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Z.-R. Wu et al. / European Journal of Medicinal Chemistry 85 (2014) 778e783
5.2.2.6. (E)-4-hydroxy-N0-[3-(4-hydroxy-3-methoxyphenyl)acryloyl]
benzohydrazide (2c). 66% yield. IR (KBr, cmꢀ1
: 3221, 3005 (NeH,
OeH); 1640 (C]O); 1609 (C]C); 1511 (CeC). 1H NMR (DMSO-d6,
5.2.2.11. (E)-N0-[3-(4-hydroxy-3,5-dimethoxyphenyl)acryloyl]-4-
methoxybenzohydrazide (4b). 84% yield. IR (KBr, cmꢀ1
3357e3266, 3008 (NeH, OeH); 1655 (C]O); 1604 (C]C); 1516
)
n
)
n:
400 MHz)
d
(ppm): 3.83 (3H, s, 3-OMe), 6.57 (1H, d, J ¼ 15.6 Hz, H-
(CeC). 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 3.74e3.96 (9H, m, 3-
8), 6.84e6.86 (3H, m, H-5, H-30, H-50), 7.08 (1H, t, J ¼ 6.4, H-6), 7.19
(1H, s, H-2), 7.47 (1H, d, J ¼ 15.6, H-7), 7.79 (2H, d, J ¼ 8.0 Hz, H-20,
H-60), 9.55 (s, OH), 9.95 (s, OH), 10.13 (s, NH), 10.23 (s, NH). 13C NMR
OMe, 5-OMe, 40-OMe), 6.62 (1H, d, J ¼ 15.6 Hz, H-8), 6.93 (2H, s, H-
2, H-6), 7.05 (2H, d, J ¼ 7.2 Hz, H-30, H-50), 7.48 (1H, d, J ¼ 15.6, H-7),
7.90 (2H, d, J ¼ 7.2 Hz, H-20, H-60), 8.91 (s, OH), 9.96 (s, NH), 10.35 (s,
(DMSO-d6, 100 MHz)
d
(ppm): 165.2, 165.1, 160.7, 148.7, 147.9, 140.6,
NH). 13C NMR (DMSO-d6, 100 MHz)
d (ppm): 164.9, 162.0, 148.6,
129.6, 126.2, 123.1, 121.7, 116.3, 115.8, 115.0, 111.2, 55.6; ESI-HRMS:
m/z 329.1126 (calcd for C17H16N2O5 þ H, 329.1132). Anal. Calcd.
for C15H13N3O2: C, 62.19; H, 4.91; N, 8.53. Found: C, 62.26; H, 4.87;
N, 8.67.
148.1, 140.9, 137.7, 129.4, 125.0, 124.6, 116.6, 113.7, 105.5, 56.0, 55.4.
ESI-HRMS: m/z 373.1393 (calcd for C19H20N2O6 þ H, 373.1394).
Anal. Calcd. for C15H13N3O2: C, 61.28; H, 5.41; N, 7.52. Found: C,
61.36; H, 5.55; N, 7.64.
5.2.2.7. (E)-N0-[3-(3,4-dihydroxyphenyl)acryloyl]benzohydrazide
5.2.2.12. (E)-4-hydroxy-N0-[3-(4-hydroxy-3,5-dimethoxyphenyl)
(3a). 55% yield. IR (KBr, cmꢀ1
)
n
: 3396, 3014 (NeH, OeH); 1677
acryloyl]benzohydrazide (4c). 64% yield. IR (KBr, cmꢀ1
)
n: 3199,
(C]O); 1606 (C]C); 1515 (CeC). 1H NMR (DMSO-d6, 400 MHz)
3004 (NeH, OeH); 1643 (C]O); 1608 (C]C); 1514 (CeC). 1H NMR
d
(ppm): 6.45 (1H, d, J ¼ 15.6 Hz, H-8), 6.77 (1H, d, J ¼ 8.0 Hz, H-5),
(DMSO-d6, 400 MHz) d (ppm): 3.81 (6H, s, 3-OMe, 5-OMe), 6.62
6.91 (1H, dd, J ¼ 8.0, 1.6 Hz, H-2), 7.02 (1H, d, J ¼ 1.2 Hz, H-6), 7.38
(1H, d, J ¼ 16.0 Hz, H-7), 7.51 (2H, t, J ¼ 7.6 Hz, H-30, H-50), 7.58 (1H, t,
J ¼ 7.2 Hz, H-40), 7.90 (2H, d, J ¼ 8.0 Hz, H-20, H-60), 9.46 (s, OH), 9.54
(s, OH), 10.07 (s, NH), 10.46 (s, NH). 13C NMR (DMSO-d6, 100 MHz)
(1H, d, J ¼ 15.6 Hz, H-8), 6.84 (2H, d, J ¼ 8.0 Hz, H-30, H-50), 6.93 (2H,
s, H-2, H-6), 7.48 (1H, d, J ¼ 16.0, H-7), 7.80 (2H, d, J ¼ 8.4 Hz, H-20,
H-60), 9.92 (br, 4H, 2 OH, 2 NH). 13C NMR (DMSO-d6, 100 MHz)
d
(ppm): 165.2, 165.0, 160.8, 148.1, 140.9, 137.7, 129.6, 125.0, 123.1,
d
(ppm): 165.9, 165.3, 148.2, 146.0, 141.2, 133.0, 132.2, 128.9, 127.9,
116.7, 115.1, 105.5, 56.0. ESI-HRMS: m/z 359.1243 (calcd for
126.4, 121.3, 116.2, 116.0, 114.2. ESI-HRMS: m/z 299.1036 (calcd for
C
18H18N2O6 þ H, 359.1238). Anal. Calcd. for C15H13N3O2: C, 60.33; H,
C
16H14N2O4 þ H, 299.1026). Anal. Calcd. for C15H13N3O2: C, 64.42; H,
5.06; N, 7.82. Found: C, 60.51; H, 4.98; N, 7.93.
4.73; N, 9.39. Found: C, 64.49; H, 4.68; N, 9.45.
5.2.3. Biological evaluation
5. 2. 2. 8. (E)-N0-[3-(3, 4-dihydroxyphenyl)acryloyl]-4-
5.2.3.1. Antiproliferative assay. The cells were obtained from the
first hospital of Lanzhou University. Culture was maintained at
37 ꢂC with 5% CO2 in a humidified atmosphere, cells were seeded in
96-well plates and then treated with different concentrations of
methoxybenzohydrazide (3b). 52% yield. IR (KBr, cmꢀ1
)
n
: 3374,
3212 (NeH, OeH); 1641 (C]O); 1606 (C]C); 1513 (CeC). 1H NMR
(DMSO-d6, 400 MHz) (ppm): 3.82 (s, 3H, OCH3), 6.44 (1H, d,
d
J ¼ 15.6 Hz, H-8), 6.77 (1H, d, J ¼ 8.0 Hz, H-5), 6.90 (1H, dd, J ¼ 8.0,
1.6 Hz, H-2), 7.02e7.05 (3H, m, H-30, H-50, H-6), 7.38 (1H, d,
J ¼ 15.6 Hz, H-7), 7.89 (2H, d, J ¼ 8.0 Hz, H-20, H-60), 9.23 (s, OH),
9.46 (s, OH), 10.01 (s, NH), 10.32 (s, NH). 13C NMR (DMSO-d6,
compounds for 24 h, 10 mL of 5 mg/mL MTT solution (dilute in
sterile PBS) diluted in serum-free media was added to each well for
4 h incubation, the solution was centrifuged for 10 min under
2000 rpm. The supernatant was mixed with 150 mL DMSO, the
absorbance at 570 nm was determined on a plate reader. IC50 values
were determined from a log plot of percent of control versus con-
centration. Assays were performed in triplicate and standard error
determined [21].
100 MHz)
d (ppm): 164.5, 161.5, 147.3, 145.2, 140.2, 132.5, 128.9,
125.6, 124.2, 120.5, 115.3, 115.2, 113.3, 113.2, 54.9. ESI-HRMS: m/z
329.1126 (calcd for C17H16N2O5 þ H, 329.1132). Anal. Calcd. for
C
15H13N3O2: C, 62.19; H, 4.91; N, 8.53. Found: C, 62.30; H, 4.84; N,
8.65.
5.2.3.2. Cell cycle analysis. The cells treated with DMSO and 1.5 mM
5. 2. 2. 9. (E)-N0-[3-(3, 4-dihydroxyphenyl)acryloyl]-4-
of compound 2c were collected after culturing for 12 and 24 h,
resuspended and fixed in 70% ethanol at 4 ꢂC. Cells were then
washed three times with ice-cold PBS, incubated for 1 h with 1 mg/
mL RNase A and 20 mg/mL PI at room temperature, and analyzed
with a flow cytometer as described previously [17,22].
hydroxybenzohydrazide (3c). 66% yield. IR (KBr, cmꢀ1
)
n
: 3402,
3212 (NeH, OeH); 1639 (C]O); 1609 (C]C); 1510 (CeC). 1H NMR
(DMSO-d6, 400 MHz)
d
(ppm): 6.44 (1H, d, J ¼ 15.6 Hz, H-8),
6.76e7.14 (4H, m, H-5, H-6, H-30, H-50), 7.18 (1H, m, H-2), 7.37 (1H,
d, J ¼ 16.0 Hz, H-7), 7.78 (2H, d, J ¼ 8.0 Hz, H-20, H-60), 9.21 (s, OH),
9.44 (s, OH), 9.95 (s, OH), 10.10 (s, NH), 10.19 (s, NH). 13C NMR
5.2.3.3. Detection of apoptosis by fluorescence microscopy.
H1299 cells were grown to about 70e80% confluence on slides and
(DMSO-d6, 100 MHz) d (ppm): 165.3, 165.0, 160.7, 147.8, 145.7, 140.7,
129.5, 126.1, 123.1, 120.9, 115.8, 115.7, 115.0, 113.8. ESI-HRMS: m/z
315.0969 (calcd for C16H14N2O5 þ H, 315.0975). Anal. Calcd. for
then treated with 2c (1.5 mM) or DMSO for 24 h. Subsequently, cells
were fixed, washed twice with PBS and stained with 1 mM AO/EB
for 30 min. Morphological changes in the apoptotic cells were
observed under a fluorescence microscope (Olympus) with an
excitation wavelength of 330e380 nm [23].
C
15H13N3O2: C, 61.14; H, 4.49; N, 8.91. Found: C, 61.33; H, 4.37; N,
8.80.
5.2.2.10. (E)-N0-[3-(4-hydroxy-3, 5-dimethoxyphenyl)acryloyl]ben-
zohydrazide (4a). 71% yield. IR (KBr, cmꢀ1
)
n
: 3352e3264, 3017
(NeH, OeH); 1657 (C]O); 1626 (C]C); 1516 (CeC). 1H NMR
(DMSO-d6, 400 MHz) (ppm): 3.81 (6H, s, 3-OMe; 5-OMe), 6.62
5.2.3.4. Western blot analysis of caspase-3 protein expression.
H1299 cells (1 ꢁ 106 cells) exposed to compound 2c were collected
into tubes and then washed with PBS. Cell lysate was normalized
and then mixed with 5 ꢁ SDS loading buffer. The samples were
then incubated at 10 ꢂC for 10 min and allowed to cool to room
d
(1H, d, J ¼ 15.6 Hz, H-8), 6.93 (2H, s, H-2, H-6), 7.48 (1H, d,
J ¼ 15.2 Hz, H-7), 7.52 (2H, d, J ¼ 7.6 Hz, H-30, H-50), 7.58 (1H, t,
J ¼ 7.2 Hz, H-40), 7.91 (2H, d, J ¼ 7.6 Hz, H-20, H-60), 8.92 (s, OH),
temperature naturally. Cell lysates containing 20 mg protein were
10.04 (s, NH), 10.51 (s, NH). 13C NMR (DMSO-d6, 100 MHz)
d
(ppm):
separated and transferred to nitrocellulose filters, the blots were
probed with the rabbit anti-human caspase-3 antibody (dilution 1:
500) and incubated at 4 ꢂC overnight, then incubated with a sec-
ondary antibody (dilution 1: 4000) at room temperature for 1 h.
The immunoblots were subsequently reprobed with a 1:5000
165.8,165.3,148.5,141.4,138.1,132.7,132.2,128.9,127.9,125.3,116.9,
105.9, 56.4. ESI-HRMS: m/z 343.1277 (calcd for C18H18N2O5þH,
343.1288). Anal. Calcd. for C15H13N3O2: C, 63.15; H, 5.30; N, 8.18.
Found: C, 63.28; H, 5.37; N, 8.31.