Antagonism of L-type Ca2+ channels CaV1.3 and Ca V1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics

Article abstract of DOI:10.1016/j.bmc.2013.04.054

The L-type calcium channel (LTCC) Ca_V1.3 is regarded as a new potential therapeutic target for Parkinson's disease. Calcium influx through Ca_V1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a Ca _V1.3 antagonist selective over Ca_V1.2 is essential because Ca_V1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of Ca_V1.3 relative to Ca_V1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of Ca_V1.3 and Ca_V1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for Ca_V1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for Ca_V1.3 over Ca_V1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2-3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in Ca_V1.3 and Ca _V1.2 LTCCs are very similar.

Full text of DOI:10.1016/j.bmc.2013.04.054

Bioorganic & Medicinal Chemistry 21 (2013) 4365–4373
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Bioorganic & Medicinal Chemistry
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Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by
1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics q
Soosung Kang a, Garry Cooper a,c, Sara Fernandez Dunne d, Chi-Hao Luan d, D. James Surmeier c,
Richard B. Silverman a,b,
⇑
a Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
b Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL
60208, USA
c Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
d High Throughput Analysis Laboratory, Northwestern University, Evanston, IL 60208, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The L-type calcium channel (LTCC) CaV1.3 is regarded as a new potential therapeutic target for Parkin-
son’s disease. Calcium influx through CaV1.3 LTCC during autonomous pacemaking in adult dopaminergic
neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative
stress in animal models. Development of a CaV1.3 antagonist selective over CaV1.2 is essential because
CaV1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous
and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent
and selective antagonists of CaV1.3 relative to CaV1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic
1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved.
The antagonism studies of CaV1.3 and CaV1.2 LTCCs using DHP mimic compounds showed that dihydro-
pyrimidines and 4H-pyrans are effective antagonists of DHPs for CaV1.3 LTCCs. Some 1,4-dihydropyrim-
idines are more selective than isradipine for CaV1.3 over CaV1.2, shown here by both calcium flux and
patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2–3. These results
support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyr-
imidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest
that the binding sites of DHP in CaV1.3 and CaV1.2 LTCCs are very similar.
Received 21 February 2013
Revised 12 April 2013
Accepted 22 April 2013
Available online 28 April 2013
Keywords:
1,4-Dihydropyrimidines
4H-Pyrans
CaV1.3 Ca2+ channel
CaV1.2 Ca2+ channel
L-type calcium channel
Parkinson’s disease
FLIPR (Fluorometric imaging plate reader)
Antagonism
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In the presence of the DHP, SNc dopamine (DA) neurons switch
to a calcium-independent form of pacemaking, lowering mito-
L-type voltage-gated calcium channels (LTCCs) are membra-
nous multimeric proteins that underlie important physiological
properties,1 such as cardiac action potential generation, calcium
homeostasis, E–C coupling, and hormone secretion. Several
isoforms2 are localized in neuronal, neurosecretory, and skeletal
muscle tissue with different pharmacological and biophysical
properties. Recently, we reported3 that an unusual engagement
of CaV1.3 LTCCs adult dopaminergic neurons of the substantia nigra
pars compacta (SNc) during pacemaking renders SNc neurons
vulnerable in animal models of Parkinson’s disease (PD). This
engagement is decreased by the application of isradipine, a
1,4-dihydropyridine (DHP) antagonist of LTCCs in adult neurons.
chondrial stress with no obvious loss of function at the cellular
or behavioral level. This strategy is neuroprotective in PD animal
models4 and clinical trials conducted in Denmark5 and United
Kingdom6 showed that by treating hypertension with DHP calcium
channel blockers the onset of Parkinson’s disease is reduced. How-
ever, treatment of presently available LTCC blockers, such as israd-
ipine, diltiazem, and verapamil, for the protection of SNc neurons
from Parkinson’s disease progression, is limited because CaV1.2
LTCCs also interact with these molecules.7 CaV1.3 and CaV1.2 are
the predominant neuronal LTCC isoforms; CaV1.2 LTCCs are the
major isoform (ꢀ90%) and are also found in abundance in cardiac
tissue of the sinoatrial node, participating heavily in cardiac pace-
making.8 Although DHPs hold the potential to ameliorate the PD
effect, they also block CaV1.2 LTCCs and might provide undesirable
effects to the cardiovascular system.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
The antagonism of CaV1.3 LTCCs has only recently attracted
attention with studies recently reported by us9,10 and other re-
search groups.11,7a As described in our earlier paper,10 our initial
effort focused on DHP analogues to verify the difference between
⇑
Corresponding author. Tel.: +1 847 491 5653.
E-mail address: r-silverman@northwestern.edu (R.B. Silverman).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.054

4366
S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
NO2
1
CaV1.3 and CaV1.2; many DHPs were synthesized and found to be
potent, but all were poorly selective or not selective for CaV1.3
channels. From this study we confirmed that ortho- or meta-nitro
phenyl substituted DHPs are more potent and slightly selective to-
ward CaV1.3. It was also found that hydrogen bonding with the
nitrogen of 1,4-dihydropyridine is essential to maintain the bioac-
tivity and selectivity toward CaV1.3 LTCCs. Since the modification
of the dihydropyridine ring was limited, our efforts to explore
DHPs were shifted to diverse alternative scaffolds, such as 1,4-
dihydropyrimidines12,13 and 4H-pyrans (Fig. 1).14 These scaffolds
are known to display similar pharmacological properties to DHP
calcium channel blockers, but have potential diverse structural
variations. Asymmetric modification of the DHP scaffold to 1,4-
dihydropyrimidines or 4H-pyrans can provide a chiral center as
well as different hydrogen-bonding donor/acceptors at or near
the pyrimidine/pyran ring. Therefore, these modifications allow
the exploration of the steric and electronic demand on the pyrim-
idine/pyran ring region for the development of small-molecule
inhibitors that can selectively antagonize CaV1.3 over CaV1.2 LTCCs.
In this study, various 1,4-dihydropyrimidines and 4H-pyrans were
synthesized and their antagonism toward voltage dependent
CaV1.3 and CaV1.2 LTCCs were studied.
NO2
'Biginelli'
CHO
5% cat. Yb(OTf)3
50~70%
R1O2C
R2
NH
R1O2C
R2
+
NH2
N
H
X
H2N
X
O
2
3
4a-e
NO2
NaH
R1O2C
R2
R4
N
formate or acyl halide
60~80%
N
X
R3
5a-l
Scheme 1. Synthesis of dihydropyrimidines via Biginelli reaction.
and urea or thiourea in the presence of Yb(OTf)3 afforded 6-al-
kyl-5-(alkoxycarbonyl)-4-(3-nitrophenyl)-3,4-dihydropyrimidin-
2(1H)-one (4a–c) or 6-alkyl-5-(alkoxycarbonyl)-4-(3-nitro-
phenyl)-3,4-dihydropyrimidin-2(1H)-thione (4d,e) in good yields.
To provide structural similarity with lead molecules isradipine or
nitrendipine, N-carbonylation or N-carbamation of Biginelli prod-
ucts 4a–e was performed with isobutyryl chloride or alkyl chloro-
formates (e.g., ClCO2Et, ClCO2iPr), in the presence of NaH or TEA, to
obtain 5a–l.
4H-Pyrans were also prepared by a three-component condensa-
tion with a cyanoacetate, an aldehyde, and a b-ketoester (Scheme 2).
Condensation of 3-nitrobenzaldehyde, cyanoacetate, and alkyl ace-
toacetate in a protic solvent afforded 3,5-diakyl 2-amino-6-methyl-
4-(3-nitrophenyl)-4H-pyran-3,5-dicarboxylates (8a–c) in good
yields. N-Acylation of 8a–c with acetyl chloride or isobutyric anhy-
dride in the presence of TEA provided 9a–c in excellent yields.
2-Substituted 1,4-dihydropyrimidines17 were prepared as
shown in Scheme 3. The strategy for the synthesis of 2-substituted
1,4-dihydropyrimidines seemed straightforward; instead of urea or
thiourea, as used in the previous Biginelli condensation, 2-methyl-
2-thiopseudourea was chosen to provide the desired products.
However, the acid catalyzed Biginelli three-component condensa-
tion was slow and led to several side products. Alternatively, con-
densation of 3-nitrobenzaldehyde with the b-ketoester first to
2. Results
2.1. Chemistry
On the basis of previous DHPs studies and lead compounds
isradipine and nitrendipine, we chose 3-nitrophenyl as the aro-
matic ring. Changes were made to the dihydropyridine scaffold
(pyrimidinone, pyrimidinethione, and hydropyran), to each of the
carboxylates (R1, R2), and to the alkyl side chain (R3) (Fig. 1);
methyl, ethyl, and isopropyl were selected for derivatization of
the carboxylates (R1, R2) or the alkyl side chain (R3). With these
substitutions, we are able to exam hydrogen-bonding acceptor/do-
nor effects and the influence on its neighboring group, especially
related to the steric bulkiness.
The synthesis of pyrimidinones and pyrimidinethiones were
similar; they were readily prepared by a Biginelli three-compo-
nent condensation with an aldehyde, a b-ketoester, and a urea
or thiourea (Scheme 1).15 Because traditional Biginelli reactions,
which are catalyzed by HCl or H2SO4 in an alcohol solvent, give
low yields with possible transesterification at high temperature,
we used Lewis acid catalyzed procedures16 in a non-alcohol sol-
vent. Condensation of 3-nitrobenzaldehyde, alkyl acetoacetate,
give
a
-benzylidene-b-keto ester 10, followed by tandem Michael
Nitrendipine
NO2
NO2
NO2
NO2
R2O2C
R3
R2O2C
R3
CO2R1
S
EtO2C
Me
CO2Me
Me
H
O
R2O2C
R3
CO2R1
O
N
N
N
N
H
N
H
N
H
N
H
H-bonding
modification
Isradipine
N
O
N
NO2
NO2
NO2
R2O2C
Me
CO2R1
R2O2C
Me
CO2R1
R2O2C
Me
CO2R1
iPrO2C
Me
CO2Me
N
N
R3
R3
S
R3
N
H
Me
N
N
N
O
N
H
H
Lead molecules
Modified molecules
Figure 1. 1,4-Dihydropyrimidines and 4H-pyrans that are mimics of DHPs and are synthesized in this study.

S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
4367
NO2
1
NO2
NO2
CHO
acyl halide, TE A
80~90%
cat. piperidine
R1O2C
CO2R2
NHR3
R1O2C
CO2R2
NH2
CO2R2
R1O2C
iPrOH
+
O
O
40~70%
O
N
9a-c
6
8a-c
7
Scheme 2. Synthesis of substituted 2-aminopyranes via three component in situ condensation and cyclization.
NO2
NO2
NO2
NO2
NH
SMe
H2N
NaH, ClCO2R2
60~80%
11
R1O2C
R1O2C
CO2R2
SMe
R1O2C
R1O2C
CO2R2
NHMe
NH2Me
NH
SMe
N
N
45~55%
NaOAc
30~45%
N
N
O
N
10
12a-b
13a-b
14a-b
Scheme 3. Synthesis of substituted dihydropyrimidines via Atwal modified Biginelli reaction.
addition and condensation with 2-methyl-2-thioseudourea sulfate
(11) in the presence of NaOAc, afforded alkyl 4-methyl-2-(methyl-
thio)-6-(3-nitrophenyl)-1,6-dihydropyrimidine-5-carboxylates
(12a–b) in moderate yields. Compounds 12a–b were allowed to re-
act with alkyl chlorofomate in the presence of NaH to give 13a–b.
Substitution of the methylthio group with methylamine proceeded
well and provided 2-methylamine substituted dihydropyrimidines
14a–b in moderate yields.
Since each enantiomer of DHPs and dihydropyrimidines is
known to have different pharmacological effects on the target cal-
cium channels, three analogues with the best combination of rela-
tive potency and selectivity (5a, 5b, 5i) were chosen, and their
racemates were resolved to greater than 97% ee using chiral
HPLC,18 with a Chiralcel OD-H column, to explore chirality-depen-
dent bioactivity.
3. Discussion
Various dihydropyrimidine alkyl esters displayed IC50 values in
the range of 0.2–100 lM at both CaV1.3 and CaV1.2 calcium chan-
nels (Table 1). Carbamates (R2) at the N3 positions of the dihydro-
pyrimidine ring appear to increase potency. Comparing the IC50
values of 4a–e (>15 lM) with other carbamates, it is apparent that
modification to a carbamate at the N3 nitrogen near the aromatic
ring leads to a gain in potency toward both CaV1.3 and CaV1.2 (Ta-
ble 1). It seems that the alkyl carbamates (R2) and esters (R3) at C5
are essential for good interactions with the calcium channels; this
result is in agreement with the previous SAR of DHPs.10 When the
alkyl ester at C5 was changed from methyl to ethyl and isopropyl,
there was little, if any, difference in potency. However, replace-
ment of the methyl group (R4) to isopropyl on C6, such as in 4c,
5c, 5d, 5h, and 5l, made the molecules less potent relative to the
methyl analogue. The more bulky steric requirement at this posi-
tion appears to be important for potency toward both CaV1.3 and
CaV1.2 LTCCs. However, these various alkyl substitutions seem
not to be strongly correlated with CaV1.3 selectivity. As observed
with the N1 acyl derivatives (5d, 5e) in Table 1, acyl substitution
of the other nitrogen on the pyrimidinone ring (5k,l) did not im-
prove the potency of either calcium channel. Replacement of the
oxygen at the C2 position of the dihydropyrimidine ring with a sul-
fur (X = S) gave an ꢀ5-fold more potent antagonist toward CaV1.3
for the dihydropyrimidinethiones (compare 5a to 5b and 5f to
5g). However, these molecules were 1.5- to 3-fold less selective
than the dihydropyrimidinones.
2.2. Biological results
The biological assay process started by preparing cultures of the
two different LTCC-containing HEK293 cells. The CaV1.3/CaV1.2
Ca2+ channel assays involved subjecting the DHP mimic molecules
to a high-throughput screen using a FLIPR™ Tetra (fluorometric
imaging plate reader, Molecular Devices) system and a calcium as-
say kit (Fluo 8). The IC50 values for each compound were deter-
mined by dose–response curves with 12 concentration points
(1 nM–50 lM) in triplicate, to obtain an IC50 value and an associ-
ated standard deviation (Table 1 through Table 4). The standard
deviations associated with the calcium channel FLIPR assay were
usually <10%, so we were confident in drawing conclusions from
the data. Of the 36 molecules put through the selectivity/potency
determination, 28 were found to have selectivity greater than
our standard compound isradipine (selectivity is 0.5 in this study),
and six molecules (5b,e,g,i; (R)-5b, (R)-5i) were shown to inhibit
2-Methylamino-1H-pyran compounds (8a–9c) containing es-
ters (R2, R3) at both the C3 and C5 positions of the pyran ring exhib-
ited moderate IC50 values (0.4–14 lM) for both CaV1.3 and CaV1.2
LTCCs, but did not display CaV1.3 selectivity (Table 2).
Acyl substitution of the 2-amino group of the pyran ring ap-
pears to be detrimental to the potency at CaV1.3 and CaV1.2. The
acylation was more detrimental to the antagonism of CaV1.2 than
to that of CaV1.3, resulting in a potency loss of ꢀ10-fold for
CaV1.2 and only ꢀ5-fold loss for CaV1.3 (9a–c). These results sug-
gest that the hydrogen bond donor near the pyrimidine ring is crit-
ical for strong binding with the target proteins in these series of
molecules. This hydrogen bonding dependence is again effective,
even after modification of the pyrimidinetriones to substituted
thioethers (Table 3. 12a–13b). These thioether molecules, which
do not have hydrogen bond donors in their scaffold, are not potent
antagonists. Methylamine substitution (14a–b) at the same
position of the pyrimidine ring provides a hydrogen bond donor
CaV1.3 Ca2+ channel with high potency (<1
lM).
To get further biological confirmation of the antagonism of dihy-
dropyrimidines, we performed whole-cell patch-clamp electro-
physiology experiments in the voltage-clamp mode on HEK293
cells stably expressing CaV1.2 and CaV1.3 LTCCs and applied com-
pound (R)-5i (Fig. 2a). Barium currents were evoked from use of a
voltage step from À80 mV to 0 mV for 100 ms. During this relatively
short voltage step, application of (R)-5i at 1 lM and 10 lM not only
significantly inhibited the peak barium current, in keeping with re-
sults from our FLIPR calcium assay, but also potently inhibited the
end barium current, which is characteristic of DHPs preference for
the inactivated state of L-type calcium channels (Fig. 2b,c).

4368
S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
Table 1
IC50 values and the selectivity of antagonism of CaV1.3 and CaV1.2 channels by compounds 4a–5l
NO2
R3O2C
R4
R2
X
N
N
R1
No.
R1
X
R2
R3
R4
IC50
(lM) CaV1.3
IC50
(lM) CaV1.2
Selectivity
Isradipine
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
5g
0.221
0.107
0.5
0.6
1.3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
S
S
O
S
O
O
O
O
S
O
O
O
O
O
H
H
H
H
Me
iPr
Me
Me
iPr
iPr
iPr
Me
Me
iPr
Me
Me
Me
iPr
iPr
iPr
Me
Me
Me
iPr
Me
Me
Me
Me
iPr
18.89 ( 0.03)
46.62 ( 0.72)
>100
11.67 ( 0.04)
58.1 ( 0.76)
>100
>100
54.97 ( 0.73)
10.7 ( 0.59)
2.48 ( 0.14)
0.35 ( 0.01)
14.84 ( 0.64)
56.58 ( 5.5)
0.50 ( 0.05)
2.48 ( 0.17)
0.17 ( 0.02)
10.4 ( 0.73)
0.64 ( 0.06)
1.93 ( 0.22)
6.13 ( 0.37)
13.92 ( 0.87)
H
15.84 ( 0.63)
2.96 ( 0.14)
0.55 ( 0.01)
24.47 ( 1.38)
>100
0.99 ( 0.04)
4.60 ( 0.5)
0.88 ( 0.01)
10.07 ( 0.39)
0.66 ( 0.02)
4.31 ( 0.43)
5.88 ( 0.48)
13.93 ( 0.02)
0.7
0.9
0.6
0.6
CO2Me
CO2Me
CO2Et
COiPr
COiPr
CO2iPr
CO2iPr
CO2iPr
CO2iPr
CO2iPr
CO2Me
CO2Et
iPr
Me
Me
Me
iPr
Me
iPr
0.5
0.6
0.2
1.0
1.0
0.5
1.1
1.0
5h
5i
5j
5k
5l
CO2Me
CO2Et
Me
iPr
Table 2
IC50 values and the selectivity of antagonism of CaV1.3 and CaV1.2 channels by
compounds 8a–9c
NO2
NO2
R3O2C
Me
CO2R2
R3O2C
Me
CO2R2
COR1
R1
N
O
O
N
H
H
8
9
No.
R1
R2
R3
IC50
(l
M) CaV1.3
IC50
(lM) CaV1.2
Selectivity
8a
8b
8c
9a
9b
9c
H
H
H
iPr
Me
Me
Et
Me
Me
iPr
Me
Me
Me
2.72 ( 0.05)
1.82 ( 0.14)
1.39 ( 0.15)
9.95 ( 0.04)
10.03 ( 0.52)
13.62 ( 0.17)
0.58 ( 0.02)
0.41 ( 0.06)
0.86 ( 0.03)
4.23 ( 0.16)
4.75 ( 0.23)
6.11 ( 0.16)
0.2
0.2
0.6
0.4
0.5
0.5
iPr
iPr
Et
Et
iPr
Figure 2. (a) Current traces from the activation of either CaV1.2 (top) or CaV1.3
(bottom) stably transfected HEK293 cells with the application of compound (R)-5i
at 1
data representing the percentage of inhibition of the peak currents (first 50 ms of
current trace) resulting from application of compound (R)-5i at 1 M (black) or
10 (red) on HEK293 cells containing either CaV1.2 (left: black, n = 5,
median = 35.3%; red, n = 5, median = 72.6%) or CaV1.3 (right: black, n = 5, med-
ian = 35%; red, n = 5, median = 63.3%) L-type calcium channels, in which the
lM (black) or 10 lM (red). The horizontal bar represents 50 ms. (b) Population
From the optically resolved compounds, it is apparent that the
(R)-isomers are more potent (>4-fold) and slightly more selective
for CaV1.3 than are the (S)-isomers. Compound (R)-5i was the most
potent analogue in the study (IC50 of CaV1.3 is 0.51 lM) and had
some selectivity for CaV1.3 channels (IC50 of CaV1.2/IC50 of
CaV1.3 = 2.1) in our FLIPR calcium assay; the IC50 of isradipine was
0.22 lM for CaV1.3 and 0.107 lM for CaV1.2. Even though (R)-5i is
ꢀ2-fold weaker of an antagonist, its CaV1.3 selectivity is 4-fold
greater than that of isradipine in the FLIPR calcium assay. Although
the ratio of % inhibition of peak current (CaV1.3/CaV1.2) with com-
l
l
M
difference of inhibition produced by 10 lM (red) treatment of compound (R)-5i
on CaV1.2 and CaV1.3 L-type calcium channels was deemed significant by the
Mann–Whitney Rank Sum Test (⁄p <0.05). (c) Population data representing the
percentage of inhibition of the end currents (last 50 ms of current trace) resulting
from application of compound (R)-5i at 1 lM (black) or 10 lM (red) on HEK293
cells containing either CaV1.2 (left: black, n = 5, median = 44.7%; red, n = 5,
median = 91.5%) or CaV1.3 (right: black, n = 5, median = 71.8%; red, n = 5, med-
ian = 68.0%). L-type calcium channels, in which neither treatment group was
deemed significant by the Mann–Whitney Rank Sum Test.
pound (R)-5i in our patch-clamp experiment is 0.99 for 1
0.87 for 10 M, this reflects a much improved selectivity than that
for DHPs like isradipine (ratio = 0.69 at 0.3
M).9 Interestingly, com-
lM and
l
l
pound (R)-5i displayed more potent inhibition during the later part
of the voltage step. The resulting end currents for both CaV1.2 and
CaV1.3 LTCCs were inhibited more potently than the peak currents,
an action most attributed to the state-dependent block by DHPs.19
and a slight gain in potency toward both CaV1.3 and CaV1.2. When
methylthio was substituted by a methyl thiocarbonate (15), the
potency was increased about 5-fold.

S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
4369
Table 3
IC50 values and the selectivity of antagonism of CaV1.3 and CaV1.2 channels by compounds 12a–15
NO2
R2O2C
Me
R1
X
N
N
No.
X
R1
R2
IC50
(
lM) CaV1.3
IC50
(l
M) CaV1.2
Selectivity
12a
12b
13a
13b
14a
14b
15
SMe
SMe
SMe
SMe
NHMe
NHMe
SCO2Me
H
H
Me
iPr
Me
iPr
Me
iPr
iPr
10.62 ( 0.21)
1.86 ( 0.11)
5.58 ( 0.81)
10.67 ( 5.95)
4.22 ( 0.3)
3.29 ( 0.38)
0.42 ( 0.08)
16.13 ( 0.09)
5.54 ( 3.5)
1.81 ( 0.29)
2.45 ( 0.37)
1.02 ( 0.08)
0.3
0.2
3.0
0.4
0.4
0.8
0.9
CO2iPr
CO2Me
CO2iPr
CO2Me
CO2Me
3.25 ( 0.01)
1.11 ( 0.1)
Table 4
IC50 values and the selectivity of antagonism of CaV1.3 and CaV1.2 channels by chiral compounds 5a–b and 5i
Name
Structure
ee %
IC50
(
l
M) CaV1.3
IC50
(
l
M) CaV1.2
Selectivity
1.2
NO2
O
O
O
O
O
(R)-(À)-5a
>99
1.91 ( 0.26)
35.10 ( 1.81)
0.56 ( 0.04)
12.8 ( 0.43)
0.51 ( 0.05)
1.96 ( 0.04)
2.36 ( 0.44)
21.74 ( 0.17)
0.50 ( 0.05)
9.87 ( 0.04)
1.02 ( 0.08)
3.07 ( 0.11)
N
N
H
NO2
O
O
O
O
O
(S)-(+)5a
(R)-(À)-5b
(S)-(+)-5b
(R)-(-)-5i
(S)-(+)-5i
>99
0.6
0.9
0.8
2.1
1.6
N
N
H
NO2
O
O
O
O
S
98
N
N
N
H
NO2
O
O
O
O
S
>99
N
H
NO2
O
O
O
O
O
97
N
N
H
NO2
O
O
O
O
O
98
N
N
H
This activity with compound (R)-5bi suggests a generalizable prop-
erty for DHPs.
antagonists for the CaV1.3 channel relative to the CaV1.2 channel
on the basis of the known LTCC antagonists isradipine and nitren-
dipine. A library of 36 DHP mimic dihydropyrimidines and 4H-pyr-
ans was synthesized, and related chiral compounds were resolved.
Antagonism studies with CaV1.3 and CaV1.2 LTCC using these DHP
mimic compounds showed that dihydropyrimidines and 4H-pyr-
ans are alternative antagonists to DHPs for CaV1.3 LTCC; however,
these DHP mimics bind almost equally to CaV1.3 and CaV1.2. The
4. Conclusions
Recently, we identified the CaV1.3 LTCC as a new potential ther-
apeutic target for Parkinson’s disease. We have explored DHP-mi-
mic analogues in an attempt to identify potent and selective

4370
S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
ratio percent antagonism ranged from 0.2 to 3. In general, the mod-
ified hydrogen bond donor/acceptors in the DHP mimic dihydro-
pyrimidines and 4H-pyrans interact similarly with their binding
sites. This also suggests that the binding sites for DHPs in CaV1.3
and CaV1.2 are very similar.
5.2.1. General procedure for the synthesis of 6-alkyl-5-
(alkyloxycarbonyl)-4-(3-nitrophenyl)-3,4-dihydropyrimidin-
2(1H)-one/thione (4a–e)
A mixture of the 3-nitrobenzaldehyde (1 mmol), b-ketoester
(1 mmol), urea or thiourea (1.5 mmol), and Yb(OTf)3 (5 mol %) in
toluene (30 mL) was heated at 100 °C overnight. After cooling to
room temperature, brine (50 mL) and ethyl acetate (50 mL) were
added, and the organic layer was partitioned, dried with MgSO4,
and concentrated by vacuum evaporation. The crude product was
purified by recrystallization using ethyl acetate and hexanes to
give 4a–e in 50–70% yields.
5. Experimental section
5.1. Bioactivity assay
IC50 values were determined using a FLIPR assay we developed
and Fluo-8 calcium dye and HEK293 cells stably expressing CaV1.3
5.2.1.1. 6-Methyl-5-(methyoxycarbonyl)-4-(3-nitrophenyl)-3,4-
and CaV1.2 LTCCs. Rat CaV1.3
AF370010), rat CaVb3 (GenBank accession number: M88751), rat
CaV 2d-1 (GenBank accession number: AF286488), and rabbit Ca-
V1.2 1C (GenBank accession number: P15381) complementary
a1D (GenBank accession number:
dihydropyrimidin-2(1H)-one (4a).
White powder, mp:
283–285 °C. 1H NMR (500 MHz, DMSO-d6) d: 9.37 (s, 1H), 8.10
(dt, J = 7.8, 1.7 Hz, 1H), 8.05 (t, J = 2.0 Hz, 1H), 7.90 (d, J = 3.5 Hz,
1H), 7.64 (m, 2H), 5.27 (d, J = 3.5 Hz, 1H), 3.51 (s, 3H), 2.25 (s,
3H). 13C NMR (125 MHz, DMSO-d6) d: 166.0, 152.3, 150.1, 148.3,
147.1, 133.4, 130.7, 122.8, 121.3, 98.5, 53.8, 51.4, 18.4. MS (ESI):
calcd for C13H13N3O5 [M+H]+, 292.09; found: 292.25.
a
a
DNAs were used for the construction. The constructs were pro-
vided by Dr. Diana Lipscombe (Brown University) and Dr. Johannes
Hell (University of Iowa). More specific protocols for transfection of
HEK293 cells with CaV1.3 and CaV1.2 constructs as well as the
FLIPR assay are as previously described in the literature.9,10
Whole-cell electrophysiology was conducted as in our previous
5.2.1.2. 6-Methyl-5-(isopropyloxycarbonyl)-4-(3-nitrophenyl)-
3,4-dihydropyrimidin-2(1H)-one (4b).
White powder, mp:
study.9 Specifically, after 24–48 h of incubation at 37 °C on poly-
D-
206–208 °C. 1H NMR (500 MHz, CDCl3) d: 8.19 (t, J = 2.0 Hz, 1H),
8.15 (ddd, J = 8.1, 2.4, 1.1 Hz, 1H), 7.70 (dt, J = 7.8, 1.3 Hz, 1H),
7.65 (s, 1H), 7.52 (t, J = 7.9 Hz, 1H), 5.82 (t, J = 2.6 Hz, 1H), 5.53
(d, J = 3.1 Hz, 1H), 4.97 (hept, J = 6.3 Hz, 1H), 2.39 (s, 3H), 1.24 (d,
J = 6.2 Hz, 3H), 1.06 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d: 164.6, 152.5, 148.3, 146.9, 145.8, 132.9, 129.9, 123.1, 122.0,
100.9, 68.0, 55.4, 22.1, 21.7, 18.9. MS (ESI): calcd for C15H17N3O5
[M+H]+, 320.12; found: 320.27.
lysine treated coverslips, stably transfected HEK-293 cells under-
went whole-cell patch-clamp electrophysiology. The external solu-
tion contained the following (in mM): 140 NaCl, 1 MgCl2, 10 BaCl2,
10 HEPES, 10 dextrose, 10 sucrose, and 20 CsCl at pH 7.4 and an
osmolarity at ꢀ320 mOSmlÀ1. The test compound stock solutions
in DMSO (100 mM or just DMSO) were diluted with the external
solution to the desired concentration (10À9 to 10À3 M), which
was perfused (2 mL/min) into the recording chamber while mea-
suring the evoked barium currents. In experiments where concen-
tration–response curves were obtained, local perfusion of the
desired concentration was employed. Barium currents were mea-
sured from whole-cell voltage patch-clamp recordings using the
Pulse 8.4 software data acquisition system (HEKA, Germany). Sig-
nals were low-pass filtered at 1 kHz, digitized (sampled) at
10 kHz, and were amplified with an Axopatch 200B patch-clamp
amplifier (Axon Instruments). Barium currents were evoked by a
depolarizing voltage step from a holding potential of À80 mV to
0 mV for 100 ms at a frequency of 0.05 Hz at room temperature
(22–25 °C). Patch pipettes were pulled from thin-wall borosilicate
glass coated with dental wax and maintained a resistance of
approximately 3–5 mO. Internal pipette solutions contained the
5.2.1.3. 6-Isopropyl-5-(methyoxycarbonyl)-4-(3-nitrophenyl)-
3,4-dihydropyrimidin-2(1H)-one (4c).
White powder, mp:
162–165 °C. 1H NMR (500 MHz, DMSO-d6) d: 9.08–9.02 (m, 1H),
8.10 (dt, J = 5.6, 2.7 Hz, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.94–7.88 (m,
1H), 7.68–7.58 (m, 2H), 5.25 (d, J = 3.5 Hz, 1H), 4.13 (hept,
J = 7.0 Hz, 1H), 3.50 (s, 3H), 1.12 (dd, J = 10.7, 7.0 Hz, 6H). 13C
NMR (125 MHz, DMSO-d6) d: 166.0, 158.4, 152.8, 148.3, 146.9,
133.4, 130.8, 122.9, 121.2, 97.4, 53.5, 51.6, 27.5, 19.6, 19.3. MS
(ESI): calcd for C15H17N3O5 [M+H]+, 320.12; found: 320.34.
5.2.1.4. 6-Methyl-5-(methyoxycarbonyl)-4-(3-nitrophenyl)-3,4-
dihydropyrimidin-2(1H)-thione (4d).
White powder, mp:
244–247 °C. 1H NMR (500 MHz, DMSO-d6) d: 10.55 (s, 1H), 9.82
(d, J = 3.7 Hz, 1H), 8.17 (dt, J = 7.4, 2.1 Hz, 1H), 8.08 (t, J = 1.9 Hz,
1H), 7.76–7.62 (m, 2H), 5.34 (d, J = 3.2 Hz, 1H), 3.57 (s, 3H), 2.33
(s, 3H). 13C NMR (125 MHz, CDCl3) d: 174.4, 165.4, 147.9, 146.3,
145.2, 133.0, 130.5, 122.8, 121.0, 99.5, 53.3, 51.3, 17.3. MS (ESI):
calcd for C13H13N3O4S [M+H]+, 308.07; found: 308.14.
following (in mM): 180 NMG (N-methyl-D-glucosamine), 40
HEPES, 4 MgCl2, 12 phosphocreatine, 0.1 leupeptin, 2 Na2ATP, 0.5
Na3GTP, 5 BAPTA, pH 7.2–7.3 and an osmolarity at ꢀ290 mOSmlÀ1
.
Electrophysiological signals were analyzed using ClampfitÒ 9.2
(Axon Instruments) and IgorProÒ6 software.
5.2. General chemistry methods
5.2.1.5.
6-Methyl-5-(isopropyloxyarbonyl)-4-(3-nitrophenyl)-
White powder,
3,4-dihydropyrimidin-2(1H)-thione (4e).
mp: 203–205 °C. 1H NMR (500 MHz, CDCl3) d: 8.14 (t, J = 2.0 Hz,
1H), 8.01 (dd, J = 8.1, 2.1 Hz, 1H), 7.67 (dt, J = 7.6, 1.3 Hz, 1H),
7.40 (t, J = 7.9 Hz, 1H), 6.77 (s, 1H), 5.14 (m, 1H), 5.08 (s.1H), 4.11
(m, 2H), 2.35 (d, J = 6.0 Hz, 6H), 1.91–1.67 (m, 4H), 1.67–1.46 (m,
4H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 167.4,
167.1, 150.2, 148.0, 145.4, 145.3, 134.5, 128.7, 123.0, 121.3,
103.1, 102.9, 60.0, 40.0, 32.8, 32.6, 23.7, 23.6, 19.3, 19.2, 14.2. MS
(ESI): calcd for C22H26N2O6 [M+H]+, 415.19; found: 415.42.
All starting reagents were purchased from Sigma–Aldrich (Mil-
waukee, WI). 1H NMR spectra were recorded on a Bruker AVANCE
III 500 MHz spectrometer with a direct cryoprobe. Chemical shifts
are reported as d values in parts per million downfield from TMS (d
0.0) as the internal standard in CDCl3. Electrospray mass spectra
were obtained on a Micromass Quattro II spectrometer. Thin-layer
chromatography was carried out on E. Merck precoated silica gel
60 F254 plates. An Agilent 971-FP flash purification system with
various Superflash 50 silica gel cartridges was used for flash col-
umn chromatography. The direct chiral resolutions of racemic 5a,
5b, and 5i were performed using a Chiralcel OD-H HPLC column
(Daicel, 250 Â 4.6 mm i.d., 5 mm). Hexanes and isopropanol were
used as the mobile phases. The operation temperature was 25 °C,
and the flow rate was 1 ml/min with 254 nm UV detection.
5.2.2. General procedure for the synthesis of 1,5-dialkyl 4-alkyl-
6-(3-nitrophenyl)-2-oxo/thioxo-2,3-dihydropyrimidine-1,5-
(6H)-dicarboxylate (5a–l)
A mixture of 4a–e (1 mmol) and NaH (60% in oil, 1.1 mmol) in
THF (30 mL) was treated with alkyl chloroformate (1.1 mmol) or

S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
4371
acyl halide (1.1 mmol) at 0 °C under argon. After stirring 5 h at
room temperature, the mixture was diluted with brine (50 mL)
and ethyl acetate (50 mL). The organic layer was partitioned, dried
with MgSO4, and concentrated by vacuum evaporation. The crude
product was purified by flash chromatography using ethyl acetate
and hexanes to afford 5a–l in 60ꢀ80% yields.
5.2.2.6. 1-Isopropyl 5-methyl 4-methyl-6-(3-nitrophenyl)-2-
oxo-2,3-dihydropyrimidine-1,5(6H)-dicarboxylate
(5f).
White powder, mp: 135–137 °C. 1H NMR (500 MHz,
CDCl3) d: 9.11 (s, 1H), 8.24 (t, J = 2.1 Hz, 1H), 8.16 (dd, J = 8.2,
2.1 Hz, 1H), 7.78–7.71 (m, 1H), 7.52 (t, J = 8.0 Hz, 1H), 6.37 (s,
1H), 5.11 (hept, J = 6.2 Hz, 1H), 3.75 (s, 3H), 2.44 (s, 3H), 1.35 (dd,
J = 18.7, 6.3 Hz, 6H). 13C NMR (125 MHz, CDCl3) d: 165.3, 152.3,
150.6, 148.4, 147.2, 142.0, 133.0, 129.8, 123.2, 122.2, 103.6, 72.6,
5.2.2.1. 1-Methyl 5-isopropyl 4-methyl-6-(3-nitrophenyl)-2-
oxo-2,3-dihydropyrimidine-1,5(6H)-dicarboxylate
(5a).
55.7, 51.9, 21.8, 21.8, 17.9. MS (ESI): calcd for
C17H19N3O7
White powder, Mp: 132–135 °C. 1H NMR (500 MHz,
[M+H]+, 400.11; found: 400.18.
CDCl3) d: 8.52 (s, 1H), 8.24 (s, 1H), 8.15 (m, 1H), 7.74 (dt, J = 7.9,
1.3 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 6.38 (s, 1H), 5.08 (hept,
J = 6.2 Hz, 1H), 3.91 (s, 3H), 2.42 (s, 3H), 1.31 (d, J = 6.2 Hz, 3H),
1.21 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 164.2,
153.6, 150.2, 148.3, 146.3, 142.1, 132.9, 129.7, 123.2, 122.0,
104.5, 68.6, 56.2, 54.7, 22.0, 21.8, 18.1. MS (ESI): calcd for
5.2.2.7. 1-Isopropyl 5-methyl 4-methyl-6-(3-nitrophenyl)-2-thi-
oxo-2,3-dihydropyrimidine-1,5(6H)-dicarboxylate
(5g).
White powder, mp: 205–206 °C. 1H NMR (500 MHz,
CDCl3) d: 9.02 (s, 1H), 8.21 (t, J = 2.1 Hz, 1H), 8.15 (ddd, J = 8.3,
2.3, 1.1 Hz, 1H), 7.73–7.69 (m, 1H), 7.54 (t, J = 4.0 Hz, 1H), 6.36 (s,
1H), 5.12 (h, J = 6.3 Hz, 1H), 3.79 (s, 3H), 2.43 (s, 3H), 1.37 (dd,
J = 22.2, 6.3 Hz, 6H). 13C NMR (125 MHz, CDCl3) d: 176.3, 165.3,
153.4, 148.4, 144.3, 141.3, 132.9, 129.8, 123.3, 121.9, 106.0, 73.7,
55.9, 52.1, 21.9, 21.8, 17.6. MS (ESI): calcd for C17H19N3O6S
[M+H]+, 394.11; found: 394.06.
C
17H19N3O7 [M+H]+, 378.13; found: 378.27. (R)-(À)-5a
tr = 11.9 min, (S)-(+)-5a tr= 23.0 min [Chiralcel OD-H column, hex-
anes/IPA 85:15, 1.0 mL/min].
5.2.2.2. 1-Methyl 5-isopropyl 4-methyl-6-(3-nitrophenyl)-2-thi-
oxo-2,3-dihydropyrimidine-1,5(6H)-dicarboxylate
(5b).
Pale yellow powder, mp: 244–247 °C. 1H NMR
5.2.2.8. 1-Isopropyl 5-methyl 4-isopropyl-6-(3-nitrophenyl)-2-
(500 MHz, CDCl3) d: 8.91 (s, 1H), 8.23 (t, J = 2.1 Hz, 1H), 8.16
(ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.74 (m, 1H), 7.53 (t, J = 8.0 Hz, 1H),
6.39 (s, 1H), 5.14 (hept, J = 6.2 Hz, 1H), 3.94 (s, 3H), 2.41 (s, 3H),
1.33 (d, J = 6.3 Hz, 3H), 1.28 (d, J = 6.3 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d: 176.3, 164.3, 154.4, 148.4, 143.6, 141.4,
132.5, 129.8, 123.2, 121.7, 107.0, 69.1, 56.2, 54.9, 22.0, 21.9, 17.7.
MS (ESI): calcd for C17H19N3O6S [M+H]+, 394.11; found: 394.22.
(R)-(À)-5b tr = 11.4 min, (S)-(+)-5b tr = 15.6 min [Chiralcel OD-H
column, hexanes/IPA 90:10, 1.0 mL/min].
oxo-2,3-dihydropyrimidine-1,5(6H)-dicarboxylate
(5h).
White powder, mp: 186–187 °C. 1H NMR (500 MHz,
CDCl3) d: 8.78 (s, 1H), 8.23 (t, J = 2.1 Hz, 1H), 8.15 (ddd, J = 8.2,
2.3, 1.0 Hz, 1H), 7.75 (dt, J = 7.9, 1.3 Hz, 1H), 7.52 (t, J = 8.0 Hz,
1H), 6.40 (s, 1H), 5.14 (hept, J = 6.3 Hz, 1H), 4.26 (hept, J = 7.0 Hz,
1H), 3.73 (s, 3H), 1.36 (dd, J = 9.0, 6.3 Hz, 6H), 1.28 (d, J = 7.1 Hz,
3H), 1.22 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 165.1,
155.9, 152.8, 150.4, 148.4, 141.9, 133.2, 129.8, 123.2, 122.1,
102.1, 72.5, 55.2, 51.9, 27.4, 21.8, 19.5, 19.5. MS (ESI): calcd for
C
19H23N3O7 [M+H]+, 428.14; found: 428.26.
5.2.2.3. 1-Ethyl 5-methyl 4-isopropyl-6-(3-nitrophenyl)-2-oxo-
2,3-dihydropyrimidine-1,5(6H)-dicarboxylate (5c).
White
5.2.2.9. 1,5-Diisopropyl 4-methyl-6-(3-nitrophenyl)-2-oxo-2,3-
dihydro-pyrimidine-1,5(6H)-dicarboxylate (5i). Colorless
powder, mp: 192–194 °C. 1H NMR (500 MHz, CDCl3) d: 8.31 (s,
1H), 8.21 (t, J = 2.1 Hz, 1H), 8.16 (ddd, J = 8.2, 2.1, 0.9 Hz, 1H),
7.75 (dt, J = 7.7, 1.2 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 6.43 (s, 1H),
4.37 (q, J = 7.2 Hz, 2H), 4.24 (hept, J = 7.0 Hz, 1H), 3.77 (m, 3H),
1.37 (t, J = 7.1 Hz, 3H), 1.24 (dd, J = 28.6, 7.0 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d: 165.0, 155.7, 153.3, 150.1, 148.4, 141.6,
133.2, 129.8, 123.3, 122.0, 102.2, 64.2, 55.3, 51.9, 27.4, 19.6, 19.5,
14.3. MS (ESI): calcd for C18H21N3O7 [M+H]+, 392.15; found:
392.34.
oil. 1H NMR (500 MHz, CDCl3) d: 8.87 (s, 1H), 8.25 (t, J = 2.0 Hz,
1H), 8.15 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.73 (dt, J = 7.8, 1.3 Hz,
1H), 7.51 (t, J = 8.0 Hz, 1H), 6.33 (s, 1H), 5.08 (dhept, J = 10.1,
6.2 Hz, 2H), 2.42 (s, 3H), 1.34 (dd, J = 18.4, 6.2 Hz, 6H), 1.25 (dd,
J = 50.5, 6.2 Hz, 6H). 13C NMR (125 MHz, CDCl3) d: 164.3, 152.3,
150.5, 148.3, 146.4, 142.4, 132.9, 129.7, 123.1, 122.3, 104.3, 72.5,
68.5, 56.0, 22.0, 21.9, 21.8, 21.7, 17.9. MS (ESI): calcd for
C
19H23N3O7 [M+H]+, 428.14; found: 428.32. (R)-(À)-5i
tr = 19.9 min, (S)-(+)-5i tr = 22.9 min [Chiralcel OD-H column, hex-
5.2.2.4. 6-Isopropyl-5-(methyoxycarbonyl)-4-(3-nitrophenyl)-3-
anes/IPA 95:5, 1.0 mL/min].
isobut -yl-3,4-dihydropyrimidin-2(1H)-one (5d).
White
powder, mp: 163–165 °C. 1H NMR (500 MHz, CDCl3) d: 8.72 (s,
1H), 8.16–8.11 (m, 2H), 7.71 (dd, J = 7.9, 1.5 Hz, 1H), 7.55–7.47
(m, 1H), 6.70 (s, 1H), 4.31 (hept, J = 7.0 Hz, 1H), 3.74 (s, 3H), 3.65
(hept, J = 6.7 Hz, 1H), 1.24 (ddd, J = 25.6, 11.7, 6.9 Hz, 12H). 13C
NMR (125 MHz, CDCl3) d: 179.3, 165.0, 155.5, 152.4, 148.4, 141.8,
133.5, 129.8, 123.1, 121.6, 102.8, 52.4, 51.9, 35.3, 27.2, 19.8, 19.7,
19.4, 19.4. MS (ESI): calcd for C19H23N3O6 [M+H]+, 390.17; found:
390.26.
5.2.2.10. 1,5-Diisopropyl 4-isopropyl-6-(3-nitrophenyl)-2-oxo-
2,3-dihy-dropyrimidine-1,5(6H)-dicarboxylate (5j).
White
powder, mp: 196–198 °C. 1H NMR (500 MHz, CDCl3) d: 8.24 (t,
J = 2.0 Hz, 1H), 8.15 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.98 (s, 1H),
7.73 (dt, J = 7.8, 1.3 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 6.34 (s, 1H),
5.08 (m, 2H), 4.22 (hept, J = 7.0 Hz, 1H), 1.37 (d, J = 6.3 Hz, 3H),
1.34 (d, J = 6.2 Hz, 3H), 1.29 (d, J = 6.2 Hz, 3H), 1.23 (d, J = 7.1 Hz,
3H), 1.21 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 6.3 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d: 164.0, 154.6, 152.6, 150.0, 148.3, 142.3,
133.0, 129.7, 123.1, 122.2, 103.0, 72.5, 68.5, 55.7, 27.3, 22.0, 21.9,
21.8, 21.8, 19.7, 19.6. MS (ESI): calcd for C21H27N3O7 [M+H]+,
456.17; found: 456.33.
5.2.2.5. 6-Methyl-5-(isopropyloxycarbonyl)-4-(3-nitrophenyl)-
3-isobu -tyl-3,4-dihydropyrimidin-2(1H)-one (5e).
White
powder, mp: 181–183 °C. 1H NMR (500 MHz, CDCl3) d: 8.98 (s,
1H), 8.21 (t, J = 2.0 Hz, 1H), 8.13 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.72
(dt, J = 7.7, 1.3 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 6.59 (s, 1H), 5.08 (hept,
J = 6.3 Hz, 1H), 3.69 (hept, J = 6.7 Hz, 1H), 2.45 (s, 3H), 1.27 (dd,
J = 39.1, 6.5 Hz, 6H), 1.21 (dd, J = 6.5, 2.4 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d: 179.4, 164.2, 152.2, 148.3, 146.1, 142.5, 133.2,
129.6, 123.0, 122.0, 104.9, 68.6, 53.7, 35.6, 22.0, 21.8, 19.8, 19.5,
17.7. MS (ESI): calcd for C19H23N3O6 [M+H]+, 390.17; found: 390.32.
5.2.2.11. 1,3-Dimethoxycarbornyl-5-(isopropyloxycarbonyl)-6-
methyl-4-(3-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one
(5k).
Pale yellow oil. 1H NMR (500 MHz, CDCl3) d: 8.28 (t,
J = 2.0 Hz, 1H), 8.23–8.14 (m, 1H), 7.75 (dt, J = 7.8, 1.2 Hz, 1H),
7.55 (t, J = 8.0 Hz, 1H), 6.41 (s, 1H), 5.11 (hept, J = 6.3 Hz, 1H),
3.97 (s, 3H), 3.93 (s, 3H), 2.50 (s, 3H), 1.32 (d, J = 6.3 Hz, 3H), 1.23

4372
S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
(d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 163.5, 153.7, 151.5,
148.5, 147.1, 145.7, 140.4, 132.9, 130.0, 123.5, 122.1, 111.9, 69.4,
55.8, 55.3, 54.9, 21.9, 21.8, 17.4. MS (ESI): calcd for C19H21N3O9
[M+H]+, 436.14; found: 436.28.
(30 mL) and stirred for 2 h at room temperature. After the reaction
mixture was diluted with brine (50 mL) and dichloromethane
(20 mL), the organic layer was partitioned, dried with MgSO4,
and concentrated by vacuum evaporation. The crude product was
purified by flash chromatography using ethyl acetate and hexanes
to afford 9a–c in 80–90% yields.
5.2.2.12.
methyl-4-(3-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one
(5l).
Colorless oil. 1H NMR (500 MHz, CDCl3) d: 8.25 (d,
1,3-Diethoxycarbornyl-5-(methoxycarbonyl)-6-
5.2.4.1. 3-Ethyl 5-methyl 2-isobutyramido-6-methyl-4-(3-nitro-
J = 2.0 Hz, 1H), 8.17 (m, 1H), 7.75 (m, 1H), 7.54 (t, J = 8.0 Hz, 1H),
6.45 (s, 1H), 4.42 (q, J = 7.1 Hz, 2H), 4.20 (m, 2H), 3.83 (s, 3H),
3.72 (h, J = 7.1 Hz, 1H), 1.41 (m, 6H), 1.26 (d, J = 7.1 Hz, 3H), 1.20
(t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 164.3, 157.6,
153.3, 150.9, 148.4, 147.3, 139.8, 133.1, 129.9, 123.5, 121.7,
116.4, 64.8, 64.7, 55.1, 52.6, 31.5, 21.1, 18.4, 14.2, 13.8. MS (ESI):
calcd for C21H25N3O9 [M + H]+, 464.17; found: 464.33.
phenyl)-4H-pyran-3,5-dicarboxylate (9a).
White powder,
mp: 142–144 °C. 1H NMR (500 MHz, CDCl3) d: 10.95 (s, 1H), 8.14
(t, J = 2.0 Hz, 1H), 8.06 (ddd, J = 8.1, 2.3, 1.1 Hz, 1H), 7.59 (dt,
J = 7.7, 1.4 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 4.92–4.88 (m, 1H), 4.13
(qd, J = 7.1, 1.1 Hz, 2H), 3.67 (s, 3H), 2.58 (hept, J = 6.9 Hz, 1H),
2.51 (d, J = 0.8 Hz, 3H), 1.26 (d, J = 6.9 Hz, 6H), 1.23 (t, J = 7.1 Hz,
3H). 13C NMR (125 MHz, CDCl3) d: 174.4, 168.1, 166.1, 158.9,
153.7, 148.0, 147.2, 134.5, 129.2, 123.5, 121.9, 108.7, 87.6, 61.1,
51.9, 37.5, 37.5, 19.2, 19.1, 18.7, 14.1. MS (ESI): calcd for
5.2.3. General procedure for the synthesis of 3,5-dialkyl 2-
amino-6-methyl-4-(3-nitrophenyl)-4H-pyran-3,5-dicarboxylate
(8a–c)
C
21H24N2O8 [M+H]+, 433.16; found: 433.2.
A mixture of the 3-nitrobenzaldehyde (1 mmol), cyanoacetate
(1 mmol), acetoacetate (1 mmol), and catalytic piperidine in iso-
propyl alcohol (20 mL) was refluxed overnight. The reaction mix-
ture was concentrated by vacuum evaporation, diluted with
brine (50 mL) and ethyl acetate (50 mL). The organic layer was par-
titioned, dried with MgSO4, and concentrated by vacuum evapora-
tion. The crude product was purified by flash chromatography
using ethyl acetate and hexanes to afford 8a–c in 40–70% yields.
5.2.4.2. 3-Isopropyl 5-methyl 2-acetacamido-6-methyl-4-(3-
nitrophenyl)-4H-pyran-3,5-dicarboxylate (9b). White pow-
der, mp: 105–110 °C. 1H NMR (500 MHz, CDCl3) d: 10.77 (s, 1H),
8.12 (t, J = 2.0 Hz, 1H), 8.06 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 7.58 (dt,
J = 7.7, 1.3 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 4.89 (d, J = 1.0 Hz, 1H),
4.12 (q, J = 7.1 Hz, 2H), 3.67 (s, 3H), 2.49 (d, J = 0.9 Hz, 3H), 2.24
(s, 3H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d:
167.8, 167.5, 166.1, 158.7, 153.0, 148.0, 147.1, 134.4, 129.2,
123.5, 122.0, 108.8, 87.6, 61.2, 51.9, 37.5, 25.6, 18.7, 14.1. MS
(ESI): calcd for C19H20N2O8 [M + H]+, 405.13; found: 405.18.
5.2.3.1. 3-Ethyl 5-methyl 2-amino-6-methyl-4-(3-nitrophenyl)-
4H-pyran-3,5-dicarboxylate (8a).
White powder, mp: 130–
134 °C. 1H NMR (500 MHz, CDCl3) d: 8.10 (t, J = 2.0 Hz, 1H), 8.02
(ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 7.56 (dt, J = 7.6, 1.4 Hz, 1H), 7.40 (t,
J = 7.9 Hz, 1H), 6.20 (s, 2H), 4.80 (d, J = 1.0 Hz, 1H), 4.05 (qd,
J = 7.1, 1.0 Hz, 2H), 3.65 (s, 3H), 2.41 (d, J = 0.9 Hz, 3H), 1.18 (t,
J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 168.5, 166.6, 158.4,
158.0, 148.6, 148.0, 134.5, 128.7, 123.4, 121.4, 109.3, 78.9, 59.8,
51.7, 37.6, 18.7, 14.3. MS (ESI): calcd for C17H18N2O7 [M+H]+,
363.12; found: 363.22
5.2.4.3.
3,5-Diisopropyl
2-acetamido-6-methyl-4-(3-nitro-
White powder,
phenyl)-4H-pyran-3,5-dicarboxylate (9c).
mp: 131–134 °C. 1H NMR (500 MHz, CDCl3) d: 10.84 (s, 1H), 8.13
(t, J = 2.0 Hz, 1H), 8.06 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.65–7.53 (m,
1H), 7.44 (t, J = 7.9 Hz, 1H), 5.01–4.89 (m, J = 6.3, 5.8 Hz, 1H), 4.86
(s, 1H), 3.67 (s, 3H), 2.49 (s, 3H), 2.23 (s, 3H), 1.31 (d, J = 6.3 Hz,
3H), 1.02 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 167.5,
167.3, 166.1, 158.6, 152.9, 147.9, 147.2, 134.5, 129.1, 123.6,
121.9, 108.7, 87.8, 69.0, 51.9, 37.6, 25.6, 22.0, 21.5, 18.7. MS
(ESI): calcd for C20H22N2O8 [M+H]+, 419.15; found: 419.21.
5.2.3.2. 3-Isopropyl 5-methyl 2-amino-6-methyl-4-(3-nitro-
phenyl)-4H-pyran-3,5-dicarboxylate (8b).
Yellow oil. 1H
NMR (500 MHz, CDCl3) d: 8.11 (t, J = 2.0 Hz, 1H), 8.01 (ddd,
J = 8.2, 2.3, 1.1 Hz, 1H), 7.57 (dt, J = 7.7, 1.4 Hz, 1H), 7.40 (t,
J = 7.9 Hz, 1H), 6.25 (s, 2H), 4.91 (hept, J = 6.3 Hz, 1H), 4.78 (d,
J = 1.1 Hz, 1H), 3.65 (s, 3H), 2.40 (d, J = 1.0 Hz, 3H), 1.27 (d,
J = 6.2 Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d: 168.0, 166.6, 158.3, 158.0, 148.8, 147.8, 134.6, 128.6, 123.5,
121.4, 109.2, 79.0, 67.1, 51.7, 37.7, 22.2, 21.7, 18.7. MS (ESI): calcd
for C18H20N2O7 [M + H]+, 399.12; found: 410.25.
5.2.5. General procedure for the synthesis of 4-(3-nitrophenyl)-
1,4-dihydro-6-methyl-2-(methylthio)-5-pyrimidinecarboxylate
(12a–b)
A mixture of the 2-[(3-nitrophenyl)methylene]-3-oxobutanoic
acid alkyl ester (1 mmol), S-methylisothiourea hemisulfate salt
(1 mmol), and sodium acetate (2 mmol) in dimethylformamide
(10 mL) was stirred at 80 °C overnight. After cooling to room tem-
perature, the mixture was diluted with brine (50 mL) and ethyl ace-
tate (50 mL). The organic layer was partitioned, washed with brine
(50 mL), dried with MgSO4, and concentrated by vacuum evapora-
tion. The crude product was purified by flash chromatography using
ethyl acetate and hexanes to afford 12a–b in 30–45% yields.
5.2.3.3. 3,5-Diisopropyl 2-amino-6-methyl-4-(3-nitrophenyl)-
4H-pyra-n-3,5-dicarboxylate (8c).
Yellow oil. 1H NMR
(500 MHz, CDCl3) d: 8.11 (t, J = 2.0 Hz, 1H), 8.01 (ddd, J = 8.1, 2.3,
1.1 Hz, 1H), 7.56 (dt, J = 7.7, 1.4 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H),
6.25 (s, 2H), 4.92 (dhept, J = 15.7, 6.2 Hz, 2H), 4.75 (d, J = 1.1 Hz,
1H), 2.39 (s, 3H), 1.26 (dd, J = 15.8, 6.2 Hz, 6H), 1.01 (dd, J = 43.9,
6.3 Hz, 6H). 13C NMR (125 MHz, CDCl3) d: 168.1, 165.6, 158.3,
157.4, 148.9, 147.6, 134.8, 128.5, 123.9, 121.2, 109.4, 78.9, 68.3,
67.0, 37.8, 22.2, 21.9, 21.7, 21.6, 18.6. MS (ESI): calcd for
5.2.5.1. 5-Methyl 4-(3-nitrophenyl)-1,4-dihydro-6-methyl-2-
(methylthio)-5-pyrimidinecarboxylate (12a).
Colorless oil.
1H NMR (500 MHz, CDCl3) d: 8.17 (t, J = 2.0 Hz, 1H), 8.11 (ddd,
J = 8.1, 2.3, 1.1 Hz, 1H), 7.70 (dt, J = 7.7, 1.4 Hz, 1H), 7.49 (t,
J = 7.9 Hz, 1H), 6.38 (s, 1H), 5.83 (s, 1H), 3.70 (s, 3H), 2.44 (s, 3H),
2.38 (s, 3H). 13C NMR (125 MHz, CDCl3) d: 166.8, 151.2, 148.4,
146.8, 145.3, 133.4, 129.2, 122.1, 122.0, 99.8, 59.2, 51.3, 19.0, 13.6.
MS (ESI): calcd for C14H15N3O4S [M+H]+, 322.09; found: 322.15.
C
20H24N2O7 [M + H]+, 405.17; found: 405.23.
5.2.4. General procedure for the synthesis of 3,5-dialkyl 2-
acylamido-6-methyl-4-(3-nitrophenyl)-4H-pyran-3,5-
dicarboxylate (9a–c)
5.2.5.2. 5-Isopropyl 4-(3-nitrophenyl)-1,4-dihydro-6-methyl-2-
The acyl halide (1.05 mmol) was added to the solution of 8a–c
(1 mmol) and triethylamine (1.2 mmol) in dichloromethane
(methylthio)-5-pyrimidinecarboxylate (12b).
Pale yellow
oil. 1H NMR (500 MHz, CDCl3) d: 8.20 (s, 1H), 8.14–8.08 (m, 1H),

S. Kang et al. / Bioorg. Med. Chem. 21 (2013) 4365–4373
4373
7.69 (dt, J = 7.7, 1.3 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 6.45 (s, 1H), 5.84
(s, 1H), 5.03 (hept, J = 6.3 Hz, 1H), 2.44 (s, 3H), 2.36 (s, 3H), 1.26 (d,
J = 6.2 Hz, 3H), 1.15 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) d:
165.8, 151.4, 148.2, 147.1, 144.9, 133.4, 129.1, 122.2, 122.1, 100.4,
67.5, 59.2, 22.1, 21.8, 18.8, 13.5. MS (ESI): calcd for C16H19N3O4S
[M+H]+, 350.12; found: 350.21.
J = 0.8 Hz, 3H), 1.29 (d, J = 6.2 Hz, 3H), 1.18 (d, J = 6.3 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 165.8, 157.5, 154.6, 148.9, 148.2, 143.5,
132.9, 129.7, 122.9, 122.1, 103.9, 67.4, 54.6, 54.3, 28.7, 23.2, 22.2,
22.0. MS (ESI): calcd for C18H22N4O6 [M+H]+, 391.16; found:
391.18.
5.2.7.3. 5-isopropyl 1-methyl 2-(acetylthio)-4-methyl-6-(3-
5.2.6. General procedure for the synthesis of 4-(3-nitrophenyl)-
1,4-dihydro-6-methyl-2-(methylthio)-5-
pyrimidinedicarboxylate (13a–b)
These molecules were prepared using the procedure for the
synthesis of 5a–l.
nitrophenyl)pyrimidine-1,5(6H)-dicarboxylate (15).
Yellow
oil. 1H NMR (500 MHz, CDCl3) d: 8.15 (s, 1H), 8.04 (ddd, J = 8.2,
2.2, 1.0 Hz, 1H), 7.64 (ddt, J = 7.8, 1.7, 0.8 Hz, 1H), 7.41 (t,
J = 8.0 Hz, 1H), 6.19 (s, 1H), 4.94 (m, 1H), 3.86 (s, 3H), 3.76 (s,
3H), 2.35 (s, 3H), 1.17 (d, J = 6.3 Hz, 3H), 1.06 (d, J = 6.2 Hz, 3H).
13C NMR (125 MHz, CDCl3) d: 174.4, 163.7, 154.6, 151.8, 148.4,
142.9, 140.3, 133.2, 129.9, 123.5, 122.3, 110.7, 69.5, 56.4, 55.8,
54.8, 21.9, 21.7, 15.9. MS (ESI): calcd for C19H21N3O8S [M+H]+,
452.11; found: 452.21.
5.2.6.1. 3-Isopropyl, 5-methyl 4-(3-nitrophenyl)-1,4-dihydro-6-
methyl-2-(methylthio)-5-pyrimidinedicarboxylate
(13a).
Colorless oil. 1H NMR (500 MHz, CDCl3) d: 8.15 (dtd,
J = 4.7, 2.3, 1.0 Hz, 2H), 7.60 (dt, J = 7.7, 1.4 Hz, 1H), 7.55–7.45 (m,
1H), 6.33 (s, 1H), 5.11 (hept, J = 6.3 Hz, 1H), 3.74 (s, 3H), 2.47 (s,
3H), 2.45 (s, 3H), 1.39 (dd, J = 21.4, 6.3 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d: 166.3, 158.7, 153.5, 152.2, 148.4, 141.8, 133.2,
129.7, 123.3, 122.4, 109.0, 72.9, 54.0, 51.8, 21.9, 21.8, 21.8, 15.6.
MS (ESI): calcd for C18H21N3O6S [M+H]+, 408.12; found: 408.16.
Acknowledgments
We thank the Michael J. Fox Foundation (Therapeutics Develop-
ment Initiative) and the RJG Foundation for financial support of the
research.
5.2.6.2. 3-Methyl, 5-isopropyl 4-(3-nitrophenyl)-1,4-dihydro-6-
methyl-2-(methylthio)-5-pyrimidinedicarboxylate
(13b).
2H), 7.61 (dt, J = 7.8, 1.4 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 6.33 (s,
1H), 5.09 (hept, J = 6.2 Hz, 1H), 3.91 (s, 3H), 2.47 (s, 3H), 2.46 (s,
3H), 1.31 (d, J = 6.2 Hz, 3H), 1.21 (d, J = 6.2 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d: 165.3, 158.0, 153.3, 152.8, 148.3, 141.9, 133.0,
129.7, 123.2, 122.1, 110.0, 68.4, 54.3, 54.3, 22.0, 21.9, 21.7, 15.5.
MS (ESI): calcd for C18H21N3O6S [M+H]+, 408.12; found: 408.18.
A. Supplementary data
Pale yellow oil. 1H NMR (500 MHz, CDCl3) d: 8.15 (m,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.054.
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A mixture of 13a,b (1 mmol), methylamine HCl salt (10 mmol),
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White powder, mp: 91–93 °C. 1H NMR (500 MHz,
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CDCl3) d: 8.20 (t, J = 2.0 Hz, 1H), 8.14 (ddd, J = 8.2, 2.3, 1.0 Hz,
1H), 7.99 (s, 1H), 7.64 (dt, J = 7.8, 1.3 Hz, 1H), 7.48 (t, J = 8.0 Hz,
1H), 6.30 (s, 1H), 5.06 (hept, J = 6.3 Hz, 1H), 3.70 (s, 3H), 3.02 (d,
J = 4.7 Hz, 3H), 2.45 (s, 3H), 1.42 (d, J = 6.2 Hz, 3H), 1.35 (d,
J = 6.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) d: 166.7, 158.2, 153.8,
149.4, 148.2, 143.5, 133.2, 129.7, 123.0, 122.5, 102.9, 72.5, 54.6,
51.2, 28.6, 23.3, 22.0, 21.7. MS (ESI): calcd for C18H22N4O6 [M +
H]+, 391.16; found: 391.11.
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5.2.7.2. 5-Isopropyl 1-methyl 4-methyl-2-(methylamino)-6-(3-
nitrophenyl)pyrimidine-1,5(6H)-dicarboxylate (14b).
Pale
yellow powder, mp: 103–105 °C. 1H NMR (500 MHz, CDCl3) d:
8.15 (s, 1H), 8.12 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 7.80 (bs, 1H), 7.63
(dt, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 6.31 (s, 1H), 5.04
(hept, J = 6.2 Hz, 1H), 3.88 (s, 3H), 3.02 (d, J = 4.3 Hz, 3H), 2.42 (d,