9374
W.M. Bloch et al. / Tetrahedron 67 (2011) 9368e9375
Found: C 68.8, H 4.8, N 21.5, C19H15N5$H2O requires: 68.85, 5.2,
21.1%; lmax/nm: 527 (
/Mꢁ1 cmꢁ1 4620).
(20 mL), n-BuLi (0.674 mL,1.28 mmol), and 2,3-dichloroquinoxaline
(8b) (0.123 g, 0.618 mmol) in THF (15 mL) and reacted in the
manner described. After stirring at room temperature, the reaction
mixture was warmed to 50 ꢀC for 1 h and then quenched. The
purple reaction mixture was subjected to the standard work-up
(using CHCl3) and yielded a dark solid, which was isolated by fil-
tration and repeatedly washed with diethyl ether until the wash-
ings were clear. This afforded the title compound as a purple solid
(0.11 g, 58%). Sublimes 280e283 ꢀC; nmax (neat, cmꢁ1) 3074, 1577,
3
4.2.6. 5-(3,5-Dimethyl-1H-pyrazol-1-yl)-8,9-dimethylindolizino[2,3-
b]quinoxaline (5c). 2-[(3,5-Dimethyl-1H-pyrazol-1-yl)methyl]pyri-
dine (7) (0.647 g, 3.46 mmol), n-BuLi (1.50 mL, 3.50 mmol), and 2,3-
dichloro-6,7-dimethylquinoxaline (8c) (0.383 g, 1.69 mmol) were
combined and reacted in the manner described in the general
procedure. The crude reaction mixture was subjected to a standard
work-up with dichloromethane. The solid obtained was suspended
in cold hexane, isolated by filtration, and thoroughly washed with
cold hexane (4ꢂ25 mL) to afford 5c as a purple solid (0.42 g, 73%).
Mp: 201e203 ꢀC; nmax (neat, cmꢁ1) 2910, 1634, 1587, 1536, 1511,
1546, 1521, 1509, 1466; 1H NMR (600 MHz/CDCl3):
d
7.86 (dd, J¼7.4,
7.8 Hz, 1H, H8/H9), 7.91 (dd, J¼7.4, 7.8 Hz, 1H, H8/H9), 7.95 (d,
J¼4.7 Hz, 1H, H1), 8.29 (d, J¼8.4 Hz, 1H, H7/H10), 8.43 (d, J¼8.4 Hz,
1H, H7/H10), 8.48 (s, 1H, H60), 8.67 (s, 1H, H50), 8.76 (d, J¼4.7 Hz, 1H,
1488; 1H NMR (600 MHz/CDCl3):
d
2.28 (s, 3H, Me0), 2.35 (s, 3H,
H2), 10.48 (s, 1H, H30), 10.50 (s, 1H, H4); 13C NMR (151 MHz/
d
Me0), 2.53 (s, 3H, Me), 2.55 (s, 3H, Me), 6.10 (s, 1H, H40), 6.67 (t, 1H,
J¼6.8 Hz, H2), 7.22 (dd, 1H, J¼9.3, 6.8 Hz, H3), 7.45 (d, 1H, J¼9.3 Hz,
H4), 7.98 (s, 1H, H7/H10), 8.04 (s, 1H, H7/H8), 8.88 (d, 1H, J¼6.8 Hz,
CDCl3): 101.1, 116.8, 127.2, 128.9, 129.0, 129.5, 129.7, 134.6, 135.4,
138.2, 138.5, 141.1, 143.7, 144.0, 144.5, 149.5, 149.6; m/z: 298.9
(MHþ); Found: C 67.9, H 3.4, N 28.4. C17H10N6 requires: C 68.4, H 3.4,
H1); 13C NMR (151 MHz/CDCl3):
d
11.6, 13.8, 20.3, 20.4, 102.1, 105.7,
N 28.2%; lmax/nm: 536, 570 (
/Mꢁ1 cmꢁ1 4760, 4200).
3
109.1,116.7,125.0, 127.4, 127.9, 129.5, 134.2,135.5,136.2,137.8, 139.2,
139.6, 142.4, 142.6, 149.7; m/z: 342.2 (MHþ); Found: C 73.0, H 5.8, N
20.1. C21H19N5$¼H2O requires: C 72.9, H 5.7, N 20.25%; lmax/nm:
4.2.10. 8,9-Dimethyl-5-(pyrazin-2-yl)-pyrazino[1,2-a]pyrrolo[2,3-b]
quinoxaline (6c). Di-2-pyrazinylmethane (10) (0.224 g, 1.30 mmol)
inTHF (20 mL), n-BuLi (0.576 mL,1.32 mmol), and 6,7-dimethyl-2,3-
dichloroquinoxaline (8c) (0.140 g, 0.616 mmol) in THF (15 mL) were
combined and reacted in the manner described. The purple reaction
mixture was worked-up as described in the general procedure,
yielding a dark solid, which was isolated by filtration and repeatedly
washed with diethyl ether until the washings were clear. This
afforded the title compound as a purple solid (0.14 g, 69%). Sublimes
278e280 ꢀC; nmax (neat, cmꢁ1) 2919,1577,1540,1507,1491,1464; 1H
NMR (600 MHz/CDCl3) 2.57 (s, 3H, Me), 2.58 (s, 3H, Me), 7.89 (d, 1H,
J¼4.8 Hz, H1), 7.94 (s, 1H, H7/H10), 8.08 (s, 1H, H7/H10), 8.45 (d, 1H,
J¼2.5 Hz, H60), 8.64 (s, 1H, H50), 8.67 (d, 1H, J¼4.8 Hz, H2), 10.40 (s,
524 (
/Mꢁ1 cmꢁ1 4800).
3
4.2.7. 2-Chloro-3-[(3,5-dimethyl-1H-pyrazol-1-yl)(pyridin-2-yl)
methyl]-6,7-dimethylquinoxaline (9c). 2-[(3,5-Dimethyl-1H-pyr-
azol-1-yl)methyl]pyridine (7) (0.397 g, 2.12 mmol), n-BuLi
(0.962 mL, 2.10 mmol), and 2,3-dichloro-6,7-dimethylquinoxaline
(0.438 g, 1.93 mmol) were combined as described in the general
procedure. The reaction mixture was allowed to stir at ꢁ78 ꢀC for
0.5 h, and then quenched with EtOH (20 mL). The solvent was re-
moved under reduced pressure and the residue was purified by
column chromatography eluting with 9:1 dichloromethane/ethyl
acetate to afford a white solid (0.25 g, 34%). Mp: 176e179 ꢀC; nmax
(neat, cmꢁ1) 1590, 1555, 1474, 1436; 1H NMR (600 MHz/CDCl3):
1H, H30), 10.41 (s, 1H, H4); 13C NMR (151 MHz/CDCl3):
d 20.5, 20.6,
100.9, 116.7, 127.0, 127.4, 128.2, 133.7, 134.9, 137.3, 137.5, 140.0, 140.4,
140.9, 143.1, 143.6, 144.4, 149,4, 149.6; Found: C 66.2, H 4.3, N 24.6.
d
2.15 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.46 (s, 3H,
CH3), 5.93 (s, 1H, H40), 7.13 (d, 1H, J¼7.7 Hz, H300), 7.18 (s, 1H, CH),
7.25 (dd, 1H, J¼4.8, 7.7 Hz, H500), 7.67 (s, 1H, H5/H10), 7.70e7.75 (m,
3H, H400, H5/H10), 8.55 (d, 1H, J¼4.8 Hz, H600); 13C NMR (151 MHz/
C19H14N6$H2O requires:C66.3, H4.3, N24.6; m/z: 327.0 (MHþ);lmax
/
nm: 531, 564 (
/Mꢁ1 cmꢁ1 4680, 3800).
3
CDCl3):
d
11.4, 13.7, 20.1, 20.4, 66.2, 106.1, 122.7, 123.4, 127.0, 128.8,
4.3. Cell line
136.9, 139.4, 140.2, 140.4, 140.6, 141.7, 146.3, 148.5 148.7, 149.9,
157.9; m/z 342.0 (MHþ) (cyclizes); Found: C 66.8, H 5.3, N 18.6.
C21H20ClN5 requires: C 66.7, H 5.4, N 18.5%.
T98G human glioblastoma multiforme cells were purchased
from the ATCC. The cells were maintained as a monolayer in min-
imum essential medium (MEM), supplemented with 10% fetal bo-
vine serum, penicillin (100 units/mL), streptomycin (100 g/mL), and
4.2.8. 5-(Pyrazin-2-yl)-pyrrolo[1,2-a:4,5-b]pyrazine
(6a). Di-2-
pyrazinylmethane (10) (0.244 g, 1.41 mmol) in THF (20 mL), n-
BuLi (0.740 mL, 1.43 mmol), and 2,3-dichloropyrazine (8a)
(0.0730 mL, 0.698 mmol) in THF (20 mL) were combined and
reacted in the manner described. The reaction mixture was stirred
at room temperature for 16 h then heated at reflux for 20 h. The
reaction mixture was quenched with methanol (2 mL). Upon
cooling to room temperature a red precipitate formed. The solvent
was removed under reduced pressure and the crude product was
isolated as described in the general procedure using CHCl3 as the
extracting solvent. The isolated product was recrystallized from
THF to afford the title compound 6a as an orange solid (0.10 g, 58%).
Mp: 262 ꢀC; nmax (neat, cmꢁ1) 3098, 1576, 1542, 1504, 1482, 1467;
L
-glutamine (2.5 mM), at 37 ꢀC in a humidified 5% CO2 atmosphere.
4.4. Cytotoxicity assays
Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay.49 Cells were
harvested with trypsin (0.1% v/v), and cell pellets were isolated by
centrifugation. Cells were then re-suspended to single cells sus-
pension, cell numbers counted using a Hemocytometer counter
(Weber) and then seeded at a density of 1ꢂ104 cells/well in 96-well
plates, in 100 mL growth medium and allowed to adhere overnight
at 37 ꢀC. Cells were incubated at 37 ꢀC in a humidified atmosphere
of 5% CO2 in the presence of compounds 1aec, 5a, 5b, and 6aec,
and vehicle (control). Serial dilutions (1:2 and 1:10) of compound
solution in cell medium were added to wells in triplicate. Com-
pounds were tested at a concentration range prescribed by the
limits of compound solubility and that produced a sigmoidal cy-
totoxic response. After 72 h of drug exposure, MTT solution in PBS
1H NMR (300 MHz/CDCl3):
d
7.97 (d, J¼4.8 Hz, 1H, H60), 8.47 (d,
J¼2.4 Hz, 1H, H1), 8.59 (d, J¼2.3 Hz, 1H, H8), 8.65e8.68 (m, 2H, H2/
H50), 9.00 (d, J¼2.3 Hz, 1H, H7), 10.18 (d, J¼1.5 Hz, 1H, H4), 10.45 (d,
J¼1.7 Hz, 1H, H30); 13C NMR (151 MHz/CDCl3): 102.7, 115.9, 128.0,
130.6, 134.3, 136.3, 137.9, 141.4, 143.7, 144.4, 144.6, 149.4, 149.4; m/z
249.2 (MHþ); Found: C 61.9, H 3.4, N 33.3. C13H8N6$¼H2O requires:
C 61.8, H 3.4, N 33.3%; lmax/nm: 464 (
3
/Mꢁ1 cmꢁ1 4200).
(20 mL, 0.25% w/v) was added to each well and the incubation
continued for 4 h. Culture medium and excess MTT solution were
removed and the resulting reduced formazan crystals dissolved by
4.2.9. 5-(Pyrazin-2-yl)-pyrazino[1,2-a]pyrrolo[2,3-b]quinoxaline
(6b). Di-2-pyrazinylmethane (10) (0.220 g, 1.27 mmol) in THF
addition of 150 mL DMSO.