New Cytotoxic Cytochalasans from a Plant-Associated Fungus Chaetomium globosum kz-19

Four new cytochalasans, phychaetoglobins A–D (1–4), together with twelve known cytochalasans (5–16), were isolated from a mangrove-associated fungus Chaetomium globosum kz-19. The new structures were elucidated on the basis of extensive 1D and 2D NMR, HR ESIMS spectroscopic analyses, and electronic circular dichroism (ECD) calculations. The absolute configuration of 2 was established by application of Mosher’s method. Compounds 4–8 exhibited moderate cytotoxicities against A549 and HeLa cell lines with the IC50 values less than 20 μM.


Introduction
Natural products from plant-associated fungi are a rich source of biologically promising lead compounds. The fungus genus Chaetomium is the rich source of cytochalasans, among which Chaetomium globosum can produce plenty of such secondary metabolites [1][2][3][4][5]. As the main secondary metabolites from C. globosum, cytochalasans are a class of compounds containing isoindolinone moieties fused to the tricyclic or tetracyclic ring systems, which have a variety of pharmacological activities, such as antitumor [6], antibacterial [7], immunomodulatory [8], and phytotoxicity [9]. The polyketo-amino acid hybrid structure of cytochalasans forms different fused macrocycles (typical 11-or 13-membered macrocycles) of cytochalasans, such as macrocycle, polycyclic, helix, epoxide, hydrogen peroxide groups and other unique structures. Biogenetically, cytochalasans are biosynthesized through a hybrid pathway of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). The che cluster in Penicillium expansum was recognized as the first gene cluster encoding PKS-NRPS in the biosynthesis of cytochalasans [10]. In addition, the oxidative tailoring enzymes of CcsB and CHGG_012343 catalyzed multiple steps involved in the process of biosynthesis [11]. In terms of anti-tumor pharmacological effects, many researchers found that cytochalasans could regulate the morphology and phenotype of tumor cells and induce apoptosis [12]. Thus, cytochalasans have the potential to be used as a supplementary chemotherapeutic drug and to decrease the incidence of drug resistance clinically in the future [13].

Structure Elucidation
Compound 1 was obtained as a pale yellow solid. Its molecular formula was deduc as C32H36N2O5 by HR ESIMS and NMR data, indicating 16 degrees of unsaturation. An ysis of the 1 H-NMR spectrum of 1 (Table 1)  , and three oxygenated groups 84.9, 59.9, 56.7), which had the characteristic skeleton of cytochalasans closely resembli chaetoglobosin C (5). Further analyses of 1 H and 13 C NMR, as well as the 2D NMR spec of 1 (Figure 2a), supported the presence of an indolyl moiety, a pyrrolidine-2-one, and cyclohexane ring in the structure, as was the case with that of 5. However, the differen between 1 and 5 were that 1 possessed two fewer ketone groups and one more ester gro than 5. The HMBC correlations from H-19 to C-17, C-18, and C-20, as well as from H-21 C-20, C-22, and C-23, revealed the presence of a lactone ring. Further HMBC correlatio  , and three oxygenated groups (δ C 84.9, 59.9, 56.7), which had the characteristic skeleton of cytochalasans closely resembling chaetoglobosin C (5). Further analyses of 1 H and 13 C NMR, as well as the 2D NMR spectra of 1 (Figure 2a), supported the presence of an indolyl moiety, a pyrrolidine-2-one, and a cyclohexane ring in the structure, as was the case with that of 5. However, the differences between 1 and 5 were that 1 possessed two fewer ketone groups and one more ester group than 5. The HMBC correlations from H-19 to C-17, C-18, and C-20, as well as from H-21 to C-20, C-22, and C-23, revealed the presence of a lactone ring. Further HMBC correlations from H-8 to C-9, C-13, C-14, and C-23, as well as from 18-CH 3 to C-17, C-18, and C-19, indicated the presence of two olefinic bonds and a ketone group on the macrocycle. The relative configuration of 1 was determined by a NOESY spectrum ( Figure  Consequently, the NOESY cross-peaks of H-3 with CH 3 -11, and CH 3 -12 with H-7, suggested that they were cofacial and in the α-orientation. Additionally, the NOESY correlations of H-22 with H-19 and H-21α, together with the coupling constant (J = 8.8 Hz) between H-19 and H-22, revealed the β-orientation of the five-numbered lactone ring. To determine the absolute configuration of 1, the theoretically calculated electronic circular dichroism (ECD) spectra were performed using time-dependent density functional theory (TDDFT). The Merck Molecular Force Field (MMFF) conformational search in an energy window of 10 kcal/mol, which was optimized at the ωB97X/TXVP level (in MeOH), yielded two lowenergy geometries with a Boltzmann population of over 1% ( Figure S9). The configurations of 1a and 1b (1b was the enantiomer of 1a) were compared using the ECD calculation at B3LYP level ( Figure S9). The experimental ECD curve of 1 was in agreement with the computed curve of 1a (Figure 2c). Therefore, the absolute structure of 1 was finally established as 3S,4R,5S,6R,7S,8R,9S,16S,19S,22R and named phychaetoglobin A.

Results and Discussion
Compound 2 was obtained as a colorless solid, and the molecular formula was determined to be C 31 H 38 N 2 O 3 based on the HRESIMS. A comparison of the 1 H and 13 C NMR spectra for 2 (Tables 1 and 2) with those of penochalasin J (14) suggested that 2 had a similar skeleton to 14. However, the signals for a double bond at C-17/C-18, a methyl at C-18, and a ketone group at C-20 in 14 were missing in the NMR spectra of 2. The information was supported by the HMBC correlations from CH 3 -16 to C-15, C-16 and C-17, and from H-18 to C-17, C-19, and C-20, as well as by the 1 H-1 H COSY spin system from H-16 to H-22. These correlations revealed the presence of three methylenes and a methine connected with a hydroxyl group at C-19. Further HMBC correlations from a pair of olefinic protons, H-21/H-22 to C-20 and C-23, also indicated the presence of an α, β-unsaturated ketone moiety at C-21, C-22, and C-23. The above observations confirmed the planar structure of 2. The relative configuration of 2 was deduced by a NOESY analysis, which was similar to that of 1. The NOESY cross-peaks of H-4/H-8/Me-11 inferred that they had the same β-orientations at these positions as 1. Moreover, Me-16 showed NOESY correlations with H-18 and H-14, which indicated the α-orientation of Me-16. The absolute configuration of C-19 was identified by the modified Mosher's method ( Figure 3). The methyl ester alcohol of 2 was treated with R-(−)and S-(+)-α-methyoxy-α-(trifluoromethyl) phenyl acetyl chloride (MTPA-Cl) to afford the Sand R-MTPA esters (2c and 2d), respectively. Analysis of the 1 H NMR and 1 H-1 H COSY spectra led to the assignment of both esters' chemical shifts in proximity at C-19. The results of ∆δ S-R values confirmed that the absolute configuration of C-19 was R (Figure 3). In addition, the ECD spectrum of 2 was determined and the Cotton effects were identical with the calculated curve of the enantiomer 2a (Figure 2c and Figure S18). Thus, compound 2 was identified and named phychaetoglobin B (Figure 1). To determine the absolute configuratio of 1, the theoretically calculated electronic circular dichroism (ECD) spectra were per formed using time-dependent density functional theory (TDDFT). The Merck Molecula Force Field (MMFF) conformational search in an energy window of 10 kcal/mol, whic was optimized at the ωB97X/TXVP level (in MeOH), yielded two low-energy geometrie with a Boltzmann population of over 1% ( Figure S9). The configurations of 1a and 1b (1 was the enantiomer of 1a) were compared using the ECD calculation at B3LYP level (Fig  ure S9). The experimental ECD curve of 1 was in agreement with the computed curve o 1a (Figure 2c). Therefore, the absolute structure of 1 was finally established a 3S,4R,5S,6R,7S,8R,9S,16S,19S,22R and named phychaetoglobin A.  1 and 2 (1a, 1b and 2a, 2b) in MeOH.
esters' chemical shifts in proximity at C-19. The results of ΔδS-R values conf absolute configuration of C-19 was R (Figure 3). In addition, the ECD spec determined and the Cotton effects were identical with the calculated curve omer 2a (Figures 2c and S18). Thus, compound 2 was identified and nam globin B (Figure 1). The molecular formula of 3 was determined to be C34H40N2O7 by HRES 16 degrees of unsaturation (Tables 1 and 2). The 13 C NMR and HSQC spect the presence of three ketones, two amide or ester groups, five methyls, an ated carbons. Analysis of the 1 H and 13 C-NMR data of 3 indicated that it structure to compound 7. However, when compared to 7, the differences we of a pair of double bonds at C-5/C-6, and the presence of a hydroxyl group group at C-6 in 3, which was implied by the HMBC correlations from Meand C-7. Additional HMBC correlations from H-7 to C-8, C-9, C-13, C-24 well as from Me-25 to C-24, revealed the presence of an ethoxycarbonyl gro NOESY correlations of H-8 with H-4 and H-5 indicated that they had an whereas the NOE correlations of CH3-12 with H-4 and CH3-11, as well as C 3, suggested the configuration of the cyclohexane ring. The absolute con was identified by comparing its experimental and calculated ECD data (Fig  3 was determined as 3S, 4R, 5S, 6R, 7S, 8R, 9S and named phychaetoglobin The molecular formula of 3 was determined to be C 34 H 40 N 2 O 7 by HRESIMS, requiring 16 degrees of unsaturation (Tables 1 and 2). The 13 C NMR and HSQC spectra of 3 showed the presence of three ketones, two amide or ester groups, five methyls, and two oxygenated carbons. Analysis of the 1 H and 13 C-NMR data of 3 indicated that it was similar in structure to compound 7. However, when compared to 7, the differences were the absence of a pair of double bonds at C-5/C-6, and the presence of a hydroxyl group and a methyl group at C-6 in 3, which was implied by the HMBC correlations from Me-12 to C-5, C-6, and C-7. Additional HMBC correlations from H-7 to C-8, C-9, C-13, C-24, and C-25, as well as from Me-25 to C-24, revealed the presence of an ethoxycarbonyl group at C-7. The NOESY correlations of H-8 with H-4 and H-5 indicated that they had an α-orientation, whereas the NOE correlations of CH 3 -12 with H-4 and CH 3 -11, as well as CH 3 -11 with H-3, suggested the configuration of the cyclohexane ring. The absolute configuration of 3 was identified by comparing its experimental and calculated ECD data ( Figure S30). Thus, 3 was determined as 3S, 4R, 5S, 6R, 7S, 8R, 9S and named phychaetoglobin C.
Compound 4 was determined as C 32 H 38 N 2 O 4 on the basis of HR ESIMS. Its NMR data suggested that 4 was an analogue of 8. However, 4 lacked the hydroxyl group on the cyclopentenone ring that was observed in 8. This finding was further confirmed by the HMBC correlations from CH 3 -18 to C-17 and C-19, as well as from H-19 to C-17, C-20, and C-21. The relative configuration of the five-membered ring from C-17 to C-21 was assigned by the NOESY spectrum. The NOE cross-peaks of H-16/H-17 and CH 3 -16/H-21 indicated that the methyl at C-16 and H-21 were in the α-orientation, whereas H-16 and H-17 were at the opposite side. The ECD spectrum was determined to confirm the absolute configuration, which was further compared to the experimental spectrum. The ECD spectrum generated for the cyclopentenone ring was 16S, 17S, 21S, which was consistent with the experimental data of 4a ( Figure S39). Therefore, the absolute configuration was established as in Figure 1 and named phychaetoglobin D.

Biological Assay
Compounds 1-16 were evaluated for their cytotoxicity against HeLa human colon adenocarcinoma cell lines and A549 human lung adenocarcinoma cells by MTT methods (Table 3). Adriamycin was used as a positive control. Compounds 5 and 7 exhibited the strongest cytotoxicity against A549 cell lines, with the IC 50 values below 10 µM. Furthermore, compounds 4-8 showed a moderate cytotoxicity against HeLa cell lines, with IC 50 values of 3.7~10.5 µM, implying that the moieties of a double bond at C-5/C-6 and a hydroxyl group at C-7 or an epoxide ring at C-6/C-7 were the characteristics primarily responsible for the cytotoxicity. Compounds 3, 10, 11, and 13-16 exhibited cytotoxic activity (IC 50 values of 12.2~33.7 µM), indicating that the presence of a ketone group other than a hydroxyl group at C-20 could increase the bioactivity. However, compound 1 displayed weak activity, which suggested that the lactone ring in 1 decreased the cytotoxicity. Compound 2 showed weak cytotoxicities, as it did not possess the functional groups related to the bioactivity. Thus, the five-membered lactone ring between C-17 and C-21 in 1 might decrease the cytotoxicity.

Fungal Material
The endophytic fungal KZ-19 was isolated from twigs of the mangrove plant Ceriops tagal, which was collected in Hainan province, China, in July 2013. The plant species was identified by Yi Sun, and the fungus was identified as a Chaetomium globosum by its rRNA gene sequence. The strain was deposited at the institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

Fermentation and Extraction
According to the previous investigation of culture conditions, the strain adopted the method of solid fermentation. The strain frozen at −80 • C was taken out and cultured on a plate of potato dextrose agar (PDA) medium at 27 ± 0.5 • C for 3 days. The mycelium was inoculated aseptically to 500 mL Erlenmeyer flasks, each containing 40 g of rice and 60 mL of distilled water. There were 100 flasks in total, and the flask cultures were incubated at 27 ± 0.5 • C for 7 days.

Isolation and Purification
After 7 days, the cultured rice with the fungus was cut into small fragments, which was then subsequently extracted with EtOAc by ultrasonication three times. The solvent was then removed under reduced pressure under vacuum to yield the total extract (4.7 g).