4102
M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
MS, 390.3254 (calcd 390.3248 for C24H38O4), 362, 344, 326, 330, 245, 175, 161,
References and notes
121, 91, 81, 55. IR mmax (CHCl3) cmꢀ1; 3205, 2824, 1650, 1580, 1450. 1H NMR
(500 MHz, DMSO-d6): d 7.30 (t, J = 8.0 Hz, 8.5Hz, H-4), 6.90 (d, J = 8.5 Hz, H-3),
1. (a) Wimo, A.; Winblad, B.; Aguero-Torres, H.; von Strauss, E. Alzheimer Dis. Assoc.
Disord. 2003, 17, 63; (b) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461; (c)
Oh, M. H.; Houghton, P. J.; Whang, W. K.; Cho, J. H. Phytomedicine 2004, 11, 544;
(d) Schulz, V. Phytomedicine 2003, 74; (e) Melzer, D. BMJ 1998, 316, 762; (f)
Mukherjeea, P. K. V.; Kumarb, M.; Malb, H. P. J. Phytomedicine 2007, 14, 289.
2. (a) Pinto, M. M. M.; Kijjoa, A.; Mondranondra, I.-N.; Gutierrez, A. B.; Herz, W.
Phytochemistry 1990, 29, 1985; (b) Hake, I.; Schonenberger, S.; Neumann, J.;
Franke, K.; Paulsen-Merker, K.; Reymann, K.; Ismail, G.; Bin din, L.; Said, I. M.;
Latiff, A.; Wessjohann, L.; Zipp, F.; Ullrich, O. J. Neuroimmunol. 2008, 91; (c)
Graham, J. G.; Quinn, M. L. F. D. S. F. N. R. J. Ethnopharmacol. 2000, 73, 347; (d)
Zeng, I.; Gu, Z.-M.; Fang, X.-P.; McLaughlin, J. L. J. Nat. Prod. 1994, 57, 376; (e)
Kijjoa, A.; Gonzalez, M. J. T. G.; Pinto, M. M. M.; Monanondra, I.-O.; Herz, W.
Planta Med. 1991, 57, 575; (f) Gonzalez, M. J. T. G.; Pinto, M. M. M.; Kijjioa, A.;
Anantachoke, C.; Herz, W. Phytochemistry 1993, 32, 433; (g) Zahir, A.; Jossang, A.;
Bodo, B.; Hadi, H. A.; Schaller, H.; Sevenet, T. J. Nat. Prod. 1993, 56, 1634; (h)
Deng, J.-Z.; Starck, S. R.; Li, S.; Hecht, S. M. J. Nat. Prod. 2005, 68, 1625; (i)
Maloney, D. J.; Deng, J.-Z.; Starck, S. R.; Gao, Z.; Sidney, M.; Hecht, S. M. J. Am.
Chem. Soc. 2005, 127, 4140; (j) Ahmad, W. Y.; Tee, S. K.; Din, L. B.; In A Scientific
Journey through Borneo Tawau Hills Park, Sabah Ismail, G., Ed.; Palahniuk
Publications: Kuala Lumpur; 1995, p 139.; (k) Gonzales, M. J.; DeOliveira, C.;
Fernandes, J.; Kijjoa, A.; Herz, W. Phytochemistry 1996, 43, 1333; (l) Pereira, J. M.;
Severino, R. P.; Vieira, P. C.; Fernandes, J. B.; Silva, M. F. G. F.; Zottis, A.; Adriano,
D.; Andricopulo, A. D.; Oliva, G.; Corrêa, A. G. Bioorg. Med. Chem. 2008, 16, 8889.
3. (a) Marston, A.; Kissling, J.; Hostettmann, K. Phytochem. Anal. 2002, 13, 51; (b)
Tanaka, S.; Yoichi, S.; Ao, L.; Matumoto, M.; Morimoto, K.; Akimoto, N.; Honda,
G.; Tabata, M.; Oshima, T.; Masuda, T.; bin Asmawi, M. Z.; Ismail, Z.; Yusof, S. M.;
Din, L. B.; Said, I. M. Phytother. Res. 2001, 15, 681; (c) Rhee, I. K.; Meent, M. V.;
Ingkaninan, K.; Verpoorte, R. J. Chromatogr. 2001, 15, 217.
4. (a) Spencer, G. F.; Tjarks, L. W.; Kleiman, R. J. Nat. Prod. 1980, 43, 724; (b) Kubo,
I.; Nihel, K.-I.; Kazuo, K. J. Agric. Food Chem. 2003, 51, 7624; (c) Du, Y.; Oshima, R.;
Yamauchi, Y.; Kumanotani, J.; Miyakoshi, T. Phytochemistry 1986, 25, 2218; (d)
Atta-ur-Rahman; Zareen, S.; Choudhary, M. I.; Akhtar, M. N.; Ngounou, F. N.
Phytochemistry 2009, 69, 2400; (e) Cheng, M.-J.; Lee, S.-J.; Chang, Y.-Y.; Wu, S.-H.;
Tsai, I.-L.; Jayaprakasam, B.; Chen, I.-S. Phytochemistry 2003, 63, 603; (f)
Carballeira, N. M.; Cruz, M. Chem. Lipids 1996, 84, 81; (g) Francis, G. W.;
Veland, K. J. Chromatogr. 1981, 219, 379.
5. (a) Franke, K.; Masaoud, M.; Schmidt, J. Planta Med. 2001, 67, 477; (b) Kato, M. J.;
Lopes, L. M. X.; Fo, H. F. P.; Yoishid, M.; Gottlieb, O. R. Phytochemistry 1985, 24,
533; (c) Liua, Y.; Abreu, P. J. M. J. Braz. Chem. Soc. 2006, 17, 527; (d) Kubo, I.; Kim,
M.; Naya, K.; Komatsu, S.; Yamagiwa, Y.; Ohashi, K.; Sakamoto, Y.; Hirakawa, S.;
Kamikawa, T. Chem. Lett. 1987, 1101; (e) Lee, J. S.; Cho, Y. S.; Park, E. J.; Kim, J.;
Oh, W. K.; Lee, H. S.; Ahn, J. S. J. Nat. Prod. 1998, 61, 867; (f) Itokawa, H.; Totsuka,
N.; Nakahara, K.; Takeya, K.; Lepoittevin, J.-P.; Asakawa, Y. Chem. Pharm. Bull.
1987, 35, 3016; (g) Green, I. R.; Tocoli, F. E.; Lee, S. H.; Niheib, K.-I.; Kubob, I.
Bioorg. Med. Chem. 2007, 15, 6236; (h) Lund, A.-K.; Lemmich, J.; Andsersen, A.;
Olsen, C. E. Phytochemistry 1997, 44, 689.
6. (a) The stem bark of K. laurina (6.2 kg) was collected from the Pasir Raja Forest
Reserve, Trengganu, Malaysia, in May 2008. The voucher specimen SK 1540/08
was deposited at the Herbarium of the Institute of Bioscience (IBS), University
Putra Malaysia. The plant was identified by the Resident Botanist of IBS. The
stem barks were cleaned, cut into small pieces and left under the shade for
5 days at room temperature. The dry pieces were ground to powder and then
steeped in distilled methanol (10 L) at room temperature for 2 days. The
methanol extract was then filtered off and the filtrate was concentrated under
reduced pressure. This procedure was repeated three times and the extracts
were combined to yield 90.1 g of crude methanolic extract. The combined
extract was then fractionated between water and organic solvents (sequentially:
hexane; dichloromethane; ethyl acetate and finally butanol) to yield the hexane
(20.2 g), DCM (3.64 g), EtOAc (7.41 g), BuOH (6.90 g) and aqueous (45.8 g)
soluble fractions. The hexane soluble fraction was subjected to silica gel column
chromatography (230–400 mesh, ASTM, Merck) using the gradient solvent
system hexane/EtOAc (8:2 to 6.5:3.5) to yield seven sub-fractions (kl-1 to kl-7).
Subfractions kl-3 (1.20 g) and kl-4 (0.09 g) were separately subjected to
repeated column chromatography (230–400 mesh) by eluting it gradiently
using hexane/EtOAc (9:5 to 5.5:4.5) as solvent system, to yield compounds 2
0
0
0
0
6.87 (d, J = 8.0 Hz, H-5), 5.50 (dt, J8
, , = 14.5,
= 14.5, 6.5 Hz, H-80), 5.36 (dt, J9
9
8
4.5 Hz, H-90), 4.47 (d, J10 ,9 = 4.5 Hz), 3.79 (s, 3H, OMe), 3.72 (s, 3H, COOMe), 2.56
(t, J = 7.5 Hz, C-10), 1.99 (m, 2H, C-70), 1.50 (m, 2H, C-110), 1.42–1.40 (br m, 6H,
3 ꢁ CH2, C-30 to C-40 and C-120), 1.31–129 (br m, 8H, 4 ꢁ CH2, C-5 to C-60 and C-
130 to C-140), 0.89 (t, J = 7.5 Hz, 3H, C-150).; (d) Bisthiomethylation of compound 1:
Compound 1 (3.0 mg) was dissolved in dimethyl disulfide (3 ml) in a 25 ml
single necked round bottom flask and iodine (10 mg) was added. The reaction
mixture was kept at 50–60 °C for overnight. The reaction mixture was diluted
with 5 ml diethyl ether and excess iodine was decomposed with 5% sodium
thiosulfate solution. The diethyl ether layer was separated and dried over
sodium sulfate anhydrous and subjected to GC–MS analysis, which showed the
peaks at m/z = 412, 396, 394, 251, 185, 161, 108, and 83 (Scheme 1).; (e) 3-
0
0
(pentadecen-100(Z)-yl)phenol (2): Colorless oil; UVMk eOH nm (log
e): 277 (2.8), 272
(2.6): 222 (3.0); IR
m
max (CHCl3) cmꢀ1, 3418–3220,m2ax928, 2809, 1610, 1455, 890.
EI-MS m/z (rel. int.): 302 [M+], HREI-MS, m/z 302.2604 (calcd 302.2610 for
C
21H34O), 302, 276, 220, 206, 175, 161, 147, 133, 121, 108 (100%), 77, 43. 1H and
13C NMR CDCl3 (125 MHz) (Table 2).; (f) 3-Methoxypentadec-(100(Z)-enyl)-
benzene (2a): Compound 2 (10 mg) was dissolved in diethyl ether at room
temperature, and into it few drops of diazomethane was added. After drying the
sample in a fume hood the compound 2b was obtained as colorless oil. EI-MS m/
z (rel. int.): 316 [M+], 302, 276, 220, 206, 175, 161, 147, 133, 123, 121, 108
(100%), 77, 55. IR m
max (CHCl3) cmꢀ1: 3252–3160, 2930–2936, 1620, 1456, 1289,
780. 1H NMR (500 MHz, CDCl3): d 7.13 (t, J = 8.0, 7.5 Hz, H-5), (d, 6.66 (br d, IH,
C-2), 6.75 (d, J = 7.5 Hz, H-4), 6.65, (br d, 1H, H-6), 3.73 (s, 3H, OMe), 2.55 (t, 7.5,
each, H-10), 2.02 (m, 4H, H-90 and H-120), 1.62 (m, 2H, H-20), 1.27–1.30 (br m,
16H, 8 ꢁ CH2, H-30 to C-80 and H-130 to H-140), 0.90 (t, 3H, J = 7.5, H-150).; (g)
Bisthiomethylation of compound 2: Dimethyl disulfide (DMDS) adduct was
prepared by taking 5 mg of compound 2 in a 25 ml round bottle flask containing
10 ml hexane. About 1.5 ml dimethyl disulfide was added into it followed by
20 mg of iodine. The reaction mixture was stirred at 50–60 °C for overnight. The
diethyl ether layer was separated and dried over sodium sulfate anhydrous and
subjected to GC–MS analysis. Fragmentation peaks observed include m/z = 396,
279, 117, 108, 107, 69, and 55.; (h) 2-Hydroxy-6-(pentadec-100(Z)-enyl)benzoic
acid (4): Colorless oil, IR m
max (CHCl3) cmꢀ1; 3510–3228, 2920, 2860, 1650, 1609,
1580, 1450. El-MS m/z (rel. int.): 346 [M+], C22H34O3, (55), 328 (29), 320 (15),
310 (22), 302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152 (50), 147
(42), 134 (37), 120 (10), 108 (50), 91 (100); 1H NMR (500 MHz, CDC13): d 7.36 (t,
J = 8.0, 8.5 Hz, each, H-4), 6.87 (dd, J = 8.0 Hz, H-3), 6.76 (dd, J = 8.5 Hz, H-5), 5.35
(centre of AB system of H-100 and H-110 J10
,
= J11
,
= 9.0 Hz,
0
0
0
0
11
10
0
0
0
0
J9
,
10
= J11
,
12
= 6.5 Hz), 2.98 (d, J = 7.5 Hz, C-10), 2.01 (m, 4H, C-90 and C-120),
1.60 (m, 2H, C-20), 1.41–1.40 (br m, 8H, 4 ꢁ CH2, C-30 to H-40 and C-130 to C-140),
1.30–1.29 (br m, 8H, 4 ꢁ CH2, C-50 to C-80), 0.91 (t, J = 7.5 Hz, 3H, C-150).; (i) 2-
Hydroxy-6-(pentadec-100(Z)-enyl)benzoic acid methyl ester (4a): Compound
4
(10 mg) was treated with ethereal solution of diazomethane and methylated
compound 4a was purified by evaporation of solution and passing through silica
gel funnel. The EI-MS showed m/z 374.2 [M+], C24H38O3, [M+], (55), 328 (29), 320
(15), 310 (22), 302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152
(50), 147 (42), 134 (37), 121 (10). 1H NMR (500 MHz CDC13): d 7.36 (t, J = 8.0,
8.5 Hz, H-4), 6.87 (dd, J = 8.5 Hz, H-3), 6.76 (dd, J = 8.0 Hz, H-5), 5.35 (centre of
AB system of H-100, J10 ,11 = J11 ,10 = 9.0 Hz, J9 ,10 = J11 ,12 = 6.5 Hz, H-100 and H-
110), 3.80 (s, 3H, OMe), 3.74 (s, 3H, COOMe), 2.98 (d, J = 7.5 Hz, 2H, C-10), 2.01 (m,
4H, C-90 and C-120), 1.60 (m, 2H, C-20), 1.41–1.39 (br m, 8H, 4 ꢁ CH2, C-30 to H-40,
C-130 to C-140), 1.31–1.29 (br m, 8H, 4 ꢁ CH2, C-50 to C-80), 0.90 (t, J = 7.5 Hz, 3H,
C-150).
0
0
0
0
0
0
0
0
7. (a) Wiesner, J.; Kriz, Z.; Kuca, K.; Jun, D.; Koca, J. J. Enzyme Inhib. Med. Chem. 2007,
22, 417; (b) Radic, Z.; Duran, R.; Vellom, D. C.; Li, Y.; Cervenansky, C.; Taylor, P. J.
Biol. Chem. 1994, 269, 11233; (c) Harel, M.; Sonoda, L. K.; Silman, I.; Sussman, J.
L.; Rosenberry, T. L. J. Am. Chem. Soc. 2008, 130, 7856; (d) Leonetti, F.; Catto, M.;
Nicolotti, O.; Pisani, L.; Cappa, A.; Stefanachi, A.; Carotti, A. Bioorg. Med. Chem.
2008, 16, 7450; (e) Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.;
Kugelmann, E. Exp. Opin. Ther. Targets 2007, 11, 161.
8. In vitro cholinesterase inhibition assay: Acetylcholinesterase inhibiting activity
was measured by the spectrophotometric method of Ellman et al.8a Electric eel
AChE (type VI-S, electric eel, Sigma Chemical Co. code: C2888), were used as
sources of the cholinesterases. Acetylthiocholine iodide (Sigma Chemical Co.
[ATCI] code: A5751) were used as substrates of the reaction, and 5,5-dithiobis-
(2-nitrobenzoic) acid (DTNB) (Sigma Chemical Co. Code: D21200, 99%) were
used for the measurement of the cholinesterase activity. In this procedure;
(45.5 mg) and
3 (57.0 mg), respectively. Using the same elution system,
subfraction kl-5 (1.80 g) was subjected to repeated silica gel column
chromatography (230–400 mesh) to obtained compounds 4 (90.4 mg) and 5
(21.0 mg). The semi pure compounds (2–5) were purified by HPLC [mobile
phase: ACN/H2O (40:60 to 45:55; UV: 254 nm; flow rate: 4.00 ml/min; column
200
ll
of 0.15 mM sodium phosphate buffer pH 7.4 with 10
ll of DTNB
(0.15 mM), 20
l
l of test compound solution, and 20 l of acetylcholinesterase
l
XterraÒ Prep MS C18 OBD™ (19 ꢁ 150 mm, 5
lm)], which led to the purification
(final concentration 0.037 U/ml in 0.1 M phosphate buffer solution) were mixed
and incubated for 10 minutes at 25 °C. The reaction was then started by the
addition of 20 ll of acetylthiocholine iodide (0.25 mM, substrate). The final
volume of the assay in each well was 270 y monitoring the formation of the
yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with
of 5 (tR: 11.2 min), 4 (tR: 11.7 min), 3 (tR: 16.3 min) and 2 (tR: 16.8 min).; (b)
Physical and spectroscopic data of compounds: (a) (+)-2-hydroxy-6-(100-
hydroxypentadec-80(E)-enyl)benzoic acid (1): colorless oil, ½a 2D5
ꢂ
+23.2, CHCl3).
UVMk eOH nm (log
e): 306 (3.9), 288 (3.3), 272 (2.3), 227 (1.8). IR mmax (CHCl3)
max
cmꢀ1; 3616–3420, 2920, 2860, 1648, 1455, 786. El-MS m/z (rel. int.): 362 [M+],
HREI-MS: 362.2667 (362.2669 calcd for C22H34O4), 344, 326 (29), 330 (15), (22),
302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152 (50), 147 (42), 134
(37), 120 (10), 108 (50), 91 (100), 82 (19), 55 (37). 1H and 13C NMR data (Table
2).; (c) (+)-2-Methoxy-6-(100-hydroxypentadec-80(E)-enyl)benzoic acid methyl
thiocholine released by the enzymatic hydrolysis of acetylthiocholine at
wavelength of 412 nm at 25 °C monitored for 3 min. Test compounds were
dissolved in analytical grade DMSO. The same volume 270 l was used for
negative control by the addition of 20 of buffer instead of sample. All
reactions were carried out in triplicate. The concentration used in M,
a
l
l
l
l
ester (1a): In
a 25 ml round bottomed flask, compound 1 (5.0 mg) was
percentage inhibition was calculated in Microsoft excel and IC50 were
calculated by using graph Pad software. (a) Ellman, G. L.; Courtney, K. D.;
Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
dissolved in CHCl3 and few drops of diazomethane were added. After drying
compound 1a was obtained. Colorless oil, El-MS m/z (rel. int.): 390 [M+], HREI-