Hydrazine-Containing Heterocycle Cytochalasan Derivatives From Hydrazinolysis of Extracts of a Desert Soil-Derived Fungus Chaetomium madrasense 375

“Diversity-enhanced extracts” is an effective method of producing chemical libraries for the purpose of drug discovery. Three rare new cytochalasan derivative chaetoglobosins B1-B3 (1–3) were obtained from chemically engineered crude broth extracts of Chaetomium madrasense 375 prepared by reacting with hydrazine monohydrate and four known metabolite chaetoglobosins (4–7) were also identified from the fungus. The structures were identified by NMR and MS analysis and electronic circular dichroism simulation. In addition, the antiproliferative activities of these compounds were also evaluated, and the drug-resistant activities of cytochalasans were evaluated for the first time. Compound 6 possessed potent activity against four human cancer cells (A549, HCC827, SW620, and MDA-MB-231), and two drug-resistant HCC827 cells (Gefitinib-resistant, Osimertinib-resistant) compared with the positive controls.


INTRODUCTION
Natural products have played an important role in the development of novel drugs because of their established structural diversity (Newman and Cragg, 2016). However, research on natural products within the pharmaceutical industry has recently declined, and it has become more and more difficult to obtain compounds bearing skeletally novel structures from natural sources (Bradshaw et al., 2001;Wolfender and Queiroz, 2012). Chemical modification of natural product extracts provides a new strategy for discovering of diverse structurally active molecules. Recently, there have been several reports (Lopez et al., 2007;Mendez et al., 2011;Salazar et al., 2011;Ramallo et al., 2012;Kikuchi et al., 2014;Wu et al., 2015;Garcia et al., 2016) on the application of chemical alteration of natural product extracts to obtain new or bioactive compounds.
Cytochalasans comprise a large group of polyketide synthase and non-ribosomal peptide synthetase-derived fungal metabolites with a wide range of biological activities (Scherlach et al., 2010). The macrocyclic structure of most cytochalasans contains carbonyl functional groups, which could react with hydrazine to form either hydrazones or acyl hydrazides that rarely emerge in nature. This transformation could increase the nitrogen content and nucleophilic character of further reactions (Feher and Schmidt, 2003). In previous studies, we reported several bioactive cytochalasans that were isolated from C. madrasense 375 derived from desert soil . To gain more novel cytochalasan-like bioactive compounds, a chemical transformation of crude broth extracts of C. madrasense 375 with hydrazine monohydrate was performed, followed by purification of reaction products, which offered three new cytochalasans derivatives chaetoglobosins B 1 -B 3 (1-3), and known chaetoglobosin B (4) (Sekita et al., 1982(Sekita et al., , 1983 chaetoglobosin D (5) (Sekita et al., 1982(Sekita et al., , 1983, chaetoglobosin E (6) (Sekita et al., 1976), and cytoglobosin A (7) (Cui et al., 2010) were also isolated from the unreactive raw extract of this fungus. In this paper, we present the isolation, structure elucidation, bioactivities, and plausible synthesis pathways of these compounds.

General Experimental Procedures
CD spectra were determined on a JASCO J-810 spectropolarimeter (JASCO Corporation), and UV data were recorded using a PERSEE UV-VIS spectrophotometer T9 (Beijing, China). NMR experiments were carried out on a Bruker AVANCE III 400 NMR spectrometer (Bruker, Germany), using tetramethylsilane (TMS) or solvent signals as an internal reference. HRESIMS data were collected on an Agilent 6250 TOF LC/MS, and ESIMS data were acquired on an Agilent 1200 series LC/MS system. Semipreparative HPLC was run on a Calmflow plus system that was equipped with a YMC Pack ODS-A column (10 mm × 250 mm 5 µm, Japan) and a 50D UV-vis Detector (Lumiere Tech Ltd) and with a flow rate of 2.0 ml/min. Packing materials for column chromatography were silica gel (200-300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), ODS (YMC, Japan), and Sephadex LH-20 (GE Healthcare BioSciences AB, Sweden). All chemicals used in the study were of analytical grade.

Fungal Material and Fermentation
The samples of the fungus C. madrasense375 (CCTCC M 2019517 CLC375) were collected from a soil sample obtain at Hotan city, XinJiang province, People's Republic of China, and identified by one of the authors (XueWei Wang). The fungus was identified as C. madrasense according to its internal transcribed spacer (ITS) sequence of ITS rDNA (Supplementary Figure 34) and beta-tubulin encoding gene (Supplementary Figure 33) from genomic DNA, as well as its morphological features. A phylogenetic tree was constructed based on the sequence of the partial beta-tubulin gene from C. madrasense and other species in the genus Chaetomium, with Aspergillus nidulans as the outgroup (Supplementary Figure 32). The sequence of the strain was deposited in GenBank with accession number KP269060.1. The fungal strain was maintained on potato dextrose agar (PDA) at 25 • C for 7 days to prepare the seed culture. Agar plugs were cut into small pieces (approximately 1 cm × 1 cm) and inoculated into four 500 ml Erlenmeyer flasks containing 200 ml of synthetic dropout Medium (SD, peptone 10.0 g/L, dextrose 40.0 g/L) and incubated at 26 • C for 10 days on a rotary shaker (120 rpm). The obtained liquid seeds were transferred into fifty 500 ml Erlenmeyer flasks, each containing 200 ml of the sterilized synthetic dropout medium and incubated at 26 • C for 35 days on a rotary shaker (120 rpm) before harvest. The culture was filtered to separate broth and mycelia, and then extracted by ethyl acetate (three times) at room temperature. The combined extracts of broth and mycelia were dried under reduced pressure to give a dark brown gum (8.3 g). The crude extract was then suspended in H 2 O and extracted with petroleum ether, EtoAc, and n-BuOH, respectively. The EtoAc extract (5.6 g) was packed with cytochalasans based on the analysis of TLC and LC-MS experiments and divided into two parts, one (4 g) prepared for chemical modification, another (1.6 g) for metabolite research directly.

Quantum-Chemical Calculation
Monte Carlo conformational searches were run with the Spartan 14 software using the Merck Molecular Force Field (MMFF). The Selected conformers, which account for more than 1% of the Boltzmann distribution, were initially optimized at the B3LYP/6-31+G (d, p) level with the conductor-like polarizable continuum model (CPCM) conductor calculation in methanol solvent. The conformers of 3 were identified via ECD simulation using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31+G (d, p) level with methanol as solvent, and the rotational strengths of 30 excited states were calculated. ECD spectra of compound 3 were obtained using the SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) software by applying Gaussian band shapes with sigma = 0.3 eV from dipole-length rotational strengths .

Antiproliferative Assay
Antiproliferative activity was performed against four human cancer cell lines (A549, SW620, MDA-MB-231, and HCC827), together with two drug-resistant (gefitinib, osimertinib) HCC827cell lines, applying the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method (Chen et al., 2014) with the DDP (cisplatin, Sigma), gefitinib, and osimertinib as positive controls, respectively. The cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 • C under a humidified atmosphere of 5% CO 2 . Cells (3 × 10 4 /well) were seeded in a 96-well plate and incubated for 24 h. The test compounds at different concentrations were added to each well and further incubated for 24 or 48 h under the same conditions. Then, 10 µl of the MTT was added to each well at a concentration of 5 mg/ml and incubated for 4 h. The medium containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Tecan Sunrise microplate reader at 490 nm. The concentration required to inhibit cell growth by 50% (IC 50 ) was calculated from inhibition curves.

RESULTS AND DISCUSSION
The ethyl acetate extract of C. madrasense 375 was obtained from the submerged fermentation liquid and divided into two parts; part 1 was treated with hydrazine monohydrazine and hexahydropyridine (catalyst) to give a chemically modified production. HPLC (Supplementary Figure 35) and 13 C NMR (Supplementary Figure 36) were performed to analyze changes in the extract. Modified production and raw extract were further separated and purified through reversed-phase C18 (ODS) column chromatography, Sephadex LH-20, as well as semipreparative HPLC to afford compounds 1-7 (Figure 1).
Chaetoglobosin B 3 (3) was obtained as a white amorphous powder. Its molecular formula was assigned as C 32 H 34 N 6 O 4 (19 unsaturations) by HR-ESI-MS (Supplementary Figure 26), which showed a protonated molecule peak at m/z 567.2738 [M+H] + . The 1 H and 13 C NMR data (Tables 1, 2;  Supplementary Figures 20, 21) suggested compounds 3 and 1 possessed the same core structure except that the heterocyclic incorporated to the 13-membered macrocyclic ring moiety. Comparing the molecular formula of 3 and 1 suggested the presence of two more nitrogen atoms and four less hydrogen atoms, indicating that two heterocyclic moieties with nitrogen atoms possibly incorporated into chaetoglobosins core structure. However, on the basis of NMR data (Tables 1, 2; Supplementary Figures 20-25) and molecular formula, there may be four possible structures (Supplementary Figure 37) of compound 3. Considering chemical synthesis, the structure of 3a accords with the reaction production of chaetoglobosin B and hydrazine monohydrate. To further verify the accurate configuration of C-19 of 3, the ECD experiments and ECD simulation (Figure 3) were both performed. The results corroborated that the configuration of C-19 was established as 19R. Thus, compound 3 was named chaetoglobosin B 3 as shown in Figure 1.
The known cytochalasan alkaloid, chaetoglobosin B (4), chaetoglobosin D (5), chaetoglobosin E (6), and cytoglobosin A (7) were identified by comparing their 1D NMR and ESI-MS data with those in the scientific literature. To make sure that 3 is not a scalemic or racemic mixture, an HPLC analysis was carried out on a Agilent 1200 (Agilent Technologies, USA) system with a chiral column Daicel Chiralcel OD-H column (Supplementary Figure 38). The result indicated that 3 is a monomeric compound.

The Plausible Synthesis Pathway
The putative synthesis pathway for compounds 1-3 was proposed starting from chaetoglobosin B through hydrazinolysis reaction. The precursor chaetoglobosin B underwent an intermolecular nuclephilic addition-elimination reaction with hydrazine, catalyzed by hexahydropyridine to give the intermediates i, followed by [1, 4] addition, ring opening, isomerization, and [1, 5] proton transfer reactions led to the formation of 1 (Scheme 1A). 2 was formed by an elimination and hydrogenation reduction of intermediates i (Scheme 1A). 3 was an unexpected production of cytochalasan and hydrazine monohydrate. There was little literature on the formation of 3 and its analogs. Herein, we surmised that 3 may be not directly produced by cytochalasan and hydrazine monohydrate. Therefore, we propound that 3 may be formed by cycloaddition of cytochalasan and the oxidation of hydrazine monohydrate. The plausible pathway of 3 is proposed as shown in Scheme 1B.

Biological Activity
Compounds 1-7 were evaluated for their inhibitory effects toward human non-small-cell lung carcinoma (A549, HCC827), human colon cancer (SW620), and human breast cancer (MDA-MB-231) cell lines. As shown in Table 3, compounds 1 and 7 exhibited selected antiproliferative activity on non-smallcell lung carcinoma A549 and human colon cancer SW620 cells, respectively. Compound 4 showed selected antiproliferative activity on three cancer cell lines, HCC827, SW620, and MDA-MB-231with IC 50 values of 11.6, 8.8, and 11.4 µM, respectively. Compound 5 displayed moderate antiproliferative activity on four cancer cell lines with the IC 50 values ranging from 6.9 to 10.6 µM. It is noteworthy that compound 6 possessed potent activity (IC 50 values ranging from 1.7 to 9.9 µM), which were all stronger than the positive control cisplatin. In addition, compound 6 showed a more significant inhibitory effect on two drug-resistant HCC827 cells (Gefitinib-resistant, Osimertinibresistant) with IC 50 values of 5.1 and 2.8 µM, respectively. However, compounds 2-3 showed no obvious inhibitory effect (IC 50 > 20 µM). According to the result of the cytotoxicity assay (Tables 3, 4), it is worth noting that the heterocyclic ring fused into the macrocyclic of compounds 1-3 may be reduced the activity by blocking the interaction with its site of action.

CONCLUSION
As a fungus, the Chaetomium genus contains more than 100 species and plenty of structural novel metabolites (including cytochalasans) with a wide range of biological activities represented from the species (Li et al., 2018). To broaden the chemical diversity of cytochalasans with interesting activity, three novel cytochalasan derivatives, termed chaetoglobosins B 1 -B 3 (1-3) were isolated from the chemical modification of an extract of C. madrasense 375, and four known metabolite chaetoglobosins (4-7) were also identified. Compounds 1-3 represented the first examples of hydrazine-containing heterocycle fused into the macrocyclic of cytochalasans. The structures of 1-3 were elucidated by means of spectroscopic data, quantum-chemical ECD simulation. In the antiproliferative

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.