Biological Activity of Oleanane Triterpene Derivatives Obtained by Chemical Derivatization

Nine new derivatives of oleanane triterpenoids isolated from Fatsia polycarpa Hayata were synthesized through chemical transformations. Acetylation was effected by reaction with acetic anhydride in pyridine to afford compounds 1–5, while compound 6 was obtained using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in CH2Cl2. The others derivatives 7–9 were obtained in reactions of the corresponding triterpenoids with EDC·HCl, 4-N,N-dimethylaminopyridine hydrochloride and 4-N,N-dimethylaminopyridine in CH2Cl2. The structures of 1–9 were elucidated from extensive spectroscopic and HRESIMS data, while the structure of 9 was further confirmed by X-ray diffraction analysis. The cytotoxic, anti-hepatitis B virus (HBV), antibacterial, hypoglycaemic and Wnt signaling activities of these derivatives were evaluated in vitro.


Results and Discussion
Compound 1 was prepared in the overnight reaction of fatsicarpain D [2] and acetic anhydride in anhydrous pyridine at 50 °C. The HRESIMS of 1 exhibited a pseudomolecular ion peak at m/z 519.3445 [M + Na] + , consistent with the molecular formula of C 32 H 48 O 4 , requiring nine degrees of unsaturation. The IR spectrum of 1 showed the diagnostic absorption band of an acetoxy functionality at 1,733 cm −1 , which was further supported by the 1 H-NMR signals at δ H 2.04 (3H, s) and at δ H 4.64 ppm for the H-3 (situated downfield from the resonance of the corresponding proton in the starting compound (δ H 3.42 ppm) [2], and 13 C-NMR signals at δ C 170.8 (qC) and 21.3 (CH 3 ). Additionally, acetylation induced significant downfield shifts of H-3 (Δδ H = 1.22 ppm) [2]. The carbonyl signal was attributed to the acetate moiety linked to C-3, as further confirmed through the crucial HMBC correlation from H-3 to the carbonyl carbon of 3-OAc. Similarly, fatsicarpain C, 3α-hydroxyolean-11en-28,13β-olide, fatsicarpain F and 3α-hydroxyolean-11,13(18)-dien-28-oic acid were submitted to acetylation with Ac 2 O in pyridine at room temperature overnight to yield 2-5, respectively. The NMR data revealed the presence of characteristic O-acetyl group signals (see Experimental). Furthermore, the structures of 2-5 were definitely confirmed by the crucial HMBC correlations of the acetyl carbonyl carbon signals in each case with H-3.
N- propyl)-N-(ethylcarbamoyl)-3α, 23-dihydroxyolean-11,13(18)-dien-28-amide (6), synthesized from fatsicarpain A and EDC·HCl in dry CH 2 Cl 2 (at 50 °C for 3 h), was obtained as a white amorphous powder. Compound 6 was analyzed for the molecular formula of C 38 H 64 O 4 N 3 by the positive HRESIMS (m/z 626.4901, [M+H] + ) coupled with the 13 C-NMR spectroscopic data (see Experimental). The NMR features of 6 were analogous to those of fatsicarpain A except that the resonances of the carboxylic acid at C-28 were replaced by those of N- (3-(dimethylamino)propyl)-N-(ethylcarbamoyl)formamide group. By interpretation of 1 H-1 H COSY correlations, it was possible to establish two partial structures of consecutive proton systems extending from H 2 -1' to H 2 -3' through H 2 -2', and from H 2 -6' to H 2 -7'. The crucial HMBC correlations from H 2 -16 to C-28 and from H 2 -6' to C-8' revealed the connectivity of the above partial structures (Figure 1). Consequently, the structure of 6 was unambiguously established. Moreover, the suggested pathway involves the rearrangement of the O-acylisourea 6a to the stable N-acylurea 6, as illustrated in Figure 2. O-acylisourea 6a is unstable in anhydrous CH 2 Cl 2 and can undergo cyclic electronic displacement (O→N acyl migration), producing the thermodynamically more stable N-acylurea 6 [17].   7, prepared by overnight reaction of fatsicarpain A with EDC·HCl, DMAP and DMAP·HCl in CH 2 Cl 2 at room temperature, was obtained as a white amorphous powder. It was analyzed for the molecular formula of C 60 H 90 O 7 by the positive HRESIMS (m/z 945.6595, [M+Na] + ) coupled with its 13 C-NMR spectroscopic data (see Experimental). The characteristic pattern for a noncyclic and saturated anhydride in the IR spectrum of 7 is the appearance of two strong bands at 1,793 and 1,772 cm −1 , and this was further supported by the 13 C-NMR signals at δ C 172.7 (qC). Anhydride coupling induced significant upfield shifts of C-28 (Δδ C = 7.3 ppm) [2]. The position of the anhydride group was confirmed by the HMBC correlations from H 2 -16 to C-28. In the same manner, compounds 8 and 9 were prepared by anhydride coupling of 3α-hydroxyolean-11,13(18)-dien-28-oic acid and 5, respectively. The IR spectra and 13 C-NMR data revealed the presence of characteristic anhydride group signals (see Experimental). In addition, compound 9 was fully characterized by the X-ray analysis ( Figure 3) which confirmed the structure and presence of anhydride group. Of four million patients worldwide infected with hepatitis B virus (HBV), about 20% are expected to develop chronic hepatitis, liver cirrhosis, or hepatocarcinoma [18]. Anti-HBV effects of compounds 1-9 were screened in vitro using the human hepatocellular carcinoma (HepG2 2.2.15) cell model system with fluorouracil as a positive control (Table 1). Preliminary cytotoxicity screening revealed that 1-6 exhibited inhibition effects on HBV replicated DNA level in the IC 50 values of 6.5, 17.9, 38.5, 24.1, 9.3 and 5.3 μM, respectively. The esterification of the hydroxy group at C-3 increased the resultant cytotoxicity against HepG2 2.2.15, as shown for the acetylated derivatives 1-5. By far, compound 6 exhibited the highest potency against HepG2 2.2.15. Particularly, compounds 7-9, possessing an anhydride group, showed no significant activity against HepG2 2.2.15 (IC 50 > 50 μM), which confirmed that the carboxylic acid at C-28 is essential to their cytotoxicity. After varying its structure at C-3 and C-28 positions, we found 1-6 to be more potent than the parent compounds (Table 1), and these findings are in accordance with the available literature data concering structure−activity relationship [13,14]. However, hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in HepG2 2.2.15 cells were not significantly inhibited by the tested compounds. Helicobacter pylori infection is associated with an increased risk for development of duodenal ulcers, gastric ulcers, gastric adenocarcinomas and gastric lymphomas. However, as other bacterial pathogens, antibiotic resistance to H. pylori is an increasing problem for eradicating infection [19]. Therefore, finding a safe and efficient treatment to decrease the need or even replace antibiotics for eradicating H. pylori infection in human becomes necessary and an important task. Preliminary anti-H. pylori activity revealed that compound 2 exhibited moderate antibacterial activity with a minimum bactericidal concentrations (MBC) of 128 μg/mL. With the exception of the above observations, the obtained negative results showed that compounds 1, 3-5 and 7-9 exhibited no discernible activity (MBC > 128 μg/mL) ( Table 2).
In the past decades, bacterial resistance to the antibiotics has emerged a serious global problem in human and veterinary medicine. The abuse of antibiotics for non-perscription application has accelerated the generation of superbacteria which makes a critical issue. According to a previous report from 1991 to 2000, the Bacillus cereus played the leading role of outbreak case of food-borne pathogens (41.2%, 113 of 171 outbreaks), followed by Staphylococcus aureus (17.9%) and Vibrio parahaemolyticus (15.7%) in central Taiwan [20]. Also, the modifiction at C-3 position of betulinic acid, oleanolic acid and ursolic acid increased antimycobacterial activity aganist Mycobacterium tubereulosis [21]. For that reason the antibacterial activities of new modified oleanane-type derivatives 1-9 were evalutaed against seven bacteria: B. cereus, E. faecalis, E. coli, L. monocytogenes, S. enterica, S. aureus and P. aeruginosa and compared with the activity of parent compounds (Table 2) [2]. As expected, the C-3 acetylated derivatives 2, 5 and 6 exhibited more potent than the parent compounds. Additionally, compounds 2, 5 and 6 revealed greater antibacterial potential than the positive control (ampicillin) against B. cereus. Particularly, compounds 5 and 6 showed significant antibacterial activity against B. cereus with MIC values at 2 and 8 µg/mL, respectively. Only 6 showed specific antibacterial activity against S. aureus and L. monocytogenes with MIC values at 16 and 32 µg/mL, respectively. It was noteworthy to mention that modification of the functional group at C-28 from carboxylic acid to N- (3-(dimethylamino)propyl)-N-(ethylcarbamoyl)formamide moiety increased the antibacterial activity against L. monocytogenes significantly. Moreover, it is thought that coupling of two active compounds would generate more activity, but anhydride derivatives 7-9 did not exhibit significant antibacterial activity against all tested pathogens. The present result suggested that the presence of the C-28 carboxylic acid moiety is important for significant activity against B. cereus.
However, none of the tested compounds had significant activity against Gram-negative pathogens, E. coli, S. enterica and P. aeruginosa. Table 2. Antibacterial activity of 1-9, fatsicarpains A, C, D, F, 3α-hydroxyolean-11-en-28,13β-olide (X) and 3α-hydroxyolean-11,13(18)-dien-28-oic acid (Y). The hypoglycaemic activities were determined only for the modified compounds 1-5 and 7-9, the exception being 6 due to insufficient quantities for testing. The hypoglycaemic activities were tested using cell-based screening method, in which glucose uptake of cells treated with the new compound is quantified and compared with that of untreated cells and cells stimulated with insulin [22,23]. It was found that compounds 1-5 but not 7-9 enhanced glucose uptake of treated cells compared with untreated, and that their effects were similar to that of insulin (Figure 4), indicating that compounds 1-5 possess insulin-like hypoglycaemic activities that can promote the glucose uptake of cells. However, the underlying mechanism is not clear. Whether it involves the activation of the insulin signaling pathway requires further investigation. The total amount of medium glucose consumed by the cells between 0 to 5 h of treatment was calculated, and data expressed as relative glucose uptake versus control (cells with no treatment). Data represent the mean ± standard deviation of triplicate. * p < 0.05 versus control by two-way ANOVA.

Minimum Bactericidal Concentrations (MBC) (μg/mL)
Wnts proteins are secreted lipoglycoproteins that function as signaling molecules to regulate embryonic development and tissue homeostatis [24]. Aberrant Wnt signaling can cause an array of human diseases, including schizophrenia, pulmonary fibrosis, rheumatoid arthritis [25,26], osteoporosis, tetraamelia syndrome, neurodegenerative diseases and various cancers [27][28][29][30][31]. To know more about the biological effects of 1-5 and 7-9, these compounds were tested on the inhibition of canonical Wnt signaling using the TCF/β-catenin-mediated luciferase activity assay [16]. Compound 6 again was not tested due to paucity of the sample. As compared to the assay performed in Wnt-3a conditioned medium without any drug treatment (set as 100%), compounds 1, 3, 5 and 8 at 1 μM of concentration specifically inhibited the Wnt signaling by 20%, 40%, 38% and 32%, respectively, while 2, 4, 7 and 9 were not effective ( Figure 5). These data showed that Wnt signaling is sensitive to 1, 3, 5 and 8 and also suggested the potential use of these compounds for the therapy of Wnt-related diseases in the future.

Plant Material
Leaves and twigs of Fatsia polycarpa (7.1 kg) were collected at Hohuan Mountain (2,105 m elevation), Taiwan, in November 2009, and identified by one of the authors (C.-H. C.). A voucher specimen (FPL) was deposited in the Research Center for Biodiversity, China Medical University, Taiwan.

Acylation
To a solution of fatsicarpain A (5.0 mg) in CH 2 Cl 2 (1.0 mL) EDC·HCl (1.0 mg) was added. The solution was stirred for 3 h at 50 °C. After the completion of the reaction the solvent was evaporated under reduced pressure to give a crude product which was subjected to a short silica gel column eluting with n-hexane-EtOAc (1:1) to yield 6 (3.2 mg).

Cytotoxicity, Anti-hepatitis B Virus (HBV) Assay and Antibacterial Activity
The experimental details of these assays were carried out according to a previously described procedure [2].

Glucose Uptake Assay
FL83B cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in F12K medium as described previously [22]. Cells were seeded in 12-well plates (4 × 10 5 cells/well), cultured overnight, washed with phosphate-buffered saline (PBS, pH 7.4), and subjected to glucose uptake assays in triplicate as described previously with modifications [22,23]. Briefly, each compound was dissolved in DMSO (dimethyl sulfoxide). Cells were incubated in 450 μL of Eagle's minimum essential medium (MEM) containing 100 nM of insulin, or 20 μM of the indicated compound. In the control, cells were incubated in MEM containing DMSO in a concentration equivalent to that contained in the other groups. At 0, 1, 2, 3, 4, and 5 h, 30 μL of the culture medium was withdrawn and centrifuged at 500 g for 5 min. Five microliters of the resulting supernatant was mixed with 250 μL of a glucose assay kit (Glucose GOD FS, DiaSys Diagnostic Systems, Holzheim, Germany) in a 96-well plate and incubated at 37 °C for 10 min. Absorbance at 500 nm was then determined using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). A standard curve was established simultaneously using solutions of glucose in concentrations between 1-10 mM. The data of glucose uptake assays were analysed against the control by two-way analysis of variance (ANOVA), with cell treatment and time (the time points at which medium glucose concentration were measured) as the two parameters. Significance was considered when the p value between groups of cells and the p value of interaction between the two parameters were both < 0.05.

Dual Luciferase Activity Assay
P19 cells (1 × 10 5 cells/well) were seeded into 24-well dish for overnight. On the next day, cells were transfected with the Wnt reporter construct pGL3-OT (Dr. Bert Vogelstein, the Johns Hopkins