4884
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.