Two new p-methoxyphenyl-type derivatives from a saline-lake derived Streptomyces sp. XZB32

Abstract Two new p-methoxyphenyl-type derivatives cytchloramol (1) and cytoxazinanone (2), along with six known compounds (3-8) were identified from the chemical investigations of a saline lake actinomycete, Streptomyces sp. XZB32. The structures of the new compounds were elucidated by extensive NMR spectroscopic analysis, HRESIMS data, GIAO (gauge-including atomic orbitals) NMR, specific optical rotation (SOR) and electronic circular dichroism (ECD) calculations. Cytotoxicity evaluation of the two new compounds showed that compound 1 exhibited significant activity against HCT-116 and MDA-MB-231 human cancer cell line with IC50 values of 2.7 ± 0.07 µM and 1.54 ± 0.14 µM, respectively. Graphical Abstract


Introduction
Natural products play a crucial role in new drugs and lead compounds research, and around 70% natural antibiotics are derived from actinomycetes (Butler 2004;Newman and Cragg 2016;Yang et al. 2019).However, the number of crucial natural products from normal soil-derived actinomycetes has decreased so that there is an increasing attention to the secondary metabolites isolated from hypersaline environment actinomycetes such as saline lakes, saline soils and salterns (Bull et al. 2000;Mincer et al. 2002;Dror et al. 2020).
As a part of our research for discovering structurally novel and bioactive structures from microorganisms, a saline lake actinomycete classified as Streptomyces sp.XZB32 was chemically investigated.As a result, one new oxazinanone derivative named cytoxazinanone (2) and one of its possible new precursors named cytchloromamol (1), along with six known homologues were identified (Figure 1).The main product was identified as cytoxazone (3), which was first reported in 1998 and identified as a selective regulator of T helper type 2 cells cytokine secretion (Kakeya et al. 1998(Kakeya et al. , 1999)).It also had been reported that cytoxazone derivatives affected the proliferation of the prostate cancer cell (Naresh et al. 2014).We herein described the details of isolation, structure elucidation, and the cytotoxicity of the new compounds.

Results and discussion
Streptomyces sp.XZB32 was separately fermented in Gause's or 2216E medium and subsequently extracted with EtOAc.The EtOAc extracts from the cultures (50 L total for Gause's and 32.5 L total for 2216E) were separated by silica gel and preparative high performance liquid chromatography purification to afford eight compounds.
Compound 1 possessed a molecular formula of C 10 H 14 NO 2 Cl based on the NMR data and the HRESIMS ion peak at m/z 216.0790 [M þ H] þ , which indicated eight index of hydrogen deficiency.The IR spectrum showed the presence of a benzene ring (1673 cm À1 ) and an hydroxy group (3069 cm À1 ).Carefully analysis of 1 H NMR spectroscopic data (Tables S1 and S2, Supporting Information) combined with HSQC correlations of 1 revealed a typical para-substituted benzene ring [d H 7.40 (2H, d  J ¼ 9.0 Hz), 4.17 (1H,ddd,J ¼ 8.6,4.6,3.3 Hz)]. 13C NMR spectrum indicated two oxygenated carbons (d C 58.52 and 55.87), one amino carbon d C 47.33, and one carbon d C 72.97 attached to chlorine atom.According to the COSY and HMBC correlations (Figure S13, Supporting Information), it is obvious that C-1 is directly connected to the benzene ring because of the strong HMBC correlations from H-1/C-1 0 , H-1/C-2 0 and H-1/C-6 0 .While a methoxy group located in the para position because of the HMBC correlation from its protons to C-4 0 and the obvious low-field chemical shifts.Besides, C-2 and C-3 are connected to C-1 in sequence due to the COSY correlations of H-1/H-2 and H-2/H-3 and the HMBC correlations of H-1/C-2, H-1/C-3, H-2/C-1, H-3/C-2 and H-3/ C-1.
GIAO (gauge-including atomic orbitals) NMR calculations for structure identification developed gradually these years (Barone, Duca, et al. 2002;Barone, Gomez-Paloma, et al. 2002).The result 13 C chemical shifts data (Table S4, Supporting Information) of the calculations with the GIAO 1 D NMR were made into linear regression curves (Figure S14, Supporting Information) and then corrected them by respective regression fit equations (Table S5, Supporting Information).Compared the corrected mean absolute deviation (CMAD) of 1a-1d (Table S5, Supporting Information), a pair of enantiomers (1a and 1 b) were excluded due to the higher CMAD values.The absolute configuration of 1 was determined by comparing the experimental and the calculated specific optical rotation data of the remaining conformers (1c and 1d), with a result showed that the calculated SOR value of 1c was À5.95 and the value of 1d was 6.35, respectively, which were corrected by fitting the calculated specific optical rotation of the several different conformations (Tables S6-S7, Supporting Information).Compared with the experimental SOR value of 6.0, compound 1 was determined as (1S,2R)-3amino-1-chloro-1-(4-methoxyphenyl)propan-2-ol, named cytchloramol.
Compound 2 had an [M þ H] þ ion peak at m/z 224.0916 in the HRESIMS data, consistent with the molecular formula C 11 H 13 NO 4 , indicating twelve index of hydrogen deficiency. 1 D NMR spectroscopic data (Tables S1 and S2, Supporting Information) and HSQC correlations of 2 obviously revealed that the para-substitution benzene ring [d H 7.32 (2H, d, J ¼ 8.7 Hz), 6.94 (2H, d, J ¼ 8.7 Hz)] and the methyl group d H 3.79 (3H, s) were similar to that of 1.In addition, two protons [d H 4.71 (1H, d, J ¼ 3.3 Hz), 3.98 (1H, dt, J ¼ 3.3, 1.8 Hz)] of two methines (C-4 d C 59.8 and C-5 dc 64.6), two protons [d H 4.46 (1H, dd, J ¼ 11.5, 1.5 Hz), 4.27 (1H, dd, J ¼ 11.5, 3.3 Hz)] of one methylene (C-6 d C 71.9), and one carbonyl carbon (C-2 d C 156.2) were observed in the spectrum.According to the 2 D NMR spectroscopic data, the COSY correlations of H-4/H-5 and H-5/H-6 meant that C-6, C-5 were connected to C-4 in sequence.Besides, C-4 was directly attached to the benzene ring, due to the HMBC correlations from H-4 to C-1 0 , C-2 0 and C-6 0 .C-6 was attached to C-2 through an oxygen atom because of the H-6/C-2 HMBC correlation and the low-field chemical shifts of H-6.There was also a secondary amino group between C-2 and C-4, which formed a six-membered ring and explained the rest two index of hydrogen deficiency.
The relative configuration of 2 was assigned based on the analysis of proton coupling constants between H-4 and H-5 (J ¼ 3.3 Hz), which indicated the cis configuration at C-4/C-5.The absolute configuration of 2 was determined by quantum chemical ECD method.As shown in Figure S15, the calculated ECD curve of (4 R,5S)-2 revealed a good agreement with the measured experimental spectrum.Thus, the absolute structure of 2 was determined as shown, and named cytoxazinanone.
Two new compounds 1 and 2 were obtained by cultivating with 2216E liquid medium instead of Gause's liquid medium.It is suspected that the carbon source and the type or concentration of inorganic salts are different between the two media.
The new compounds were evaluated for their cytotoxicity against the HCT-116 and the MDA-MB-231 cancer cell lines (Table S3, Supporting Information).As a results, compound 1 exhibited significant cytotoxicity against HCT-116 cells and MDA-MB-231 cells with IC 50 values of 2.75 ± 0.07 mM and 1.54 ± 0.14 mM, respectively.

General experimental procedures
Shimadzu UV-1800 spectrophotometer and Thermo Fisher Nicolet IS10 spectrophotometer were used for measuring UV and IR spectra.High resolution mass data were detected by Agilent 1260 HPLC-6230 TOF tandem spectrometer.Shimadzu DGU 20 A 5 system equipped with an Agilent Pursuit XRs-C18 column (10 lm, 4.6 Â 250 mm) was applied to HPLC analysis.1 D and 2 D NMR were recorded on a JEOL JNM-ECZR instrument using TMS as an internal standard.Specific optical rotations were determined with a Rudolph API-35201 automatic polarimeter.ECD spectra were collected on a JASCO J-1500-150ST circular dichroism spectormeter.Crude extract was separated by silica gel CC (100-200 or 300-400 mesh, Qingdao Haiyang Chemical Company) and Sephadex LH-20 (Amersham Pharmacia Biotech) column chromatography and purified by using Shimadzu LC-20AP system equipped with an Agilent Pursuit C-18 column (10 lm, 21.2 Â 250 mm) was applied to further purification.

Actinomycic material
Streptomyces sp.XZB32 was isolated from the saline lake (Tibet Autonomous Region, China) by using a standard dilution plating method.The strain was maintained in Gause's solid medium consisting of (per liter) 20 g of soluble starch, 1 g of KNO 3 , 0.5 g of K 2 HPO 4 , 0.5 g of MgSO 4 7H 2 O, 0.01 g of FeSO 4 7H 2 O, 20 g of agar, 20 g of artificial sea salt, pH ¼ 7.2-7.4and subcultured monthly.Moreover, the strain was also maintained in 2216E solid medium consisting of (per liter) 5 g of peptone, 1 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of NaCl, 5.98 g of MgCl 2 , 3.24 g of Na 2 SO 4 , 1.8 g of CaCl 2 , 0.55 g of KCl, 0.16 g of Na 2 CO 3 , 0.08 g of KBr, 0.034 g of SrCl 2 , 0.022 g of boric acid, 0.004 g of sodium silicate, 0.0024 g of NaF, 0.0016 g of NH 4 NO 3 , 0.008 g of Na 2 HPO 4 .The strain was identified using 16S rDNA sequence analysis by Sangon Biotech (Shanghai, China), and its DNA sequence was compared to the GenBank database using BLAST (nucleotide sequence comparison).The 16S rDNA sequence of strain XZB32 has been deposited in GenBank (accession number ON197357).A voucher strain (Streptomyces sp.XZB32) is preserved at the Laboratory of Institute of Marine Biology Pharmacology, Ocean College, Zhejiang University, China.

Extraction and isolation
Colonies of the strain XZB32 growing on Gause's agar plate or 2216E agar plate were inoculated into 500 mL Erlenmeyer flasks, containing 250 mL of Gause's liquid medium or 250 mL of 2216E liquid medium, and then incubated at 28 C for 8 days on a rotary shaker (180 rpm).A total of two-hundred Erlenmeyer flasks for Gause's liquid medium and a hundred and thirty Erlenmeyer flasks for 2216E liquid medium were prepared for this study.

Computational details
The systematic random conformational analysis of compounds was performed in the GMMX program by using MMFF94 molecular force field.The conformers were optimized using a DFT force field at the B3LYP/def2-TZVP level of theory in CH 3 OH by using Gaussian 16 software (Frisch et al. 2016).The optimized conformers were used B3LYP/6-311G(2d,p) level of theory with the TDDFT model.The ECD curves were all weighted by the Boltzmann distribution.The calculated ECD spectra of (4 R,5S)-2 and (4S,5R)-2 were compared with the experimental data.The ECD spectra were produced by SpecDis 1.70.1 software (Bruhn et al. 2017).The NMR shielding constants of the optimized stable conformers were calculated with the GIAO method at the B3LYP/ def2-TZVP level of theory in CH 3 OH.The NMR chemical shifts were calculated for each conformer according to the Boltzmann distribution and relative energies.Excluded a pair of highly deviated enantiomers by CMAD.The specific optical rotation values of the conformers generated for NMR calculations were calculated at the B3LYP/def2-TZVP level of theory in CH 3 OH.The calculated specific optical rotation data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy, and the fitted SOR values of 1c and 1d were obtained.

Cytotoxic assay
Compounds 1 and 2 were tested for cytotoxic activity against HCT-116 and MDA-MB-231 cancer cell lines in triplicate, using sulforhodamine B method.The IC 50 value is calculated through SPSS software.Detailed procedures were previously described (Zhou et al. 2018).