2492
K.-K. Bai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2488–2493
CONHCH2), 5.34 (s, 1H, H-12), 4.49 (s, 1H, H-3), 3.88 (s, 1H, HNCHCON), 3.40 (s,
1H, Ar-CH2), 3.30 (s, 2H, NH2), 3.16 (s, 1H,Ar-CH2), 3.12 (t, 2H,
COHNCH2CH2NHCO), 3.10 (t, 2H, COHNCH2CH2NHCO), 2.05 (s, 3H, CH3COO),
1.07, 0.94, 0.93, 0.87, 0.86, 0.85, 0.74 (s, 21H, 7ꢁCH3); Calcd for C43H65N3O4: C
75.07, H 9.52, N 6.11; Found: C 74.52, H 10.03, N 6.12.
Open-foundation of the Fujian Key Lab of Medical Instrument and
Pharmaceutical Technology, China (No.09003).
Supplementary data
N-(3b-hydroxy-urs-12-en-28-oyl)-1-amino, N-glycyl-2-aminoethane (UA-
11a): white crystalline powder. Yield 87.1%; mp 205–208 °C; ESI-MS: m/z
556.5 [M+H]+; UV–Vis (Methanol), kmax = 208 nm; IR (KBr): 3412, 3087, 2926,
Supplementary data associated with this article can be found, in
include MOL files and InChiKeys of the most important compounds
described in this article.
1683, 1641, 1251, 1044, 997 cmꢂ1 1H NMR (600 MHz, DMSO-d6): 8.56 (s, 1H,
;
CONHCH2), 7.39 (s, 1H, CONHCH2), 5.21 (s, 1H, H-12), 4.31 (s, 1H, HO-3), 4.03–
4.02 (d, 1H, J = 6.3 Hz, H2NCH2CON), 3.46 (s, 1H, H2NCH2CON), 3.35 (br s, 2H,
NH2), 3.10 (s, 1H, CONHCH2H2NHCO), 3.03–2.99 (m, 1H, CONHCH2CH2NHCO),
2.18 (d, 1H, J = 10.1 Hz, H-18), 1.02, 0.91, 0.88, 0.84, 0.82, 0.67, 0.67 (s, 21H,
7ꢁCH3); Calcd for C34H57N3O3: C 73.47, H 10.34, N 7.56; Found: C 73.29, H 9.91,
N 7.53.
References and notes
N-(3b-hydroxy-urs-12-en-28-oyl)-1-amino,
N-(2-amino-4-methylthio-1-
1. Liu, J. J. Ethnopharmacol. 2005, 100, 92.
2. Ma, C. M.; Cai, S. Q.; Cui, J. R.; Wang, R. Q.; Tu, P. F.; Masao, H.; Mohsen, D. Eur. J.
Med. Chem. 2005, 40, 582.
3. Hsu, H. Y.; Yang, J. J.; Lin, C. C. Cancer Lett. 1997, 111, 7.
4. Meng, Y. Q.; Liu, D.; Cai, L. L.; Chen, H.; Cao, H.; Wang, Y. Z. Bioorg. Med. Chem.
2009, 17, 848.
5. Shao, J. W.; Dai, Y. C.; Xue, J. P.; Wang, J. C.; Lin, F. P.; Guo, Y. H. Eur. J. Med. Chem.
2011, 46, 2652.
butyl-)-2-aminoethane (UA-11b): white powder. Yield 89.2%; mp 99–101 °C;
ESI-MS: m/z 630.6 [M+H]+; UV–Vis (Methanol), kmax = 210 nm; IR (KBr): 3378,
2924, 2854, 1641, 1260, 1093, 1028, 802 cmꢂ1 1H NMR (600 MHz, DMSO-d6):
;
8.01 (s, 1H, CONHCH2), 7.28 (s, 1H, CONHCH2), 5.28 (s, 1H, H-12), 4.34 (s, 1H,
HO-3), 3.38 (br s, 2H, NH2), 3.25 (s, 1H, HNCHCON), 3.15 (s, 1H,
CONHCH2CH2NHCO), 3.04 (s, 1H, CONHCH2CH2NHCO), 2.17 (d, 1H,
J = 10.1 Hz, H-18), 2.08 (s, 3H, SCH3), 1.63 (t, 2H, J = 12.4 Hz, CH2CH2SCH3),
1.08, 0.96, 0.94, 0.90, 0.87, 0.72, 0.72 (s, 21H, 7ꢁCH3); Calcd for C37H63N3O3S: C
70.54, H 10.08, N 6.67, S 5.09; Found: C 70.05, H 9.92, N 6.26, S 4.62.
6. Wang, P.; Wang, J.; Guo, T. T.; Li, Y. X. Carbohydr. Res. 2010, 345, 607.
7. Jin, I. J.; Ko, Y., III; Kim, Y. M.; Han, S. K. Arch. Pharm. Res. 1997, 20, 269.
8. Bai, K. K., P.R. China. Patent. CN201010115902, 2010.
9. Properties of the novel UA derivatives: N-[3b-Hydroxy-urs-12-en-28-oyl]-2-
amino-1,5-pentanedioic acid (UA-4): white crystalline powder. Yield 95.3%;
mp 317–320 °C; ESI-MS: m/z 610.9 [M+K]+; UV–Vis (Methanol), kmax = 210 nm;
N-(3b-hydroxy-urs-12-en-28-oyl)-1-amino,
N-(2-amino-3-phenyl-1-
propionyl-)-2-aminoethane (UA-11c): white powder. Yield 96.8%, mp 154–
147 °C; ESI-MS: m/z 668.6, [M+Na]+; UV–Vis (Methanol), kmax = 205, 242 nm; IR
(KBr): 3355, 2926, 2869, 1673, 1638, 1538, 1455, 1251, 1042, 746, 700,
663 cmꢂ1 1H NMR (600 MHz, DMSO-d6): 8.49 (s, 1H, CONHCH2) , 7.35 (s, 1H,
;
IR (KBr): 3384, 2976, 2936, 2872, 1712, 1635 cmꢂ1 1H NMR (600 MHz, DMSO-
;
CONHCH2), 7.30–7.24 (5H, Ar-H), 5.20 (s, 1H, H-12), 4.31 (s, 1H, HO-3), 3.79 (s,
1H, HNCHCON), 3.38 (s, 1H, Ar-CH2), 3.06 (s, 1H, Ar-CH2), 3.34 (br s, 2H, NH2),
3.03–3.00 (m, 1H, CONHCH2CH2NHCO), 2.91–2.88 (m, 1H, CONHCH2CH2NHCO),
2.16 (d, 1H, J = 10.2 Hz, H-18), 1.02, 0.90, 0.89, 0.85, 0.81, 0.67, 0.67 (s, 21H,
7ꢁCH3); Calcd for C41H63N3O3: C 76.23, H 9.83, N 6.51; Found: C 75.59, H 9.37,
N 6.47.
d6): 7.22 (d, 1H, J = 7.3 Hz, NH), 5.19 (t, 1H, J = 3.6 Hz, H-12), 4.24 (s, 1H, OH-3),
4.13–4.09 (m, 1H, NHCHCOOH), 2.99 (s, 1H, H-3), 2.22 (t, 2H, J = 7.3 Hz,
CH2COOH), 0.83 (d, 3H, J = 6.4 Hz, CH3), 1.03, 0.91, 0.89, 0.85, 0.67, 0.65 (s, 18H,
6ꢁCH3); Anal. Calcd for C34H53NO6: C 71.42, H 9.34, N 2.45; Found: C 71.60, H
9.26, N 2.27.
N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-1,4-butanedioic
acid
(UA-5):
10. MTT assay: The anti-proliferative activities of the molecules were determined
by the MTT assay. UA, Taxol and UA derivatives were initially dissolved in
DMSO at 20 mM and serially diluted with culture medium to various
concentrations. Cells were seeded in 96-well plates at a density of 1 ꢁ 104
cells per well. After 24 h, the cells were treated with different concentrations of
drugs for another 48 h. Each compound was tested at six different
white crystalline powder. Yield 93.6%; mp 302–305 °C; ESI-MS: m/z 624.8
[M+K]+, 1194.4 [2 M+Na]+; UV–Vis (Methanol), kmax = 204 nm; IR (KBr): 3417,
2971, 2928, 2871, 1731, 1636 cmꢂ1 1H NMR (600 MHz, DMSO-d6): 7.27 (d, 1H,
;
J = 7.2 Hz, NH), 5.20 (t, 1H, J = 3.6 Hz, H-12), 4.33–4.29 (m, 1H, NHCHCOOH),
4.24 (s, 1H, OH-3), 2.99 (s, 1H, H-3), 2.22 (t, 2H, J = 7.3 Hz, CH2COOH), 0.83 (d,
3H, J = 6.4 Hz, CH3), 1.02, 0.90, 0.89, 0.84, 0.67, 0.65 (s, 18H, 6ꢁCH3); Anal. Calcd
for C35H55NO6: C 71.76, H 9.46, N 2.39; Found: C 71.77, H 9.34, N 2.07.
concentrations, 10, 20, 40, 60, 80 and 100
replicated in six wells. The negative control (0
the same amount of DMSO as that of the highest test concentration. Next,
excess MTT was added, and the culture was incubated for additional 4 h. The
resultant formazan formed by metabolically viable cells was well dissolved in
l
l
M, and each concentration was
M of test compound) contained
N-[3b-acetoxy-urs-12-en-28-oyl]-2-aminoethylamine
(UA-6):
white
crystalline powder. Yield 75.5%; mp 140–142 °C; ESI-MS: m/z 541.5 [M+H]+;
UV–Vis (Methanol), kmax = 208 nm; IR (KBr): 3365, 2950, 2853, 1728, 1631,
1252, 1026 cmꢂ1 1H NMR (600 MHz, CDCl3): 6.32 (s, 1H, CONHCH2), 5.33 (s,
;
100 lL of DMSO. The absorbance was then measured on a microplate reader
1H, H-12), 4.49 (t, 1H, J = 7.6 Hz, H-3), 3.41–3.38 (m, 1H, CONHCH2CH2NH2),
3.06–3.04 (m, 1H, CONHCH2CH2NH2), 2.79 (s, 2H, CH2NH2), 2.05 (s, 3H, CH3CO),
1.09, 0.95, 0.94, 0.86, 0.85, 0.84, 0.79 (s, 21H, 7ꢁCH3); Anal. Calcd for
apparatus (TECAN DNA Expert, Switzerland) at 570 nm. The average
absorbance of the six duplicates was applied to calculate the inhibitory
effects by subtracting the absorbance measured at the same wavelength from
DMSO treated cells. The concentration of the compound, which gives the 50%
C34H56N2O3: C 75.51, H 10.44, N 5.18; Found: C 75.76, H 10.71, N 4.94.
N-[3b-hydroxy-urs-12-en-28-oyl]-2-aminoethylamine(UA-7):
white
growth inhibition value, corresponds to the IC50
.
crystalline powder. Yield 86.7%; mp 145–147 °C; ESI-MS: m/z 499.5 [M+H]+;
UV–Vis (Methanol), kmax = 206 nm; IR (KBr): 3362, 2925, 2868, 1640, 1043,
11. Burton, R. F. Camp. Biochem. Physiol. 1995, 111A, 125.
12. Ishizuka, K.; Sahara, N. C.; Murayama, M.; Yoshiike, Y.; Takashima, A. Neurobiol.
Aging. 2004, 25, S149.
13. Stella, V. J.; NtiAddae, K. W. Adv. Drug. Del. Rev. 2007, 59, 677.
14. Desino, K. E.; Pignatello, R.; Guccione, S.; Basile, L.; Ansar, S.; Michaelis, M. L.;
Ramsay, R. R.; Kenneth, L. A. Biochem. Pharm. 2009, 78, 1412.
15. Ma, C. M.; Wu, X. H.; Masao, H.; Wang, X. J.; Kano, Y. J. Pharm. Pharmaceut. Sci.
2009, 12, 243.
999 cmꢂ1 1H NMR (600 MHz, DMSO-d6): 7.13 (s, 1H, CONHCH2), 5.20 (s, 1H, H-
;
12), 3.5–3.1 (br s, OH), 3.02ꢄ2.98 (m, 2H, CONHCH2CH2NH2), 2.97–2.92 (m, 2H,
CONHCH2CH2NH2), 2.15(d, 1H, H-18), 0.82 (d, 3H, J = 5.2 Hz , CH3), 1.03, 0.91,
0.89, 0.85, 0.69, 0.67 (s, 18H, 6ꢁCH3); Anal. Calcd for C32H54N2O2: C 77.06, H
10.91, N 5.63; Found: C 76.56, H 10.54, N 5.27.
N-(3b-acetoxy-urs-12-en-28-oyl)-1-amino, N-glycyl-2-aminoethane (UA-9a):
white powder. Yield 88.5%; mp 147–149 °C; ESI-MS: m/z 498.5 [M+H]+; UV–Vis
(Methanol), kmax = 210 nm; IR (KBr): 3397, 2925, 2854, 1736, 1681, 1640, 1246,
16. Mar, A. A.; Szotek, E. L.; Koohang, A.; Flavin, W. P.; Eiznhamer, D. A.; Flavin, M.
T.; Xu, Z. Q. Bioorg. Med. Chem. Lett. 2010, 20, 5389.
1027, 805 cmꢂ1 1H NMR (600 MHz, CDCl3): 7.62 (s, 1H, CONHCH2), 6.31 (s, 1H,
;
17. Cell cycle analysis: AGS cells were used to analyze the cell cycle effects of the
compounds. Cells were seeded at 1 ꢁ 105 cells per well in six-well culture
CONHCH2), 5.29 (s, 1H, H-12), 4.42 (d, 1H, J = 7.4 Hz, H-3), 3.54–3.52 (m, 1H,
HNCHCON), 3.10–3.08 (m, 1H, HNCHCON), 3.36, 3.30 (s, 1H, each,
COHNCH2CH2NHCO), 3.27 (s, 1H, COHNCH2CH2NHCO), 3.22 (d, 1H,
J = 13.4 Hz, COHNCH2CH2NHCO), 1.97 (s, 3H, CH3COO), 1.01, 0.86, 0.86, 0.80,
0.78, 0.78, 0.69 (s, 21H, 7ꢁCH3); Calcd for C36H59N3O4: C 72.32, H 9.95, N 7.03;
Found: C 71.95, H 9.94, N 6.87.
plates. After 24 h, the cells were treated with tested compounds at 10 lM or at
concentrations equivalent to their IC50 values, and incubated for an additional
48 h. The cells were harvested and fixed with 70% ethanol at 4 °C overnight,
then treated with RNAse (100 lg/mL) for 20 min, stained with propidium
iodide (Sigma, USA) for 10 min, and finally analyzed using a flow cytometer
(Beckman Coulter, EPICS XL, USA). The percentages of cells in G0/G1, S, and G2/
M phases were determined by CellQuest software (Becton, Dickinson and
Company). All experiments were performed in triplicate and gave the similar
results.
N-(3b-acetoxy-urs-12-en-28-oyl)-1-amino,
N-(2-amino-4-methylthio-1-
butyl)-2-aminoethane (UA-9b): white powder. Yield 89.2%; mp 96ꢄ98 °C;
ESI-MS: m/z 672.6 [M+H]+; UV–Vis (Methanol), kmax = 208 nm; IR (KBr): 3355,
2925, 2871, 1736, 1650, 1246, 1027, 804 cmꢂ1 1H NMR (600 MHz, CDCl3): 7.67
;
(s, 1H, CONHCH2), 6.34 (s, 1H, CONHCH2), 5.28 (s, 1H, H-12), 4.42 (d, 1H,
J = 7.4 Hz, H-3), 3.43,3.39 (s, 2H, each, COHNCH2CH2NHCO), 3.31,3.27 (s, 2H,
each, COHNCH2CH2NHCO), 3.11ꢄ3.10 (m, 1H, HNCHCON), 2.54 (m, 2H,
CH3SCH2), 2.04 (s, 3H, CH3CO), 1.97 (s, 3H, SCH3), 1.65 (t, 2H, J = 12.4 Hz,
CH2CH2SCH3), 1.01, 0.88, 0.87, 0.80, 0.79, 0.78, 0.69 (s, 21H, 7ꢁCH3); Calcd for
18. Tu, H. Y.; Huang, A. M.; Wei, B. L.; Gan, K. H.; Hour, T. C.; Yang, S. C.; Pu, Y. S.;
Lin, C. N. Bioorg. Med. Chem. 2009, 17, 7265.
19. Annexin V/propidium iodide (AV/PI) dual staining: AGS cells were treated with 0,
5, 10, 20 lM UA-7 for 24 h, washed and resuspended in PBS buffer. Apoptotic
cells were identified by double staining with recombinant fluorescein
isothiocyanate (FITC)-conjugated Annexin V and propidium iodide, by using
the Annexin V-FITC apoptosis detection Kit (KeyGEN, China) following the
manufacturer’s instructions. Flow cytometric analysis was performed
immediately after staining. Data acquisition and analysis were performed by
using CellQuest software.
C
39H65N3O4S: C 69.70, H 9.75, N 6.25, S 4.77; Found: C 69.29, H 9.66, N 6.24, S
4.27.
N-(3b-acetoxy-urs-12-en-28-oyl)-1-amino,
N-(2-amino-3-phenyl-1-
propionyl-)-2-aminoethane (UA-9c): white powder. Yield 96.8%; mp
82ꢄ84 °C, ESI-MS: m/z 688.6 [M+H]+; UV–Vis (Methanol), kmax = 205,
241 nm; IR (KBr): 3363, 2924, 2853, 1735, 1648, 1246, 1095, 1027, 803, 746,
20. Dadashzadeh, S.; Mirahmadi, N.; Babaei, M. H.; Vali, A. M. J. Control Release
2010, 148, 177.
701, 665 cmꢂ1 1H NMR (600 MHz, CDCl3): 8.63 (s, 1H, CONHCH2), 6.86 (s, 1H,
;