd, J = 9.2, 20.9 Hz, 2H),
2.90 (t, J = 7.8 Hz, 2H), 2.66−2.58 (m, 1H), 2.37 (t, J = 4.5 Hz, 1H),
2.22 (m, 1H), 1.81−1.65 (m, 2H), 1.59−1.50 (m, 1H), 1.47 (m, 1H),
1.45−1.37 (m, 2H), 1.36−1.31 (m, 1H); 13C NMR (126 MHz,
CDCl3) δ 165.90, 164.52, 152.15, 139.84, 134.29, 129.82, 129.09,
127.15, 126.93, 59.65, 42.82, 42.08, 40.90, 37.73, 37.53, 33.66, 29.49,
28.77, 23.86. HRMS (ESI): calculated for C19H21ClN2O3 [M − H]−,
2,3-Dimethyl-N-((S)-1-phenylethyl)cyclopentanamine (29d).
Diastereomeric mixture 29d was prepared (yield 63%) from 2,3-
dimethylcyclopentanone and (S)-1-phenylethanamine following a
1
similar reaction procedure described for 18a. H NMR (500 MHz,
359.1168; found, 359.1172. 49, HPLC purity: 99.0%; [α]25 (c 2,
D
CDCl3) δ 7.49−7.14 (m, 5H), 3.82−3.70 (m, 1H), 3.00 (dt, J = 7.7,
6.6 Hz, 1H), 1.85−1.78 (m, 1H), 1.73 (m, 1H), 1.69−1.53 (m, 4H),
1.35 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.7 Hz,
3H); 13C NMR (126 MHz, CDCl3) δ 146.62, 128.73, 128.31, 126.72,
58.85, 56.57, 43.21, 40.09, 31.83, 31.14, 24.53, 20.90, 14.02. MS (ESI):
calculated for C15H23N [M + H]+, 218.19; found, 218.28.
CHCl3) = +9.4 chiral HPLC (Daicel OD-RH, 4.6−150 mm, 5 μm;
60% acetonitrile/40% 0.1 M aqueous KPF6; 0.5 mL/min; 268 nm) tR
= 26.400 min, >99% ee. 51, HPLC purity: 99.0%; [α]25D (c 2, CHCl3)
= −9.1, chiral HPLC (Daicel OD-RH, 4.6−150 mm, 5 μm; 60%
acetonitrile/40% 0.1 M aqueous KPF6; 0.5 mL/min; 268 nm) tR
=
22.867 min, 97.2% ee.
General Procedure for 19−22, 24−27, and 30−36. (Method
B). To a solution of an N-(1-phenylethyl)cyclopentylamine (18a,b,
23a,b, 29a−d; 1 mmol) in dry dichloromethane (10 mL) was added
an isocyanate (1 mmol), and the mixture was stirred at room
temperature for 3 h. The solvent was removed by vacuum distillation,
and 95% TFA in water (5 mL) was added to the reaction mixture.
After being stirred for 1 h at room temperature, the reaction mixture
was concentrated under vacuum, redissolved in toluene, and then
dried by vacuum distillation. After the sample was dissolved in dry
dichloromethane (40 mL), malonyl chloride (1.1 mmol) was added
dropwise under vigorous stirring at room temperature for 5 min. The
resulting pale yellow solution was stirred for an additional 1 h and
washed with brine (50 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated at reduced pressure to a small
volume. The resultant reaction mixture was purified with silica gel (24
g) using an Agilent 971-FP purification system to give analytically pure
compounds (50−80% yield).
1 -( (1R , 2S , 4 S ) - B i c yc lo [ 2 . 2 . 1 ] h e p t an -2 - yl ) - 3 - ( 4 -
chlorophenethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (50) and
1-((1S,2R,4R)-Bicyclo[2.2.1]heptan-2-yl)-3-(4-chlorophenethyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (52). White powder; mp 121−
123 °C; 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.4 Hz, 2H), 7.18
(d, J = 8.3 Hz, 2H), 4.60 (m, 1H), 4.06 (m, 2H), 3.61 (dd, J = 21.1,
10.1 Hz 2H), 2.87 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 3.8 Hz, 1H), 2.35 (t,
J = 4.5 Hz, 1H), 2.19 (m, 1H), 1.78−1.61 (m, 2H), 1.58−1.47 (m,
1H), 1.47−1.29 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 165.90,
164.53, 152.17, 136.27, 132.56, 130.33, 128.68, 59.64, 42.91, 42.06,
40.92, 37.72, 37.54, 33.35, 29.44, 28.76, 23.82. HRMS (ESI):
calculated for C19H21ClN2O3 [M − H]−, 359.1168; found, 359.1183.
50, HPLC purity: 99.0%; [α]25 (c 2, CHCl3) = +14.5, chiral HPLC
D
(Daicel OD-RH, 4.6−150 mm, 5 μm; 60% acetonitrile/40% 0.1 M
aqueous KPF6; 0.5 mL/min; 268 nm) tR = 25.367 min, >99% ee. 52,
HPLC purity: 100%; [α]25 (c 2, CHCl3) = −14.7, chiral HPLC
D
(Daicel OD-RH, 4.6−150 mm, 5 μm; 60% acetonitrile/40% 0.1 M
aqueous KPF6; 0.5 mL/min; 268 nm) tR = 23.367 min, 97.0% ee.
1-Cyclopentyl-3-(3,5-dichlorophenethyl)pyrimidine-2,4,6-
Spectral Data of the Most Active Compounds. (S)-1-(3-
Chlorophenethyl)-3-(1-cyclohexylethyl)pyrimidine-2,4,6-
(1H,3H,5H)-trione (43) and (R)-1-(3-Chlorophenethyl)-3-(1-
cyclohexylethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (45). Color-
less oil; 1H NMR (500 MHz, CDCl3) δ 7.26−7.07 (m, 4H), 4.50 (m,
1H), 4.09 (m, 2H), 3.60 (s, 2H), 2.90 (m, 2H), 2.04 (m, 1H), 1.87 (m,
1H), 1.76 (m, 1H), 1.66 (m, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.32−1.06
(m, 4H), 0.86 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 164.85,
164.64, 150.91, 139.75, 134.27, 129.81, 129.13, 127.21, 126.95, 51.35,
42.50, 39.88, 39.20, 33.57, 30.83, 29.83, 26.07, 25.79, 25.71, 16.29.
HRMS (ESI): calculated for C20H25ClN2O3 [M − H]−, 375.1481;
1
(1H,3H,5H)-trione (85). White powder; mp 144−146 °C; H NMR
(500 MHz, CDCl3) δ 7.25 (t, J = 1.9 Hz, 1H), 7.16 (d, J = 1.9 Hz,
2H), 5.15 (m, 1H), 4.09−4.02 (m, 2H), 3.66 (s, 2H), 2.92−2.80 (m,
2H), 1.95 (m, 4H), 1.85 (m, 2H), 1.66−1.52 (m, 2H); 13C NMR (126
MHz, CDCl3) δ 164.69, 164.49, 150.85, 141.12, 135.02, 127.49,
127.05, 54.39, 42.36, 40.11, 33.51, 28.72, 25.57. HRMS (ESI):
calculated for C17H18Cl2N2O3 [M − H]−, 367.0622; found, 367.0637.
HPLC purity: 94.5%.
found, 375.1494. 43, HPLC purity: 99.2%, [α]25 (c 2, CHCl3) =
1-Cyclopentyl-3-(3,4-dichlorophenethyl)pyrimidine-2,4,6-
D
1
(1H,3H,5H)-trione (86). White powder; mp 131−133 °C; H NMR
−6.8, chiral HPLC (Daicel AD-RH, 4.6−150 mm, 5 μm; 70%
acetonitrile/30% 0.05 M pH 2 phosphate buffer; 0.4 mL/min; 268
nm) tR = 30.933 min; >99% ee. 45, HPLC purity: 99.9%, [α]25D (c 2,
CHCl3) = +7.0; chiral HPLC (Daicel AD-RH, 4.6−150 mm, 5 μm;
70% acetonitrile/30% 0.05 M pH 2 phosphate buffer; 0.4 mL/min;
268 nm) tR = 28.017 min; >99% ee.
(500 MHz, CDCl3) δ 7.43−7.32 (m, 2H), 7.12 (d, J = 8.2 Hz, 1H),
5.24−5.08 (m, 1H), 4.13−4.03 (m, 2H), 3.66 (s, 2H), 2.93−2.78 (m,
2H), 2.04−1.90 (m, 4H), 1.89−1.79 (m, 2H), 1.67−1.54 (m, 2H); 13C
NMR (126 MHz, CDCl3) δ 164.71, 164.52, 150.87, 138.03, 132.50,
130.91, 130.85, 130.52, 128.40, 54.36, 42.48, 40.12, 33.18, 28.71,
25.56. HRMS (ESI): calculated for C17H18Cl2N2O3 [M − H]−,
367.0622; found, 367.0637. HPLC purity: 98.0%.
(S)-1-(4-Chlorophenethyl)-3-(1-cyclohexylethyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (44) and (R)-1-(4-Chlorophenethyl)-3-
(1-cyclohexylethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (46). Col-
1-(3,5-Bis(trifluoromethyl)phenethyl)-3-cyclopentylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (87). White powder; mp 163−166 °C;
1H NMR (500 MHz, CDCl3) δ 7.78 (s, 1H), 7.73 (s, 2H), 5.27−4.99
(m, 1H), 4.21−4.04 (m, 2H), 3.66 (s, 2H), 3.17−2.93 (m, 2H), 2.00−
1.75 (m, 6H), 1.66−1.51 (m, 2H); 13C NMR (126 MHz, CDCl3) δ
164.57, 164.54, 150.83, 140.26, 131.84 (q, J = 33.3 Hz), 129.19 (m),
123.23 (q, J = 272.8 Hz), 120.88 (m), 54.38, 42.16, 40.03, 33.66,
28.68, 25.53. HRMS (ESI): calculated for C19H18F6N2O3 [M − H]−,
435.1149; found, 435.1169. HPLC purity: 98.5%.
1
orless oil; H NMR (500 MHz, CDCl3) δ 7.25 (d, J = 8.3 Hz, 2H),
7.16 (d, J = 8.3 Hz, 2H), 4.49 (m, 1H), 4.08 (m, 2H), 3.60 (s, 2H),
2.88 (m, 2H), 2.04 (m, 1H), 1.87 (m, 1H), 1.76 (m, 1H), 1.66 (m,
2H), 1.35 (d, J = 6.9 Hz, 3H), 1.31−1.06 (m, 4H), 0.85 (m, 2H); 13C
NMR (126 MHz, CDCl3) δ 164.85, 164.65, 151.06, 136.19, 132.56,
130.37, 128.66, 51.34, 42.56, 39.88, 39.19, 33.27, 30.81, 29.85, 26.06,
25.79, 25.70, 16.28. HRMS (ESI): calculated for C20H25ClN2O3 [M −
H]−, 375.1148; found, 375.1494. 44, HPLC purity: 97.8%, [α]25D (c 2,
J
dx.doi.org/10.1021/jm4005048 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1-((1-(4-Chlorophenyl)cyclopropyl)methyl)-3-cyclopentyl-
C19H20Cl2N2O3 [M − H]−, 393.08; found, 393.26. HPLC purity:
98.1%.
1
pyrimidine-2,4,6(1H,3H,5H)-trione (96). Colorless oil; H NMR
(500 MHz, CDCl3) δ 7.21 (s, 4H), 5.00 (p, J = 8.7 Hz, 1H), 4.06 (s,
2H), 3.51 (s, 2H), 1.92−1.81 (m, 2H), 1.79−1.66 (m, 4H), 1.59−1.43
(m, 2H), 1.07−0.98 (m, 2H), 0.87−0.78 (m, 2H); 13C NMR (126
MHz, CDCl3) δ 164.75, 150.82, 140.85, 132.80, 131.52, 128.32, 54.21,
48.83, 40.03, 28.56, 25.49, 25.04, 12.05. HRMS (ESI): calculated for
C19H21ClN2O3 [M − H]−, 359.1168; found, 359.1186. HPLC purity:
96.1%.
1-(( )-exo-Norbornyl)-3-(3-(trifluoromethyl)phenethyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (106), 1-((1R,2S,4S)-Bicyclo-
[2.2.1]heptan-2-yl)-3-(3-(trifluoromethyl)phenethyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (107), and 1-((1S,2R,4R)-
Bicyclo[2.2.1]heptan-2-yl)-3-(3-(trifluoromethyl)phenethyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (108). White powder; mp 65−
69 °C; 1H NMR (500 MHz, CDCl3) δ 7.51−7.26 (m, 4H), 4.59−4.45
(m, 1H), 4.10−3.93 (m, 2H), 3.55 (dd, J = 21.0, 12.9 Hz, 2H), 2.89 (t,
J = 7.8 Hz, 2H), 2.52 (d, J = 3.7 Hz, 1H), 2.27 (d, J = 4.4 Hz, 1H),
2.11 (m, 1H), 1.71−1.54 (m, 2H), 1.49−1.41 (m, 1H), 1.40−1.21 (m,
4H); 13C NMR (126 MHz, CDCl3) δ 165.90, 164.58, 152.17, 138.80,
132.45, 130.84 (q, J = 32.0 Hz), 129.05, 125.69 (q, J = 3.7 Hz), 124.08
(q, J = 272.4 Hz), 123.63 (d, J = 4.1 Hz), 59.64, 42.74, 42.09, 40.86,
37.74, 37.54, 33.77, 29.50, 28.77, 23.84. HRMS (ESI): calculated for
C20H21F3N2O3 [M − H]−, 393.1432; found, 393.1448. 106, HPLC
purity: 97.9%. 107, HPLC purity: 96.8%, [α]25D (c 2, CHCl3) = −7.5,
chiral HPLC (Daicel OD-RH, 4.6−150 mm, 5 μm; 60% acetonitrile/
40% 0.1 M aqueous KPF6; 0.5 mL/min; 268 nm) tR = 18.800 min,
1-(2-(3-Chlorophenyl)-2-methylpropyl)-3-cyclopentylpyri-
1
midine-2,4,6(1H,3H,5H)-trione (98). Colorless oil; H NMR (500
MHz, CDCl3) δ 7.46−7.00 (m, 4H), 5.20−4.97 (m, 1H), 4.08 (s, 2H),
3.61 (s, 2H), 1.97−1.73 (m, 6H), 1.61−1.49 (m, 2H), 1.36 (s, 6H);
13C NMR (126 MHz, CDCl3) δ 165.00, 164.77, 151.22, 148.51,
134.09, 129.42, 126.69, 126.59, 124.55, 54.29, 51.06, 40.22, 40.15,
28.67, 26.94, 25.54. MS (ESI): calculated for C19H23ClN2O3 [M −
H]−, 361.13; found, 361.23. HPLC purity: 99.1%.
1-Cyclopentyl-3-(2-methyl-2-(3-(trifluoromethyl)phenyl)-
1
propyl)pyrimidine-2,4,6(1H,3H,5H)-trione (99). Colorless oil; H
NMR (500 MHz, CDCl3) δ 7.75−7.59 (m, 2H), 7.59−7.39 (m, 2H),
5.08 (p, J = 8.7 Hz, 1H), 4.12 (s, 2H), 3.61 (s, 2H), 1.89 (m, 2H),
1.85−1.72 (m, 4H), 1.58−1.48 (m, 1H), 1.42 (s, 6H); 13C NMR (126
MHz, CDCl3) δ 164.98, 164.66, 151.15, 147.28, 130.37, 129.88,
128.61, 123.32, 123.06, 122.05, 54.27, 51.05, 40.25, 40.09, 28.65,
27.01, 25.51. MS (ESI): calculated for C20H23F3N2O3 [M − H]−,
395.16; found, 395.28. HPLC purity: 95.3%.
1-(2-(3-Chlorophenyl)propyl)-3-(( )-endo-norbornyl)-
pyrimidine-2,4,6-(1H,3H,5H)-trione (102). Colorless oil; 1H NMR
(500 MHz, CDCl3) δ 7.27−7.05 (m, 4H), 4.64−4.49 (m, 1H), 4.11
(m, 1H), 3.97 (m, 1H), 3.67−3.44 (m, 2H), 3.36−3.17 (m, 1H),
2.68−2.45 (m, 1H), 2.37−2.28 (m, 1H), 2.14 (m, 1H), 1.85−1.57 (m,
3H), 1.57−1.49 (m, 1H), 1.46−1.35 (m, 2H), 1.34−1.17 (m, 5H); 13C
NMR (126 MHz, CDCl3) δ 165.89, 165.85, 164.69, 152.33, 152.27,
145.13, 145.08, 134.24, 134.21, 129.74, 129.71, 127.79, 127.09, 125.71,
125.67, 59.62, 59.60, 47.87, 47.77, 47.76, 42.04, 41.96, 40.91, 40.81,
37.87, 37.81, 37.74, 37.70, 37.55, 37.49, 29.69, 29.35, 28.76, 23.83,
23.71, 18.53. MS (ESI): calculated for C20H23ClN2O3 [M − H]−,
373.13; found, 373.10. HPLC purity: 99.3%.
96.9% ee. 108, HPLC purity: 99.0%; [α]25 (c 2, CHCl3) = +7.7;
D
chiral HPLC (Daicel OD-RH, 4.6−150 mm, 5 μm; 60% acetonitrile/
40% 0.1 M aqueous KPF6; 0.5 mL/min; 268 nm) tR = 21.583 min;
>99% ee.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectroscopic and analytical data of all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 847-491-5653. E-mail: Agman@chem.
northwestern.edu.
Notes
1-(1-(3-Chlorophenyl)propan-2-yl)-3-(( )-endo-norbornyl)-
pyrimidine-2,4,6-(1H,3H,5H)-trione (103). Colorless oil; 1H NMR
(500 MHz, CDCl3) δ 7.48−7.10 (m, 4H), 4.70−4.49 (m, 1H), 4.20−
4.02 (m, 2H), 3.73−3.51 (m, 2H), 2.54 (d, J = 3.8 Hz, 1H), 2.34 (d, J
= 4.6 Hz, 1H), 2.13−2.02 (m, 1H), 1.77−1.60 (m, 2H), 1.58−1.48 (m,
1H), 1.48−1.38 (m, 2H), 1.37 (s, 3H), 1.35 (s, 3H), 1.35−1.22 (m,
2H); 13C NMR (126 MHz, CDCl3) δ 165.87, 165.01, 152.60, 148.54,
134.08, 129.41, 126.68, 126.60, 124.58, 59.72, 51.28, 41.94, 41.00,
40.24, 37.74, 37.52, 29.35, 28.75, 27.03, 26.92, 23.82. MS (ESI):
calculated for C21H25ClN2O3 [M − H]−, 387.15; found, 387.28.
HPLC purity: 96.9%.
1-(3,5-Bis(trifluoromethyl)phenethyl-3-(( )-endo-
norbornyl)pyrimidine-2,4,6(1H,3H,5H)-trione (104). White pow-
der; mp 74−77 °C; 1H NMR (500 MHz, CDCl3) δ 7.78 (s, 1H), 7.73
(s, 2H), 4.72−4.57 (m, 1H), 4.22−4.07 (m, 2H), 3.77−3.56 (m, 2H),
3.06 (t, J = 7.8 Hz, 2H), 2.61 (d, J = 4.1 Hz, 1H), 2.36 (t, J = 4.6 Hz,
1H), 2.18 (m, 1H), 1.83−1.63 (m, 2H), 1.60−1.49 (m, 1H), 1.49−
1.28 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 165.76, 164.60, 152.11,
140.31, 131.83 (q, J = 33.4 Hz), 129.20 (d, J = 3.7 Hz), 123.25 (q, J =
272.8 Hz), 120.88 (p, J = 3.7 Hz), 59.70, 42.36, 42.09, 40.80, 37.72,
37.52, 33.67, 29.52, 28.74, 23.81. HRMS (ESI): calculated for
C21H20F6N2O3 [M − H]−, 461.1305; found, 461.1321. HPLC purity:
96.4%.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Michael J. Fox Foundation (Therapeutics
Development Initiative) and the RJG Foundation for financial
support of this research.
ABBREVIATIONS USED
■
LTCC, L-type calcium channel; PD, Parkinson’s disease; PYT,
pyrimidinetrione; SNc, substantia nigra pars compacta; FLIPR,
fluorometric imaging plate reader; DMEM, Dulbecco’s
modified Eagle medium
REFERENCES
■
(1) Casamassima, F.; Hay, A. C.; Benedetti, A.; Lattanzi, L.; Cassano,
G. B.; Perlis, R. H. L-type calcium channels and psychiatric disorders, a
brief review. Am. J. Med. Genet., Part B 2010, 153B, 1373−1390.
(2) Catterall, W. A.; Perez-Reyes, E.; Snutch, T. P; Striessnig, J.
International Union of Pharmacology. XLVIII. Nomenclature and
structure−function relationships of voltage-gated calcium channels.
Pharmacol. Rev. 2005, 57, 411−425.
1-(2(3,5-Dichlorophenyl)ethyl)-3-(( )-endo-norbornyl)-
pyrimidine-2,4,6(1H,3H,5H)-trione (105). White powder; mp
(3) Lipscombe, D.; Helton, T. D; Xu, W. L-type calcium channels:
the low down. J. Neurophysiol. 2004, 92, 2633−2641.
1
150−151 °C; H NMR (500 MHz, CDCl3) δ 7.26 (s, 1H), 7.17 (s,
2H), 4.71−4.58 (m, 1H), 4.13−4.01 (m, 2H), 3.65 (dd, J = 21.2, 8.7
Hz, 2H), 2.87 (t, J = 7.8 Hz, 2H), 2.66−2.58 (m, 1H), 2.42−2.34 (m,
1H), 2.25−2.15 (m, 1H), 1.81−1.66 (m, 2H), 1.61−1.51 (m, 1H),
1.48−1.32 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 165.85, 164.55,
152.13, 141.15, 135.01, 127.51, 127.05, 59.70, 42.53, 42.11, 40.90,
37.76, 37.55, 33.50, 29.55, 28.80, 23.88. MS (ESI): calculated for
(4) Sinnegger-Brauns, M. J.; Huber, I. G.; Koschak, A.; Wild, C.;
Obermair, G. J.; Einzinger, U.; Hoda, J.-C.; Sartori, S. B.; Striessnig, J.
Expression and 1,4-dihydropyridine-binding properties of brain L-type
calcium channel isoforms. Mol. Pharmacol. 2009, 75, 407−414.
(5) Zuccotti, A.; Clementi, S.; Reinbothe, T.; Torrente, A.; Vandael,
D. H.; Pirone, A. Structural and functional differences between L-type
K
dx.doi.org/10.1021/jm4005048 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
calcium channels: crucial issues for future selective targeting. Trends
Pharmacol. Sci. 2011, 32, 366−375.
(25) Eda, M.; Takemoto, T.; Ono, S.-I.; Okada, T.; Kosaka, K.;
Gohda, M.; Matzno, S.; Nakamura, N.; Fukaya, C. Novel potassium-
channel openers: preparation and pharmacological evaluation of
racemic and optically active N-(6-amino-3-pyridyl)-N′-bicycloalkyl-
N″-cyanoguanidine derivatives. J. Med. Chem. 1994, 37, 1983−1990.
(26) (a) Bollu, V.; Boren, B. C.; Dalgar, J. E.; Flatt, B. T.; Haq, N.;
Hudson, S.; Mohan, R.; Morrissey, M.; Pratt, B.; Wang, T.-L. Triazole
and Imidazole Derivatives for Use as TGR5 Agonists in the Treatment
of Diabetes and Obesity. PCT Int. Appl. 2011071565, Jun 16, 2011.
(b) Meier, H.; Bender, E.; Brueggemeier, U.; Flamme, I.; Karthaus, D.;
Kolkhof, P.; Meibom, D.; Schneider, D.; Voehringer, V.; Fuerstner, C.;
Keldenich, J.; Lang, D.; Pook, E.; Schmerk, C. Preparation of 4-
Arylimidazol-2-ones and 5-Aryl-1,2,4-triazolones as Vasopressin
Receptor Inhibitors. PCT Int. Appl. 2007134862, Nov 29, 2007.
(27) Nugent, T. C.; Ghosh, A. K.; Wakchaure, V. N.; Mohanty, R. B.
Asymmetric reductive amination: convenient access to enantioen-
riched alkyl-alkyl or aryl-alkyl substituted α-chiral primary amines. Adv.
Synth. Catal. 2006, 348, 1289−1299.
(28) Moss, N.; Gauthier, J.; Ferland, J.-M. The enantioselective
synthesis of neopentylamine derivatives. Synlett 1995, 2, 142−144.
(29) (a) Frahm, A. W.; Knupp, G. Asymmetric Synthesis of cis-2-
substituted cyclohexanamines with high optical purity. Tetrahedron
Lett. 1981, 22, 2633−2636. (b) Knupp, G.; Frahm, A. W. Synthesis
and absolute configuration of 2-substituted cyclohexylamine. Chem.
Ber. 1984, 117, 2076−2098.
(30) Handrock, R.; RaoSchymanski, R.; Klugbauer, N.; Hofmann, F.;
Herzig, S. Dihydropyridine enantiomers block recombinant L-type
Ca2+ channels by two different mechanisms. J. Physiol. 1999, 521.1,
31−42.
(6) Striessnig, J.; Bolz, H. J.; Koschak, A. Channelopathies in CaV1.1,
CaV1.3, and CaV1.4 voltage-gated L-type Ca2+ channels. Pfluegers Arch.
2010, 460, 361−374.
(7) Olson, P. A.; Tkatch, T.; Hernandez-Lopez, S.; Ulrich, S.; Ilijic,
E.; Mugnaini, E.; Zhang, H.; Bezprozvanny, I.; Surmeier, D. J. G-
protein-coupled receptor modulation of striatal Cav1.3 L-type Ca2+
channels is dependent on a Shank-binding domain. J. Neurosci. 2005,
25, 1050−1062.
(8) Chan, C. S.; Guzman, J. N.; Ilijic, E.; Mercer, J.; Rick, C.; Tkatch,
T.; Meredith, G.; Surmeier, D. J. Rejuvenation protects neurons in
mouse models of Parkinson’s disease. Nature 2007, 447, 1081−1086.
(9) Riederer, P.; Wuketich, S. Time course of nigrostriatal
degeneration in Parkinson’s disease. A detailed study of influential
factors in human brain amine analysis. J. Neural Transm. 1976, 38,
277−301.
(10) Fearnley, J. M.; Lees, A. J. Ageing and Parkinson’s disease:
substantia nigra regional selectivity. Brain 1991, 114, 2283−2301.
(11) Guzman, J. N.; Sanchez-Padilla, J.; Wokosin, D.; Kondapalli, J.;
Ilijic, E.; Schumacker, P. T.; Surmeier, D. J. Oxidant stress evoked by
pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature
2010, 468, 696−700.
́
(12) Guzman, J. N.; Sanchez-Padilla, J.; Chan, C. S.; Surmeier, D. J.
Robust pacemaking in substantia nigra dopaminergic neurons. J.
Neurosci. 2009, 29, 11011−11009.
(13) Jankovic, J. Parkinson’s disease: clinical features and diagnosis. J.
Neurol., Neurosurg. Psychiatry 2008, 79, 368−376.
(14) Epstein, M. Calcium antagonists: still appropriate as first line
antihypertensive agents. Am. J. Hypertens. 1996, 9, 110−121.
(15) Sullivan, E.; Tucker, E. M.; Dale, L. Measurement of [Ca2+]
using the fluorometric imaging plate reader (FLIPR). Methods Mol.
Biol. 1999, 114, 125−133.
(16) Minta, A.; Kao, J. P. Y; Tsien, R. Y. Fluorescent indicators for
cytosolic calcium based on rhodamine and fluorescein chromophores.
J. Biol. Chem. 1989, 264, 8171−8178.
(17) Triggle, D. J. 1,4-Dihydropyridines as calcium channel ligands
and privileged structures. Cell. Mol. Neurobiol. 2003, 23, 293−303.
(18) Triggle, D. J.; Hawthorn, M.; Gopalakrishnan, M.; Minarini, A.;
Avery, S.; Rutledge, A.; Bangalore, R.; Zheng, W. Synthetic organic
ligands active at voltage-gated calcium channels. Ann. N.Y. Acad. Sci.
1991, 635, 123−138.
(19) Chang, C.-C.; Cao, S.; Kang, S.; Kai, L.; Tian, X.; Pandey, P.;
Fernandez, D. S.; Luan, C.-H.; Surmeier, D. J.; Silverman, R. B.
Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates
toward voltage-dependent L-type Ca2+ channels CaV1.3 and CaV1.2.
Bioorg. Med .Chem. 2010, 18, 3147−3158.
(20) Kang, S.; Cooper, G.; Dunne, S. F.; Luan, C.-H.; Surmeier, D. J.;
Silverman, R. B. Antagonism of L-type Ca2+ channels CaV1.3 and
CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine
mimics, Bioorg. Med. Chem. [Online early access]. DOI: 10.1016/
j.bmc.2013.04.054. Published Online: Apr 28, 2013.
(21) Kang, S.; Cooper, G.; Dunne, S. F.; Dusel, B.; Luan, C.-H.;
Surmeier, D. J.; Silverman, R. B. CaV1.3-selective L-type calcium
channel antagonists as potential new therapeutics for Parkinson’s
disease. Nat. Commun. 2012, 3, 1146.
(22) Wohler, F. Ueber kunstliche bildung des harnstoffs. Ann. Phys.
̈
̈
Chem. 1828, 88, 253.
(23) Biltz, H.; Wittek, H. Alkylierte und acylierte barbitursauren. Ber.
1921, 54B, 1035.
(24) (a) Poll, T.; Sobczak, A.; Hartmann, H.; Helmchen, G.
Diastereoface-discriminative metal coordination in asymmetric syn-
thesis: D-pantolactone as practical chiral auxiliary for Lewis acid
catalyzed Diels−Alder reactions. Tetrahedron Lett. 1985, 26, 3095−
3098. (b) Poll, T.; Abdel Hady, A. F.; Karge, R.; Linz, G.; Weetman, J.;
Helmchen, G. N-Substituted hydroxysuccinhides from (S)-malic acid
as new reagents for asymmetric Diels−Alder additions to enoates.
Tetrahedron Lett. 1989, 30, 5595−5598.
L
dx.doi.org/10.1021/jm4005048 | J. Med. Chem. XXXX, XXX, XXX−XXX