Shamsuzzaman et al. / Journal of Photochemistry and Photobiology B: Biology 129 (2013) 36–47
37
OH
2.3. General method for the synthesis of cholest-5-en-7-one
derivatives (1b–3b)
OH
A solution of tert-butyl chromate [tert-butyl alcohol (30 mL),
CrO (10 g), acetic acid (42 mL) and acetic anhydride (5 mL)] was
added at 0 °C to a solution of cholest-5-ene derivatives (1a–3a)
4 g) in CCl (75 mL), glacial acetic acid (15 mL) and acetic anhy-
3
H C
3
(
4
dride (5 mL) [15]. The contents were refluxed for 3 h and then di-
luted with water. The organic layer was washed with sodium
bicarbonate solution (5%) and water and dried over anhydrous Na2-
CN
HN
N
4
SO . Evaporation of solvents under reduced pressure provided oil
which was crystallized from methanol to give cholest-5-en-7-one
O
NH2
derivatives (1b–3b).
Fig. 1. Substituted pyran derivative-an inhibitor of human Chk-1 Kinase.
2.4. General method for the synthesis of steroidal 4H-pyran derivatives
have also been screened for in vitro cytotoxicity against different
(1c–3c)
human cancer and non-cancer cell lines.
To a solution of cholest-5-en-7-one (1b–3b) (1 mmol) in abso-
lute ethanol (20 mL) was added malononitrile in equimolar ratio
followed by piperidine (1.5 mL). The reaction mixture was refluxed
for 11 h. The progress of reaction was monitored by TLC. After com-
pletion of reaction, excess solvent was removed to three fourths of
the original volume. The reaction mixture was taken in ether,
washed with water and dried over anhydrous sodium sulfate.
Evaporation of solvents and recrystallization from methanol affor-
ded respective products (1c–3c) (see Scheme 1).
2
. Experimental
2.1. Materials and methods
All chemicals were purchased from Sigma–Aldrich (India) and
Merck (India). Melting points were determined on a Kofler appara-
tus and are uncorrected. The IR spectra were recorded on KBr pel-
lets with Perkin Elmer RXI Spectrometer and values are given in
ꢁ1
1
13
cm . H and C NMR spectra were run in CDCl
400 MHz) instrument with TMS as internal standard and values
are given in ppm (d). Mass spectra were recorded on a JEOL SX
02/DA-6000 Mass Spectrometer. Carbon, hydrogen and nitrogen
3
on a JEOL Eclipse
0
0
2
.4.1. 3b-Acetoxy-2 -amino-3 -cyanocholest-6-eno[5,7-de]4H-pyran
(
(
1c)
White powder, yield 70%, m.p. 163–164 °C; IR (KBr,
340 (NH ), 2203 (CN), 1713 (OCOCH
CAO), 1328 (CAN); H NMR (CDCl
.7 (1H, m, C -H, W ½ = 15 Hz), 2.5 (2H, brs, NH
with D O), 2.05 (3H, s, OCOCH ), 1.2 (3H, s, C13-CH
10-CH
ꢁ1
m
max/cm ):
1
3
(
4
2
3
), 1625, 1620 (C@C), 1065
, d, ppm): 5.69 (1H, s, C H),
, exchangeable
), 1.14 (3H, s,
contents were determined on Carlo Erba Analyzer Model 1106.
Thin layer chromatography (TLC) plates were coated with silica
gel G and exposed to iodine vapors to check the homogeneity as
well as the progress of reaction. Sodium sulfate (anhydrous) was
used as a drying agent. Super coiled pBR322 DNA was purchased
from GeNei (India) and was used for the agarose gel experiment
without further purification. Double-stranded calf thymus DNA,
purchased from Sigma, was dissolved in a 0.1 M Tris-buffer. The
purity of DNA was verified by monitoring the ratio of absorbance
at 260 nm to that of 280 nm, which was in the range 1.8–1.9.
The concentration of the DNA was determined spectrophotometri-
1
3
6
3
a
2
2
3
3
13
C
3
), 1.04 and 1.02 (other methyl protons); C NMR (CDCl
3
,
0
d, ppm): 173.1 (OCOCH
(
H, 9.26, N, 5.44. Found: C, 75.59, H, 9.44, N, 5.51. MS: m/z 508 [M ].
3
), 168 ðC Þ, 157.2 (C
7
), 132.2 (CN), 111.6
2
0
6
C ), 72.2 (C
3
), 67.2 ðC Þ; Anal. Calcd for C32
H
48
N
2
O
3
%: C, 75.43,
3
+Å
0
0
2
(
.4.2. 3b-Chloro-2 -amino-3 -cyanocholest-6-eno[5,7-de]4H-pyran
2c)
Yellow powder, yield 80%, m.p. 143–144 °C; IR (KBr,
m
max
/
ꢁ1
ꢁ1
cally using
e
260 = 6600 M cm [12]. The human cancer cell lines
ꢁ1
2
cm ): 3396 (NH ), 2259 (CN), 1630, 1625 (C@C), 1116 (CAO),
327 (CAN), 742 (C-Cl); H NMR (CDCl
used for the cytotoxicity experiment were SW480, A549, HepG2,
HL-60, MCF-7, HeLa and DU-145 which were obtained from Na-
tional Cancer Institute (NCI), biological testing branch, Federick Re-
search and Development Centre, USA. The treated and control
cancer cells were viewed with a FluoView FV1000 (Olympus, To-
kyo, Japan) confocal laser scanning microscope (CLSM) equipped
with argon and HeNe lasers. 2-Thiobarbituric acid (TBA) and tri-
chloroacetic acid (TCA) were purchased from Merck (India). T4
DNA ligase enzyme was purchased from CalBioChem and was uti-
lized as received.
1
1
3
, d, ppm): 5.3 (1H, s, C
-H, W ½ = 17 Hz), 2.72 (2H, brs, NH , exchange-
), 1.16 (3H, s, C10-CH
6
H), 3.9 (1H, m, C
able with D
1
1
3
a
2
2
O), 1.23 (3H, s, C13-CH
.02 (other methyl protons); C NMR (CDCl
), 129.2 (CN), 114.6 (C
O%: C, 74.26, H, 9.12, N, 5.61. Found: 74.38, H,
.29, N, 5.78. MS: m/z 484/486 [M ].
3
3
), 1.04 and
13
0
3
, d, ppm): 166 ðC Þ,
2
0
55.2 (C
7
6
), 50.6 ðC Þ, 50.2 (C
3
). Anal. Calcd.
3
for C30
H
45ClN
2
+Å
9
2
3
0
0
.4.3. 2 -Amino-3 -cyanocholest-6-eno[5,7-de]4H-pyran (3c)
White powder, yield 75%, m.p. 149–150 °C; IR (KBr, max/cm ):
363 (NH ), 2234 (CN), 1630, 1617 (C@C), 1078 (CAO), 1327
, d, ppm): 5.26 (1H, s, C H), 2.67 (2H, brs,
, exchangeable with D O), 1.2 (3H, s, C13-CH ), 1.17 (3H, s,
ꢁ1
m
2
1
2.2. Synthesis of cholest-5-ene derivatives (1a–3a)
(CAN); H NMR (CDCl
NH
10-CH
3
6
2
2
3
1
3
3b-Acetoxycholest-5-ene (1a) was synthesized by heating mix-
C
3
), 1.04 and 1.02 (other methyl protons); C NMR (CDCl
3
,
0
ture of cholesterol (25 mg), pyridine (37 mL freshly distilled over
KOH) and freshly distilled acetic anhydride (25 mL) on a water
bath for 2 h [13]. 3b-Chlorocholest-5-ene (2a) was synthesized
by gently heating freshly purified thionyl chloride (10 mL) and
cholesterol (12.5 g) on water bath for 1 h [14]. Cholest-5-ene (3a)
was synthesized by dissolving 3b-chlorocholest-5-ene (5 g) in
warm amyl alcohol (115 mL) and sodium metal (10 g) was added
in small portions to the solution with continuous stirring over
the period of 8 h [15].
d, ppm): 164 ðC Þ, 154.3 (C
7
), 134.4 (CN), 113.3 (C
6 3
), 66.7 (C ’),
2
23.2 (C
3
); Anal. Calcd. for C30
46
H N
2
O%: C, 79.96, H, 10.12, N, 6.17
+
Å
found: C, 80.0, H, 10.22, N, 6.22; MS: m/z 450 [M ].
2.5. DNA binding experiments
2.5.1. Absorption and emission spectroscopy
The DNA binding experiments of compounds (1c–3c) were car-
ried out by using absorption titration and emission spectroscopy