Synthesis, cytotoxicity and pro-apoptosis of novel benzoisoindolin hydrazones as anticancer agents

Article abstract of DOI:10.1248/cpb.58.1324

A series of benzoisoindolin hydrazones as analogues of natural lignan diphyllin were synthesized and the structures of these compounds were established by ¹H-NMR, ¹³C-NMR, Mass and high resolution (HR)-MS. The compounds were evaluated for in vitro cytotoxicity against KB, A549 and HCT-116 cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Compound 4 possessed the highest growth inhibitory effect. Significant apoptosis of HCT-116 cells treated with compound 4 was observed by Hoechst33342-propidium iodide (PI) and acridine orange (AO)-ethidium bromide (EB) staining assay. Western blot analysis disclosed that compound 4 induced apoptosis via the mitochondrial pathway accompanied by an increased expression of Bax and a decreased expression of Bcl-2.

Full text of DOI:10.1248/cpb.58.1324

1324
Regular Article
Chem. Pharm. Bull. 58(10) 1324—1327 (2010)
Vol. 58, No. 10
Synthesis, Cytotoxicity and Pro-apoptosis of Novel Benzoisoindolin
Hydrazones as Anticancer Agents
c
Yu ZHAO,a Jie HUI,a Dan WANG,a Li ZHU,*,a Jing-Huai FANG,b and Xiao-Dong ZHAO
a Institute of Nautical Medicine, Nantong University; b School of Science, Nantong University; Nantong 226001, People’s
Republic of China: and c School of Electromechanical Engineering, Zhejiang Ocean University; Zhoushan 316000,
People’s Republic of China. Received February 25, 2010; accepted July 20, 2010; published online July 22, 2010
A series of benzoisoindolin hydrazones as analogues of natural lignan diphyllin were synthesized and the
structures of these compounds were established by 1H-NMR, 13C-NMR, Mass and high resolution (HR)-MS. The
compounds were evaluated for in vitro cytotoxicity against KB, A549 and HCT-116 cancer cell lines by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compound 4 possessed the highest growth
inhibitory effect. Significant apoptosis of HCT-116 cells treated with compound 4 was observed by
Hoechst33342-propidium iodide (PI) and acridine orange (AO)-ethidium bromide (EB) staining assay. Western
blot analysis disclosed that compound 4 induced apoptosis via the mitochondrial pathway accompanied by an
increased expression of Bax and a decreased expression of Bcl-2.
Key words diphyllin; synthesis; apoptosis; isoindolin; anticancer
Cancer is now the leading cause of death worldwide. lactone moitey of diphyllin. On the other hand, many publi-
There is an urgent need to develop novel agents for human cations reported that the azomethine moieties showed inter-
cancer therapy.1) Because the resistance to apoptosis is a hall- esting inhibitory activity against tumoral cells.13) It might be
mark of cancer cells, the crucial approach to anticancer drug of great interest to combine diphyllin to azomethine groups
discovery is the activation of apoptotic cascades.2) A large to prepare novel derivatives. Herein, we report the synthesis,
number of anticancer drugs have been found to induce the cytotoxicity and apoptosis inducing activity of these ben-
apoptotic process in cancerous cells. Thus, discovery of po- zoisoindolin hydrazones as analogues of diphyllin (2—11,
tent apoptosis inducer for cancer treatment is an attractive Chart 1).
and prospective strategy.
In searching for novel anticancer agents, various plants Results and Discussion
provided a large number of lead compounds for further de-
Synthesis of Benzoisoindolin Hydrazones The syn-
velopment.3) Naturally occuring lignan lactones such as thetic route used to synthesize title compounds is outlined in
podophyllotoxin exhibit a wide range of pharmacological ac- Chart 1. Diphyllin was prepared according to the procedure
tivities (Fig. 1).4,5) Two podophyllotoxin derivatives, etopo- reported previously.14) Nextly, hydrazide 1 was obtained as
side and teniposide, are currently in clinical use as antineo- yellow solid in 87% yield by the reaction of diphyllin with
plastic agents due to their abilities to inhibit human DNA- excess hydrazine hydrate in ethanol over a 6 h reflux period.
topoisomerase II.6) Structure–activity relationships (SAR) of The structure of compound 1 was confirmed by mass spec-
1
podophyllotoxin disclose that the trans-fused-g-lactone ring troscopy and H-NMR. It gave a [MϩH]-ion peak at m/z
is not essential although it contributes to potency.7,8)
395.3 in the electrospray ionization (ESI)-MS in accord with
Diphyllin, an arylnaphthalene lignan lactone isolated from the molecular formula C21H19N2O6. The 1H-NMR spectra in-
many traditional medicinal plants, has been reported to pos- dicated the chemical shift of the NH2 proton at dϭ4.46 ppm
sess anticancer and antiviral activities.9,10) Previously, we in the form of singlet peak. Schiff base reactions of 1 with
have successfully synthesized several natural diphyllin glyco- respective aromatic aldehydes were performed in the pres-
sides and their analogues.11,12) Biological results showed that ence of a catalytic amount of acetic acid in ethanol to give
they were effective apoptosis inducers and cell proliferation hydrazone derivatives 2—11 in the good yields of 79—87%.15)
inhibitors, which encouraged us to synthesize more com-
Inhibitory Effects of Compounds 1—11 on Prolifera-
pounds to fully discuss the SAR of diphyllin. To our best tion of Cancer Cells The title compounds 2—11 and inter-
knowledge, there is no such effort addressed to modify the mediate 1 were evaluated for their cytotoxic activity against
three cancer cell lines using a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) growth inhibition
assay. Etoposide was used as a positive control. IC50 values
were summarized in Table 1 and represent the concentration
inducing a 50% decrease of cell growth after 3 d incubation.
In general, these derivatives displayed potent antiprolifera-
tive activity against three cancer cell lines. These results sug-
gested that lactone ring was not essential to diphyllin to keep
potency. Substituting lactone ring with isoindolin hydrazones
did not show substantial loss in their antiproliferative. The
hydrazone 4 was the most effective derivative of all the
Fig. 1. Natural Antitumor Lignans and Etoposide
newly synthesized compounds, showing IC50 values in the
∗ To whom correspondence should be addressed. e-mail: zhulili65@163.com
© 2010 Pharmaceutical Society of Japan

October 2010
1325
Chart 1. Synthesis of Benzoisoindolin Hydrazones
Table 1. Antiproliferative Activity against Cancer Cell Lines of Hydra-
zones 1—11 and Etoposide
IC50 (mM)
Compound
KB
HCT-116
A549
Ͼ50
Ͼ50
Ͼ50
10.4
Ͼ50
17.4
Ͼ50
35.5
Ͼ50
Ͼ50
Ͼ50
Ͼ50
Ͼ50
1
2
3
4
5
6
Ͼ50
28.7
Ͼ50
7.2
Ͼ50
Ͼ50
Ͼ50
Ͼ50
10.4
16.6
Ͼ50
12.4
11.3
5.3
10.0
5.8
5.6
Ͼ50
19.8
19.8
Ͼ50
Ͼ50
24.3
Ͼ50
48.1
13.5
7
8
9
10
11
Fig. 2. Morphological Study of HCT-116 Cells Treated with Compound 4
in 2 mM, 4 mM Respectively for 72 h by Hoechst33342-PI, AO/EB Double
Staining
Hoechst/PI (A—C): bright blue cells showed early apoptotic, pale red cells were later
apoptotic. AO/EB (D—F): vacuolation and DNA fragmentation was obvious, orange
cells indicate later apoptosis.
Diphyllin
Etoposide
range of 5.6—10.4 mM in three cancer cell lines. Compound
4 has a methyl group at 4-position of benzaidehyde skeleton
showed the best antiproliferative activities. The results
showed that compounds 1 and 3 were highly active only
against HCT-116 cell line. However, the precurosor diphyllin
showed lower activities, which suggestted there is different
modes of action of these two compounds.
Induction of Apoptosis in HCT-116 Cells by Compound
4
On the basis of the above results, further biological evalu-
Fig. 3. Bar Diagram for Comparison of Percent Cells in Apoptosis as Re-
vealed by AO/EB, Hoechst/PI Staining Followed by Manual Counting in
Different Fields of Vision for 3 Times
ations have been focused on compound 4. HCT-116 cells
were stained with Hoechst33342-propidium iodide (PI) and
acridine orange-ethidium bromide (AO-EB) to verify the
type of cell death. Without compound 4 treatment, the nuclei clear that most of cells treated with compound 4 exhibited
of control cells showed uniform blue fluorescence, which in- typical characteristics of apoptotic cells like plasma mem-
dicated the cells were healthy and the nuclei were intact. brane blebbing. This result also indicates that cells death oc-
However, after treatment with compound 4 for 72 h, most of curred primarily through apoptosis. Data collected from the
nucleis were stained pale red by PI, which indicated cells manual counting of cells with normal and apoptotic nuclear
were apoptotic (Fig. 2). These results showed that cells death features are shown in Fig. 3.
occurred primarily through apoptosis.
Effect of Compound 4 on Apoptosis-Related Proteins
The results obtained from the AO-EB staining are as Apoptosis is usually controlled by two major pathway, the
shown in Fig. 2. Viable cells with intact DNA and nucleus mitochondrial pathway and membrane death receptor path-
showed homogeneous bright green. Early apoptotic cells way.16) To reveal the possible mechanisms responsible for the
showed green nuclei, but perinuclear chromatin condensation apoptotic effects of compound 4, we nextly evaluate its ef-
was visible as bright green patches or fragments. Late apop- fects on the expression level of a series of proteins associated
totic cells DNA were fragmented and stained orange. It was with apoptosis. Bcl-2 can prevent the release of cytochrome

1326
Vol. 58, No. 10
tive) m/z: 535.1488 [MϩNa]ϩ (Calcd for C29H24N2O7Na 535.1481). 152 °C
turn red, then decomposed.
N-(2-Hydroxybenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,
7؅-dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone (3)
1
White solid, yield 83%. H-NMR (DMSO-d6) d: 8.28 (1H, s), 7.59 (1H, s),
7.56 (1H, dd, Jϭ8.1, 1.5 Hz), 7.26 (1H, t, Jϭ7.8 Hz), 6.98 (1H, d, Jϭ7.8 Hz),
6.94—6.90 (3H, m), 6.84 (1H, d, Jϭ1.5 Hz), 6.73 (1H, dd, Jϭ8.1, 1.5 Hz),
6.08 (2H, s), 4.78 (2H, s), 3.89 (3H, s), 3.60 (3H, s). 13C-NMR (DMSO-d6)
d: 163.6, 157.4, 150.4, 149.8, 147.1, 146.8, 145.8, 144.1, 131.6, 130.1,
130.0, 129.6, 129.5, 129.1, 124.2, 124.1, 123.1, 119.9, 119.3, 116.8, 115.4,
111.7, 108.1, 105.9, 101.4, 55.9, 55.5, 44.6. MS (ESI) m/z: 499 (Mϩ). HR-
ESI-MS (positive) m/z: 521.1324 [MϩNa]ϩ (Calcd for C28H22N2O7Na
521.1325). 251 °C turn red, then decomposed.
Fig. 4. Western Blot Analysis of Protein Levels of Bcl-2, Bax in A549
Cells Treated with Compound 4
Compound 4 treated cell extracts were obtained and equal amounts of protein were
resolved by SDS-PAGE. Separated protein bands were transferred onto PVDF mem-
branes and incubated with specific primary and secondary antibodies, respectively.
Western blots were developed with enhanced chemiluminescence reagent. b-Actin was
used normalize total proteins.
N-(4-Methylbenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,7؅-
dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone
(4)
1
White solid, yield 87%. H-NMR (DMSO-d6) d: 8.10 (1H, s), 7.63 (1H, s),
7.60 (2H, s), 7.24 (1H, s), 7.21 (1H, s), 6.98 (1H, d, Jϭ8.1 Hz), 6.90 (1H, s),
6.82 (1H, d, Jϭ1.5 Hz), 6.72 (1H, dd, Jϭ7.8, 1.5 Hz), 6.08 (2H, s), 4.77 (2H,
s), 3.90 (3H, s), 3.60 (3H, s), 2.29 (3H, s). 13C-NMR (DMSO-d6) d: 163.9,
150.4, 149.8, 147.1, 146.7, 145.7, 143.7, 140.1, 132.5, 130.1, 129.8, 129.7,
128.9, 127.5, 124.8, 124.2, 123.1, 115.6, 111.7, 108.1, 106.1, 101.4, 101.3,
56.0, 55.5, 45.4, 21.5. MS (ESI) m/z: 497 (Mϩ). HR-ESI-MS (positive) m/z:
519.1522 (Calcd for C29H24N2O6Na 519.1532). 139 °C turn red, then decom-
posed.
c from the mitochondria during apoptosis mediated by the
mitochondrial pathway. In contrast, Bax can induce the re-
lease of cytochrome c from the mitochondria.17) The present
results revealed that compound 4 induced apoptosis via the
mitochondrial pathway accompanied by an increased expres-
sion of Bax and a decreased expression of Bcl-2 (Fig. 4).
N-(3,4,5-Trimethoxybenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-
6؅,7؅-dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone
(5) White solid, yield 80%.1H-NMR (DMSO-d6) d: 8.11 (1H, s), 7.64 (1H,
s), 7.07 (2H, s), 6.99 (1H, d, Jϭ8.1 Hz), 6.93 (1H, s), 6.84 (1H, d, Jϭ1.5
Hz), 6.73 (1H, dd, Jϭ8.1, 1.5 Hz), 6.08 (2H, d, Jϭ3.9 Hz), 4.80 (2H, s), 3.91
(3H, s), 3.81 (6 H, s), 3.68 (3H, s), 3.62 (3H, s). 13C-NMR (DMSO-d6) d:
163.9, 153.6, 150.4, 149.9, 147.1, 146.8, 145.8, 143.6, 139.5, 130.7, 130.1,
129.7, 128.9, 124.7, 124.2, 123.1, 115.8, 111.7, 108.1, 106.0, 104.8, 101.5,
101.3, 60.6, 56.4, 56.3, 56.0, 55.5, 45.6. MS (ESI) m/z: 573 (Mϩ). HR-ESI-
MS (positive) m/z: 595.1708 (Calcd for C31H28N2O9Na 595.1693). 155 °C
Conclusion
In the present study, we have described the preparation and
identification of benzoisoindolin hydrazones from diphyllin
in two steps. These derivatives displayed potent cytotoxic ac-
tivity against three human cancer cell lines. Compound 4
showed the highest cytotoxic potency. Apoptosis of HCT-116
cells induced by compound 4 was observed by Hoechst33342-
PI staining assay, AO-EB staining assay and Western blot
analysis. Compound 4 induced apoptosis via the mitochondr- turn red, then decomposed.
N-(3,4-Dimethoxybenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,7؅-
dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone (6)
ial pathway accompanied by an increased expression of Bax
and a decreased expression of Bcl-2. Our preclinical data in-
dicate that compound 4 is a potential therapeutic agent for
cancer.
White solid, yield 79%.1H-NMR (DMSO-d6) d: 8.06 (1H, s), 7.60 (1H, s),
7.34 (1H, d, Jϭ1.8 Hz), 7.19 (1H, dd, Jϭ8.4, 1.5 Hz), 6.98 (1H, d, Jϭ1.8
Hz), 6.95 (1H, s), 6.90 (1H, s), 6.81 (1H, d, Jϭ1.8 Hz), 6.71 (1H, dd, Jϭ7.8,
1.5 Hz), 6.07 (2H, d, Jϭ3.3 Hz), 4.76 (2H, s), 3.89 (3H, s), 3.75 (6H, s), 3.59
(3H, s). 13C-NMR (DMSO-d6) d: 163.8, 150.9, 150.3, 149.8, 149.4, 147.1,
146.7, 145.9, 143.6, 130.1, 129.6, 128.7, 127.9, 124.9, 124.2, 123.0, 122.1,
115.7, 111.9, 111.8, 108.7, 108.1, 105.9, 101.4, 101.3, 56.1, 56.0, 55.7,
55.5, 45.5. MS (ESI) m/z: 543 (Mϩ). HR-ESI-MS (positive) m/z: 565.1573
(Calcd for C30H26N2O8Na 565.1587). 151 °C turn red, then decomposed.
N-(4-Nitrobenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,7؅-
Experimental
Syntheses Solvents were purified in the usual way. TLC was performed
on precoated Merck silica Gel 60 F254 plates. Flash chromatography was per-
1
formed on silica gel (100—200 mesh, Qingdao, China). H- and 13C-NMR
spectra were taken on a Bruker 300 MHz spectrometer with tetramethylsi-
lane (TMS) as an internal standard, and chemical shifts were recorded in val-
ues. The high resolution spectra were obtained on a Q-TOF Global Mass
(ESI-MS).
dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone
(7)
Yellow solid, yield 87%. 1H-NMR (DMSO-d6) d: 8.27 (1H, s), 8.24 (1H, s),
8.20 (1H, s), 7.95 (1H, s), 7.92 (1H, s), 7.63 (1H, s), 7.01 (1H, d, Jϭ7.8 Hz),
6.92 (1H, s), 6.85 (1H, s), 6.75 (1H, d, Jϭ8.1 Hz), 6.11 (2H, s), 4.83 (2H, s),
3.91 (3H, s), 3.63 (3H, s). 13C-NMR (DMSO-d6) d: 164.1, 150.6, 149.9,
148.0, 147.2, 146.8, 146.0, 141.6, 140.9, 130.0, 129.7, 129.3, 128.2, 124.5,
124.3, 124.2, 123.4, 115.9, 111.7, 108.1, 106.1, 101.5, 101.4, 56.0, 55.6,
45.6. MS (ESI) m/z: 528 (Mϩ). HR-ESI-MS (positive) m/z: 550.1235 (Calcd
for C28H21N3O8Na 550.1226). 165 °C turn red, then decomposed.
4؅-(3؆,4؆-Methylenedioxyphenyl)-6؅,7؅-dimethoxyl-1؅-hydroxyl-benzyl-
2,3-dihydro-isoindol-1-one-hydrazine (1) To a stirred solution of diphyllin
(190 mg, 0.5 mmol) and in EtOH (30 ml) was added hydrazine hydrate (2 ml,
80%, 32 mmol). The mixture was maintained under reflux for 6 h, until TLC
indicated the end of reaction. The reaction mixture was cooled and the solid
was collected by filtration and washed with cool ethanol to afford a pale yel-
low solid 1 (170 mg, 87%). 1H-NMR (DMSO-d6) d: 7.59 (1H, s), 6.96 (1H,
d, Jϭ7.8 Hz), 6.91 (1H, s), 6.78 (1H, d, Jϭ1.5 Hz), 6.69 (1H, dd, Jϭ7.8, 1.5
Hz), 6.08 (2H, s), 4.81 (2H, s), 4.46 (2H, s), 3.91 (3H, s), 3.62 (3H, s). MS
(ESI) m/z: 395 (Mϩ). High resolution (HR)-ESI-MS (positive) m/z:
417.1066 [MϩNa]ϩ (Calcd for C21H18N2O6Na: 417.1063).
The hydrazones 2—11 were prepared as the following method: a solution
of 6 mmol of substituted aldehydes in ethanol was added to a solution of 5
mmol of 1 in 50 ml of ethanol. The mixture was refluxed on a water bath for
2—2.5 h. After cooling, the precipitate was filtered, dried and recrystallized
from ethanol.
N-(4-Fluorobenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,7؅-
dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone
(8)
White solid, yield 83%. 1H-NMR (DMSO-d6) d: 8.14 (1H, s), 7.78—7.73
(2H, m), 7.61 (1H, s), 7.29—7.23 (2H, m), 6.97 (1H, d, Jϭ7.8 Hz), 6.90
(1H, s), 6.82 (1H, d, Jϭ1.8 Hz), 6.71 (1H, dd, Jϭ7.8, 1.5 Hz), 6.11 (2H, s),
4.78 (2H, s), 3.89 (3H, s), 3.60 (3H, s). 13C-NMR (DMSO-d6) d: 165.0,
163.9, 150.3, 149.8, 147.1, 146.8, 145.9, 131.9, 130.1, 129.7, 129.5, 129.4,
128.9, 124.7, 124.2, 123.1, 116.5, 116.2, 115.7, 111.7, 108.1, 106.0, 101.4,
101.3, 56.0, 55.5, 45.5. MS (ESI) m/z: 501 (Mϩ). HR-ESI-MS (positive)
m/z: 523.1281 (Calcd for C28H21N2O6FNa 523.1281). 157 °C turn red, then
decomposed.
N-(3-Methoxybenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,7؅-
dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone
(2)
1
White solid, yield 82%. H-NMR (DMSO-d6) d: 8.15 (1H, s), 7.66 (1H, s),
7.34—7.33 (3H, m), 7.02 (1H, s), 6.99 (1H, s), 6.95 (1H, s), 6.85 (1H, d, Jϭ
1.5 Hz), 6.75 (1H, dd, Jϭ7.8, 1.5 Hz), 6.10 (2H, d, Jϭ1.5 Hz), 4.85 (2H, s),
3.93 (3H, s), 3.79 (3H, s), 3.64 (3H, s). 13C-NMR (DMSO-d6) d: 163.9,
160.0, 150.4, 149.8, 147.1, 146.8, 145.9, 143.2, 136.7, 130.4, 130.1, 129.7,
128.9, 124.7, 124.2, 123.1, 120.4, 116.3, 115.8, 111.7, 111.6, 108.1, 106.0,
101.4, 56.5, 56.0, 55.5, 45.8. MS (ESI) m/z: 513 (Mϩ). HR-ESI-MS (posi-
N-(4-Chlorobenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅,
7؅-dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone (9)
1
White solid, yield 84%. H-NMR (DMSO-d6) d: 8.12 (1H, s), 7.73 (1H, s),
7.71 (1H, s), 7.62 (1H, s), 7.50 (1H, s), 7.47 (1H, s), 6.97 (1H, d, Jϭ7.8 Hz),
6.90 (1H, s), 6.82 (1H, s), 6.72 (1H, d, Jϭ7.8Hz), 6.08 (2H, s), 4.80 (2H, s),
3.89 (3H, s), 3.60 (3H, s). 13C-NMR (DMSO-d6) d: 163.9, 150.3, 149.8,
147.1, 146.8, 145.9, 142.0, 134.6, 134.2, 130.1, 129.7, 129.3, 128.9, 124.5,

October 2010
1327
124.2, 123.1, 115.7, 111.7, 108.1, 105.9, 101.4, 56.0, 55.5, 45.5. MS (ESI) polyvinylidene difluoride (PVDF) membrane was incubated in blocking
m/z: 517 (Mϩ). HR-ESI-MS (positive) m/z: 539.0986 (Calcd for C28H21N2O6 buffer (TBS containing 0.1% Tween 20 and 5% nonfat milk) for 1 h at room
NaCl 539.0982). 149 °C turn red, then decomposed.
temperature. Then the membrane was incubated with the appropriate pri-
N-(4-Bromobenzylidenyl)-4؅-(3؆,4؆-methylenedioxyphenyl)-6؅, mary antibody overnight at 4 °C or 2 h at room temperature with gentle
7؅-dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1-hydrazone (10) shaking. The membrane was washed thrice with rinsing buffer for 15 min
White solid, yield 81%. 1H-NMR (DMSO-d6) d: 8.09 (1H, s), 7.65—7.58 and then incubated with the corresponding peroxidase-conjugated secondary
(5H, m), 6.97 (1H, d, Jϭ8.1 Hz), 6.90 (1H, s), 6.82 (1H, d, Jϭ1.5 Hz), 6.71
antibody for 1 h at room temperature. After repeating the washes in tripli-
(1H, dd, Jϭ7.8, 1.5 Hz), 6.07 (2H, s), 4.77 (2H, s), 3.89 (3H, s), 3.60 (3H, cate, the protein of interest was detected by enhanced chemiluminescence
s). 13C-NMR (DMSO-d6) d: 163.9, 150.4, 149.8, 147.1, 146.8, 145.9, 142.1, reagents from EC3 imaging system (UVP, U.S.A.).
134.5, 132.2, 130.1, 129.7, 129.2, 128.9, 124.6, 124.2, 123.3, 123.1, 115.7,
Statistical Analyses Data were presented as meansϮS.E. and analyzed
111.7, 108.1, 106.0, 101.4, 56.0, 55.5, 45.5. MS (ESI) m/z: 561 (Mϩ). HR- by SPSS software. Picture were processed with Photoshop software. Mean
ESI-MS (positive) m/z: 583.0494 (Calcd for C28H21N2O6NaBr 583.0481). values were obtained from at least three indepent experiments.
159 °C turn red, then decomposed.
N-(3-Hydroxy-4-methoxybenzylidenyl)-4؅-(3؆,4؆-methylened-
Acknowledgements The studies in the laboratories were supported by
ioxyphenyl)-6؅,7؅-dimethoxyl-1؅-hydroxyl-benzyl-2,3-dihydro-isoindol-1- National Program on Key Basic Research Project (2009CB930300), Foun-
hydrazone (11) White solid, yield 80%. 1H-NMR (DMSO-d6) d: 8.03 dation of Jiangsu Educational Committee (08KJD350004) and Program of
(1H, s), 7.62 (1H, s), 7.23 (1H, d, Jϭ1.8 Hz), 7.10 (1H, dd, Jϭ8.7, 1.8 Hz),
6.98 (1H, d, Jϭ3.3 Hz), 6.95 (1H, d, Jϭ3.6 Hz), 6.90 (1H, s), 6.81 (1H, d,
Department of Education of Zhejiang Province (Y200803102).
Jϭ1.5 Hz), 6.71 (1H, dd, Jϭ8.1, 1.8 Hz), 6.07 (2H, s), 4.79 (2H, s), 3.90 References
(3H, s), 3.77 (3H, s), 3.60 (3H, s). 13C-NMR (DMSO-d6) d: 163.7, 150.3,
150.0, 149.8, 147.2, 147.1, 146.7, 145.9, 144.0, 130.2, 129.7, 128.7, 128.2,
125.0, 124.2, 123.0, 120.6, 115.7, 112.9, 112.3, 111.7, 108.1, 106.0, 101.4,
56.5, 56.0, 55.5, 45.6. MS (ESI) m/z: 529 (Mϩ). HR-ESI-MS (positive) m/z:
551.1440 (Calcd for C29H24N2O8Na 551.1430). 129 °C turn red, then decom-
posed.
Cell Culture Three human cell lines, KB (oral squamous), A549 (lung),
HCT-116 (colon) were cultured on RPMI-1640 medium supplemented with
fetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 mg/
ml) in 25 cm2 culture flasks at 37 °C in a humidified atmosphere with 5%
CO2.
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 5ϫ104—1ϫ105 cells/ml in fresh medium. After
overnight growth, cells were treated with compounds (predissolved in di-
methyl sulfoxide (DMSO)) at selected concentrations for a period of 3 d.
The medium was then discarded and replaced with MTT dye. Plates were in-
cubated at 37 °C for 4 h. The resulting formazan crystals were solubilized in
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