Alkaloids from the stems of Fissistigma maclurei Merr. inhibit the proliferation of synoviocytes

Abstract Two new aporphine-derived alkaloids, aporaloids C and D (1 and 2), along with eight known biogenetically related alkaloids (3–10) were isolated from the stems of Fissistigma maclurei Merr. Their structures were elucidated by detailed analysis of NMR, HRESIMS, MS, IR, UV and Optical rotations data. Compounds 1 and 2 represent a rare example of N-methylol aporphine-derived alkaloids from natural sources. The inhibitory effect of all compounds on the proliferation of primary synovial cells was evaluated. Compound 3 showed potent inhibitory effect on the proliferation of synoviocytes with an IC50 value of 4.8 μM. Compounds 1, 2, 6–9 and 10 exhibited moderate inhibitory activity on synoviocytes, with IC50 values of 36.8, 37.1, 31.2 μM, 32.5, 36.3, 36.8 and 18.2 μM, respectively. Graphical Abstract


Introduction
The genus Fissistigma Griff.(Annonaceae) consists of 61 species, which are mainly distributed in Australia and Asia.Fissistigma maclurei Merr. is a climbing shrub, which is mainly distributed in Hainan, Guangxi, Yunnan province of China (Fu et al. 2021;Wu et al. 2022;Zhong et al. 2022).Its dried stem and roots are widely used in Li medicine in Hainan province of China (SFDA 1997).Decoctions of this Li medicine are used to treat the bruises sprains and rheumatism.Scientific evidence indicates that rheumatoid arthritis (RA) is a type of autoimmune disease and that it is related to synoviocytes induction (Hasler and Zouali 2001;Shevach 2004;Wicks et al. 1994).Alkaloids, sesquiterpenoids and flavonol glycosides were reported as the constituents of this plant (Van et al. 2020;Gao et al. 2021;Luo et al. 2022).In our previous investigations of active natural products from F. maclurei, three new sesquiterpenes were obtained from the stems of F. maclurei (Gao et al. 2021;Luo et al. 2022).The stems were obtained from a 3-year old F. maclurei and the diameters of these stems are about four centimeters.The mature stems of F. maclurei surrounded by bark with wrinkled shapes are grayish brown, and the young branches have dark brown fuzzes
Compound 2 was also obtained as white powder, with the molecular formula C 20 H 23 NO 5 from HRESIMS data (m/z 358.1643 for 2 [M + H] + ; calc'd 358.1649) combined with 1 H and 13 C NMR spectroscopic data.The first preliminary investigation of its 1 H and 13 C NMR showed that compound 2 was closely related to 5, except for the presence of an N-methylol group (δ H 4.61, 2H, s; δ C 89.9) in 2. This was corroborated by the HMBC correlations from H-12 to C-5/6a.Detailed analysis of 2D NMR (HSQC, 1 H-1 H COSY and HMBC) spectra confirmed that the other part of the molecule were the same as those of 5. Compound 2 also showed a positive optical rotation [α] 25 D +84.Thus, compound 2 has a 6aS configuration.Compound 2 represent a rare example of N-methylol aporphine-derived alkaloids from natural sources.
All compounds were evaluated for an inhibitory effect on the proliferation of synoviocytes.Cell growth inhibition was evaluated using an MTT assay and a lactate dehydrogenase (LDH) assay (Zhou et al. 2017).Based on the LDH assay, no cytotoxicity was observed in compounds 1-10 treated cells (cell viability > 95%).Based on the MTT assay, compound 3 exhibited potent inhibitory activity on synoviocytes, with an IC 50 value of 4.8 μM (methotrexate was used as a positive control, with an IC 50 value of 1.2 μM).Compounds 1, 2, 6-9 and 10 exhibited moderate inhibitory activity on synoviocytes, with IC 50 values of 36.8, 37.1, 31.2, 32.5, 36.3, 36.8 and 18.2 μM, respectively.The IC 50 values of compound 4 and 5 higher than 100 μM.These results indicated that the α,β-unsaturated ketone of porphine alkaloids was important for the inhibitory activity on synoviocytes.In addition, comparing structure and activity of compounds 1, 2, 4 and 5 indicated that the carbonyl and N-methylol group was important for inhibitory activity on synoviocytes.Thus, we suggest that aporphine-derived alkaloids may play an important role in the anti-RA of F. maclurei and may deliver benefits in the treatment of anti-RA diseases.Further work is here warranted.

General
IR spectra were recorded on a Nicolet 6700 spectrophotometer.1D and 2D NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for 1 H and 100 MHz for 13 C) with residual solvent peaks as references (δ H 3.310 and δ C 49.000 for MeOD-d 4 ).TMS was used as an internal standard.MeOD-d 4 was used as solvents.HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer.Optical rotations were measured on a JASCO P-1020 digital polarimeter.The mass spectra were acquired using a TripleTOF TM 5600+ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA) in negative and positive ESI mode.Semi-Preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector (detection wavelength: 210, 230, 254, and 269 nm) using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 µm) (flow phase: MeCN and H 2 O, veiocity of flow: 2.5 mL/min).Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh), octadecylsilyl silica gel (YMC; 12 nm-50 μm) were used for column chromatography (CC).Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography (TLC).

Plant material
The stems of F. maclurei was collected from Bawangling National Nature Reserve, Changjiang County, Hainan Province (Northern latitude 18°491538′, East longitude 108°906186), China in June 2018.The plant was collected under the guidance of the local forestry bureau and we identified it as F. maclurei by comparing with the description of this plant in the literature (Editorial Committee of flora of China, 1979).A voucher specimen (No.MGFM20180701) has been deposited at the Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University.

Biological assays
All compounds were evaluated for an inhibitory effect on the proliferation of synoviocytes.Cell growth inhibition was evaluated using an MTT assay.Primary synovial cells were cultured from the synovium tissue of SD rats (Zhou et al. 2017).The SD rats were offered by the Animal Care and Use Committee, Fujian University of Traditional Chinese Medicine (license no.SYXK (Min) 2009-0001).

Conclusions
In this study, ten alkaloids including two new aporphine-derived alkaloids, aporaloids C and D were isolated from the stems of F. maclurei for the first time.These compounds can help to extend the phytochemical knowledge of the genus Fissistigma.
Furthermore, the inhibitory effects on the proliferation of synoviocytes of all compounds were evaluated.Aporphine-derived alkaloids were firstly found to have anti-RA activity.It can provide scientific basis for the development of this Li medicine.