Artificial Erythrina Alkaloids from Three Erythrina Plants, E. variegata, E. crista-galli and E. arborescens.

Fourteen unprecedented artificial Erythrina alkaloids were isolated from the Erythrina variegata, E. crista-galli and E. arborescens (Fabaceae). The structures of these alkaloids were determined by spectroscopic analyses. Their possible formations were proposed. All isolated compounds showed no cytotoxicity and hypoglycemic activity at cell screening bioassay.

Alkaloid 5 displayed a hydrogen adduct ion at m/z 354.1709 [M+H] + (calc. for C 21 H 23 NO 4 , 354.1707). The 1D NMR spectroscopic data of compound 5 were similar to those of 4 except for the following differentiations: in the 1 H NMR spectrum, the signal displayed at δ H 4.72 in 4 which was assigned to the active hydrogen in the hydroxy was disappeared in compound 5. Correspondingly, the methine signal at δ C 64.7 (C-11) in compound 4 was replaced with a methylene (δ C 26.0) in 5. Thus, compound 5 was an analogue of 4 without the hydroxy moiety and determined to be 8-(2-oxopropyl)-erythraline.
The HRESIMS of 6 gave a hydrogen adduct ion at m/z 384.1806 [M+H] + , indicative of a molecular formula of C 22 H 25 NO 5 . In comparing with those of 4, the 1 H NMR spectrum of 6 gave signal of an addional methyoxyl group (δ H 3.55, s, 3H), and its 13 C NMR spectrum showed an downfield chemical shift δ C 74.8. These findings suggested the C-11 of 6 was substituted by a methoxy rather than a hydroxy. Thus, the structure of 6 was determined to be 8-(2-oxopropyl))-11-methoxy-erythraline.  The molecular formula of 7 was determined to be C 21 H 25 NO 5 from the HRESIMS m/z at 394.1626 [M+Na] + . Its 1 H NMR spectrum showed two aromatic singlet protons (δ H 6.84 and 6.76), three conjugate olefin signals (δ H 6.60, 6.10 and 5.67), and four methoxy groups (δ H 3.28, 3.70, 3.78 and 3.97). The 13 C NMR spectrum of 7 showed three methylenes (δ C 24.6, 43.3, 44.4), two methines (δ C 76.9 and 70.5) and a carbonyl (δ C 172.1). These data suggested 7 might be a carbomethoxyl derivative of erysotrine [17]. The HMBC correlations from δ H 3.78 (OCH 3 ) and δ H 4.33(H-8) to δ C 172.1 (C=O) assigned the carbomethoxy at C-8. The molecular formula of 8 was determined to be C 20 H 21 NO 6 from the HRESIMS m/z at 372.1444 [M+H] + . The 1 H and 13 C NMR the structural pattern of 8 was identical to that of 5, and the additional carbomethoxyl moiety was identical to that of 7. Accordingly, the structures of 7 and 8 were determined to be 8-carbomethoxyerysotrine and 8-carbomethoxyerythranine, respectively.
Alkaloid 9 showed molecular ion peaks at m/z 370.1652 [M+H] + , suggesting the molecular formulae C 21 H 24 NO 5 . In comparing with compound 5, the 1 H and 13 C NMR signal of δ H 2.13 (CH 3 ) and δ C 207.7(CH 2 COCH 3 ) in 5 were changed to δ H 3.61 (OCH 3 ) and δ C 172.5(CH 2 COOCH 3 ) in 9, respectively. The remaining NMR data were almost identical to those of 5. Thus, the structure of 9 was determined to be 8-acetatemethoxyerythraline.
The molecular formulas of compounds 10 and 11 were deduced to be C 21 H 23 NO 6 13 C NMR data of both 10 and 11 are very similar to those of 9 except that the methylene signal was replaced by an oxymethine signal at the C-11 position. Further, in the 13 C NMR, the signal for C-11 appeared at 64.8 and 74.7 ppm for compounds 10 and 11, similar to that of 4 and 6, respectively. Thus, 10 was identified as 8-acetatemethoxyerythranine. 11 had an extra methoxy and was identified as 8-acetatemethoxy-10β-methoxyerythraline.
The molecular formula 12 was established as C 22 H 27 NO 5 based on the HRESIMS m/z = 386.1964 [M+H] + . From the 1 H and 13 C NMR data, the structure of 12 was very similar to 9 except for the replacement of methylenedioxy group by two methoxys at C-15 and C-16. This was confirmed from the HMBC and HSQC spectra. Alkaloid 12 was thus identified as 8-acetatemethoxyerysotrine.
The HRESIMS m/z at 394.1628 [M+Na] + of 13 assigned the molecular formula to be C 21 H 25 NO 5 , 58 mass units higher than that of erysotrine. Its 13 C NMR spectrum gave an additional methylene (δ C 34.9) and a carbonyl (δ C 172.3) signals, indicating the existence of an acetyl group. The HMBC correlations from δ H 2.51 (CH 2 CO) and δ H 4.14 (H-8) to δ C 172.3 (C=O) suggested that the acetyl group was located at C-8. Accordingly, the structure of 13 was determined to be 8-acetylerythsotrine. The molecular formula of 14 was dirermined to be C 20 H 21 NO 5 by the HRESIMS m/z at 378.1313 [M+Na] + . The 1D NMR spectrum gave signals similar to that of erythraline expect for the replacement of a methylene by an acetyl group (δ C 35.4 and δ C 172.3). In the HMBC spectrum, the correlations from δ H 4.07 (H-8) to δ C 172.3 (COOH) and δ H 2.74 (CH 2 COOH) to δ C 65.4 (C-8) and δ C 172.3 (COOH) confirmed that 14 was an 8-acetyl derivative of erythraline.
Since N-containing compounds were main candicates of anticancer and hypoglycemic drugs, so alkaloids 1-14 were evaluated for their cytotoxicity against human A-549 lung cancer, SGC-7901 gastric cancer, and HeLa cell lines using the MTT method. In addition, their hypoglycemic activity on 3T3-L1 myoblasts cell were screened. Unfortunately, none of them showed positive activity. Alkaloids 1-14 possessed acetonyl, acetyl methyl, acetate, or methyl formate groups, which indicated they were artificial products. Without considering the artifitial units, these alkaloids are known. Duing the extraction and isolation, methanol, acetone, petroleum ether, especial ethyl acetate, were used as solvents. Accordingly, acetone and residual of acetic acid, methyl acetate and methyl formate in above solvents would become reaction reagents. Alkaloids 1-6 and 9-14 were formed firstly through an iminium immediate by oxidation, then by nucleophilic attack from carbanion of acetone, acetic acid, and methyl acetate in base condition. On the other hand, the iminium immediate could be tautomerized to inmine and attacked to methyl formate, generating the carbomethoxy substitued products (7-8) (Fig. 3).

General Experimental Procedures
Optical rotations were measured with a Jasco p-1020 digital polarimeter. UV spectra were recorded on a Shimadzu 2401PC spectrophotometer. IR spectra were obtained on a Bruker Tensor 27 infrared spectrophotometer with KBr pellets. 1 H, 13 C and 2D NMR spectra were obtained on Bruker AV-600, AVANCE III-500 and 400 MHz spectrometers with SiMe 4 as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. MS data were recorded on an UPLC-IT-TOF MS. Column chromatography (CC) was performed on either silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) or RP-18 silica gel (20-45 μm, Fuji Silysia Chemical Ltd., Japan). Fractions were monitored by TLC on silica gel plates (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized with Dragendorff's reagent spray. MPLC was performed using a Buchi pump system coupled with RP-18 silica gelpacked glass columns (15 × 230 and 26 × 460 mm, respectively). HPLC was performed using Waters 1525 pumps coupled with analytical or preparative Sunfire C 18 columns (4.6 × 150 and 19 × 250 mm, respectively). The HPLC system employed a Waters 2998 photodiode array detector and a Waters fraction collector III.

Extraction and Isolation
The dried and powdered flowers of E. variegata (10.0 kg) were extracted with 90% MeOH (25 L) for three times. The extracts were concentrated under reduced pressure, and then dissolved in 2% acetic acid to adjust pH to 2-3 and then partitioned twice with EtOAc. The aqueous layers were basified with NH 3 ·H 2 O to adjust pH to 8-9 and then extracted with EtOAc to give a crude alkaloid fraction (110 g). The crude alkaloid was subjected to column chromatography (CC) over silica gel with gradient CHCl 3 -Acetone (1:0 to 1:1) to afford seven fractions (Fr. I-Fr. VII). Fr. I (6.1 g) was divided into 2 subfractions (Fr. I-1-Fr. I-2) by using RP-MPLC eluting Flowers of E. crista-galli (11 kg) were powdered and extracted with 90% MeOH (25 L) for three times. The extract was concentrated in vacuo to give a brown residue. The crude alkaloid (90 g) were obtained using the same acid-base treatment method described above, and then subjected to column chromatography (CC) over silica gel and eluted with gradient    Tables 3 and  4

Cytotoxicity
The human A-549 lung cancer, SGC-7901 gastric cancer, and HeLa cell lines were used in the cytotoxic assay. These cells were grown in DMEM media (HyClone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) at 37 °C in 5% CO 2 . The cytotoxicity of all alkaloids were determined based on the MTT method in 96-well microplates. In short, 100 µL adherent cells were seeded into each well and incubated for 12 h before the addition of the test alkaloids/drug. At the same time, the suspended cells were seeded at an initial density of 1 × 10 5 cells/mL just before the addition of the alkaloids/drug. Each tumor cell line was exposed to a test compound at concentration 20 μM in DMSO in triplicate for 48 h, with camptothecin as the positive control. After treatment, cell viability was assessed.

Hypoglycemic Activity
3T3-L1 myoblasts cells were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM supplemented with 10% FBS or CS (for 3T3-L1 cells), 100 units/ml penicillin and 100 mg/ml streptomycin in 10 cm diameter dishes in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C. Cells were maintained in continuous passages by trypsinization of subconfluent cultures and fed fresh medium every 48 h. For differentiation, L6 myoblasts were transferred to DMEM with 2% FCS in tissue culture plates for 5-6 days, 3T3-L1 cells were exposed to 0.5 mM IBMX, 1 mM dexamethasone, 1 mM rosiglitazone and 1 mg/mL insulin for 3 days, and 1 mg/ml insulin for the other day. For glucose uptake assay, cells were serum starved for 4 h in 96-well plates, followed by incubated with insulin and alkaloids 1-14 for 24 h. Finally, the supernatants of cultured cells were collected and subjected to glucose assay using a commercially kit. The quantified values were normalized based on the results of the MTS assay.  Entry δ C (4) a δ C (5) a δ C (6) a δ C (7) b δ C (8) a δ C (9) a δ C (10) a δ C (11) a δ C (12) a δ C (13) b δ C (14)