Synthesis and discovery of 18α-GAMG as anticancer agent via down
expression of p65 protein
Tang, Wen-jian; Yang, Yong-an; Xu, He; Shi, Jing-bo; Liu, Xin-hua*
School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China;
Xin-Hua Liu, Tel.: +86 551 65161115; fax: +86 551 65161115. E-mail:
xhliuhx@163.com
Synthesis
General Chemistry Methods
1
H (500/300 MHz) and
13
C (125/75 MHz) NMR spectra were recorded on a
Bruker AV500/300 spectrometer using Me4Si as an internal standard unless otherwise
noted. LC/HRMS was performed on an Agilent 1260-6221 TOF LC/MS system in
positive electrospray ionization mode (ESI+). Melting points were uncorrected. Silica
gel (200–300 mesh, Huanghai, Qingdao). TLC: pre-coated silica gel F254 plates.
Optical rotations: polar 3002 polarimeter. Aspergillus sp. Ts-1 was isolated from soil
collected in Kashi of Xinjiang Uygur Autonomous Region (China) and selectively
hydrolyzed the terminal–glucuronyl linkage of 18β-GA to yield glycyrrhetic acid
3-O-mono-β-D-glucuronide (18β-GAMG). Its subculture and 18β-GA were provided
by Jiangsu Tian Sheng Pharmaceutical Co. Ltd. All materials were obtained from
commercial suppliers, were of analytical reagent grade and used without further
purification.
Synthesis of 18β-GAMG from 18β-GA via biotransformation
Aspergillus sp. Ts-1 on glucose yeast agar slant was inoculated into a 250 mL
Erlenmeyer flask containing 100 mL of the seed medium consisting of 1.0 g glucose,
0.2 g yeast, 1.0 g agar, 0.1 g KH2PO4 and 0.025g MgSO4 in distilled water (pH 7.0).
The culture media were sterilized at 121 ºC for 20 min and the fermentation was
carried out at 30 ºC on a rotary shaker at 200 rpm. After 24 h of inoculation, 30 mL
sterilized medium was inoculated into a 1000 mL Erlenmeyer flask containing 300
mL pre-culture sample consisting of 15 g GA, 0.30 g KH2PO4, 3.0 g urea and 0.24 g
MgSO4 in distilled water and the pH value was adjusted to 6.0. The culture media
were sterilized at 121 ºC for 20 min and the fermentation was carried out at 30 ºC on a
rotary shaker at 250 rpm. After 72 h of inoculation, the culture solution was filtered
and the filtrate was extracted with ethyl acetate. The extract was concentrated under
the reduced pressure. The residue (14.5 g) was applied to a silica gel column (800 g,
5.0 × 100 cm) and eluted with 0 to 30% MeOH in CHCl3. By TLC analysis, fractions
I–IX was obtained. Fractions VI–VIII was concentrated in vacuo and recrystallization
from aqueous MeOH to give 18β-GAMG (6.35 g, 54% yield) as a white crystalline
powder. Mp 237−239 °C; [α]D20 = +91 (c = 1.0, MeOH); 1H NMR (500 MHz,
DMSO-d6) δ 0.76 (s, 3H, 24-CH3), 0.77 (s, 3H, 28-CH3), 0.99 (s, 3H, 23-CH3), 1.06 (s,
2×3H, 25-CH3, 26-CH3), 1.10 (s, 3H, 29-CH3), 1.34 (s, 3H, 27-CH3), 2.34 (s, 1H,
9-H), 3.01 (m, 1H, 4'-H), 3.08 (dd, 1H, J=4.8, 11.2 Hz, 3-H), 3.15 (t, 1H, J=9.0 Hz,
3'-H), 3.30 (m, 1H, overlapped, 2'-H), 3.58 (d, 1H, J=9.7 Hz, 5'-H), 4.25 (d, 1H, J=7.8
Hz, 1'-H), 5.40 (s, 1H, 12-H);
13
C NMR (125 MHz, DMSO-d6) δ 16.2, 16.4, 16.9,
18.3, 23.0, 25.6, 25.8, 26.1, 27.4, 27.8, 28.4, 30.4, 31.5, 32.1, 36.3, 37.5, 38.4, 39.1,
40.6, 42.9, 43.1, 44.9, 48.0, 54.1, 61.0, 71.5, 73.7, 75.6, 76.1, 87.7, 105.5, 127.3,
169.7, 170.5, 177.6, 198.9. TOF-HRMS: m/z [M + Na]+ calcd for C36H54NaO10:
669.3609; found: 669.3608.
Figure S1. The isomerization reaction was monitored by 13C-NMR spectroscopy