M. Jayanthi and P. Rajakumar
Vol 000
completion of the reaction, it was extracted with CHCl3
(3 × 100 mL), washed with aq., NaHCO3 (100 mL), and
brine (100 mL), dried over Na2SO4, and evaporated. The
residue was purified by column chromatography using
CHCl3/hexane (2:3) as eluent.
129.4, 132.7, 134.6, 143.7, 159.9; Anal. Calcd for
165H132N24O21: C, 71.11; H, 4.77; found: C, 70.81;
H, 4.52.
C
3-(Bromomethyl)-1H-indazole.
Colorless
liquid;
BIOLOGY
1
lachrymatory. Yield: 72%; H NMR (300 MHz, CDCl3):
δH 4.79 (s, 2H), 7.22–7.31 (m, 2H), 7.35–7.38 (m, 1H),
7.47–7.49 (m, 1H); 13C NMR (75 MHz, CDCl3): δC
62.9, 127.0, 128.8, 128.9, 129.4, 132.8, 138.2; m/z: 212
[M + 1].
Materials and methods. Cell preparation and culturing.
The A549 lung adenocarcinoma cell line was procured
from the National Centre for Cell Science (NCCS),
Pune, India, with the passage number of 11. Cells were
maintained in Dulbecco’s Minimum Essential Media
(DMEM) supplemented with 10% Fetal Bovine Serum
(FBS), with 100 units/mL penicillin and 100 μg/mL
Dendritic bromo compound 6 (G1-CH2Br). White solid;
1
mp: 76–79°C; yield: 74%; H NMR: (300 MHz, CDCl3)
δH 4.65 (s, 2H), 5.15 (s, 4H), 6.57 (s, 1H), 6.66 (s, 2H),
7.26–7.31 (m, 5H), 7.38–7.41 (m, 3H), 7.54–7.55 (m,
2H); 13C NMR: (75 MHz, CDCl3) δC 65.3, 67.2, 101.3,
106.0, 127.0, 128.9, 129.1, 129.4, 132.7, 134.6, 143.6,
159.9; Anal. Calcd for C23H19BrN4O2: C, 69.62; H, 4.13;
found: C, 59.51; H, 3.94; m/z: 464 [M + 1].
streptomycin. Cells were cultured in
a humidified
atmosphere with 5% CO2 at 37°C. Cells were grown
in 75 cm2 culture flask, and after a few passages, cells
were seeded for experiments. The experiments were
carried out at 70 to 80% confluence. Upon reaching
confluence, cells were detached using 0.25% Trypsin-
EDTA solution.
Dendritic bromo compound 9 (G2-CH2Br). White solid;
1
mp: 94–98°C; yield: 71%; H NMR: (300 MHz, CDCl3)
δH 4.40 (s, 4H), 5.01 (s, 2H), 5.12 (s, 8H), 6.56–6.57 (m,
2H), 6.65–6.66 (m, 5H), 7.25–7.26 (m, 8H), 7.27–7.28
(m, 9H), 7.37–7.40 (m, 5H); 13C NMR: (75 MHz,
CDCl3) δC 65.5, 67.3, 70.2, 102.2, 108.4, 127.1, 127.6,
128.2, 128.7, 128.9, 129.2, 129.5, 132.7, 134.4, 140.0,
159.9; Anal. Calcd for C53H43BrN8O6: C, 65.77; H, 4.48;
found: C, 65.52; H, 4.22; m/z: 968 [M + 1].
Cell proliferation assay or MTT assay. Proliferation of
A549 cells was assessed by MTT assay [22]. The
proliferation test is based on the color reaction of
mitochondrial dehydrogenase in living cells by MTT.
Cells were plated in 96-well plate at a concentration of
5 × 104 cells/well 24 h after plating. After 24 h of cells
incubation, the medium was replaced with 100-μL
medium containing MJ–MU–G0 and MJ–MU–G2 at
different concentrations (0.98–500 μM/well) and
incubated for 24 h. Untreated cells served as control and
received only 0.1% DMSO in which the drug was
prepared. At the end of treatment period, media from
control and drug-treated cells was discarded and 20 μL of
MTT (5 mg/mL PBS) was added to each well. Cells were
then incubated for 4 h at 37°C in CO2 incubator. MTT
was then discarded and the coloured crystals of produced
formazan were dissolved in 200 μL of DMSO and mixed
Following the general procedure A, the dendrimers 1–3
were synthesized in good yield by reacting three equivalents
the
corresponding
dendritic
bromide,
namely,
3-(bromomethyl)-1H-indazole, 6 and 9 with one equivalent
of 1,3,5-trihydroxybenzene in DMF at RT for 24 h.
G0 dendrimer 1. Off-white solid; mp: 88–90°C; yield:
1
82%; H NMR: (300 MHz, CDCl3) δH 5.13 (s, 6H), 6.31
(s, 3H), 7.25–7.31 (m, 6H), 7.38–7.41 (m, 3H),
7.53–7.56 (m, 3H); 13C NMR: (75 MHz, CDCl3) δC
67.3, 95.1, 126.9, 128.9, 129.0, 129.4, 132.7, 134.5,
160.4, 162.6; Anal. Calcd for C30H24N6O3: C, 69.76; H,
4.68; found: C, 69.52; H, 4.34; m/z: 517 [M + 1].
effectively
by
pipetting
up
and
down.
Spectrophotometrical absorbance of the purple blue
formazan dye was measured using an ELISA reader
(BIORAD) at 570 nm. Optical density of each sample
was compared with control optical density and graphs
were plotted.
Flow cytometry. To investigate the effect of dendrimer
1 and dendrimer 3 on the cell cycle distribution, A549
cells (1 × 105 cells/mL) were treated with 26 and 52 μM
of dendrimer 1 and 75 and 150 μM of dendrimer 3
cultured for 24 h. The treated cells were harvested,
washed with phosphate-buffer saline (PBS), and fixed in
75% ethanol at 4°C overnight. After washing twice with
cold PBS, cells were suspended in PBS containing
40 μg/mL propidium iodide (PI) and 0.1 mg/mL RNase A
followed by shaking at37°C for 30 min. The stained cells
G1 dendrimer 2. Off-white solid; mp: 96–98°C; yield:
1
72%; H NMR: (300 MHz, CDCl3) δH 4.63 (s, 6H), 5.13
(s, 12H), 6.56 (s, 3H), 6.65 (s, 6H), 7.22–7.27 (m, 13H),
7.37–7.40 (m, 7H), 7.52–7.55 (m, 7H); 13C NMR:
(75 MHz, CDCl3) δC 65.2, 67.2, 101.3, 106.0, 127.0,
128.9, 129.1, 129.4, 132.7, 134.6, 143.6, 160.0;
Anal. Calcd for C75H60N12O9: C, 70.74; H, 4.75; found:
C, 70.49; H, 4.48; m/z: 1273 [M + 1].
G2 dendrimer 3. Off-white solid; mp: 88–91°C; yield:
70%; 1H NMR: (300 MHz, CDCl3) δH 4.76 (s, 12H),
5.07 (s, 6H), 5.13 (s, 24H), 6.56 (s, 6H), 6.65 (s, 14H),
7.22–7.29 (m, 27H), 7.33–7.40 (m, 27H), 7.52–7.55 (m,
16H); 13C NMR: (75 MHz, CDCl3) δC 65.2, 67.2, 70.1,
101.3, 106.0, 127.0, 127.6, 128.0, 128.6, 128.9, 129.1,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet