Daphmacrodins A and B, alkaloids from Daphniphyllum macropodum

Two new daphnicyclidin-type Daphniphyllum alkaloids, daphmacrodins A and B (1 and 2) were isolated from the leaves and stems of Daphniphyllum macropodum. Their structures were elucidated by extensive spectroscopic techniques, including 2D NMR spectroscopy and mass spectrometry. The relative configuration of 1 was further confirmed by a single-crystal X-ray diffraction analysis. Their cytotoxic activities against five human cancer cell lines, pesticidal activities against brine shrimp (Artemia salina), and antibacterial activities against five standard bacterial and fungal strains were evaluated. The structure of 1 was successfully transformed to 2 by a chemical method.


Introduction
Daphniphyllum alkaloids are a family of diversified and complex polycyclic natural products elaborated by plants of the genus Daphniphyllum. 1 Their unique structural features have attracted great interest as challenging targets for total synthesis 2 and biosynthetic research 3 for several decades. In recent years, quite a number of new Daphniphyllum alkaloids have been isolated and identified, and some of them possessed novel skeletons. 4 In our continuing search for structurally unique Daphniphyllum alkaloids, 4b-4d two new daphnicyclidintype compounds, 5 daphmacrodins A and B (1 and 2) with rare cyclopentadienyl anion 6 were isolated from the leaves and stems of D. macropodum. We report herein the isolation, structural elucidation, and the bioactivities of these two alkaloids.
Daphnicyclidin B (2), possesses a molecular formula of C 22 H 25 NO 4 determined by positive HRESIMS (m/z 368.1861 [M + H] + , calcd for C 22 H 26 NO 4 , 368.1861) and 11 degrees of unsaturation. The NMR data showed high similarity to those of 1, except that the quaternary carbon C-4 ( C 196.7) was replaced by a methine carbon at  C 66.4. This assignment was confirmed by 1 H-1 H COSY and HMBC spectra (Fig. 2), especially by the HMBC correlations from H-7 ( H 3.81, t, J = 11.0 Hz; 2.73, m) and H-19 ( H 3.16, m) to C-4 ( C 66.4) (Fig.  2B). The β-oriented H-4 ( H 3.53, br. s) was fixed by the ROESY correlations between H-4 and Me-21 ( H 1.36, s). Further detailed analysis of the 2D NMR (HSQC, 1 H-1 H COSY, and HMBC experiments) revealed that the rest part of 2 was in agreement with 1. Hence, the structure of 2 was established as shown in Fig. 1.
To investigate the structural relationship between 1 and 2, compound 1 was quantificationally transformed into 2 by reduction of the imine group in 1 according to reported method 7 (Scheme 1).
Compounds 1 and 2 were assayed in vitro for pesticidal activity against brine shrimp (Artemia salina) by the previously described Microwell method, 8 for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480) using the MTT method 9 with cisplatin and paclitaxel as positive controls, and for bacteriostatic activity against (ATCC25923, ATCC27853, MRSA 92, and MRSA 98 by two-fold dilution method, 10 while no positive results were observed at concentrations of 50 and 100 μg/mL.

Experimental Section
General Experimental Procedures. Melting points were measured on a Yuhua X-4 digital microdisplaying Melting Point Apparatus. Optical rotations were measured with a Jasco P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. CD spectra were recorded with an Applied Photophysics Chirascan spectrometer. A Tenor 27 spectrophotometer was used for IR spectra as KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-500 spectrometer with TMS as internal standard. HRESIMS was performed on an API QSTAR time-of-flight  Extraction and Isolation. The air-dried, powdered leaves and stems (34 kg) of D. macropodum were extracted three times with 95% EtOH. The extract was adjusted with saturated tartaric acid to pH 2-3 and then defatted with petroleum ether (PE). Next, the aqueous phase was adjusted to pH 10 with saturated Na 2 CO 3 and extracted with CHCl 3 to obtain the crude alkaloid fraction (260 g). The total alkaloid was subjected to normal phase Si gel (200-300 mesh; CHCl 3 /MeOH, 1:0→0:1) to obtain five major fractions . Fraction 4 (Fr. 4, 30 g) was further chromatographed over a reverse phase medium pressure column (MeOH/H 2 O, 1:1→1:0) to give four fractions (Fr. 4A-4E). Fraction 4E (8.0 g) was subjected to normal phase Si gel (300-400 mesh; CHCl 3 /MeOH = 25:1) to obtain seven fractions (Fr. 4E1-4E7). Fr. 4E7 was again separated by normal phase Si gel (300-400 mesh; CHCl 3 /MeOH = 5:1) to give compounds 1 (30 mg) and 2 (4 mg).

Reduction of 1 into 2:
A solution of 1 (5.0 mg) in anhydrous isopropanol (2 mL) was treated with sodium borohydride (1.6 mg, 3.08 eq.) The reaction mixture was stirred at 85 C for 24 h in sealed tube. Then 0.1 mL saturated ammonium chloride aqueous was added and stirred for 1 h. The isopropanol was removed under diminished pressure and the residue was partitioned in CHCl 3 /H 2 O = 1:1 (total 10 mL). The organic extracts were combined and the solvent was removed to give solid 2 (5.0 mg, 99.4%) which was determined by TLC analysis and negative ESIMS spectrum comparison with that of natural source of daphmacrodin B.
X-ray Crystal Structure Analysis. Colorless crystals of 1 were obtained from MeOH. Intensity data were collected at room temperature on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Mo Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT Software. The structure was solved by direct methods using SHELXL-97. 11 Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H-atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystal data: C 23 H 27 NO 5 (containing one MeOH molecular), tetragonal space group P2 1 , a = 7.0149(9) Å，b = 11.4350(14) Å ， c = 12.2778(15) Å, α = γ = 90  = 95.218(2), V = 980.8(2) Å 3 , Z = 2, D x = 1.346 kg m 3 . Crystallographic data (excluding structure factor tables) for 1 were deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 865586. Copies of the data can be obtained free of charge by application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44 (0) (1223) 336 033; e-mail: deposit@ccdc.cam.ac.uk]

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0095-z and is accessible for authorized users.  Mingming Cao (2012-2015 is appreciated. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.