3918
H. O. Saxena et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3914–3918
(b) Negi, A. S.; Chattopadhyay, S. K.; Srivastava, S.; Bhattacharya, A. K. Synth.
Commun. 2005, 35, 15; (c) Srivastava, V.; Negi, A. S.; Kumar, J. K.; Faridi, U.;
indanone derivatives (5–100 lg/ml) at 37 °C for 60 min. The concentrations of
indanones were chosen higher than the concentrations at which the
compounds showed anticancer activity.
Sisodia, B. S.; Darokar, M. P.; Luqman, S.; Khanuja, S. P. S. Bioorg. Med. Chem.
Lett. 2006, 16, 911; (d) Srivastava, V.; Saxena, H. O.; Shanker, K.; Kumar, J. K.;
Luqman, S.; Gupta, M. M.; Khanuja, S. P. S.; Negi, A. S. Bioorg. Med. Chem. Lett.
2006, 16, 4603; (e) Srivastava, V.; Darokar, M. P.; Fatima, A.; Kumar, J. K.;
Chowdhury, C.; Dwivedy, G. R.; Shrivastava, K.; Gupta, V.; Chattopadhyay, S. K.;
Luqman, S.; Saxena, H. O.; Gupta, M. M.; Negi, A. S.; Khanuja, S. P. S. Bioorg. Med.
Chem. 2007, 13, 518.
Aliquots of saline solutions of decreasing concentration (from 10 to 1 g/L) were
prepared as described earlier.19,20 The test compound treated erythrocytes
were then transferred to tubes containing decreasing concentrations of saline
solutions. After careful mixing, the cell suspensions were left to equilibrate for
30 min and then centrifuged at 3000 rpm for 5 min. The absorbance of
supernatants was read at 540 nm, with the standardised against an assay blank
(the 10 g/L saline supernatant corresponds to 0% haemolysis). The recorded
optical density (OD) of the supernatant reflects the degree of haemolysis of the
erythrocytes. The percentage lysis was calculated by dividing the OD of the
supernatant obtained from a particular saline concentration by the OD of
the standard (1 g/L) representing 100% haemolysis.21 Osmotic fragility curves
were constructed by plotting the percentage lysis against the concentration of
saline solutions. The MEF50 (mean erythrocyte fragility) value, which is the
saline concentration at which 50% of the cells haemolyse at standard pH and
temperature, was then obtained from the curve.
9. Srivastava, V.; Negi, A. S.; Kumar, J. K.; Gupta, M. M.; Khanuja, S. P. S. Bioorg.
Med. Chem. 2005, 13, 5892.
10. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchel, A. R.. In Book, Vogel’s Text
Book of practical organic chemistry; Addison-Wesley Longman, Inc.: Reading,
MA, 1998; p 1034.
11. Lawrence, N. J.; Armitage, E. S. M.; Greedy, B.; Cook, D.; Ducki, S.; McGown, A. T.
Tetrahedron Lett. 2006, 47, 1637.
12. Saxena, H. O.; Faridi, U.; Kumar, J. K.; Luqman, S.; Daroker, M. P.; Shanker, K.;
Chanotiya, C. S.; Gupta, M. M.; Negi, A. S. Steroids 2007, 72, 892.
13. (a) Syntheses: General procedure for preparing indanone derivatives from
16. Woerdenbag, H. J.; Moskal, T. A.; Pras, N.; Malingré, T. M.; Farouk, S.; EI-Feraly,
H.; Kampinga, H.; Konings, A. W. T. J. Nat. Prod. 1993, 56, 849.
chalcones: Synthesis of
3-(30,40,50-Trimethoxyphenyl)-4,5,6-trimethoxy-
indan-1-one (10). In a Borosil test tube, chalcone 9 (150 mg, 0.39 mmol) was
taken in trifluoroacetic acid (0.5 ml) and the tube was sealed carefully with
flame. The reaction mixture was heated at 120 °C for 4 h. The reaction mixture
was poured into crushed ice and extracted with ethyl acetate, organic layer
was washed with water, dried over anhydrous sodium sulphate and
evaporated in vacuo. The crude residue thus obtained was purified through
column chromatography on silica gel using ethylacetate–hexane as eluent. The
desired indanone 10 was obtained as a solid. It was recrystallised with
chloroform–hexane (1:3) to get 10 as a light brown solid.
17. Selected physical data:Compound 10: Yield = 64%; mp = 107–110 °C; IR (KBr,
cmꢂ1): 2938, 1705, 1591, 1509, 1500, 1129. 1H NMR(CDCl3, 300 MHz) d 2.58–
2.65 (dd, 1H, 2-CH, J = 2.58, 19.29 Hz), 3.13–3.22(dd, 1H, 2-CH, J = 7.98,
19.26 Hz), 3.42 (s, 3H, OCH3), 3.78 (s, 6H, 2ꢁ OCH3), 3.81 (s, 3H, OCH3), 3.91
(s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.50–4.53 (dd, 1H, 3-CH, J = 7.95, 2.49 Hz),
6.31 (s, 2H, aromatic protons), 7.09 (s, 1H, aromatic proton); 13C NMR(CDCl3,
75.46 MHz) d 42.30, 47.48, 56.58, 56.68, 56.68, 60.44, 61.14, 61.14, 100.81,
105.03, 132.65, 137.75, 140.42, 140.42, 144.38, 149.21, 150.89, 153.84, 153.84,
155.45, 205.08. EI Mass GC–MS (CH3CN): 388 [M+], 373, 357, 181.
(b) Synthesis of
3-(30,40,50-Trimethoxyphenyl)-4,5,6-trimethoxy-indan (20).
Compound 12: Yield = 71%; mp = 119–123 °C; IR (KBr, cmꢂ1): 2936, 2838, 1704,
1599, 1498, 1470, 1101. 1H NMR(CDCl3, 300 MHz) d 2.54–2.61 (dd, 1H, 2-CH,
J = 1.99, 19.07 Hz), 3.07–3.16 (dd, 1H, 2-CH, J = 8.03, 19.09 Hz), 3.38 (s, 3H,
OCH3), 3.69 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 4.75–4.78 (br d, 1H, -CH, J = 7.95, 2.49 Hz), 6.53–6.58
(br s, 2H, aromatic protons), 7.08 (s, 1H, aromatic proton); EI mass GC–MS
(CH3CN): 388 [M+], 357, 373, 358, 342.
Indanone 10 (100 mg, 0.26 mmol) was taken in trifluoroacetic acid (2.5 ml) and
the reaction flask was stirred in an ice-bath. After stirring for 5 min sodium
borohydride (100 mg, 2.6 mmol) was added in portions with maintaining the
bath temperature 0–15 °C for an hour. After that the reaction mixture was
stirred at room temperature for 6 h. On completion, 10 ml water was added to
reaction mixture and it was extracted with ethyl acetate, organic layer was
washed with water, dried over anhydrous sodium sulphate and evaporated in
vacuo. The crude residue thus obtained was purified through column
chromatography on silica gel using ethyl acetate–hexane as eluent. The
desired indan 20 was obtained as oil.
Compound 13: Yield: 48%; mp = 112–115 °C; 1H NMR(CDCl3, 300 MHz) d 2.58–
2.65 (dd, 1H, 2-CH, J = 2.42, 19.26 Hz), 3.14–3.23(dd, 1H, 2-CH, J = 7.97,
19.27 Hz), 3.39 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 4.54–4.58 (dd, 1H, –CH, J = 2.36 and 7.90 Hz), 6.66–7.23 (m, 8H,
aromatic protons); EI mass GC–MS (CH3CN): 328 [M+], 313, 207.
(c) 2-Hydroxy,
3-(30,40,50-trimethoxyphenyl)-4,5,6-trimethoxy-ind-2-en-1-one
(16). Indanone 10 (100 mg, 0.26 mmol) was taken in a round-bottomed flask
with 1,4-dioxane (10 ml) and selenium dioxide (290 mg, 2.6 mmol). The
reaction mixture was refluxed for 6 h. On completion, reaction mixture was
filtered and filtrate was evaporated in vacuo. The crude mass thus obtained
was purified through column chromatography on silica gel using ethyl
acetate–hexane as eluent. The cyclised product 17 was first obtained
followed by the desired derivative 16.
Compound 16: Yield = 67%; mp = oil; IR (KBr, cmꢂ1): 3444, 2939, 1727, 1594,
1499, 1466, 1126. 1H NMR(CDCl3, 300 MHz) d 3.33 (s, 3H, OCH3), 3.78 (s, 6H,
2ꢁ OCH3), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.65 (s, 2H, aromatic protons),
6.97 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75 MHz) d 42.30, 47.48, 56.58,
56.68, 56.68, 60.44, 61.14, 61.14, 100.81, 105.03, 132.65, 137.75, 140.42,
140.42,144.38, 149.21, 150.89, 153.84, 153.84, 155.45, 205.08. EI Mass GC–MS
(CH3CN): 402 [M+], 387, 195.
14. In-vitro anticancer activity using MTT assay. In-vitro cytotoxicity testing was
performed as per reported method.16 2 ꢁ 103 cells/well were incubated in the
5% CO2 incubator for 24 h to enable them to adhere properly to the 96-well
polystyrene microplates (Grenier, Germany). Test compound dissolved in
dimethyl sulphoxide (DMSO, Merck, Germany), in at least five concentrations,
were added into the wells and left for 4 h. After the incubation, the compound
plus media was replaced with fresh media and the cells were incubated for
another 48 h in the CO2 incubator at 37 °C. The concentration of DMSO were
Compound 17: Yield = 16%; mp = 145–148 °C; IR (KBr, cmꢂ1): 2933, 1697, 1474,
1384, 1096. 1H NMR(CDCl3, 300 MHz) d 3.90 (s, 3H, OCH3), 3.95 (s, 3H, OCH3),
3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.07 (s, 3H, OCH3),
7.04 (s, 1H, aromatic proton), 8.07 (s, 1H, aromatic proton); 13C NMR(CDCl3,
75 MHz) d 56.55, 56.93, 61.10, 61.37, 61.51, 62.09, 105.96, 106.96, 127.09,
130.38, 133.06, 136.87, 137.10, 141.73, 147.17, 147.52, 149.42, 153.86, 154.26,
156.33, 187.84. EI Mass GC–MS (CH3CN): 400 [M]+.
Compound 19: Yield = 58%; mp = 132–136 °C ; IR (KBr, cmꢂ1): 3516, 2941, 1593,
1503, 1463, 1419, 1335, 1120. 1H NMR(CDCl3, 300 MHz) d 1.92–2.01 (m, 1H, 2-
CH), 2.94–2.99 (m, 1H, 2-CH), 3.45 (s, 3H, OCH3), 3.79 (s, 12H, 4ꢁ OCH3), 3.90
(s, 3H, OCH3), 4.22–4.27 (m, 1H, 3-CH), 5.16–5.20 (m, 1H, 1-CH), 6.48 (s, 2H,
aromatic protons), 6.81 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75.46 MHz) d
46.35, 47.58, 56.45, 56.53, 60.21, 60.42, 60.94, 60.98, 75.35, 103.54, 105.24,
always kept below 1.25%, which was found to be non-toxic to cells. Then, 10 lL
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was
added to each well and plates were incubated at 37 °C for 4 h. DMSO
(100 lL) was added to all wells and mixed thoroughly to dissolve the dark blue
crystals. The plates were read on SpectraMax 190 Microplate reader (Molecular
Devices Inc. USA) at 570 nm within 1 h of DMSO addition.
105.88, 130.49, 137.01, 141.54, 142.30, 142.82, 150.36, 153.31, 154.46. EI Mass
28
15. Determination of osmotic haemolysis of erythrocytes: Blood from healthy human
male volunteers (n = 3) with informed consent was collected for experiments
using heparin (10 U/ml) as the anti-coagulant. The collected blood was stored
at 4 °C and was used for experiments within 4 h of collection.18 Experiments
were carried in-vitro by adding heparinised blood to hypotonic solutions of
varying concentrations of phosphate buffered saline (0.85% to 0.10%).
Phosphate-buffered saline stock (10%) was prepared by dissolving 5 g of
sodium chloride, 1.3655 g of disodium hydrogen orthophosphate and 0.243 g
of sodium dihydrogen orthophosphate in 100 ml of autoclaved double distilled
water. From this stock, working standards of 0.85% to 0.10% were prepared. The
tubes were incubated at 37 °C for 60 min with mild shaking and the extent of
haemolysis was measured colorimetrically at 540 nm.19 Results are expressed
in terms of mean erythrocyte fragility (MEF50), which is the level of haemolysis
of the erythrocytes at 50% saline concentrations. Similarly, prior to the
experiment, heparinised blood was incubated with effective concentration of
GC–MS (CH3CN): 390 [M+], 372, 357. ½
a
ꢃ
þ 6:99ꢄ (1.04, MeOH).
589
Compound 20: Yield = 72%; mp = 71–74 °C ; 1H NMR(CDCl3, 300 MHz) d 1.99–
2.06 (m, 1H, 2-CH), 2.52–2.59 (m, 1H, 2-CH), 2.85–2.89 (m, 1H, 1-CH), 2.99–
3.06 (m, 1H, 1-CH), 3.51 (s, 3H, OCH3), 3.77 (s, 6H, 2ꢁ OCH3), 3.80 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.37–4.42 (m, 1H, 3-CH), 6.33 (s, 2H,
aromatic proton), 6.63 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75.46 MHz) d
22.66, 29.67, 31.93, 49.43, 56.35, 56.35, 60.15, 60.73, 60.81, 104.01, 105.19,
130.84, 139.95, 142.38, 150.29, 153.93, 153.76. EI Mass GC–MS (CH3CN): 390
[M+], 372, 357.
18. Luqman, S.; Rizvi, S. I. Asia Pacific J. Pharmacol. 2004, 16, 53.
19. Luqman, S.; Obli Prabu, K. V.; Pal, A.; Saikia, D.; Darokar, M. P.; Khanuja, S. P. S.
Nat. Prod. Commun. 2006, 1, 481.
20. Luqman, S.; Kumar, N.; Rizvi, S. I. University Allahabad Studies 2004, 3, 29.
21. Dacie, J. V.; Lewis, S. M. Practical Hematology, 1984; Orient Longman,
p
152.