Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus

Abstract Callisalignenes G–I (1–3), three new meroterpenoids of β-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 μM, respectively. Graphical Abstract Cytotoxic Acylphloroglucinol Derivatives from Callistemon salignus 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.

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][2][3][4][5][6][7][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.

Results and Discussion
Compound 1 was isolated as a colorless gum and its molecular formula was assigned as C 25 H 38 O 3 by an ion peak at m/z 409.2714 [M ? Na] ? (calcd for C 25 H 38 O 3 Na, 409.2713) in the HRESIMS and 13 C 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 1 H NMR spectrum displayed signals for two olefinic protons at d H 5.75 (dd, J = 9.9, 1.8 Hz, H-2 0 ) and 5.98 (dd, J = 9.9, 4.4 Hz, H-3 0 ), five tertiary methyls at d H 1.26 (s, Me-7 0 ), 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 d H 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-10 0 ), and 0.99 (d, J = 6.8 Hz, Me-9 0 ). The 13 C NMR and HSQC spectra of 1 exhibited 25 carbon signals corresponding to nine methyls, two methylenes, seven methines (including two olefinic carbons at d C 130.9 and 135.2), and seven quaternary carbons (including two carbonyls at d C 198.6 and 213.4, two olefinic carbons at d C 110.3 and 167.0, and one oxygenated carbon at d C 76.2). The above-mentioned NMR data were similar to those of callistiviminene M [7], except for the presence of an additional methylene. The 1 H-1 H 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 (d C 213.4) and an oxygenated olefinic carbon C-1 (d C 167.0) and from Me-14 and Me-15 to the carbonyl carbons C-3 (d C 213.4) and C-5 (d C 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 (d H 2.97) to C-6 (d C 110.3) and the substructure of H-7-H 2 -8-H-9-Me-10/Me-11 suggested by 1 H-1 H COSY spectrum (Fig. 2). Similarly, the HMBC correlations from Me-7 0 to C-1 0 (d C 76.2), C-2 0 (d C 130.9), and C-6 0 (d C 33.7), as well as the substructure indicated by 1 H-1 H 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-1 0 and C-7-C-6 0 bonds. The relative configuration of 1 was defined by a ROESY experiment. The ROESY correlations (Fig. 2) of Me-7 0 with H-6 0 , of H-8 0 with H-6 0 , and of H-7 with Me-7 0 suggested that these protons were cofacial. Finally, the absolute configuration of 1 was unambiguously determined as 7S,1 0 R,4 0 R,6 0 R from a positive Cotton effect at 265 (De ?36.23) in its experimental ECD spectrum in  (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 C 25 H 38 O 3 Na, 409.2713) and 13 C NMR data. The 1 H and 13 C 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 (d H 0.82) to C-8 (d C 39.2), and from Me-9 (d H 0.95) to C-7 (d C 36.4) and C-10 (d C 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 1 H and 13 C NMR data for 2 by HMBC and 1 H-1 H COSY spectra (Fig. 2), the gross structure of the other parts was the same as 1. However, the carbon signals for C-1 (d C 79.4), C-4 0 (d C 36.7), and C-6 (d C 39.8) of 2 were very different, implying that 2 was an isomer of 1. The ROESY correlations of Me-7 0 with H-6 0 and H-5 0 a indicated that these protons were in the same plane, while those of H-5 0 b with H-4 0 , and of H-4 0 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,1 0 R,4 0 R,6 0 R by comparing the calculated ECD spectrum with its experimental values (Fig. 3). Thus, the structure of callisalignene H was determined as 2.
Compound 3  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-9 0 (d H 0.99) and Me-10 0 (d H 1.31) to C-2 0 (d C 52.9), C-4 0 (d C 40.4) and C-8 0 (d C 32.6), from H-2 0 (d H 2.04), H-6 0 (d H 1.88) and H-7 0 (d H 1.89) to C-1 0 , and the fragment (H-2 0 - (Fig. 2). In the ROESY spectrum of 3, correlations of H-2 0 a with H-7 0 b and of H-7 0 a 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,1 0 S,2 0 S,4 0 R 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, CHCl 3 ), 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,7 0 S for (-)-4.

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

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

Quantum Chemical ECD Calculations
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].

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.

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]. VP-16 (etoposide) was used as a positive control.