Zanthoisobutylamides A − C: rare dimeric C-6 substituent dihydrobenzophenanthridine alkaloids from the roots of Zanthoxylum nitidum

Abstract Three new dihydrobenzophenantridine alkaloids, zanthoisobutylamides A–C (2–4), consisting of a rare 6-alkylamide dihydrochelerythrine moiety, and two new small molecules of the unsaturated alkylamide, zanthoxylumamide J (1) and of phenylpropanoid, methyl 2-hydroxy-3,4-dimethoxycinnamate (5) together with 44 known compounds were isolated from the roots of Zanthoxylum nitidum. The structures of these compounds were established by analysis of spectroscopic data and comparison of their spectroscopic data with those previously published data. Some isolated compounds were evaluated for their cytotoxic activities. Graphical Abstract


Introduction
Zanthoxylum nitidum has been used to treat many symptoms of disease and was reported to be a rich source of bioactive compounds (Kan 1970;Hu et al. 2007;Yang et al. 2008). The secondary metabolites of Z. nitidum mainly involve benzophenanthridine alkaloids, unsaturated alkylamides, coumarins, lignans, phenylpropenoids and quinolines (Yang et al. 2008;Zhu et al. 2015;Wang et al. 2015). In 2019, we investigated the chemical constituents of a CH 2 Cl 2 extract of the stem of Z. nitidum and evaluated their antimicrobial activities (Chakthong et al. 2019). In continuing, the roots of Z. nitidum was investigated and evaluated for their cytotoxic activities. The results of these investigations are presented in this report. In particular, we present the first report of monomeric and dimeric C-6 substituent dihydrobenzophenanthridine alkaloids linked with unsaturated alkylamide units (2-4) together with an unsaturated N-alkylamide compound 1. Furthermore, one new phenylpropanoid (5) and 44 known compounds were isolated from this plant ( Figure 1 and Supplementary material1, Figure S1). Some of these constituents were evaluated for their cytotoxic activities.

Results and discussion
The extraction and purification of a CH 2 Cl 2 extract from the roots of Z. nitidum resulted in the isolation of one new alkylamide (1), three novel dihydrobenzophenanthridine alkaloids (2-4), and one new phenylpropanoid (5), together with 44 known compounds (6-49) ( Figure 1 and Supplementary material, Figure S1). The structures of these compounds were established by spectroscopic data analysis.
Compound 2 was isolated as an orange viscous solid, [a] 25 D ¼ þ20.6 (c 0.1, CHCl 3 ). Its UV spectrum showed absorption bands at 208, 229, 278 and 325 nm. The IR spectrum showed absorption bands of NH stretching at 3362 cm À1 and conjugated carbonyl stretching at 1672, 1638 and 1629 cm À1 . A molecular ion in the HRESIMS at m/z 625.2914 [M þ H] þ corresponded to a molecular formula of C 37 H 40 N 2 O 7 . The 13 C NMR spectrum of 2 displayed resonances for one methyl, two methoxy, one dioxymethylene, seven methines and ten quaternary carbons as well as the 1 H NMR spectrum showed resonances for two sets of ortho aromatic protons (J ¼ 8.7 Hz) at d H 7.51 (H-12) and 7.75 (H-11), and d H 6.98 (H-9) and 7.57 (H-10); two singlet aromatic protons at d H 7.07 and 7.32; two doublets for non-equivalent protons of a methylenedioxy substituent at d H 5.97 and 5.94 (J ¼ 0.9 Hz); a doublet of doublet for benzylic proton at d H 5.05 (dd, J ¼ 3.9, 11.4 Hz, H-6); two singlets of methoxyl groups at d H 3.98 (7-OMe) and 3.94 (8-OMe) and a singlet of a N-methyl at d H 2.59, presumed to be a 6-substituted dihydrochelerythrine moiety (Supplementary material, Table S2). The comparison of 1 H and 13 C NMR data of 2 with chelerythrine (42) clearly showed that they have the same benzophenanthridine alkaloid core skeleton (Supplementary material, Table S2). The presumption was supported by the fragmentation pattern from ESIMS which showed a base peak at m/z 348. Furthermore, the remaining resonances in 1 H NMR and the 13 C NMR spectral data included the resonances of unsaturated isobutylamide which were similar to compound 1 except for the absence of the methyl group protons present in 1 at d H 2.34 (H 3 -12), and the presence in 2 of non-equivalent methylene protons (H A -1 0 and H B -1 0 ) resonating at d H 2.87 (dd, J ¼ 11.4, 13.5 Hz) and 2.34 (dd, J ¼ 3.9, 13.5 Hz). The correlation between the dihydrochelerythrine moiety and the unsaturated alkylamide skeleton was confirmed by the correlations of H-6 (d H 5.05) to C-1 0 (d H 43.2) and of H 2 -1 0 (d H 2.87 and d H 2.34) to C-6 (d H 56.7) (Supplementary material, Figure S2). Thus, the structure of 2 was a 6-isobutylamide dihydrochelerythrine, and it was named zanthoisobutylamide A. The specific rotation of 2 f[a] 25 D þ20.6 (c 0.1, CHCl 3 )g was of the same sign as that of (þ)-6-acetonyldihydrochelerythrine f[a] 25 D þ137.5 (c 0.04, MeOH)g, which was isolated from Macleaya cordata by using chiral resolution (Sai et al. 2016). Therefore, the configuration of zanthoisobutylamide A (2) was assigned as 6S. Zanthoisobutylamide B (3) was isolated as an orange viscous solid, [a] 25 D À41.2 (c 0.008, CHCl 3 ). Its UV spectrum showed absorption bands at 229, 277 and 325 nm. The IR spectrum showed absorption bands of NH stretching at 3345 cm À1 and conjugated carbonyl stretching at 1717, 1673 and 1653 cm À1 . A molecular ion detected by HRESIMS at m/z 972.4070 [M þ H] þ corresponded to a molecular formula of C 58 H 57 N 3 O 11 . The fragmentation base peak in ESIMS at m/z 348 pointed to a dihydrochelerythrine moiety. The 1 H and 13 C NMR spectra of compound 3 were similar to the spectra of compound 2 except for the presence of the resonances for another dihydrochelerythrine unit (Supplementary material, Table S2). The second dihydrochelerythrine unit was confirmed by the 1 H NMR resonances at d H 6.92 (s, H-1 000 ), 7.24 (s, H-4 000 ), 7.30 (d, J ¼ 8.7 Hz, H-12 000 ), 7.70 (d, J ¼ 8.7 Hz, H-11 000 ), 6.99 (d, J ¼ 8.7 Hz, H-9 000 ), 7.58 (d, J ¼ 8.7 Hz, H-10 000 ), 5.93 (d, 3.97 (s, 7 000 -OMe), 3.94 (s, 8 000 -OMe), 2.52 (s, N-Me,) and 4.75 (d, 10.5, H-6 000 ). The HMBC correlations of the non-equivalent methylene protons at d H 2.23 and 2.77 (H 2 -1 0 ) to C-6 (d C 56.9) and of the benzylic proton at H-6 (d H 5.00) to C-2 0 (d C 199.3) and C-1 0 (d C 43.8), supported the connection between the first unit of the dihydrochelerythrine moiety and the unsaturated alkylamide skeleton via the methylene group at C-1 0 (Supplementary material, Figure S2). In addition, HMBC experiment showed the correlations of H-8 0 (d H 3.05) to C-6 000 (d C 61.7) and of H-6 000 (d H 4.75) to C-8 0 (d C 49.2), C-9 0 (d C 32.2) and C-7 0 (d C 203.5) confirmed the connection of the second unit of the dihydrochelerythrine moiety to C-8 0 of the unsaturated alkylamide.
Compound 4 was isolated as an orange viscous solid with the molecular formula C 58 H 57 N 3 O 11 determined by HRESIMS (m/z found 994.3891 [M þ Na] þ ) which was the same as that of 3. Detailed comparison of NMR data indicated that compound 4 had the same two units of dihydrochelerythrines and the unsaturated alkylamide which characterised to the isomeric compound of 3 (Supplementary material, Table S2). There are significant differences on the 1 H and 13 C NMR data (Supplementary material, Table S2). The 1 H NMR spectrum of 4 showed downfield shifts by D d þ0.09 from H-10 0 , þ0.10 from H-9 0 , þ0.28 from H-6 0 , þ0.18 from H-5 0 , and þ0.12 from non-equivalent methylene H A -1 0 and high field shifts by D d À0.08 from H-4 and À0.15 from H-4 000 (Supplementary material, Table S2). Therefore, compounds 3 and 4 are diastereomers, differing the relative configuration at C-8 0 .
The absolute configuration of dihydrochelerythrine derivatives at C-6 have been reported by comparing the calculated electronic circular dichroisms (ECD) via a quantum method with the experimental ECD data of the chiral resolution isolated compounds (Sai et al. 2016). Due to the complexity of the molecules and ECD curve patterns, therefore, the absolute configuration of compounds 3 and 4 were not determined.
As far as we know, C-6 substituent dihydrobenzophenanthridine alkaloids and dimericbenzophenanthridine alkaloids have been isolated and reported in genus Zanthoxylum and the genera Bocconia, Chelidonium, Corlidalis, Fagara, Macleaya, Sanguinaria and Toddalia (Krane et al. 1984;Yang et al. 2014;Qing et al. 2017). For example, 8-acetonyldihydronitidine and 8-acetonyldihydroavicine are related C-6 substituent dihydrobenzophenanthridine alkaloids reported in the genus Zanthoxylum which were isolated from the stem bark of Z. teraspermum and showed good significant growth inhibition of Staphylococcus aureus (Nissanka et al. 2001). Buesgeniine, a C-6 substituent dihydrobenzophenanthridine alkaloid linked with a benzylic derivative via a methylene bridge, was isolated from stem bark of of Z. rhoifolium (Tane et al. 2005). Zanthomuurolanine, epi-zanthomuurolanine, zanthocadinanine A, zanthocadinanine B, epi-zanthocadinanine A and epi-zanthocadinanine B are dihydrochelerythrine alkaloids and cadinane-type sesquiterpenes linked by a methylene bridge that were all isolated from stem bark of Z. nitidum (Yang et al. 2008).
Although benzophenanthridine alkaloids, C-6 substituent benzophenanthridine alkaloids and unsaturated alkylamides from Z. nitidum and other species in the genus have been reported Zhu et al. 2015;Chakthong et al. 2019), C-6 substituent dihydrobenzophenanthridine alkaloids connected with an unsaturated alkylamide unit are reported here for the first time. Compound 2 comprised one unit of dihydrochelerythrine connected with an unsaturated alkylamide via C-6 to C-1 0 whereas compounds 3 and 4 were unique benzophenanthridine dimers with linkage structures at C-6 to C-1 0 and C-6 000 to C-8 0 .
Compound 5 was obtained as a white powder. The UV spectrum showed maximum absorptions at 201, 241 and 306 nm. The IR spectrum showed the absorption bands of OH stretching at 3378 cm À1 and of conjugated ester carbonyl stretching at 1715 cm À1 . A molecular ion in the HRESIMS at m/z 261.0739 corresponded to the molecular formula C 12 H 14 O 5 . The 1 H NMR and 13 C NMR spectroscopic data of 5 revealed resonances very similar to those of methyl 2,3,4-trimethoxycinnamate (Peterson et al. 1988) (Supplementary material, Table S3). The difference was the presence in 5 of a singlet 2-OH group at d H 6.38 instead of 2-OMe present in methyl-2,3,4-trimethoxycinnamate. The hydroxyl group was in the ortho position of 3-OMe (d 3.91, s) and the meta position of H-6 (d H 7.18, d, 9.0). Consequently, 3-OMe and 2-OH showed cross peaks to C-3 (d C 135.5) as well as H-6, and 2-OH showed correlations to C-2 (d C 149.3). Therefore, 5 was assigned as methyl 2-hydroxy-3,4-dimethoxycinnamate.

General experimental procedures
Optical rotations were measured using a JASCO P-200 polarimeter. Ultraviolet spectra were measured in methanol solution on a UV-160A spectrophotometer (SHIMADZU). The IR spectra were recorded in neat on a Perkin À Elmer FTS FT À IR spectrophotometer. The 1 H and 13 C NMR spectra were recorded on a FT À NMR Bruker Ultra ShieldTM 300 and 500 MHz and a Unity Inova Varian 500 MHz spectrometer using tetramethylsilane (TMS) as the internal standard. Low-resolution ESI mass spectra were recorded on a Micromass spectrometer with Waters Alliance 2690/LCT. High-resolution ESI mass spectra were recorded on a Waters LCT Premier time-of-flight (TOF) mass spectrometer. Quick column chromatography (QCC) was carried out on silica gel 60 GF254 (Merck). Column chromatography (CC) was performed using silica gel 100 (70-230 Mesh ASTM, Merck). Thin À layer chromatography (TLC) and preparative TLC were performed on silica gel 60 F254 (Merck). Solvents for extraction and chromatography were distilled at their boiling ranges prior to use.

Plant material
The dried stems of Z. nitidum were collected from Amphoe Khuan Khanun, Phatthalung province in southern Thailand in August 2016. A voucher specimen S.

Cell proliferation assay
Cell proliferation was analysed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The cancer cell lines LoVo (human colon adenocarcinoma cell), A431 (human epidermoid carcinoma cells), SW1353 (human chondrosarcoma cell), PC-3 (human prostate cancer cell) and MDA-MB-231 (human breast cancer cell) were obtained from the American Type Culture Collection. Cells were seeded into 96well plates for 24 h and then treated with the bioactive compounds at different concentrations (0, 0.5, 1.5, 3 and 5 mg/mL) for 24 h. After incubation, 0.5 mg/mL of MTT solution was added to each well and incubated for 2 h at 37 C. The supernatant was removed and the formazan crystal was dissolved in 100 mL of DMSO. The absorbance was measured using an Epoch TM Microplate spectrophotometer at 570 nm and cell proliferation percentage (%) was calculated relative to the control.