Cytotoxic grifolin derivatives isolated from the wild mushroom Boletus pseudocalopus (Basidiomycetes)

Article abstract of DOI:10.1002/cbdv.200800217

Activity-guided purification of a MeOH extract of the Korean wild mushroom Boletus pseudocalopus afforded three new grifolin derivatives, 1-3, along with four known phenolic compounds 4-7. Their structures were established by a combination of ¹

Full text of DOI:10.1002/cbdv.200800217

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Cytotoxic Grifolin Derivatives Isolated from the Wild Mushroom Boletus
pseudocalopus (Basidiomycetes)
by Junsik Song, Md. Maniruzzaman Manir, and Surk-Sik Moon*
Department of Chemistry, Kongju National University, Kongju 314-701, Republic of Korea
(phone: þ82-41-850-8495; fax: þ82-41-850-8479; e-mail: ssmoon@kongju.ac.kr)
Activity-guided purification of a MeOH extract of the Korean wild mushroom Boletus pseudoca-
lopus afforded three new grifolin derivatives, 1–3, along with four known phenolic compounds 4–7. Their
structures were established by a combination of 1H- and 13C-NMR, NOESY, and extensive two-
dimensional NMR spectroscopic experiments such as gCOSY, gHSQC, gHMBC, and ROESY. The
major metabolites 4 and 5 were subjected to reduction to provide the side chain-reduced compounds 8
and 9 for biological testing. All of the compounds except compound 6 showed anticancer activities in the
range of IC50 3.5–11.0 mg/ml against human lung carcinoma A549 and mouse melanoma B16F1 cell lines.
In addition, all compounds showed moderate radical-scavenging activities determined by DPPH assay.
Introduction. – Mushrooms (macrofungi) accumulate a variety of cytotoxic
secondary metabolites including polyphenols, terpenoids, and alkaloids [1–5]. Medicinal
mushrooms such as Ganoderma lucidum, Phellinus linteus, and Coriolus versicolor,
have an established history of use in traditional Asian therapies [6][7]. Several
polysaccharides and polysaccharide conjugates have been commercialized for the
clinical treatment of patients undergoing anticancer therapy. Irofulven, derived from
antitumor sesquiterpene illudins, possesses strong cytotoxic effects against a wide
variety of solid tumors [2][8].
In the search for biologically active compounds from natural sources [9][10], we
have collected hundreds of wild mushroom species in the mountainous areas during the
hot humid summer and prepared MeOH extracts from them for anticancer-activity
tests. Among them, the extracts from Boletus pseudocalopus showed strong cytotoxic
activities against human lung cancer, mouse melanoma, and human melanoma cell lines
with IC50 values of 20, 15, and 40 mg/ml, respectively, as evaluated by the sulforhod-
amine B (SRB) colorimetric method. This mushroom is recognized by its red fruiting
bodies and the blue staining, when the fruit-bodies are cut or brushed, presumably due
to the characteristic bolete pigments like xerocomic acid, variegatic acid, and
variegatorubin [11][12]. Phytochemicals in this species have been rarely investigated,
while the Boletales like B. satanas, B. erythropus, and B. curtisii have been reported to
produce cytotoxic glycoprotein bolesatine, which shows inhibitory effect of protein
synthesis in radiation-induced thymic lymphosarcoma cell line (IC50 0.6 mg/ml) [13], a
water-soluble b-glucan [14], and yellow pigments curtisins [15], respectively.
Here, we report the isolation and structure determination of new cytotoxic
metabolites 1–3 together with known grifolin derivatives 4–7 from the wild Korean
mushroom B. pseudocalopus. In addition, their cytotoxic effects against cancer cell lines
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
(human lung, human melanoma, and mouse melanoma) and radical-scavenging
activities are reported.
Results and Discussion. – 1. Isolation and Structure Elucidation. Fresh wild B.
pseudocalopus mushrooms (1.1 kg) were extracted twice with MeOH. The extracts
were fractionated by a silica-gel flash column chromatography, and their cytotoxicities
against human lung cancer A549, mouse melanoma B16F1, and human melanoma SK-
Mel-2 cell lines evaluated by the SRB colorimetric cell-growth inhibition measure-
ments. The active fractions were further purified by C18 MPLC, followed by preparative
C18 HPLC to afford 5.0, 3.1, 4.6, 2.9, and 5.8 mg of compounds 1, 2, 3, 6, and 7 as minor
metabolites, respectively, and 2.0 and 1.3 g of compounds 4 and 5 as major metabolites,
respectively (Fig. 1).
Fig. 1. Chemical structures of grifolin derivatives 1–9 isolated from Boletus pseudocalopus, and vitamin
E (a-tocopherol)
Compound 1 was obtained as a pale yellow oil with a specific optical rotation of
þ2.8 (c¼0.36, MeOH). The molecular formula was determined to be C25H38O3 as the
high-resolution time-of-flight mass spectrum (HR-TOF-MS) showed its deprotonated
molecular ion [MꢀH]ꢀ at m/z 385.2740. The IR spectral absorptions at n˜max 3345, 1622,
and 1600 cmꢀ1, and the UV absorption (MeOH) at lmax 231 and 273 nm indicated the
presence of a phenolic group. The 1H-NMR spectrum in CDCl3 gave signals for five Me
groups as singlets at d 1.58, 1.59, 1.67, 1.81, and 2.15, for one Me group as doublet at d

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1437
1.47 (J¼6.8), and signals of a MeO group at d 3.36, of CH H-atom at d 4.69 (q, J¼6.8),
of three olefinic H-atoms at d 5.06–5.08 (m, 2 H) and 5.29 (tq, J ¼ 6.0, 0.8, 1 H), and of
an aromatic H-atom as singlet at d 6.20. The 13C-NMR spectrum showed the presence of
two O-bearing aromatic C-atoms (d 154.3 and 155.1). The characteristic appearance of
a farnesyl group was observed in the 1H- and 13C-NMR spectra (Table 1). In the HMBC
spectrum, important long-range C,H correlations were observed, which served to locate
the side chains and the MeO group: HꢀC(9) to C(3), C(4), C(5), and C(8); HꢀC(1’)
to C(2), C(3), C(2’), and C(3’); Me(7) to C(4), C(5), and C(6); and the MeO H-atoms
to C(9) (Fig. 2). The interpretation of the 2D-NMR spectra (1H,1H-COSY, HSQC,
HMBC, and NOESY) allowed all 1H- and 13C-NMR assignments; thus, the structure of
compound 1 was determined to be 4-(1-methoxyethyl)-5-methyl-2-[(2E,6E)-3,7,11-
trimethyldodec-2,6,10-trienyl]benzene-1,3-diol.
Fig. 2. HMBC Correlations (H!C) in 1–3
Compound 2 was obtained as a pale yellow oil ([a]2D0 ¼ þ0.97 (c¼0.31, MeOH)).
The molecular formula was determined to be C26H40O3 by negative-ion HR-TOF-MS
(m/z 399.2869 ([MꢀH]ꢀ ). The IR and UV spectra of compound 2 were observed to be
almost identical with those of compound 1. The 1H- and 13C-NMR spectra of compound
2 were similar with those of compound 1, except the signals of the EtO group observed
at d(H) 1.26 (d(C) 15.6) and d(H) 3.53–3.61 (d(C) 65.1) of compound 2 instead those
of the MeO group of compound 1. Thus, the structure of compound 2 was deduced to be
4-(1-ethoxyethyl)-5-methyl-2-[(2E,6E)-3,7,11-trimethyldodec-2,6,10-trienyl]benzene-
1
1,3-diol. All H and 13C signals were assigned from the interpretation of extensive 2D
NMR (HSQC, HMBC, and ROESY) experiments (Table 1).
Compound 3 was obtained as a pale yellow oil with a molecular formula of C26H38O3
determined by the HR-TOF-MS spectrum. It showed a specific optical rotation of
[a]D20 ¼ ꢀ3.8 (c¼0.42, MeOH). The IR (n˜max 3330, 1604, and 1450 cmꢀ1) and UV (lmax
235 and 283 nm) spectra of compound 3 indicated the presence of a OH group and an
aromatic ring. The 1H-NMR spectrum of 3 in CDCl3 showed the characteristic signals
for a farnesyl group and one aromatic H-atom signal. In the 1H,1H-COSY spectrum, the
CH H-atom (d 3.05 (br. quint., J¼7.2)) was observed to be coupled with the Me H-
atoms (d 1.24 (d, J¼7.2)) and the CH2 H-atoms (d 1.78–1.85 (m) and 2.02–2.05 (m)),

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 1. 1H- and 13C-NMR Data of the Compounds 1–3 in CDCl3. d in ppm; J in Hz.
Position
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
1
2
3
4
5
6
7
8
9
155.1
112.6
154.3
116.4
154.8
112.4
154.2
116.9
153.9
112.3
150.8
117.6
134.7
110.8
18.9
21.8
27.0
36.7
134.1
133.7
6.20 (s)
2.15 (s)
109.6 6.20 (s)
19.4 2.16 (s)
20.8 1.48 (d, J¼6.8)
78.2 4.80 (q, J¼6.8)
109.4 6.30 (s)
19.5 2.22 (s)
21.1 1.24 (d, J¼7.2)
76.0 3.05 (br. q, J¼7.2)
2.02–2.05 (m),
1.78–1.85 (m)
5.53 (br. d, J¼8.8)
22.4 3.40 (d, J¼6.8)
1.47 (d, J¼6.8)
4.69 (q, J¼6.8)
10
11
1’
2’
3’
4’
5’
6’
7’
8’
92.0
22.6
3.41 (d, J¼7.2)
22.2 3.42 (d, J¼7.0)
5.29 (tq, J¼6.0, 0.8) 122.4 5.31 (tq, J¼7.0, 1.0) 122.3 5.25 (br. t, J¼7.0) 122.4
138.5
138.3
138.2
40.5
26.6
2.04–2.07 (m)
2.08–2.11 (m)
5.06–5.08 (m)
40.0 2.04–2.08 (m)
26.7 2.10–2.13 (m)
124.0 5.08–5.10 (m)
135.7
39.9 1.96–2.00 (m)
26.9 2.03–2.06 (m)
124.6 5.08–5.10 (m)
131.5
40.1 2.04–2.08 (m)
26.8 2.07–2.12 (m)
123.9 5.07 (br. t, J¼6.8) 124.0
135.5
135.7
39.9
26.9
1.94–1.98 (m)
2.02–2.05 (m)
5.06–5.08 (m)
40.0 1.94–1.98 (m)
27.0 2.02–2.05 (m)
9’
10’
11’
12’
13’
14’
15’
124.5 5.09 (br. t, J¼6.8) 124.6
131.3
131.6
25.9
17.9
16.3
16.5
1.67 (br. s)
1.59 (s)
1.58 (br. s)
1.81 (br. s)
25.9 1.69 (d, J¼1.2)
26.0 1.67 (s)
18.0 1.59 (s)
16.4 1.57 (s)
16.6 1.80 (s)
17.9 1.61 (s)
16.2 1.60 (d, J¼0.8)
16.4 1.82 (d, J¼1.0)
57.1
MeOꢀC(9) 3.36 (s)
EtOꢀC(9)
3.53–3.61 (m),
1.26 (t, J¼7.0)
65.1
15.6
which were in turn coupled with the CH H-atom (d 5.53 (br. d, J¼8.8)). Based on the
coupling constants, the two CH H-atoms were assigned to be axial. These H-atoms
were correlated with the corresponding C-atoms in the HSQC spectrum as follows:
d(H) 3.05 to d(C) 27.0; d(H) 1.24 to d(C) 21.8; d(H) 1.78–1.85 and 2.02–2.05 to d(C)
36.7; and d(H) 5.53 to d(C) 92.0. In the HMBC experiment, the H-atom with the signal
at d 3.05 (HꢀC(9)) was correlated with the C-atoms with signals at d 21.8, 36.7, 117.6,
134.7, and 150.8; other important correlations were observed between CH2(10) and
C(8), C(9), and C(11); and HꢀC(1’) and C(1), C(2), C(3), C(2’), and C(3’) (Fig. 2).
Thus, the structure of compound 3 was determined to be (2R*,4R*)-3,4-dihydro-4,5-
dimethyl-8-[(2E,6E)-3,7,11-trimethyldodec-2,6,10-trienyl]-2H-[1]benzopyran-2,7-diol.
Compound 4, the major metabolite of this mushroom, was obtained as a brown
solid. Its molecular formula was deduced to be C22H31O2 based on the positive-ion HR-
TOF-MS spectrum (m/z 329.2482, [MþH]þ ). Upon extensive spectroscopic analysis,
it was determined to be grifolin, previously isolated from the inedible mushroom

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1439
Grifola confluens with antibiotic activities [16][17] and from Albatrellus ovinus with
antioxidant activities [18]. The second major metabolite 5 was obtained as a brown
solid and determined to be neogrifolin [18], an isomer of grifolin. Compound 6 was
obtained as a brown oil and determined to be 2-(4,8-dimethylnona-3,7-dienyl)-3,4-
dihydro-2,7-dimethyl-2H-[1]benzopyran-3,5-diol [19]. Compound 7 was obtained as a
brown oil and determined to be 2-((2E,6E)-4,8-dimethylnona-3,7-dienyl)-2,7-dimeth-
yl-2H-[1]benzopyran-5-ol, a dehydrated form of compound 6, which was previously
described in the literature as confluentin isolated from Albatrellus spp. [20].
To see the effect of the relatively rigid structural feature of the farnesyl groups on
the biological activity, compounds 8 and 9 were prepared from the hydrogenation of
compounds 4 and 5 with H2 gas in the presence of Pd/C catalyst at room temperature in
MeOH, respectively.
2. Biological Studies. The cytotoxic effects of the isolates 1–7, and the reduced
compounds 8 and 9 were evaluated by SRB colorimetric method against human lung
carcinoma, mouse melanoma, and human melanoma cell lines. The IC50 values of the
compounds, except compound 6, were within a range of 5.0–11.0 and 3.5–7.3 mg/ml
against human lung carcinoma A549 and mouse melanoma B16F1, respectively,
comparable to cisplatin, which was used as a positive control (Table 2). The new
isolates 1–3 showed relatively stronger cytotoxic effects against human melanoma SK-
Mel-2 than the other compounds. It is interesting to note that vitamin E (a-tocopherol),
a structurally related antioxidant, showed no cytotoxic effects against all the cancer cell
lines tested below a concentration of 40 mg/ml. The radical-scavenging activities of
compounds 1–9 were also evaluated by using the diphenyl-p-picrylhydrazyl (DPPH)
assay method. All compounds showed slightly lower radical-scavenging activities
compared to vitamin E (Table 2). Hydrogenated compounds 8 and 9 did not
significantly increase or decrease cytotoxicity or radical-scavenging activities over
the mother compounds, grifolin (4) and neogrifolin (5). Recently, 4 was reported to
Table 2. Cytotoxic and Free-Radical-Scavenging Activities of Compounds 1–9 (IC50 [mg/ml]ꢁSD)a)
Compound
Cell lineb)
DPPH Radicalc)
A549
B16F1
SK-Mel-2
1
2
3
4
5
6
7
8
8.5ꢁ0.6
9.0ꢁ0.9
5.0ꢁ0.7
10.5ꢁ1.3
10.5ꢁ0.7
>40
11.0ꢁ1.1
7.5ꢁ0.8
5.0ꢁ0.3
>40
7.3ꢁ0.7
16.9ꢁ1.8
11.9ꢁ1.4
13.7ꢁ1.2
>40
>40
>40
72.4ꢁ7.8
63.1ꢁ6.2
67.4ꢁ4.5
43.8ꢁ6.8
11.1ꢁ1.8
75.9ꢁ3.3
67.4ꢁ5.2
28.6ꢁ3.5
14.6ꢁ2.6
4.8ꢁ0.8
–
4.3ꢁ0.2
3.5ꢁ0.7
6.1ꢁ0.2
5.4ꢁ0.4
>40
6.0ꢁ0.8
6.5ꢁ1.1
4.1ꢁ0.4
8.0ꢁ1.1
>40
>40
9
a-Tocopherol
Cisplatin
>40
>40
10.0ꢁ0.9
9.5ꢁ0.5
5.2ꢁ0.3
a) Values are means of three independent experiments with standard deviations. b) A549: Human lung
carcinoma; B16F1: mouse melanoma; SK-Mel-2: human skin cancer cell line. c) DPPH: Diphenyl-p-
picrylhydrazyl; ꢂ–ꢃ indicates that the sample was not tested.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
inhibit tumor cell growth by inducing apoptosis in vitro [21] and induce cell-cycle arrest
in the G1 phase via the ERK1/2 pathway [1]. The derivatives of 4 with cytotoxic effects
as well as radical-scavenging activities would serve as antitumor compounds.
This work was supported by grants from the Korea Science and Engineering Foundation (Grant No.
KOSEF R01-2003-000-10458-0).
Experimental Part
General. TLC: silica-coated plastic plates (Merck, Kieselgel 60 F254, 0.25 mm); spots were detected
under a UV lamp (254 nm) or by heating after spraying with a soln. of anisaldehyde-H2SO4 in EtOH.
Column chromatography (CC): silica gel 60 (Merck, 70–230 mesh). C18 Medium-pressure liquid
chromatography (MPLC): YAMAZEN F540 pump system using C18 LiChroprep RP-18 (25–40 mm,
30 i.d.ꢂ300 mm, Merck) at a flow rate of 10 ml/min. Prep. HPLC: Waters 600 system with a photodiode
array detector 996 using C18 Pegasil (Senshu Pak, 20 i.d.ꢂ250 mm); flow rate 7 ml/min and detection at
210 nm. Optical rotation: Perkin-Elmer 341 LC polarimeter. UV Spectra: Shimadzu UV-2401 PC
1
spectrometer; lmax in nm (log e). IR Spectra: Perkin-Elmer BX FT-IR spectrometer; n˜max in cmꢀ1. H-
and 13C-NMR spectra: Varian Mercury 400 NMR spectrometer at 400 and 100 MHz, resp., in CDCl3,
referenced in residual solvent signals (d(H) 7.26 and d(C) 77.24 for CDCl3; d(H) 3.30 and d(C) 49.0 for
CD3OD). HR-TOF-MS: Waters LCT Premier mass spectrometer on an electron spray ionization (ESI)
mode coupled to a Waters AQUITY UPLC system and data acquisition using MassLynx software, version
4.0; in m/z. Optical density for 96-well microplates: Tecan Sunrise microplate reader.
Plant Material. The fruiting bodies of the mushroom Boletus pseudocalopus were collected in the
Jirisan National Forest in the republic of Korea in July, 1998. The mushroom was identified by Dr. Yang
Seob Kim at the National Institute Agricultural Science and Technology in Suwon, Republic of Korea. A
voucher specimen (No. SM 5345) was deposited with the natural-product chemistry laboratory of Kongju
National University, Republic of Korea.
Extraction and Isolation. The fresh fruiting bodies of the fresh mushroom (1.0 kg) were soaked in
MeOH (1 l) and left for two weeks at r.t. The extract was decanted, and this was repeated one more time.
The combined extracts were concentrated under vacuum to yield a brown oily syrup (46.5 g). The crude
extract was chromatographed on a silica-gel column (50 i.d.ꢂ350 mm) with elution by a mixture of
hexane and AcOEt of increasing polarity to yield 17 fractions. Fr. 3 (1.1 g; hexane/AcOEt 8 :2) was
further purified by C18 MPLC (MeOH/H2O 6 :4 to 10 :0 for 200 min) to yield ten subfractions. Fr. 3.5
(26 mg) was purified by C18 HPLC (MeOH/H2O 9 :1 to 10 :0 for 80 min) to yield 1 (5.0 mg), eluting at
22.5 min. Fr. 3.7 (50 mg) was purified by C18 HPLC (MeOH/H2O 9 :1 to 10 :0 for 80 min) to yield 2
(3.1 mg), eluting at 37.7 min. Fr. 4 (3.2 g, hexane/AcOEt 8 :2) was further purified using C18 MPLC
(MeOH/H2O 6 :4 to 10 :0 for 200 min) to yield four subfractions. The concentration of the Fr. 4.2 yielded
4 (¼ grifolin; 2.0 g), and the Fr. 4.4 (271 mg) was purified by C18 HPLC (MeOH/H2O 9 :1 to 10 :0 for
140 min) to provide 6 (2.9 mg), eluting at 22.4 min, and compound 7 (5.8 mg), eluting at 40.0 min. Fr. 5
(800 mg, hexane/AcOEt 7:3) was further purified using C18 MPLC (MeOH/H2O 6 :4 to 10 :0 for
200 min) to yield eight subfractions. The Fr. 5.4 (33 mg) was purified by C18 HPLC (MeOH/H2O 9 :1 to
10 :0 for 80 min) to yield 3 (4.6 mg) eluting at 12.3 min. Fr. 6 (3.6 g, hexane/AcOEt 6 :4) was subjected to
C18 MPLC (MeOH/H2O 6 :4 to 10 :0) to yield four subfractions. Concentration of Fr. 6.3 yielded 5
(¼ neogrifolin; 1.7 g).
Hydrogenation of 4. Compound 4 (54.6 mg, 0.166 mmol) was stirred at r.t. overnight under a H2
atmosphere in the presence of Pd/C (5 mg) in MeOH (5 ml). Filtration through a short column of silica
gel and concentration provided 8 (54.3 mg).
Hydrogenation of 5. The reaction was carried out in the same way as described for compound 4
except for the use of 5 (42.4 mg, 0.129 mmol) to provide 9 (42.1 mg).
4-(1-Methoxyethyl)-5-methyl-2-[(2E,6E)-3,7,11-trimethyldodec-2,6,10-trienyl]benzene-1,3-diol (1).
Pale yellow oil. [a]D20 ¼ þ2.8 (c¼0.36, MeOH). UV (MeOH): 204 (4.37), 231 (sh., 3.7), 273 (2.79). IR

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1441
(neat, KBr plate): 3345, 2945, 1622, 1600, 1449, 1269, 1027, 739. 1H- and 13C-NMR: see Table 1. HR-TOF-
MS: 385.2740 ([MꢀH]ꢀ , [C25H38O3 ꢀH]ꢀ ; calc. 385.2743).
4-(1-Ethoxyethyl)-5-methyl-2-[(2E,6E)-3,7,11-trimethyldodec-2,6,10-trienyl]benzene-1,3-diol (2).
Pale yellow oil. [a]2D0 ¼ þ0.97 (c¼0.31, MeOH). UV (MeOH): 207 (4.66), 234 (sh., 3.9), 274 (2.91).
IR (neat, KBr plate):3317, 2974, 1622, 1600, 1450, 1226, 1164, 1028, 832. 1H- and 13C-NMR: see Table 1.
HR-TOF-MS: 399.2869 ([MꢀH]ꢀ , [C26H40O3 ꢀH]ꢀ ; calc. 399.2899).
3,4-Dihydro-4,5-diemethyl-8-[(2E,6E)-3,7,11-trimethyldodec-2,6,10-trienyl]-2H-[1]benzopyran-2,7-
diol (3). Pale yellow oil. [a]2D0 ¼ ꢀ3.8 (c¼0.42, MeOH). UV (MeOH): 208 (4.78), 235 (sh., 3.01), 283
(3.15). IR (neat, KBr plate): 3330, 2943, 1604, 1450, 1146, 1096, 1027, 737. 1H- and 13C-NMR: see Table 1.
HR-TOF-MS: 397.2725 ([MꢀH]ꢀ , [C26H38O3 ꢀH]ꢀ ; calc. 397.2743).
5-Methyl-2-(3,7,11-trimethyldodec)benzene-1,3-diol (8). Pale yellow solid. M.p. 638. 1H-NMR
(CD3OD): 6.11 (s, 2 H); 2.53–2.50 (m, 2 H); 2.12 (s, 3 H); 1.52 (sept., J¼6.0, 1 H); 1.37–1.33 (m,
2 H); 1.48–1.21 (m, 10 H); 1.18–1.12 (m, 2 H); 1.12–1.06 (m, 2 H); 0.92 (d, J¼6.0, 3 H); 0.87 (d, J¼6.4,
6 H); 0.85 (d, J¼6.8, 3 H). 13C-NMR (CD3OD): major isomer: 157.2 (2 C); 137.0; 114.7; 108.6 (2 C);
40.7; 38.72; 38.63; 38.62; 37.6; 37.5; 34.3; 34.1; 29.3; 26.1; 25.6; 23.3; 23.2; 21.5; 20.5; 20.3. HR-TOF-MS:
333.2779 ([MꢀH]ꢀ , [C22H37O2 ꢀH]ꢀ ; calc. 333.2794).
3-Methyl-2-(3,7,11-trimethyldodec)benzene-1,3-diol (9). Pale yellow solid. M.p. 408. 1H-NMR
(CD3OD): 6.12 (d, J¼2.0, 1 H); 6.10 (d, J¼2.8, 1 H); 2.53–2.50 (m, 2 H); 2.16 (s, 3 H); 1.52 (sept, J¼
6.0, 1 H); 1.36–1.33 (m, 2 H); 1.48–1.21 (m, 10 H); 1.18–1.12 (m, 2 H); 1.12–1.06 (m, 2 H); 0.95 (d, J¼
6.4, 3 H); 0.88 (d, J¼6.0, 6 H); 0.86 (d, J¼6.0, 3 H). 13C-NMR (CD3OD): major isomer: 157.1; 156.2;
138.7; 120.8; 109.5; 101.3; 40.7; 38.7; 38.6; 38.5; 37.9; 37.8; 34.5; 34.1; 29.3; 26.1; 25.6; 23.3; 23.2; 20.4; 20.3;
19.9. HR-TOF-MS: 333.2771 ([MꢀH]ꢀ , [C22H37O2 ꢀH]ꢀ ; calc. 333.2794).
Cytotoxicity Assay. The cytotoxicities of compounds 1–9 against human lung cancer cells A549,
mouse melanoma B16F1, and human melanoma SK-Mel-2 cell lines were determined by a colorimetric
sulforhodamine B (SRB) assay [9]. Briefly, cancer cell lines were placed at a density of 5ꢂ104, 2ꢂ104,
and 1ꢂ105 cells/ml for A-549, B16F1, and SK-Mel-2, resp., in RPMI-1640 medium (100 ml) in a 96-well
plate. After 24 h incubation at 378 under humidified 5% CO2, serially-diluted test solns. (100 ml in RPMI
media) were added to the wells and incubated for another 48 h. Cell viability was determined by the
relative optical density of the bound SRB dye, compared to the control, which was measured at 520 nm in
a microplate reader. The results were expressed as the concentration at which there was 50% inhibition
(IC50).
Free Radical Scavenging Assay. Radical scavenging activities were determined by 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay [22]. Serially diluted solns. (20 ml) of test samples were added to an EtOH
soln. (80 ml) of DPPH (59 mg/ml) in a 96-well plate. After incubation for 30 min with shaking at 248,
changes in absorbance were measured in a 96-well microplate reader at 517 nm. The concentration
required for a 50% decrease in the absorbance of a control soln. of DPPH was expressed as IC50
.
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Received June 19, 2008