Mumic acids A–E: new diterpenoids from mumiyo

Five new diterpenoids belonging to labdane and isopimarane skeletons, mumic acids A–E (1–5), have been isolated from mumiyo. Their structures and absolute configurations were elucidated on the basis of spectroscopic data and chemical derivatization.


Introduction
Mumiyo, also known as mumijo, mumie, or shilajit, is a material often found as crusts in rock cracks or interstices in the alpine region of Central Asia. Mumiyo has been used as a traditional medicine in the former Soviet Union, India, and Tibet for more than 3000 years, and is currently available in numerous countries as a food supplement [1]. Although there are many claims on the activity of mumiyo, scientific evidences on the chemical components and bioactivity are lacking [1]. In our search for new bioactive compounds [2][3][4][5][6][7][8][9][10][11], the mumiyo in Kyrgyzstan was investigated, resulting in the isolation of five new diterpenoids, mumic acids A-E (1)(2)(3)(4)(5) and agathic acid (6) [12]. The structure elucidation of 1-5 are reported herein (Fig. 1 (Table 2) revealed 22 carbon resonances due to 3 carbonyls, 2 sp 2 quaternary carbons, 2 sp 3 quaternary carbons, 1 sp 2 methines, 3 sp 3 methines, an sp 2 methylene, 6 sp 3 methylenes, and 4 methyls. The 1 H and 13 C NMR data (Tables 1 and 2) of 1 showed similarities to those of agathic acid (6) isolated in this study, suggesting the structure of 1 as a labdane type of diterpenoid related to 6.
The relative configuration of 1 was determined by analyses of the NOESY correlations (  (Table 1) of 3 suggested the presence of a sugar moiety. The differences in the 1 H and 13 C NMR data (Tables 1 and 2) of 3 and 6 are reminiscent to the differences observed between 1 and 2. Thus, 3 was assumed to be a 19-O-b-glucuronic acid derivative of agathic acid (6). Acid hydrolysis of 3 gave 6 and a sugar, which was identified as D-glucuronic acid on the basis of HPLC analysis with chiral detector.
The configuration of 5 was determined as follows. The b-orientation of C-17, C-19, and C-20 was deduced from the ROESY correlations of H 3 -20/H 3 -17 and H 3 -19. The 1 H-1 H coupling constant value of H-3/H-2a and H-2b (11.3 and 3.1 Hz) indicated H-3 to be a-oriented. C-3 was determined to be of S configuration based on the advanced Mosher's method [13]. The vicinal coupling constant value of H-14/H-15a and H-15b (Hz, respectively) and the CE signs in the CD spectrum , 229 (0), and 221 (?6.9) nm] of the 3,15,16-tribenzoyl-18-methyl-derivative of 5 [14] indicated the absolute configuration of C-15 of the terminal 1,2-diol to be R. Thus, 5 was deduced to be a new isopimarane diterpenoid with hydroxyl group at C-3, C-15, and C-16, a carboxylic acid at C-18, and C-8-C-14 double bond.
Compounds 1-5 were tested for cytotoxic activity against the HL-60 (human promyelocytic leukemia) cell line, LPS-induced NO production inhibitory activity on the RAW264.7 (murine leukemic monocyte macrophage) cell line, melanin-production inhibitory activity on the B16F10 (murine melanoma) cell line, lipid-droplet accumulation inhibitory activity on the MC3T3-G2/PA6 (mouse pre- adipocyte) cell line, and vasorelaxant activity on rat aortic artery. All compounds gave negative results for these bioactivity assays.

Experimental section
General experimental procedures Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer and IR spectra on a JASCO FT/IR-4100 spectrophotometer. CD spectra were recorded on a JASCO J-820 polarimeter. High-resolution ESI MS were obtained on an LTQ Orbitrap XL (Thermo Scientific). 1 H and 2D NMR spectra were measured on a 400-or 700-MHz spectrometer at 300 K, while 13 C NMR spectra were measured on a 125-or 175-MHz spectrometer. The residual CD 3 OD chemical shift used as an internal standard are d H 3.31 and d C 49.0 and for CDCl 3 are d H 7.26 and d C 77.0. Standard pulse sequences were used for the 2D NMR experiments.

Material
The natural mumiyo samples were collected from Kyrgys. To 1 kg of natural mumiyo, 4 L of water was added, and the mixture was then stirred and heated to boiling for about 40 min. The mixture was cooled, and the precipitates were separated from the supernatant. The precipitates were then extracted with water repeatedly to obtain the water extract. The supernatant and the water extract were combined and subjected to centrifugation for 10 min. The supernatant was collected and concentrated by an evaporator. The concentrated solution was again subjected to centrifugation, and the resulting supernatant was concentrated by an evaporator (until a water content of 30 %). The final concentrated solution (50 g) was cooled and then packed as commercial mumiyo. The mumiyo sample used in this study is stored at the Department of Pharmacognosy, Hoshi University, as sample HOSHI12001.
The rest of the n-BuOH-soluble materials (7.5 g) were subjected to ODS silica gel column chromatography glucuronic acid in the aqueous layer of hydrolysate of 2 was 4.9 min, with positive intensity. 3 (1.0 mg) was subjected to a similar treatment as 3, and the retention time of glucose in the aqueous layer of hydrolysate of 3 was 4.9 min, with positive intensity.
Synthesis of 2,14,15-tri-O-acyl-19-methyl-4 and 3,14,15-tri-O-acyl-18-methyl-5 To a solution of 4 (0.8 mg in 100 lL MeOH), 20 lL of TMS-diazomethane (10 % in n-hexane) was added and left at room temperature. After 10 min, the reaction mixture was dried under an N 2 stream, and the resulting residue (0.8 mg) was dissolved in 150 lL of CH 2 Cl 2 . To the CH 2 Cl 2 solution, a catalytic amount of 4-(dimethylamino)pyridine and 2 lL of triethylamine were added, and the mixture was then separated into three containers (50 lL each). Into the container, (R)-MTPA chloride, (S)-MTPA chloride, or benzoyl chloride was added, and the solutions were allowed to stand at room temperature overnight. The residue obtained under an N 2 stream was subjected to SiO 2 column chromatography (CHCl 3 ) to obtain the tri-(S)-MTPA, tri-(R)-MTPA, and tri-benzoyl derivatives of 19-methyl-4. The same procedure was used to obtain tri-(S)-MTPA, tri-(R)-MTPA, and tri-benzoyl derivatives of 19-methyl-5.