Nine new cytochalasan alkaloids from Chaetomium globosum TW1-1 (Ascomycota, Sordariales)

Chemical investigation on the methanol extract of Chaetomium globosum TW1-1, a fungus isolated from the common pillbug (Armadillidium vulgare), has resulted in the isolation of nine new highly oxygenated cytochalasan alkaloids, armochaetoglobins S–Z (1 and 3–9) and 7-O-acetylarmochaetoglobin S (2), together with eight structurally related known analogues (10–17). Their structures and absolute configurations were elucidated by spectroscopic analyses. Among them, compound 2 presents to be the first member of chaetoglobosin family with an acetyl group, and compounds 3 represents the first chaetoglobosin characterized by an 2′,3′-epoxy-indole moiety. The discovery of these new compounds revealed the largely untapped chemical diversity of cytochalasans and enriched their chemical research. Compounds 1–9 were evaluated for their cytotoxic activities against five human cancer cell lines, and compounds 8 and 9 exhibited significant cytotoxic activities with IC50 values ranging from 10.45 to 30.42 μM.

The carbon profile and characteristic 1 H NMR signals (Table 1), as well as 2D NMR spectra of 1 revealed that it has a similar indole based cytochalasan skeleton as that of chaetoglobosin Q 14 . Comparison of the NMR data (Tables 1 and 2) of 1 with those of chaetoglobosin Q suggested their structural similarities. The main difference of the two compounds is the fragment from C-19 to C-22 in the macrocycle ring. The olefinic carbons C-21 and C-22 in chaetoglobosin Q were replaced by two methylene signals in the spectra of 1, which was suggested by 1 (Fig. 3) of 1 with that of armochaetoglobin F 26 , whose absolute configuration was undoubtedly identified by X-Ray diffraction analysis. The final name of armochaetoglobin S was assigned to 1.
The molecular weight of compound 2 was 42 mass units more than that of 1 as revealed by HRESIMS ion peak at m/z 613.2859 ([M + Na] + , cacld for C 34 H 42 N 2 O 7 Na, 613.2890). Comparison of the NMR data (Tables 1 and 2) for 2 and 1 revealed a high level of similarity with the only significant difference of the presence of an additional acetyl (δ C 20.8 and 172.3; δ H 1.98, 3H, s). The acetyl group was located at C-7 based on HMBC correlations from H-7 to C-1′′ . Further analyses of 2D NMR data ( 1 H-1 H COSY, HMBC, and NOESY) of 2 confirmed the structure and relative configuration of 2, and it was given the trivial name 7-O-acetylarmochaetoglobin S. To the best of our knowledge, compound 2 presents to be the first acetylated chaetoglobosin.
Compound 3 was obtained as colorless amorphous powder. The HRESIMS data showed a quasi-molecular ion peak at m/z 569.2628 ([M + Na] + ), corresponding to a molecular formula of C 32 H 38 N 2 O 6 with 15 degrees of unsaturation. Comparison of the NMR data (Tables 1 and 2) of 3 with those of chaetoglobosin F(10) 28 suggested their structural similarities, however, with higher degrees of oxidation than that of 10. The unexpected epoxy ring fused to the indole ring in compound 3 was evidenced by the chemical shifts of C-2′ (δ C 82.4) and C-3′ (δ C 91.9) and HMBC correlations from H-2′ to C-10, C-3′ , C-1′ a, and C-3′ a. The overall NMR data, including HSQC, 1 H-1 H COSY, and HMBC spectra, allowed the elucidation of the planar structure of 3. Compound 3 represents the first example of naturally occurring chaetoglobosin characterized by 2′ ,3′ -epoxy-indole moiety. The configuration for the core skeleton of 3 was assigned to be as the same as that of 10 by a NOESY experiment and further confirmed by comparison of their NMR data. Based on the identical ECD curves (Fig. 3) of 3 and 1, the stereochemistry of 3 was elucidated.
The molecular formula of compound 4 was determined as C 32 Na, 569.2628). Exhaustive interpretation of the 1 H and 13 C NMR data (Tables 1 and 2) revealed that 4 was also a chaetoglobosin derivative. Moreover, its NMR data closely resembled those of 1 according to the consequent comparison, in which the major difference of the chemical shifts were noticed in the position of C-20 in the macrocyclic ring. A detailed comparison of the 13 C NMR spectra of 4 and 1 suggested that the oxygenated methine (C-20, δ C 72.6) in 1 was replaced by a carbonyl group (δ C 205.9) in 4. Furthermore, the carbon signal of the methine (C-6) at δ C 74.0 in 1 shifted downfield to δ C 77.7 in 4, which could only be rationalized by supposing the change of configuration of C-6. This hypothesis was evidenced by NOESY correlation of Me-12/H-8 and the absence of the NOESY cross-peak between Me-12 and Me-11. Additional supporting evidences for the structure of 4 were provided by 1 H-1 H COSY, HMBC, NOESY, and ECD spectra, which allowed the confirmation of the planar structure and configuration of 4.
Armochaetoglobin V (5) had the same molecular formula as that of 1, suggesting that 5 is an isomer of 1. Comparison of its NMR spectra (Tables 1 and 2) with that of 1 disclosed that the 6,7-diol in 1 was substituent by a 5,7-diol in 5, which was evidenced by chemical shifts of C-5 and C-6 (δ C 40.1 and 74.0 for 1; δ C 74.8 and 49.3 for 5), and further confirmed by 1 H-1 H COSY cross-peak of H-6/H-7 and HMBC correlations from Me-12 to C-5, C-6, and C-7. The configurations of the two hydroxyls were assigned to be co-facial and β-oriented as revealed by NOESY correlations of Me-3 to Me-11 and Me-12, and of Me-12 to H-7. As the first member of chaetoglobosin with a hydroxyl at C-5, compound 5 was outstanding from the chaetoglobosin family.
Compound 6 was obtained as white amorphous powder, which possessed a molecular formula of C 32 H 38 N 2 O 5 , with 15 degrees of unsaturation, based on the [M + Na] + peak at m/z 553.2660 (calcd for 553.26784) in the positive HRESIMS. The 1 H and 13 C NMR data of 6 (Tables 1 and 2) resembled those of 1, which indicated that they share the same chaetoglobosin skeleton. However, the downfield shifted chemical shifts of H-7 (δ H 5.43) and H-12 (δ H 3.89) in compound 6, suggested the vicinal diol at C-6 and C-7 in 1 were replaced by a Δ 6 double bond in 6. Moreover, the methyl (C-12) in 1 was oxygenated to be a hydroxymethyl (δ C 61.4). These speculations were further supported by combination of HRESIMS and 2D NMR data ( 1 H-1 H COSY, HSQC, and HMBC). Further analyses of its NOESY spectrum and ECD curve established the absolute configuration of 6 and the trivial name armochaetoglobin W was given.
HRESIMS analysis of 7 indicated that it possessed the molecular formula of C 32 H 38 N 2 O 6 . The 1 H and 13 C NMR data for 7 (Tables 1 and 2) were similar to those of chaetoglobosin U (11) 20 . The major differences of them were the chemical shifts of C-6 and C-7 (δ C 73.5 and 74.2 in 7; δ C 57.5 and 60.9 in 11). The above analyses, combined with the HRESIMS, suggested that the epoxy ring of 11 was hydrolyzed to be a vicinal diol at C-6 and C-7 in compound 7. The observed HMBC correlations from H-4 to C-6, from H-11 to C-4, C-5, and from C-6, and H-12 to C-5, C-6, and C-7 supported the above deduction. In addition, the similar NOESY spectra and identical ECD curves of the two compounds allowed the assignment of the absolute configuration of 7. Therefore, the structure of compound 7 was established as shown and named armochaetoglobin X.
The HRESIMS data of 8 exhibited an ion peak at m/z 535.2509 (calcd for C 32   www.nature.com/scientificreports/ general similar to those of 11, especially in rings A-C. However, inspection of the 1 H NMR spectrum of 8 revealed a new olefinic proton at δ H 5.91 that was not observed for 11. In addition, the associated carbon signal of C-18 was downfield shifted from 147.2 to 183.5 ppm. Examination of the 2D NMR data of 8 confirmed that an additional olefinic proton was assigned to C-18 of the trisubstituted double bond in 8, which replaced the hydroxyl at the tetrasubstituted double bond in 11. The relative configuration of 8 was established by a NOESY experiment, in  The absolute configuration of 8 was assigned comparison of its ECD spectrum ( Fig. 4) with that of armochaeglobine C 25 , whose absolute configuration was determined by X-ray diffraction analysis and reported by us previously.
Armochaetoglobin Z (9) was obtained as a colorless amorphous powder. Its molecular formula was determined as C 32 H 36 N 2 O 4 by HRESIMS. Detailed comparison of the NMR data of 9 with that of 8 suggested a similar structure for them. The main differences of the two compounds were that the epoxy group in 8 was substituted by a double bond at C-5 and a hydroxyl at C-7 in 9, which was evidenced by analyses of their NMR data and HMBC correlations from Me-11 to C-4, C-5, and C-6, from Me-12 to C-5, C-6, C-7, and from H-7 to H-8 and H-13. The 7-OH was revealed to β-oriented according to NOESY correlations of H-7 to Me-11 and Me-12. In addition, the ECD curve of 9 (Fig. 4) was nearly identical to that of armochaeglobine C and 8, leading to the final assignment of its absolute configuration.

Evaluation of cytotoxicity in human cell lines.
Cytochalasans are commonly reported to exhibit cytotoxic effects and some of them have been considered as starting molecular for anti-tumor drug candidates. Therefore, in this case, compounds 1-9 were all evaluated for their cytotoxic activities against five human tumor cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480) and the immortalized non-cancerous Beas-2B human bronchial epithelial cell line, in vitro, by the MTS method (Table S1, SI). According to the reported structure-activity relationships, the epoxide at C-6/C-7 and the aromatic substituent at C-10 were required for cytotoxicity 1 , and our results were consistent with literature. Compound 8 exhibited most potent cytotoxic activities toward HL-60, A-549, SMMC-7721, and SW-480 cell lines, and in contrast, compound 9, with the epoxide hydrolysed, only exhibited cytotoxicity against HL-60 and A-549 cell lines. Moreover, compound 3 was inactive at a concentration of 40 μM, which might be due to the destruction of the aromatic feature of the substituent at C-10 induced by epoxidation at the indole ring.
In summary, the fungus Chaetomium globosum has provided a rich spectrum of indole-based cytochalasans. In this study, nine new highly oxygenated cytochalasan derivatives (1-9), along with eight known chaetoglobosins, were isolated from the rice culture of Chaetomium globosum, isolated from the common pillbug (Armadillidium