6810
H. N. Nagesh et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6805–6810
117.76, 78.64, 76.89, 58.72, 56.21 50.63. EI-MS m/z 302.12 (M+H)+; HRMS: (ESI
(8a) Yield = 74%; white solid, mp 151–152 °C; 1H NMR (400 MHz, CDCl3) d 8.52 (d,
J = 8.4 Hz, 1H), 8.43 (d, J = 7.6, Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.93 (d,
J = 9.2 Hz, 1H), 7.78–7.32 (m, 9H), 3.92 (s, 2H), 3.54 (m, 4H), 2.90 (m, 4H). 13C NMR
(100.61 MHz, CDCl3) d 174.12, 151.76, 142.18, 139.84, 137.84, 135.51, 134.42,
130.80, 129.47, 128.13, 127.69, 126.61, 124.60, 123.11, 121.64, 120.43, 119.92,
114.85, 108.16, 58.18, 50.74, 45.27. EI-MS m/z 485.12 (M+H) +; HRMS: (ESI m/z)
for C26H25N6O2S calcd.: 485.1759, found: 485.1764 (M+H)+.
m/z) for C20H20N3 calcd: 302.3929, found: 302.3933 (M+H)+.
30. Synthesis of 6-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)phenan-
thridine (7a): 6-(4-(prop-2-ynyl)piperazin-1-yl)phenanthridine (0.6571 mmol)
was dissolved in 1:2 ratio of water and t-BuOH (3 mL). Then CuSO4Á5H2O
(0.1314 mmol), sodium ascorbate (0.1314 mmol) and benzyl azide
(0.7228 mmol) was added. Resultant mixture was stirred at RT for 3 h.
Completion of the reaction was monitored by TLC using 2% MeOH in DCM as
mobile phase. After the reaction was complete, volatile was evaporated in
vacuo and the compound was extracted using EtOAc (3 Â 5 mL). Combined
organic layers were washed with saturated brine solution, dried over
anhydrous sodium sulphate and evaporated in vacuo. Column
chromatography of the residue using 1–2% MeOH in DCM gave 6-(4-
((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)phenanthridine (7a)
Yield = 91%; white solid, mp 132–133 °C; 1H NMR (400 MHz, CDCl3) d 8.55
(d, J = 8.4 Hz, 1H), 8.43 (d, J = 7.2, Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.00 (s,
1H),7.88 (d, J = 9.2 Hz, 1H), 7.78–7.32 (m, 9H), 4.98 (s, 2H), 3.92 (s, 2H), 3.55 (m,
4H), 2.90 (m, 4H). 13C NMR (100.61 MHz, CDCl3) d 172.46, 148.76, 141.28,
139.84, 138.32, 135.51, 134.42, 130.80, 129.47, 128.13, 127.69, 126.61, 124.60,
123.11, 122.24, 121.64, 120.43, 119.92, 116.85, 60.16, 58.18, 50.74, 45.27.
EI-MS m/z 435.18 (M+H)+; HRMS: (ESI m/z) for C27H27N6 calcd: 435.2297,
found: 435.2289 (M+H)+.
31. Synthesis of 6-(4-((1-(phenylsulfonyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)
phenanthridine (8a): 6-(4-(prop-2-ynyl)piperazin-1-yl)phenanthridine (0.6571
mmol) was dissolved in toluene (5 mL). Then CuTC (0.0657 mmol), and
benzene sulfonyl azide (0.7228 mmol) was added. Resultant mixture was
stirred at RT for 1 h. Completion of the reaction was monitored by TLC using 2%
MeOH in DCM as mobile phase. After the reaction was complete, saturated aq
NH4Cl (5 mL) was added and the compound was extracted using EtOAc
(3 Â 5 mL). Combined organic layers were washed with saturated brine
solution, dried over anhydrous sodium sulphate and evaporated in vacuo.
Column chromatography of the residue using 1–2% MeOH in DCM gave 6-(4-
((1-(phenylsulfonyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)phenanthridine
32. 200
sterile 96-well plates to minimize evaporation of the medium in the test wells
during incubation. The wells in rows B–G in columns 3–11 received 100 L of
7H9GC broth. 100
L of 2Â drug solutions were added to the wells in rows B–G
in columns 2 and 3. By using a multichannel pipette, 100 L was transferred
from column 3 to column 4, and the contents of the wells were mixed well.
Identical serial 1:2 dilutions were continued through column 10, and 100 L of
excess medium was discarded from the wells in column 10. 100 L of MTB
inoculum was added to the wells in rows B–G in columns 2–10. Add 100 L of
medium to B11 and C11 (media control), 100 L of Mtb inoculum to D11 and
E11 and 100 L of MTB inoculum with 3–5% DMSO to F11 and G11 (solvent
control). The plates were sealed with parafilm and were incubated at 37 °C for
5 days. 50
L of a freshly prepared 1:1 mixture of 10Â Alamar Blue (Accumed
lL of sterile deionized water was added to all outer-perimeter wells of
l
l
l
l
l
l
l
l
l
International, Westlake, Ohio) reagent and 10% Tween 80 was added to well
D11. The plates were reincubated at 37 °C for 24 h. If well D11 turned pink, the
reagent mixture was added to all wells in the microplate (if the well remained
blue, the reagent mixture would be added to another control well and the
result would be read on the following day). The microplates were resealed with
parafilm and were incubated for an additional 24 h at 37 °C, and the colors of
all wells were recorded. A blue color in the well was interpreted as no growth,
and a pink color was scored as growth. A few wells appeared violet after 24 h of
incubation, but they invariably changed to pink after another day of incubation
and thus were scored as growth (while the adjacent blue wells remained blue).
The MIC was defined as the lowest drug concentration which prevented a color
change from blue to pink.