154
K. Lahbib et al. / Journal of Molecular Structure 1091 (2015) 152–158
Fig. 3. Synthesis of 3-keto-1,5-bisphosphonates 3.
Thus, the reaction of bis-Mannich base hydrochlorides 2 with an
excess of triethyl phosphite (5 equivalents), performed under
reflux, for 6 h, in solvent-free conditions, led to 3-keto-1,5-bisphos-
phonates 3.
Antioxidant, pro-oxidant and chelating investigation
Compounds 1–3 were tested for their in vitro antioxidant
activities by DPPH, H2O2, hydroxyl radical and Ferric Reducing
Power (FRP) methods. The ratio of pro-oxidant to the antioxidant
activity (ProAntidex) was also examined and allowed us to evalu-
ate the net antioxidant potential of these compounds. Ferrous, cal-
cium and magnesium ion chelating abilities were also evaluated.
All the tests, for the determination of antioxidant activity, were
carried out in triplicate and the results averaged. Absorbance was
recorded using an UV–Vis spectrophotometer (Jinway UV–Vis
6405).
General procedure for the synthesis of compounds 3. A mixture of
bis-Mannich base hydrochloride 2 (0.02 mol) and triethyl phos-
phite (0.1 mol) was heated under reflux for 6 h. After cooling, the
excess of triethyl phosphite was removed under reduced pressure,
then CHCl3 (100 mL) was added. The organic phase was washed
with water (2 ꢁ 50 mL), dried over Na2SO4 and concentrated under
vacuum. The obtained residue was chromatographed on a silica gel
column using a mixture of ether and hexane (3:1) as eluent.
IC50 values were calculated by linear regression. Means SD
were calculated. The data were analyzed for statistical significance
using one-way ANOVA followed by Tukey post test.
Spectral data for the synthesized compounds
The structures of compounds 1, 2 and 3 were confirmed by
infrared (IR) and nuclear magnetic resonance (NMR) spectro-
scopies. 1H, 31P, and 13C NMR spectra were recorded with CDCl3
as the solvent for compounds 3 or in a mixture of CDCl3 and
DMSO-d6 for Mannich bases 1 and 2, on a Bruker AC-300 spec-
trometer operating at 300.1 MHz for 1H, 121.5 MHz for 31P and
75.5 MHz for 13C. The chemical shifts are reported in ppm relative
to TMS (internal reference) for 1H and 13C NMR and relative to 85%
H3PO4 (external reference) for 31P NMR. The coupling constants are
reported in Hz. For the 1H NMR, the multiplicities of signals are
indicated by the following abbreviation: s: singulet; d: doublet;
t: triplet; q: quartet; quint: quintet; m: multiplet. IR spectra were
DPPH radical scavenging activity
The most common methods to determine antioxidant activity in
a practical, rapid and accurate manner are those that involve a
radical chromophore, simulating the reactive oxygen species
(ROS), and the free radical DPPH, of purple coloration that absorbs
at 517 nm, is one of the most widely used radical chromophores for
in vitro evaluation of antioxidant activity.
The DPPH radical scavenging assay employed here is as
described by Braca et al. [21]. The reduction capability of DPPH
radical is determined by the decrease in absorbance at 517 nm
induced by antioxidants. Ascorbic acid (AA) is the reagent used
as standard. The sample is able to reduce the stable radical DPPH
to the yellow-colored diphenylpicrylhydrazine. Experimentally,
various dilutions of the methanolic solution of compounds 1–3
or standard (0.003–0.3 mg/mL, in triplicate) were added to DPPH
solution (0.035 mg/mL). The absorbance of the mixture was taken
at 517 nm with methanol as blank. A control sample with no added
test compounds was also analyzed. Radical scavenging activity was
expressed as a percentage and calculated using the formula: %
Scavenging = [(Acont ꢂ Atest)/Acont] ꢁ 100, where Acont is the absor-
bance of the control, and Atest is the absorbance of the sample in
the presence of test compound 1–3. The result was presented as
IC50 (the concentration of test compound required for scavenging
50% of the DPPH radical).
The results of these experiments are summarized in (Fig. 4). It
was found that bis-Mannich bases 2 have the highest DPPH radical
scavenging activity with an IC50 at 1.73 mg/mL for 2b followed by
2a with an IC50 at 2.52 mg/mL when compared with other
compounds. The remaining compounds exhibited DPPH radical
scavenging activity in the following order: 1a (IC50: 3.83 mg/mL),
3a (IC50: 5.73 mg/mL), 3b (IC50: 7.03 mg/mL), 1b (IC50: 9.09
mg/mL), and compared with ascorbic acid (IC50: 0.0396 mg/mL).
recorded on
a Nicolet IR200 spectrometer (Thermo Electron
Scientific Instruments LLC, Madison, WI, USA).
Spectral data for compounds 1 (a,b) and 3 (a,b) were mentioned
in our previous articles [19,20].
Spectral data for compounds 2 (a,b) are as follows:
2a: White solid; M = 271.23 g molꢂ1
;
mp = 180–182 °C;
yield = 79%; IR (neat):
m
c@o = 1740 cmꢂ1
;
m
NH = 3448 cmꢂ1 1H
;
NMR: d = 1.75–2.48 (m, 4H, CH2ACH2); 2.91 (s, 3H,
CH3ANACH3); 2.93 (s, 3H, CH3ANACH3); 3.07–3.72 (m, 3H, CH
and CH2AN); 9.57 (br s, 1H, NAH); 13C NMR: d = 10.1 (s,
CH2ACH2); 20.2 (s, CH2ACH2); 34.6 (s, CH3ANACH3); 36.8 (s,
CH3ANACH3); 44.6 (s, CHACH2); 58.0 (s, CH2AN); 216.2 (s, C@O).
2b: White solid; M = 285.14 g molꢂ1
;
;
mp = 175–176 °C;
yield = 77%; IR (neat):
m
c@o = 1728 cmꢂ1
m
NH = 3450 cmꢂ1
;
1HNMR: d = 1.18–2.86 (m, 6H, (CH2)3); 2.76 (s, 3H, CH3ANACH3);
2.78 (s, 3H, CH3ANACH3); 2.94–3.66 (m, 3H, CH and CH2AN);
10.70 (br s, 1H, NAH); 13CNMR: d = 22.3 (s, CH2ACH2ACH2); 27.2
(s, CH2ACH2ACH2); 32.6 (s, CH2ACH2ACH2); 40.4 (s,
CH3ANACH3); 41.2 (s, CH3ANACH3); 49.4 (s, CHACH2); 56.4 (s,
CH2AN); 209.1 (s, C@O).