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V. Pawar et al. / Bioorg. Med. Chem. 18 (2010) 3198–3211
complete. During the heating period, some crystals of isonitroso-
acetanilide was separated. On cooling the solution in running
water, the remainder crystallized, and was filtered with suction
and air-dried. Percent Yield: 84.26% (Solid). Melting point (uncor-
rected) 160.
–CH2); 2.40 (s, 3H, –CH3); 4.19 (s, 2H, –CH2); 7.38–7.62 (m, 3H, –
Ph); 8.87 (s, 2H, –CH); ESIMS m/z 324.14 [M+1]. Anal. Calcd for
C17H20N6O: C, 62.95; H, 6.21; N, 25.91. Found: C, 62.96; H, 6.23;
N, 25.92.
4.4.4.4. 3-(4H-1,2,4-Triazol-4-ylimino)-5-methyl-1-((piperazin-
1-yl) methyl) indolin-2-one (N 21). Percent Yield: 66.22% (Solid).
Melting point (uncorrected) 269 °C. FTIR (KBr): cmꢀ1 3438 (N–H
Stretch); 3058 (Ar C–H Stretch); 1698 (C@O Stretch); 1488 (C@N
Stretch); 1269 (Cyclic C–N Stretch); 1H NMR (300 MHz, DMSO, d
ppm) 1.62 (s, 1H, –NH); 2.18–2.28 (t, 4H, –CH2); 2.391–2.48 (t,
4H, –CH2); 2.80 (s, 3H, –CH3); 3.82 (s, 2H, –CH2); 7.57–7.78 (m,
3H, –Ph); 8.59 (s, 2H, –CH); ESIMS m/z 325.25 [M+1]. Anal. Calcd
for C16H19N7O: C, 59.06; H, 5.89; N, 30.13. Found: C, 59.09; H,
5.90; N, 30.14.
4.4.2. Step II—synthesis of 5-methyl Isatin28
40.24 ml of concentrated sulfuric acid (sp. gr. 1.84) was warmed
to 50 °C in a 250 ml round-bottomed flask fitted with an efficient
mechanical stirrer and to this, 10 g (0.05 mol) of dry isonitrosoace-
top-toluidine was added at such a rate as to keep the temperature
between 60 °C and 70 °C but not higher. External cooling was ap-
plied at this stage so that the reaction can be carried out more rap-
idly. After the addition of the isonitroso compound, the solution
was heated to 80 °C and this temperature was kept for about
10 min to complete the reaction. Then the reaction mixture was
cooled to room temperature and poured upon 10–12 times its vol-
ume of cracked ice. After standing for about one-half hour, the isat-
in was filtered with suction, washed several times with cold water
to remove the sulfuric acid, and then dried in the air. Recrystalline
Solvent—Glacial acetic acid, Percent Yield: 86.90% (Solid). Melting
point (uncorrected) 178 °C.
4.4.4.5. (Z)-3-(4H-1,2,4-Triazol-4-ylimino)-5-methyl-1-(methyl-
(phenyl)amino)methyl)indolin-2-one (N 43). Percent Yield:
60.16% (Solid). Melting point (uncorrected) 270 °C. FTIR (KBr):
cmꢀ1 3118 (Ar C–H Stretch); 1724 (C@O Stretch); 1520 (C@N
Stretch); 1312 (Ar C–N Stretch); 1182 (Aliphatic C–N Stretch) 1H
NMR (300 MHz, DMSO, d ppm): 2.34 (s, 3H, –CH3); 3.06 (s, 3H, –
CH3); 5.03 (s, 2H, –CH2); 6.79–7.26 (m, 5H, Ph); 7.16–8.21 (m,
3H, Ph); 8.97 (s, 2H, –CH); ESIMS m/z 346.39 [M+1]. Anal. Calcd
for C19H18N6O: C, 65.88; H, 5.24; N, 24.26. Found: C, 65.89; H,
5.24; N, 24.28.
4.4.3. Step III—synthesis of Schiff’s base10,12,19
Equimolar quantities (0.06 mol) of 5-methyl isatin (9.66 g) and
1,2,4-triazole (5.04 g) were dissolved in 75 ml of warm alcohol
containing 1 ml of glacial acetic acid. The reaction mixture was re-
fluxed for 4 hours and set aside. The resulting mixture was washed
with dilute alcohol, dried. Recrystalline Solvent—ethanol/chloro-
form mixture, Percent Yield: 77.51% (Solid). Melting point (uncor-
rected) 199 °C.
4.5. RT inhibiting assay
In a separate reaction tube, 4–6 ng recombinant HIV-1-RT di-
luted in lysis buffer (20
HIV-1-RT added was used as a negative control. 20
itors were diluted in lysis buffer and 20 l reaction mixture (solu-
ll/well) was added. Lysis buffer with no
ll of RT inhib-
4.4.4. Step IV—synthesis of Mannich base (N14, N18, N19, N20
and N43)10,12,19
l
tion 3a or 3b) per reaction tube and incubated for 1 h at 37 °C. For
the number of micro plate (MP) modules to be used, enough foil
bags were opened and MP modules were put into the frame in
the correct orientation. MP modules were ready to use and need
not be rehydrated prior to addition of the samples.
To
a slurry consisting (Z)-3-(4H-1,2,4-triazol-4-ylimino)-5-
methylindolin-2-one (0.04 mol), 50% ethanol and 37% formalin
1 ml was added to the secondary amine (0.04 mol) dropwise with
cooling and shaking. The reaction mixture was allowed to stand at
room temperature for 1 h with occasional shaking. The solid prod-
uct was separated out, filtered and recrystallised.
The samples (60 ll) were transferred into the wells of the MP
modules. MP modules were covered with a cover foil and incu-
bated for 1 h at 37 °C. The solution was removed completely and
4.4.4.1. 3-(4H-1,2,4-Triazol-4-ylimino)-1-((dimethylamino) methyl)-
rinsed five times with 250
6) for 30 s each and washing buffer was removed carefully.
200 l of anti-DIG-POD working dilution (200 mU/ml, solution
5a) were added per well, MP modules were covered with a cover
foil and incubated for 1 h at 37 °C. The solution was completely re-
ll of washing buffer per well (solution
5-methylindolin-2-one (N 14).
Percent Yield: 54.11% (Solid). Melt-
ing point (uncorrected) 250 °C. FTIR (KBr): cmꢀ1 3115 (C–H Stretch);1697
(C@O Stretch); 1492 (C@N Stretch); 1317 (Ar C–N Stretch); 1178 (Ali-
phatic C–N Stretch) 1H NMR (300 MHz, DMSO, d ppm): 2.26 (s, 6H, –
CH3); 2.34 (s, 3H, –CH3); 4.03 (s, 2H, –CH2); 7.16–8.02 (m, 3H, Ph); 8.97
(s, 2H, –CH); ESIMS m/z 285.5 [M+1]; Anal. Calcd for C14H16N6O: C,
59.14; H, 5.67; N, 29.56. Found: C, 59.16; H, 5.68; N, 29.57.
l
moved. MP modules were rinse five times with 250
buffer per well (solution 6) for 30 s each and washing buffer was
carefully removed. 200 l of ABTS substrate solution (solution 7)
ll of washing
l
was added per well and incubated at +15 to +25 °C until color
development (green color) was sufficient for photometric detec-
tion (10–30 min). Using microplate (ELISA) reader, absorbance of
the samples at 405 nm was measured (reference wavelength: ap-
prox. 490 nm).
4.4.4.2. 3-(4H-1,2,4-Triazol-4-ylimino)-5-methyl-1-(morpholi-
nomethyl) indolin-2-one (N 18). Percent Yield: 66.00% (Solid).
Melting point (uncorrected) 240 °C. FTIR (KBr): cmꢀ1 3042 (Ar C–
H Stretch); 2827 (Aliphatic C–H Stretch); 1707 (C@O Stretch);
1506 (C@N Stretch); 1059 (Cyclic C–O–C Stretch) 1H NMR
(300 MHz, DMSO, d ppm): 2.30 (s, 3H, –CH3); 2.74–2.87 (t, 4H, –
CH2); 3.21–3.38 (t, 4H, –CH2); 4.16 (s, 2H, –CH2); 7.78–8.02 (m,
3H, Ph); 8.76 (s, 2H, –CH); ESIMS m/z 326.15 [M+1]. Anal. Calcd
for C16H18N6O2: C, 58.88; H, 5.56; N, 25.75. Found: C, 58.87; H,
5.58; N, 25.76.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
4.4.4.3. 3-(4H-1,2,4-Triazol-4-ylimino)-5-methyl-1-((piperidin-
1-yl) methyl) indolin-2-one (N 19). Percent Yield: 66.66% (Solid).
Melting point (uncorrected) 252 °C. FTIR (KBr): cmꢀ1 3105 (Ar C–H
Stretch); 1683 (C@O Stretch); 144 1(C@N Stretch); 1271 (Ar C–N
Stretch); 1H NMR (300 MHz, DMSO, d ppm) 1.86–2.17 (m, 10H,
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