Vol. 32, No. 1 (2020) Synthesis and Enzyme Inhibition Activity of Organophosphorus Trichalcones Containing Core-Phosphate Moiety 61
TABLE-1
SYNTHESIS OF ORGANOPHOSPHORUS TRI-CHALCONES (5a-h)
Product
R
H
H
H
H
H
H
H
H
R1
H
H
F
OMe
H
Cl
H
H
R2
H
Cl
F
OMe
Br
H
NO2
OMe
R3
H
H
F
OMe
H
H
H
H
R4
H
H
H
H
H
H
H
H
Yield (%) (method)
80(A)
Observed m.p. (°C)
197-201
5a
5b
5c
5d
5e
5f
75(A)
82(B)
70(B)
77(A)
78(A)
87(B)
80(B)
225-228
184-189
210-213
198-205
174-176
207-212
182-188
5g
5h
d6) ppm: δ 187.8 (C=O), 155.6 (P-O-C-Ar), 143.7 (C=C), 134.6
(-C-Ar), 133.2 (C-Ar), 130.5 (C-Ar), 128.8 (C-Ar), 128.8 (C-
Ar), 122 (C-Ar), 119.9 (C=C), 119.8 (C-Ar).
Tris(4-(3-(4-Methoxyphenyl)acryloyl)phenyl)phos-phate
(5g): IR (KBr, νmax, cm-1): 3160-2900 broad (olefinic =C-H,
Ar=C-H), 1660 (conj. C=O str.), 1560 (-C=C- str.), 1340 (P=O
str.), 1080 (P-O-C- str.). 1H NMR (DMSO-d6) ppm: δ 3.81 (s,
9H, Ar-OCH3), 6.88 (d, 6H, Ar-H, J = 9 Hz), 7.01 (d, 6H, Ar-H,
J = 9 Hz), 7.65 (d, 3H, O=C-CH=), 7.74-7.84 (m, 9H Ar-H,
olefinic CH), 8.05 (d, 6H, Ar-H, J = 9 Hz). 13C NMR (DMSO-
d6) ppm: δ 187 (C=O), 161.9 (C-Ar-OMe), 156 (P-O-C-Ar),
145.1 (=C-Ar), 131.3 (C-Ar), 130.5 (C-Ar), 130.2 (C-Ar), 127.5
(C-Ar), 121.3 (C=C), 116.4 (C-Ar), 114.2 (C-Ar), 55.8 (O-CH3).
Tris(4-(3-(4-Chlorophenyl)acryloyl)phenyl)phosphate
(5b): IR (KBr, νmax, cm-1): 3135 broad (olefinic =C-H, Ar=C-
H), 1680 (conj. C=O str.), 1595 (-C=C- str.), 1331 (P=O str.),
1109 (P-O-C- str.). 1H NMR (DMSO-d6) ppm: δ 7.37 (d, 6H,
Ar-H, J = 8 Hz), 7.44 (d, 6H, Ar-H, J = 8 Hz), 7.69 (d, 3H, Ar-
CH=, J = 16 Hz), 7.78-7.85 (m, 9H, Ar-H, O=C-CH=), 8.13
(d, 6H, Ar-H, J = 8 Hz). 13C NMR (DMSO-d6) ppm: δ 187.6
(C=O), 156.2 (P-O-C-Ar), 142.1 (C=C), 135 (C-Ar), 133.6
(C-Ar), 132.8 (C-Ar), 130.5 (C-Ar), 128.9 (C-Ar), 122.7 (C-
Ar), 119.9 (C=C), 119.8 (C-Ar).
Tris(4-(3-(3,4,5-fluoro-phenyl)acryloyl)phenyl)phos-
phate (5c): IR (KBr, νmax, cm-1): 3160 broad (olefinic =C-H,
Ar=C-H), 1670 (conj. C=O str.), 1600 (-C=C- str.), 1340 (P=O
str.), 1130 (P-O-C- str.). 1H NMR (DMSO-d6) ppm: δ 6.88-
6.91 (m, 12H, Ar-H), 7.55-7.63 (m, 3H, O=C-CH=), 7.76 (d,
6H , Ar-CH=, J = 8 Hz), 7.91 (d, 6H, Ar-H, J = 8 Hz), 7.95-8.10
(m, 6H, Ar-H).
Biological activity
Amylase activity determination: α-Amylase activity was
determined according to the method as described by Ishimoto
et al. [40]. The reaction mixture contain purified insect amylase
solution (0.02 mL) in a test tube with 1 % starch solution (1 mL)
and 0.02 M phosphate buffer (1 mL). This mixture was incu-
bated at 40 ºC for 10 min. To a mixture, 3,5-dinitrosalicylic
acid (DNSA) reagent (1mL) was added and incubated in a boiling
water bath for 5 min. The contents were cooled and diluted
with volume (3 mL) by distilled water. Absorbance was read
at 540 nm.All the spectrophotometric measurements were per-
formed on a UV-visible recording spectrophotometer (Shimadzu,
Model-UV-1800). One unit (U/mL) of α-amylase activity is
defined as the amount of protein (α-amylase) required for liber-
ating 1 µmol of reducing sugar (maltose) from starch/min under
assay conditions.
α-Amylase inhibitor assay: α-Amylase inhibitory activity
was determined according to the method described in the litera-
ture [39]. With some modifications, 20 µL of enzyme (Tribolium
castaneum amylase) and the synthetic compounds 100 µL were
mixed and incubated for 30 min at room temperature followed
by addition of 1 mL of 0.02 M sodium phosphate buffer
and1 mL of 1 % starch solution in all the test tubes. After 10
min the reaction was terminated with addition of 1 mL of
DNSA colour reagent followed by boiling in water bath for 5
min. The mixture was cooled to room temperature and diluted
with 3 mL of distilled water and the absorbance measured at
540 nm (Shimadzu-UV-VIS Spectrophotometer). The control
samples were also prepared accordingly without any synthetic
compound and compared with the test samples. The results
were expressed as % inhibition calculated using the formula:
Tris(4-(3-(3,4,5-OMe-phenyl)acryloyl)phenyl)phos-
phate (5d): IR (KBr, νmax, cm-1): 3180-2900 broad (olefinic
=C-H,Ar=C-H), 1660 (conj. C=O str.), 1597 (-C=C- str.), 1320
1
(P=O str.), 1125 (P-O-C str.). H NMR (DMSO-d6) ppm: δ
3.70 (s, 9H, Ar-OMe), 3.86 (s, 18H, Ar-OMe), 6.89-6.92 (d,
6H, Ar-H, J = 12 Hz), 7.20 (s, 6H, Ar-H), 7.60-7.65 (d, 3H,
O=C-CH=, J = 20 Hz), 7.83-7.88 (d, 3H, Ar-CH=, J = 20 Hz),
8.06-8.09 (d, 6H, Ar-H, J = 12Hz).
Tris(4-(3-(4-Bromophenyl)acryloyl)phenyl)phosphate
(5e): IR (KBr, νmax, cm-1): 3135 broad (olefinic =C-H, Ar=C-
H), 1670 (conj. C=O str.), 1596 (-C=C- str.), 1331 (P=O str.),
1
1109 (P-O-C- str.); H NMR (DMSO-d6) ppm: δ 6.88 (d, 6H,
Ar-H, J = 8 Hz), 7.33-8.12 (m, 24H, Ar-H, Ar-CH=, O=C-
CH=).
Tris(4-(3-(3-Chlorophenyl)acryloyl)phenyl)phosphate
(5f): IR (KBr, νmax, cm-1): 3150 (olefinic C-H, HC=Ar), 1665
(conj. C=O str.), 1560 (-C=C- str.), 1350 (P=O str.), 1160 (P-O-
C- str.); 1H NMR (DMSO-d6) ppm: δ 6.89 (d, 6H, Ar-H, J = 9
Hz), 7.36-7.48 (m, 6H, Ar-H), 7.65 (d, 3H, O=C-CH=, J = 12
Hz), 7.78-7.81 (m, 3H, olefinic C-H), 7.97-8.11 (m 12H, Ar-
CH).
Tris(4-(3-(4-Nitrophenyl)acryloyl)phenyl)phosphate
(5h): IR (KBr, cm-1): 3160 broad (olefinic =C-H, Ar=C-H),
1670 (conj. C=O str.), 1336 (P=O str.), 1105 (P-O-C- str.), 1590
(-C=C- str.). 1H NMR (DMSO-d6) ppm: δ 6.91 (d, 6H, Ar-H,
J = 9 Hz), 7.75 (d, 3H, Ar-CH=, J = 18 Hz), 8.08-8.16 (m, 15H,
Ar-H, O=C-CH=), 8.27 (d, 9H, Ar-H, J = 9 Hz).
Abs (control) − Abs (compound)
Inhibition activity (%) =
×100
Abs (control)
Antimicrobial activity: Antimicrobial activities of the
synthesized trichalcones (5a-h) were determined by disc diff-
usion method. The compounds were evaluated for antibacterial