Synthesis and characterization of novel PUFA esters exhibiting potential anticancer activities: An in vitro study

Article abstract of DOI:10.1016/j.ejmech.2011.07.044

Polyunsaturated fatty acids (PUFAs) have been reported to play a regulatory role in tumour growth progression. In the present study, we have synthesized ester derivatives of two important PUFA viz., linoleic acid (LA) and arachidonic acid (AA) with propof

Full text of DOI:10.1016/j.ejmech.2011.07.044

European Journal of Medicinal Chemistry 46 (2011) 4878e4886
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and characterization of novel PUFA esters exhibiting potential
anticancer activities: An in vitro study
,
Azmat Ali Khan a, Mahboob Alam b, Saba Tufail a, Jamal Mustafa c, Mohammad Owais a
*
a Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
b Department of Chemistry, Faculty of Science, Aligarh Muslim University, Aligarh 202002, India
c University Polytechnic, Aligarh Muslim University, Aligarh 202002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyunsaturated fatty acids (PUFAs) have been reported to play a regulatory role in tumour growth
progression. In the present study, we have synthesized ester derivatives of two important PUFA viz.,
linoleic acid (LA) and arachidonic acid (AA) with propofol, a widely used general anaesthetic-sedative
agent. The novel propofol ester analogues have been found to inhibit various cancer cell lines in
a dose-dependent manner. Moreover, the compounds have been found to induce apoptotic cell death by
enhancing the release of cytochrome c and expression of caspase-3. The data of the present study suggest
that novel propofolePUFA esters have strong potential to emerge as effective anticancer agents.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 13 January 2011
Received in revised form
26 July 2011
Accepted 26 July 2011
Available online 3 August 2011
Keywords:
Linoleic acid
Arachidonic acid
Propofol
Anticancer
Apoptosis
1. Introduction
well as linolenic acids can be considered essential fatty acids for
mammals. In general, linoleic acid (LA) is obtained from plant based
Polyunsaturated fatty acids (PUFAs) perform multiple tasks in
cell functioning and intracellular signalling [1e4]. Their role as
dietary supplements in the prevention and treatment of chronic
disorders have attracted due interest in recent past [2,3]. Preclinical
studies demonstrate effectiveness of PUFA derivatives in growth
inhibition and cytotoxicity against cancer cells in vitro [1e4]. The
possible role of PUFA in inhibition of tumour cell proliferation,
induction of apoptosis and improved sensitivity against cancer cells
at par with other chemotherapeutic agents [5,6] has led to the
development of fatty acid based anticancer compounds [7e11].
Although several PUFA derivatives have been thoroughly investi-
gated in experimental models, however, to the best of our knowl-
edge derivatives of the two important fatty acids viz., arachidonic
acid and linoleic acid have till date not been explored for their
anticancer potential.
dietary sources. LA by itself has been reported to show anti-tumour
activity on various neoplastic cells in culture [12,13]. It has also
been reported to exert differential effects on the kinetics of various
anticancer drugs [14].
LA may be converted to certain other PUFAs including arach-
idonic acid. Arachidonate inturn is precursor of regulatory lipids,
the eicosanoids, which are very potent biological signalling mole-
cules that can act as short-range messengers. Arachidonic acid (AA)
metabolism has apparently been observed to play a key role in
cancer biology where its ability to induce cancer cell apoptosis has
become an interesting strategy in cancer therapy. Arachidonic acid,
is basically an u-6 fatty acid and is primarily found in fatty parts of
red meat and fish. The presence of four cis double bonds are
primarily responsible for its flexibility, keeping the pure AA in
liquid state, even at subzero temperatures. It is also supposed to
help mammalian cell membranes to attain correct fluidity at
physiological temperature [15]. It is one of the essential PUFAs and
contrary to other more abundant PUFAs, level of unesterified AA is
stringently controlled within mammalian cells [16]. Interestingly,
the anticancer potential of AA has been established against various
cell lines and animal models [17e19].
While mammalian hepatocyte can readily introduce double
bonds at Δ9 position of fatty acid but cannot introduce additional
double bonds between C-10 and the methyl terminal end. Being
necessary precursors for the synthesis of other products, linoleic as
Propofol (Diisopropylphenol) is
phenolic compound that is widely used as an intravenous
a low molecular weight
* Corresponding author. Tel.: þ91 571 2720388; fax: þ91 571 2721776.
E-mail address: owais_lakhnawi@yahoo.com (M. Owais).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.044

A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
4879
sedative-hypnotic agent in humans and animals. Preliminary
3.2.2. FT-IR spectroscopy
studies demonstrated that LD50 of propofol in mouse was 170 mg/
kg [20], while it was 386 mg/kg in human beings [21]. It has
a structural analogy with the vitamin E, an antioxidant vitamin,
a feature that partly explains its antioxidant properties [22].
Interestingly, clinically relevant concentrations of propofol are
reported to decrease the metastatic potential of human cancer cells
[23] and have also been shown to induce apoptosis through both
extrinsic and intrinsic pathways [24]. The propofol also entertains
certain advantages such as rapid clearance, minimal side effects
and non-toxicity to humans at high doses that render it suitable as
a future anticancer drug [25].
In the present study, we have synthesized various ester
analogues of LA and AA with propofol. Both LA and AA have been
conjugated with one of the two isomers of propofol viz.
2,4-Diisopropylphenol (2,4-propofol) or 2,6-Diisopropylphenol
(2,6-propofol) separately. To establish the structure of the synthe-
sized compounds; various analytical methods such as UV spectra,
FT-IR, 1H NMR, 13C NMR and high-resolution FAB-MS have been
exploited. The cytotoxic effect of novel propofolePUFA analogues
was assessed against various cancer cell lines. In vitro anticancer
potential was further established by assessing lipid peroxidation
level and induction of apoptosis in treated cancer cells.
The infrared absorption spectra of propofolePUFA analogues are
provided as supplementary file (Supplementary Fig. 3). In the
following write-up, respective resulting spectroscopic values for
2,4-propofolePUFA analogues are given in bracket.
3.2.2.1. PropofoleLA analogues. FT-IR spectra of 2,6P-LA showed
two strong absorption bands at 1757.85 (1755.51) and 1137.00
(1147.02) cmꢀ1 that are attributable to
n(C]O) and n(CeO) bond,
respectively, indicating the presence of an ester. The spectra also
revealed a short band at 2931.26 (2925.99) cmꢀ1, a characteristic of
an aromatic CeH bond (propofol), and the band at 2866.02
(2858.87) cmꢀ1, a characteristic of aliphatic CeH bonds. No
hydroxyl (eOH) absorption band was seen, indicating the absence
of non-esterified propofol.
3.2.2.2. PropofoleAA analogues. The FT-IR spectra showed two
strong absorption bands at 1750.23 (1756.21) and 1143.30 (1134.42)
cmꢀ1 that are attributable to
n(C]O) and n(CeO) bond, respec-
tively, indicating the presence of an ester group in newly synthe-
sized compounds. The spectra also revealed a short band at 2957.69
(2949.86) cmꢀ1 a characteristic of an aromatic CeH bond (propo-
fol), and the band at 2908.39 (2913.53) cmꢀ1, a characteristic of
aliphatic CeH bonds. No hydroxyl (eOH) absorption band was seen,
indicating the absence of non-esterified propofol.
2. Chemistry
3.3. Cytotoxic effect of propofolePUFA analogues against cancer
cells
The two isomers of propofol viz. 2,4-propofol and 2,6-propofol
were conjugated with LA and AA separately, employing synthesis
pattern introduced by Siddiqui et al. [10] with some modifications.
The four novel compounds were obtained by esterification of
hydroxyl (1CeOH) group of propofol with the terminal carboxylic
group of PUFA in the presence of N,N-dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP). The reaction
sequences are outlined in Scheme 1 and 2.
The cytotoxic effect of propofolePUFA derivatives against
various cancer cell lines was examined by evaluating the metabolic
status of cells after exposing them with increasing concentrations
of various synthesized compounds (0e15 mM). Inhibition of cancer
cell growth was observed to be in a dose-dependent manner
(Supplementary Figs. 6a and 6b). All tested compounds showed
significant (p < 0.05) anticancer activity in comparison to vehicle
control. The anticancer property of the synthesized compounds
was compared with standard drug (Table 1). It was observed that
esterification with the 2,4-propofol/2,6-propofol resulted in an
increase in the anticancer activity of the parent PUFA. The effec-
tiveness of compounds, however, varied with respect to cell lines
where inhibition was seen in the order:
3. Results
3.1. Synthesis of propofolePUFA analogues
The chemical synthesis setup resulted in synthesis of ester
derivatives of propofol (Scheme 1 and 2). The synthesis of novel
drug product was confirmed by assessing Rf value using solvent
system with composition 1:1 n-hexane and diethyl ether. The new
product was observed at locations having a new Rf value on TLC
plates when compared to both PUFA and propofol as substrates
(Supplementary Fig. 1). The compounds synthesized in good yield
were colourless viscous liquid (oily) at room temperature.
2,4P-LA/2,6P-LA: MDA-MB-361
> HepG2 > SK-MEL-1 >
A549 > HL-60 (Supplementary Fig. 6a)
2,4P-AA/2,6P-AA: HepG2 > HL-60 > A549 > MDA-MB-361 >
SK-MEL-1 (Supplementary Fig. 6b)
Results on normal HFL1 cell line were observed to be quite
distinct where propofoleLA analogues were found to spare killing
of normal cells. In contrast, propofoleAA analogues showed w15%
killing thus seemed to be less potent than parent AA.
3.2. Characterization of propofolePUFA analogues
Note: Further in vitro evaluation of anticancer efficacy of newly
synthesized compounds was done on the cell lines that were found
to be most susceptible to the cytotoxic effect. Therefore, MDA-MB-
361 (human ductal carcinoma, breast) cell line for propofoleLA
analogues and HepG2 (human liver hepatocellular carcinoma) cell
line for propofoleAA analogues were used.
3.2.1. UV spectroscopy
3.2.1.1. PropofoleLA analogues. The spectra of parent LA
(Supplementary Fig. 2) revealed absorption peak at 267 nm.
Whereas 2,6-propofol and 2,4-propofol showed absorption peaks
at 292 and 281 nm, respectively. These peaks were shifted to 268
and 267 nm, for 2,6P-LA and 2,4P-LA compounds, respectively.
3.4. Evaluation of lipid peroxidation
3.2.1.2. PropofoleAA analogues. The spectra of parent AA
(Supplementary Fig. 2) showed absorption peak at 272 nm whereas
for 2,6-propofol and 2,4-propofol the absorption peaks were found
to be centred at 293 and 281.5 nm, respectively. These peaks were
shifted to 282 and 280 nm, for 2,6P-AA and 2,4P-AA compounds,
respectively.
Treatment of HepG2 cells with propofoleAA analogues (Fig. 1)
increased the malondialdehyde (MDA) production after 24 h in
a dose-dependent manner. The compounds significantly caused
more lipid peroxidation even at relatively low doses
(2.27e3.40 nmol MDA produced per 106 cells). Among parent
reactant controls, AA showed increase in MDA production with

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A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
Scheme 1. Schematic representation of chemical synthesis of propofoleLA analogues.
increase in concentration whereas no effect was observed with
propofol alone treatment. PropofoleLA analogues were unable to
increase the lipid peroxidation level (0.25e0.34 nmol MDA
produced per 106 cells) significantly in MDA-MB-361 cells (Fig. 1A).
Similar effect was recorded for other parent reactant molecules.
blotting after 24 h of treatment. Exposure with PropofoleLA
analogues induced upregulation of apoptosis in MDA-MB-361
cancer cells at 15
mM concentration as evident from significant
increase (p < 0.01) in expression of cytochrome c and caspase-3
(Fig. 2, lanes 4 and 5). Although propofoleAA analogues were able
to initiate the release of cytochrome c but no expression of caspase-3
was recorded (Fig. 3). Mean densitometric values of individual
bands of cytochrome c and caspase-3 were quantified using UVI doc
software. Each value was mean of three experiments. In propo-
foleLA analogues; 35.2% and 36.6% cytochrome c release and 32.6%
and 33.7% caspase-3 expression was observed after treatment with
3.5. Apoptosis induction by propofolePUFA analogues
Both HepG2 and MDA-MB-361 cancer cells were incubated with
novel propofol derivatives for 24 h. The expression of various
apoptotic proteins in two cell lines was screened by Western

A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
4881
Scheme 2. Schematic representation of chemical synthesis of propofoleAA analogues.
2,4P-LA and 2,6P-LA, respectively (Fig. 2B,C). The propofoleAA
analogues induced 24.7% and 24.2% cytochrome c release after
treatment with 2,4P-AA and 2,6P-AA, respectively (Fig. 3B).
of dietary PUFAs in relation to tumour suppression [26,27].
Recently, clinical applications of such compounds in cancer treat-
ment have also been reported [28]. For example, the
u-hydroxy
PUFAs are reported to prevent cancer metastasis and also inhibit
cancer [6]. It has also been suggested that exogenous unsaturated
PUFA may increase anticancer activity of various existing cancer
chemotherapeutic agents [29]. Earlier studies involving chemically
modified PUFA molecules have demonstrated their specific and
potent biological activity against a range of therapeutic targets.
Since last decade, several attempts have been made to synthesize
novel anticancer compounds by conjugating fatty acids with
various anticancer drugs [9,28].
4. Discussion
Most of the presently available anticancer drugs are associated
with toxicity and other related adverse effects. Besides, some other
limitations such as widespread systemic distribution in the recip-
ient, as well as their rapid elimination, further aggravate toxicity
related issues and eventually restrict their usage as potential
chemotherapeutic agents. Keeping into consideration various
untoward manifestations associated with most of the presently
available anticancer drugs, it is desirable to opt for suitable
substitutes for them. There have been many reports concerning role
In the present study, synthesis of four novel propofolePUFA
analogues was achieved by coupling of the hydroxyl function
(1CeOH) present in the propofol with the terminal carboxylic group
Table 1
Cytotoxicity of propofolePUFA analogues (1e4) in a panel of cell lines.
PropofolePUFA analogues
Cell lines (IC50
,
m
M)
MDA-MB-361
HepG2
SK-MEL-1
A549
HL-60
HFL1
2,4-Diisopropylphenol-linoleic acid (2,4P-LA)
2,6-Diisopropylphenol-linoleic acid (2,6P-LA)
2,4-Diisopropylphenol-arachidonic acid (2,4P-AA)
2,6-Diisopropylphenol-arachidonic acid (2,6P-AA)
Doxorubicin
4.1
3.3
3.7
3.1
2.17
3.2
2.76
7.5
7.21
2.03
4.9
4.1
LA
LA
LA
LA
5.3
4.6
3.6
LA
LA
4.4
3.9
NA
NA
LA
LA
NA
3.092
3.137
The highest concentration tested was 15
mM. Values are means of three observations. LA, less active; NA, not active; SK-MEL-1, human skin malignant melanoma; HepG2,
human liver hepatocellular carcinoma; MDA-MB-361, ductal breast carcinoma; A549, human lung carcinoma; HL-60, human leukaemia; HFL1, human lung fibroblast.

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A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
Fig. 1. Modulation of lipid peroxidation by propofoleLA analogues in MDA-MB-361 breast cancer cells and propofoleAA analogues in HepG2 liver cancer cells. Cells were exposed
to vehicle alone (control or C) or 25, 50 or 100
M of individual compounds for 24 h. Lipid peroxidation is expressed as nmol malondialdehyde (MDA) produced/106 cells. Data are
presented as means ꢁ SD from five experiments.
m
of PUFA to synthesize a specific ester. Quantitative formation of the
compounds was achieved with the help of DCC as a coupling reagent
and DMAP as a catalyst (Scheme 1 and 2). As a catalyst, DMAP is
essential for esterification, especially in a concentration range of
10 mol%. The DCC/DMAP method is a more suitable synthetic route
with mild reaction conditions, higher yield and convenient product
purification. Spectrophotometric characterization involving UV
absorption, infrared, NMR and mass spectroscopy established
synthesis of the ester based compounds. In infrared absorption
spectra, the presence of an ester bond, aromatic CeH absorbance,
and absence of free eOH group absorbance, confirmed the forma-
tion of the specific ester. Elemental analysis of compounds showed
the values for ‘CHN’ within ꢁ0.4% of the theoretical ones. The
presence of carbon and hydrogen ions in structure of newly formed
compounds was determined by 1H NMR and 13C NMR spectra. When
NMR signal of the compounds were compared with the parent
propofol [10], the hydroxyl proton signal at about 4.82 ppm in 2,6-
propofol was not observed in the spectra of compounds. The
absence of signal of hydroxyl group in 1H spectra of the compounds
confirms the synthesis of new products, which differ from parent
compounds. Overall shift in signals indicated that the ester bond was
formed at C10 hydroxyl group of the propofol (Supplementary Figs.
4a–d). Results of mass spectra identified products with molecular
mass values which were very close to the calculated molecular
mass values of 440 Da/464.47 Da for propofoleLA/propofoleAA
analogues, respectively (Supplementary Figs. 5a–d).
Keeping in view the fact that PUFAs play a regulatory role in
tumour growth suppression on one hand, and propofol enhances the
Fig. 2. A: Immunoblots showing relative distribution of cytochrome c, caspase-3 and
b-actin (loading control) in MDA-MB-361 cells after treatment with (1) control; (2) linoleic
acid; (3) propofol; (4) 2,4P-LA; (5) 2,6P-LA. B,C: Densitographs showing relative density of cytochrome c and caspase-3 respectively, in the nitrocellulose blot. Results are mean
values ꢁ SD in triplicates.

A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
4883
present study, role of these compounds on induction of apoptotic
factors was therefore analyzed by determining the release of
cytochrome c and activation of caspase-3. Cytochrome c has
a function in the intrinsic pathway of apoptosis and leads to the
activation of caspase-3, which is a downstream enzyme in the
apoptosis process and is involved in the execution phase of the
death pathway. In propofoleLA analogues; 2,6P-LA showed
increased expression of both cytochrome c (22.5%) and caspase-3
(17.3%) when compared with 2,4P-LA (Fig. 2BeC). On the other
hand, 2,4P-AA upregulated the release of cytochrome c slightly
more as compared to 2,6P-AA (Fig. 3B). Surprisingly no signals were
recorded for caspase-3 expression in HepG2 cells when treated by
propofoleAA analogues. Effective induction of apoptosis by pro-
pofoleAA and propofoleLA analogues is similar to the effects
shown by other PUFA derivatives viz. propofoleDHA and propo-
foleEPA [10]. Overall, the compounds were found to induce
caspase-3 mediated apoptosis in cancer cell lines that eventually
led to the death of cancer cells. As apoptotic cell death is accom-
panied by the release of cytochrome c from mitochondria to the
cytosol followed by activation of caspase-3 that is suggestive of
mitochondrial mode for observed programmed cell death.
Fig. 3. A: Immunoblots showing relative distribution of cytochrome c and
b-actin
Conclusively, chemical synthesis followed in the present study
has introduced four novel propofolePUFA analogues. The novel
compounds are colourless viscous liquid (oily) at room tempera-
ture, conforming to the common physical state of PUFA esters. The
structural characterization of novel compounds has also been
achieved successfully. The in vitro studies suggest that novel
compounds possess potential anticancer efficacy. The increased
anti-proliferating effect of novel compounds can be attributed to
their unique structures wherein PUFA with strong hydrophobic
nature is rapidly translocated across the plasma membrane, besides
propofol, being a partly lipophilic agent, also facilitates uptake of
PUFAepropofol based compounds by cancer cells. It seems volatile
nature and a very short plasma half-life of propofol also help in its
rapid removal from membranes. It is speculated that conjugation of
propofol with LA and AA will result in increased lipid-solubility,
bioavailability, activity, and decreased side-effects of the novel
compounds. Moreover, the two structural moieties of the
compound (propofol/PUFA), which should be released in the cell
via enzymatic actions, were expected to have synergistic effect so
that an optimum increase in their chemotherapeutic index is
possible. Experiments are under way to establish efficacy of the
compounds against various types of cancer in animal models.
(loading control) in HepG2 cells after treatment with (1) control; (2) arachidonic acid;
(3) propofol; (4) 2,4P-AA; (5) 2,6P-AA. B: Densitographs showing relative density of
cytochrome c in the nitrocellulose blot. Results are mean values ꢁ SD in triplicates.
cytotoxicity of anticancer agent on the other; all the four novel
propofolePUFA analogues were evaluated for their anticancer effi-
cacy against various cancer cell lines. The cytotoxic potential of
various PUFA and propofol based esters were evaluated against
a panel of cancer cells. The compounds showed significant growth
inhibition of cancer cells in a dose-dependent manner. The differ-
ence in inhibition activity was observed within a given PUFA
derivative where both 2,6-propofolePUFA were found to be more
potent than 2,4-propofol analogues. Nevertheless, the novel
compounds were able to inhibit the cancer cells at concentration
where parent reactants alone were not effective, thereby, estab-
lishing their potent anticancer properties. Interestingly, the
synthesized test compounds (except slight toxicity shown by pro-
pofoleAA analogues) did not induce any structural alterations in
non-cancerous HFL1 cells under experimental conditions while they
were able to inhibit cancerous cells, indicating that toxicity is not
due to PUFA metabolism per se but rather due to the effect of the
ester based compounds. Overall, the inhibitory effects of novel
compounds were more or less similar to the results obtained for
docosahexaenoic acidepropofol esters recently reported by Harvey
et al. [11]. However, considering wide availability of both PUFAs used
in the present study, the synthesized compounds are likely to be
more economical when used as anticancer agent in clinical settings.
In order to identify the mechanism likely to be involved in test
compounds-induced cell growth inhibition, the possibility of
involvement of lipid peroxidation pathway was investigated. For
propofoleLA analogues, negligible MDA production was counted on
MDA-MB-361 cells at 24 h. Interestingly, results are in accordance
with those reported for exogenous AA [30]. Both newly synthesized
propofoleAA analogues induced a dose-dependent increase of MDA
production at 24 h time period in HepG2 cells. However, the pres-
ence of propofol, an antioxidant, in the newly synthesized test
compounds, seems to down-regulate lipid peroxidation level in
comparison to parent AA (Fig. 1). The data of the present study
suggest that in contrast to LA, AA influences the growth of cancer
cells by increasing lipid peroxidation products [31].
5. Conclusions
The high abundance of PUFA in various natural sources offers an
economic and cost effective strategy for synthesis of propofolePUFA
derivatives. The present study focuses on synthesis of four novel
propofolePUFA analogues. The synthesized novel compounds
reveal dose-dependent growth inhibition against a panel of cancer
cell lines. The mechanism underlying the antiproliferative effect of
LA and AA appears to be involving apoptotic pathways despite the
production of MDA derived from lipid peroxidation by propofoleAA
analogues. From the data of the present study it could be suggested
that the PUFA-propofol based ester compounds are more active,
possess unique anticancer activities than the parent reactant
controls and are able to inhibit tumour growth.
6. Experimental protocols
The anticancer effects of PUFA upon tumour cells have been
attributed to their ability to trigger programmed cell death by
6.1. Materials
apoptosis. The induction of apoptosis by
u-6 PUFAs has been
Linoleic acid, arachidonic acid, 2,6-Diisopropylphenol, 2,4-
extensively studied against various tumour types [32]. In the
Diisopropylphenol, N,N-dicyclohexylcarbodiimide (DCC), 4-

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A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
dimethylaminopyridine (DMAP), Rosewell Park Memorial Institute
(RPMI)-1640 medium, Eagle’s Minimum Essential Medium
(EMEM); 2-[4-(-2 hydroxyethyl)piperazine-1-yl]ethane sulfonic
acid (HEPES); phenylmethylsulfonyl fluoride (PMSF); EDTA;
dithiothreitol (DTT); 3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-
tetrazolium bromide (MTT), thiobarbituric acid (TBA), sodium
dodecyl sulphate (SDS) were acquired from SigmaeAldrich. Foetal
calf serum (FCS) was procured from Bio-Whittaker. Thin-layer
chromatographic plates (60A, 0.2 mm thick) and silica gel (60e120
mesh) were purchased from Fisher Scientific. Dichloromethane
(DCM), methanol, ethanol, chloroform, n-hexane, diethyl ether,
sucrose, KCl, MgCl2, dimethyl sulphoxide (DMSO) and acetic acid
were procured from Merck.
6.3.2. 2,4-Diisopropylphenol-linoleic acid (2,4P-LA)
Yield 84%; 1H NMR (CDCl3, dH; ppm; J, Hz): 0.88 (3H, q, J 6.8 Hz,
H20), 1.22 (12H, m, 2AreC e(CH3)2), 1.44e1.33 (14H, m, 7ꢃ CH2),
1.78e1.75 (2H, t, J 7.6 Hz, eCOCH2CH2e), 2.07 (4H, q, J 6.8 Hz,
2eCH2eCH]), 2.58 (2H, t, J 6.4 Hz, eCOCH2e), 2.79 (2H, t, J 6.4 Hz,
]CHeCH2eCH]), 3.00 and 2.90 (2H, t and t, Are2CHe), 6.87 (1H,
d, J 8.1, AreH60) and 7.05 (1H, dd, J 6.0 and 2.4 Hz, AreH50) and 7.13
(1H, d, J 2.2 Hz, AreH30); 13C NMR (CDCl3; , ppm): 25.0 (C3*),
d
29.1e29.7 (C4, C5, C6, C7, C14, C15,), 27.2 (C8), 130.2 (C9), C10
(128.1), C11 (25.6), C12 (127.9), C13 (130.0), C16 (31.5), C17 (22.6),
14.1 (C18), 33.8 (AreCe(C)2e)*, 27.5 and 23.0 (Are2C)*, 121.9
(Aromatic carbon C60),124.3 (Aromatic carbon C50),124.6 (Aromatic
carbon C30), 139.5 (Aromatic carbon C20), 146.0 (Aromatic carbon
C40) 146.5 (Aromatic C10) and 172.2 (CO).
Monoclonal antibodies such as anti-caspase-3, anti-cytochrome
*
c, anti-
b
-actin were procured from BD Biosciences (San Diego, CA).
Note: Asterisks denote assignments that may be interchanged
The secondary anti-mouse peroxidase-conjugated antibody was
from Amersham Pharmacia Biotech. Chemiluminescence detection
kit was purchased from GE healthcare.
due to merging of peaks.
6.3.3. 2,6-Diisopropylphenol-linoleic acid (2,6P-LA)
Yield 86%; 1H NMR (CDCl3, dH; ppm; J, Hz): 0.88 (3H, q, J 6.3 Hz,
H20), 1.20 (12H, d, J 6.8, 2AreCe(CH3)2), 1.35e1.26 (14H, m, 7ꢃ
CH2), 1.88 (2H, q, J 7.5 Hz eCOCH2CH2e), 2.06 (4H, q, J 6.8 Hz,
2eCH2eCH), 2.63 (2H, t, J 7.6 Hz, eCOCH2e), 2.24 (2H, d, J 6.8 Hz, ]
CHeCH2eCH]), 3.18 (2H, m, AreCHe), 7.15 (1H, t, J 6.4, AreH40)
and 7.20 (2H, d, J 6.0 Hz, AreH30 and H50; AreH60) and 7.05 (1H, dd,
J 6.0 and 2.4 Hz, AreH50) and 7.13 (1H, d, J 2.2 Hz, AreH30); 13C NMR
6.2. Cell lines and culture conditions
Cell lines SK-MEL-1 (ATCC# HTB-67), HepG2 (ATCC# HB-8065),
MDA-MB-361 (ATCC# HTB-27), A549 (ATCC# CCL-185), HL 60
(ATCC# CCL-240) and non-cancerous HFL1 (ATCC# CCL-153) were
obtained from American Type Culture Collection (Rockville, MD).
SK-MEL-1 and HepG2 cell lines were maintained in EMEM
whereas MDA-MB-361, A549, HL-60 and HFL1 were maintained in
RPMI medium. The complete growth mediums were supple-
(CDCl3; d
, ppm): 25.0 (C3*), 29.2e29.7 (C4, C5, C6, C15, C17*), 31.9
(C7), 27.2 (C8), 130.6 (C9), 128.1 (C10), C11 (27.1), C12 (127.9), C13
(130.2), C14 (29.2), 14.1 (C18), 27.5 (AreCe(C)2e)*, 26.5 (AreC)*,
123.4e123.8 (Aromatic carbon C30 and C50), 126.7 (Aromatic carbon
C40), 140.3 (Aromatic carbon C20 and C60), 145.6 (Aromatic C10) and
172.4 (CO).
mented with 10% (v/v) heat-inactivated FCS, 2 mM
L-glutamine,
100 U/ml penicillin and 100
mg/ml streptomycin. All cells were
maintained at 37 ꢂC in a 95% humidified atmosphere containing
5% CO2. Cells were screened periodically for mycoplasma
contamination.
6.3.4. 2,4-Diisopropylphenol-arachidonic acid (2,4P-AA)
Yield 82%; 1H NMR (CDCl3, dH: ppm): 0.88 (q, J 6.8 Hz, 3H), 1.22
(m, 2AreCe(CH3)2, 12H), 1.25e1.30 (m, 6H), 1.32e1.35 (m, 2H), 1.85
(t, J 7.4 Hz, 2H), 2.06 (dt, J 6.6 Hz, 2H), 2.22 (dd, J 6.2 Hz, 6.8 Hz, 2H),
2.58 (t, J 7.6 Hz, 2H), 2.84 (m, 6H), 2.87 (m, AreCHe, 2H), 5.35e5.44
(m, 8H), 6.87 (d, J 8.1 Hz, AreH60, 1H) and 7.05 (dd, J 6.0 Hz, 2.4 Hz,
6.3. Synthesis and purification of propofolePUFA analogues
Synthesis of the compounds was accomplished using method
of Siddiqui et al. [10] with some modifications. Firstly, 1 mmol of
a respective PUFA was dissolved in 5 ml dichloromethane (DCM).
Subsequently, 0.45 mmol of coupling reagent N,N-dicyclohex-
ylcarbodiimide (DCC) was added and the reaction mixture was
stirred for 10 min at room temperature (23e25 ꢂC). Finally,
1 mmol of one of the propofol isomer (2,6-propofol/2,4-propofol)
was dissolved and reaction mixture was esterified in the pres-
ence of a catalyst, 0.152 mmol 4-dimethylaminopyridine (DMAP).
The reaction mixture was stirred for a period of 10 h in dark.
Reaction was stopped by filtration and the filtrate was concen-
trated under reduced pressure to get the product. The progres-
sion of reaction and synthesis of product was visualized by thin
layer chromatography (TLC) followed by exposure to iodine
vapour. Finally, the synthesized compound was purified by silica
gel column chromatography with solvent system n-hexane and
diethyl ether (1:1).
AreH50, 1H) and 7.13 (d, J 2.2 Hz, AreH30, 1H); 13C NMR (CDCl3; dC
:
ppm): 14.1 (C20), 22.6 (C19), 23.0 (AreCe(C)2e)*, 24.1 (C3), 24.9
(C4), 25.6 (C7), 26.6 (C10), 27.2 (C13) *, 27.5 (AreC), 29.3 (C16), 29.3
(C17), 31.5 (C18), 33.7 (Ar40eC), 33.6 (C2), 121.8 (Aromatic carbon
C60), 124.4 (Aromatic carbon C50), 124.6 (Aromatic carbon C30)
127.8e130.7 (C5, C6, C8, C9, C11, C12, C14 and C15), 139.5 (Aromatic
carbon C20), 146.0 (Aromatic carbon C40) 146.5 (Aromatic C10) and
172.2 (CO). FAB-MS: m/z ¼ 487 (6%), m/z ¼ 439 (63%), m/z ¼ 263
(78%), m/z ¼ 261 (16%), m/z ¼ 245 (9%), m/z ¼ 205 (6%), m/z ¼ 178
(100%), m/z ¼ 163 (97%), m/z ¼ 137 (8%), m/z ¼ 135 (28%), m/z ¼ 95
(66%), 91 (53%), 82 (34%) and lower mass fragments (less than 6%).
*
Note: Asterisks denote assignments that may be interchanged
due to unhindered behaviour of 2,4-propofol as comparison of
2,6-propofol.
6.3.5. 2,6-Diisopropylphenol-arachidonic acid (2,6P-AA)
Yield 85%; 1H NMR (CDCl3, dH: ppm): 0.88 (q, J 7.0 Hz, 3H), 1.20
(d, J 6.8 Hz, 2AreCe(CH3)2, 12H), 1.25e1.36 (m, 6H), 1.88 (t, J 7.3 Hz,
2H), 2.06 (dis, J 6.8 Hz, 2H), 2.24 (d, J 6.0 Hz, 6.8 Hz, 2H), 2.63 (t, J
7.4 Hz, 2H), 2.85 (m, 6H), 2.88 (m, AreCHe, 2H), 5.35e5.91 (m, 8H),
7.15 (t, J 6.4 Hz, AreH40, 1H) and 7.20 (d, J 6.0 Hz, AreH30 and H50,
2H); 13C NMR (CDCl3; dC: ppm): 14.1 (C20), 22.6 (C19), 22.7, (C3),
24.9 (C4), 25.6 (C7), 26.7 (C10), 27.1 (C13), 27.2 (AreCe(C)2e), 27.5
(C16), 29.3 (AreC), 29.7 (C17), 30.9 (C18), 33.6 (C2), 123.4e123.9
(Aromatic carbon C30 and C50), 126.7 (Aromatic carbon C40)
127.8e130.7 (C5, C6, C8, C9, C11, C12, and C14), 133.6 (C15), 140.3
(Aromatic carbon C20 and C60), 145.5 (Aromatic C10) and 172.2 (CO).
6.3.1. Elemental analysis
The values found for CHO of synthesized compounds using
elemental analysis, were within ꢁ0.4% of the theoretical ones.
Values are as follows: 2,4P-LA: analysis calculated for C30H48O2: C,
81.76; H, 10.97; O, 7.27. Found: C, 81.75; H, 10.99; O, 7.26 2,6P-LA:
analysis calculated for C30H48O2: C, 81.76; H, 10.97; O, 7.27. Found:
C, 81.73; H,11.01; O, 7.26, 2,4P-AA: analysis calculated for C32H48O2:
C, 82.69; H, 10.42; O, 6.89. Found: C, 82.75; H, 10.50; O, 6.75 and
2,6P-AA: analysis calculated for C32H48O2: C, 82.69; H, 10.42; O,
6.89. Found: C, 82.72; H, 10.43; O, 6.85.

A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
4885
FAB-MS: m/z ¼ 510 (Mþ2Na)þ (3%), m/z ¼ 418 (74%), m/z ¼ 401
6.7. Preparation of post-nuclear fraction for apoptosis assay
(13%), m/z ¼ 375 (68%), m/z ¼ 373 (6%), m/z ¼ 333 (29%), m/z ¼ 285
(6%), m/z ¼ 271 (100%), m/z ¼ 269 (48%), m/z ¼ 239 (65%), m/
z ¼ 237 (13%), m/z ¼ 219 (6%), m/z ¼ 178 (87%), m/z ¼ 163 (42%), m/
z ¼ 137 (35%), m/z ¼ 135 (13%), m/z ¼ 111 (10%), 95 (16%), 91
(Characteristics peak), 82 (13%) and lower mass fragments (less
than 6%).
Cancer cells (1 ꢃ107 cells per well) were grown in 6-well plates
in serum free culture medium (respective to the cell line used) in
a humidified CO2 incubator at 37 ꢂC. After 24 h, confluent cells
were treated with control (ethanol only) or parent control (PUFA
only/propofol only) or test compound and further incubated for
24 h. After stipulated time period, the cells were harvested by
trypsinization and washed twice in PBS. The cells were suspended
6.4. Spectroscopic characterization of propofolePUFA analogues
in 50
100 mm NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1
0.5 mM EGTA, 200 M PMSF, 0.5 mM DTT and 1 g/ml of leupeptin
m
l of ice-cold TNN buffer containing 50 mM TriseHCl pH 7.4,
mg/ml pepstatin,
The formation of propofolePUFA analogues was confirmed by
various spectrophotometric studies. The presence of propofol in the
synthesized compounds was assessed by UV spectroscopy on UV
Mini-1240 spectrophotometer. The absorption spectra were
measured between 200 and 600 nm. The absorbance was read and
spectral scanning curves were made. The infrared spectra of the
compounds were recorded on Nikolet-6700 FT-IR. Ten microlitre
m
m
and homogenized in a Teflon homogenizer. A post-nuclear fraction
was prepared by centrifugation for 5 min at 2000 rpm at 4 ꢂC. The
supernatant was further centrifuged for 20 min at 10,000 g at 4 ꢂC
and the resultant cytosolic fraction was used for detecting the
expression of two apoptotic factors; viz. cytochrome c and cas-
pase-3.
(1 mg/ml) of the compound was deposited exactly within the cell
limit and was run as a thin film after evaporation of solvent. For
data acquisition, a resolution of 32 was used. The analysis was
performed in triplicate with data spacing of 15.428 cmꢀ1. Elemental
analyses of compounds were carried out with a Carlo Erba EA-1108
analyzer. The formation of new compounds was elucidated with 1H
and 13C NMR spectra on BRUKER AVANCE II 400 NMR spectrometer.
The molecular mass of the compounds was determined by FAB-MS
spectrum on a JEOL SX 102 Mass Spectrometer/Data System using
Argon/Xenon (6 kV, 10 mA) as the FAB gas.
6.8. Protein determination
Protein content was determined with the BCA method [35]. The
mixture of solutions A and B (1:49) of BCA reagent was added to the
protein sample and further incubated at 37 ꢂC for 45 min. The
absorbance was measured at 562 nm and the protein concentration
was calculated using a standard curve of BSA.
6.9. Western blotting
6.5. Growth inhibition of cancerous cell lines by propofolePUFA
analogues
The samples with equal amounts of protein were subjected to
10% SDS-polyacrylamide gel electrophoresis [34]. Immunoblotting
of resolved proteins was achieved using nitrocellulose membrane.
Non-specific binding on the nitrocellulose membrane was mini-
mized by its blocking for 1 h at room temperature with PBS-T [PBS
(pH 7.5) and 0.05% Tween-20] containing 5% (w/v) non-fat skim-
med milk. The treated membrane was washed in PBS-T and then
incubated overnight at 4 ꢂC with specific primary antibodies
(monoclonal anti-cytochrome-c or monoclonal anti-caspase-3) in
PBS-T containing 5% (w/v) BSA. The membranes were again washed
in PBS-T, anti-mouse peroxidase-conjugated secondary antibodies
in PBS-T were added for 2 h, and immunoreactive bands were
detected by enhanced chemiluminescence detection kit. Blots were
The novel compounds were examined for their cytotoxicity
against five different types of cancer cell lines viz., SK-MEL-1
(human skin malignant melanoma), HepG2 (human liver hepato-
cellular carcinoma), MDA-MB-361 (human ductal carcinoma,
breast), A549 (human lung carcinoma), HL-60 (human leukaemia,
acute promyelocytic) as well as one non-cancerous HFL1 (human
lung fibroblast) using a standard 3-4, 5-dimethylthiazol-2-yl-2,
5-diphenyl-tetrazolium bromide (MTT) reduction assay. Cells in
exponential growth were seeded into 96-well plates at a concen-
tration of 5 x 105 cells/200
ml/well and allowed to grow in specific
medium containing 5% FCS. After 24 h, cells were treated with
various concentrations of test compound or parent reactant
controls (PUFA only/propofol only) at a concentration range of
re-probed with an antibody for b-actin to control for equal protein
loading and transfer. Densitometric values of protein bands were
quantified using UVI-doc Imaging Software.
0e15
mM. Vehicle control (ethanol only) and positive control
(doxorubicin) cells were cultured using the same conditions.
Following 94 h incubation, the medium was removed and replaced
with fresh medium. MTT reagent (5 mg/ml in PBS) was added to
each well at a volume of 1:10 and incubated for 2e3 h at 37 ꢂC. After
6.10. Statistical data analysis
Results are expressed as the mean ꢁ SD of three experiments for
each treatment and were plotted accordingly. Individual treat-
ments were tested against the control by using Student-t tests.
Significant differences from control were considered at p < 0.05.
Analyses were conducted with SPSS version 13.0.
treatment, 100 ml of DMSO was added to each well after carefully
aspirating the supernatants. Absorbance was measured at 620 nm
in a multi-well plate reader. Triplicate wells were prepared for each
individual concentration. Dose-response curves were plotted as
percentages of the cell absorbances. Drug sensitivity was expressed
in terms of the concentration of drug required for a 50% reduction
of cell viability (IC50).
Acknowledgements
We are thankful to Prof. M. Saleemuddin, Co-ordinator, Inter-
disciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh,
India for providing the central instrumentation facility to carry out
the experiments. We also thank Sophisticated Analytical Instru-
mentation facility (SAIF) of Central Drug Research Institute, Luck-
now, India for providing NMR and mass spectra facility. The study
was conducted in financial support to Mr. Azmat Ali Khan from
University Grants Commission (India).
6.6. Lipid peroxidation
Lipid peroxidation was measured spectrophotometrically by
determining malondialdehyde (MDA) production by the thio-
barbituric acid assay [33]. Data were expressed as nmol of MDA
produced per 106 cells.

4886
A.A. Khan et al. / European Journal of Medicinal Chemistry 46 (2011) 4878e4886
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