Indole Alkaloids and Chromones from the Stem Bark of Cassia alata and Their Antiviral Activities

The Cassia (Leguminosae) genus has attracted a lot of attention as a prolific source of alkaloids and chromones with diverse structures and biological properties. The aim of this study is to screen the antiviral compounds from Cassia alata. The extract of the stem bark of this plant was separated using silica gel, MCI, ODS C18, and Sephadex LH-20 column chromatography, as well as semi-preparative HPLC. As a result, three new indole alkaloids, alataindoleins A–C (1–3); one new chromone, alatachromone A (4); and a new dimeric chromone-indole alkaloid, alataindolein D (5) were isolated. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Interestingly, alataindolein D (5) represents a new type of dimeric alkaloid with an unusual N-2−C-16’ linkage, which is biogenetically derived from a chromone and an indole alkaloid via an intermolecular nucleophilic substitution reaction. Compounds 1–5 were tested for their anti-tobacco mosaic virus (TMV) and anti-rotavirus activities, and the results showed that compounds 2–4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates were higher than those of the positive control (with inhibition rate of 32.8%). Compounds 1 and 5 also showed potential anti-TMV activities with inhibition rates of 26.5% and 31.8%, respectively. In addition, compounds 1–5 exhibited potential anti-rotavirus activities with therapeutic index (TI) values in the range of 9.75~15.3. The successful isolation and structure identification of the above new compounds provided materials for the screening of antivirus drugs, and contributed to the development and utilization of C. alata.


Introduction
Cassia Linn, a cosmopolitan genus of the legume family (Fabaceae) includes 600 species, some of which (C. siamea, C. occidentalis, C. obtusifolia, and C. alata, etc.) are used in the folk medicine of the Chinese Dai tribe, notably for the treatment of rheumatoid arthritis, skin diseases, diabetes, malaria, skin trauma and constipation [1][2][3]. Among them, C. alata locally known as "Ya-La-Meng-Long", is commonly used as an effective treatment for hypertension, some gastrointestinal diseases, and skin diseases in clinics and drug stores of the subtropical region of Yunnan Province, China [3][4][5].
In our ongoing research on biologically active metabolites from the genus Cassia, we are reinvestigating the chemical constituents of the stem bark of C. alata collected in Yingjiang County, Dehong Prefecture, which led to the isolation and identification of three new indole alkaloids, alataindoleins A-C (1-3); one new chromone, alatachromone A (4); and a new dimeric chromone-indole alkaloid, alataindolein D (5). Among them, alataindolein D, a novel-type dimeric alkaloid, was biosynthesized from an indole alkaloid and a chromone with an unusual coupling pattern (N-2−C-16' linkage). In addition, compounds 2-4 showed high anti-TMV activities, and compounds 1-5 also exhibited potential anti-rotavirus activities. Herein, we report the isolation and structural elucidation of the above new compounds, as well as their antivirus properties.
1568, 1459 cm −1 , indicating the presence of carbonyl and aromatic rings. The 1 H, 13 C, and HSQC NMR data ( Table 1) of 1 show resonances due to a 1,2,3,5-tetrasubstituted benzene ring (C-4~C-9, H-5, and H-7), an acyl carbon (C-1), a methylene carbon (C-3 and H2-3), one methoxy group (C 56.2 q, H 3.78 s), and two methyl groups (C-10, C-11, H3-10, and H3-11). Based on its molecular formula, the acyl and methylene carbons should be connected by a nitrogen atom to form an isoindolin-1-one nucleus [17,18] to support the 6 degrees of unsaturation. This deduction was also supported by the HMBC correlations ( Figure 2) from H2-3 to C-1, C-4, C-8, and C-9, and from H-7 to C-1, C-8, and C-9. Since the nucleus of compound was determined, the additional carbons (two methyl and one methoxy group) accounted for the remaining substituents. The location of the methoxy group was assigned to the C-4 position based on the HMBC correlations from methoxy protons (δH 3.78) to C-4. Two methyl groups located at C-6 and N-2 were supported by the HMBC correlations from H3-11 to C-5, C-6, and C-7; from H-5 and H-7 to C-11; from H3-10 to C-1, C-3; and from H2-3 to C-10, respectively. Thus, the structure of 1 was established as 4methoxy-2,6-dimethyl-isoindolin-1-one, and it was given a trivial name of alataindolein A. Compound 2 was also obtained as yellow gum. A molecular formula C11H13NO3 was assigned from HRESIMS (m/z: 230.0790 [M+Na] + , calcd 230.0793). The 1 H and 13 C NMR data of 2 (Table 1) were highly similar to those of 1. The obvious differences resulted from Compound 2 was also obtained as yellow gum. A molecular formula C 11 H 13 NO 3 was assigned from HRESIMS (m/z: 230.0790 [M+Na] + , calcd 230.0793). The 1 H and 13 C NMR data of 2 (Table 1) were highly similar to those of 1. The obvious differences resulted from the replacement of the methyl group in 1 by the hydroxylmethyl group in 2. The location of the hydroxylmethyl group at C-6 was also supported by the HMBC correlations of the hydroxylmethyl proton (H 2 -11) with C-5, C-6, and C-7, of H-5, and H-7 with C-11. Accordingly, the structure of 6-(hydroxymethyl)-4-methoxy-2-methylisoindolin-1-one (2) was established, and its trivial name became alataindolein B.
Regarding 6-(3-Hydroxypropyl)-4-methoxy-2-methylisoindolin-1-one (3), a yellow gum, its 1 H and 13 C NMR spectra data were also similar to those of 1. Chemical shift differences resulted from the disappearance of a methyl group and the appearance of 3-hydroxypropyl group signals (-CH 2 CH 2 CH 2 -OH, C-11~C-13, H 2 -11~H 2 -13) [17,19] in compound 3. This indicated that the methyl group at C-6 in 1 was converted into a 3-hydroxypropyl group in compound 3. The HMBC correlations of H 2 -11 with C-5, C-6, and C-7, of H 2 -12 with C-6 also indicated this structure change. Thus, the structure of 3 was established as shown, and it was given a trivial name of alataindolein C.
Compound 5 was obtained as yellow gum. Its molecular formula C 25 H 27 NO 5 (Table 3) were assignable to four methyls, four methylenes, four aromatic methines, a methoxy (δ C 56.2 q), and twelve quaternary carbons (including three carbonyls and eight aromatic carbons). Among them, three carbonyls and twelve olefinic carbons account for 8 degrees of unsaturation, suggesting that compound 5 is a highly aromatized C-25 nitrogen-containing structure with a tetracyclic ring system. The 1 H and 13 C NMR spectra data of compound 5 were highly similar to those of 1 in C-1~C-10 (part a), and highly similar to those of compound 4 in C-2'~C-15' (part b). The obvious chemical shift differences resulted from the disappearance of a N-methyl group resonance (C-10, H 3 -10) in 1 and hydroxymethyl group (C-16, H 2 -16) group in compound 5, and appearance of a N-methylene (C-16', H 2 -16'). The above information indicated that compound 5 should be a heterodimer comprising a benzoisoindolin-1-one moiety (part a) and a gem-dimethyl chromone moiety (part b), and two moieties were connected by a N-atom and C-16'. The connection from C-16' to the N-atom was supported by the HMBC correlations (Figure 2), which formed H 2 -16' to C-3, C-1, C-6', C-7', and C-8'; from H-6' to C-16'; and from H-3 to C-16'. The existence of gem-dimethyl chromone, benzoisoindolin-1-one and the substituent positions, can also be confirmed by a further analysis of its HMBC correlations. Accordingly, the structural assignment of 5 is depicted in Figure 1 and given a trivial name of alataindolein D. To the best of our knowledge, this compound has a new carbon skeleton with a N-2−C-16' linkage, which formed between an indole alkaloid and a chromone via an intermolecular nucleophilic substitution reaction ( Figure 3).  (Figure 2), which formed H2-16' to C-3, C-1, C-6', C-7', and C-8'; from H-6' to C-16'; and from H-3 to C-16'. The existence of gem-dimethyl chromone, benzoisoindolin-1-one and the substituent positions, can also be confirmed by a further analysis of its HMBC correlations. Accordingly, the structural assignment of 5 is depicted in Figure 1 and given a trivial name of alataindolein D. To the best of our knowledge, this compound has a new carbon skeleton with a N-2−C-16' linkage, which formed between an indole alkaloid and a chromone via an intermolecular nucleophilic substitution reaction ( Figure 3).

Anti-TMV Activities
Since certain alkaloids and chromones from the Cassia genus exhibit potential anti-TMV activities [7,10,12,[15][16][17][18], the anti-TMV activities for compounds 1-5 were tested using the half-leaf method [21,22] at a concentration of 20 μM. Ningnanmycin (a commercial product for plant disease in China, with inhibition rate of 32.8%) was used as a positive control. The results (Table 4 and Figure S17) reveal that compounds 2-4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates are higher than those of the positive control (with inhibition rate of 32.8%). Compounds 1 and 5 also showed potential anti-TMV activities with inhibition rates of 26.5%

Anti-TMV Activities
Since certain alkaloids and chromones from the Cassia genus exhibit potential anti-TMV activities [7,10,12,[15][16][17][18], the anti-TMV activities for compounds 1-5 were tested using the half-leaf method [21,22] at a concentration of 20 µM. Ningnanmycin (a commercial product for plant disease in China, with inhibition rate of 32.8%) was used as a positive control. The results (Table 4 and Figure S17) reveal that compounds 2-4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates are higher than those of the positive control (with inhibition rate of 32.8%). Compounds 1 and 5 also showed potential anti-TMV activities with inhibition rates of 26.5% and 31.8%, respectively. In addition, the IC 50 values of five new compounds were also obtained, and the results (Table 4) reveal that compounds 1-5 showed IC 50 in the range of 14.2~55.3 µM. Since compounds 2-4 exhibit potential anti-TMV activities, the protective effects of compounds 2-4 on TMV were also evaluated by pretreating the tobacco plant with 20 µM solutions of compounds or a solution of DMSO for 6 h before inoculation with TMV. The results show that compounds 2-4 demonstrated protective effects against host plants with inhibition rates of 40.8%, 68.2%m and 49.3% ( Figure S18), respectively. These results indicated that pretreatment with compounds 2-4 could greatly increase the resistance of the host plant to TMV infection.

Anti-Rotavirus Activities
In order to study whether the chromones and indole alkaloids from the stem bark of C. alata had more broad antiviral activities, compounds 1-5 were also tested for their antirotavirus activity. Their ability to prevent the cytopathic effects of rotavirus in MA104 cells was tested according to our previous literatures [23], and their effects were measured in parallel with the determination of antiviral activity using ribavirin as a positive control. The results (Table 5) revealed that compounds 1-5 exhibited potential anti-rotavirus activities with therapeutic index (TI) values in the range of 9.75~15.3.

Plant Materials
The stems of C. alata Linn. were collected from Yingjiang County, Dehong Prefecture of Yunnan Province, People's Republic of China, in September 2020. The identification of plant material was verified by Prof. Ning Yuan. A voucher specimen (Ynni-12-20-52) was deposited in Key Laboratory of Chemistry in Ethnic Medicinal Resources, Yunnan Minzu University, P. R. China.

Anti-TMV Assays
The anti-TMV activities were tested using the half-leaf method according to our previous studies [21,22], and Ningnanmycin (C 16  For compounds with significant activities in half-leaf method assays, their protective effects on TMV were also evaluated by pretreating a tobacco plant with 20 µM solutions of compounds in DMSO, or a solution of DMSO for 6 h before inoculation with TMV.

Anti Rotavirus Assay
The anti rotavirus activities were tested according to our previous literature [23]. Human rotavirus Wa group was used to infect the cell culture MA104 in vitro, the 50% cytotoxicity concentration (CC 50 ) and half maximal effective concentration (EC 50 ) were evaluated, and ribavirin (C 8 H 12 N 4 O 5 , CAS No. 1646818-35-0, a broad-spectrum antiviral drug) was used as a positive control. MA-104 cells (1 × 10 5 cells per well) were grown in 96-well plates for 48 h. The media were removed and replaced by new media containing serial dilutions of compounds under test. After incubation for 72 h, the media were discarded, and 5.0 µL of MTT solution was added to each well. Plates were then incubated at 37 • C for 4 h. The solution was removed, and 100 µL of 0.04 mol/L HCl-isopropanol was added to each well to dissolve formazan crystals. Using a microplate reader, the absorbance of each well was measured at 540 nm. After subtracting the background absorbance at 655 nm, the 50% CC 50 of each compound was estimated by regression analysis.
In the mixed treatment assay, each compound was mixed with a 0.01 multiplicity of infection (MOI) of the rotaviruses at various concentrations (1-160 µg/mL) and incubated at 4 • C for 1 h. The mixtures were inoculated in triplicates onto near confluent MA-104 cell monolayers (1 × 10 5 cells per well) for 1 h with occasional rocking. The solution was removed, and the cells were replaced with eagles minimum essential medium (EMEM) containing 1 µg/mL trypsin. The cells were incubated for 72 h at 37 • C under 5% CO 2 atmosphere until the cells in the control showed complete viral cytopathic effect (CPE) by light microscopy. EC 50 was estimated by regression analysis.

Conclusions
With the aim of continuing to explore bioactive metabolites from Cassia Linn, chemical investigations on C. alata were carried out. As expected, three new indole alkaloids, alataindoleins A-C (1-3); one new chromone, alatachromone A (4); and a new dimeric chromone-indole alkaloid, alataindolein D (5) were isolated. To the best of our knowledge, alataindolein D (5) represents a new type of dimeric alkaloid with an unusual N-2−C-16' linkage, which is biogenetically derived from a chromone and an indole alkaloid via an intermolecular nucleophilic substitution reaction. The anti-TMV assay revealed that compounds 2-4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates are higher than that of positive control. In addition, compounds 1-5 exhibited potential anti-rotavirus activities with therapeutic index (TI) values in the range of 9.75~15.3. The successful isolation and structure identification of the above new compounds provide materials for the screening of antivirus drugs and contribute to the development and utilization of C. alata.

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Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from thecorresponding author.