Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus

Natural Products and Bioprospecting, Jun 2017

Xu-Jie Qin, Tong Shu, Qian Yu, Huan Yan, Wei Ni, Lin-Kun An, Pan-Pan Li, Yin-E Zhi, Afsar Khan, Hai-Yang Liu

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://link.springer.com/content/pdf/10.1007%2Fs13659-017-0138-6.pdf

Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus

Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus Xu-Jie Qin . Tong Shu . Qian Yu . Huan Yan . Wei Ni . Lin-Kun An . Pan-Pan Li . Yin-E Zhi . Afsar Khan . Hai-Yang Liu 0 1 2 3 4 0 X.-J. Qin T. Shu H. Yan W. Ni P.-P. Li Y.-E. Zhi H.-Y. Liu Yunnan Key Laboratory of Medicinal Chemistry , Kunming 650201 , People's Republic of China 1 X.-J. Qin T. Shu H. Yan W. Ni P.-P. Li Y.-E. Zhi H.-Y. Liu (&) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming 650201 , People's Republic of China 2 A. Khan Department of Chemistry, COMSATS Institute of Information Technology , Abbottabad 22060 , Pakistan 3 Q. Yu L.-K. An Institute of Medicinal Chemistry and Chemical Biology, School of Pharmaceutical Sciences, Sun Yat-sen University , Guangzhou 510006 , People's Republic of China 4 T. Shu University of Chinese Academy of Sciences , Beijing 100049 , People's Republic of China Callisalignenes G-I (1-3), three new meroterpenoids of b-triketone and monoterpene, along with two known analogues (4 and 5), were isolated from Callistemon salignus. Their structures and absolute configurations were unambiguously established by a combination of NMR and MS analysis and electronic circular dichroism (ECD) evidence. Callisalignenes H (2) and I (3) have a rare sec-butyl moiety at C-7. Meroterpenoids 1-3 exhibited cytotoxicity against HCT116 cells with IC50 values of 8.51 ± 1.8, 9.12 ± 0.3, and 16.33 ± 3.3 lM, respectively. Graphical Abstract Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus Callistemon salignus - Xu-Jie Qin, Tong Shu, Qian Yu have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13659-017-0138-6) contains supplementary material, which is available to authorized users. 1 Introduction Plants of genus Callistemon (Myrtaceae) are evergreen shrubs or small trees native to Australia and have been popularly cultivated in southern China as ornamental species with bottle brush inflorescence. Recently, acylphloroglucinols and their derivatives, including adducts of a phloroglucinol moiety coupled with its derivative or a terpenoid unit, have been extensively obtained from this genus [1–8]. Many of these compounds exhibited insecticidal [1], antibacterial [3], epoxide hydrolase inhibitory [4], and cytotoxic [5] effects. We have previously reported nine new acylphloroglucinol derivatives, callisalignones A–C and callisalignenes A–F, and 18 known analogues with antimicrobial and cytotoxic activities from the leaves and twigs of Callistemon salignus [9]. An extensive phytochemical investigation on petroleum ether extract of C. salignus resulted in the isolation of three acylphloroglucinol derivatives, named callisalignenes G–I (1–3), along with two known analogues (4 and 5) (Fig. 1). All the isolates were evaluated for their antimicrobial and cytotoxic activities. Herein, details of the isolation, structure elucidation, and bioactivity of these isolates are described. 2 Results and Discussion Compound 1 was isolated as a colorless gum and its molecular formula was assigned as C25H38O3 by an ion peak at m/z 409.2714 [M ? Na]? (calcd for C25H38O3Na, 409.2713) in the HRESIMS and 13C NMR data. The UV absorption maximum at 265 nm combined with the IR absorption bands at 1721 and 1651 cm-1 revealed the presence of a,b-unsaturated keto group. The 1H NMR spectrum displayed signals for two olefinic protons at dH 5.75 (dd, J = 9.9, 1.8 Hz, H-20) and 5.98 (dd, J = 9.9, 4.4 Hz, H-30), five tertiary methyls at dH 1.26 (s, Me-70), 1.28 (s, Me-12), 1.29 (s, Me-15), 1.31 (s, Me-14), and 1.35 (s, Me-13), and four secondary methyls at dH 0.94 (d, J = 6.9 Hz, Me-11), 0.95 (d, J = 6.9 Hz, Me-10), 0.98 (d, J = 6.8 Hz, Me-100), and 0.99 (d, J = 6.8 Hz, Me-90). The 13C NMR and HSQC spectra of 1 exhibited 25 carbon signals corresponding to nine methyls, two methylenes, seven methines (including two olefinic carbons at dC 130.9 Fig. 1 Structures of 1–5 obtained from C. salignus and 135.2), and seven quaternary carbons (including two carbonyls at dC 198.6 and 213.4, two olefinic carbons at dC 110.3 and 167.0, and one oxygenated carbon at dC 76.2). The above-mentioned NMR data were similar to those of callistiviminene M [7], except for the presence of an additional methylene. The 1H–1H COSY spectrum disclosed the presence of a single spin system. The HMBC correlations from Me-12 and Me-13 to the carbonyl carbon C-3 (dC 213.4) and an oxygenated olefinic carbon C-1 (dC 167.0) and from Me-14 and Me-15 to the carbonyl carbons C-3 (dC 213.4) and C-5 (dC 198.6) revealed the presence of a b-triketone moiety. The isopentyl group was attached to the b-triketone, based on the HMBC correlations from H-7 (dH 2.97) to C-6 (dC 110.3) and the substructure of H-7– H2-8–H-9–Me-10/Me-11 suggested by 1H–1H COSY spectrum (Fig. 2). Similarly, the HMBC correlations from Me-70 to C-10 (dC 76.2), C-20 (dC 130.9), and C-60 (dC 33.7), as well as the substructure indicated by 1H–1H COSY spectrum and the molecular formula information, verified that the monoterpenoid moiety (a-phellandrene) and the b-triketone unit were connected via C-1–O–C-10 and C-7–C-60 bonds. The relative configuration of 1 was defined by a ROESY experiment. The ROESY correlations (Fig. 2) of Me-70 with H-60, of H-80 with H-60, and of H-7 with Me-70 suggested that these protons were cofacial. Finally, the absolute configuration of 1 was unambiguously determined as 7S,10R,40R,60R from a positive Cotton effect at 265 (De ?36.23) in its experimental ECD spectrum in Fig. 2 Key 1H–1H COSY, HMBC, and ROESY correlations of 1–3 comparison with the theoretical spectrum (Fig. 3). Therefore, structure of callisalignene G was established as 1. Compound 2 was assigned the same molecular formula as that of 1 by HRESIMS (m/z 409.2708 [M ? Na]?, calcd for C25H38O3Na, 409.2713) and 13C NMR data. The 1H and 13C NMR spectra of 2 were similar to those of 1 except for the signals of a side chain at C-7. The observed HMBC correlations from Me-11 (dH 0.82) to C-8 (dC 39.2), and from Me-9 (dH 0.95) to C-7 (dC 36.4) and C-10 (dC 24.7) supported that a sec-butyl group was attached to C-7 in 2 rather than that of isobutyl in 1. After the full assignment of 1H and 13C NMR data for 2 by HMBC and 1H–1H COSY spectra (Fig. 2), the gross structure of the other parts was the same as 1. However, the carbon signals for C-1 (dC 79.4), C-40 (dC 36.7), and C-6 (dC 39.8) of 2 were very different, implying that 2 was an isomer of 1. The ROESY correlations of Me-70 with H-60 and H-50a indicated that these protons were in the same plane, while those of H-50b with H-40, and of H-40 with H-7 suggested that they were co-facial. The assignment of 8S configuration could be explained in view of its proposed biosynthetic pathway [9]. Furthermore, the absolute configuration of 2 was determined as 7R,8S,10R,40R,60R by comparing the calculated ECD spectrum with its experimental values (Fig. 3). Thus, the structure of callisalignene H was determined as 2. Compound 3 had the same molecular formula C25H38O3 as those of 1 and 2 by HRESIMS (387.2899 [M ? H]?, calcd for C25H39O3, 387.2894) and 13C NMR data. Comparison of the NMR data of 3 (Table 1) with those of 2 suggested that they both shared the same b-triketone moiety with an isobutyl group at C-7. The remaining ten carbon signals for the monoterpene unit corresponded to two tertiary methyls, four methylenes, two methines, and two quaternary carbons (including an oxygenated one) revealed the presence of b-pinene unit. This conclusion was further confirmed by HMBC correlation from Me-90 (dH 0.99) and Me-100 (dH 1.31) to C-20 (dC 52.9), C-40 (dC 40.4) and C-80 (dC 32.6), from H-20 (dH 2.04), H-60 (dH 1.88) and H-70 (dH 1.89) to C-10, and the fragment (H-20– H2-30–H-40–H2-50–H2-60) determined by 1H–1H COSY spectrum (Fig. 2). In the ROESY spectrum of 3, correlations of H-20a with H-70b and of H-70a with H-7 indicated that H-7 was b-oriented. From biosynthetic considerations, the absolute configuration of C-8 in 3 was also assigned as S. The absolute configuration of 3 was assigned as 7R,8S,10S,20S,40R by comparison of its experimental and calculated ECD spectra (Fig. 3). Accordingly, the structure of callisalignene I was established as 3. Besides the three new meroterpenoids, two known compounds were identified as (–)-callistenone F (4) [10] and viminalin B (5) [5] by the comparison of their experimental data and reported values. It is to be noted that callistenone K has been reported as a racemic mixture with a specific rotation value of ?3.6 (c 1.0, CHCl3), but in the current study 4 was isolated as a single diastereomer with a negative specific rotation value [-133.0 (c 0.1, MeOH)]. Further analysis of its experimental and calculated ECD spectra (Fig. 3) validated the absolute configurations of 8S,70S for (–)-4. All the isolates were evaluated for their antimicrobial effects toward three bacterial (S. aureus, E. coli, and P. aeruginosa) and three fungal strains (A. fumigatus, C. parapsilosis, and C. albicans). None of them showed antimicrobial effects (MIC [250 lg/mL). Additionally, cytotoxicities of 1–5 against six human cancer cells (HCT116, Huh7, Hela, CCRF-CEM, DU145, and A549) were also conducted and the results were summarized in Table 2. 1–3 exhibited cytotoxicity against HCT116 cells with IC50 values of 8.51 ± 1.8, 9.12 ± 0.3, and 16.33 ± 3.3 lM, respectively, compared to that of positive control (VP-16, 20.26 ± 0.5 lM). Moreover, 1 and 3 displayed cytotoxicity against A549 cells with IC50 values of 12.85 ± 8.2 and 10.03 ± 3.2 lM (VP-16, 25.79 ± 6.2 lM), respectively. 3 Experimental 3.1 General Experimental Procedures Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV2401 PC spectrophotometer. IR spectra were Fig. 3 Calculated and experimental ECD spectra of 1–4 determined on Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr discs. ECD spectra were recorded on an Applied Photophysics spectropolarimeter. 1D and 2D NMR spectra were recorded on Bruker AV 600 or 800 MHz spectrometers using TMS as an internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals, and coupling constant (J) values were reported in Hz. HRESIMS data were measured using an Agilent 1290 UPLC/6540 Q-TOF mass spectrometer. Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), Si gel (200–300 mesh, Qingdao Marine Chemical Co., Qingdao, People’s Republic of China), and RP-18 (50 lm, Merck, Germany) were used for column chromatography (CC). Semi-preparative HPLC was performed on an Agilent 1260 instrument with a ZORBAX SB-C18 column (9.4 9 250 mm, 5 lm). Fractions were monitored by Si gel GF254 (Qingdao Marine Chemical Co., China) or RP-18 F254 (Merck, Darmstadt, Germany) plates. Spots were visualized under UV light and by spraying with 10% H2SO4 in EtOH followed by heating. 3.2 Plant Material Twigs and leaves of C. salignus were collected from Kunming City, Yunnan Province, P. R. China, in February 2016 and identified by Dr. Rong Li (Kunming Institute of Botany, Chinese Academy of Sciences). A voucher specimen (HY0025) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 3.3 Extraction and Isolation Air-dried and powdered twigs and leaves of C. salignus (10.0 kg) were percolated with petroleum ether (PE) at room temperature three times (3 9 24 h; 50 L) and then filtered. After removal of solvent under reduced pressure, the crude extract (130 g) was subjected to silica gel CC, eluted with PE-EtOAc (100:1 ? 1:1, v/v) to yield five fractions A–E. Fraction A (25 g) was applied to Sephadex Table 1 1H (800 MHz) and 13C (200 MHz) NMR data for 1–3 in CDCl3 dH (mult., J in Hz) 5.75, dd (9.9, 1.8) 5.98 dd (9.9, 4.4) dH (mult., J in Hz) 2.68, dd (5.2, 3.4) 1.60, overlapped 5.26, dd (10.2, 2.3) 5.69, dd (10.2, 2.3) dH (mult., J in Hz) LH-20 column (CHCl3–MeOH 1.5:1, v/v) to give A3 (3 g), which was further separated on an RP-18 column and eluted with a gradient of MeCN–H2O (80:20 ? 95:5, v/v) to obtain five subfractions (A3-1–A3-5). Subfraction A3-4 (225 mg) was subsequently purified by semi-preparative HPLC with MeCN–H2O (90:10 ? 100:0 v/v, 5 mL/min) as mobile phase to afford 1 (5 mg), 2 (4 mg), 3 (8 mg), and 5 (35 mg). Similarly, fraction C (42 g) was separated by Sephadex LH-20 column (CHCl3–MeOH 1.5:1, v/v) to give three subfraction (C1–C3). After repeated purification by RP-18 column with MeCN–H2O (60:40 ? 75:25, v/v), 4 (18 mg) was obtained from C2 (0.5 g). 3.3.1 Callisalignene G (1) Colorless gum; [a] ?188.3 (c 0.1, MeOH); UV (MeOH) kmax (log e) 265 (4.18) nm; CD (MeOH) 265 (D e ? 36.23), 306 (De -7.57) nm; IR (KBr) vmax 3440, 2932, 1721, 1651, 1469, 1248 cm–1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 409.2714 [M ? Na]? (calcd for C25H38O3Na, 409.2713). 3.3.2 Callisalignene H (2) Colorless gum; [a] -221.4 (c 0.1, MeOH); UV (MeOH) kmax (log e) 266 (4.20) nm; CD (MeOH) 267 (De -31.35), Table 2 Cytotoxicities with IC50 values (lM) of meroterpenoids 1–5 1 2 3 4 5 VP-16 309 (De ? 4.11) nm; IR (KBr) vmax 3439, 2930, 1720, 1649, 1467, 1247 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 409.2708 [M ? Na]? (calcd for C25H38O3Na, 409.2713). 3.3.3 Callisalignene I (3) Colorless gum; [a] ?164.7 (c, MeOH); UV (MeOH) kmax (log e) 267 (4.24) nm; CD (MeOH) 267 (De ?27.50), 306 (De -6.02) nm; IR (KBr) vmax 3428, 2962, 1718, 1651, 1467, 1384, 1174-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 387.2899 [M ? H]? (calcd for C25H39O3, 387.2894). 3.3.4 (–)-Callistenone K (4) Colorless gum; [a] -133.0 (c 0.1, MeOH); CD (MeOH) 235 (De ?5.45), 248 (De ?1.77), 286 (De ?15.10), 317 (De -13.95) nm. The conformations generated by the MM2 force field in Chem-Bio3D software overlaid with key correlations observed in the ROESY spectrum were subjected to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program [11]. The corresponding minimum geometries were further optimized by Density Functional Theory (DFT) calculations at the B3LYP/6-31?G(d) level Table 2. The theoretical calculations of ECD were performed using Time Dependent DFT at B3LYP/6-311??G(2d,p) level in MeOH. The calculated ECD curves were generated by SpecDis (version 1.63) software [12]. 3.5 Antibacterial Assay The antimicrobial activities compounds 1–5 were carried out against three bacterial strains (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) and three fungal strains (Aspergillus fumigatus, Candida parapsilosis, and C. albicans) using the antimicrobial susceptibility assay [13]. The compounds were dissolved in DMSO, serially diluted to the concentration ranges of 250–0.061 lg/mL, and tested in a 96-well plate. Gentamycin (for bacteria) and voriconazole (for fungi) were used as positive controls. The experiments were conducted for three independent replicates. The MIC was determined as the lowest concentration that inhibited the visible growth of bacteria and fungi. 3.6 Cytotoxicity Assay All the compounds were evaluated for their cytotoxicities against six tumor cell lines, including HCT116 (human colorectal cancer cell line), Huh7 (human hepatoma cell line), Hela (human cervical cancer cell line), CCRF-CEM (human acute lymphocytic leukemia cell line), DU145 (human prostatic cancer cell line), and A549 (human lung cancer cell line), by MTT assay in 96-well plates [14]. VP16 (etoposide) was used as a positive control. Acknowledgements This work was financially supported by National Natural Science Foundation of China (Nos. 31600283 and 31570363) and fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (No. P2017-ZZ04) from Kunming Institute of Botany, Chinese Academy of Sciences. Compliance with Ethical Standards Conflict of interest The authors declare no conflict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 1. B.P.S. Khambay , D.G. Beddie , A.M. Hooper , M.S.J. Simmonds , P.W.C. Green , J. Nat . Prod. 62 , 1666 - 1667 ( 1999 ) 2. J.Q. Cao , X.J. Huang , Y.T. Li , Y. Wang , L. Wang , R.W. Jiang , W.C. Ye , Org. Lett. 18 , 120 - 123 ( 2016 ) 3. S. Rattanaburi , W. Mahabusarakam , S. Phongpaichit , A.R. Carroll , Tetrahedron 69 , 6070 - 6075 ( 2013 ) 4. P.N. Khanh , H.V. Duc , T.T. Huong , N.T. Son , V.T. Ha , D.T. Van , B.H. Tai , J.E. Kim , A.R. Jo , Y.H. Kim , N.M. Cuong , Fitoterapia 109 , 39 - 44 ( 2016 ) 5. L. Wu , Y.L. Zhang , X.B. Wang , Y.M. Zhang , M.H. Yang , J. Luo , L.Y. Kong , Tetrahedron 73 , 1105 - 1113 ( 2017 ) 6. L. Wu , J. Luo , Y.L. Zhang , M.D. Zhu , X.B. Wang , J.G. Luo , M.H. Yang , B.Y. Yu , H.Q. Yao , Y. Dai , Q.L. Guo , Y.J. Chen , H.B. Sun , L.Y. Kong , Tetrahedron Lett . 56 , 229 - 232 ( 2015 ) 7. L. Wu , X.B. Wang , R.J. Li , Y.L. Zhang , M.H. Yang , J. Luo , L.Y. Kong , Phytochemistry 131 , 140 - 149 ( 2016 ) 8. L. Wu , J. Luo , X.B. Wang , R.J. Li , Y.L. Zhang , L.Y. Kong , RSC Advances 5 , 93900 - 93906 ( 2015 ) 9. X.J. Qin , H. Liu , Q. Yu , H. Yan , J.F. Tang , L.K. An , A. Khan , Q.R. Chen , X.J. Hao , H.Y. Liu , Tetrahedron 73 , 1803 - 1811 ( 2017 ) 10. H.X. Liu , Y.C. Chen , Y. Liu , W.M. Zhang , J.W. Wu , H.B. Tan , S.X. Qiu , Fitoterapia 114 , 40 - 44 ( 2016 ) 11. M.J. Frisch et al. , in Gaussian 09 Revision C.01, ed. by Gaussian, Inc. ( Wallingford CT , USA, 2010 ) 12. T. Bruhn , A. Schaumlo¨ffel , Y. Hemberger , G. Bringmann , Chirality 25 , 243 - 249 ( 2013 ) 13. X.J. Qin , H. Yan , W. Ni , M.Y. Yu , A. Khan , H. Liu , H.X. Zhang , L. He , X.J. Hao , Y.T. Di , H.Y. Liu , Sci. Rep. 6 , 32748 ( 2016 ) 14. T. Mosmann , J. Immunol . Methods 65 , 55 - 63 ( 1983 )


This is a preview of a remote PDF: https://link.springer.com/content/pdf/10.1007%2Fs13659-017-0138-6.pdf

Xu-Jie Qin, Tong Shu, Qian Yu, Huan Yan, Wei Ni, Lin-Kun An, Pan-Pan Li, Yin-E Zhi, Afsar Khan, Hai-Yang Liu. Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus, Natural Products and Bioprospecting, 2017, 315-321, DOI: 10.1007/s13659-017-0138-6