Two Novel Norwithasteroids with Unusual Six- and Seven-Membered Ether Rings in Side Chain from Flos Daturae

Chemical investigation of 50% ethanol eluate fraction of macroporous resin for the flower of Datura metel L. collected in Jiangsu province of China resulted in the isolation of two novel naturally occurring norwithasteroids, baimantuoluoline I (1) and baimantuoluoside J (2). Their structures were elucidated as 5α, 6β, 12β-trihydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid (1) and 5α, 6β, 12β, 25-tetrahydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid (2) on the basis of extensive spectroscopic analysis, including 1D, 2D-NMR, and HR-ESI-MS. According to the literatures, this study represents the first report of the norwithasteroids in the side chain with unusual six- and seven-membered ether rings instead of those with an unmodified skeleton (δ-lactone or δ-lactol side chain) and a modified skeleton (γ-lactone or γ-lactol side chain) in the family of withanolides. Meanwhile, compounds 1 and 2 were evaluated for their immunosuppressive activity against mice splenocyte proliferation in vitro.


Introduction
Flos Daturae is the dry flower of Datura metel L. (Solanaceae), which widely distributed in Jiangsu, Guangdong, and Fujian province of China [1]. It has been used in traditional Chinese medicine for the treatment of asthma, convulsions, pain, and rheumatism for centuries [2]. It is said that it was almost one of the most important anesthetic of the ancient times. It has a very long history and is referred to in many early writings in China. For instance, according to the legend as early as 200 AD, a Chinese skilled doctor, Huatuo, once made use of "mafeisan" on patients in surgery [2,3]. A recent study found that it has an obvious effect on the treatment of psoriasis [4,5]. Moreover, it had been for clinical use in the first affiliated hospital of Heilongjiang University of Chinese Medicine, China [6,7]. Our research group had done some work on its pharmacological actions for psoriasis and extraction and isolation of active constituents [5][6][7][8]. As a part of a continuing project to study the active constituents of Flos Daturae against psoriasis [3][4][5], we investigated 50% ethanol eluate fraction of macroporous resin for the flowers of D. metel L., which resulted in the discovery of two novel ergostane derivatives ( Figure 1).
Naturally occurring withanolides are not widely distributed in the plant kingdom and isolated mainly from flowers, leaves, and seeds of Solanaceae plants [9][10][11]. Most of the withanolides may be subdivided into two subgroups: those with an unmodified skeleton ( -lactone or -lactol side chain) and a modified skeleton ( -lactone or -lactol side chain) [12,13]. In this paper, we present the isolation and structural characterization of the two novel naturally occurring norwithasteroids on the basis of the interpretation of spectral data, including 1 H-NMR, 13 C-NMR, DEPT, 1 H-1 H-COSY, HSQC, HMBC, NOESY, and HR-ESI-MS. According to the literatures, this study represents the first report of naturally occurring norwithasteroids in the side chain with unusual six-and seven-membered ether rings formed rather than those with an unmodified skeleton ( -lactone or -lactol side chain) and a modified skeleton ( -lactone or -lactol side chain) in the family of withanolides. Meanwhile, compounds

Con A-Induced Mouse Splenocyte Proliferation.
Mouse splenocyte proliferation was assayed by MTT method as previously described [14]. Splenocytes were seeded into a 96well flat-bottom microtiter plate at 1 × 10 8 cells/mL in 100 L of complete medium. Serial drug dilutions were prepared in medium immediately prior to each assay. Thereafter, 100 L aliquots of serial dilution of each test compound were added (parallel triplicate wells were set), and then the cells were incubated in the absence or presence of Concanavalin A (Con A, final concn. 5 g/mL) for 44 h in the humidified 5% CO 2 incubator at 37 ∘ C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) in phosphate buffered saline (PBS) at 5 mg/mL (10 L) was added to each well, plates were incubated at 37 ∘ C for 4 h, and the formazan crystals formed were dissolved through addition of 100 L of DMSO/well. The absorption of the samples was measured using an ELISA reader (Uniscan Titertec) at a wavelength of 570 nm. Cyclosporine was used for positive control. The immunosuppressive activity of each compound was expressed as the concentration that inhibited splenocyte proliferation to 50% (IC 50 ) of the control value.   (Table 1)  there is a double doublet at 3.44 (1H, dd, J = 10.8, 4.4 Hz) in 1 H-NMR spectrum. Its coupling constants and splitting pattern were characteristic of 12 -hydroxywithanolide [3][4][5]. The 13 C-NMR spectrum of 1 showed resonances for all 30 carbons ( Table 1). The carbon signals from C-1 to C-21 were easily assigned according to 2D-NMR and the comparison of similar substituted patterns of withanolides from the A ring to the D ring. The characteristic downfield signals at 207.3 were due to ketone carbonyl, along with the characteristic doublets at 128.9 and 144.0 for the vinylic carbons at C-2 and C-3, respectively, in the ring A. The signals appearing at 16.2 and 9.2 were assigned to the Me-19 and Me-18, respectively. The typical signals at 78.3, 75.2, 79.8.0, and 65.5 were assigned to the oxygenated carbons at C-5, C-6, C-12, and C-21, respectively. The chemical shifts of the C-5 and C-6 further indicate the presence of a 5 , 6 -dihydroxyl steroid residue because their chemical shifts and multiplicity agreed with a similar substitution pattern [4]. The downfield chemical shifts of C-21 suggested that there may be such a 21, 24 epoxy structure segment in the side chain. In that case, a tetrahydropyrane ring was proposed, including C-20 ( 44.9), C-21 ( 65.5), C-22 ( 68.3), C-23 ( 40.8), and C-24 ( 75.5). This tetrahydropyrane ring plays an important role in connection of steroid mother nucleus and the side chain. Further support this assumption was obtained that a serial of long-range correlations between H-21 [ 3.47 (1H, br. t, J = 11.6 Hz) and 3.86 (1H, dd, J = 11.6, 5.  Figure 2). Taking into account the NMR spectral data and the 9 degrees of unsaturation calculated from the empirical formula of compound 1, it was suggested that it is possible to be presence of another ether ring except for an , -unsaturation carbonyl group, four rings of steroid skeleton, one tetrahydropyrane ring, and one carboxyl. In that case, another seven-membered-ether ring was proposed as shown in Figure 1.  Figure 2. Therefore, the structure of 1 was deduced as 5 , 6 , 12 -trihydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid, which was named baimantuoluoline I. The 1 H-NMR spectrum of 2 (Table 1) showed distinct resemblance to those of baimantuoluoside I. The only notable difference was the change of H-25 signal, which was missing. The 13 C-NMR (DEPT) spectrum showed an additional downfield C-atom signal at 82.2 in 2 (Table 1), which was affirmatively assigned to the C-25, indicating that C-25 was substituted by a hydroxyl group. On the basis of previous data, the structure of 2 was identified to be 5 , 6 , 12 , 25-tetrahydroxy-1-oxo-2-en-ergosta-21,24;22,29diepoxy-26-carboxylic acid, which was named baimantuoluoline J.

Possible Biosynthetic Pathway to Proposed Structures.
The biosynthetic pathway of common withanolides in plants is derived from phytosterol, which possibly experienced a sequential oxidation, hydroxylation, and cyclization in the side chains and finally formed the basic skeleton of withanolides (Figure 3(a)) [12]. For compounds 1 and 2, they have two more carbon atoms than the common withanolide compounds in the side chain, which is never to be seen in other phytosterol compounds. A logical biosynthetic pathway of compounds 1 and 2 is postulated as shown in Figure 3(b). The compounds 1 and 2 may be derived from the ordinary phytosterol compounds as precursors that firstly experience  Figure 3: Possible biosynthetic pathway to common withanolides (a) and proposed structures (b). The order of a and b may be interchangeable. hydroxylation and oxidation and along with an acetatemalonate (AA-MA) pathway resulting in the side chains increase two carbon atoms and finally involve a complex process such as reduction, cyclization, and oxidation to form this kind of unusual six-and seven-membered ether rings in side chain. This type of biosynthetic pathway is rarely seen, and detailed evidences also need to be further confirmed.

Effect of Compounds on the Splenocyte Proliferation of
Mouse. The colorimetric assay using MTT for cell proliferation was then carried out to evaluate Con A-stimulated mouse splenocyte in the presence of various concentrations of 1 and 2. The results of this assay showed that compounds 1 and 2 possess the obvious immunosuppressive activity with the IC 50 values of 10.2 nM and 13.5 nM (Table 2), respectively. However, their activities were much lower than that of the positive medicine cyclosporine. Compounds 1 and 2 did not show cytotoxicity in the range of tested concentrations, indicating that the immunosuppressive effects observed in vitro may be not due to the toxicity of compounds.

Conclusion
Over the period of August 1996 to March 2010, 360 new naturally occurring withanolides were isolated largely from the flowers, leaves, and seeds of Solanaceae plants such as Withania somnifera, W. cagalans, W. aristata, Physalis pubescens, P. peruviana, P. minima, D. ferox, D. fastuosa, D. inoxia, D. metel, and D. stramoniam, [9][10][11]. The characteristic feature of their skeleton of withanolides can be summarized as the two subgroups: those with an unmodified skeleton (lactone or -lactol side chain) and a modified skeleton (lactone or -lactol side chain) [12], which have been shown to be associated with many biological activities including cytotoxic, anti-inflammatory, antioxidant, antiarthritic, anticholinesterase, immunoprotective, trypanocidal, antimalarial, leishmanicidal, and diuretic [13]. However, till now, no studies on ergostane derivatives with unusual six-and seven-membered ether rings formed in the side chain have been found in the family of Solanaceae. At the same time, these two naturally occurring norwithasteroids have been found to possess immunosuppressive activity by mice splenocyte proliferation in vitro. It is worthy of mentioning that tropane alkaloids have for a long time been considered as characteristic ingredients of Flos Daturae [12]. This finding revealed another kind of biological