Synthesis and cytotoxic evaluation of a series of resveratrol derivatives modified in C2 position

Article abstract of DOI:10.1016/j.ejmech.2006.08.006

Eleven C2-substituted derivatives of resveratrol (trans-3,4′,5-trihydroxystilbene, RES) were prepared by partial synthesis from RES and evaluated for their cytotoxic activities against a human nasopharyngeal epidermoid tumor cell line KB. Among them, comp

Full text of DOI:10.1016/j.ejmech.2006.08.006

European Journal of Medicinal Chemistry 42 (2007) 263e267
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and cytotoxic evaluation of a series of resveratrol
derivatives modified in C2 position
Xian-Feng Huang, Ban-Feng Ruan, Xiao-Ting Wang, Chen Xu,
*
Hui-Ming Ge, Hai-Liang Zhu , Ren-Xiang Tan
*
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
People’s Republic of China
Received 24 February 2006; received in revised form 20 August 2006; accepted 21 August 2006
Available online 25 September 2006
Abstract
Eleven C2-substituted derivatives of resveratrol (trans-3,40,5-trihydroxystilbene, RES) were prepared by partial synthesis from RES and eval-
uated for their cytotoxic activities against a human nasopharyngeal epidermoid tumor cell line KB. Among them, compounds 2 and 3 were more
active than 5-fluorouracil (5-FU), an anticancer drug, and compound 5f exhibited similar activity to 5-FU. On the basis of the biological results,
structureeactivity relationships were discussed.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Resveratrol derivatives; Nasopharyngeal; Antitumor activities; Structureeactivity relationships
1. Introduction
be used as an antitumor drug. Therefore, modifications of
RES, keeping its stilbene backbone, are necessary.
Resveratrol (trans-3,40,5-trihydroxystilbene, RES, 1), a phy-
toalexin with a stilbene structure is present in medicinal plants,
grape skin, peanuts, and red wine [1]. Stilbene-based com-
pounds are widely represented in nature and have become of
particular interest to chemists and biologists because of their
wide range of biological activities [2e5]. RES has been dis-
covered as a potential cancer chemopreventive agent based
on its striking inhibitory effects on cellular events associated
with cancer initiation, promotion, and progression [6]. In addi-
tion to the anticancer-promoting activity, RES has displayed in
vitro growth inhibition in a number of human cancer cell lines
[7]. The simplicity of resveratrol, associated with its interest-
ing anticancer activity, offers promises for the rational design
of new chemotherapeutic agents. However, due to its photo-
sensitivity and metabolic instability [8e10], RES itself cannot
Recently, many RES derivatives by total synthesis have
been reported, as well as the activities including ceramide-me-
diated proapoptotic, antineoplastic, apoptosis-inducing, anti-
fungal, antihyperglycemic and lipid modulating [11e14].
These totally synthesized RES analogues mainly focused on
changing the replaced groups and its positions on two aro-
matic rings of RES. However, there are few reports about par-
tially synthesized derivatives starting from RES [15]. In this
paper, 11 new compounds were prepared by convenient syn-
thesis methods (Scheme 1) and first reported. Their cytotoxic
activities against a human nasopharyngeal epidermoid tumor
cell line KB were also evaluated.
2. Chemistry
Though many totally synthesized RES analogues in the last
five years were reported, there are few derivatives that are par-
tially synthesized [15]. Compound 2 was isolated from plants
[16], but was not easily available in the market. In our labora-
tory, at the beginning compound 2 was synthesized by the
* Corresponding authors. Tel.: þ86 25 8359 2085; fax: þ86 25 8330 2728.
E-mail addresses: zhuhl@nju.edu.cn (H.-L. Zhu), rxtan@nju.edu.cn (R.-X.
Tan).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.08.006

264
X.-F. Huang et al. / European Journal of Medicinal Chemistry 42 (2007) 263e267
direct treatment of RES with (CH3)2SO4 in 10% NaOH solu-
tion under nitrogen atmosphere in order to prevent the oxida-
tion of compound 1. Better yield (88%) of methylation of RES
was obtained by using CH3I instead of (CH3)2SO4. Because
(CH3)2SO4 is a much cheaper methylated agent than CH3I,
the former was preferred. Vilsmeier formylation of compound
2 by treatment with slight excess of POCl3 in DMF at 0 ꢀC
yielded aldehyde 3. The Vilsmeier reaction conditions were in-
vestigated and two procedures were used: compound 2 was
dissolved in N,N-dimethylformamide (DMF) or N-methylfor-
manilide (MFA) and POCl3 was added slowly to the solution.
The reaction mixtures were monitored by TLC. After com-
pound 2 disappeared, the mixtures were added to iceewater,
followed by extraction with CHCl3. When the solvent was
changed from DMF to MFA the product mixture was more
complex and the yield was lower. A side chain containing ni-
trogen atoms is a common structural feature of many classes
of drugs. So in the next step, condensation reaction of com-
pound 3 with a series of amines containing various functional
groups (tyramine, morpholine, furfurylamine, etc.), which are
cheap and available easily in the market afforded the Schiff
bases (Fig. 1). Reduction of the latter with NaBH4 afforded
the corresponding saturated alkyl amines (Fig. 2). Although
the marked deepening of the color indicated a progressive for-
mation of the chromophore C]Ne as the reaction mixtures
were heated, only two Schiff bases, compounds 4a and 4b,
were isolated (Fig. 1). In most instances, no crystal products
of Schiff bases could be obtained.
R
H3CO
NH
OCH3
5
H3CO
5a R=
5b
5d
R=
R=
5c
OH
O
R=
F
R=
5f
N
5e
R=
O
5g
R=
Fig. 2. Structures of compounds 5ae5g.
electron-withdrawing effect of eCHO in compound 3 is
essential for its cytotoxicity. In another modification, the
eCHO on site C2 was replaced with a series of groups con-
taining nitrogen atoms (4a and 4b, 5ae5g). Among the com-
pounds, compounds containing alkyl ring amino side chains
(4a, 4b, 5a, 5b, and 5f) showed better activities against KB
than those containing aromatic ring amino side chains (5c,
5d, 5e, and 5g). This result may be explained because the for-
mer had better lipophilicity than the latter. Two Schiff bases
(4a and 5a) exhibited similar activity with their reduced prod-
ucts (4b and 5b). When the alkyl ring was morpholine the
compound (5f, IC50 ¼ 13.1 mM) showed the highest cytotoxic
activity, which was even comparable with 5-fluorouracil
(IC50 ¼ 13.4 mM). However, it remains to be further investi-
gated as to how the change of side chains influences the cyto-
toxic activity. In conclusion, among all the synthesized RES
analogues, compounds 2 and 3 were more active than 5-fluo-
rouracil (5-FU) and compound 5f exhibited similar activity to
5-FU. In addition, the lipophilicity was found to play a very
important role in the cytotoxic potency and compounds 2
and 3 had a common feature of polar structure. These results
provided us a better understanding of the preliminary
structureeactivity relationships of resveratrol as a potential
antitumor agent, the design and the synthesis of RES
derivatives with superior activities.
3. Pharmacological evaluation and discussion
The cytotoxic activities of the compounds against a human
nasopharyngeal epidermoid tumor cell line KB are summa-
rized in Table 1. The results indicate that methylation of three
hydroxyl groups of compound 1 can remarkably improve the an-
titumor activity. This modification is likely to make the
compounds more lipophilic, which may increase the perme-
ability of the cell membrane. As a result, all of the derivatives
showed higher potency against KB than RES. When the C2 H
of the phenyl ring of compound 2 was replaced by a eCHO
group, the activity was not influenced in a significant way
(IC50 of compounds 2 and 3 ¼ 10.3 and 9.2 mM, respectively),
then the eCHO of compound 3 was reduced and this modifi-
cation considerably decreased the cytotoxic activity, implying
Table 1
Cytotoxic activities of compounds 1e6 against a human nasopharyngeal epi-
dermoid tumor cell line KB
R
Compound
IC50
(mM)
Compound
IC50
(mM)
H3CO
N
1
82.8 ꢁ 1.55
10.3 ꢁ 0.59
9.2 ꢁ 0.37
17.2 ꢁ 0.7
18.9 ꢁ 1.2
15.1 ꢁ 0.46
17.2 ꢁ 0.54
5c
28.8 ꢁ 2.6
29.8 ꢁ 1.4
31.7 ꢁ 0.74
13.1 ꢁ 0.67
30.3 ꢁ 1.5
43.3 ꢁ 0.6
13.4 ꢁ 0.2
2
5d
5e
3
OCH3
4a
4b
5a
5b
5f
4
5g
6
H3CO
5-FU
R=
4b R=
4a
IC50 ¼ 50% inhibitory concentration represents the mean ꢁ S.D. from dosee
Fig. 1. Structures of compounds 4a and 4b.
response curves of at least three experiments.

X.-F. Huang et al. / European Journal of Medicinal Chemistry 42 (2007) 263e267
265
4. Conclusions
5.2. Experimental procedure for the synthesis
of trans-3,40,5-trimethoxystilbene (2)
In summary, a eCHO was first introduced into the phenyl
ring of RES and 11 new compounds were prepared and first
reported. Their cytotoxic activities against a human nasopha-
ryngeal epidermoid tumor cell line KB were evaluated. Com-
pounds 2 and 3 showed more potent inhibitory activities than
5-fluorouracil and compound 5f also exhibited similar activity
to it. At present we are exploring de-methylation of these RES
derivatives selectively and economically, and expected that
these modifications may result in more potent anticancer
agents.
Dimethyl sulfate (4.9 ml) was dropped into a solution of
456 mg (2 mmol) of compound 1 in 5 ml of aq NaOH (10%) un-
der an atmosphere of nitrogen while cooling with an iceewater
bath, keeping the dropping velocity at which the temperature of
the reaction solution was under 40 ꢀC. The mixture was stirred
for 2 h, and extracted with 10 ml of EtOAc twice. The organic
layer was washed with water, dried over Na2SO4, and evapo-
rated under vacuum. Purification by silica gel afforded com-
pound 2 (colorless crystalline, 410 mg, 1.52 mmol, yield
1
76%). Mp: 54e56 ꢀC, ESI MS: 271.3 [M þ H]þ, H NMR
(DMSO-d6): 3.77 (s, 9H), 6.39 (s, 1H), 6.74 (s, 2H), 6.94 (d,
2H, J ¼ 8.4 Hz), 7.02 (d, 1H, J ¼ 16.4 Hz), 7.21 (d, 1H,
J ¼ 16.4 Hz), 7.53 (d, 2H, J ¼ 8.4 Hz) (Scheme 1).
5. Experimental protocols
5.1. Chemistry
RES was bought from Xi’an Mingzhu Company, Xi’an
1
5.3. Experimental procedure for the synthesis
of trans-2-formyl-3,40,5-trimethoxystilbene (3)
China. H NMR and 13C NMR spectra were recorded at 300
and 500 MHz on Bruker spectrometers in DMSO-d6. MS spec-
tra were recorded with a mariner system 5304 mass spectrom-
eter. Melting points were obtained by using a Boetius micro
melting point apparatus. Elemental analyses were performed
on a CHNeO-Rapid instrument. Flash-column chromatogra-
phies were performed on silica gel Merck Kieselgel 60
(230e400 mesh). Thin-layer chromatographies were per-
formed on silica gel plates (GF254, Merk); spots were detected
visually by ultraviolet irradiation (254 nm).
To a solution of 270 mg (1 mmol) of compound 2 in 5 ml of
DMF was added dropwise 200 ml (0.21 g, 1.0 mmol) of POCl3
while cooling with an iceewater bath. The reaction mixture
was stirred for 30 min at room temperature. The solution
was added to a mixture of ice and water, and the yellow
solution was extracted with three 10 ml portions of dichloro-
methane. The combined organic layers were washed with
water, dried over anhydrous sodium sulfate, filtered,
H3CO
HO
a
OCH3
OH
H3CO
HO
1
2
b
R
N
H3CO
H3CO
CHO
c
OCH3
OCH3
4
3
H3CO
H3CO
H3CO
H3CO
d
d
R
NH
OH
OCH3
OCH3
5
6
H3CO
H3CO
Scheme 1. Synthesis of the resveratrol analogs. Reagents and conditions: (a) (CH3)2SO4, 10% NaOH, 0e40 ꢀC, 76%; (b) POCl3, DMF, 0 ꢀC, 69%; (c) RNH2,
ethanol, reflux, 94e95%; (d) NaBH4, ethanol, 40 ꢀC, 74e95%.

266
X.-F. Huang et al. / European Journal of Medicinal Chemistry 42 (2007) 263e267
evaporated, and recrystallized from dichloromethane, and af-
forded 3 (yellow solid, 206 mg, 0.69 mþmol, yield 69%). Mp:
2H), 1.61 (m, 2H), 1.71 (m, 2H), 1.38 (m, 2H), 3.06 (m,
1H), 3.73 (s, 6H), 3.77 (s, 2H), 3.80 (s, 3H), 3.92 (s, 3H),
6.47 (s, 1H), 6.81 (s, 1H), 6.95 (d, 2H, J ¼ 8.3 Hz), 7.11 (d,
1H, J ¼ 16.2 Hz), 7.37 (d, 1H, J ¼ 16.2 Hz), 7.52 (d, 2H,
J ¼ 8.3 Hz). Anal. Calc for C23H29NO3: C, 75.17; H, 7.95;
N, 3.81%. Found: C, 75.12; H, 8.08; N, 3.76%.
1
108e109 ꢀC, ESI MS: 299.3 [M þ H] , H NMR (DMSO-
d6): 3.78 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 6.63 (s, 1H),
6.91 (s, 1H), 6.97 (d, 2H, J ¼ 7.9 Hz), 7.21 (d, 1H,
J ¼ 16.2 Hz), 7.50 (d, 2H, J ¼ 7.9 Hz), 7.95 (d, 1H,
J ¼ 16.2 Hz), 10.41 (s, 1H). Anal. Calc for C18H18O4: C,
72.74; H, 6.08%. Found: C, 72.56; H, 6.11%.
5.5.2. Synthesis of 2-(N-cyclohexylaminomethyl)-3,40,
5-trimethoxystilbene (5b)
5.4. General experimental procedure for the synthesis
of 4a and 4b
White powder, yield 86%, mp: 58e60 ꢀC, ESI MS: 382.2
1
[M þ H]þ, H NMR (DMSO-d6): 1.05e1.22 (m, 6H), 1.63
(m, 2H), 1.84 (m, 2H), 2.42 (m, 2H), 3.77 (m, 8H), 3.80 (s,
3H), 6.47 (d, 1H, J ¼ 1.9 Hz), 6.80 (d, 1H, J ¼ 1.9 Hz), 6.95
(d, 2H, J ¼ 8.5 Hz), 7.11 (d, 1H, J ¼ 16.3 Hz), 7.37 (d, 1H,
J ¼ 16.3 Hz), 7.51 (d, 2H, J ¼ 8.5 Hz). Anal. Calc for
C24H31NO3: C, 75.56; H, 8.19; N, 3.67%. Found: C, 75.61;
H, 8.16; N, 3.72%.
Compound 2 (1 mmol) was dissolved in 5 ml of ethanol/di-
chloromethane (1:1), amine was added to the solution. The re-
action mixture was refluxed for 30 min, and cooled to room
temperature, and afforded the crystal products.
5.4.1. Synthesis of 2-(N-cyclopentyliminomethyl)-3,40,
5-trimethoxystilbene (4a)
5.5.3. Synthesis of 2-(N-benzylaminomethyl)-3,40,
5-trimethoxystilbene (5c)
Colorless crystals, yield 94%, mp: 108e109 ꢀC, ESI MS:
1
366.3 [M þ H]þ, H NMR (DMSO-d6): 1.63 (m, 4H), 1.85
White powder, yield 78%, mp: 71e73 ꢀC, ESI MS: 390.2
1
(m, 2H), 1.88 (m, 2H), 3.31 (m, 1H), 3.77 (s, 3H), 3.82 (s,
3H), 3.86 (s, 3H), 6.53 (d, 1H, J ¼ 1.6 Hz), 6.91 (d, 1H,
J ¼ 1.6 Hz), 6.94 (d, 2H, J ¼ 8.5 Hz), 7.10 (d, 1H, J ¼
16.4 Hz), 7.43 (d, 2H, J ¼ 8.5 Hz), 8.08 (d, 1H, J ¼
16.4 Hz), 8.61 (s, 1H). Anal. Calc for C23H27NO3: C, 75.59;
H, 7.45; N, 3.83%. Found: C, 75.36; H, 7.39; N, 3.86%.
[M þ H]þ, H NMR (DMSO-d6): 3.77 (s, 3H), 3.80 (s, 3H),
3.84 (s, 3H), 4.11 (m, 2H), 4.23 (m, 2H), 6.55 (d, 1H,
J ¼ 2.0 Hz), 6.89 (d, 1H, J ¼ 2.0 Hz), 6.98 (d, 2H,
J ¼ 8.7 Hz), 7.18 (d, 2H, J ¼ 8.7 Hz), 7.45e7.48 (m, 3H),
7.52e7.58 (m, 4H). Anal. Calc for C25H27NO3: C, 77.09; H,
6.99; N, 3.60%. Found: C, 77.21; H, 7.06; N, 3.62%.
5.4.2. Synthesis of 2-(N-cyclohexyliminomethyl)-3,40,
5-trimethoxystilbene (4b)
5.5.4. Synthesis of 2-(N-(2-(4-hydroxyphenyl)ethyl)
aminomethyl)-3,40,5-trimethoxystilbene (5d)
Colorless crystals, yield 95%, mp: 145e146 ꢀC, ESI MS:
White powder, yield 74%, mp: 164e165 ꢀC, ESI MS:
420.2 [M þ H]þ, 1H NMR (DMSO-d6): 2.59 (t, 2H,
J ¼ 7.0 Hz), 2.71 (t, 2H, J ¼ 7.0 Hz), 3.73 (s, 3H), 3.75 (s,
2H), 3.78 (s, 3H), 3.80 (s, 3H), 3.80 (s, 3H), 6.45 (d, 1H,
J ¼ 2.0 Hz), 6.61 (d, 2H, J ¼ 8.6 Hz), 6.82 (d, 2H, J ¼ 2.0 Hz),
6.94 (m, 2H), 6.96 (m, 2H), 7.10 (d, 1H, J ¼ 16.2 Hz), 7.32
(d, 1H, J ¼ 16.2 Hz), 7.49 (d, 2H, J ¼ 8.6 Hz), 9.15 (s, 1H).
Anal. Calc for C26H29NO4: C, 74.44; H, 6.97; N, 3.34%.
Found: C, 74.32; H, 7.07, N, 3.28%.
1
380.3 [M þ H]þ, H NMR (DMSO-d6): 1.22e1.60 (m, 6H),
1.63e1.79 (m, 4H), 3.17 (m, 1H), 3.77 (s, 3H), 3.82 (s, 3H),
3.86 (s, 3H), 6.53 (d, 1H, J ¼ 1.9 Hz), 6.92 (d, 1H,
J ¼ 1.9 Hz), 6.95 (d, 2H, J ¼ 8.6 Hz), 7.10 (d, 1H, J ¼
16.4 Hz), 7.43 (d, 2H, J ¼ 8.6 Hz), 8.07 (d, 1H, J ¼
16.4 Hz), 8.64 (s, 1H). Anal. Calc for C24H29NO3: C, 75.96;
H, 7.70; N, 3.69%. Found: C, 76.17; H, 7.61; N, 3.63%.
5.5. General experimental procedure for the synthesis
of 5ae5g
5.5.5. Synthesis of 2-(N-(4-fluorobenzyl)
aminomethyl)-3,40,5-trimethoxystilbene (5e)
White powder, yield 88%, mp: 77e79 ꢀC, ESI MS: 408.2
Compound 2 (1 mmol) was dissolved in 5 ml of ethanol/di-
chloromethane (1:1), amine was added to the solution. The re-
action mixture was refluxed for 30 min, and cooled to room
temperature. NaBH4 of 0.5 mmol was added to the reaction
solution slowly, and stirred at room temperature for 2 h. The
mixture was evaporated under vacuum, and dissolved in di-
chloromethane (5 ml). The solution was washed with saturated
NaCl solution and water, respectively, dried over anhydrous
sodium sulfate, and evaporated. Purification by silica gel af-
forded pure products.
1
[M þ H]þ, H NMR (DMSO-d6): 3.66 (s, 3H), 3.69 (s, 3H),
3.84 (m, 5H), 4.13 (s, 2H), 6.31 (d, 1H, J ¼ 2.1 Hz), 6.55e
6.57 (m, 3H), 6.76 (d, 2H, J ¼ 8.7 Hz), 6.96 (d, 2H,
J ¼ 8.7 Hz), 7.28e7.32 (m, 2H), 7.53e7.57 (m, 2H). Anal.
Calc for C25H26FNO3: C, 73.69; H, 6.43; N, 3.44%. Found:
C, 73.56; H, 6.63; N, 3.49%.
5.5.6. Synthesis of 2-(N-(2-(morpholin-4-yl)ethyl)
aminomethyl)-3,40,5-trimethoxystilbene (5f)
White powder, yield 87%, mp: 86e88 ꢀC, ESI MS: 413.5
1
5.5.1. Synthesis of 2-(N-cyclopentylaminomethyl)-3,40,
5-trimethoxystilbene (5a)
White powder, yield 82%, mp: 46e48 ꢀC, ESI MS: 368.2
[M þ H]þ, 1H NMR (DMSO-d6): 1.38 (m, 2H), 1.45 (m,
[M þ H]þ, H NMR (DMSO-d6): 2.23 (m, 4H), 2.35 (t, 2H,
J ¼ 5.9 Hz), 2.62 (t, 2H, J ¼ 5.9 Hz), 3.40 (m, 4H), 3.77 (s,
6H), 3.79 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 6.48 (d, 1H,
J ¼ 2.1 Hz), 6.82 (d, 1H, J ¼ 2.1 Hz), 6.95 (d, 2H,

X.-F. Huang et al. / European Journal of Medicinal Chemistry 42 (2007) 263e267
267
J ¼ 8.7 Hz), 7.13 (d, 1H, J ¼ 16.2 Hz), 7.32 (d, 1H,
J ¼ 16.2 Hz), 7.55 (d, 2H, J ¼ 8.7 Hz). Anal. Calc for
C24H32N2O4: C, 69.88; H, 7.82; N, 6.79%. Found: C, 69.67;
H, 7.73; N, 6.77%.
reference. After 48 h exposure period, 40 ml of PBS containing
2.5 mg mlꢃ1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) was added to each well. And 4 h later
the medium was replaced by 150 ml DMSO to solubilize the
purple formazan crystals produced. The absorbance at
570 nm of each well was measured on an ELISA plate reader.
And the IC50 value was defined as the concentration at which
50% survival of cells was allowed.
5.5.7. Synthesis of 2-(N-(furan-2-yl)
methylaminomethyl)-3,40,5-trimethoxystilbene (5g)
White powder, yield 84%, mp: 70e72 ꢀC, ESI MS: 380.4
1
[M þ H]þ, H NMR (DMSO-d6): 3.70 (s, 2H), 3.72 (s, 2H),
3.77 (s, 3H), 3.78 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 6.28
(d, 1H, J ¼ 2.9 Hz), 6.52 (m, 1H), 6.48 (d, 1H, J ¼ 2.0 Hz),
6.82 (d, 1H, J ¼ 2.0 Hz), 6.96 (d, 2H, J ¼ 8.6 Hz), 7.09 (d,
1H, J ¼ 16.3 Hz), 7.25 (d, 1H, J ¼ 16.3 Hz), 7.46 (d, 2H,
J ¼ 8.6 Hz), 7.58 (m, 1H). Anal. Calc for C23H25NO4: C,
72.80; H, 6.64; N, 3.69%. Found: C, 72.75; H, 6.81; N, 3.73%.
Acknowledgment
The work was co-financed by grants (Projects 20432030 &
30570010) from National Natural Science Foundation of
China.
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5.6. Experimental procedure for the synthesis of
2-(hydroxymethyl)-3,40,5-trimethoxystilbene (6)
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dissolved in dichloromethane (5 ml). The solution was washed
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1
[M þ Na]þ, H NMR (DMSO-d6): 3.77 (s, 3H), 3.78 (s, 3H),
3.81 (s, 3H), 4.56 (s, 2H), 6.46 (d, 1H, J ¼ 2.1 Hz), 6.84 (d,
1H, J ¼ 2.1 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.13 (d, 1H,
J ¼ 16.3 Hz), 7.45 (d, 1H, J ¼ 16.3 Hz), 7.53 (d, 2H,
J ¼ 8.4 Hz). Anal. Calc for C18H20O4: C, 71.98; H, 6.71%.
Found: C, 71.83; H, 6.68%.
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5.7. Cytotoxicity study using KB cells
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The cytotoxicity was evaluated as described elsewhere [17]
with some modifications. Briefly, target tumor cells were
grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 ꢂ 104
cells mlꢃ1 with the complete medium, 100 ml of the obtained
cell suspension was added to each well of 96-well culture
plates. The subsequent incubation was permitted at 37 ꢀC,
5% CO2 atmosphere for 24 h before the cytotoxicity assess-
ments. Tested samples at pre-set concentrations were added
to 6 wells with 5-fluorouracil co-assayed as a positive
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1098e1106.