Spiroleiferthione A and Oleiferthione A: Two Unusual Isothiocyanate-Derived Thioketone Alkaloids from Moringa oleifera Lam. Seeds

Spiroleiferthione A (1), with a 2-thiohydantoin a heterocyclic spiro skeleton, and oleiferthione A (2), an imidazole-2-thione derivative, were isolated from the aqueous extract of Moringa oleifera Lam. seeds. The unprecedented structures of 1 and 2 were elucidated by extensive spectroscopic data, X-ray diffraction, and gauge-independent atomic orbital (GIAO) NMR calculation, as well as electronic circular dichroism (ECD) calculation. The structures of 1 and 2 were determined to be (5R,7R,8S)-8-hydroxy-3-(4′-hydroxybenzyl)-7-methyl-2-thioxo-6-oxa-1, 3-diazaspiro [4.4] nonan-4-one, and 1-(4′-hydroxybenzyl)-4,5-dimethyl-1,3-dihydro-2H-imidazole-2-thione, respectively. Biosynthetic pathways for 1 and 2 have been proposed. Compounds 1 and 2 are considered to have originated from isothiocyanate and then undergone a series of oxidation and cyclization reactions to form 1 and 2. Compounds 1 and 2 demonstrated weak inhibition rates of NO production, 42.81 ± 1.56% and 33.53 ± 2.34%, respectively, at a concentration of 50 μM. Additionally, Spiroleiferthione A demonstrated moderate inhibitory activity against high glucose-induced human renal mesangial cell proliferation in a dosage-dependent manner. A wider range of biological activities, and the diabetic nephropathy protective activity of Compound 1 in vivo and its mechanism of action, need further investigation after the sufficient enrichment of Compound 1 or total synthesis.


Proposed Biosynthetic Pathway of 1 and 2
Biosynthetic pathways for 1 and 2 are proposed in Scheme 1. The typical isothiocyanate derived from phenylalanine was considered to be the starting unit [24]. The initial head-to-tail cyclization of isothiocyanate (A) with methyl alanine would produce B with the removal of a methoxy moiety [25]. The oxidation of B would yield key intermediate C. SAM-dependent methyltransferase would catalyze the conversion of C into E, which would dehydrate to afford 2. The oxidation of key intermediate C would give D. Finally, the cyclization of D with methyl lactate would produce 1 in a transformation similar to that producing B.

Proposed Biosynthetic Pathway of 1 and 2
Biosynthetic pathways for 1 and 2 are proposed in Scheme 1. The typical isothiocyanate derived from phenylalanine was considered to be the starting unit [24]. The initial headto-tail cyclization of isothiocyanate (A) with methyl alanine would produce B with the removal of a methoxy moiety [25]. The oxidation of B would yield key intermediate C. SAM-dependent methyltransferase would catalyze the conversion of C into E, which would dehydrate to afford 2. The oxidation of key intermediate C would give D. Finally, the cyclization of D with methyl lactate would produce 1 in a transformation similar to that producing B.

Biological Activity Evaluation of 1 and 2
In bioassay experiments, Compounds 1 and 2 were evaluated for their antimicrobial activity and their inhibitory activities against lipopolysaccharide (LPS)-activated nitric oxide (NO) production in RAW 264.7 cells and high glucose-induced human renal mesangial cell (HRMC) proliferation. Compounds 1 and 2 displayed no antimicrobial activity against four bacteria, including Escherichia coli, Staphylococcus aureus subsp. Aureus, Salmonella enterica subsp. Enterica, and Pseudomonas aeruginosa at a concentration of 100 µM. In the antimicrobial assay, the positive control group (Ceftazidime) displayed inhibition rates of 99.853 ± 0.129% and 100.04 ± 0.069% against E. coli and P. aeruginosa, respectively. Penicillin G sodium salt displayed inhibition rates of 100.232 ± 0.201% and 99.942 ± 0.084% against S. aureus and S. enterica, respectively. Compounds 1 and 2 showed a weak inhibition of NO production with 42.81 ± 1.56% and 33.53 ± 2.34%, respectively, at a concentration of 50 µM. The positive control (L-NG-monomethylarginine) demonstrated an inhibition of NO production with 59.31 ± 2.19%. However, as shown in Figure 6, Compound 1 showed the moderate inhibition of high glucose-induced HRMC proliferation in a dosage-dependent manner. This result suggests that Compound 1 can potentially exert protective effects against the progression of diabetic nephropathy.

Biological Activity Evaluation of 1 and 2
In bioassay experiments, Compounds 1 and 2 were evaluated for their antimicrobial activity and their inhibitory activities against lipopolysaccharide (LPS)-activated nitric oxide (NO) production in RAW 264.7 cells and high glucose-induced human renal mesangial cell (HRMC) proliferation. Compounds 1 and 2 displayed no antimicrobial activity against four bacteria, including Escherichia coli, Staphylococcus aureus subsp. Aureus, Salmonella enterica subsp. Enterica, and Pseudomonas aeruginosa at a concentration of 100 μM. In the antimicrobial assay, the positive control group (Ceftazidime) displayed inhibition rates of 99.853 ± 0.129% and 100.04 ± 0.069% against E. coli and P. aeruginosa, respectively. Penicillin G sodium salt displayed inhibition rates of 100.232 ± 0.201% and 99.942 ± 0.084% against S. aureus and S. enterica, respectively. Compounds 1 and 2 showed a weak inhibition of NO production with 42.81 ± 1.56% and 33.53 ± 2.34%, respectively, at a concentration of 50 μM. The positive control (L-NG-monomethylarginine) demonstrated an inhibition of NO production with 59.31 ± 2.19%. However, as shown in Figure 6, Compound 1 showed the moderate inhibition of high glucose-induced HRMC proliferation in a dosagedependent manner. This result suggests that Compound 1 can potentially exert protective effects against the progression of diabetic nephropathy.

Discussion
To date, no more than 50 naturally occurring thiohydantoin derivatives have been reported in the literature [2][3][4][5][6][7][8][9][10]. Spiroleiferthione A is a rare 2-thiohydantoin with an unprecedented heterocyclic spiro skeleton that differs from these reported thiohydantoin derivatives. Thus far, only two imidazole-2-thione derivatives derived from ergothioneine have been isolated from a plant extract, although all previously reported ergothioneine-derived natural products are from fungal and animal sources [11]. Oleiferthione A is a novel naturally occurring imidazole-2-thione derivative, which is the third identified plant origin of imidazole-2-thione derivatives. Spiroleiferthione A showed moderate inhibitory activity against high glucose-induced human renal mesangial cell proliferation in a dosage-dependent manner. However, on account of the limitations, the diabetic-nephropathy-protective activity of Compound 1 in vivo and its mechanism of action were not further investigated. Although it has been reported that natural imidazole-2-thione derivatives possess biological activities, such as antioxidant, hepatoprotective, and epithelial-mesenchymal transition inhibition properties [12][13][14], and natural 2-thiohydantoin derivatives showed anti-neuroinflammatory [3,4,8], antibacterial [9], anticancer [8,26], hypolipidemic [2], anticarcinogenic [2], antimutagenic [2], and antithyroidal [2] activities, a wider range of biological effects was not further evaluated due to the limitations of the two compounds. Although various bioactivities of 2-thiohydantoin derivatives have been reported, the antidiabetic nephropathy activity was investigated in this study for the first time. In order to comprehensively investigate its bioactivities and mechanisms of action, the total synthesis of 2-thiohydantoin derivatives may be an effective approach to produce sufficient yields.

General Experimental Procedures
Optical rotations were measured using an INESA SGW-3 automatic polarimeter (Shanghai INESA Physico Optiacal Instrument, Shanghai, China). UV data were recorded using a Cary 300 spectrometer (Agilent Technologies, Santa Clara, CA, USA). CD data were measured using a JASCO J-815 CD spectrometer (JASCO, Tokyo, Japan). IR data were recorded using a Nicolet IS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were obtained at 500 MHz or 600 MHz for 1 H, and 125 MHz or 150 MHz for 13 C on Bruker 500 MHz or 600 MHz spectrometers (Bruker, Billerica, MA, USA) in CD 3 OD or DMSO-d 6 , respectively, with the tetramethyl silane (TMS) peak used as a reference. HR-ESIMS data were measured using an Agilent Q-TOF 6545 spectrometer (Agilent Technologies). Column chromatography (CC) was performed using silica gel (200−300 mesh, Qingdao Marine Chemical, Qingdao, China), MCI gel (CHP20P) (Mitsubishi Chemical, Tokyo, Japan), and macroporous adsorbent resin (HPD-300) (Ainuo chemical technology Co., LTD, Zhengzhou, China). HPLC separation was performed using a system consisting of an Agilent 1260, an Agilent 1260 pump, and an Agilent 1260 wavelength absorbance detector with an Agilent (250 × 9.4 mm) semipreparative column packed with C 18 (5 µm) (Agilent Technologies). TLC separations were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical). Spots were visualized under UV light (254 or 356 nm), or by spraying with 10% H 2 SO 4 in 90% EtOH followed by heating.

Antimicrobial Assay
The microorganisms used in antibacterial assays were obtained from the China General Microbiological Culture Collection Center (CGMCC) (Escherichia coli ATCC25922, Staphylococcus aureus subsp. Aureus ATCC29213, Salmonella enterica subsp. enterica ATCC14028, and Pseudomonas aeruginosa ATCC27853. Lysogeny broth (LB, Huankai Microbial, Guangzhou, China) culture media were used for culturing bacteria, while Mueller-Hinton broth (MHB, Huankai Microbial, Guangzhou, China) was used to determine the minimum inhibitory concentration. The inocula of bacteria was prepared from 24 h-old agar cultures and incubated to logarithmic phase. Ceftazidime (Yuanye biotechnology Co., LTD, Shanghai, China) was used as a positive reference substance. We measured the minimum inhibitory concentration (MIC) of Compounds 1 and 2 using the broth microdilution method in 96-well microtiter plates, as described in the literature [27]. The MIC was defined as the lowest concentration without any colony growth after incubating the fungus at 28 • C for 16 h. All tests were performed in triplicates.

Anti-Inflammatory Assay
Murine RAW 264.7 cells were purchased from the Shanghai cell bank of the Chinese Academy of Sciences (Shanghai, China). These cells were incubated in RPMI1640 medium plus 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate in a humidified incubator with 5% CO 2 . The cells were treated with LPS (1 µL/mL) and the test compounds for 48 h. L-NMMA was used as a positive control. Accumulated nitrite in the culture supernatants, an indicator of NO synthase activity, was measured by the Griess reaction [28]. Cell viability was examined by MTS assay (Beyotime Inst. Biotech. Shanghai, China) according to the manufacturer's instructions. The absorbance was examined at 570 nm using a Spectra Max microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). All tests were performed in triplicates. The results were expressed as a percentage of the response of the related LPS-treated groups that were designated as 100%.

Inhibitory Assay of High Glucose-Induced HRMC Proliferation
HRMCs were cultured in DMEM (Sigma, St. Louis, MO, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA) and 1% double antibiotic (penicillinstreptomycin, Beyotime Biotechnology Co., Ltd., Shanghai, China) at 37 • C in a humidified environment containing 5% CO 2 . Next, 20 µL of L-DMEM was added to the blank and control wells, and 10 µL each of L-DMEM and 250 mM glucose (0.26 g of D-(+)-glucose dissolved in 8 mL of phosphate-buffered saline (PBS) and filtered using a 0.22 µm membrane) was added to the other wells to a final glucose concentration of 30 mM, which were then incubated for 24 h [22]. Different concentrations of Compounds 1 and 2 (10, 20, and 50 µM) were added to the designated wells, and the Cell Counting Kit-8 (CCK8, Dojindo, Kyushu Island, Japan) assay was used to detect optical density after 24 h of incubation. We used L-DMEM containing only HRMCs and no intervention drugs as a control group.

Statistical Analysis
One-way ANOVAs and LSD t-tests were used to analyze the groups of samples using SPSS 19.0. The data are expressed as the mean ± SD (standard deviation). All the data shown are representative of at least three independent experiments. p < 0.05 was considered to be statistically significant.

Computational Section
Crest software was used to search the conformers of (5R,7R,8S)-1 and (5S, 7R, 8S)-1 on the GFNFF level of theory [29,30], following the optimization of GFN2-XTB level with a 4 kcal/mol energy window to remove high-energy conformers [31]. Optimization and frequency calculations of each conformer were performed on B3LYP-D3(BJ)/TZVP (IEF-PCM, CH 3 OH) level of theory. DFT GIAO 13 C NMR calculations were performed on the ωB97xD/6-31G * (IEFPCM, CH 3 OH) level, and data processing followed the reported STS protocol [23]. The calculated shielding tensors of conformers were Boltzmann averages based on the Gibbs free energy. Theoretical ECD was calculated by time-dependent density functional theory (TDDFT) at the mPW1PW91/6-311g(d) level with the IEF-PCM solvent model (MeOH). SpecDis v1.71 was used to simulate the ECD for (5R,7R,8S)-1 curve with Gaussian band shape at 0.28 eV. The calculated ECD curve of each conformer was Boltzmann-averaged based on its Gibbs free energy. All the DFT calculations were performed using the Gaussian 16 software package.

Conclusions
Two isothiocyanate-derived thioketone alkaloids, Spiroleiferthione A and Oleiferthione A, were isolated from the aqueous extract of M. oleifera seeds. The novel structures of 1 and 2 were elucidated by extensive spectroscopic data, gauge-independent atomic orbital (GIAO) NMR calculation, ECD, and X-ray diffraction. Spiroleiferthione A is a rare 2-thiohydantoin with an unprecedented heterocyclic spiro skeleton. Oleiferthione A is a novel, naturally occurring imidazole-2-thione derivative. Spiroleiferthione A demonstrated moderate inhibitory activity against high glucose-induced human renal mesangial cell proliferation in a dosage-dependent manner. This study has enriched the structural diversity of M. oleifera seeds, especially the isothiocyanate-derived, sulfurcontaining compounds. It provides clues for the subsequent study of thioketone derivatives and their activities.