C.-C. Chang et al. / Bioorg. Med. Chem. 18 (2010) 3147–3158
3157
ice cold water and extracted with ethyl acetate (3 ꢂ 5 mL). The or-
ganic layer was washed with sodium thiosulfate, with water, dried,
and concentrated in vacuo. The crude products were purified by
column chromatography using silica gel (60–120 mesh) and eluted
with ethyl acetate/hexane (1:3) to afford 1,4-dihydropyridines in
41–98% yields.
calcd for C18H22NO4S (M+H+): 348.1270; found: 348.1267; Anal.
Calcd for C18H21NO4S (347.11): C, 62.23; H, 6.09; N, 4.03. Found:
C, 62.06; H, 5.95; N, 3.89.
5.3. General procedure for the synthesis of various 4-aryl
(alkyl)-1,4-dihydro-2,6-dialkyl-3,5-pyridinedicarboxylates
(parallel synthesis in Table 3).20
5.2.1. Methyl ethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4)
To a suspension of compound 70 (200 mg) in 3 mL of dichloro-
methane was added acetic anhydride (171 lL) at room tempera-
ture. The reaction mixture was stirred at room temperature for
2 h followed by addition of two drops of acetyl chloride and then
1.1 equiv of various alcohols. The reaction mixture was stirred
overnight (18 h) and then purified by flash chromatography to af-
ford the desired product.
Yellow solid; mp 78–81 °C; 1H NMR (CDCl3, 500 MHz): d = 1.16
(t, J = 7.0 Hz, 3H, CH3), 2.28 (s, 6H, 2CH3), 3.63 (s, 3H, OCH3), 4.01–
4.14 (m, 2H, OCH2), 5.72 (s, 1H, CH), 5.79 (s, 1H, NH), 7.24–7.71 (m,
4H, C6H4); Anal. Calcd for C18H20N2O6 (360.13): C, 59.99; H, 5.59;
N, 7.77. Found: C, 60.11; H, 5.40; N, 7.77.
5.2.2. Diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyri-
dine-3,5-dicarboxylate (12)
5.3.1. Ethyl methoxyethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (77)
Yellow solid; mp 158–160 °C; 1H NMR (CDCl3, 400 MHz):
d = 1.22 (t, J = 7.2 Hz, 6H, 2CH3), 2.37 (s, 6H, 2CH3), 4.03–4.13 (m,
4H, 2OCH2), 5.09 (s, 1H, CH), 5.74 (s, 1H, NH), 7.36–8.13 (m, 4H,
C6H4); 13C NMR (CDCl3, 125 MHz): d = 14.5, 19.8, 40.2, 60.1,
103.5, 121.6, 123.2, 128.8, 134.8, 145.0, 148.1, 150.2, 167.4; HRMS
(ES): m/z calcd for C19H21N2O6 (MꢁH+): 373.1400, found 373.1396;
Anal. Calcd for C19H22N2O6 (374.14): C, 60.95; H, 5.92; N, 7.48.
Found: C, 60.98; H, 5.98; N, 7.36.
Yellow solid; mp 83–85 °C; 1H NMR (CDCl3, 500 MHz): d 1.22 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.36 (s, 6H, CH3 ꢂ 2), 3.35 (s, 3H, OCH3),
3.51–3.56 (m, 2H, OCH2), 4.04–4.12 (m, 2H, OCH2CH3), 4.12–4.22
(m, 2H, OCH2), 5.11 (s, 1H, CHAr), 5.99 (br s, 1H, NH), 7.38 (t,
J = 8.0 Hz, 1H, Ar-H), 7.67 (d, J = 8.0 Hz, 1H, Ar-H), 8.00 (d,
J = 8.0 Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H); 13C NMR (CDCl3,
125 MHz): d 14.2, 19.5, 19.6, 39.9, 58.8, 60.0, 63.0, 70.5, 103.0,
103.4, 121.3, 123.1, 128.6, 134.7, 144.7, 145.3, 148.1, 149.9,
167.0, 167.1; HRMS (ESI): m/z calcd for C20H25N2O7 (M+H+):
405.1662; found: 405.1666; Anal. Calcd for C20H24N2O7 (404.15):
C, 59.40; H, 5.98; N, 6.93. Found: C, 59.33; H, 6.11; N, 6.83.
5.2.3. Diethyl 2-methyl-6-n-propyl-4-(3-nitrophenyl)-1,4-dihy-
dropyridine-3,5-dicarboxylate (56)
Yellow solid; mp 97–98 °C; 1H NMR (CDCl3, 500 MHz): d 1.00 (t,
J = 7.0 Hz, 3H, CH3), 1.20–1.25 (two t overlapped at 1.22 and 1.24,
J = 7.0 Hz, 6H, OCH2CH3 ꢂ 2), 1.58–1.72 (m, 2H, CH2), 2.36 (s, 3H,
CH3). 2.65–2.75 (m, 2H, CH2), 4.05–4.15 (m, 4H, OCH2CH3 ꢂ 2),
5.11 (s, 1H, CHAr), 5.95 (br s, 1H, NH), 7.38 (t, J = 8.0 Hz, 1H. Ar-
H), 7.65 (d, J = 8.0 Hz, 1H, Ar-H), 8.00 (d, J = 8.0 Hz, 1H, Ar-H),
8.14 (s, 1H, Ar-H); 13C NMR (CDCl3, 125 MHz): d 14.0, 14.2, 14.3,
19.6, 2.0, 34.6, 39.9, 60.0, 102.9, 103.1, 121.3, 123.1, 128.6, 134.5,
144.9, 148.1, 149.2, 150.0, 166.8, 167.2; HRMS (ESI): m/z calcd
for C21H27N2O6 (M+H+): 403.1864; found: 403.1874; Anal. Calcd
for C21H26N2O6 (402.17): C, 62.67; H, 6.51; N, 6.96. Found: C,
62.61; H, 6.64; N, 6.88.
5.3.2. Methyl cyanoethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (80)
Yellow solid; mp 134–137 °C; 1H NMR (CDCl3, 500 MHz): d 2.38
(s, 3H, CH3), 2.39 (s, 3H, CH3), 2.65–2.69 (m, 2H, OCH2CH2CN), 3.66
(s, 3H, OCH3), 4.21–4.31 (m, 2H, OCH2CH2CN), 5.10 (s, 1H, CHAr),
6.04 (br s, 1H, NH), 7.41 (t, J = 7.5 Hz, 1H, Ar-H), 7.68 (d,
J = 7.5 Hz, 1H, Ar-H), 8.02 (d, J = 7.5 Hz, 1H, Ar-H), 8.10 (s, 1H, Ar-
H); 13C NMR (CDCl3, 125 MHz): d 18.1, 19.5, 20.0, 39.6, 51.2,
58.4, 102.0, 103.6, 117.1, 121.6, 122.8, 128.9, 134.4, 144.7, 146.6,
148.4, 149.3, 166.3, 167.4; HRMS (ESI): m/z calcd for C19H20N3O6
(M+H+): 386.1352; found: 386.1354; Anal. Calcd for C19H19N3O6
(385.12): C, 59.22; H, 4.97; N, 10.90. Found: C, 59.25; H, 5.04; N,
10.73.
5.2.4. Ethyl n-pentyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihy-
dropyridine-3,5-dicarboxylate (82)
Yellow viscous oil; 1H NMR (CDCl3, 500 MHz): d 0.84–0.90 (m,
3H, OCH2CH2CH2CH2CH3), 1.19–1.32 (m, overlapped with t at
1.24, J = 8.0 Hz, 7H, CH2 ꢂ 2 and CH3), 1.56–1.62 (m, 2H,
OCH2CH2CH2CH2CH3), 2.36 (s, 3H, CH3), 2.37 (s, 3H, CH3), 3.98–
4.10 (m, 4H, OCH2 ꢂ 2), 5.10 (s, 1H, CHAr), 6.04 (br s, 1H, NH),
7.38 (t, J = 8.0 Hz, 1H, Ar-H), 7.65 (d, J = 8.0 Hz, 1H, Ar-H), 8.01 (d,
J = 8.0 Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H); 13C NMR (CDCl3,
125 MHz): d 14.0, 14.2, 19.6, 22.3, 28.2, 28.3, 39.9, 60.0, 64.2,
103.2, 103.3, 121.3, 123.0, 128.6, 134.5, 144.9, 145.0, 148.1,
149.9, 167.1, 167.2; HRMS (ESI): m/z calcd for C22H29N2O6
(M+H+): 417.2026; found: 417.2026; Anal. Calcd for C22H28N2O6
(416.19): C, 63.45; H, 6.78; N, 6.73. Found: C, 63.18; H, 6.75; N,
6.65.
5.4. Method for transfection of tsA201 cells with CaV1.3 and
CaV1.2
5.4.1. Constructs
Rat Cav1.3a1D, CaVb3, and CaVa2d-1 cDNA were gifts of Dr. D.
Lipscombe, Brown University, Providence, RI. Sequence alignments
and RT-PCR from brain tissue revealed a few point mutations in
CaV1.3
a1D, which were corrected by site-directed mutagenesis.
Rabbit CaV1.2
a1C cDNA was a gift of Dr. Johannes Hell, University
of Iowa.
5.4.2. Transfection of tsA201 cells
5.2.5. Dimethyl 2,6-dimethyl-4-(2-methylthiophenyl)-1,4-dihy-
dropyridine-3,5-dicarboxylate (113)
tsA201 cells were maintained in D-MEM medium supple-
mented with 10% fetal bovine serum (Invitrogen) without antibiot-
Compound 113: White solid; mp 168–170 °C; 1H NMR (CDCl3,
500 MHz): d 2.31 (s, 6H, CH3 ꢂ 2), 2.49 (s, 3H, SCH3), 3.62 (s, 6H,
OCH3 ꢂ 2), 5.46 (s, 1H, CHAr), 5.66 (br s, 1H, NH), 7.06 (t,
J = 8.0 Hz, 1H, Ar-H), 7.10 (t, J = 8.0 Hz, 1H, Ar-H), 7.26 (d,
J = 8.0 Hz, 1H, Ar-H), 7.32 (d, J = 8.0 Hz, 1H, Ar-H); 13C NMR (CDCl3,
125 MHz): d 18.10, 19.6 ꢂ 2, 37.1, 50.9 ꢂ 2, 104.6 ꢂ 2, 126.0, 126.8,
128.1, 129.9, 136.6, 143.7ꢂ, 147.8, 168.1 ꢂ 2; HRMS (ESI): m/z
ics. A mixture of CaV1.3 a1D or CaV1.2 a1C, CaV b3, and CaVa2d-1
cDNA at a molar ratio of 1:1:1 together with 1/40 (w/w) GFP cDNA
(Invitrogen) were transfected into tsA201 cells using Geneporter
reagent (Genetic Therapy Systems, San Diego, CA) according to
the manufacturer’s protocol. Cells were trypsinized 48 h later and
plated on poly-D-lysine-coated coverslips. GFP-labeled cells were
recorded after attachment.