S. A. Dake et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2527–2532
2531
for 20 min. All the plates incubated at respective growth tempera-
ture. The standard antibiotic discs of Ampicillin directly placed on
inoculated agar plates and incubated which would be served as po-
sitive antibacterial control. Negative control was prepared using
m
= 3356.07, 2928.70, 1641.98, 1407.37, 1251.16, 1017.92, 818.37,
447.09 cmÀ1 1H NMR (200 MHz, CDCl3) d ppm: 1.26–1.33 (t,
;
J = 8.0 Hz, 6H), 4.06–4.19 (q, J = 5.9 Hz, 4H), 3.5–3.9 (d, 1H), 4.88–
5.04 (d, 1H), 7.2–7.3 (d, 1H), 7.2 (dd, 2H), 8.23 (dd, 2H), 7.59 (s,
1H), 7.80 (s, 1H); 13C NMR (50.32 MHz, CDCl3) d ppm: 136.40,
126.43, 115.61, 56.68, 16.41.
Diethyl(4-chloro-2-nitrophenylamino)(4-chlorophenyl)methyl-
phosphonate: Table 1, entry 4d. M.F. C17H19Cl2N2O5P; FT-IR (KBr):
sterile disc loaded with 10 ll of DMSO. After 24 h of incubation
cultured bacteria with zone of inhibition equal to or greater than
7 mm were considered susceptible to compounds.19
Antiproliferative activity of 4j:
m
= 3357.09, 2975.35, 1617.46, 1408.34, 1252.37, 1056.30, 781.32,
H
656.07 cmÀ1 1H NMR (400 MHz, CDCl3) d ppm: 1.24–1.33 (t,
;
N
NO2
J = 8.0 Hz, 6H), 3.5–3.9 (d, 1H), 4.00–4.14 (q, J = 7.9 Hz, 4H), 4.77–
4.92 (d, 1H), 8.20 (dd, 2H), 6.53–6.58 (dd, 2H), 7.24–7.38 (m,
3H); 13C NMR (50.32 MHz, CDCl3) d ppm: 136.23, 128.90, 115.96,
77.03, 16.44; MS (EI, 70 eV): [m+Na]+ = 456.15, [m+H]+ = 434.14.
31P NMR (300 MHz; CDCl3) d ppm: 17.48.
H
C
P
O
H
N
Cl
OEt
EtO
4j
Diethyl(4-chloro-2-nitrophenylamino)(2-hydroxyphenyl) methyl-
phosphonate: Table 1, entry 4e. M.F. C17H20ClN2O6P; FT-IR (KBr):
We used all the compounds for the in vitro antiproliferative assay.
But unfortunately, only 4j from Table 1 was found antiproliferative
in nature against the melanoma cells. The other compounds
showed zero/negligible antiproliferative activity (results not dis-
cussed here). Therefore we used 4j for antiproliferative assay.
The viability assay and time dependent assays were performed
for the study.
A-549 and SK-MEL2 cell lines: A549 and SK-MEL2 cell lines
were obtained from the National Centre for Cell Science, NCCS
Pune-411007, India and seeded at a density of 104–105 cells/well
on a 12-well plate Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5–10% FBS, 100 U/ml penicillin (or 1% penicil-
m
= 3357.36, 2928.14, 1614.99, 1407.09, 1252.11, 1021.21, 816.36,
783.61, 563.25 cmÀ1 1H NMR (200 MHz, CDCl3) d ppm: 1.33–
;
1.40 (t, J = 8.0 Hz, 6H), 3.5–3.9 (d, 1H), 4.13–4.29 (q, J = 5.9 Hz,
4H), 5.40–5.56 (d, 1H), 6.66–6.85 (m, 3H), 7.01–7.27 (m, 3H),
8.16 (s, 1H), 8.93 (br s, 1H); 13C NMR (50.32 MHz, CDCl3) d ppm:
155.22, 136.42, 125.87, 116.22, 77.08, 16.43; 31P NMR (500 MHz;
CDCl3) d ppm: 21.56.
Spectral data for Table 2: Diethyl(4-iodo-2-nitrophenylamino)(4-
hydroxyphenyl) methylphosphonate: Table 2, entry 4a. M.F.
C
17H20IN2O6P; FT-IR (KBr):
m
= 3353.67, 3100.00, 2955.00,
1608.64, 1247.69, 1018.13, 555.95 cmÀ1
;
1H NMR (400 MHz,
CDCl3) d ppm: 1.06–1.09 (t, J = 8.0 Hz, 6H), 4.07–4.10 (q, J = 8.0 Hz,
4H), 4.12 (dd, 1H), 4.02 (dd, 1H), 5.91 (dd, J = 8.0 Hz, 3H), 7.10–
7.27 (m, 3H), 8.2 (s, 1H), 5.0 (br s, 1H); 13C NMR (50.32 MHz, CDCl3)
lin–streptomycin) and 4 mM -glutamine, at 37 °C in a humidified
L
5% CO2-containing atmosphere. The plates, seeded 24 h before pro-
ceed to experiment with 10,000–100,000 cells per well, were
washed 2–3 times with serum-free medium.
Viability assay: Cells were seeded at 30,000 cells/well in 12-well
plates, 0, 2, 4, 8, 10, 15 and 20 lM 4j was added to wells separately
d
ppm: 164.71, 129.70, 127.77, 125.39, 60.61, 13.70; MS (EI, 70 eV):
[m+Na]+ = 529.48, [m+H]+ = 506.00. 31P NMR (300 MHz; CDCl3) d
ppm: 18.37.
Diethyl(4-iodo-2-nitrophenylamino)(3-nitrophenyl)methylphos-
phonate: Table 2, entry 4b. M.F.C17H19IN3O7P; FT-IR (KBr):
and maintained at 37 °C in an atmosphere of 5% CO2 and 95% rel-
ative humidity in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% foetal bovine serum, 1% non-essential
amino acids. Following 24 h incubation at 37 °C viability was
checked with trypan blue method.
Time dependence and sensitivity assay: The cells were seeded at
30,000 cells/well in 12-well plates. The fixed concentration of
20 lM was added to each wells and incubated for 48 h. After an
m
= 3377.54, 2925.00, 1610.53, 1553.29, 1493.22, 1231.56, 1080,
860.12, 808.93, 693.00, 519.11 cmÀ1 1H NMR (500 MHz, CDCl3) d
;
ppm: 1.37–1.41 (t, J = 8.0 Hz, 6H), 4.21–4.29 (q, J = 7.9 Hz, 4H),
5.49–5.55 (dd, 1H), 7.06 (d, 1H), 7.21–7.24 (dd, J = 5.0 Hz, 2H),
8.18 (s, 1H), 6.71–6.73 (dd, 2H), 6.83 (s, 1H), 8.97 (br s, 1H); 13C
NMR (50.32 MHz, CDCl3) d ppm: 155.28, 142.57, 136.33, 133.05,
129.71, 127.23, 125.85, 121.51, 120.35, 116.21, 77.05, 63.90,
16.34; MS (EI, 70 eV): [m+K]+ = 553.81, m+ = 535.23. 31P NMR
(300 MHz; CDCl3) d ppm: 16.50.
interval of 6 h, the % survival of each well was calculated.
Spectral data of selected compounds: Diethyl(4-chloro-2-nitro-
phenylamino)(4-hydroxy phenyl) methylphosphonate: Table 1, entry
Diethyl(4-iodo-2-nitrophenylamino)(4-hydroxy-3-methoxy-
phenyl)methylphosphonate: Table 2, entry 4c. M.F.C18H22IN2O7P;
4a. M.F. C17H20ClN2O6P; FT-IR (KBr):
m
= 3355.88, 2984.04, 1613,
1467.86, 1343.89, 1245.24, 1052.65, 747.86 cmÀ1
;
1H NMR
FT-IR (KBr):
m
= 3475.84, 3357.36, 2930.00, 1614.99, 1504.02,
(400 MHz, CDCl3) d ppm: 1.25–1.27 (t, J = 8.0 Hz, 3H), 1.33–1.31 (t,
J = 3.9 Hz, 3H), 4.01 (d, 1H), 4.06–4.15 (q, J = 8.0 Hz, 4H), 4.80–4.88
(d, J = 7.9 Hz, 1H), 6.67–6.72 (dd, J = 8.0 Hz, 3H), 7.20–7.28 (m, 3H),
8.17 (s, 1H), 8.88 (br s, 1H); 13C NMR (50.32 MHz, CDCl3) d ppm:
157.36, 142.65, 136.22, 133.06, 128.64, 125.95, 123.86, 121.60,
116.37, 77.41, 77.08, 16.34; MS (EI, 70 eV): [m+Na]+ = 437.26,
m+ = 414; 31P NMR (81.01 MHz; CDCl3) d ppm: 20.21.
1458.13, 1252.11, 1021.21, 816.86, 763.61 cmÀ1
;
1H NMR
(200 MHz, CDCl3) d ppm: 1.21–1.31 (t, J = 8.0 Hz, 6H), 3.87 (s,
3H), 3.91–4.12 (q, J = 3.9 Hz, 4H), 3.9 (dd, 1H), 4.80 (dd, 1H), 6.66
(s, 1H), 6.78 (dd, 1H), 6.40 (dd, 1H), 8.17 (s, 1H), 7.21 (dd, 1H),
6.54 (dd, 1H), 5.03 (s, 1H); 13C NMR (50.32 MHz, CDCl3) d ppm:
155.22, 142.66, 136.42, 125.87, 121.53, 116.22, 77.08, 64.53,
16.43; 31P NMR (300 MHz; CDCl3) d ppm: 18.31.
Diethyl(4-chloro-2-nitrophenylamino)(4-hydroxy-3-methoxy-
phenyl)methylphosphonate: Table 1, entry 4b. M.F. C18H22ClN2O7P;
Diethyl(4-iodo-2-nitrophenylamino)(2-hydroxyphenyl)
phosphonate: Table 2, entry 4d. M.F. C17H20IN2O6P; FT-IR (KBr):
= 3195.41, 2928.9, 1606.88, 1460.58, 1258.65, 1025.30, 761.77,
561.07 cmÀ1 1H NMR (400 MHz, CDCl3) d ppm: 1.27–1.3 (t,
methyl-
FT-IR (KBr):
m
= 3475.84, 3357.36, 2930.00, 1614.99, 1504.02,
1458.13, 1252.11, 1021.21, 775.25 cmÀ1
;
1H NMR (200 MHz,
m
;
CDCl3) d ppm: 1.21–1.31 (t, J = 8.0 Hz, 6H), 3.87 (s, 3H), 3.91–4.12
(q, J = 4.0 Hz, 4H), 3.9 (d, 1H), 4.80 (d, 1H), 6.57 (s, 1H), 6.67 (dd,
1H), 6.45 (dd, 1H), 7.15 (s, 1H), 7.21 (dd, 1H), 6.65 (dd, 1H), 5.09
(s, 1H); 13C NMR (50.32 MHz, CDCl3) d ppm: 155.22, 142.66,
136.42, 127.25, 116.22, 82.5, 77.08, 64.53.
J = 8.0 Hz, 6H), 4.19–4.23 (q, J = 7.9 Hz, 4H), 4.10 (dd, 1H), 5.3 (dd,
1H), 6.46 (d, 1H), 7.2 (m, 3H), 7.55 (dd, 1H), 8.5 (s, 1H), 8.80 (m,
1H); 31P NMR (500 MHz; CDCl3) d ppm: 21.08.
Diethyl(4-iodo-2-nitrophenylamino)(4-chlorophenyl)
phosphonate: Table 2, entry 4e. M.F. C17H19ClIN2O5P; FT-IR (KBr):
= 3376.9 2927.5, 1609.62, 1494.60, 1295.29, 1249.10, 1064.80,
methyl-
Diethyl(4-chloro-2-nitrophenylamino)(3-nitrophenyl)methylphos-
phonate: Table 1, entry 4c. M.F. C17H19ClN3O7P; FT-IR (KBr):
m