Synthesis of α-hydroxyphosphonates and their antioxidant properties

Article abstract of DOI:10.1002/ardp.201200192

A series of α-hydroxyphosphonates were synthesized from the reaction of aldehyde (1) with triethylphosphite (2) in the presence of oxone and evaluated for their antioxidant properties against lipid peroxidation, reduced glutathione, superoxide dismutase,

Full text of DOI:10.1002/ardp.201200192

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
1
Full Paper
Synthesis of a-Hydroxyphosphonates and Their Antioxidant
Properties
Kalla Reddi Mohan Naidu1, Krishnammagari Suresh Kumar2, Palanisamy Arulselvan3,
Chinnappareddy Bhupendra Reddy4, and Ola Lasekan1
1 Department of Food Technology, Universiti Putra Malaysia, UPM 43400, Serdang, Malaysia
2 Department of Chemistry, Sri Venkateswara University, Tirupati, India
3 Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, UPM
43400, Serdang, Selangor, Malaysia
4 Department of Polymer Science and Engineering, Dankook University, South Korea
A series of a-hydroxyphosphonates were synthesized from the reaction of aldehyde (1) with
triethylphosphite (2) in the presence of oxone and evaluated for their antioxidant properties
against lipid peroxidation, reduced glutathione, superoxide dismutase, and catalase. The majority
of the compounds showed promising antioxidant activity. Diethyl anthracen-9-yl (hydroxy)
methylphosphonate (3n) is the most potent and biologically active compound against free radicals.
Keywords: Abramov reaction / Antioxidant activity / a-Hydroxyphosphonates / Oxone
Received: May 7, 2012; Revised: July 27, 2012; Accepted: July 31, 2012
DOI 10.1002/ardp.201200192
Introduction
an understanding of the importance of free radicals in caus-
ing cellular pathologies, and the role of antioxidants in
preventing this, shows that an imbalance of ROS and anti-
oxidant defense systems may lead to chemical modifications
of biologically relevant macromolecules.
Free radicals are fundamental to any biochemical process
and represent an important part of aerobic life and metab-
olism. Free radicals and their related oxygen species have
attracted a great deal of attention in recent years [1]. The free
radicals are mainly derived from reactive oxygen and nitro-
gen species (ROS and RNS), which are continuously produced
in the living system via various enzymatic and non-enzymatic
cross-reactions like the respiratory chain reaction, phago-
cytosis, prostaglandin synthesis, the cytochrome P450 system,
and oxidative phosphorylation in the mitochondria [2–4].
Oxygen free radicals are detrimental to the integrity of
important tissues and mediate their toxicity/injury. The
mechanism of injury/damage involves lipid peroxidation
(LPO), which destroys the structural integrity of the cells,
lipids, proteins, and nucleic acids [5, 6].
Antioxidants are substances that neutralize ROS/free
radicals or their adverse actions [8]. Specifically enzymatic
antioxidants and non-enzymatic antioxidant defense systems
minimize the harmful effects of ROS by various antioxidant
systems. Nature has endowed us with protective antioxidant
systems: superoxide dismutase (SOD), catalase (CAT), gluta-
thione, glutathione peroxidase, and reductase, vitamin E,
vitamin C, apart from many synthetic and dietary com-
ponents. There is epidemiological evidence correlating a
higher intake of dietary components/foods with the ability
of antioxidants to lower the incidence of various human
morbidities and/or mortalities [8]. Based on this information,
we aimed to evaluate the effects of synthesized title com-
pounds on NADPH-dependent LPO and the associated
mechanism.
Lipids, when reacted with free radicals, can undergo the
highly damaging chain reaction of LPO, leading to both
direct and indirect effects. The damage caused by LPO is
highly detrimental to the functioning of the cell [7].
Consideration of the basic biomedical science that underlies
It is well known that a-hydroxyphosphonates are struc-
tural analogs of a-hydroxyphosphonic acids [9, 10]. a-
Hydroxyphosphonates act as wide-range enzyme inhibitors
for farnesyl protein transferase (FPT) [11], human renin
[12, 13], human protein tyrosine phosphatase (PTP)
[14–16], purine nucleoside phosphorylase (PNP) [17], and
Correspondence: Kalla Reddi Mohan Naidu, Department of Food
Technology, Universiti Putra Malaysia, UPM 43400, Serdang, Malaysia
E-mail: drrmnaidu@gmail.com
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2
K. R. M. Naidu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
5-enolpyruvylshikimate-3-phosphate synthase (EPSP) [18].
They also showed anticancer activity against various human
cancer cell lines [19, 20] and inhibition of the human immu-
nodeficiency virus (HIV) [21].
O
P
O
Oxone
P
OEt
R
+
OEt
OEt
OEt
EtO
Methanol,
R
H
_
rt, 2 10 min
OH
_
_
1a n
2
3a n
Various methods based on the reaction of aldehydes or
ketones with dialkyl phosphites in the presence of various
bases [22, 23] and acids [24] have been used to synthesize a-
hydroxyphosphonates (Pudovik reaction). However, there are
few reports describing the reaction of trialkyl phosphite with
aldehydes or ketones in the presence of acid catalysts such as
TMSCl [25], HCl.Et2O [26], LiClO4.Et2O and TMSCl [27]; guani-
dine hydrochloride [28] has also been tried (Abramov reac-
tion). But none of them were found to be satisfactory. Hence,
the search continues for a better catalyst for the synthesis of
a-hydroxyphosphonates in terms of operational simplicity
and economic viability. Herein, we report the use of oxone as
water-soluble protic acid (HSO4ꢀ) that meets the demands for
an economic catalyst. This is the first finding of utilizing
oxone as a catalyst for the synthesis of a-hydroxyphospho-
nates. Oxone (2KHSO5.KHSO4.K2SO4) is a commercially avail-
able reagent and has been used for many organic synthetic
transformations [29, 30]. Recently, we have reported on the
synthesis, characterization, and antioxidant properties of
some substituted-1,3,2-diazaphosphole 1-oxides containing
various substituted phenols and various amino acid esters
at the phosphorus atom [31].
Compound
R
Compound
R
OMe
OMe
CH3
CH3
3a
3h
Cl
3b
3c
3i
3j
Cl
CH3
CH3
N
3k
3l
3d
CH3
NO2
N
3e
3f
O2N
3m
3n
O
NO2
3g
OMe
In this present investigation, we synthesized a-hydroxy-
phosphonates and evaluated their possible antioxidant prop-
erties compared to the known natural antioxidant vitamin C.
Scheme 1. Oxone-catalyzed synthesis of the a-hydroxyphos-
phonates 3a–n.
Results and discussion
Till date, this is the first feasible application of the Abramov
reaction for the synthesis of a-hydroxyphosphonates (3a–n)
from various aromatic and heteroaromatic aldehydes (1a–n)
and triethylphosphite (2) in the presence of oxone in meth-
anol (Scheme 1). This is a highly advantageous method as it
requires simple and mild experimental conditions and less
reaction time, and is cost effective and applicable to various
substituted aryl, heterocyclic substrates.
Table 1. Conversion of aldehydes to the a-hydroxyphosphonates
3a–n
Entry Product
Time
(min)
Yield
(%)
Melting point (8C)
Found
Literature
The chemical structures of the synthesized compounds are
shown in Table 1. It was found that electron-withdrawing
aromatic aldehydes proved to be more reactive and to afford
high product yields. This effect is more pronounced in case of
compounds substituted at the para position with more aro-
matic rings (Table 1; 3l, 3m). Even ortho-substituted com-
pounds (3b, 3e) with electron-withdrawing moieties also
experience this effect due to hydrogen bonding with the
aldehydic hydrogen located at the ortho position. These moi-
eties, as in the carbonyl compounds, contributed to higher
electrophilicity due to extending the delocalization of the
electron cloud over the sp2 carbon cyclic frame with/without
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
6
10
8
8
7
7
6
4
7
5
5
2
2
84
91
90
91
93
94
83
85
82
84
90
94
96
95
74–75
75–77
67–68
80–81
114–116
86–87
121–122
96–97
95–96
Liquid
81–82
75–76 [40, 41]
74–75 [40, 41]
67–68 [40, 41]
81–82 [40]
114–116 [40]
87–88 [40–42]
120–121 [40, 41]
95–97 [43]
94–95 [40, 41]
–
9
10
11
12
13
14
80–81 [40]
–
–
149–151 [44]
132–133
65–66
150–151
4
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Antioxidant Properties of a-Hydroxyphosphonates
3
heteroatom and substitutions facilitating nucleophilic phos-
phite addition to the carbonyl compounds. The heteroaro-
matic aldehyde also proved to be more reactive and obtained
high yields.
IR, 1H, 13C, 31P and mass spectrometry and elemental
analyses of compounds 3a–n confirmed the proposed struc-
tures. The IR spectra of 3a–n showed the expected absorption
bands at 3280–3271, 1249–1238, and 1048–1040 cmꢀ1 for
1
–
the –OH, P O and P–O–C stretching vibrations. In the H NMR
–
spectra, compounds 3a–n gave a multiplet in the range of d
8.66–6.59 for aromatic protons and a singlet at d 5.29–5.11 for
the –OH proton. The 31P chemical shifts were observed as
Figure 3. Antioxidant nature of structural analogs of a-hydroxyphos-
phonic acids (3a–n) against SOD activity.
–
singlet at d 24.20–20.80 corresponding to P O for all the
–
compounds 3a–n.
Antioxidant properties of a-hydroxyphosphonates
(3a–n)
As depicted in Figs. 1–4, most of our designed compounds
exhibited good antioxidant activities against LPO, GSH, SOD,
Figure 4. Antioxidant nature of structural analogs of a-hydroxyphos-
phonic acids (3a–n) against CAT activity.
and CAT. Among all these analogues, 3n showed the best
biological activity, which is comparable to that of the known
natural antioxidant vitamin C. However, in most cases, the
unsubstituted benzaldehyde had no distinct influence on
their antioxidant activities. In addition, the introduction
of electron-withdrawing groups (3b–f) resulted in higher
antioxidant potential than the corresponding analogous
electron-donating groups (3g–3k). Compounds substituted
with benzyloxy, 2-pyridyl (3l, 3m) showed even lower inhibi-
tory efficiency on the oxidative proxy, which is possibly due,
in part, to the relatively rigid structure of this substituent.
LPO is part of the oxidative degradation of polyunsaturated
fatty acids and involves the formation of various lipid
radicals, leading to cell membrane damage. Free radicals
induce LPO in polyunsaturated lipid-rich vital organs, especi-
ally brain and liver [32]. Peroxidation of lipid molcules is a
natural phenomenon and takes place on their exposure to
oxygen. Recently, free radical-induced LPO has gained more
importance because of its contribution towards several path-
ologies, such as wound healing, oxygen toxicity, liver dis-
orders, and inflammation [33]. Various researchers have
Figure 1. Antioxidant nature of structural analogs of a-hydroxyphos-
phonic acids (3a–n) against LPO production.
Figure 2. Antioxidant nature of structural analogs of a-hydroxyphos-
phonic acids (3a–n) against GSH levels.
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4
K. R. M. Naidu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
shown that numerous natural and synthetic antioxidants
can be used to prevent LPO and its associated complexes
[33]. In the present study, the synthesized a-hydroxyphospho-
nates were evaluated for their antioxidant nature.
General synthetic procedure for the title compounds (3a–n)
To a mixture of aldehyde (0.001 mol) and triethylphosphite
(0.0013 mol) in methanol (5 mL), oxone (0.05 mmol) was added.
The mixture was stirred at room temperature for the appropriate
time (see Table 1) until TLC showed that the reaction was com-
plete. The insoluble oxone was then filtered; water (15 mL) was
added to the filtrate to quench the reaction and extracted with
ethyl acetate (2–10 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered and then evaporated. The crude
product was purified by silica gel flash column chromatography
and eluted with an ethyl acetate/hexane mixture to afford the
a-hydroxyphosphonates.
Natural or synthetic antioxidants are essential to endoge-
nous defense systems against injury triggered by LPO and
harmful reactions induced by free radicals (ROS), which are
frequently produced in the body during normal metabolic
processes [34]. The depletion levels of thiobarbituric acid-
reactive substances (TBARS) and increased activities of anti-
oxidant molcules in the tissue homogenates treated with
the synthesized a-hydroxyphosphonates might be due to
decreased LPO end-products and/or an improvement of the
antioxidant defense system.
Diethyl (hydroxy) (phenyl) methylphosphonate (3a)
Yield 84%; m.p. 74–758C; IR (KBr) ymax (cmꢀ1): 3274 (brs, OH),
1240 (P¼O), 1043 (P–O–C); 1H NMR (CDCl3) d (ppm): 7.36–7.18
(m, 5H, Ar–H), 5.25 (s, 1H, –OH), 4.64 (d, 1H, 2JP-H ¼ 10.7 Hz, –CH),
4.00–3.90 (m, 4H, O–CH2–CH3), 1.26–1.17 (m, 6H, O–CH2–CH3);
Conclusion
2
13C NMR (CDCl3) d (ppm): 147.5 (d, JP-C ¼ 3.0 Hz), 134.0, 12.8,
1
2
127.1, 70.3 (d, JP-C ¼ 157.0 Hz), 62.9 (d, JP-C ¼ 7.1 Hz), 16.0
(d, JP-C ¼ 6.0 Hz); 31P NMR data: d 22.82; ESI-MS: m/z 244
3
An efficient synthesis of a-hydroxyphosphonates was
achieved by a one-pot, two-component reaction between sub-
stituted aromatic aldehydes and triethylphosphite in the
presence of oxone. This procedure is simple, inexpensive,
and effective, making it an attractive alternative protocol
over the existing procedures for the synthesis of a-hydroxy-
phosphonates. Some of the new a-hydroxyphosphonates
exhibited promising antioxidant activity. Based on the anti-
oxidant properties of the a-hydroxyphosphonates, further
studies will be conducted to determine the structure–activity
relationships.
(MþH)þ; Anal. calcd. for C11H17O4P: C, 54.10; H, 7.02. Found:
C, 53.98; H, 7.00.
Diethyl (2-chlorophenyl) (hydroxy) methylphosphonate
(3b)
Yield 85%; m.p. 75–778C; 1H NMR (CDCl3) d (ppm): 7.46–7.21
2
(m, 4H, Ar–H), 5.17 (s, 1H, –OH), 4.89 (d, JP-H ¼ 10.6 Hz,
1H, -CH), 4.32–4.10 (m, 4H, O–CH2–CH3), 1.38–1.29 (m, 6H,
(ppm): 149.4, 138.3,
O–CH2–CH3); 13C NMR (CDCl3)
d
1
128.1, 127.5, 126.3, 126.4, 70.4 (d, JP-C ¼ 151.0 Hz), 64.4
2
2
3
(d, JP-C ¼ 6.2 Hz), 63.0 (d, JP-C ¼ 6.2 Hz), 16.9 (d, JP-C
¼
3
5.9 Hz), 16.6 (d, JP-C ¼ 5.9 Hz).
Experimental
Diethyl (4-chlorophenyl) (hydroxy) methylphosphonate
(3c)
Yield 82%; m.p. 67–688C; IR (KBr) ymax (cmꢀ1): 3272 (brs, OH),
1243 (P¼O), 1040 (P–O–C); 1H NMR (CDCl3) d (ppm): 7.46–7.23
(m, 4H, Ar–H), 5.15 (s, 1H, –OH), 4.79 (d, 2JP-H ¼ 10.9 Hz, 1H, –CH),
Chemistry
Chemicals procured from Sigma–Aldrich and Merck were used as
such without further purification. The melting points (m.p.)
determined in open capillary tubes on a Mel-Temp apparatus
(Tempo Instruments and Equip Pvt. Ltd., Mumbai, India) are
expressed in degrees centigrade (8C) and are uncorrected.
Infrared (IR) spectra were obtained on a Nicolet (San Diego,
CA, USA) 380 Fourier transform infrared (FT-IR) spectropho-
tometer at the Environmental Engineering Laboratory,
Sri Venkateswara University, Tirupati, India, and samples were
4.26–3.99 (m, 4H, O–CH2–CH3), 1.35–1.24 (m, 6H, O–CH2–CH3). 13
C
NMR (CDCl3, 100.57 MHz) d (ppm): 150.4, 140.3, 129.1, 128.1, 72.4
1
2
3
(d, JP-C ¼ 159.0 Hz), 63.79 (d, JP-C ¼ 6.8 Hz), 16.9 (d, JP-
¼ 5.8 Hz), 16.4 (d, 3JP-C ¼ 5.8 Hz). 31P NMR (CDCl3,
C
161.9 MHz): d 20.8. ESI-MS: m/z 279 (MþH)þ; Anal. calcd. for
C11H16ClO4P: C, 47.41; H, 5.79. Found: C, 47.38; H, 5.64.
analyzed as potassium bromide (KBr) disks; absorptions (ymax
)
Diethyl (hydroxy) (3-nitrophenyl) methylphosphonate (3d)
Yield 85%; m.p. 80–818C; 1H NMR (CDCl3) d (ppm): 8.18
(d, J ¼ 8.2 Hz, 2H, Ar–H), 7.49 (d, J ¼ 8.2 Hz, 2H, Ar–H), 5.26
were reported in wave numbers (cmꢀ1). The 1H, 13C, and
31P nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker (Ettlingen, Germany) AMX 400 MHz NMR spec-
trometer operating at 400 MHz for 1H NMR, 100.57 MHz for
13C NMR, and 161.9 MHz for 31P NMR, and are expressed in
parts per million (ppm). All compounds were dissolved in
CDCl3 and chemical shifts were referenced to TMS in 1H NMR
and 13C NMR and 85% H3PO4 in 31P NMR. Mass spectra
were recorded on a Jeol SX 102DA/600 (Tokyo, Japan) mass
spectrometer using argon/xenon (6 keV, 10 mA) as the FAB
gas. Microanalysis was performed with a Thermo Finnigan
(Courtaboeuf, France) Flash EA 1112 I instrument at the
University of Hyderabad, Hyderabad, India.
2
(s, 1H, –OH), 4.97 (d, JP-H ¼ 10.6 Hz, 1H, –CH), 4.38–4.26
(m, 4H, O–CH2–CH3), 1.41–1.28 (m, 6H, O–CH2–CH3); 13C NMR
(CDCl3) d (ppm): 152.3, 148.9, 128.1, 127.7, 12.9, 126.3, 70.5
1
2
2
(d, JP-C ¼ 153.0 Hz), 64.7 (d, JP-C ¼ 6.7 Hz), 63.4 (d, JP-C
¼
3
3
6.7 Hz), 16.5 (d, JP-C ¼ 6.0 Hz), 16.2 (d, JP-C ¼ 6.0 Hz).
Diethyl (hydroxy) (2-nitrophenyl) methylphosphonate (3e)
Yield 87%; m.p. 114–1168C; 1H NMR (CDCl3) d (ppm): 8.21
(d, J ¼ 8.2 Hz, 2H, Ar–H), 7.50 (d, J ¼ 8.2 Hz, 2H, Ar–H), 5.29
2
(s, 1H, –OH), 4.99 (d, JP-H ¼ 10.6 Hz, 1H, –CH), 4.38–4.24
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Antioxidant Properties of a-Hydroxyphosphonates
5
(m, 4H, O–CH2–CH3), 1.39–1.28 (m, 6H, O–CH2–CH3); 13C NMR
(CDCl3) d (ppm): 151.9, 148.1, 128.0, 127.6, 126.8, 126.1, 70.4
287 (MþH)þ; Anal. calcd. for C14H23O4P: C, 58.73; H, 8.10.
Found: C, 58.66; H, 8.04.
1
2
2
(d, JP-C ¼ 153.0 Hz), 64.3 (d, JP-C ¼ 6.7 Hz), 63.2 (d, JP-C
¼
3
3
6.7 Hz), 16.3 (d, JP-C ¼ 6.0 Hz), 16.0 (d, JP-C ¼ 6.0 Hz).
Diethyl (4-(dimethylamino)phenyl) (hydroxy)
methylphosphonate (3k)
Yield 84%; IR (KBr) ymax (cmꢀ1): 3273 (brs, OH), 1240 (P¼O), 1042
Diethyl (hydroxy) (4-nitrophenyl) methylphosphonate (3f)
Yield 84%; m.p. 86–878C; 1H NMR (CDCl3) d (ppm): 8.20
(d, J ¼ 8.2 Hz, 2H, Ar–H), 7.48 (d, J ¼ 8.2 Hz, 2H, Ar–H), 5.28
1
(P–O–C); H NMR (CDCl3) d (ppm): 7.38 (d, J ¼ 8.4 Hz, 2H, Ar–H),
7.19 (d, J ¼ 8.4 Hz, 2H, Ar–H), 5.19 (s, 1H, –OH), 4.94
2
2
(s, 1H, –OH), 4.99 (d, JP-H ¼ 10.6 Hz, 1H, –CH), 4.39–4.25
(d, JP-H ¼ 10.8 Hz, 1H, –CH), 4.08–3.92 (m, 4H, O–CH2–CH3),
(m, 4H, O–CH2–CH3), 1.40–1.29 (m, 6H, O–CH2–CH3); 13C NMR
3.12–2.98 (s, 6H, N–(CH3)2), 1.26–1.17 (m, 6H, O–CH2–CH3);
1
(CDCl3) d (ppm): 152.4, 148.8, 128.2, 127.9, 70.8 (d, JP-C
2
13C NMR (CDCl3) d (ppm): 148.7 (d, JP-C ¼ 3.0 Hz), 134.0, 127.1,
¼
2
2
1
2
153.0 Hz), 64.9 (d, JP-C ¼ 6.7 Hz), 63.5 (d, JP-C ¼ 6.7 Hz), 16.9
126.2, 70.6 (d, JP-C ¼ 159.0 Hz), 63.2 (d, JP-C ¼ 7.0 Hz), 63.0
3
3
2
3
3
(d, JP-C ¼ 6.0 Hz), 16.5 (d, JP-C ¼ 6.0 Hz).
(d, JP-C ¼ 7.0 Hz), 33.8, 16.3 (d, JP-C ¼ 6.0 Hz), 16.2 (d, JP-C
¼
6.0 Hz); 31P NMR: d 22.10; ESI-MS: m/z 287 (MþH)þ; Anal. calcd.
for C13H22NO4P: C, 54.35; H, 7.72; N, 4.88. Found: C, 54.24;
H, 7.64; N, 4.80.
Diethyl (hydroxy) (4-methoxyphenyl) methylphosphonate
(3g)
1
Yield 83%; m.p. 121–1228C; H NMR (CDCl3;) d (ppm): 7.31–7.01
(m, 4H, Ar–H), 5.19 (s, 1H, –OH), 4.63 (d, 2JP-H ¼ 10.4 Hz, 1H, –CH),
4.20–4.02 (m, 4H, O–CH2–CH3), 3.87 (s, 3H, –O–CH3), 1.28–1.19 (m,
6H, O–CH2–CH3); 13C NMR (CDCl3;) d (ppm): 147.4, 137.3, 122.1,
Diethyl (hydroxyl) (4-(pyridin-2-yl)phenyl)
methylphosphonate (3l)
Yield 96%; m.p. 132–1338C; IR (KBr) ymax (cmꢀ1): 3280 (brs, OH),
1208 (P¼O), 1033 (P–O–C); 1H NMR (CDCl3) d (ppm): 8.66
(d, J ¼ 6.8 Hz, 1H, Ar–H), 7.92 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.73
(d, J ¼ 7.2 Hz, 1H, Ar–H), 7.69 (d, J ¼ 8.0, 1H, Ar–H), 7.55
(d, J ¼ 7.2 Hz, 2H, Ar–H), 7.22 (d, J ¼ 4.8 Hz, 1H, Ar–H), 5.24
1
2
121.3, 69.4 (d, JP-C ¼ 149.0 Hz), 62.4 (d, JP-C ¼ 6.1 Hz), 62.1
2
3
3
(d, JP-C ¼ 6.1 Hz), 57.3, 16.0 (d, JP-C ¼ 6.1 Hz), 15.7 (d, JP-C
¼
5.9 Hz).
2
(brs, 1H, –OH), 5.05 (d, JP-H ¼ 12.0 Hz, 1H, -CH), 4.07–4.03
Diethyl (3,4-dimethoxyphenyl) (hydroxy)
(m, 4H, O–CH2–CH3), 1.27–1.21 (m, 6H, O–CH2–CH3); 13C NMR
methylphosphonate (3h)
2
(CDCl3) d (ppm): 157.13, 149.5, 138.8 (d, JP-C ¼ 3.0 Hz), 137.9,
Yield 91%; m.p. 96–978C; IR (KBr) ymax (cmꢀ1): 3257 (brs, OH),
1212 (P¼O), 1011 (P–O–C);1H NMR (CDCl3) d (ppm): 7.07 (s, 1H,
Ar–H), 6.93 (d, J ¼ 8.3 Hz, 1H, Ar–H), 6.78 (d, J ¼ 8.3 Hz, 1H,
1
136.8, 127.4, 127.0, 122.1, 120.6, 70.5 (d, JP-C ¼ 159.0 Hz), 63.2
(d, 2JP-C ¼ 8.0 Hz), 63.0 (d, 2JP-C ¼ 8.0 Hz), 16.4 (d, 3JP-C ¼ 6.0 Hz),
3
16.3 (d, JP-C ¼ 6.0 Hz); 31P NMR: d 22.37; ESI-MS: m/z 321
2
Ar–H), 5.11 (s, 1H, –OH), 4.87 (d, JP-H ¼ 10.0 Hz, 1H, –CH),
(MþNa)þ; Anal. calcd. for C16H20NO4P: C, 59.81; H, 6.27; N,
4.09–3.90 (m, 4H, O–CH2–CH3), 3.86 (s, 6H, –OCH3), 1.28
(t, J ¼ 7.2 Hz, 3H, O–CH2–CH3), 1.21 (t, J ¼ 7.2 Hz, 3H,
O–CH2–CH3); 13C NMR (CDCl3): d (ppm): 150.2, 148.1, 128.9
4.36. Found: C, 59.89; H, 6.22; N, 4.33.
2
1
Diethyl (4-(benzyloxy)phenyl) (hydroxy)
(d, JP-C ¼ 3.0 Hz), 120.1, 116.4, 113.9, 70.3 (d, JP-C ¼ 161.0 Hz),
2
2
methylphosphonate (3m)
62.9 (d, JP-C ¼ 7.1 Hz), 61.9 (d, JP-C ¼ 7.1), 56.2, 55.9, 16.1
3
3
(d, JP-C ¼ 7.0 Hz), 15.8 (d, JP-C ¼ 7.0 Hz); 31P NMR: d 23.2;
ESI-MS: m/z 304 (M)þ; Anal. calcd. for C13H21O6P: C, 51.31; H,
6.96. Found: C, 51.23; H, 6.92.
Yield 96%; m.p. 65–668C; IR (KBr) ymax (cmꢀ1): 3257 (brs, OH),
1212 (P¼O), 1011 (P–O–C);1H NMR (CDCl3) d (ppm): 7.43–7.30
(m, 7H, Ar–H), 6.97 (d, J ¼ 8.8 Hz, 2H, Ar–H), 5.20 (s, 1H, –OH),
5.06 (s, 2H, –O–CH2), 4.94 (d, 2JP-H ¼ 10.0 Hz, 1H, –CH), 4.09–3.92
(m, 4H, O–CH2–CH3), 1.27 (t, J ¼ 7.2 Hz, 3H, O–CH2–CH3), 1.81
(t, J ¼ 7.2 Hz, 3H, O–CH2–CH3); 13C NMR (CDCl3) d (ppm): 158.8,
136.9, 128.7,128.60, 128.4 (d, 2JP-C ¼ 6.0 Hz), 128.0, 127.4, 114.9,
Diethyl (hydroxy) (p-tolyl) methylphosphonate (3i)
Yield 82%; m.p. 95–968C. 1H NMR (CDCl3) d (ppm): 7.29–7.00
2
(m, 4H, Ar–H), 5.26 (s, 1H, –OH), 4.58 (d, JP-H ¼ 10.1 Hz, 1H,
1
2
70.6 (d, JP-C ¼ 160.0 Hz), 70.13, 63.1 (d, JP-C ¼ 7.0 Hz), 63.0
–CH), 4.21–4.02 (m, 4H, O–CH2–CH3), 2.32 (s, 3H), 1.27–1.19
(m, 6H, O–CH2–CH3); 13C NMR (CDCl3) d (ppm): 145.4, 137.1,
2
3
3
(d, JP-C ¼ 7.0 Hz), 16.4 (d, JP-C ¼ 6.0 Hz), 16.3 (d, JP-C
¼
6.0 Hz); 31P NMR: d 22.70; ESI-MS: m/z 351 (MþH)þ; Anal. calcd.
1
2
122.2, 121.4, 69.1 (d, JP-C ¼ 150.0 Hz), 62.6 (d, JP-C ¼ 6.0 Hz),
for C18H23O5P: C, 61.71; H, 6.62. Found: C, 61.78; H, 6.58.
2
3
61.3 (d, JP-C ¼ 6.0 Hz), 21.3, 16.0 (d, JP-C ¼ 5.8 Hz), 15.4
3
(d, JP-C ¼ 5.8 Hz).
Diethyl anthracen-9-yl (hydroxy) methylphosphonate (3n)
Yield 95%; m.p. 150–1518C; IR (KBr) ymax (cmꢀ1): 3273 (OH), 1240
(P¼O), 1042 (P–O–C); 1H NMR (CDCl3) d (ppm): 8.42 (s, 1H, Ar–H),
7.96 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.51–7.41 (m, 6H, Ar–H), 6.59
Diethyl (hydroxy) (4-isopropylphenyl) methylphosphonate
(3j)
Yield 84%; IR (KBr) ymax (cmꢀ1): 3273 (brs, OH), 1240 (P¼O), 1042
(d, JP-H ¼ 16.0 Hz, 1H, –CH), 5.27 (s, 1H, –OH), 4.02–3.96
2
1
(P–O–C); H NMR (CDCl3) d (ppm): 7.38 (d, J ¼ 8.4 Hz, 2H, Ar–H),
(m, 2H, O–CH2–CH3), 3.88–3.78 (m, 1H, O–CH2–CH3), 3.68–3.58
(m, 1H, O–CH2–CH3), 1.17 (t, J ¼ 7.2 Hz, 3H, O–CH2–CH3), 0.89
(t, J ¼ 7.2 Hz, 3H, O–CH2–CH3); 13C NMR (CDCl3) d (ppm): 131.6,
2
7.19 (d, J ¼ 8.4 Hz, 2H, Ar–H), 5.14 (s, 1H, –OH), 4.94 (d, JP-H
¼
10.8 Hz, 1H, –CH), 4.08–3.92 (m, 4H, O–CH2–CH3), 2.92–2.85
(m, 1H, CH(CH3)2), 1.26–1.17 (m, 12H, O–CH2–CH3, and
2
130.4, 129.1 (d, JP-C ¼ 4.0 Hz), 129.0, 127.6, 127.5, 125.8, 124.9,
CH(CH3)2); 13C NMR (CDCl3) d (ppm): 148.7 (d, JP-C ¼ 3.0 Hz),
68.2 (d, JP-C ¼ 163.0 Hz), 63.1 (d, JP-C ¼ 7.0 Hz), 62.8 (d, JP-C ¼
2
1
2
2
1
2
3
3
134.0, 127.1, 126.2, 70.6 (d, JP-C ¼ 159.0 Hz), 63.2 (d, JP-C
¼
¼
7.0 Hz), 16.3 (d, JP-C ¼ 6.0 Hz), 16.0 (d, JP-C ¼ 6.0 Hz); 31P NMR:
d 24.20; ESI-MS: m/z 345 (MþNa)þ; Anal. calcd. for C19H21O4P:
C, 66.27; H, 6.15. Found: C, 66.19; H, 6.12.
2
3
7.0 Hz), 63.0 (d, JP-C ¼ 7.0 Hz), 33.8, 23.8, 16.3 (d, JP-C
6.0 Hz), 16.2 (d, JP-C ¼ 6.0 Hz); 31P NMR: d 22.83; ESI-MS: m/z
3
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
K. R. M. Naidu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Biological antioxidant assays
– Inhibition of GSH:
Net rate ꢀ Intercept
GSH ¼
ꢁ Dilution factor
Animals
Slope
Adult Wistar albino rats (200–250 g) were purchased from
the Faculty of Science, Sri Venkateswara University, India,
and were acclimatized under standard laboratory conditions
with a natural dark/light cycle for 1 week, with free access to
standard food (Hindustan Lever Ltd., India) and water
ad libitum. Animals were acclimatized for at least 7 days to
adapt to the laboratory prior to experiments. All animal
procedures were approved by the Institutional Animal
Care and Use Committee (IACUC), Sri Venkateswara
University, India.
– Inhibition of SOD:
0:93 ꢁ ðVs=VcÞ ꢀ 1
Superoxide dismutase ¼
1:073 ꢀ 0:073 ꢁ ðVs=VcÞ
Here, Vs is the rate of sample containing SOD and Vc is the
average rate of the blank sample (SOD ¼ 0).
– Inhibition of CAT:
OD of the sample ꢁ Volume of assay
CAT ¼
Preparation of rat brain homogenate
13:6 ꢁ Volume of enzyme source ꢁ mg of protein
In the present study, rats were anesthetized with ether and
sacrificed by decapitation, and the whole brain tissue was
dissected, placed on ice and weighed. Brain tissues were
immediately homogenized in 0.1 M phosphate buffer
(pH 7.4) containing 1 mM EDTA. The tissue homogenates
were centrifuged for 10 min at 3000 ꢁ g; the pellet was
discarded and the low-speed supernatant was collected.
The low-speed supernatants from the tissue homogenates
were used to evaluate the beneficial effects of different
concentrations of the synthesized title compounds 3a–n
on various oxidative stress biomarkers.
where OD ¼ optical density.
Statistical analysis
The experimental data were statistically expressed as
mean ꢃ SD of three analyses.
The authors are grateful to the Research University Grants Scheme (RUGS),
Universiti Putra Malaysia, UPM 43400, Serdang, Malaysia.
References
Induction of oxidative stress and experimental design
Brain tissue homogenates and 0.1 mL of the synthesized title
compounds 3a–n and ascorbic acid (positive control) were
mixed and incubated in a water bath at 378C for 5 min;
0.15 mL H2O2 was added to induce LPO, and the mixture
was reincubated at 378C for 15 min. Then, various oxidative
stress biomarkers were analyzed with standard methods.
Levels of LPO and reduced glutathione (GSH) and the activi-
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acid assay was based on the oxidation of ascorbic acid to
dehydroascorbic acid followed by treatment with 2,4-dinitro-
phenylhydrazine to form the bis-dinitrophenylhydrazone
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