JOURNAL OF CHEMICAL RESEARCH 2009 647
Table 2 The inhibition for P-388 and A-549 growth
Entry
Inhibition of P-388/%
concentration (mol L-1)
Inhibition of A-549/%
concentration (mol L-1)
10-4
10-5
10-6
10-7
10-8
10-4
10-5
10-6
10-7
10-8
1b
1c
1d
2b
5a
5d
5e
0
0
0
0
0
0
0
0
31.1
30.8
36.2
32.4
26.1
29.5
25.7
2.4
1.0
0
7.1
2.2
6.8
2.7
0
3.4
0
12.1
1.2
8.6
0
9.9
1.5
1.9
4.2
0
23.9
0
7.1
23.4
25.9
0
0
0
16.1
7.3
6.3
0
0
5.9
1.2
13.8
0
0
1.4
1.4
4.8
2.9
0
0
15.0
10.7
4.9
0
10.1
19.5
28.9
1.2
0
7.7
0
8.8
Dibutyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate (1d): 1.5 g, 26%,
m.p. 53–54°C, (lit.10 56–61°C). IR nmax(KBr)/cm-1: 3336 (NH), 2979
(CH), 1719 (C=O). 1H NMR (CDCl3, TMS, 400 MHz)/dppm: 7.49 (s,
2H), 4.31 (t, J = 7.2 Hz, 4H), 1.75–1.68 (m, 4H), 1.44–1.38 (m, 4H), 0.95
(t, J = 6.8 Hz, 6H). EI-MS (m/z): 284 (M+). Anal. Calcd for C12H20N4O4:
C, 50.7; H, 7.1; N, 19.7; Found: C, 50.9; H, 7.3; N, 19.7%.
as the 3,6-aryl substituted 1,2,4,5-tetrazine derivatives which
we had previously tested before.5-7
Conclusion
Unbranched dialkyl dihydro-1,2,4,5-tetrazine-3,6-dicarb-
oxylates were synthesised by Boger's method. A crystal
structure of 1b showed that the central six-membered tetrazine
ring has a 1,4-dihydro structure with a boat conformation.
Branched dialkyl esters of 1,4-dihydro-1,2,4,5-tetrazine-
3,6-dicarboxylic acid were synthesised by transesterification
with the corresponding alcohols and this was accompanied
3,6-asymmetric substituted esters. Amidation was developed
to obtain dialkyl amides of 1,4-dihydro-1,2,4,5-tetrazine-3,6-
dicarboxylic acid with good to excellent yields. Some selected
compounds were evaluated in vitro against P-388 and A-549
cancer cell lines, but showed poor inhibitory activities.
General procedure for the synthesis of 2a–b, 3a, 4a
To a solution of dialkyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxy-
late (20 mmol) in the corresponding alcohol (30 mL), was added
p-toluenesulfonic acid (0.3 g, 2 mmol). The mixture was refluxed for
2–4 h and the crude product was purified by column chromatography
to obtain the corresponding ester of 1,4-dihydro-1,2,4,5-tetrazine.
Diisopropyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate (2a):
1.6 g, 31%, m.p. 56–57°C. IR nmax(KBr)/cm-1: 3330 (NH), 2981
(CH), 1718 (C=O). 1H NMR (CDCl3, TMS, 400 MHz)/dppm: 7.53 (s,
2H), 5.23–5.17 (m, 2H), 1.36 (d, J = 6.8 Hz, 12H). EI-MS (m/z): 256
(M+). Anal. Calcd for C10H16N4O4: C, 46.9; H, 6.3; N, 21.9; Found: C,
46.95; H, 6.3; N, 21.8%.
Diisobutyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate (2b):
1.2 g, 21%, m.p. 94–96°C. IR nmax(KBr)/cm-1: 3348 (NH), 2974
(CH), 1717 (C=O). 1H NMR (CDCl3, TMS, 400 MHz)/dppm: 7.52 (s,
2H), 4.09 (d, J = 7.2 Hz, 4H), 2.07–2.04 (m, 2H), 0.97 (d, J = 6.8 Hz,
12H). EI-MS (m/z): 284 (M+). Anal. Calcd for C12H20N4O4: C, 50.7;
H, 7.1; N, 19.7; Found: C, 51.0; H, 7.1; N, 19.7%.
Experimental
Melting points were taken on XRC-1 apparatus and are uncorrected.
IR spectra were obtained on a PK-6000 spectrophotometer or a
Thermo Nicolet Avatar 370 FT-IR spectrophotometer. 1H NMR
spectra were recorded on a Bruker AC 400 spectrometer operating at
400 MHz or a Bruker AVANCE III spectrometer at 500 MHz using
TMS as the internal standard. MS spectra were run on an HP5989B
instrument or a Waters GCT Premier with EI source. Elemental
analyses of C, H, N were performed on a Thermo Finnigan Flash
EA 1112 instrument. All the chemicals and solvents were analytical
reagent grade and were used as received.
3-Isopropyl6-methyl1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate
(3a): 2.0 g, 44%, m.p. 83–84°C. IR nmax(KBr)/cm-1: 3325 (NH),
1
2974 (CH), 1715 (C=O). H NMR (CDCl3, TMS, 400 MHz)/dppm:
7.52 (s, 1H), 7.47 (s, 1H), 5.22–5.19 (m, 1H), 3.92 (s, 3H), 1.36 (d,
J = 6.8 Hz, 6H). EI-MS (m/z): 228 (M+). Anal. Calcd for C8H12N4O4:
C, 42.1; H, 5.3; N, 24.55; Found: C, 42.3; H, 5.4; N, 24.65%.
3-Ethyl6-isopropyl1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate
(4a): 1.7 g, 35%, m.p. 80–82°C. IR nmax(KBr)/cm-1: 3381 (NH), 2996,
General procedure for the synthesis of 1a–d according to Boger's
method8
To the corresponding alcohol (25 mL) with thionyl chloride (3.8 mL)
at –20°C was added 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylic
acid (3.4 g, 20 mmol) suspended in the alcohol (25 mL) in portions.
Other procedures were identical to Boger's method.
1
2978, 2941 (CH), 1714 (C=O). H NMR (CDCl3, TMS, 400 MHz)/
dppm: 7.50 (s, 1H), 7.47 (s, 1H), 5.06–5.01 (m, 1H), 3.94–3.91 (m, 2H),
1.38–1.32 (m, 9H). EI-MS (m/z): 242 (M+). Anal. Calcd for C9H14N4O4:
C, 44.6; H, 5.8; N, 23.1; Found: C, 44.5; H, 5.9; N, 23.3%.
General procedure for the synthesis of 5a–e
Dimethyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate (1a): 2.5 g,
To
a
solution of dimethyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-
63%; m.p. 171–172°C, (lit.8 171–172°C). IR nmax(KBr)/cm-1: 3361
dicarboxylate 1a (4.0 g, 20 mmol) in ethanol (50 mL), was added
amine (50 mmol). The mixture was stirred at 60–70°C for 0.5–1 h
and then cooled. The precipitate was collected and recrystallised to
afford the corresponding amide of 1,4-dihydro-1,2,4,5-tetrazine.
Amide 5a was prepared by adding saturated ammonia in ethanol.
1,4-Dihydro-1,2,4,5-tetrazine-3,6-dicarboxamide (5a): 3.3 g, 97%,
m.p. > 280°C. IR nmax(KBr)/cm-1: 3391, 3274, 3219 (NH), 1686 (C=O).
1H NMR (CDCl3 + DMSO-d6, TMS, 500 MHz)/dppm: 7.96 (br, 4H),
7.59 (s, 2H). EI-MS (m/z): 170 (M+). Anal. Calcd for C4H6N6O2: C,
28.2; H, 3.55; N, 49.4; Found: C, 28.1; H, 3.5; N, 49.0%.
1
(NH), 2961 (CH), 1723 (C=O). H NMR (CDCl3, TMS, 400 MHz)/
dppm: 7.49 (s, 2H), 3.92 (s, 6H). EI-MS (m/z): 200 (M+).
Diethyl 1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate (1b): 1.4 g,
31%, m.p. 101–102°C, (lit.9 101–102°C). IR nmax(KBr)/cm-1: 3378
1
(NH), 2983 (CH), 1717 (C=O). H NMR (CDCl3, TMS, 400 MHz)/
dppm: 7.55 (s, 2H), 4.41–4.35 (m, 4H), 1.38 (t, J = 6.8 Hz, 6H).
EI-MS (m/z): 228 (M+). Anal. Calcd for C8H12N4O4: C, 42.1; H, 5.3;
N, 24.55; Found: C, 42.4; H, 5.4; N, 25.0%. Crystal data: Monoclinic,
P21/c, a = 8.4740 (10) Å, b = 13.4510 (15) Å, c = 10.159 (3) Å,
b = 109.090 (17)°, V = 1094.3 (4) Å3, Z = 4, Dx = 1.385 Mg m-3, Mo
Ka radiation, m = 0.11 mm-1, T = 298 (2) K, 2262 measured reflections,
1954 independent reflections, Rint = 0.014, Final R1 = 0.050,
wR(F2) = 0.169.
CCDC 255702 contains the supplementary crystallographic
data_request.cif.
N3,N6-Dipropyl-1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxamide
(5b): 4.0 g, 79%, m.p. 235–238°C. IR nmax(KBr)/cm-1: 3338, 3272
1
(NH), 2965 (CH), 1663 (C=O). H NMR (CDCl3, TMS, 500 MHz)/
dppm: 7.57 (s, 2H), 6.88 (br, 2H), 3.29 (q, J = 7.0 Hz, 4H), 1.58 (q,
J = 7.0 Hz, 4H), 0.94 (t, J = 7.5 Hz, 6H). EI-MS (m/z): 254 (M+).
Anal. Calcd for C10H18N6O2: C, 47.2; H, 7.1; N, 33.05; Found: C,
47.4; H, 7.0; N, 33.2%.
N3,N6-Dibutyl-1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxamide (5c):
5.0 g, 89%, m.p. 230–233°C. IR nmax(KBr)/cm-1: 3341, 3273 (NH),
2957 (CH), 1661 (C=O). 1H NMR (CDCl3, TMS, 500 MHz)/dppm: 7.76
(s, 1H), 7.61 (s, 1H), 6.90 (br, 1H), 6.78 (br, 1H), 3.33 (q, J = 7.0 Hz,
4H), 1.52 (m, J = 7.0 Hz, 4H), 1.37 (m, J = 7.5 Hz, 4H), 0.93 (t,
J = 7.5 Hz, 6H). EI-MS (m/z): 282 (M+). Anal. Calcd for C12H22N6O2:
C, 51.05; H, 7.85; N, 29.8; Found: C, 50.8; H, 8.0; N, 29.7%.
Dipropyl1,4-dihydro-1,2,4,5-tetrazine-3,6-dicarboxylate(1c):1.4g,
27%, m.p. 78–79°C, (lit.10 78–81°C). IR nmax(KBr)/cm-1: 3313 (NH),
1
2962 (CH), 1717 (C=O). H NMR (CDCl3, TMS, 400 MHz)/dppm:
7.56 (s, 2H), 4.27 (t, J = 7.2 Hz, 4H), 1.79–1.74 (m, 4H), 0.98 (t,
J = 6.8 Hz, 6H). EI-MS (m/z): 256 (M+). Anal. Calcd for C10H16N4O4:
C, 46.9; H, 6.3; N, 21.9; Found: C, 46.9; H, 6.4; N, 22.2%.