Synthesis and cytotoxic evaluation of novel 2,3-di-O-alkyl derivatives of l-ascorbic acid

Article abstract of DOI:10.1007/s00044-013-0714-1

A new series of 2,3-di-O-alkyl derivatives of 5,6-O-isopropylidene-l- ascorbic acid were synthesized using phase transfer catalysis in aqueous media. These derivatives were screened for their superoxide radical scavenging activity and anticancer activity against human breast cancer cell line (MCF-7), leukemic cell line (HL-60), and cervical cell line (HeLa). All these derivatives exhibited enhanced scavenging effect than l-ascorbic acid except for the 4-fluorobenzyl or 2/4-chlorobenzyl alkyl group either at 3-O and/or 2-O position displayed pro-oxidant activity. These pro-oxidant derivatives (2c-e, m) exhibited potent anticancer activities against all the cell lines (IC ₅₀ = 25.79-57.21 μM). However, these compounds were also cytotoxic to human normal leukemic macrophages THP-1. On the other hand, antioxidant derivatives displayed albeit slight (2k, IC₅₀ = 57.96-63.45 μM), but selective inhibitory effect toward all tumor cell lines. Thus, pro-oxidant and antioxidant properties can be used to predict the cytotoxic selectivity of drug against normal and cancer cells.

Full text of DOI:10.1007/s00044-013-0714-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1257–1266
DOI 10.1007/s00044-013-0714-1
ORIGINAL RESEARCH
Synthesis and cytotoxic evaluation of novel 2,3-di-O-alkyl
derivatives of L-ascorbic acid
•
Santosh R. Kote Ratnakar Mishra
•
•
Ayesha A. Khan Shankar R. Thopate
Received: 6 June 2013 / Accepted: 10 August 2013 / Published online: 30 August 2013
ꢀ Springer Science+Business Media New York 2013
Abstract A new series of 2,3-di-O-alkyl derivatives of
5,6-O-isopropylidene-L-ascorbic acid were synthesized
using phase transfer catalysis in aqueous media. These
derivatives were screened for their superoxide radical
scavenging activity and anticancer activity against human
breast cancer cell line (MCF-7), leukemic cell line (HL-
60), and cervical cell line (HeLa). All these derivatives
exhibited enhanced scavenging effect than L-ascorbic acid
except for the 4-fluorobenzyl or 2/4-chlorobenzyl alkyl
group either at 3-O and/or 2-O position displayed pro-
oxidant activity. These pro-oxidant derivatives (2c–e,
m) exhibited potent anticancer activities against all the cell
lines (IC50 = 25.79–57.21 lM). However, these com-
pounds were also cytotoxic to human normal leukemic
macrophages THP-1. On the other hand, antioxidant
derivatives displayed albeit slight (2k, IC50 = 57.96–
63.45 lM), but selective inhibitory effect toward all tumor
cell lines. Thus, pro-oxidant and antioxidant properties can
be used to predict the cytotoxic selectivity of drug against
normal and cancer cells.
Introduction
It is now generally accepted that oxygen radicals play a
vital role in vivo not only as a mediator of signal trans-
duction but also as causative species for oxidative damage
of membranes and tissues, eventually resulting in a variety
of diseases, cancer, aging, etc. (Amorati et al., 2011; Valko
et al., 2007). Antioxidant enzymes such as superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxi-
dase or low molecular organic molecules such as Vitamin
C, E and glutathione are known to scavenge the oxidant
species by hydrogen donation or breaking of an oxidation
propagation chain. L-Ascorbic acid (Vitamin C, AsA) is
also an important biomolecule widely distributed in aerobic
organisms. It is a biological cofactor that plays a role in
numerous biological pathways, fundamental to cellular
function (Uttara et al., 2009). Being a strong reducing
agent at physiological pH, ascorbate anion acts as radical
quencher directly or by recycling other antioxidants such as
a-tocopherol or glutathione. In the presence of trace metals
it reacts with O2 to form H2O2 and dehydroascorbic acid,
•-
Keywords L-Ascorbic acid derivatives ꢀ
via the formation of O2 and hence exhibits prooxidant
Free radical scavenging activity ꢀ Anticancer activity ꢀ
Mitochondrial assay ꢀ DNA cleavage assay
effects (Burton and Ingold, 1990). For this reason, high
doses of intravenous ascorbate have been recently proven
to deliver hydrogen peroxide to tissue fluids and to retard
tumor growth in numerous animal models (Chen et al.,
2008; Verrax and Calderon, 2009). Recent studies have
suggested that cellular levels of H2O2 play, directly or
indirectly, a key role in malignant transformation, but also
sensitize cancer cells to H2O2-induced cell death. This dual
role of H2O2 in cancer might be exploitable for develop-
ment of cancer chemopreventive and therapeutic agents.
Several reports have appeared in the literature to address
the above problem by synthesizing various L-ascorbic acid
derivatives with enhanced stability and activity. For
S. R. Kote ꢀ S. R. Thopate (&)
Department of Chemistry, Dr. John Barnabas Post Graduate
School for Biological Studies, Ahmednagar College,
Station Road, Ahmednagar 414001, Maharashtra, India
e-mail: srthopate@gmail.com
R. Mishra ꢀ A. A. Khan
Institute of Bioinformatics & Biotechnology, University of Pune,
Pune 411007, Maharashtra, India
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Med Chem Res (2014) 23:1257–1266
Table 1 Synthesis of 2,3-di-O-alkyl derivatives of 5,6-O-isopropylidene-L-ascorbic acid
O
O
Methoda A / B
O
O
O
O
O
O
HO
OH
RO
OR1
1
2
Comp. no.
R
R1
% yielda
2a
2b
2c
2d
2e
2f
Methyl
Benzyl
Methyl
Benzyl
94
80
74
60
71
76
66
64
65
61
68
59
58
63
4-Fluorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
Naphthylmethyl
Methyl
4-Fluorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
Naphthylmethyl
2-Fluorobenzyl
4-Fluorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
4-Nitrobenzyl
4-Fluorobenzyl
Methyl
2g
2h
2i
Methyl
Methyl
2j
Methyl
2k
2l
Methyl
4-Chlorobenzyl
4-Chlorobenzyl
Naphthylmethyl
2m
2n
a
Methyl
Isolated yield. Method A: 2.1 eq. RX, KOH, 10 mol% TBAI, H2O:EtOAc (1:1), RT, 17–24 h. Method B: (a). 1.1 eq. RX, KOH, 10 mol%
TBAI, H2O:EtOAc (1:1), RT, 17–24 h. (b). 1.1 eq. R1X, KOH, 10 mol% TBAI, H2O:EtOAc (1:1), RT, 17–24 h
Table 2 Pro-oxidant behavior
of L-ascorbic acid derivatives
(2c–e and 2l)
Time in minute
Control
2c
2d
2e
2l
0
0.04901
0.04234
0.04457
0.04598
0.04741
0.04871
0.04993
0.05101
0.05193
0.05302
0.05418
1.07386
1.06524
1.05818
1.0526
0.17924
0.13093
0.13015
0.1318
0.10266
0.10407
0.10609
0.10789
0.10965
0.11137
0.111
0.08408
0.082989
0.08221
0.08202
0.08118
0.080769
0.080519
0.07986
0.079229
0.07902
0.07905
0.5
1
1.5
2
1.04844
1.045
0.13424
0.13664
0.1387
2.5
3
1.04196
1.03974
1.03692
1.03502
1.03358
3.5
4
0.14036
0.14264
0.14438
0.14606
0.11245
0.11383
0.11502
0.11642
The absorbance values exceed
the control depicting generation
of more superoxide radicals
4.5
5
example, 5,6-O-modified L-ascorbic acid derivatives have
been found to be effective anti-tumor agents against various
human cancers, and induce apoptosis in tumor cells (Tanuma
et al., 1993; Gazivoda et al., 2007; Wittine et al., 2012),
2-C alkyl derivatives have been shown to have immuno-
stimulant activity (Veltri et al., 1986; Woolverton et al.,
1986; Sakagami et al., 1996), 2-O and 3-O-alkylated deriv-
atives are known to protect against peroxidation of lipids of
the bio-membranes (Nihro et al., 1991; Kato et al., 1988; El-
Demerdash et al., 2005). Among all the L-ascorbic acid
derivatives reported in the literature, only 5,6-O-modified
derivatives have been tested as a anti-tumor agents for var-
ious human cancers by inducing apoptosis in tumor cells
(Tanuma et al., 1993; Gazivoda et al., 2007; Wittine et al.,
2012). Although, 2-O and/or 3-O-alkylated derivatives of
L-ascorbic acid have shown ROS scavenging activity (Nihro
et al., 1991; Kato et al., 1988), their anticancer activity have
not been evaluated. As an ongoing project in our laboratory
to explore the utility of L-ascorbic acid in synthetic organic
chemistry, we have developed a simple and user-friendly
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Med Chem Res (2014) 23:1257–1266
1259
SOD Activity
method for synthesis of 2,3-di-O-alkyl derivatives of L-
ascorbic acid (Thopate and Kote, 2012). Moreover, these
derivatives can be synthesized easily as compared to prep-
aration of 5,6-di-O-modified derivatives. In this paper, we
report synthesis of new 2,3-di-O-alkyl derivatives of 5,6-O-
isopropylidene-L-ascorbic acid and preliminary studies
concerning scavenging of superoxide anion and antitumor
activity against various human cancer cells. Further, the
mechanism of cell death has been proposed.
14
12
10
8
6
4
2
0
Fig. 1 SOD activity IC50 values of the ascorbic acid derivatives. The
SOD activities of compounds were determined spectrophotometri-
cally by the NBT assay method in triplicate and are presented as the
mean % inhibition of NBT reduction. One unit of SOD activity was
defined as the concentration of the test substrate required for 50 %
inhibition of NBT reduction by superoxide anion
Results and discussion
Chemistry
Synthesis of 2-O- and/or 3-O-alkyl derivatives of L-ascor-
bic acid, is a complex issue, as it is fairly sensitive to the
reaction conditions leading to a mixture of 3-O-alkyl, 2-O-
alkyl, 2,3-di-O-alkyl, and/or 2-C-alkyl derivatives. Most of
the methods reported in the literature require anhydrous
reaction conditions and give mixture of products, while in
highly polar solvents like water; 2-C-alkylation is favored
(Thopate and Kote, 2012). We synthesized various new
2,3-di-O-alkyl derivatives of 5,6-O-isopropylidene-L-
ascorbic acid by employing recently reported methodology
from our laboratory which allows us to prepare 2,3-di-O-
alkyl derivatives of 5,6-O-isopropylidene-L-ascorbic acid
selectively even in aqueous medium under phase transfer
catalysis (PTC) (Thopate and Kote, 2012). The 2,3-di-O-
alkyl derivatives 2a–f of 5,6-O-isopropylidene-L-ascorbic
acid 1 were synthesized under the PTC conditions in uni-
formly excellent yields (Table 1), using 2.1 eq. alkylating
agent. Furthermore, differentially protected 2,3-di-O-alkyl
derivatives 2g–n were obtained in good yields (Table 1) in
a one-pot reaction by adding two different alkylating
agents in a sequential manner.
radical scavenging as compared to L-ascorbic acid (AsA)
(IC50 = 11.27 lM). In 2,3-di-O-alkyl derivatives, com-
pound 2f with di-2-(methyl)naphthyl group also showed
promising scavenging ability with IC50 of 5.98 lM. Di-O-
methyl 2a or di-O-benzyl 2b derivatives exhibited good
activity with IC50 of 7.4 lM and with IC50 of 6.8 lM,
respectively. These results indicate that the activity
increases as the electron withdrawing ability of alkyl group
increases. While in differentially protected 2,3-di-O-alkyl
derivatives, compound 2k revealed powerful superoxide
radical scavenging ability with IC50 of 5.5 lM. This can be
explained on the basis of above result as 4-nitrobenzyl at
2-O-position is a powerful electron withdrawing group.
Replacing 4-nitrobenzyl by either 4-chlorobenzyl 2j or
4-fluorobenzyl group 2h, slightly decreases the radical scav-
enging activity (2j, IC50 = 6.4 lM, 2h, IC50 = 6.98 lM,
respectively). Changing the position of chloro or fluoro group
from para to ortho position on benzene ring decreases the
activity further (2i, IC50 = 7.04 lM, 2g, IC50 = 8.13 lM,
respectively). Furthermore, interchanging the alkyl groups
between 2-O and 3-O positions retains the superoxide radi-
cal scavenging ability, for example compound 2j (IC50
6.4 lM) and 2m (IC50 = 6.5 lM) exhibits almost similar
activity.
=
Biological investigation
The SOD activities
In vitro cytotoxicity
The newly synthesized derivatives of 5,6-O-isopropyli-
dene-L-ascorbic acid were first examined for their super-
oxide scavenging activity using riboflavin–nitro blue
tetrazolium (NBT) assay. Among the tested compound, 2c–
e, l were found to be pro-oxidant. The results (Table 2)
showed that the derivatives with 4-fluorobenzyl or 2/4-
chlorobenzyl alkyl group either at 3-O and/or 2-O position
exhibits pro-oxidant activity. The remaining compounds,
2a, b, f–k, m, n, were found to be antioxidants (Fig. 1).
The examination of SOD-like activities of the ascorbic
acid derivatives were found to have enhanced ability of
With promising SOD activity shown by new L-ascorbic
acid derivatives 2a–n, these derivatives were further
evaluated for their antiproliferative activity against human
breast cancer (MCF-7) cell line, leukemic (HL-60) cell
line, cervical (HeLa) cell line, and human leukemic mac-
rophages (THP-1) cell line taken as control using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) colorimetric assay (Table 3). For comparative
purpose, L-ascorbic acid was also included in assay. Of all
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Med Chem Res (2014) 23:1257–1266
IC50 = 63.45 lM) with some selectivity toward Hela cell line
compared to MCF-7 and HL-60 cell lines. Incorporation of
chloro or flouro group either at para or meta position in benzene
ring led to a decrease of activity of the resultant analogs 2g–
j. Other interesting results were obtained with interchange of
alkyl groups between 2-O and 3-O position. Compound 2j
Table 3 Cytotoxic activity of 2,3-di-O-alkyl derivatives of 5,6-O-
isopropylidene-L-ascorbic acid
Comp.
no.
Cytotoxic activity, IC50 (lM)a
Hela
(SD 0.042)
MCF-7
(SD 0.064)
HL-60
(SD 0.054)
2a
2b
2c
2d
2e
2f
75.26
68.54
25.79
48.26
51.06
59.33
82.12
78.32
69.57
66.78
57.96
38.66
65.89
82.32
130.23
74.24
70.14
28.31
50.09
52.22
61.13
83.23
70.14
70.57
67.18
59.96
41.04
66.29
83.32
128.54
80.27
78.22
32.19
55.77
57.21
64.87
85.46
73.98
75.33
68.09
63.45
45.63
69.56
86.46
135.76
and 2m maintained similar cytotoxic level (IC50
65.89–69.56 lM and 66.78–68.09 lM, respectively).
=
The above results depicted that these new L-ascorbic acid
derivatives were found to have higher activity in the human
cervical cancer cell line HeLa as compared to MCF-7 and
HL-60 cell lines. Recently, accumulative evidence suggests
that induction of ROS formation and redox dysregulation is
causatively involved in the chemotherapeutic efficacy of
many established anticancer drugs that have traditionally not
been associated with free radical mechanism of action. It has
also been demonstrated that cisplatin apoptogenicity
depends on formation of ROS and occurs independent of
nuclear DNA damage, suggesting that apoptogenic oxidative
stress is the crucial mechanism of cisplatin-induced cancer
cell death (Wondrak 2009). A combination therapy
employing a chemotherapeutic drug with an experimental
pro-oxidant thus provides a promising therapeutic strategy
for initiation of cancer cell cytotoxicity.
2g
2h
2i
2j
2k
2l
2m
2n
AsA
a
Data represent mean values for four independent determinations
evaluated derivatives, compound 2c showed the best ani-
tumor activity against all evaluated tumor cell lines
(IC50 = 25.79–32.19 lM). Similarly, compounds 2d–e,
l exhibited rather marked inhibitory activity against the
growth of all tumor cell lines (IC50 within the concentra-
tion range of 38.66–57.21 lM). However, these derivatives
were also cytotoxic to normal human leukemic macro-
phages THP-1 cell line, which can be correlated with their
pro-oxidant property.
Morphological studies
In order to elucidate the mechanism of cell death, we
carried out morphological analysis (Fig. 2). The micro-
scopic observation of normal and drug-treated MCF-7 cell
lines under Carl Ziess inverted microscope revealed typical
apoptotic features such as chromatin condensation and
membrane blebbing for compounds (2a, b, f–n) having
antioxidant activity. The pro-oxidant compounds (2c–e,
l) had necrotic features with cell disruption. The apoptotic
mode of cell death was further confirmed by the mito-
chondrial assay.
Other derivatives with anti-oxidant activity exhibited
enhanced antiproliferative activity against all tested tumor cell
linescompared to L-ascorbic acid (IC50 = 130.23–135.76 lM)
(Table 3) without any noticeable cytotoxic effect on normal
human leukemic macrophages THP-1 cell line. These results
also shows some structure activity relationship as observed in
SOD activity. First, alkylation at 2-O and 3-O position
increases the potency by 1.6 to 5.0-fold compared to L-ascorbic
acid. Second, substituted benzyl group is more effective than
the aliphatic methyl group. In symmetrically substituted 2,3-di-
O-alkyl derivatives, di-O-2-(methyl)naphthyl derivative 2f
exhibited good cytotoxic activity with IC50 = 59.33–
64.87 lM. Di-O-benzyl and di-O-methyl derivatives 2b and
The mitochondria were extracted from normal and drug-
treated MCF-7 cell lines and observed under Carl Zeiss
microscope (Fig. 3). For the compounds (2c–e, l), mito-
chondria remained intact which confirms necrotic cell
death, while for the remaining compounds, there was dis-
ruption of mitochondria as seen for 2m.
We propose here the cell death is due to the presence of
H2O2 generated within the cell upon drug treatment. For
the antioxidant compounds the amount of H2O2 is minimal
which brings about apoptotic death, while for the pro-
oxidant compounds the sudden burst of H2O2 leads to
necrosis. This was further confirmed by DNA fragmenta-
tion studies. DNA was treated with synthesized compounds
along with L-ascorbic acid and fragmentation study was
carried out using gel electrophoresis (Fig. 4). In the
absence of N-acetylcysteine (NAC), all derivatives
2a, respectively, showed decreased efficiency (IC50
=
68.54–78.22 and 75.26–80.27 lM, respectively). The most
promising antitumor activity was observed from differentially
protected 2,3-di-O-alkyl derivative series. Compound 2k with
4-nitrobenzyl group at 2-O position and methyl group at
3-O position exhibited potent antitumor activity (Hela,
IC50 = 57.96 lM; MCF-7, IC50 = 59.96 lM; HL-60,
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Med Chem Res (2014) 23:1257–1266
1261
Control
AsA
2a
2b
2f
2g
2h
2i
2j
2k
2m
2n
2c
2d
2e
2l
Fig. 2 A photomicrograph of morphological changes induced in response to treatment of MCF-7 cells for 48 h. The treated and untreated cells
were observed under Carl Ziess inverted microscope and photographed, as described in Materials and Methods
Control
2c
2d
2e
2l
2m
Fig. 3 Mitochondrial assay. The mitochondria were extracted from normal and drug-treated MCF-7 cell lines and observed under Carl Ziess
inverted microscope and photographed, as described in Materials and Methods
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Med Chem Res (2014) 23:1257–1266
Fig. 4 Electrophoretic analysis of internucleosomal DNA fragmen-
tation of treated and untreated cells after 48 h. Lane 1 control DNA, 2
DNA?L-ascorbic acid, 3 DNA?2a, 4 DNA?2b, 5 DNA?2n, 6
DNA?2e, 7 DNA?2l, 8 DNA?2d, 9 DNA?2c, 10 DNA?2c?SOD,
11 DNA?2c?NAC
revealed a classical internucleosomal DNA laddering (lane
3–10), while in the presence of NAC even most powerful
pro-oxidant compound 2c did not show any fragmentation.
Since NAC is a known inhibitor of H2O2, it is observed
from the above figure that DNA cleavage is restored (lane
11) and is similar to the control. These results confirmed
that the H2O2 is a produced within the cell upon drug
treatment.
To the best of our knowledge, we report here for the
first time 2,3-di-O-alkyl derivatives of 5,6-O-isopropyli-
dene-L-ascorbic acid as antitumor agents. The facile
synthesis, good potency, and selectivity could facilitate
further optimization of 2,3-di-O-alkyl derivatives of L-
ascorbic acid as antitumor agents and this series of
compounds provided a promising starting point for further
drug optimization and development as novel anticancer
agents.
Conclusions
Experimental
We report the synthesis and biological evaluation of a new
series of symmetrically and differentially substituted
derivatives of 5,6-O-isopropylidene-L-ascorbic acid. SOD-
like activity showed that symmetrically substituted deriv-
atives with 4-fluorobenzyl or 2/4-chlorobenzyl alkyl group
either at 3-O and/or 2-O position exhibited pro-oxidant
activity. Mono-substituted derivatives of the above alkyl
groups along with methyl, benzyl, naphthylmethyl deriva-
tives at 3-O or 2-O position showed antioxidant activity.
The antitumor study of these compounds signified that the
antioxidant compounds illustrated potent activity against
MCF-7, HL-60, and HeLa cell lines. Compounds with pro-
oxidant activity showed even larger effect on these three
cell lines but these compounds were also cytotoxic to
normal human cell.
General information
All solvents and reagents were used as obtained from
commercial source and were used without further purifi-
cation unless otherwise indicated. Thin-layer chromatog-
raphy (TLC) was performed on E. Merck silica gel 60F254
(0.25 mm thickness) plates and were visualized under short
(254 nm) and long (365 nm) UV light. Column chroma-
tography was performed using silica gel (Acme) 200–400
mesh. Melting points (mp) were determined in open cap-
illary tubes using paraffin oil bath and are uncorrected.
Infra red (IR) spectra were recorded on Perkin Elmer
Model 1600 series Fourier Transform (FT) instrument.
Optical rotations were recorded on JASCO DIP Digital 181
Polarimeter at 25 ꢁC. 1H NMR and 13C NMR were
recorded on Varian Mercury 300 and 75 MHz respectively
in CDCl3 solution and tetramethylsilane (TMS) as internal
reference (d scale). High-resolution mass spectra (HRMS)
were performed with a QTOF Micromass Mass Spec-
trometer by electro spray ionization mode. The purity of all
newly synthesized compounds were analyzed by HPLC,
with the purity all being higher than 95 %. Analytical
HPLC was performed on Varian Inc, USA 410 Prostar
Binary LC System chromatograph equipped with UV
Preliminary results about the mechanism of action indi-
cate that the mode of cell death is apoptotic for the antioxi-
dant compound while necrosis was observed for the pro-
oxidant compounds as seen in both morphological and
mitochondrial assay. DNA fragmentation analysis con-
firmed production of varying amount of reactive oxygen
species-like H2O2 which causes cell death. Compounds
which produce less amount of H2O2 induce apoptosis and
these were antioxidants in nature. On the other hand, pro-
oxidant derivatives produce excessive amount of H2O2,
which causes necrosis. Thus, the amount of reactive oxygen
species generated could be fine tuned with the chemical
structures to design appropriate cytotoxicity of drugs.
detector (220 nm) using
a AD-026 C-18 (5 mm,
250 mm 9 4.6 mm reverse phase) column to detect the
purity of the product. Methanol:acetonitrile (1:1) was used
as mobile phase at a flow rate of 1.0 mL/min.
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Med Chem Res (2014) 23:1257–1266
1263
(m, 3H), 7.48 (m, 1H); 13C NMR (75 MHz, CDCl3) d 25.6,
25.9, 65.2, 70.6, 71.0, 73.9, 74.6, 110.2, 121.4, 126.9,
126.9, 129.4, 129.5, 129.6, 129.9, 130.0, 131.0, 133.0,
133.5, 133.6, 134.0, 156.1, 168.8; HRMS [MH?] calcd for
C23H22O6Cl2, 465.0872; found, 465.0883.
General procedure for the preparation of 2,3-di-O-alkyl
derivatives of 5,6-O-isopropylidene-L-ascorbic acid
under PTC 2a–f
To a stirred solution of 1 (1.00 g, 4.6 mol, 1 eq.) and tetra
n-butyl ammonium iodide (TBAI) (10 mol%) in water:-
ethyl acetate (3 mL each), potassium hydroxide (KOH)
(9.66 mol) was added followed by addition of corre-
sponding alkylating agent (9.66 mol). Stirring was con-
tinued at room temperature for 24 h. Product was extracted
with ethyl acetate (3 9 10 mL). Combined ethyl acetate
extracts were dried over anhydrous sodium sulfate. Ethyl
acetate was removed under reduced pressure and the crude
product was purified on silica gel column chromatography
using ethyl acetate–hexane (1:4) solvent system.
5,6-O-Isopropylidene-2,3-di-O-4-chlorobenzyl-L-ascorbic
acid (2e) White solid; mp 82–83 ꢁC; [a]2D5 ? 54.34
(c 0.94 MeOH); IR (KBr)/cm-1: 2894, 1761, 1676, 958,
1
815; H NMR (300 MHz, CDCl3) d 1.36 (s, 3H), 1.39 (s,
3H), 4.03 (m, 1H), 4.11 (m, 1H), 4.27 (dt, J = 6.7 &
2.7 Hz, 1H), 4.54 (d, J = 2.7 Hz, 1H), 5.06 (AB q,
J = 11.4 Hz, 2H), 5.17 (AB q, J = 12.9 Hz, 2H), 7.15 (m,
2H), 7.26–7.35 (m, 6H); 13C NMR (75 MHz, CDCl3) d
25.6, 25.8, 65.2, 72.6, 72.7, 73.5, 74.4, 110.3, 121.1, 128.8,
128.8, 128.9, 130.3, 133.7, 134.4, 134.5, 134.6, 156.1,
168.8. HRMS [MH?] calcd for C23H22O6Cl2, 465.0872;
found, 465.0882.
5,6-O-Isopropylidene-2,3-di-O-methyl-L-ascorbic acid (2a)
White solid; mp 100–102 ꢁC; [a]2D5 ? 11.34 (c 0.7 CHCl3); IR
1
(Nujol)/cm-1 :3002, 2920, 1760, 1679; H NMR (300 MHz,
CDCl3) d 1.36 (s, 3H), 1.39 (s, 3H), 3.85 (s, 3H), 4.01–4.13 (m,
2H), 4.15 (s, 3H), 4.25–4.31 (m, 1H), 4.51 (d, J = 3 Hz, 1H);
13C NMR (75 MHz, CDCl3) d 25.5, 25.7, 59.4, 60.3, 65.1,
73.8, 74.4, 110.3, 123.1, 156.7, 168.9.
5,6-O-Isopropylidene-2,3-di-O-2-naphthylmethyl-L-ascorbic
acid (2f) White solid; mp 140–142 ꢁC; [a]2D5 ? 14.25
(c 0.7 CHCl3); IR (KBr)/cm-1:2984, 1747, 1678, 835, 767;
1H NMR (300 MHz, CDCl3) d 1.35 (s, 3H), 1.40 (s, 3H),
4.05 (m, 2H), 4.2 (dt, J = 6.6 & 2.7 Hz, 1H), 4.55 (d,
J = 2.7 Hz, 1H), 5.26 (s, 2H), 5.32 (q, J = 8.4 Hz, 2H),
7.20–7.26 (m, 2H), 7.47–7.53 (m, 6H), 7.67 (m, 1H), 7.75
(m, 1H), 7.81–7.87 (m, 4H).
5,6-O-Isopropylidene-2,3-di-O-benzyl-L-ascorbic acid (2b)
White solid; mp 127–128 ꢁC; [a]2D5 ? 23.4 (c 2 CHCl3); IR
(Nujol)/cm-1
:
2923, 2860, 1750, 1676; 1H NMR
(300 MHz, CDCl3) d 1.36 (s, 3H), 1.40 (s, 3H), 4.07 (m,
2H), 4.23 (m, 1H), 4.52 (d, J = 3.0 Hz, 1H), 5.04–5.21 (m,
4H), 7.18–7.40 (m, 10H); 13C NMR (75 MHz, CDCl3) d
25.6, 25.8, 65.2, 73.5, 73.8, 74.6, 110.2, 121.1, 127.7,
128.3, 128.5, 128.6, 128.6, 129.1, 130.1, 135.3, 135.9,
156.6, 169.1.
General procedure for differentially protected 2,3-di-O-
alkyl derivatives of 5,6-O-isopropylidene-L-ascorbic acid
in a one pot reaction 2g–n
To a stirred solution of 1 (1.00 g, 4.6 mol, 1 eq.) and TBAI
(10 mol%) in water: ethyl acetate (3 mL each), KOH
(4.6 mol) was added followed by addition of the corre-
sponding alkylating agent (4.6 mol). Stirring was contin-
ued at room temperature for 24 h (TLC check). Then, KOH
(4.6 mol) was added followed by addition of next alkyl-
ating agent (4.6 mol). Stirring was continued at room
temperature for further 24 h. Product was extracted with
ethyl acetate (3 9 10 mL). Combined ethyl acetate
extracts were dried over anhydrous sodium sulfate. Ethyl
acetate was removed under reduced pressure and the crude
product was purified on silica gel column chromatography
using ethyl acetate–hexane (1:4) system.
5,6-O-Isopropylidene-2,3-di-O-4-fluorobenzyl-L-ascorbic acid
(2c) White solid; mp 60–62 ꢁC; [a]2D5 ? 43.77 (c 0.7
CHCl3); IR (KBr)/cm-1: 2955, 2933, 2891, 1753, 1678,
1
1605; H NMR (300 MHz, CDCl3) d 1.35 (s, 3H), 1.39 (s,
3H), 3.98–4.12 (m, 2H), 4.26 (m, 1H), 4.52 (d, J = 2.7 Hz,
1H), 5.03–5.21 (m, 4H), 7.04 (m, 4H), 7.20 (m, 2H), 7.38
(m, 2H), 13C NMR (75 MHz, CDCl3) d 25.5, 25.8, 65.1,
72.7, 72.8, 73.6, 74.5, 110.2, 115.3, 115.4, 115.7, 121.1,
129.6, 129.8, 130.9, 131.1, 156.3, 164.4, 168.9; HRMS
[MH?] calcd for C23H22O6F2, 433.1463; found, 433.1447.
5,6-O-Isopropylidene-2,3-di-O-2-chlorobenzyl-L-ascorbic acid
(2d) White solid; mp 68–69 ꢁC; [a]2D5 ? 55.73 (c 0.52
MeOH); IR (KBr)/cm-1: 2990, 1759, 1680, 938, 824, 754;
1H NMR (300 MHz, CDCl3) d 1.35 (s, 3H), 1.40 (s, 3H),
4.02 (m, 1H), 4.10 (t, J = 6.6 Hz, 1H), 4.27 (dt, J = 6.6 &
3.3 Hz, 1H), 4.58 (d, J = 3.3 Hz, 1H), 5.27 (s, 2H), 5.39
(AB q, J = 12.3 Hz, 2H), 7.22–7.28 (m, 4H), 4.31–4.39
5,6-O-Isopropylidene-2-O-2-fluorobenzyl-3-O-methyl-L-ascor-
bic acid (2g) White solid; mp 59–60 ꢁC; [a]2D5 ? 26.10
(c 0.78 MeOH); IR (KBr)/cm-1: 2991, 2956, 2429, 2340,
1
1755, 1682, 956, 847, 725; H NMR (300 MHz, CDCl3) d
1.35 (s, 3H), 1.40 (s, 3H), 3.96 (s, 3H), 4.00 (m, 1H), 4.10
(t, J = 6.6 Hz, 1H), 4.23 (dt, J = 6.6 & 3.3 Hz, 1H), 4.49
(d, J = 3.3 Hz, 1H), 5.20 (s, 2H), 7.12 (m, 2H), 7.28–7.46
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Med Chem Res (2014) 23:1257–1266
(m, 2H); 13C NMR (75 MHz, CDCl3) d 25.5, 25.8, 59.5,
65.1, 67.3, 73.8, 74.5, 110.2, 120.9, 124.1, 124.2, 130.7,
130.8, 131.7, 131.8, 157.6, 169.0. HRMS [MH?] calcd for
C17H19O6F, 339.1244; found, 339.1246.
HRMS [MH?] calcd for C17H19O8N, 366.1189; found,
366.1184.
5,6-O-Isopropylidene-2-O-4-fluorobenzyl-3-O-4-chlorobenzyl-
L-ascorbic acid (2l) White solid; mp 72–73 ꢁC;
[a]2D5 ? 55.01 (c 0.55 MeOH); IR (KBr)/cm-1: 2993, 1763,
5,6-O-Isopropylidene-2-O-4-fluorobenzyl-3-O-methyl-L-ascor-
bic acid (2h) White solid; mp 58–59 ꢁC; [a]2D5 ? 34.29
(c 1.42 MeOH); IR (KBr)/cm-1: 2989, 2931, 2893, 2442,
1780, 1690, 1600, 837, 772, 715; 1H NMR (300 MHz,
CDCl3) d 1.36 (s, 3H), 1.40 (s, 3H), 3.91 (s, 3H), 4.05 (m,
1H); 4.12 (m, 1H), 4.22 (dt, J = 6.7 & 2.7 Hz, 1H), 4.50
(d, J = 2.7 Hz, 1H), 5.09 (AB q, J = 11.4 Hz, 2H), 7.05
(m, 2H), 7.38 (m, 2H); 13C NMR (75 MHz, CDCl3) d 25.5,
25.8, 59.4, 65.1, 73.0, 73.7, 74.4, 110.3, 115.3, 115.6,
120.9, 131.0, 131.8, 157.5, 169.0; HRMS [MH?] calcd for
C17H19O6F, 339.1244; found, 339.1248.
1
1676, 854, 819; H NMR (300 MHz, CDCl3) d 1.36 (s,
3H), 1.39 (s, 3H), 4.02–4.13 (m, 2H), 4.24–4.28 (dt,
J = 6.6 & 2.7 Hz, 1H), 4.53 (d, J = 2.7 Hz, 1H);
5.02–5.21 (m, 4H), 7.02–7.08 (m, 2H), 7.11–7.21 (m, 2H),
7.26–7.38 (m, 4H); 13C NMR (75 MHz, CDCl3) d 25.6,
25.8, 65.2, 72.5, 73.5, 74.4, 110.3, 115.3, 115.6, 121.1,
128.8, 129.0, 130.9, 131.0, 131.8, 133.7, 134.6, 156.1,
168.8; HRMS [MH?] calcd for C23H22O6ClF, 449.1167;
found, 449.1156.
5,6-O-Isopropylidene-2-O-methyl-3-O-4-chlorobenzyl-L-
ascorbic acid (2m) White solid; mp 158–160 ꢁC;
[a]2D5 ? 37.75 (c 0.48 MeOH); IR (KBr)/cm-1: 2985, 1754,
1679, 1445, 1337, 810; 1H NMR (300 MHz, CDCl3) d 1.35
(s, 3H), 1.38 (s, 3H), 3.77 (s, 3H), 4.01–4.14 (m, 2H), 4.30
(dt, J = 5.7 & 2.1 Hz, 1H), 4.54 (d, J = 2.1 Hz, 1H), 5.42
(s, 2H), 7.27–7.38 (m, 4H); 13C NMR (75 MHz, CDCl3) d
25.5, 25.7, 59.8, 65.2, 72.5, 73.7, 74.4, 110.3, 123.2, 128.8,
129.0, 133.9, 134.5, 154.9, 168.62; HRMS [MH?] calcd
for C17H19O6Cl, 355.0948; found, 355.0946.
5,6-O-Isopropylidene-2-O-2-chlorobenzyl-3-O-methyl-L-ascor-
bic acid (2i) White solid; mp 81–82 ꢁC; [a]2D5 ? 30.85
(c 0.91 MeOH); IR (KBr): 2989, 1761, 1680, 1465, 1374,
1254, 1133, 1110, 960, 884, 766 cm-1
;
1H NMR
(300 MHz, CDCl3) d 1.34 (s, 3H), 1.39 (s, 3H), 3.96 (s,
3H), 3.97–4.07 (m, 1H), 4.08–4.12 (m, 1H), 4.24 (m, 1H),
4.49 (d, J = 3.0 Hz, 1H), 5.23 (AB q, J = 9 Hz, 2H), 7.25
(m, 2H), 7.39 (m, 1H), 7.49 (m, 1H); 13C NMR (75 MHz,
CDCl3) d 25.6, 25.9, 59.7, 65.2, 70.8, 73.8, 74.6, 110.3,
121.0, 127.0, 129.5, 130.0, 131.2, 133.7, 134.1, 157.5,
169.0; HRMS [MH?] calcd for C17H19O6Cl, 355.0948;
found, 355.0945.
5,6-O-Isopropylidene-2-O-methyl-3-O-(2-methyl)naphtyl-
L-ascorbic acid (2n) Faint yellow solid; mp 118–120 ꢁC;
[a]2D5 ? 43.16 (c 0.5 CHCl3); IR (KBr)/cm-1: 2986, 1755,
5,6-O-Isopropylidene-2-O-4-chlorobenzyl-3-O-methyl-L-
ascorbic acid (2j) White solid; mp 74–75 ꢁC;
[a]2D5 ? 34.46 (c 0.56 MeOH); IR (KBr)/cm-1: 2989, 1769,
1681, 1375, 1349, 953, 846, 804; 1H NMR (300 MHz,
CDCl3) d 1.36 (s, 3H), 1.40 (s, 3H), 3.93 (s, 3H), 4.04 (m,
1H), 4.12 (t, J = 6.6 Hz, 1H), 4.25 (dt, J = 6.6 & 2.4 Hz,
1H), 4.50 (d, J = 2.4 Hz, 1H), 5.11 (AB q, J = 10.2 Hz,
2H), 7.34 (m, 4H); 13C NMR (75 MHz, CDCl3) d 25.6,
25.8, 59.5, 65.1, 72.9, 73.6, 74.4, 110.3, 120.9, 128.7,
130.3, 134.4, 134.5, 157.5, 169.0; HRMS [MH?] calcd for
C17H19O6Cl, 355.0948; found, 355.0956.
1
1681, 884, 859, 797; H NMR (300 MHz, CDCl3) d 1.35
(s, 3H), 1.38 (s, 3H); 3.78 (s, 3H), 4.02–4.14 (m, 2H), 4.32
(m, 1H), 4.56 (d, J = 3 Hz, 1H), 5.61 (s, 2H), 7.44–7.53
(m, 3H), 7.83–7.88 (m, 4H); 13C NMR (75 MHz, CDCl3) d
25.5, 25.8, 60.0, 65.2, 73.5, 73.8, 74.6, 110.3, 123.2, 125.0,
126.5, 126.5, 126.8, 127 0.7, 127.9, 128.5, 132.8, 133.0,
133.2, 155.3, 168.8; HRMS [MH?] calcd for C21H22O6,
371.1495; found, 371.1479.
Riboflavin/NBT method
5,6-O-Isopropylidene-2-O-4-nitrobenzyl-3-O-methyl-L-ascor-
bic acid (2k) Faint yellow solid; mp 108–110 ꢁC;
[a]2D5 ? 12.00 (c 0.5 CHCl3); IR (KBr)/cm-1: 2985, 2446,
1759, 1674, 1607, 1518, 1464, 1371, 1342, 854, 829, 743;
1H NMR (300 MHz, CDCl3) d 1.37 (s, 3H), 1.40 (s, 3H),
4.02–4.07 (m, 4H), 4.15 (m, 1H), 4.29 (m, 1H), 4.54 (d,
J = 3 Hz, 1H), 5.25 (q, J = 12.3 Hz, 2H), 7.59 (d,
J = 8.4 Hz, 2H), 8.24 (d, J = 8.4 Hz, 2H); 13C NMR
(75 MHz, CDCl3) d 25.5, 25.8, 59.6, 65.2, 72.3, 73.5, 74.5,
110.4, 121.1, 123.7, 128.8, 143.3, 147.8, 157.4, 168.8;
The reduction of NBT to the blue formazan at 560 nm, in a
mixture of phosphate buffer (pH 7.8), EDTA-cyanide, NBT,
and extract. After 6 min incubation of small glass tubes
which contained the above mixture in a light box, riboflavin
was added successively to each tube at timed intervals. The
absorbance was read after 6, 12, 18, and 24 min from adding
the riboflavin. One unit of SOD was defined as that amount of
enzyme causing 50 % inhibition of the initial rate of reduc-
tion of the NBT (Janknegt et al., 2007).
123

Med Chem Res (2014) 23:1257–1266
1265
Cytotoxicity assay
DNA cleavage assay
The cytotoxicity of the compounds was assessed against
human breast cancer cell line MCF-7, leukemic cell HL-60,
cervical cell line HeLa, and human leukemic macrophages
THP-1 cell line taken as control procured from the National
Centre for Cell Sciences (NCCS) repository. The cell
viability was assessed by the MTT assay. The number of
viable cells remaining after appropriate treatment was
determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT; Sigma Chemical CO.) assay
(Chen et al., 2004). Briefly, cells were plated (4,000 cells/
well per 0.2 ml RPMI 1640 medium) in 96 well microtiter
plates and incubated overnight. The test agent was then
added at indicated concentrations to quadruplicate wells.
After 24, 48, and 72 h, MTT was added to each well at a
final volume of 0.5 mg/ml and the microplates were
incubated at 37 ꢁC for 3 h. After the supernatant was
removed, the formazan salt resulting from the reduction of
MTT was solubilized in dimethyl sulfoxide (DMSO, Sigma
Chemical Co.) and the absorbance was read at 570 nm
using an automatic plate reader (Molecular Devices Cor-
poration, Sunnyvale CA). The cell viability was extrapo-
lated from optical density (OD)570 values and expressed as
percent survival using this formula and the graph was
plotted.
1.0 mL of 1 9 105 cells/mL of the cell culture was taken
and centrifuged at 10,000 rpm for 15 min. To the super-
natant, 100 lL lysis buffer (without SDS) was added and
kept at room temperature for 5 min and mixed on a cy-
clomixer (vortex). 300 lL of lysis buffer (with SDS) was
added and gently mixed by inversion. This was followed by
the addition of 66 lL of 5 M NaCl. The mixture was
centrifuged at 10,000 rpm for 15–30 min at room temper-
ature, and the supernatant transferred to a new microcen-
trifuge tube. Equal volume of chloroform was added and
mixed gently till it turned milky. The mixture was treated
with RNase for 5 min. Ice-cold isopropanol was added and
incubated for 1 h on ice. The mixture was centrifuged at
10,000 rpm for 15 min and the supernatant was decanted.
The DNA pellet was air dried to remove traces of isopro-
panol and re-suspended in about 50 lL of Tris-ethylene-
diaminetetraacetic acid (EDTA) buffer. The DNA isolated
was run on a gel and absorbance at 260/280 nm was noted
to see the quality of extracted DNA.
Acknowledgments We thank Department of Science and Tech-
nology (DST), New Delhi for financial support (Grant No. SR/S1/OC-
19/2007), Professor Mukund G. Kulkarni (Department of Chemistry,
University of Pune), and Dr. R. J. Barnabas (Ahmednagar College,
Ahmednagar) for helpful discussion and suggestions. Santosh R Kote
thank University Grant Commission (UGC), New Delhi for awarding
fellowship.
ðODÞ570 of drug treated sample ꢁ 100
% Cell viability ¼
ðODÞ570 of drug untreated sample
Optical density mean and standard deviation was graphed
versus log concentration, and IC50 was calculated by a
sigmoidal dose response curve using prism software
(GraphPad, San Diego, CA).
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