RSC Advances PAPER View Article Online View Journal | View Issue Calycindaphines A–J, Daphniphyllum alkaloids from the roots of Daphniphyllum calycinum† Cite this: RSC Adv.,2021,11,9057 Ji Yang,a Xin Liu,ab Jing Fu,a Hao-Yuan Lyu,a Li-Ping Bai, a Zhi-Hong Jiang *a and Guo-Yuan Zhu *a Ten new Daphniphyllum alkaloids, calycindaphines A–J(1–10), together with seventeen known alkaloids were isolated from the roots of Daphniphyllum calycinum. Their structures were established by extensive spectroscopic methods and compared with data from literature. Compound 1 is a novel alkaloid with a new rearrangement C22 skeleton with the 5/8/7/5/5 ring system. Compound 2 represents the second example of calyciphylline G-type alkaloids. Compound 10 is the first example of secodaphniphylline-type alkaloid absent of the oxygen-bridge between C-25/C-29. The possible biogenetic pathways of 1 and 2 were also proposed. All the isolated compounds were evaluated for their bioactivities in three cell Received 6th January 2021 models. Compounds 22, 23, and 26 showed significant NF-kB transcriptional inhibitory activity at Accepted 22nd February 2021 a concentration of 50 mM. Compounds 16 and 18 exhibited significant TGF-b inhibitory activity in HepG2 Creative Commons Attribution 3.0 Unported Licence. DOI: 10.1039/d1ra00107h cells. Compounds 24 and 26 induced autophagic puncta and mediated the autophagic marker LC3-II rsc.li/rsc-advances conversion in HEK293 cells. Introduction isolated from the roots of D. calycinum. In this paper, we re- ported the isolation, structural elucidation, and bioactivities of The Daphniphyllum alkaloids were characteristically distributed these isolates. in plants of the Daphniphyllum family.1–4 To date, more than 200 Daphniphyllum alkaloids with nearly 20 skeletons have been This article is licensed under a isolated from ten species. Daphniphyllum alkaloids not only Results and discussion provided abundant structural types but also enlighten novel Compound 1, a white amorphous powder, has a molecular strategies for the total synthesis and biosynthesis of unusual Open Access Article. Published on 01 March 2021. Downloaded 9/24/2021 7:04:15 PM. – formula of C H O N as established by its HRESIMS data (m/z skeletons.5 10 Daphniphyllum calycinum, an evergreen tree native 23 31 3 370.2375 [M + H]+, calcd for 370.2377), with 9 degrees of unsa- to southern China, provided an abundant resource of Daphni- 11,12 turation. IR absorptions implied the presence of an ester phyllum alkaloids. The leaves, stems, and roots of D. calyci- À À carbonyl (1733 cm 1) and a lactam (1644 cm 1).14 The 1H NMR num are used in Traditional Chinese Medicine to treat several data (Table 1) of 1 exhibited proton resonances of a methoxy (d symptoms including fever, asthma, inammation, and inu- H – 3.61, s), a methyl singlet (d 1.10, s), a methyl doublet (d 1.15, enza.11 13 Previous phytochemical investigations on the leaves H H d, J ¼ 6.7 Hz), an olenic proton (d 5.42, d, J ¼ 5.2 Hz). Its 13C and stems of D. calycinum led to the isolation of more than 50 H NMR, DEPT, and HSQC spectra showed the occurrence of 23 Daphniphyllum alkaloids with 15 different skeletons. However, carbon resonances (Table 2), which consisted of three methyls the chemical from the roots of the D. calycinum has not been (a methoxy at d 50.9), nine methylenes (two N-methylenes at d reported. To seek potential bioactive alkaloids from D. calyci- C C 46.8 and 52.5), ve methines (an olenic methine at d 118.9), num, the roots of D. calycinum were rst deeply studied. As C six quaternary carbons (two carbonyls at d 182.9 and 175.2, and a result, ten new Daphniphyllum alkaloids, named calycinda- C three olenic quaternary carbons at d 140.6, 139.0, and 135.5). phines A–J(1–10), as well as 17 known alkaloids (11–27) were C The above-mentioned spectroscopic data suggested that 1 possesses two carbonyls and two double bonds which aState Key Laboratory of Quality Research in Chinese Medicine, Guangdong-Hong accounting for four out of nine indices of hydrogen de ciency, Kong-Macao Joint Laboratory of Respiratory Infectious Disease, Macau Institute for and the remaining ve degrees of unsaturation were speculated Applied Research in Medicine and Health, Macau University of Science and for the presence of a pentacyclic system in 1. The 1H–1H COSY Technology, Macau, People's Republic of China. E-mail: [email protected]; spectrum of 1 suggested three proton-bearing structural moie- [email protected] ties: C-4/C-3/C-2/C-18/C-19/20, C-7/C-1/C-12/C-11, and C-13/C- bBiology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China 14/C-15/C-16/C-17 (Fig. 2). These three fragments, quaternary † Electronic supplementary information (ESI) available. See DOI: carbons, and the nitrogen atom were then connected by the 10.1039/d1ra00107h detailed HMBC analysis (Fig. 2). The HMBC correlations from © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2021,11,9057–9066 | 9057 View Article Online RSC Advances 9058 1 Table 1 H NMR (600 MHz) spectroscopic data of compounds 1–10 in CDCl3 | No. 12345678910 RSC Adv. 1 3.80 (d, 4.2) 3.03 (s) 2.99 (s) 3.11 (s) 2a 2.24 (m) 2.15 (m) 2.65 (m) 3.59 (m) 1.40 (m) 1.69 (m) 1.03 (m) 0.96 (m) 1.09 (m) ,2021, 2b 0.84 (m) 1.32 (m) 3a 2.51 (m) 2.00 (m) 2.03 (m) 2.06 (m) 1.60 (m) 1.70 (m) 1.50 (m) 1.89 (m) 1.55 (m) 1.50 (m) 11 3b 1.94 (m) 1.66 (dd, 14.4, 4.4) 1.74 (m) 1.66 (m) 2.00 (m) 1.42 (m) 1.40 (m) 1.29 (m) ,9057 Creative Commons Attribution 3.0 Unported Licence. 4a 2.02 (m) 4.07 (dd, 10.5, 4.4) 3.22 (t, 3.1) 2.00 (m) 1.61 (m) 1.83 (m) 1.81 (m) 1.17 (m) 1.67 (m) 1.59 (m) 4b 1.63 (m) 1.42 (m) 1.76 (m) 1.35 (m) 1.38 (m) 1.60 (m) 1.15 (m) – 6 2.33 (m) 2.01 (m) 2.55 (m) 2.79 (m) 2.38 (m) 1.91 (t, 5.3) 1.99 (t, 5.3) 1.91 (t, 5.1) 9066 © 2021 The Author(s). Published by the Royal Society of Chemistry 7a 4.32 (t, 13.1) 2.88 (dd, 14.2, 8.9) 3.40 (dd, 14.3, 9.8) 4.98 (m) 3.36 (dd, 13.6, 10.1) 2.78 (m) 5.97 (s) 2.30 (d, 5.9) 2.60 (t, 4.6) 2.56 (d, 3.5) 7b 2.48 (m) 3.14 (m) 3.73 (dd, 13.6, 8.2) 9 1.72 (m) 1.75 (m) 1.03 (t, 3.4) 10 2.54 (m) 11a 5.32 (d, 5.2) 2.42 (m) 2.17 (m) 1.83 (m) 2.24 (m) 1.97 (m) 2.55 (dd, 12.9, 5.8) 1.65 (m) 1.68 (m) 1.67 (m) 11b 1.99 (m) 1.29 (m) 1.74 (m) 2.21 (m) 1.48 (m) 1.55 (m) 1.49 (m) 12a 2.67 (m) 2.01 (m) 1.90 (m) 2.07 (m) 1.60 (m) 2.31 (m) 2.38 (m) 1.59 (m) 1.79 (m) 1.59 (m) 12b 1.97 (m) 1.61 (m) 1.61 (m) 1.87 (m) 2.24 (m) 1.97 (m) 1.40 (m) 1.61 (m) 1.41 (m) This article is licensed under a 13a 3.45 (m) 2.79 (m) 2.53 (m) 1.84 (m) 2.31 (m) 2.65 (dd, 13.8, 7.9) 2.27 (m) 2.04 (m) 1.65 (m) 1.69 (m) 13b 2.89 (d, 15.5) 2.35 (m) 2.28 (dd, 13.5, 8.8) 1.68 (m) 2.61 (m) 2.30 (m) 2.07 (m) 1.96 (m) 1.54 (m) 14a 3.18 (t, 7.6) 2.93 (m) 3.18 (m) 2.74 (m) 3.27 (dt, 11.3, 8.1) 3.41 (dt, 12.2, 7.0) 3.61 (m) 2.92 (m) 2.86 (m) 1.30 (m) 14b 1.28 (m) 2.65 (m) 1.41 (m) Open Access Article. Published on 01 March 2021. Downloaded 9/24/2021 7:04:15 PM. 15a 3.63 (m) 2.93 (m) 3.62 (m) 5.48 (m) 3.38 (m) 2.78 (m) 2.75 (q, 8.0) 1.78 (m) 1.69 (m) 1.67 (m) 15b 1.72 (m) 1.61 (m) 1.78 (m) 16a 1.80 (m) 1.79 (m) 1.83 (m) 2.18 (m) 1.22 (m) 1.78 (m) 2.29 (m) 1.73 (m) 1.73 (m) 1.75 (m) 16b 1.00 (m) 1.17 (m) 1.17 (m) 1.88 (dt, 12.1, 6.8) 1.48 (m) 2.05 (m) 1.44 (m) 1.46 (m) 1.45 (m) 17a 2.68 (m) 2.53 (m) 2.49 (m) 2.09 (m) 2.21 (m) 2.35 (m) 5.54 (m) 1.66 (m) 1.69 (m) 1.47 (m) 17b 2.46 (m) 2.23 (m) 2.21 (m) 1.42 (m) 2.46 (m) 1.90 (m) 1.54 (m) 1.56 (m) 1.16 (m) 18 2.49 (m) 2.28 (m) 2.35 (t, 6.3) 2.24 (m) 2.15 (m) 2.15 (m) 1.49 (m) 1.47 (m) 1.51 (m) 19a 3.32 (d, 9.4) 3.22 (m) 4.42 (s) 2.29 (dd, 13.6, 8.6) 4.04 (dd, 13.1, 7.9) 4.37 (dd, 13.2, 5.6) 0.90 (d, 6.5) 0.91 (d, 6.5) 0.94 (d, 6.5) 19b 3.27 (d, 9.4) 2.02 (m) 4.46 (dd, 13.6, 7.7) 3.89 (dd, 13.1, 8.9) 2.31 (m) 20 1.15 (d, 6.7) 1.02 (d, 7.1) 1.13 (d, 6.7) 1.49 (s) 0.99 (d, 6.7) 0.93 (d, 6.9) 0.87 (d, 6.8) 0.89 (d, 6.5) 0.90 (d, 6.5) 0.89 (m) 21a 1.10 (s) 1.22 (s) 0.97 (s) 1.47 (s) 1.16 (s) 1.22 (s) 1.08 (s) 0.78 (s) 3.72 (d, 10.6) 0.76 (s) 21b 3.49 (d, 10.6) 22 3.94 (m) 1.68 (m) 23 3.61 (s) 3.61 (s) 3.62 (s) 3.65 (s) 3.70 (s) 3.68 (s) 24 3.25 (s) 0.59 (s) 0.78 (s) 0.89 (s) 25a 3.58 (dd, 11.8, 1.5) 4.25 (d, 12.1) 3.65 (m) 25b 3.51 (d, 11.8) 3.52 (d, 12.1) 3.49 (d, 10.3) 26 4.49 (d, 6.0) 4.66 (d, 6.9) 3.74 (dd, 11.2, 4.0) 27a 2.07 (m) 2.07 (m) 1.74 (m) 27b 1.96 (m) 1.03 (m) 1.50 (m) 28a 2.05 (m) 2.07 (m) 1.76 (m) 28b 1.83 (m) 1.87 (m) 1.47 (m) 30 1.47 (s) 1.43 (s) 1.20 (s) Paper View Article Online Paper RSC Advances 13 Table 2 C NMR (150 MHz) spectroscopic data of compounds 1–10 in CDCl3 No.