Macathiohydantoin L, a Novel Thiohydantoin Bearing a Thioxohexahydroimidazo [1,5-a] Pyridine Moiety from Maca (Lepidium meyenii Walp.)

Five new thiohydantoin derivatives (1–5) were isolated from the rhizomes of Lepidium meyenii Walp. NMR (1H and 13C NMR, 1H−1H COSY, HSQC, and HMBC), HRESIMS, and ECD were employed for the structure elucidation of new compounds. Significantly, the structure of compound 1 was the first example of thiohydantoins with thioxohexahydroimidazo [1,5-a] pyridine moiety. Additionally, compounds 2 and 3 possess rare disulfide bonds. Except for compound 4, all isolates were assessed for neuroprotective activities in corticosterone (CORT)-stimulated PC12 cell damage. Among them, compound (−)-3 exhibited moderate neuroprotective activity (cell viability: 68.63%, 20 μM) compared to the positive control desipramine (DIM) (cell viability: 88.49%, 10 μM).


Introduction
Hydantoin, imidazolidine-2,4-dione, is a five-membered heterocycle that is one of the oxidized forms of imidazolidine with a cyclic urea core. The hydantoin scaffold has been enhanced in clinical use, for example, phenytoin, nitrofurantoin, and ethotoin. Thiohydantoin, an isosteric analogue of hydantoin, similarly possesses versatile biological activities, such as fungicidal, herbicidal [1], immunomodulating [2], and anticancer activities [3]. Based on enzalutamide, Xu et al. designed and synthesized a tetrahydroisoquinoline thiohydantoin scaffold. Several new analogues displayed improved antagonistic effect against the androgen receptor (AR) while maintaining the higher selective toxicity toward LNCaP cells (AR-rich) versus DU145 cells (AR-deficient) compared to enzalutamide [4]. However, (thio)hydantoin derivatives were rarely isolated from nature before 2017.

The specific rotation value [α]
26 D -7.07 (c 0.130, MeOH) of 1 suggested that it could be an enantiomer mixture, which was further substantiated by a chiral analysis. In order to determine the absolute configuration of enantiomers (+)-1 and (-)-1, electronic circular dichroism (ECD) calculations were carried out. The predicted ECD spectrum of (4S)-1 agreed well with the experimental CD spectrum of (+)-1, leading to the unambiguous assignment of the absolute configuration of 4S for (+)-1 and 4R for (-)-1, respectively ( Figure 3). The specific rotation value [α] 26 D -7.07 (c 0.130, MeOH) of 1 suggested that it could be an enantiomer mixture, which was further substantiated by a chiral analysis. In order to determine the absolute configuration of enantiomers (+)-1 and (-)-1, electronic circular dichroism (ECD) calculations were carried out. The predicted ECD spectrum of (4S)-1 agreed well with the experimental CD spectrum of (+)-1, leading to the unambiguous assignment of the absolute configuration of 4S for (+)-1 and 4R for (-)-1, respectively ( Figure 3).

Structure Determination of Macathiohydantoin M (2)
Macathiohydantoin M (2) was isolated as colorless oil. The molecular formula of 2 was assigned as C 14 H 16 N 2 OS 3 by HRESIMS data ([M + Na] + , m/z 347.0318, calcd 347.0317) with eight degrees of unsaturation. The 1 H NMR spectrum (Table 1) of 2 displayed signals of five aromatic protons at δ H 7.52 (2H, d, (J = 7.2 Hz), H-3a and H-7a), δ H 7.26 (m, H-5a), and δ H 7.30 (m, H-4a and H-6a) for monosubstituted phenyl moiety and one singlet methyl at δ H 2.11 (s, H 3 -9). Additionally, four quaternary carbons (including two carbonyl groups) and four methylenes were assigned based on the 13 C-DEPT spectra and the HSQC correlations. The aforementioned information showed that the structure of 2 was similar with that of macathiohydantoin D [29]. Simultaneously, the observed HMBC correlations ( Figure 2) of H 2 -7 with C-1, C-4, C-5, and C-6; H 2 -5 with C-3, C-4, C-6, and C-7; and of H 2 -1a to C-1, C-3, C-2a, and C-3a, together with the 1 H-1 H COSY correlations of H 2 -5/H 2 -6/H 2 -7, further confirmed the above deduction. However, detailed comparison of their 13 C NMR data displayed that the chemical shift of C-4 obviously shifted high-field in 2 (δ C 80.3 for 2, δ C 92.5 for macathiohydantoin D). Considering two additional sulfur atoms and one singlet methyl in the molecular formula of 2, a methyl disulfide bond was established and located at C-4.
Similarly, 2 was found to be also a pair of enantiomers through chiral analysis. The subsequent chiral HPLC resolution of 2 gave the anticipated enantiomers (-)-2 and (+)-2, whose experimental CD curves were opposite. Thus, as depicted in Figure 3, the absolute configurations of (-)-2 and (+)-2 were deduced to be 4R and 4S by comparing with the calculated ECD curve of 4S-2.

Structure Determination of Macathiohydantoin M (2)
Macathiohydantoin M (2) was isolated as colorless oil. The molecular formula of 2 was assigned as C14H16N2OS3 by HRESIMS data ([M + Na] + , m/z 347.0318, calcd 347.0317 with eight degrees of unsaturation. The 1 H NMR spectrum (Table 1) (Table 1) indicated a high similarity between 2 and 3, except for an additional methoxyl and the replacement of monosubstituted phenyl by disubstituted phenyl in 2. Further evidence was established from the HMBC correlations ( Figure 2) of H 3 -OMe to C-4a and H 2 -1a to C-1, C-3, C-2a, C-3a.

Structure Determination of Macathiohydantoin O (4)
Macathiohydantoin O (4) was isolated as colorless oil with the molecular formula of Compound 4 was also identified as a thiohydantoin derivative based on its 1D NMR data, which were similar with those of macathiohydantoin E [29] with the only difference in the methoxyl at C-4 in 4 instead of the hydroxyl in macathiohydantoin E. Furthermore, the HMBC correlation from H 3 -OMe to C-4 confirmed that methoxyl was located at C-4. Due to the specific rotation value of 4 being [α] 26 D +30.93 (c 0.120, MeOH) similar with (+)-macathiohydantoin E [+49.00 (c 0.007, MeOH)], the absolute configuration of (+)-4 was directly deduced to be 4S.

General Experimental Procedures
Optical rotations were obtained with a Rudolph Autopol VI polarimeter in MeOH. A Shimadzu UV-2700 spectrometer was used to obtain UV spectra. 1 H and 13 C NMR spectra were acquired on Bruker AV-600 and AV-800 instruments (Bruker, Zurich, Switzerland) using tetramethylsilane (TMS) as an internal standard for chemical shifts in CDCl 3 . Chemical shifts (δ) were expressed in ppm and referenced to the TMS resonance. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were performed on an UPLC system (1260, Agilent) coupled to a quadrupole time-of-flight mass spectrometer (Agilent 6540 Q-TOF, Agilent Technologies, Foster City, CA, USA). Infrared spectra were recorded on a Bruker Tensor-27 instrument by using KBr pellets. An Agilent 1100 series instrument equipped with an Agilent ZORBAX SB-C18 column (5 µm, 9.4 mm × 250 mm) was used for high-performance liquid chromatography (HPLC) analysis. Chiral chromatography using a CHIRALCEL AD-H column (5 µm, 4.6 mm × 150 mm) was used to resolve enantiomers.

Plant Material Extraction and Isolation
The air-dried and powered maca rhizomes (37 kg) were extracted three times with acetone at room temperature and evaporated to remove solutions to yield the crude extract.

Conclusions
In summary, five new thiohydantoin derivatives (1)(2)(3)(4)(5) were isolated from the rhizomes of L. meyenii. Specifically, compound 1 possesses thioxohexahydroimidazo [1,5-a] pyridine moiety. Additionally, compounds 2 and 3 possess the rare disulfide bonds, and compound (-)-3 exhibited moderate neuroprotective activity compared with desipramine (DIM) as a positive control. Our research not only enriches the structural types of compounds in Maca but also provides a material basis for Maca as a potential health food to treat neurodegenerative diseases.
Supplementary Materials: The following are available online. 1D and 2D NMR spectra of all isolated compounds. Detailed information for each material is given in the Supplementary Material.
Author Contributions: R.Z. and J.L. have jointly planned and carried out the isolation and structure determination of the reported five compounds, while R.Z. wrote the manuscript; H.Y. carried out the biological assays; M.Q. supervised the work of R.Z. and J.L., revised the manuscript, and designed the project, while X.P. supervised the work and L.Z. designed the project. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable, as the study did not involve humans or animals.
Informed Consent Statement: Not applicable, as the study did not involve humans or animals.
Data Availability Statement: All data are available in this publication and in the Supplementary Materials.