Synthesis of dihydrosterculic acid-based monoglucosyl diacylglycerol and its analogues and their biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.12.048

In the present study, Lactobacillus plantarum glycolipid (GL1) molecule in β-configuration and its fatty acid analogues were synthesized using trichloroacetimidate methodology. The β-configuration of the GL1 molecule was unambiguously assigned by NMR studies using 2D-ROESY (NOE) and J-coupling analysis. Dihydrosterculic acid was synthesized using Furukawa's reagent and the selective esterification of dihydrosterculic acid at C-3 position of glycerol was achieved with EDC-HCl at 0 °C. In vitro cytotoxicity of the GL1 molecule and its fatty acid analogues was evaluated against DU145, A549, SKOV3 and MCF7 cell lines. Among all the synthesized molecules, the GL1 molecule and compound 7d showed moderate activity, while the compound 7b showed promising activity against all the tested cell lines with IC₅₀ values of 20.1, 18.2, 19.1 and 17.6 ?1/4M, respectively. In addition, all tested compounds showed poor cytotoxicity against normal HUVEC cells. The MCF7 cells when treated with compound 7b showed lower bromodeoxyuridine incorporation levels as compared to untreated cells, suggesting that the compound 7b was highly effective and inhibited the cell proliferation. In addition, the compounds showed significant increase in caspases 3 and 9 levels by inducing apoptosis in MCF 7 cells.

Full text of DOI:10.1016/j.ejmech.2015.12.048

European Journal of Medicinal Chemistry 109 (2016) 134e145
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis of dihydrosterculic acid-based monoglucosyl diacylglycerol
and its analogues and their biological evaluation
,
,
,
, d
Vudhgiri Srikanth a d, R.B.N. Prasad a d, Y. Poornachandra b d, V.S. Phani Babu c
,
C. Ganesh Kumar b d, B. Jagadeesh c d, Ram Chandra Reddy Jala a
,
,
, d, *
a Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
c Centre for NMR & Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
d Academy of Scientific and Innovative Research, New Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, Lactobacillus plantarum glycolipid (GL1) molecule in
acid analogues were synthesized using trichloroacetimidate methodology. The
b
-configuration and its fatty
-configuration of the
Received 26 September 2015
Received in revised form
2 December 2015
Accepted 24 December 2015
Available online 31 December 2015
b
GL1 molecule was unambiguously assigned by NMR studies using 2D-ROESY (NOE) and J-coupling
analysis. Dihydrosterculic acid was synthesized using Furukawa's reagent and the selective esterification
of dihydrosterculic acid at C-3 position of glycerol was achieved with EDC-HCl at 0 ꢀC. In vitro cytotoxicity
of the GL1 molecule and its fatty acid analogues was evaluated against DU145, A549, SKOV3 and MCF7
cell lines. Among all the synthesized molecules, the GL1 molecule and compound 7d showed moderate
activity, while the compound 7b showed promising activity against all the tested cell lines with IC50
Keywords:
Dihydrosterculic acid
Glycolipid-1
values of 20.1, 18.2, 19.1 and 17.6
mM, respectively. In addition, all tested compounds showed poor
cytotoxicity against normal HUVEC cells. The MCF7 cells when treated with compound 7b showed lower
bromodeoxyuridine incorporation levels as compared to untreated cells, suggesting that the compound
7b was highly effective and inhibited the cell proliferation. In addition, the compounds showed signif-
icant increase in caspases 3 and 9 levels by inducing apoptosis in MCF 7 cells.
Furukawa's reagent
Cytotoxicity
Caspases
Cell proliferation
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
exhibiting various biological activities [10,11,16,17]. Recently, Sau-
vageau and coworkers [18] isolated three new glycolipids, GL1, GL2
Among the available broad spectrum of lipids, glycoglycer-
olipids have gained significant attention in view of their diverse
properties. They are major constituents present in chloroplast
membranes of photosynthetic cells and in bacterial cell walls [1e3].
Various glycoglycerolipid types were earlier isolated from different
sources like bacteria [4], algae [5] and plants [6]. Glycolipids are
well-studied molecules exhibiting different biological activities
such as antitumor [4e9], antimicrobial [10], antiviral [11,12], anti-
inflammatory [13], immunosuppressive [14] and oxygen scav-
enging [15] activities. However, all these activities are strictly
dependent on their fatty acyl chain length [16,17], olefinic nature
[11], and all these bioactive glycoglycerolipids were found to exist
and GL3 from a probiotic L. plantarum strain which exhibited
immunomodulatory properties. Moreover, the major benefits
exhibited by Lactobacilli as a probiotic organism contributes to the
health improvement by exhibiting immunomodulatory properties
which is of significance in the area of glycolipids. In GL1, the fatty
acids like dihydrosterculic acid (9, 10-methyleneoctadecanoic acid)
and oleic acids are present at sn-1 and sn-2 positions, respectively.
Cyclopropenoid fatty acids such as sterculic and malvalic acids are
present in many seed oils of Malvaceae family [19], and lactobacillic
acid is present in Lactobacillus arabinosus and specifically the
cyclopropane fatty acids with C12, C19 and C21 are present in both
Gram-negative and Gram-positive bacteria [20] exhibiting notable
biological activities such as antibiotic, antiviral, antibacterial, anti-
fungal and antitumor activities [19e21]. Further, the oleic acid and
its analogues are known to exhibit antitumor activity [22]. Earlier
in b-configuration [4e8, 10,11,13,14]. Earlier studies indicated that
the fatty acid composition of glycoglycerolipids plays a key role in
reports suggest that many biologically active glycolipids exist in b-
configuration [4e14].
* Corresponding author. Centre for Lipid Research, CSIR-Indian Institute of
Chemical Technology, Uppal Road, Hyderabad 500007, India.
E-mail addresses: jrcreddy10@gmail.com, ramchandra.iict@gov.in (R.C.R. Jala).
http://dx.doi.org/10.1016/j.ejmech.2015.12.048
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
135
However, Sauvageau and coworkers [18] earlier reported the
isolation and characterization of dihydrosterculic acid-based gly-
afford 14 with 100% yield. The formation of cyclopropane ring was
confirmed by 1H NMR analysis, the signals were observed at
colipids from L. plantarum (GL1) with
a
-configuration (Fig. 1a).
d
ꢁ0.33 ppm (q) and disappearance of double bond signals at
Considering the above facts and the biological importance of
dihydrosterculic acid and the significant role of various fatty acid
chain substituents, we herein report the synthesis of dihy-
d
5.34 ppm (m). Dihydrosterculic acid 15 was obtained by hydro-
lysis of methyl dihydrosterculate 14 by lithium hydroxide mono-
hydrate (LiOH.H2O) in THF:H2O (7:3, v/v). Then the diol 5 was
selectively esterified at C-3 OH of glycerol with dihydrosterculic
acid 15 (1 eq.) by using EDC-HCl (1 eq.) and DMAP (cat.) at 0 ꢀC for
6 h which exclusively gave 8 with 98% yield. Under these experi-
mental conditions, 2-acyl glycerol derivative was not observed.
Usually this reaction was reported with DCC [11]; however, the
byproduct DCC-urea was difficult to separate from the product, so
this was esterified with EDC-HCl. At the same time to avoid the acyl
migration of fatty acid from C-3 OH to C-2 OH of glycerol, simul-
taneously oleic acid, EDC-HCl and DMAP were added in equimolar
ratio to the same reaction mixture and stirred for overnight at RT
which gave 9 with 89% yield. For the synthesis of other fatty acid
analogues (6a-6d), EDC-HCl was used at RT for overnight to get
88e92% yields. The selective removal of acetyl groups on the sugar
unit was achieved with hydrazine monohydrate [29], which
drosterculic acid-based glycolipids with b-configuration (Fig. 1b)
using trichloroacetimidate method. Later, these compounds were
evaluated for their cytotoxicity in different cancer cell lines. In
addition, the active compounds were also investigated for the
activation of caspases (both 3 and 9) and the incorporation of
bromodeoxyuridine in human breast carcinoma (MCF 7) cell line.
2. Results and discussion
2.1. Chemistry
In Scheme 1, the synthesis of glucosyl diacyl glycerides started
with acylation of glucose with Ac2O and NaOAc under reflux con-
ditions for 4 h, the acylated product 1 was purified (excess starting
materials and reagents were removed by water wash) by simple
recrystalization [23] with 92% yield. The chemoselective removal of
the acetyl group at anomeric position was achieved by hydrazine
acetate [24] in DMF to give 2 with 92% yield. Subsequently, hemi-
acetal 2 was reacted with trichloroacetonitrile in the presence of
resulted in the formation of b-glucosyl diacylglcerols (10, 7a-7d)
with 75e78% yields. The relative cis stereochemistry [33] of the
cyclopropane ring in compound 10 and 7a was clearly identified by
1H NMR spectrum, the cyclopropane ring methylene protons
appeared as two individual peaks at
d
ꢁ0.34 and 0.56 ppm. The
diastereomers formed in compound (10, 7a), when dihy-
drosterculic acid linked with sugar moiety were inseparable, and
the individual diastereomers [31] were not observed in the NMR
and ROESY (Fig. 3) spectra.
DBU at RT, exclusively to give a-trichloroacetimidate [25] 3 due to
anomeric effect. The coupling of Imidate 3 with (S)-(þ)eSolketal
[26] 12 (which was obtained from mannitol di-acetonoid as shown
in
fluoromethanesulfonate [27] (TMSOTf) at RT, overnight, led to
formation of 4 in econfiguration with 82% yield.
This
-orientation was achieved by NGP of C-2ʹ acetate group of
Scheme
2)
in
presence
of
trimethylsilyl
tri-
b
2.2. NMR spectroscopic studies of compounds 10 and 7b
b
sugar with C-1ʹ carbon of sugar; it allowed only equatorial attack of
The structures of compounds 10 and 7b were unequivocally
established from the 1D-(1H and 13C) and 2D-NMR (1He1H DQF-
COSY, 1He1H ROESY, 13Ce1H HSQC, and 13Ce1H HMBC) spectro-
scopic analysis. The NMR spectral data for compound 10 and
compound 7b are presented in Tables 1 and 2, respectively. In the
1H NMR spectrum of compounds 10 and 7b, the anomeric proton
acceptor. 1H NMR analysis [11] revealed that the signals of H-1ʹ
proton was observed as dd at
d
¼ 4.61 ppm, J ¼ 7.9, 10.52 Hz. Diol 5
was obtained through selective hydrolysis of isopropylidene group
of 4 by zinc nitrate hexahydrate in acetonitrile [28]. Further in the
present study, the reaction times were reduced; the purification
methodologies were simplified by using newer reagents as
compared to those mentioned in the earlier report [29].
H-1ʹ appeared as a doublet at
which clearly revealed the existence of a
[11,34]. In addition, the -configuration of the glucopyranose in
d
4.31 ppm with JH1ʹ-H2ʹ ¼ 7.7 Hz,
b
-linked glucopyranose
Based on Scheme 3, the synthesis of dihydrosterculic (9, 10-
methyleneoctadecanoic) acid was started with preparation of
methyl oleate 13 from oleic acid with 2% methanol-sulphuric acid
(MeOHeH2SO4). This was followed by the reaction using Furukawa
reagent [30e32] to obtain methyl oleate 13 with diethyl zinc
((C2H5)2Zn) and diiodomethane (CH2I2) by stirring overnight at RT.
Then the reaction was quenched with saturated NH4Cl solution
followed by extraction with DCM and further purified by flash
column chromatography (to remove excess diiodomethane only) to
b
compounds 10 and 7b was unambiguously confirmed from the
observed characteristic ROESY correlations between H-1ʹ and H-3ʹ,
between H-3ʹ and H-5ʹ, and between H-5ʹ and H-1ʹ protons (Figs. 2
and 4). The ROESY correlation between H-2ʹ and H-4ʹ in compounds
10 and 7b indicated that the protons are in spatial proximity (1, 3-
diaxial orientation). The sugar to the glycerol linkage was estab-
lished from the observed HMBC spectrum between H-1ʹ and C-1
and H-1 and C-1ʹ. The NOE correlations in ROESY spectrum
Fig. 1. Structures Of GL1 and target molecule.

136
V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
Scheme 1. Reagents and conditions: i) Ac2O, NaOAc, reflux 4 h, 92%; ii) Hydrazine Acetate, DMF, 50 ꢀC, 2 h, 92%, iii) CCl3CN,DBU, DCM, rt, 3 h, 77%; iv) 12, TMSOTf, DCM, 4 Aꢀ MS.,
0
ꢀC to rt, overnight, 82%; v) Zn(NO3)2$6H2O, MeCN, 50 ꢀC, 6 h, 75%; vi) EDC-HCl, DMAP, DCM, Fatty acid, 0 ꢀC to rt, overnight, 88e92%; vii) N2H4$H2O,aq EtOH (85%), 44 ꢀC, 6 h,
75e78%; viii) EDC-HCl, DMAP, DCM, Fatty acid(1 eq.), 0 ꢀC, 6 h, 98%.
Scheme 2. Reagents and conditions: i) Anhydrous ZnCl2, Acetone, K2CO3, 2.5 h, rt, 90%; ii) NalO4, NaHCO3, DCM, rt, 45 min; iii) EtOH, NaBH4, NH4Cl, 2 h, rt, 85%.
between H-1ʹ and H-1a, H-1ʹ and H-1b, and H-1ʹ and H-2 protons
strongly support the presence of linkage between sugar and glyc-
erol in compounds 10 and 7b. In the 1H NMR spectrum of com-
pound 10, the methylene protons of the cyclopropane ring
Furthermore, the relative cis stereochemistry of the cyclopropane
in compound 10 was unambiguously established from the observed
ROESY correlations between H-13a, H-11 and H-13a and H-15
protons (Fig. 3). The observed ROESY correlations between H-13b
and H-12 and between H-13b and H-14 protons clearly indicated
that the protons were in the same plane of cyclopropane ring.
appeared as two individual peaks at
d
ꢁ0.34 and 0.56 ppm, which
clearly suggests the cis stereochemistry of the cyclopropane [33].

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
137
Scheme 3. Reagents and conditions: i) MeOHeH2SO4, rt, 3 h, 98%; ii) (C2H5)2Zn, CH2I2, DCM, ꢁ5 ꢀC to rt, overnight, 100%; iii) LiOH, THF:H2O (7:3), overnight, rt, 99%.
Fig. 2. 1He1H ROESY (Rotating-frame Overhauser Effect Spectroscopy) spectrum of compounds 10 and 7b (CDCl3, 298 K).
2.3. Biology
2.3.1. In vitro cytotoxicity evaluation
Glycoglycerolipid analogues have received increased attention
from a cancer chemoprevention perspective due to their promising
inhibitory effect on tumour growth. The fatty acyl chain length, its
position, and the nature of the sugar moiety are some of the
important features that influence the cytotoxicity [35,36]. In the
present study, the cytotoxic activities of all the synthesized com-
pounds (10, 9, 6a-6d, 7a-7d) were determined on a panel of four
human tumour cell lines, namely DU145, A549, SKOV3 and MCF7.
The data to this regard are presented in Table 3. A comparison of the
unsaturated vs. saturated compounds showed that enhanced
cytotoxicity values were noticed for unsaturated compounds. It was
observed that the target molecule 10 showed moderate cytotoxicity
against all the tested cell lines with IC50 values ranging between
35.6 and 41.6 mM and this may plausibly be due to the presence of a
mixture of unsaturated and saturated fatty acids. Among all the
tested analogues, the oleic acid analogue 7b exhibited good cyto-
toxicity against all the cell lines, DU145, A549, SKOV3 and MCF7
which showed IC50 values ranging between 17.6 and 20.1 mM. Based
on these results, it was noticed that the oleoyl moiety contributed
significantly to the anti-tumour activity. This observation corrob-
orates with the earlier findings of Nagatsu and coworkers [17].
However, cytotoxicity values exhibited by the dihydrosterculic acid
Fig. 3. Expanded region of 1He1H ROESY (Rotating-frame Overhauser Effect Spec-
troscopy) spectrum of compound-10 (CDCl3, 298 K).

138
V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
Table 1
1H and13C NMR spectral data for compound 10 (in CDCl3).
Position
d1H (ppm)
d13C (ppm)
COSY (1He1H)
HMBC (1He13C)
1ʹ
2ʹ
3ʹ
4ʹ
5ʹ
6ʹ
1a
1b
2
3a
3b
4
23
5
24
6
25
*7-10
*25-29
11
15
12
14
13a
13b
*16-20
*34-38
21
39
22
40
30
33
31
32
4.31 (d, J ¼ 7.7 Hz)
103.3
73.3
76.2
69.4
75.7
61.4
68.1
68.1
70.0
62.7
62.7
173.8
173.5
34.1
34.1
24.8
24.8
e
2ʹ
1ʹ, 3ʹ
2ʹ, 4ʹ
3ʹ, 5ʹ
4ʹ, 6ʹ
5ʹ
1b, 2
1a, 2
1a, 1b, 3a, 3b
2, 3b
2, 3a
e
C-1
C-1ʹ
C-4ʹ
C-3ʹ
3.37 (m)
3.53 (m)
3.59 (m)
3.31 (m)
3.83 (m)
e
e
3.70 (m)
C-1ʹ, C-2, C-3
C-1ʹ, C-2, C-3
C-1, C-23
C-1, C-2, C-4
C-1, C-2, C-4
e
3.90 (ABX, J ¼ 6.1, 11.2 Hz)
5.26 (m)
4.18 (ABX, J ¼ 6.7, 12.1 Hz)
4.38 (m)
e
e
e
e
2.3 (t, J ¼ 7.3 Hz)
6
25
5, 7
24, 26
C-4, C-5, C-6
C-23, C-24, C-25
C-4, C-5
C-23, C-24
2.3
1.59 (m)
1.59
e
e
e
e
e
e
e
1.1 (m)
1.1
0.64 (m)
0.64
29.0
29.0
15.5
15.5
10.8
10.8
e
10, 12
14, 16
11, 13a, 13b
13a, 13b, 15
13b, 12, 14
13a, 12, 14
C-11, C-12
C-14, C-15
e
e
ꢁ0.34 (q, J ¼ 4.5 Hz)
C-11, C-12, C-14, C-15
C-11, C-12, C-14, C-15
0.56 (m)
e
e
e
e
e
e
e
1.28 (m)
1.28
e
22
40
21
39
29, 31
32, 34
30
e
e
e
0.88 (t, J ¼ 6.5 Hz)
14.1
14.1
27.1
27.1
129.7
129.7
e
0.88
e
2.0 (m)
2.0
5.33 (m)
5.33
C-31
C-32
C-30, C-31
C-32, C-33
33
*Overlapped signals.
analogue 7a on all the tested cell lines were very low due to the
presence of saturated fatty acids and the acetylated analogues (6a,
6b and 6d) showed no cytotoxicity against all the tested cell lines.
Since the synthesized dihydrosterculic acid-based monoglucosyl
diacylglycerol and its analogues are strong amphiphilic derivatives,
and they may be usually toxic for the cell via membrane perme-
ation. Considering this fact, we further checked all the compounds
for cytotoxicity against normal HUVEC cell line and it was observed
that the test compounds showed poor cytotoxicity against these
normal cells, which suggests that the tested compounds are
biocompatible in nature towards the normal cells.
the absorbance values were measured at 450 nm for estimating the
amount of bromodeoxyuridine incorporated within the MCF-
7 cells. The cells treated with compound 7b showed lower bro-
modeoxyuridine incorporation levels as compared to untreated
cells which indicated that the compound was highly effective and
inhibited the cell proliferation (Fig. 5).
2.3.3. Caspase activity
Apoptosis is an intricate process involving a cascade mechanism
that uses many proteins and enzymes. However, the key enzymes
in this process are the caspases, a family of cysteine proteases that
control and mediate the apoptotic response. Caspases are a family
of genes important for maintaining homeostasis and provide crit-
ical links in cell regulatory networks through the regulation of or-
dered processes such as cell death and inflammation. Some of the
caspases like 3, 6, 7, 8, and 9 are known to play a key role in the
induction of apoptosis [38]. In addition, these enzymes have gained
a renewed interest in the recent past to understand the involve-
ment of cytotoxicity with that of apoptosis by the test compound.
The initiator caspases activate executioner caspases that subse-
quently coordinate their activities to demolish key structural pro-
teins and activate other enzymes. The initiator caspase-9 is
responsible for the intrinsic apoptotic pathway [39]. Among all
caspases, caspase 3 is one of the most important effector caspase
which is activated by caspase 9 due to the proteolytic cleavage of
procaspase 3. In addition, caspase activation is required for tumor
cell death and elimination of oncogenic potential. In the absence of
caspase activation, the tumor cells show long-term survival and
growth in the biological system. Considering these facts, the MCF7
2.3.2. Bromodeoxyuridine cell proliferation assay
Based on the cytotoxicity results, further investigations were
carried out on active compound 7b by performing the bromo-
deoxyuridine cell proliferation assay [37], which mainly detects the
incorporation of bromodeoxyuridine into the cellular DNA during
cell proliferation using an anti-BrdU antibody conjugated to
peroxidase as a probe. When the cells are cultured with the labeling
medium that contains BrdU, this pyrimidine analog is incorporated
in place of thymidine into the newly synthesized DNA of the
proliferating cells. The enzyme horseradish peroxidase catalyzes
the conversion of the chromogenic substrate tetra-
methylbenzidine (TMB) from
a colorless solution to a blue
colored solution, the intensity of which is proportional to the
amount of incorporated BrdU in the cells. After treatment of MCF-
7 cells with compound 7b, the bromodeoxyuridine incorporated in
the newly synthesized DNA of actively proliferating cancer cells
was quantified. After 24 h incubation with the test compound 7b,

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
139
Table 2
1H and13C NMR spectral data for compound 7b (in CDCl3).
No
d1H (ppm)
d13C (ppm)
COSY (1He1H)
HMBC (1He13C)
1ʹ
2ʹ
3ʹ
4ʹ
5ʹ
6ʹ
1a
1b
2
4.31 (d, J ¼ 7.7 Hz)
103.3
73.3
76.2
69.4
75.7
61.4
68.1
68.1
70.0
62.7
62.7
173.8
173.5
34.0
34.0
24.6
24.6
e
2ʹ
C-1
C-1ʹ
C-4ʹ
C-3ʹ
3.37 (m)
1ʹ, 3ʹ
2ʹ, 4ʹ
3ʹ, 5ʹ
4ʹ, 6ʹ
5ʹ
1b, 2
1a, 2
1a, 1b, 3a, 3b
2, 3b
2, 3a
e
3.53 (m)
3.59 (m)
3.31 (m)
3.83 (m)
3.70 (m)
3.90 (m)
5.26 (m)
e
e
C-1ʹ, C-2, C-3
C-1ʹ, C-2, C-3
C-1, C-23
C-1, C-2, C-4
C-1, C-2, C-4
e
3a
3b
4
22
5
23
6
24
*7-10
*25-28
11
14
29
32
12
13
30
31
*15-19
*33-37
20
38
21
39
4.18 (ABX, J ¼ 6.61, 12.13 Hz)
4.38 (m)
e
e
e
e
2.31 (m)
2.31
1.60 (m)
1.60
6
24
5, 7
23, 25
e
C-4, C-5, C-6
C-22, C-23, C-24
C-4, C-5, C-6
C-22, C-23, C-24
e
e
e
e
e
e
2.01 (m)
2.01
2.01
2.01
5.34 (m)
5.34
5.34
5.34
27.2
27.2
27.2
27.2
129.8
129.8
129.8
129.8
e
10, 12
13, 15
28, 30
31, 33
11
14
29
32
e
C-11, C-12
C-13, C-14
C-29, C-30
C-31, C-32
C-11, C-12
C-13, C-14
C-29, C-30
C-31, C-32
e
e
e
e
e
e
1.29 (m)
1.29
e
21
39
20
38
e
e
e
0.88 (t, J ¼ 6.8 Hz)
14.1
14.1
e
0.88
e
*Overlapped signals.
cells when treated with compound 7b along with doxorubicin
(positive control) at two different concentrations (5 and 10 M),
the cell proliferation. In addition, the compound 7b showed a sig-
nificant increase in the levels of both caspases 3 and 9.
m
showed activation in the levels of both caspase 9 (Fig. 6a) and
caspase 3 (Fig. 6b). The estimated caspase levels observed were
nearly 2e3 folds higher as compared to the untreated cells. This
activation in caspase 3 and caspase 9 levels suggests that the
compound 7b has the ability to induce apoptosis in MCF7 cells via
an intrinsic pathway.
4. Experimental
4.1. Materials and general experimental procedures
All the chemicals used in these schemes were of analytical grade
and they were obtained from different commercial sources and
were used without any further purification. All the dry reactions
were carried out under nitrogen atmosphere using anhydrous
freshly distilled solvents and 4 Å molecular sieves in flame dried
glassware using standard gas-light syringes, cannulas and septa.
Reactions were monitored on micro TLC plates (coated with TLC
grade silica gel, obtained from Merck) and the spots were detected
by iodine vapors. Column chromatography was performed by using
silica gel (100e200 mesh) procured from Qualigens (India) using
freshly distilled solvents. All the 1H NMR and 13C NMR spectra were
recorded with a Bruker Avance (operated for 1H NMR at 300 MHz,
500 MHz, 600 MHz and for 13C NMR at 75 MHz, 125 MHz, 175 MHz)
spectrometer, using TMS (
standard for chemical shifts (
values are presented in ppm (parts per million) units. Mass spectra
were recorded with HR-ESI-MS. IR spectra were recorded in chlo-
roform on a PerkineElmer FT-IR spectrum BX. The melting points
were determined on a Barnstead Electro Thermal 9200 Instrument.
3. Conclusions
In conclusion, we have synthesized the L. plantarum GL1
molecule with beconfiguration using trichloroacetimidate meth-
odology with good yields for the first time. The purification process
was simplified and the main fatty acid, dihydrosterculic acid in GL1
was synthesized from oleic acid using Furukawa reagent with 100%
yield. We also refined the complicated purification process by
replacing N,N'-dicyclohexylcarbodiimide adopted in earlier studies
with N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride as a coupling reagent for esterification to obtain water
soluble byproducts. Further, the selective esterification of primary
hydroxyl group of glycerol was achieved with the same reagent at
0 ꢀC with high yield. The in vitro cytotoxicity evaluation of the target
molecule and its fatty acid analogues exhibited good to moderate
anti-tumor activity. Among the tested compounds, the oleic acid
analogue 7b exhibited promising cytotoxicity against all the tested
cell lines. In addition, all tested compounds showed poor cytotox-
icity against normal HUVEC cells. It was observed that unsaturation
in fatty acids played a significant role in contributing to the cyto-
toxicity. The bromodeoxyuridine cell proliferation assay also
showed that the compound 7b was highly effective and inhibited
d
¼ 0 ppm) and
d 77.00 ppm as internal
d
) in CDCl3 at 25 ꢀC. The chemical shift
4.2. 1, 2, 3, 4, 6-penta-O-acetyl-b-D-glucopyranoside (1)
A mixture of dextrose (5.0 g, 27.77 mmol) and sodium acetate
(6.83 g, 83.33 mmol) were dissolved in acetic anhydride (43 mL,

140
V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
Fig. 4. Schematic representation of the characteristic NOEs (shown as curves) that supported b-configuration of the glucopyranose residue of compounds 10 and 7b
Table 3
In vitro cytotoxicity evaluation of different glycoglycerolipid analogues.
Test compound
DU145
A549
SKOV3
MCF7
HUVEC
10
7a
7b
7c
7d
9
6a
38.9 0.44
209.7 0.55
20.1 0.26
119.3 0.62
54.9 0.48
186.0 0.63
e
41.6 0.34
123.6 0.62
18.2 0.28
105.8 0.54
35.6 0.46
ea
35.6 0.33
112.5 0.63
19.1 0.22
89.6 0.48
44.4 0.22
e
38.2 0.28
99.8 0.58
17.6 0.18
32.5 0.42
32.6 0.32
94.2 0.49
e
99.8 0.42
119.8 0.35
110.2 0.12
126.3 0.43
96.1 0.24
121.5 0.28
e
e
e
6b
e
e
e
e
e
6c
6d
33.0 0.18
e
30.2 0.38
e
29.4 0.33
e
25.6 0.29
e
118.9 0.38
e
Doxorubicin (Control)
0.7 0.11
0.8 0.09
0.7 0.12
0.9 0.08
79.6 0.32
a
¼
No activity, DU145: human prostate cancer cells (ATCC No. HTB-81), A549: human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), SKOV3: human ovarian
cancer cell line (ATCC No. HTB-77), MCF-7: human breast adenocarcinoma cells (ATCC No. HTB-22), HUVEC: normal human umbilical vein epithelial cells (ATCC No. CRL-1730).
20.5, 20.4; IR (CHCl3) 3027, 2969, 2951, 2907,1756, 1743, 1422, 1371,
1322, 1225, 1067,1048, 912, 756, 704, 641,599 cm ꢁ1; HRMS (ESI) m/
z [MþNa]-calc for C16H22O11Na 413.10543 found 413.10449.
4.3. 2, 3, 4, 6-O-tetra acetyl-glucopyranose hemiacetal (2)
Hydrazine acetate (1.04 g, 11.28 mmol) was added to a solution
of 1, 2, 3, 4, 6-penta-O-acetyl-b-D-glucopyranoside (1) (4 g,
10.25 mmol) in DMF (40 mL) at 50 ꢀC and stirred the reaction for 2 h
under N2. Using TLC (hexane-EtOAc, 1:1, v/v) the formation of
product was observed with the disappearance of starting material;
the mixture was diluted with EtOAc, washed with aqueous 5% NaCl
and water, dried over anhydrous Na2SO4 and concentrated to give
yellow oil. This crude oil was subjected to silica gel column chro-
matography. The required product was eluted in hexane: EtOAc
(2:1, v/v) solvent mixture as syrup (92%, 3.28 g). 1H NMR (500 MHz,
Fig. 5. Effect of compound 7b on the Bromodeoxyuridine content incorporated in
MCF7 cell line. Doxorubicin was used as a positive control. Statistical analysis was
performed using GraphPad PRISM software version 3.0. All experimental data were
compared using Student's t-test. All the experiments were performed on triplicates
and each value is represented as mean SD (n ¼ 3).
CDCl3)
d
5.54 (t, J ¼ 9.9 Hz, 1H), 5.46 (d, J ¼ 2.8 Hz, 1H), 5.09 (t,
J ¼ 9.4 Hz, 1H), 4.8e4.9 (m, 1H), 4.22e4.29 (m, 2H), 4.10e4.16 (m,
1H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H); 13C NMR
458 mmol). The reaction mixture was refluxed for 4 h at 90 ꢀC. After
the reaction time, the reaction mixture was cooled to RT and then
poured into the beaker containing crushed ice (250 mL) under
stirring conditions. The penta-acetate was precipitated. The pre-
cipitation was filtered and washed with ice-cold water until the
odour of the acetic acid was disappeared. The crude product was
purified by recrystalization from MeOH to afford the title com-
pound (9.96 g, 92%) as a white crystalline solid. mp 130e135 ꢀC, lit.
(75 MHz, CDCl3)
d 171.06,170.5170.3, 169.7, 95.3, 89.9 72.9, 72.4,
71.8, 71.1, 69.9, 68.4, 66.9, 61.9, 60.5, 30.1, 29.6, 20.68, 20.6; IR
(CHCl3) 3460, 3024, 1748, 1369, 1235, 1038, 756 cmꢁ1; HRMS (ESI)
m/z [MþNa]-calc for C14H20O10Na ¼ 371.09487 found 371.09459.
4.4. 2, 3, 4, 6-tetra-O-acetyl-
(3)
a-glucopyranosyl trichloroacetimidate
132e133.5 ꢀC; 1H NMR (500 MHz, CDCl3)
d
5.72 (d, J ¼ 8.3 Hz, 1H),
2,3,4,6-O-tetra-acetyl-glucopyranose hemiacetal (2) (3 g,
8.62 mmol) was treated with trichloroacetonitrile (8.6 mL,
86.2 mmol) and DBU (0.26 mL, 3.44 mmol) in anhydrous DCM
(30 mL) and stirred for 2 h at RT. After 2 h, the reaction mixture was
concentrated under reduced pressure and purified by silica gel
5.26 (t, J ¼ 9.3 Hz, 1H), 5.11e5.16 (m, 2H), 4.29 (dd, J ¼ 4.5, 12.5 Hz,
1H), 4.11 (dd, J ¼ 2.1, 12.5 Hz, 1H), 3.83e3.86 (m, 1H), 2.11 (s, 3H),
2.09 (s, 3H), 2.03 (s, 6H), 2.01 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
170.4169.9, 169.2, 169, 168.7, 91.5, 72.6, 72.5, 70, 67.5, 61.3, 20.6,

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
141
Fig. 6. Effect of compound 7b and doxorubicin (positive control) at different concentrations on MCF-7 cells on (a) Caspase 9 and (b) Caspase 3 activity. The enzymatic activity was
assessed using AFC conjugated Ac-DEVD (for caspase 3) and Ac-LEHD (for caspase 9) substrates. Statistical analysis was performed using GraphPad PRISM software version 3.0. The
results are expressed as means of fluorescence intensity standard deviation (SD), (n ¼ 3) of three independent experiments performed in triplicates.
column chromatography. The required product was eluted in hex-
ane: EtOAc (4:1, v/v) solvent mixture with 77% yield (3.26 g). 1H
solid (1.64 g, 75%). 1H NMR (500 MHz, CDCl3):
d
5.22 (t, J ¼ 9.6 Hz,
1H), 5.07 (t, J ¼ 9.9 Hz,1H), 5.0 (t, J ¼ 9.4 Hz,1H), 4.55 (d, J ¼ 7.93 Hz,
NMR (300 MHz, CDCl3)
d
8.6 (s, 1H), 6.61 (d, J ¼ 3.02 Hz, 1H), 5.5 (br
1H), 4.17e4.24 (m, 2H), 3.59e3.88 (m, 6H), 2.1 (s, 3H), 2.06 (s, 3H),
s, 1H), 5.34e5.46 (m, 2H), 4.45 (t, J ¼ 6.7 Hz, 1H), 4.06e4.2 (m, 2H),
2.03 (s, 3H), 2.01 (s, 3H); 13C NMR (75 MHz, CDCl3)
d
170.7, 170.1,
2.17 (s, 3H), 2.04e2.03 (2s, 9H); IR (CHCl3) 3478, 3347, 2135, 1748,
169.6, 169.4, 101.3, 101.2, 72.5, 71.9, 71.2, 70.4, 68.3, 63.3, 61.8, 20.6,
1676, 1371, 1218, 1073, 756 cmꢁ1
.
20.5; IR (CHCl3) 3482, 2945, 1753, 1369, 1230, 1041, 757 cmꢁ1
;
HRMS (ESI) m/z [M þ NHþ4 ]-calc for C17H26O12NHþ4 ¼ 440.17625
b-D
-20,30,40,6'-tetra-O-acetyl-
found 440.17527.
4.5. 1, 2eisopropylidene-3-O-(
glucopyranosyl)-sn-glycerol (4)
4.7. Gen0eral 0procedures for synthesis of mono acid 1,2-O-diacyl-3-
Imidate (3) (3.2 g, 6.5 mmol), (S)-(þ)-Solketal (1.28 g,
9.75 mmol) and 4 Aꢀ molecular sieves were dissolved in freshly
distilled DCM (35 mL). To this reaction mixture TMSOTf (0.35 mL,
1.95 mmol) was added drop wise at 0 ꢀC and the reaction mixture
was slowly allowed to RT and stirred for overnight. After all the
starting materials have been used, the reaction mixture was filtered
and dissolved in EtOAc and water. The organic layer was extracted
with aq. NaHCO3 solution, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude mixture was pu-
rified by silica gel column chromatography. The required spot was
eluted in hexane: EtOAc (3:1, v/v) solvent mixture as a white solid
O-(
6d)
b-D
-2 ,30,4 ,6'-tetra-O-acetyl-glucopyranosyl)-sn-glycerols (6a-
3-O-(b
-D-20,30,40,6'-tetra-O-acetyl-glucopyranosyl)-sn-glycerol
(5) (0.23 mmol) was dissolved in anhydrous dichloromethane
(5 mL), fatty acid (0.47 mmol), N-(3-Dimethylaminopropyl)-
N0ethylcarbodiimide hydrochloride (EDC) (0.47 mmol) and DMAP
(catalytic amount) were added under nitrogen at 0 ꢀC. The reaction
mixture was stirred for overnight at RT. After all the starting ma-
terials have been used, the reaction mixture was dissolved in CHCl3
and washed with water and brine solution. The organic layer was
dried over anhydrous sodium sulphate and evaporated under
reduced pressure. The mixture was purified by silica gel column
chromatography using hexane: EtOAc (4: 1, v/v) solvent mixture to
give title compounds (6a-6d) in 88e92% yields.
(2.46 g, 82%). 1H NMR (500 MHz, CDCl3):
d 5.19 and 5.21 (dd,
(apparent t), J ¼ 9.4 and 9.4 Hz, each 1H), 5.05e5.1 (m, 1H),
4.97e5.01 (m, 1H), 4.61 (dd, J ¼ 7.93, 10.52 Hz, 1H), 4.27e4.21 (m,
2H), 4.14 (dd, J ¼ 2.4, 12.3 Hz, 1H), 3.99e4.05 (m, 1H), 3.87 (dd,
J ¼ 4.2, 10.6 Hz, 1H), 3.78e3.81 (m, 1H), 3.68e3.74 (m, 1H), 3.64 (dd,
J ¼ 5.8,10.6 Hz,1H), 2.09 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 2.0 (s, 3H),
4.7.1. 1,2-O-di-dihydrosterculoyl-3-O-(
acetyl-glucopyranosyl)-sn-glycerol (6a)
b
-D-20,30,40,6'-tetra-O-
1.34 (s, 3H), 1.41 (s, 6H); 13C NMR (75 MHz, CDCl3)
d 170.6, 170.2,
169.4, 169.3, 109.5, 109.3, 100.8, 100.7,74.3, 74.2, 72.7, 72.6, 71.7,
71.09, 70.4, 69.09, 68.3, 66.6, 66.06, 61.8, 26.7, 26.5, 25.3, 25.1, 20.7,
The title compound was obtained in hexane: EtOAc (4: 1, v/v)
solvent mixture with 88% yield (0.203 g). 1H NMR (500 MHz,
20.6, 20.5; IR (CHCl3) 2987, 2943, 1755, 1371, 1222, 1041, 757 cmꢁ1
;
CDCl3):
d
5.21 (dd, (apparent t), J ¼ 9.6, 9.4 Hz, overlapped, 1H), 5.18
HRMS (ESI) m/z [MþNa]-calc for C20H30O12Na ¼ 485.16295 found
(dd, (apparent t), overlapped, 1H), 5.07 (t, J ¼ 9.6 Hz, 1H), 4.96e5.01
(m, 1H), 4.53 (t, J ¼ 7.0 Hz, 1H), 4.24e4.31 (m, 2H), 4.07e4.14 (m,
2H), 3.95 (dd, J ¼ 5.01, 11.1 Hz, 1H), 3.66e3.71 (m, 2H), 2.3 (t,
J ¼ 8.5 Hz, 4H), 2.09 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 2.0 (s, 3H),
1.58e1.63 (p, 4H), 1.27e1.37 (m, 46H), 1.1e1.16 (m, 4H), 0.88 (t,
J ¼ 6.8 Hz, 6H), 0.62e0.67 (m, 4H), 0.54e0.58 (m, 2H), -0.33 (q,
485.16118.
4.6. 3-O-(
(5)
b
-
D
-20,30,40,6'-tetra-O-acetyl-glucopyranosyl)-sn-glycerol
-20,30,40,6'-tetra-O-acetyl-glucopyr-
1,2eIsopropylidene-3-O-(
b-
D
J ¼ 5.03 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 173.28, 172.8, 170.6,
170.2, 169.3, 169.2, 101, 72.5, 71.8, 70.9, 69.8, 69.5, 68.3, 68.2, 67.6,
67.5, 62.1, 61.7, 34.2, 34, 31.8, 30.1, 29.6, 29.3, 29.1, 28.6, 24.8, 22.6,
20.7, 20.5, 15.6, 15.7, 14.1, 10.8; IR (CHCl3) 2924, 2853, 1751, 1460,
anosyl)-sn-glycerol (4) (2.4 g, 5.19 mmol) was dissolved in aceto-
nitrile (25 mL) and 5 equiv. zinc nitrate hexahydrate (7.72 g,
25.97 mmol) was added, the reaction mixture was stirred for 6 h at
50 ꢀC. After completion of reaction, the starting materials and the
organic solvent was evaporated under reduced pressure and the
mixture was portioned in water and chloroform. The organic phase
was dried over anhydrous sodium sulphate and evaporated. The
crude reaction mixture was purified by silica gel column chroma-
tography using hexane: EtOAc (5:1, v/v) solvent mixture to give
1223, 1041 cmꢁ1
;
HRMS (ESI) m/z [M
þ
NHþ4 ]-calc for
C
55H94O14NHþ4 ¼ 996.69818 found 996.69614.
4.7.2. 1,2-O-dioleoyl-3-O-(b-D
-20,30,40,6'-tetra-O-acetyl-
glucopyranosyl)-sn-glycerol (6b)
The title compound was obtained in hexane: EtOAc (4: 1, v/v)

142
V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
solvent mixture with 90% yield (0.202 g). 1H NMR (300 MHz,
CDCl3):
overlapped, 1H), 5.17 (overlapped, 1H), 5.08 (t, J ¼ 9.8 Hz, 1H),
4.95e5.02 (m, 1H), 4.53 (dd, J ¼ 4.5, 8.3 Hz, 1H), 4.23e4.32 (m, 2H),
4.05e4.15 (m, 2H), 3.95 (dd, J ¼ 4.5, 10.5 Hz, 1H), 3.64e3.72 (m, 2H),
2.29 (t, J ¼ 7.5 Hz, 4H), 2.1 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s,
3H), 2.02 (8H, overlapped), 1.58e1.66 (p, 4H), 1.28 (m, 40H), 0.88 (t,
4.8.1. 1,2-O-di-dihydrosterculoyl-3-O-
glycerol (7a)
The title compound was obtained in CHCl3: MeOH (19: 1, v/v)
solvent mixture with 76% yield (0.125 g). 1H NMR (300 MHz, CDCl3):
b-D-glucopyranosyl-sn-
d
5.3e5.38 (m, 4H), 5.2 (dd, (apparent t), J ¼ 9.6, 9.4 Hz,
d
5.26 (br s,1H), 4.39 (br t, J ¼ 9.8 Hz,1H), 4.32 (d, J ¼ 7.5 Hz,1H), 4.17
(ABX, J ¼ 6.9, 12.1 Hz, 1H), 3.9 (ABX, J ¼ 5.2, 10.5 Hz, 1H), 3.82 (m,
2H), 3.65e3.71 (m, 1H), 3.48e3.6 (m, 2H), 3.28e3.39 (m, 2H), 2.29
(br t, J ¼ 7.3 Hz, 4H), 1.58e1.66 (p, 4H), 1.25e1.35 (m, 40H),
1.09e1.15 (m, 4H), 0.88 (t, J ¼ 6.7 Hz, 6H), 0.6e0.67 (m, 4H),
0.51e0.58 (m, 2H), -0.34 (q, J ¼ 4.5 Hz, 2H); 13C NMR (75 MHz,
J ¼ 6.7 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 173.2, 172.9, 172.8, 170.6,
170.2, 169.3, 169.2, 129.9, 129.6, 101, 72.5, 71.8, 71, 69.6, 69.5, 68.1,
67.6, 67.5, 62.1, 61.7, 34.1, 34, 31.8, 29.7, 29.6, 29.4, 29.2, 29.1, 27.18,
27.14, 24.8, 22.6, 20.7, 20.5, 14.09; IR (CHCl3) 2925, 2855, 1752, 1375,
1226, 1045, 905 cmꢁ1; HRMS (ESI) m/z [M þ NHþ4 ]-calc for
CDCl3)
d 173.8, 173.7, 173.5, 173.4, 103.4, 103.2, 76.1, 75.7, 73.2, 73.1,
70, 69.2, 68, 62.9, 62.7, 62.5, 61.2, 61.1, 60.9, 34.2, 34.1, 31.9, 30.1,
29.6, 29.5, 29.4, 29.3, 29.1, 28.6, 24.8, 22.6, 15.6, 14.1, 10.9; IR (CHCl3)
3403, 2924, 2854, 1739, 1460, 1165, 1076, 721 cmꢁ1; HRMS (ESI) m/z
[M þ NHþ4 ]-calc for C47H86O10NHþ4 ¼ 828.65592 found 828.65492.
C
53H90O14NHþ4 ¼ 968.66688 found 968.66527.
4.7.3. 1,2-O-di-10-undecenoyl-3-O-(
glucopyranosyl)-sn-glycerol (6c)
b-D
-20,30,40,6'-tetra-O-acetyl-
4.8.2. 1,2-O-dioleoyl-3-O-b-D-glucopyranosyl-sn-glycerol (7b)
The title compound was obtained in hexane: EtOAc (4: 1, v/v)
The title compound was obtained in CHCl3: MeOH (19: 1, v/v)
solvent mixture with 92% yield (0.164 g). 1H NMR (500 MHz,
solvent mixture with 78% yield (0.128 g). 1H NMR (600 MHz,
CDCl3):
d
5.76e5.84 (m,2H), 5.2 (dd, (apparent t), J ¼ 9.6, 9.4 Hz,
CDCl3):
d 5.37e5.31 (m, 4H), 5.27 (m, 1H), 4.4-4.37 (m,1H), 4.31 (d,
overlapped, 1H), 5.18 (dd, (apparent t), overlapped, 1H), 5.08 (t,
J ¼ 9.9 Hz, 1H), 4.96e5.01 (m, overlapped, 3H), 4.91e4.94 (m,
overlapped, 2H), 4.53 (t, J ¼ 7.7 Hz, 1H), 4.23e4.31 (m, 2H),
4.06e4.14 (m, 2H), 3.95 (dd, J ¼ 4.8, 10.9 Hz, 1H), 3.65e3.71 (m, 2H),
2.29 (t, J ¼ 9 Hz, 4H), 2.09 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s,
3H), 2.02 (overlapped, 4H) 1.57e1.63 (p, 4H), 1.34e1.39 (m, 4H),
J ¼ 7.7 Hz, 1H), 4.18 (ABX, J ¼ 6.61, 12.13 Hz, 1H), 3.92-3.88 (m, 1H),
3.83 (m, 2H), 3.73e3.68 (m, 1H), 3.57 (m, 1H), 3.53 (m, 1H), 3.38 (m,
1H), 3.31 (m, 1H), 2.31 (m, 4H), 2.01 (m, 8H), 1.59 (m, 4H), 1.25e1.29
(m, 44H), 0.88 (t, J ¼ 6.8 Hz, 6H); 13C NMR (175 MHz, CDCl3)
d 173.8,
173.7, 173.5, 173.4, 129.9, 129.6, 103.4, 76.1, 75.8, 73.2, 73.1, 70, 69.1,
68, 62.9, 62.8, 61.2, 34.2, 34.1, 31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1,
27.2, 24.8, 22.6, 14.1; IR (CHCl3) 3396, 2924, 2854, 1739, 1460, 1166,
1.25e1.3 (m, 22H); 13C NMR (75 MHz, CDCl3)
d 173.2, 172.7, 170.6,
1077, 722 cmꢁ1
C
;
HRMS(ESI) m/z [M
þ
NHþ4 ]-calc for
170.1, 169.3, 169.1, 139.4, 114.1, 100.9, 72.5, 71.8, 70.9, 69.7, 68.1, 67.6,
67.5, 62.1, 61.7, 34.1, 33.9, 33.7, 31.8, 29.6, 29.2, 29.1, 28.9, 28.8, 24.7,
22.6, 20.66, 20.5; IR(CHCl3) 2927, 2855, 1753, 1374, 1229, 1040,
45H82O10NHþ4 ¼ 800.62462 found 800.62385.
908 cmꢁ1
C
;
HRMS (ESI) m/z [M
þ
NHþ4 ]-calc for
4.8.3. 1,2-O-di-10-undecenoyl-3-O-b-D-glucopyranosyl-sn-glycerol
39H62O14NHþ4 ¼ 772.44778 found 772.44793.
(7c)
The title compound was obtained in CHCl3: MeOH (19: 1, v/v)
solvent mixture with 78% yield (0.096 g). 1H NMR (300 MHz,
4.7.4. 1,2-O-didodecanoyl-3-O-(
glucopyranosyl)-sn-glycerol (6d)
b
-D-20,30,40,6'-tetra-O-acetyl-
CDCl3):
d 5.72e5.86 (m, 2H), 5.25 (m, 1H), 4.9e5.01 (m, 4H), 4.39
(m,1H), 4.32 (d, J ¼ 7.3 Hz, 1H), 4.16 (ABX, J ¼ 7.1, 12 Hz, 1H), 3.88
(ABX, J ¼ 5.2,10.7 Hz,1H), 3.82 (m, 2H), 3.65e3.71 (m, 1H), 3.47e3.6
(m, 2H), 3.36 (m, 1H), 3.29 (m, 1H), 2.26e2.32 (m, 4H), 2.03 (q,
J ¼ 6.9 Hz, 4H) 1.58 (m, 4H),1.25e1.27 (m, 20H); 13C NMR (125 MHz,
The title compound was obtained in hexane: EtOAc (4: 1, v/v)
solvent mixture with 90% yield (0.167 g). 1H NMR (500 MHz,
CDCl3):
d
5.22 (dd, (apparent t), J ¼ 9.6, 9.4 Hz, overlapped, 1H), 5.17
(dd, (apparent t), overlapped, 1H), 5.07 (t, J ¼ 9.6 Hz, 1H), 4.95e5.02
(dd, (apparent t), J ¼ 8.1, 9.8 Hz, 1H), 4.53 (dd, J ¼ 4.1, 7.9 Hz, 1H),
4.23e4.32 (m, 2H), 4.05e4.15 (m, 2H), 3.95 (dd, J ¼ 4.9, 10.9 Hz, 1H),
3.64e3.72 (m, 2H), 2.3 (t, J ¼ 7.1 Hz, 4H), 2.09 (s, 3H), 2.05 (s, 3H),
2.02 (s, 3H), 2.01 (s, 3H), 1.58e1.63 (p, 4H), 1.26 (s, 32H), 0.88 (t,
CDCl3)
d 173.8, 173.7, 173.5, 173.3, 139, 114.1, 103.4, 103.2, 76.1, 75.7,
73.2, 70, 69.2, 68, 62.8, 62.7, 61.3, 34.2, 34.1, 33.7, 31.8, 29.6, 29.5,
29.3, 29.2, 29.1, 29, 28.8, 24.8, 22.6; IR (CHCl3) 3394, 2924, 2854,
1737, 1459, 1164, 1072, 907, 626 cmꢁ1; HRMS (ESI) m/z [M þ NH4þ]-
calc for C31H54O10NHþ4 ¼ 604.40552 found 604.40484.
J ¼ 6.4 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 173.3, 172.8, 170.7, 170.2,
169.4, 169.2, 101.03, 100.8, 72.67, 72.61, 71.8, 70.9, 69.6, 68.2, 67.6,
62.1, 61.7, 34.2, 34.06, 31.9, 29.6, 29.3, 29.4, 29.2, 29.1, 24.8, 22.6,
4.8.4. 1,2-O-didodecanoyl-3-O-(b-D-2ʹ,3ʹ,4ʹ,6ʹ-tetra-O-acetyl-
20.5, 14.1; IR (CHCl3) 2926, 2853, 1751, 1375, 1217, 1044, 752 cmꢁ1
;
glucopyranosyl)-sn-glycerol (7d)
The title compound was obtained in CHCl3: MeOH (19: 1, v/v)
solvent mixture with 77% yield (0.097 g). 1H NMR (300 MHz,
HRMS (ESI) m/z [M þ NHþ4 ]-calc for C41H70O14NHþ4 ¼ 804.51038
found 804.50954.
CDCl3):
d
5.26 (br s, 1H), 4.41 (br d, J ¼ 9.4 Hz,1H), 4.36 (d, J ¼ 7.6 Hz,
1H), 4.17 (ABX, J ¼ 6.7, 12.3 Hz, 1H), 3.9 (ABX, J ¼ 6.8, 11.2 Hz, 1H),
3.84 (m, 2H), 3.68e3.74 (m, 1H), 3.57e3.63 (m, 2H), 3.3e3.41 (m,
2H), 2.28e2.32 (m, 4H), 1.58e1.66 (p, 4H), 1.26 (m, 32H), 0.88 (t,
4.8. General procedure for synthesis of 1,2-O-diacyl-3-O-
glucopyranosyl-sn-glycerols (7a-7d)
b-D-
J ¼ 6.7 Hz, 6H); 13C NMR (125 MHz, CDCl3)
d 173.8, 173.5, 173.4,
1,2-O-Diacyl-3-O-(
b
-D-20,30,40,6'-tetra-O-acetyl-glucopyr-
103.2,103.1, 75.9, 75.6, 73.2, 70, 69.2, 68.5, 62.9, 62.8, 61.1, 34.2, 34.1,
31.8, 29.6, 29.5, 29.3, 29.1, 24.8, 22.6, 14.08; IR (CHCl3) 3376, 2920,
2852, 1737, 1462, 1072, 721 cmꢁ1; HRMS (ESI) m/z [MþNa]-calc for
anosyl)-sn-glycerols (6a-6d) (0.1 mmol) was dissolved in 85% aq.
EtOH (5 mL) and hydrazine hydrate (1.2 mmol) was added. The
reaction mixture was stirred for 6 h at 44 ꢀC. After all the starting
materials have been used, aq. EtOH was evaporated under reduced
pressure. Then the mixture was dissolved in CHCl3 (50 mL) and
poured into ice-cold water, extracted with CHCl3 (2 ꢂ 50 mL). The
combined organic layers were dried over anhydrous sodium sul-
phate and evaporated to give a syrupy mixture. The crude mixture
was purified by silica gel column using a solvent mixture of CHCl3:
MeOH (19: 1, v/v) to give title compounds (7a-7d) in 75e78% yields.
C33H62O10Na ¼ 641.42352 found 641.42311.
4.9. 1-O-dihydrosterculoyl-3-O-(b-D-2ʹ,3ʹ,4ʹ,6ʹ-tetra-O-acetyl-
glucopyranosyl)-sn-glycerol (8)
3-O-(b
-D-20,30,40,6'-tetra-O-acetyl-glucopyranosyl)-sn-glycerol
(5) (0.1 g, 0.23 mmol) was dissolved in anhydrous dichloromethane
(5 mL), dihydrosterculic acid (0.07 g, 0.23 mmol), N-(3-

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
143
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC)
(0.045 g, 0.23 mmol) and DMAP (pinch) were added under nitrogen
at 0 ꢀC. The reaction mixture was stirred for 6 h at 0 ꢀC. After all the
starting materials were used, the reaction mixture was dissolved in
CHCl3 and washed with water and brine solution. The organic layer
was dried over anhydrous sodium sulphate and evaporated under
reduced pressure. The mixture was purified by silica gel column
chromatography using a solvent mixture of hexane: EtOAc (2: 1, v/
v) to give title compound (0.162 g, 98%). 1H NMR data (500 MHz,
sulphate and evaporated to give a syrupy mixture. The crude
mixture was purified by silica gel column chromatography using a
solvent mixture of CHCl3: MeOH (19: 1, v/v) to give title compound
(0.123 g, 75%). 1H NMR (500 MHz, CDCl3):
d 5.30e5.37 (m, 2H), 5.26
(m, 1H), 4.39 (m,1H), 4.32 (d, J ¼ 7.7 Hz, 1H), 4.18 (ABX, J ¼ 6.7,
12.05 Hz, 1H), 3.91 (ABX, J ¼ 6.1, 11.2 Hz, 1H), 3.83 (m, 2H),
3.67e3.73 (m, 1H), 3.57e3.6 (m, 1H), 3.52e3.54 (m, 1H), 3.37 (m,
1H), 3.31 (m, 1H), 2.31 (t, J ¼ 7.3 Hz, 4H), 2.01 (m, 4H), 1.58e1.66 (m,
4H), 1.25e1.37 (m, 42H), 1.1e1.16 (m, 2H), 0.88 (t, J ¼ 6.5 Hz, 6H),
CDCl3):
d
5.21 (dd, (apparent t), J ¼ 9.6, 9.4 Hz, 1H), 5.07 (t,
0.63e0.68 (m, 2H), 0.53e0.57 (m, 1H), -0.34 (q, J ¼ 4.5 Hz, 1H); 13
C
J ¼ 9.8 Hz, 1H), 5.0 (br d, J ¼ 9.4 Hz, 1H), 4.56 (dd, J ¼ 4.3, 7.93 Hz,
1H), 4.18e4.26 (m, 2H), 3.65e4.12 (m, 6H), 2.34 (t, J ¼ 7.5 Hz, 2H),
2.09 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.58e1.66 (p, 2H),
1.27e1.36 (m, 22H), 1.1e1.17 (m, 2H), 0.88 (t, J ¼ 6.22 Hz, 3H),
NMR (125 MHz, CDCl3) d 173.8, 173.7, 173.4, 173.3, 129.9, 129.6,
103.4, 76.1, 75.7, 73.2, 73.1, 70, 69.2, 68.0, 62.8, 62.7, 61.2, 34.2, 34.1,
31.9, 30.1, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 28.6, 27.1, 24.8, 22.6, 15.6,
14, 10.9; IR (CHCl3) 3403, 2924, 2854, 1738, 1460, 1076, 721 cmꢁ1
;
0.58e0.64 (m, 2H), 0.52e0.58 (m, 1H), -0.33 (q, J ¼ 3.96 Hz, 1H); 13
C
HRMS (ESI) m/z [M þ NHþ4 ]-calc for C46H84O10NHþ4 ¼ 814.64027
NMR (75 MHz, CDCl3)
d
173.9170.6, 170.1, 169.3, 169.4, 101.3, 101.2,
found 814.63944.
72.5, 72.2, 71.8, 71.1, 68.8, 68.2, 64.9, 64.7, 61.8, 59.8, 34.08, 31.8,
30.1, 30.12, 29.6, 29.4, 29.37, 29.3, 29.1, 28.6, 24.8, 22.6, 20.5, 15.7,
4.12. 1,2:5,6-di-O-isopropyliidene-D-mannitol (11)
14.1, 10.8; IR (CHCl3) 3493, 2924, 2854, 1754, 1460, 1226, 1041 cmꢁ1
;
HRMS (ESI) m/z [M þ NHþ4 ]-calc for C36H60O13NHþ4 ¼ 718.43722
Acetone (50 mL) was saturated with anhydrous ZnCl2 (10.5 g,
76.84 mmol), the milky solution so formed was allowed to settle
down. The saturated solution was transferred under N2 pressure
through cannula to the round bottom flask containing D-mannitol
(5 g, 27.44 mmol). The three necked flask was fitted with a CaCl2
drying tube and N2 balloon, septa and its contents were stirred at
RT until the solution became clear (2e2.5 h). The mixture was
poured into saturated K2CO3 solution (40 mL) and stirred at RT for
0.5 h and filtered. The filtrate and residue were extracted with
CHCl3 (50 mL ꢂ 3), and the organic layer was dried over anhydrous
Na2SO4, concentrated under reduced pressure. This crude mixture
was recrystallized with hot hexane: DCM (9:1, v/v) solvent mixture
to give the title compound (6.47 g, 90%) as a white crystalline solid.
found 718.43721.
4.10. 1-O-dihydrosterculoyl-2-O-oleoyl-3-O-(b-D-2ʹ,3ʹ,4ʹ,6ʹ-tetra-
O-acetyl-glucopyranosyl)-sn-glycerol (9)
3-O-(b
-D-20,30,40,6'-tetra-O-acetyl-glucopyranosyl)-sn-glycerol
(5) (0.1 g, 0.23 mmol) was dissolved in anhydrous dichloromethane
(5 mL), dihydrosterculic acid (0.07 g, 0.23 mmol), N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC)
(0.045 g, 0.23 mmol) and DMAP (pinch) were added under nitrogen
at 0 ꢀC. The reaction mixture was stirred for 6 h at 0 ꢀC. After all the
starting materials were used, equimolar amounts of oleic acid, EDC
and a little amount of DMAP were added subsequently. The reac-
tion mixture was stirred at RT for overnight. Then the reaction
mixture was dissolved in CHCl3 and washed with water and brine
solution. The organic layer was dried over anhydrous sodium sul-
phate and evaporated under reduced pressure. The mixture was
purified by silica gel column chromatography using a solvent
mixture of hexane: EtOAc (4: 1, v/v) to give title compound (0.205 g,
mp 120e121 ꢀC, lit. 120e122 ꢀC. 1H NMR (500 MHz, CDCl3)
d 4.19
(dd, J ¼ 6.2, J ¼ 12.2 Hz, 2H), 4.12 (t, J ¼ 8.5 Hz, 2H), 3.98 (dd, J ¼ 5.6,
8.5 Hz, 2H), 3.75 (t, J ¼ 6.5 Hz, 2H), 2.68 (bs, 2H), 1.42 (s, 6H), 1.36 (s,
6H); 13C NMR (125 MHz, CDCl3)
d 109.3, 75.9, 70.9, 66.6, 26.6, 25.1;
IR (CHCl3) 3290, 2990, 2936, 2893, 1643, 1416, 1383, 1373, 1246,
1216, 1158, 1126,1064, 943, 857, 758, 667 cm ꢁ1; HRMS (ESI) m/z
[MþNa]-calc for C12H22O6Na 285.13022 found 285.13026; Specific
20
90%). 1H NMR (300 MHz, CDCl3):
d
5.28e5.39 (m, 2H), 5.22 (dd,
rotation: [
a]
¼ (þ) 6.5, (C ¼ 1, CHCl3).
D
(apparent t), J ¼ 9.6, 9.4 Hz, overlapped, 1H), 5.16 (overlapped, 1H),
5.07 (t, J ¼ 9.6 Hz, 1H), 4.95e5.02 (m, 1H), 4.53 (dd, J ¼ 4.1, 7.9 Hz,
1H), 4.23e4.32 (m, 2H), 4.05e4.15 (m, 2H), 3.95 (dd, J ¼ 5.09,
10.9 Hz, 1H), 3.64e3.72 (m, 2H), 2.3 (t, J ¼ 7.5 Hz, 4H), 2.1 (s, 3H),
2.05 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.01(4H, overlapped),
1.58e1.66 (p, 4H), 1.27e1.3 (m, 42H), 1.04e1.16 (m, 2H), 0.88 (t,
J ¼ 6.22 Hz, 6H), 0.6e0.68 (m, 2H), 0.52e0.59 (m, 1H), -0.34 (q,
4.13. (S)-(þ)-1,2-Isopropylideneglycerol or (S)-(þ)-Solketal (12)
To a stirred solution of diacetonoid (11) (6 g) in DCM (60 mL),
sat. NaHCO3 solution (2 mL) and NaIO4 (3.3 g) was added portion
wise in 2e3 min. The reaction mixture was stirred for 2 h at RT.
After 2 h, anhydrous sodium sulphate (2 g) was added and the
reaction mixture was stirred for 1 h at RT. After completion of re-
action time, the reaction mixture was filtered through vacuum filter
and filtrate was concentrated on rotary evaporator at 28 ꢀC with
cool water circulation. The crude (S)-(þ)-glyceraldehyde was dis-
solved in ethyl acetate (40 mL) and ethanol (5 mL) followed by
gradual addition of NaBH4 (0.65 g) at 0 ꢀC. This reaction mixture
was stirred at RT for 3 h. At that stage, acetone was added (5 mL) at
0 ꢀC and allowed to stir at RT for 15 min. Then the reaction mixture
was cooled to 0 ꢀC and saturated NH4Cl solution was added drop
wise with the dropper until there was no more evolution of
hydrogen gas. After this quenching process, the reaction mixture
was transferred to a separating funnel. The two layers were sepa-
rated, from this the organic layer was separated and the aqueous
layer was extracted with diethyl ether twice. The combined organic
layers were washed with brine solution and dried over anhydrous
Na2SO4 and concentrated under vaccum at 45 ꢀC to obtain a crude
product. The crude product was purified by silica gel column
chromatography using a solvent mixture of hexane: EtOAc (4: 1, v/
J ¼ 3.96 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 173.2, 172.9, 172.7,
170.6, 170.1, 169.3, 169.2, 129.9, 129.6, 101, 100.7, 72.6, 71.8, 71, 70.1,
69.7, 69.6, 68.2, 67.6, 67.5, 62.1, 61.7, 34.1, 34, 31.8, 30.1, 29.7, 29.6,
29.4, 29.3, 29.1, 28.6, 27.1, 24.8, 22.6, 20.6, 20.5, 15.7, 14.08, 10.8; IR
(CHCl3) 2924, 2853,1751,1371,1223,1041, 723 cmꢁ1; HRMS (ESI) m/
z [M þ NHþ4 ]-calc for C54H92O14NHþ4 ¼ 982.68253 found 982.68253.
4.11. 1-O-dihydrosterculoyl-2-O-oleoyl-3-O-
b-
D-glucopyranosyl-
sn-glycerol (10)
1-O-dihydrosterculoyl-2-O-oleoyl-3-O-(
b
-D-20,30,40,6'-tetra-O-
acetyl-glucopyranosyl)-sn-glycerol (9) (0.2 g, 0.21 mmol) was dis-
solved in 85% aq. EtOH (5 mL) and hydrazine hydrate (1.2 mmol)
was added. The reaction mixture was stirred at 44 ꢀC for 6 h. After
all the starting materials were used, aq. EtOH was evaporated under
reduced pressure. Then the mixture was dissolved in CHCl3 (50 mL)
and poured into ice-cold water, extracted with CHCl3 (2 ꢂ 50 mL).
The combined organic layers were dried over anhydrous sodium

144
V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
v) to obtain a title compound (2.56 g, 85%) with oil like character-
istics. 1H NMR (300 MHz, CDCl3)
4.19e4.27 (m, 1H), 4.04 (t,
and concentrated on rotary evaporator. The crude mixture was pu-
rified bysilica gel columnchromatography using a solvent mixture of
hexane:EtOAc(19:1, v/v)toobtainawhitesolid (1.9g,100%).1H NMR
d
J ¼ 8.1 Hz, 1H), 3.78 (t, J ¼ 8.1 Hz, 1H), 3.69 (d, J ¼ 11.3 Hz, 1H), 3.59
(dd, J ¼ 4.9, 11.1 Hz, 1H), 2.9(Bs, OH), 1.43 (s, 3H), 1.37 (s, 3H); 13C
data (300 MHz, CDCl3)
d
2.35 (t, J ¼ 7.3 Hz, 2H), 1.58e1.66 (p, 2H),
NMR (125 MHz, CDCl3)
d
109.2, 76.1, 65.6, 62.8, 26.5, 25.1; IR (CHCl3)
1.27e1.37 (m, 22H), 1.12e1.14 (m, 2H), 0.88 (t, J ¼ 6.7 Hz, 3H),
3420, 2987, 2936, 2884, 1457, 1372, 1214, 1157, 1051, 845 cmꢁ1
;
0.62e0.65 (m, 2H), 0.52e0.58 (m, 1H), -0.33 (q, J ¼ 3.96 Hz, 1H); 13
C
20
Specific rotation: [
a
]
¼ (þ)10.28, (C ¼ 5, MeOH).
NMR (75 MHz, CDCl3) d 180.5, 34.1, 31.9, 30.22, 30.12, 29.69, 29.37,
D
29.29, 29.07, 28.71, 28.66, 24.68, 22.7, 15.7, 14.12, 10.89; IR (CHCl3)
3420, 2926, 2854,1711,1463,1285, 942 cmꢁ1; HRMS (ESI) m/z [M-H]-
calc for C19H35O2 ¼ 295.26316 found 295.26467.
4.14. Synthesis of methyl oleate (13)
Oleic acid (2 g, 7.09 mmol) was dissolved in 2% MeOHeH2SO4
solution (50 mL) and the reaction mixture was refluxed on water
bath for 6 h. The reaction was monitored with micro TLC. After
completion of the reaction, the organic solvent was evaporated
under reduced pressure. The mixture was portioned in water and
ethyl acetate and the organic phase was washed with water until
the acidic nature was disappeared. The organic layer was dried over
anhydrous sodium sulphate and evaporated. The crude reaction
mixture was purified by silica gel column chromatography using a
solvent mixture of hexane: EtOAc (97: 3, v/v) to give the title
4.17. Biological activities
4.17.1. In vitro cytotoxicity evaluation
The cytotoxicity of the prepared dihydrosterculic acid-based
monoglucosyl diacylglycerol and its analogues was on the basis of
measurement of in vitro growth of tumor cell lines in 96 well plates
by cell mediated reduction of tetrazolium salt to water insoluble
formazan crystals using doxorubicin as a standard control. The
cytotoxicity was assessed against a panel of four different tumor
cell lines: DU145 derived from human prostate cancer cells (ATCC
No. HTB-81), A549 derived from human alveolar adenocarcinoma
epithelial cells (ATCC No. CCL-185), SKOV3 derived from human
ovarian cancer cell line (ATCC No. HTB-77) and MCF-7 derived from
human breast adenocarcinoma cells (ATCC No HTB-22) using the
MTT assay [40]. The IC50 values (50% inhibitory concentration) were
calculated from the plotted absorbance data for the dose response
compound (2.05 g, 98%). 1H NMR (500 MHz, CDCl3)
d 5.37e5.32 (m,
2H), 3.64 (s, 3H), 2.3 (t, J ¼ 7.6 Hz, 2H), 2.01 (m, 4H), 1.63 (p, 2H), 1.3
(m, 20H), 0.89 (t, J ¼ 6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 173.8,
129.7,129.5, 51.1, 33.8, 31.7, 29.6, 29.5, 29.4, 29.1, 29.03, 28.99, 28.94,
27.05, 27, 24.8, 22.5, 13.9; IR (CHCl3) 2926, 2855, 1744,
1463,1436,1196,1170 cmꢁ1
.
4.15. Synthesis of methyl dihydrosterculate (14)
curves. IC50 values (in mM) are expressed as the average of two
independent experiments.
To a stirred solution of oleic acid methyl ester (13) (2 g,
6.77 mmol), in DCM (40 mL), 1 M n-hexane solution of diethyl zinc
(4.17 mL, 40.54 mmol), was added under N2 atmosphere, were
placed in a cooling ice bath (ꢁ5 ꢀCe0 ꢀC) and stirred for 1 h. Then
diiodomethane (6.53 mL, 81.08 mmol) was added. After 10 min, a
milky solution was formed. Then the reaction mixture was stirred
for overnight at RT. After completion of the reaction, saturated
NH4Cl (20 mL) was added to the reaction mixture to break the white
solid. After this quenching process, the reaction mixture was
transferred to a separating funnel. The two layers were separated,
from this the organic layer was separated and the aqueous layer was
extracted with CHCl3 (30 mL ꢂ 2). The combined organic layers were
washed with brine solution and dried over anhydrous Na2SO4 and
concentrated under vacuum. Here the 100% starting material was
converted to the product. The crude product contained excess
diiodomethane only. This was purified by silica gel column chro-
matography using pure hexane followed by a solvent mixture of
hexane: EtOAc (97:3, v/v) to give title compound as oil (2.09 g,100%).
4.17.2. Bromodeoxyuridine cell proliferation assay
The effect of test compounds on the quantification of DNA
synthesis in actively proliferated cancer cells was investigated by
using bromodeoxyuridine cell proliferation assay kit (Abcam) ac-
cording to manufacturer's instructions. Initially, the cells with a
density of 1 ꢂ 104 were seeded and allowed to grow for 24 h. Later,
bromodeoxyuridine was added and allowed to incorporate into the
cells for 5 h followed by the addition of test compounds at 5 and
10 mM concentrations for 24 h and doxorubicin was run in parallel
as a positive control. Fixation was done for 30 min at room tem-
perature. Later the cells were washed with PBS and then anti-
bromodeoxyuridine antibody was added which binds to bromo-
deoxyuridine that was incorporated in the cells. After 1 h incuba-
tion, 100 mL anti-bromodeoxyuridine goat anti-mouse-horse radish
peroxidase (HRP)-conjugated secondary antibody (1:2000) was
added and incubated for another 30 min. The washing procedures
were followed according to the manufacturer's instructions. The
1H NMR (500 MHz, CDCl3)
d
3.66 (s, 3H), 2.3 (t, J ¼ 7.6 Hz, 2H),
TMB substrate (100 mL) was added and then incubated for another
1.59e1.64 (p, 2H), 1.23e1.37 (m, 22H), 1.11e1.17 (m, 2H), 0.88 (t,
J ¼ 6.7 Hz, 3H), 0.63e0.66 (m, 2H), 0.53e0.57 (m, 1H), -0.33 (q,
30 min at room temperature. Later the absorbance values were
recorded at a wavelength of 450 nm. Lower absorbance values
reflect lower bromodeoxyuridine concentrations in the sample and
thus an indirect measure of a low cell proliferation rate.
J ¼ 4.12 Hz,1H); 13C NMR (75 MHz, CDCl3)
d 174.1, 51.3, 34, 31.9, 30.16,
30.07, 29.63, 29.47, 29.39, 29.26, 29.1, 28.6, 27.13, 24.8, 22.6, 15.6, 14,
10.8; IR (CHCl3) 2926, 2855, 1736, 1460, 1437, 1215, 759 cmꢁ1
.
4.17.3. Caspase activity
4.16. Synthesis of dihydrosterculic acid (9,10-
methyleneoctadecanoic acid) (15)
Caspases are cysteine aspartic proteases which play a major role
in the apoptotic cell death. The activity of caspases-9 and 3 by the
test compounds was measured using AFC conjugated Ac-LEHD and
Ac-DEVD as substrates, respectively [41]. Briefly, the MCF7 cells
were seeded in 6 well plates with the confluence of 1.5 ꢂ 105 per
well and incubated for 24 h. Later, the cells were treated with the
test compound along with doxorubicin as a positive control. After
treatment, the cells were trypsinized and centrifuged at 2500 ꢂ g
for 10 min at 4 ꢀC. Cells were lyzed using lysis buffer (HEPES
250 mM, CHAPS 25 mM, DTT 25 mM) followed by incubation on ice
for 20e30 min. The lysate was centrifuged at 16,000 ꢂ g for
Methyl dihydrosterculate (14) (2 g, 6.45 mmol) was dissolved in
20 mL of THF: H2O (7:3, v/v) and added 3 equivalents of LiOH.H2O.
The reaction mixture was stirred at RT for 16 h. After completion of
reaction, the reaction mixture was concentrated under vacuum till a
white ppt was formed. This white precipitate was neutralized with
1 N HCl and the reaction mixture was extracted with ethyl acetate.
Thecombinedorganiclayerswerewashed withwatertoremoveacid
traces. The organic layer was dried over anhydrous sodium sulphate

V. Srikanth et al. / European Journal of Medicinal Chemistry 109 (2016) 134e145
145
20 min at 4 ꢀC and the obtained supernatant was used for mea-
surement of caspase activity. In a 96 well black polystyrene plate,
galactolipid from rose hip (Rosa canina) that inhibits chemotaxis of human
peripheral blood neutrophils in vitro, J. Nat. Prod. 66 (2003) 994e995.
[15] K. Nakata, High resistance to oxygen radicals and heat is caused by a gal-
actoglycerolipid in Microbacterium sp M874, J. Biochem. 127 (2000) 731e737.
[16] D. Colombo, L. Franchini, L. Toma, F. Ronchetti, N. Nakabe, T. Konoshima,
H. Nishino, H. Tokuda, Anti-tumor-promoting activity of simple models of
galactoglycerolipids with branched and unsaturated acyl chains, Eur. J. Med.
Chem. 40 (2005) 69e74.
2
ml of caspase-3 substrate and 1:1 ratio of supernatant and 2x
assay buffer (200 mM HEPES, 1% CHAPS, 20 mM EDTA, 50 mM DTT)
were taken. The reaction was allowed to take place for 1 h. The
fluorescence generated by the release of the fluorogenic group AFC
on cleavage by caspases-9 and 3 were measured by excitation at
400 nm and emission at 505 nm wavelength at every 5 min dura-
tion over a period of 1 h. The liberated fluorescence was normalized
to cell lysate protein using Bradford's protein estimation method.
Statistical analysis was performed using GraphPad PRISM software
version 3.0 (GraphPad Software, Inc, La Jolla, CA, USA). All experi-
mental data were compared using Student's t-test. In all compari-
sons, p < 0.05 was considered statistically significant. The results
are expressed as means of fluorescence intensity standard devi-
ation (SD), (n ¼ 3) of three independent experiments performed in
triplicates.
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Acknowledgements
V. Srikanth Y. Poornachandra and V. S. Phani Babu acknowledge
Council of Scientific and Industrial Research (CSIR), New Delhi, In-
dia, for financial support in the form of Senior Research Fellowship
(SRF).
Appendix A. Supplementary data
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