Synthesis and bioevaluation of novel arylnaphthalene lignans as anticancer agents

Article abstract of DOI:10.1007/s00044-012-0245-1

Novel arylnaphthalene lignans were synthesized and their structures were established by ¹H NMR, ¹³C NMR, and HRMS. These compounds were evaluated for their in vitro cytotoxicity against cancer cell lines by MTT assay. Compound 5d possessed the highest cytotoxicity against KB cells. Apoptosis of KB cells treated with 5d was observed by acridine orange and ethidium bromide double staining assay. Western blot analysis disclosed that 5d induced apoptosis via mitochondrial pathway accompanied by an increased expression of Bax and a decreased expression of Bcl-2.

Full text of DOI:10.1007/s00044-012-0245-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2505–2510
DOI 10.1007/s00044-012-0245-1
ORIGINAL RESEARCH
Synthesis and bioevaluation of novel arylnaphthalene lignans
as anticancer agents
•
Yu Zhao Jie Hui Li Zhu
•
Received: 8 February 2012 / Accepted: 14 September 2012 / Published online: 25 September 2012
Ó Springer Science+Business Media New York 2012
Abstract Novel arylnaphthalene lignans were synthe-
sized and their structures were established by 1H NMR, 13
Arylnaphthalene lignans represent a significant subclass
of lignans. Many of these arylnaphthalene lignans are
derived from traditional herbs and have received increasing
attention over the past decades for their interesting bioac-
tivities (Foley et al., 2010); Fig. 1).
C
NMR, and HRMS. These compounds were evaluated for
their in vitro cytotoxicity against cancer cell lines by MTT
assay. Compound 5d possessed the highest cytotoxicity
against KB cells. Apoptosis of KB cells treated with 5d
was observed by acridine orange and ethidium bromide
double staining assay. Western blot analysis disclosed that
5d induced apoptosis via mitochondrial pathway accom-
panied by an increased expression of Bax and a decreased
expression of Bcl-2.
Diphyllin, an arylnaphthalene lignan isolated from many
traditional medicinal plants, has been reported to possess
anticancer and antiviral activities (Beers et al., 1988; Day
et al., 2002). Previously, we have prepared a series of
diphyllin derivatives and analogs (Zhao and Li, 2007; Zhao
et al., 2008; Hui et al., 2012). Biologic assays disclosed
that several compounds possessed potent anticancer activ-
ities and could induce apoptosis in various cancer cells.
These results encouraged us to synthesize more compounds
to fully discuss the SAR of diphyllin. In this paper, we
report the synthesis, cytotoxicity, and apoptosis inducing
activity of novel analogs of diphyllin (5a–5j, Scheme 1).
Keywords Arylnaphthalene lignans Á Synthesis Á
Cytotoxicity Á Anticancer Á Apoptosis
Introduction
There is an urgent need to develop novel agents for human
cancer therapy (Jemal et al., 2005). Apoptosis is an
important phenomenon in cancer chemotherapy, and the
resistance to apoptosis is a hallmark of cancer cells. The
crucial approach to anticancer drug discovery is selectively
inducing apoptosis in cancer cells without causing exces-
sive damage to normal cells (Fesik, 2005). A great number
of anticancer drugs have been found to induce the apoptotic
process in cancerous cells. Thus, discovery of more potent
apoptosis regulators for cancer treatment is an attractive
and prospective strategy (Huang, 2002).
Results and discussion
Synthesis of arylnaphthalene lignans
The lignans 5a–5j were synthesized as shown in Schemes 1
(Charlton et al., 1996; Ogiku et al., 1995). Compound 1 and
ethylene–glycol were refluxed in benzene with p-TsOHÁH2O
under a Dean-Stark trap to obtain acetal (2). The crude acetal
2 was dissolved in dry THF at -78 °C for half an hour and
then warmed to room temperature for another 2.5 h to get
3a–3j, which were used immediately in the following Diels–
Alder reactions. They were heated with diethyl acetylene-
dicarboxylate (DEADC) and acetic acid at 140 °C for 1 h.
The analogs (5a–5j) were prepared by one pot reduction and
cyclization of 4a–4j with NaBH4 in methanol and subse-
quently with 10 % HCl (Scheme 1).
Y. Zhao Á J. Hui Á L. Zhu (&)
Institute of Nautical Medicine, Nantong University,
Nantong 226001, China
e-mail: zhulili65@163.com
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Med Chem Res (2013) 22:2505–2510
Table 1 Cytotoxicities against cancer cells and HEK 293 cell line of
5a–5j
Compounds
IC50 (lM)
A549 SW480 KB
HCT-116 HEK 293
5a (diphyllin) 8.0
3.4
[100
5.3
48.5
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
5b
97.2
23.2
[100
5c
[100 [100
[100 [100
5.1 29.3
5d
[100
[100 [100
[100 22.8
5.6
5e
[100 [100
[100 [100
[100 88.5
Fig. 1 Diphyllin and podophyllotoxin
5f
5g
[100 [100
[100 [100
[100 [100
[100 [100
Cytotoxicities of compounds 5a–5j against cancer cells
and HEK 293 cell
5h
20.6
15.1
[100
[100
5i
5j
[100 [100
8.3 13.5
Compounds 5a–5j were evaluated for their cytotoxic
activities against three cancer cells and HEK 293 cell by a
MTT growth inhibition assay. IC50 values were summarized
in Table 1 and it represented the concentration inducing a
50 % decrease of cell growth after 3-days incubation.
In general, these derivatives displayed significant loss of
cytotoxicities compared to diphyllin (5a). These results
suggested that substitute pattern of D-ring was essential to
their potency. Compound 5d showed the best cytotoxicity
among all the newly synthesized derivatives with the IC50
values of 5.1 lM in KB cell and 5.6 lM in SW480 cell. All
these compounds did not possess apparent cytotoxicities
against normal HEK 293 cell.
Etoposide
3.9
6.4
The results obtained from the AO/EB staining are as
shown in Fig. 2. Viable cells with intact DNA and nucleus
showed homogeneous bright green. Early apoptotic cells
showed green nuclei, but perinuclear chromatin condensa-
tion was visible as bright green patches or fragments. Late
apoptotic cells’ DNA was fragmented and stained orange. It
was clear that most of the cells treated with compound 5d
exhibited typical characteristics of apoptotic cells like
plasma membrane blebbing. This result also indicates that
cell death occurred primarily through apoptosis.
Induction of apoptosis in KB cells by compound 5d
Effect of compound 5d on apoptosis-related proteins
On the basis of the above results, further biologic evalua-
tions have been focused on compound 5d. KB cells were
stained with AO/EB to verify the type of cell death.
Apoptosis is usually controlled by two major pathways—the
mitochondrial pathway and membrane death receptor
Scheme 1 Synthesis of
arylnaphthalene lignans.
Reagents and conditions:
a glycol, benzene,
p-TsOHÁH2O, quant; b ArCHO,
n-BuLi, THF, -78 °C, quant;
c DEADC,CH2Cl2, AcOH,
(70–75 %), d NaBH4, MeOH,
then 10 % HCl, 65 %
(60–70 %)
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Med Chem Res (2013) 22:2505–2510
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Fig. 2 Morphological study of
KB cells treated with compound
5d in 0, 5, and 10 lM for 72 h
by AO/EB double staining. AO/
EB (A–C) vacuolation and
DNA fragmentation was
obvious, orange cells indicate
later apoptosis (Color figure
online)
internal standard and chemical shifts were recorded in
values. The Mass spectra were obtained on a Shimadzu
LCMS-2010EV (ESIMS).
General procedure for the synthesis of hydroxyl acetals
Acetal 2 was prepared according to literature. 2 (1.5 g,
5.4 mmol) was dissolved in dry THF (40 mL) under nitro-
gen, cooled to -78 °C, and n-BuLi (2.30 mL, 5.6 mmol)
was added dropwise over 5 min. The mixture was stirred for
another 15 min, followed by the dropwise addition of
benzaldehyde (5.11 mmol) in THF (10 mL). After stirring
for 30 min, the solution was gradually warmed to room
temperature and stirred for another 2.5 h, followed by the
addition of H2O (30 mL). The resulting mixture was
extracted with Et2O (39 30 mL), dried (MgSO4), and con-
centrated to give a colorless solid (quant). These crude
products were not further purified or characterized, but were
used immediately in the following Diels–Alder reactions.
Fig. 3 Protein levels of cleaved-caspase-3, Bcl-2, and Bax in KB
cells treated with compound 5d were analyzed by Western blot.
b-Actin was used normalize total proteins
pathway (Hegardt et al., 2001). To reveal the possible
mechanisms responsible for the apoptotic effects of com-
pound 5d, we next evaluate its effects on the expression level
of a series of proteins associated with apoptosis. Bcl-2 can
prevent the release of cytochrome c from the mitochondria
during apoptosis mediated by the mitochondrial pathway. In
contrast, Bax can induce the release of cytochrome c from the
mitochondria (Liu et al., 2006). The present results revealed
that compound 5d induced apoptosis via the mitochondrial
pathway accompanied by an increased expression of Bax and
a decreased expression of Bcl-2 (Fig. 3).
General procedure for the synthesis of arylnaphthalenes
Glacial acetic acid was added to a mixture of hydroxyac-
etal 3a–3j (0.5 mmol) and DEADC (0.12 g, 0.7 mmol) in a
minimum amount of CH2Cl2 and the temperature of the
mixture was quickly brought up to 140 °C. The mixture
was heated for 1 h. The cooled mixture was diluted with
CH2Cl2 (10 mL), washed with 5 % sodium bicarbonate
solution (39 10 mL), dried (MgSO4), and concentrated
under vacuum. The residue was purified by flash chroma-
tography (1:4, EtOAc-petroleum ether) to give arylnaph-
thalenes 4a–4j (70–75 %).
Conclusions
In summary a series of arylnaphthalene lignans have been
synthesized by substituting D-ring of diphyllin with various
aromatic rings. These analogs displayed moderate cytotox-
icities against cancer cell lines. Apoptosis of KB cells induced
by compound 5d was observed by AO/EB staining assay and
Western blot analysis. Compound 5d induced apoptosis via
the mitochondrial pathway accompanied by an increased
expression of Bax and a decreased expression of Bcl-2.
1-(3,4,5-Trimethoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4b)
1
Yield 72 %, obtained as white solid: H NMR(CDCl3): d
Experimental
12.43 (s, 1H, ArOH), 7.71 (s, 1H, ArH), 6.78 (s, 1H, ArH),
6.54 (s, 2H, ArH), 4.38 (dd, 2H, J = 11.4, 7.2 Hz, CH2),
4.04 (s, 3H, OCH3), 4.00 (dd, 2H, J = 14.4, 6.9 Hz, CH2),
3.88 (s, 3H, OCH3), 3.80 (s, 6H, OCH3), 3.74 (s, 3H,
OCH3), 1.33 (t, 3H, J = 7.2 Hz, CH3), 0.96 (t, 3H,
J = 7.2 Hz, CH3); ESI–MS: m/z, 537.5 [M?23].
General methods
1H NMR and 13C NMR spectra were taken on a Bruker
300 MHz spectrometer with tetramethylsilane (TMS) as an
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1-(3,4-Dimethoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4c)
1-(4-Methoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4h)
1
1
Yield 72 %, obtained as white solid: H NMR (CDCl3): d
Yield 73 %, obtained as white solid: H NMR(CDCl3): d
12.42 (s, 1H, ArOH), 7.70 (s, 1H, ArH), 7.21 (d, 1H,
J = 6.6 Hz, ArH), 6.94 (d, 1H, J = 9.0 Hz, ArH), 6.67
(s, 1H, ArH), 4.37 (dd, 2H, J = 14.4, 7.2 Hz, CH2), 4.01
(s, 3H, OCH3), 3.94 (dd, 2H, J = 14.1, 7.2 Hz, CH2), 3.84
(s, 3H, OCH3), 3.70 (s, 3H, OCH3), 1.33 (t, 3H,
J = 6.9 Hz, CH3), 0.96 (t, 3H, J = 7.2 Hz, CH3);
ESI–MS: m/z, 477.4 [M?23].
12.46 (s, 1H, ArOH), 7.74 (s, 1H, ArH), 6.96 (q, 3H,
J = 7.8 Hz, ArH), 6.76 (s, 1H, ArH) 4.44 (q, 2H, J = 7.2 Hz,
CH2), 4.05 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 3.74 (s, 3H, OCH3), 1.39 (t, 3H, J = 7.2 Hz, CH3),
1.03 (t, 3H, J = 7.2 Hz, CH3); ESI–MS: m/z, 507.4 [M?23].
1-(3,5-Dimethoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4d)
1-(4-Methylphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4i)
1
Yield 71 %, obtained as white solid: H NMR (CDCl3): d
12.50 (s, 1H, ArH), 7.74 (s,1H, ArH), 6.82 (s, 1H, ArH),
6.51 (s, 3H, ArH), 4.45 (q, 2H, J = 7.2 Hz, CH2), 3.79
(s, 6H, CH3), 3.77 (s, 3H, CH3), 1.39 (t, 3H, J = 7.2 Hz,
CH3); ESI–MS: m/z, 507.4 [M?23].
1
Yield 75 %, obtained as white solid: H NMR (CDCl3): d
12.44 (s, 1H, ArOH), 7.75 (s, 1H, ArH), 7.27 (q, 4H, ArH),
6.72 (s, 1H, ArH), 4.42 (dd, 2H, J = 7.2 Hz, CH2), 4.05 (s,
3H, OCH3), 3.73 (dd, 2H, J = 7.2 Hz, CH2), 2.43 (s, 3H,
CH3), 1.38 (t, 3H, J = 7.2 Hz,CH3), 0.99 (t, 3H,
J = 7.2 Hz, CH3); ESI–MS: m/z, 461.4 [M?23].
1-(2,5-Dimethoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4e)
1
Yield 73 %, obtained as white solid: H NMR (CDCl3):
1-Phenyl-4-hydroxy-6,7-dimethoxy-naphthalene-2,3-
dicarboxylate (4j)
12.47 (s, 1H, ArOH), 7.73 (s, 1H, ArH), 6.94 (s, 2H, ArH),
6.80 (s, 1H, ArH), 6.63 (s, 1H, ArH), 4.43 (m, 2H, CH2),
4.07 (s, 3H, OCH3), 3.99 (q, 2H, CH2), 3.77 (s, 3H, OCH3),
3.74 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 1.38 (t, 3H,
J = 7.2 Hz, CH3), 1.00 (s, 3H, J = 7.2 Hz, CH3); ESI–
MS: m/z, 507.4 [M?23].
1
Yield 75 %, obtained as white solid: H NMR (CDCl3):
12.45 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.44 (d, 3H,
J = 7.2 Hz, ArH), 7.34 (d, 2H, J = 7.8 Hz, ArH), 6.67
(s, 1H, ArH), 4.44 (dd, 2H, J = 6.9 Hz, CH2), 4.05 (s, 3H,
OCH3), 3.99 (dd, 2H, J = 7.2 Hz, CH2), 3.71 (s, 3H,
OCH3), 1.38 (t, 3H, J = 7.2 Hz, CH3), 0.96 (t, 3H,
J = 7.2 Hz, CH3); ESI–MS: m/z, 447.3 [M?23].
1-(2-Methoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4f)
1
Yield 74 %, obtained as white solid: H NMR (CDCl3): d
General procedure for the synthesis of arylnaphthalene
lignans
12.46 (s, 1H, ArOH), 7.77 (s, 1H, ArH), 7.40 (t, 1H,
J = 8.1, ArH), 7.19 (d, 1H, J = 1.8 Hz, ArH), 7.05 (dd,
2H, J = 15.3 Hz, 7.2 Hz, ArH), 6.58 (s, 1H, ArH), 4.42
(m, 2H, CH2), 4.04 (s, 3H, OCH3), 4.00 (m, 2H, CH2), 3.71
(s, 3H, OCH3), 3.69 (s, 3H, OCH3), 1.37 (t, 3H,
J = 7.2 Hz, CH3), 0.96 (t, 3H, J = 7.2 Hz, CH3); ESI–
MS: m/z, 477.4 [M?23].
4a–4j (0.20 mmol), NaBH4 (380 mg, 10 mmol), and
MeOH (20 mL) were stirred under nitrogen at r.t. for 6 h.
10 % HCl (6 mL) was added cautiously and the mixture
was stirred for 1 h, followed by the extraction with ether.
The solution was concentrated to give a yellow solid which
was then recrystallized from EtOH to afford pure 5a–5j
(65–72 %).
1-(3-Methoxyphenyl)-4-hydroxy-6,7-dimethoxy-
naphthalene-2,3-dicarboxylate (4g)
1-(3,4,5-Trimethoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-
6,7-dimethoxynaphthalene-2-carboxylic Acid Lactone (5b)
1
Yield 74 %, obtained as white solid: H NMR(CDCl3): d
12.42 (s, 1H, ArOH), 7.72 (s, 1H, ArH), 7.35 (q, 1H,
J = 7.8 Hz, ArH), 6.94(q, 3H, J = 9.0 Hz, ArH), 6.72 (s,
1H, ArH), 4.42 (dd, 1H, J = 14.4, 7.2 Hz, CH2), 4.03 (s,
3H, OCH3), 3.97 (dd, 1H, J = 7.2 Hz, CH2), 3.79 (s, 3H,
OCH3), 3.71 (s, 3H, OCH3), 1.365 (t, 3H, J = 7.2 Hz,
CH3), 0.98 (t, 3H, J = 7.2 Hz, CH3); ESI–MS: m/z, 477.4
[M?23].
Yield 72 %, obtained as pale yellow solid: 1H NMR (DMSO-
d6): d 10.37 (s, 1H, ArOH), 7.59 (s, 1H, ArH), 7.01 (s, 1H,
ArH), 6.58 (s, 2H, ArH), 5.32 (s, 2H, CH2), 3.90 (s, 3H,
OCH3), 3.73 (s, 3H, OCH3), 3.71 (s, 6H, 29 OCH3), 3.63 (s,
3H, OCH3); 13C NMR (DMSO-d6): d 170.0, 152.8, 152.7,
152.6, 151.0, 150.2, 145.3, 131.0, 130.4, 129.7, 122.2, 118.9,
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Med Chem Res (2013) 22:2505–2510
2509
1-(3-Methoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-6,7-
dimethoxynaphthalene-2-carboxylic Acid Lactone (5g)
108.7, 67.0, 60.6, 56.4, 56.3, 56.1, 55.6; HRMS calcd. for
C23H23O8 427.1393, found 427.1388; 224 °C (dec).
Yield 71 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6): d 7.62 (s, 1H, ArH), 7.41 (t, 1H, J = 8.1 Hz,
ArH), 7.02 (d, 1H, J = 8.1 Hz, ArH), 6.90 (s, 1H, ArH),
6.87 (s, 2H, ArH), 5.35 (s, 2H, CH2), 3.93 (s, 3H, OCH3),
3.77 (s, 3H, OCH3), 3.60 (s, 3H, OCH3); 13C NMR
(DMSO-d6): d 170.2, 159.0, 150.9, 150.1, 145.2, 132.9,
132.5, 130.4, 129.7, 127.5, 123.4, 122.4, 118.3, 113.8,
113.6, 105.5, 101.2, 67.0, 56.5, 55.8, 55.5; HRMS calcd.
for C21H18O6Na 389.0996, found 389.0998; 238 °C (dec).
1-(3,4-Dimethoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-
6,7-dimethoxynaphthalene-2-carboxylic Acid Lactone (5c)
Yield 69 %, obtained as pale yellow solid: 1H NMR (DMSO-
d6): d 7.62 (s, 1H, ArH), 7.05 (m, 2H, ArH), 6.89 (m, 2H,
ArH), 5.35 (s, 2H, CH2), 3.93 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 3.72 (s, 3H, OCH3), 3.63 (s, 3H, OCH3); 13C NMR
(DMSO-d6): 170.7, 150.9, 150.1, 148.6, 145.2, 130.5, 129.9,
127.8, 123.8, 123.3, 122.2, 119.0, 115.0, 111.6, 106.2, 101.2,
66.6, 60.2, 56.1, 56.0, 55.9, 55.6; HRMS calcd. for
C22H20O7Na 419.1101, found 419.1112; 246 °C (dec).
1-(4-Methoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-6,7-
dimethoxynaphthalene-2-carboxylic Acid Lactone (5h)
1-(3,5-Dimethoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-
6,7-dimethoxynaphthalene-2-carboxylic Acid Lactone (5d)
Yield 65 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6): d 10.32 (s, 1H, ArOH), 7.58 (s, 1H, ArH),
7.19 (d, 2H, J = 8.7 Hz, ArH), 6.99 (d, 2H, J = 8.7 Hz,
ArH), 6.89 (s, 1H, ArH), 5.32 (s, 2H, CH2), 3.89 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 3.58 (s, 3H, OCH3); 13C
NMR (DMSO-d6): d 170.2, 159.0, 150.9, 150.1, 145.2,
132.1, 132.0, 130.3, 129.9, 127.5, 123.7, 122.2, 118.9,
113.7, 113.6, 105.9, 101.2, 67.0, 56.0, 55.5, 55.4; HRMS
calcd. for C21H18O6Na 389.0996, found 389.0988; 218 °C
(dec).
Yield 70 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6): d 10.40 (s, 1H, ArOH), 7.61 (s, 1H, ArH),
6.95 (s, 1H, ArH), 6.57 (s, 1H, ArH), 6.46 (s, 2H, ArH),
5.35 (s, 2H, CH2), 3.93 (s, 3H, OCH3), 3.75 (s, 3H, OCH3),
3.63 (s, 3H, OCH3), 3.34 (s, 3H, OCH3); 13C NMR
(DMSO-d6): 169.9, 160.3, 150.9, 150.1, 145.4, 137.8,
130.1, 129.5, 123.6, 122.1, 118.9, 109.1, 105.9, 101.1,
100.0, 67.1, 60.2, 56.1, 56.0, 55.8, 55.6; HRMS calcd. for
C22H20O7Na 419.1101, found 419.1121; 260 °C (dec).
1-(2,5-Dimethoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-
6,7-dimethoxynaphthalene-2-carboxylic Acid Lactone (5e)
1-(4-Methylphenyl)-4-hydroxy-3-(hydroxymethyl)-6,7-
dimethoxynaphthalene-2-carboxylic Acid Lactone (5i)
Yield 68 %, obtained as pale yellow solid: 1H NMR (DMSO-
d6): 7.61 (s, 1H, ArH), 7.05 (q, 2H, ArH), 6.77 (d, 2H,
J = 15.3 Hz, ArH), 5.37 (s, 2H, CH2), 3.93 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 3.60 (s, 3H, OCH3), 3.53 (s, 3H, OCH3),
13C NMR (DMSO-d6): 170.0, 153.2, 151.7, 150.9, 150.1,
145.4, 129.7, 126.7, 125.3, 123.7, 122.1, 119.5, 117. 8, 114.3,
112.6, 105.8, 101.1, 67.1, 56.2, 56.0, 55.8, 55.5; HRMS calcd.
for C22H20O7Na 419.1101, found 419.1111; 216 °C (dec).
Yield 72 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6) 7.59 (s, 1H, ArH), 7.26 (m, 4H, ArH), 6.87
(s, 1H, ArH), 5.32 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 3.36
(s, 3H, OCH3), 2.37 (s, 3H, CH3); 13C NMR (DMSO-d6):
170.1, 150.9, 150.1, 145.3, 136.9, 132.6, 130.8, 130.5,
129.7, 128.9, 123.7, 122.2, 118.9, 105.9, 101.2, 100.0,
67.0, 60.2, 56.0, 55.5, 21.4; HRMS calcd. for C21H18O5Na
373.1046, found 373.1055; 244 °C (dec).
1-(2-Methoxyphenyl)-4-hydroxy-3-(hydroxymethyl)-6,7-
dimethoxynaphthalene-2-carboxylic Acid Lactone (5f)
1-Phenyl-4-hydroxy-3-(hydroxymethyl)-6,7-
dimethoxynaphthalene-2-carboxylic Acid Lactone (5j)
Yield 69 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6): 10.36 (s, 1H, ArOH), 7.62 (s, 1H, ArH), 7.46
(t, 1H, J = 7.2 Hz, ArH), 7.15 (t, 2H, J = 7.2 Hz, ArH),
7.06 (t, 1H, J = 6.9 Hz, ArH), 6.74 (s, 1H, ArH), 5.37
(s, 2H, CH2), 13C NMR (DMSO-d6): 170.0, 157.6, 150.9,
150.0, 145.3, 132.0, 129.8, 126.9 124.3, 123.7, 122.1,
120.5, 119.6, 111.6, 105.7, 101.2, 100.0, 67.1, 56.0, 55.7,
55.4; HRMS calcd. for C21H18O6Na 389.0996, found
389.0990; 237 °C (dec).
Yield 70 %, obtained as pale yellow solid: 1H NMR
(DMSO-d6): 7.63 (s, 1H, ArH), 7.48 (s, 3H, ArH), 7.31 (d,
2H, J = 8.7 Hz, ArH), 6.85 (s, 1H, ArH), 5.371 (s, 2H,
CH2), 3.94 (s, 3H, OCH3), 3.58 (s, 3H, OCH3) 13C NMR
(DMSO-d6): 170.1, 150.9, 150.1, 145.6, 135.7, 130.9,
130.3, 129.7, 128.3, 128.1, 127.9, 127.1, 123.7, 122.1,
118.9, 105.8 101.2, 67.1, 56.5, 56.0, 55.4; HRMS calcd. for
C20H16O5Na 359.0890, found 359.0896; 239 °C (dec).
123

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Med Chem Res (2013) 22:2505–2510
Cell culture
Statistical analyses
Four human cell lines, KB (oral squamous), A549 (lung),
SW-480 (colon), and HEK 293 (renal) were cultured on
RPMI-1640 medium supplemented with fetal bovine serum
(10 %), penicillin (100U/mL), and streptomycin (100 lg/
mL) in 25-cm2 culture flasks at 37 °C in a humidified
atmosphere with 5 % CO2.
Data were presented as Mean SE and analyzed by SPSS
software. Picture were processed by means of Photoshop
software. Mean values were obtained from at least three
independent experiments.
Acknowledgments The studies in the laboratories were supported
by the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD), The natural science foundation of
Jiangsu Province (BK2011390).
Cell viability
Cell viability was assessed by the MTT assay. Cells were
harvested from the culture during the exponential growth
phase, and seeded into multiwell culture plates at 5 9 104–
1 9 105 cells/mL in fresh medium. After overnight growth,
cells were treated with compounds (predissolved in DMSO)
at selected concentrations for a period of 3 days. The
medium was then discarded and replaced with MTT dye.
Plates were incubated at 37 °C for 4 h. The resulting for-
mazan crystals were solubilized in Lysis buffer (SDS 10 g,
DMF 25 mL, H2O 25 mL, acetic acid 1 mL, pH 4.7), and
the optical density was read at 570 nm using a microplate
reader (Biotek synergy 2).
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