Cytotoxic and Optically Active Pyrisulfoxins From the Endophytic Streptomyces albolongus EA12432

R-Pyrisulfoxin C (1), S-pyrisulfoxin D [(+)-2], R-pyrisulfoxin D [(–)-2], pyrisulfoxin E (13), S-pyrisulfoxin F [(+)-14], and R-pyrisulfoxin F [(–)-14], six new caerulomycin derivatives with a 2,2′-bipyridine skeleton, were obtained from the cultures of the endophytic Streptomyces albolongus EA12432 with Aconitum carmichaeli (Ranunculaceae). Additionally, the racemic pyrisulfoxins A [(±)-3] and B [(±)-4] were further purified as optically pure compounds and identified the configurations for the first time. The racemic pyrisulfoxin D [(±)-2] displayed significant cytotoxicity against a series of cancer cell lines with IC50 values ranging from 0.92 to 9.71 μM. Compounds 7, 8, and (±)-3 showed cytotoxicity against the HCT-116, HT-29, BXPC-3, P6C, and MCF-7 cell lines. Notably, compounds 7 and 8 have a strong inhibition both on the proliferation of human colon cancer cells HCT-116 and HT-29 with IC50 values ranging from 0.048 to 0.2 μM (doxorubicin, 0.21 and 0.16 μM), and compound 1 showed a selective inhibition on the proliferation of the gastric carcinoma cell lines, N87, with an IC50 value of 8.09 μM. Optically pure compounds R(–)-14 and S(+)-14 showed weak cytotoxicity against HCT-116 and MCF-7 cell lines with the IC50 values of 14.7 μM and 10.4 μM, respectively. Interestingly, compounds 1 and (±)-2 didn't show cytotoxic activity against two human normal cell lines, HEK-293F and L02, with IC50 values >100 μM.


General Experimental Procedures
Melting points were obtained on X-4 digital display micromelting point measuring instrument. Optical rotations were measured with POLAX-L polarimeter. Ultraviolet (UV) spectra were recorded on a Thermo Fisher Scientific NanoDrop One micro-spectrophotometer. IR spectra were taken on a Nicolet Nexus 470 spectrophotometer using KBr pellets. ECD spectra were measured on a JASCO-815 spectrometer (JASCO, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra of compounds 1, 2, and 4-13 were recorded on a Bruker Avance 500 MHz spectrometer while 2a, 3, and 14, were measured on a JEOL JNM-ECP 600 spectrometer with TMS as an internal standard. Chemical shift (δ) was expressed in ppm with reference to the solvent signals. Mass spectra were recorded on an Agilent 6200 Q-TOF MS system. Thin layer chromatography (TLC) was performed on plates precoated with silica gel GF 254 (10-40 µm). Column chromatography (CC) was performed on silica gel (100-200 mesh, 200-300 mesh, 300-400 mesh, Qingdao Marine Chemical Ltd., Qingdao, People's Republic of China), , and Sephadex LH-20 (Amersham Biosciences). Medium-pressure liquid chromatography (MPLC) was performed on a LC3000 equipped with a P3000A pump modules, and columns packed with RP-18 gel. Semipreparative high-performance liquid chromatography (HPLC) was performed using an octadecyl silica (ODS) column [YMCpack ODS-A, 10 × 250 mm, 5 µm, 4 ml/min].

Fermentation and Extraction
Spores were inoculated into 500 ml Erlenmeyer flasks containing 150 ml liquid medium that was prepared by dissolving soluble starch (20 g), KNO 3 (1 g), K 2 HPO 4 ·3H 2 O (0.5 g), MgSO 4 ·7H 2 O (0.5 g), FeSO 4 (0.01 g), and NaCl (0.5 g) in sea water (1L). The flasks were incubated at 180 rpm and 28 • C for 5 days as seed culture (OD 600 1.375), which was then inoculated into 200 × 1,000 ml Erlenmeyer flasks, each containing 80 g rice and 40 ml sea water. All the media were statically cultured at 28 • C for 30 d. The culture broth was extracted with ethyl acetate (EtOAc) four times (30 L each). The EtOAc extracts were concentrated under reduced pressure to yield a dark brown gum 30 (40.2 g).
Spores were inoculated into 500 ml Erlenmeyer flasks containing 150 ml liquid medium that was prepared by dissolving soluble starch (20 g), KNO 3 (1 g), K 2 HPO 4 ·3H 2 O (0.5 g), MgSO 4 ·7H 2 O (0.5 g), FeSO 4 (0.01 g), and NaCl (0.5 g) in sea water (1L). The flasks were incubated at 180 rpm and 28 • C for 5 days as seed culture (OD 600 1.504), which was then inoculated into 1,000 ml Erlenmeyer flasks containing 80 g rice and 40 ml sea water. The flasks were incubated at room temperature for 90 d. The culture broth was soaked and extracted with ethyl acetate (EtOAc) four times (30 L each). The EtOAc extracts were concentrated under reduced pressure to yield a dark brown gum 90 (10 g).

Oxidation of Compound 5 to (±)-2
Compound 5 (9.0 mg) was dissolved into 2.25 ml of THF and 0.45 ml of water. The mixture was cooled in an ice bath, and 120 µl of aqueous solution of potassium peroxomonosulfate (Oxone) (0.1 g/ml) was added dropwise. The resulting mixture was stirred at 0 • C for 2.5 h. Then, 1 ml of aqueous solution of NaHCO 3 (pH 8) was added to quench the reaction. The reaction product was extracted by n-BuOH to afford 9.2 mg (96.8% yield) of (±)-2, which was identified by the same MS ( Figure S22) and co-HPLC retention time (t R 10.5 min, Figure S39) and NMR (Figures S23, S24, Tables 1, 3) to those of natural one.

ECD Calculation
The calculations were performed by using the density functional theory (DFT) as carried out in the Gaussian 09 (Frisch et al., 2010). The preliminary conformational distributions search was performed by HyperChem 8.0 software. All groundstate geometries were optimized at the B3LYP/6-31G(d) level (Stephens et al., 2007) (Tables S4-S6). Time-dependent DFT (TDDFT) at B3LYP/6-31G(d) was employed to calculate the electronic excitation energies and rotational strengths in MeOH (Casida, 1995) (Tables S7-S9). The overall calculated ECD curves were weighted by Boltzmann distribution with a halfbandwidth of 0.30 eV and UV corrections of compounds (S)-2, (R)-3, and (S)-14 were −15, 0, and 0 nm, respectively. The calculated ECD spectra were produced by SpecDis 1.70.1 software (Bruhn et al., 2017). Solvent effects of MeOH were evaluated at the same DFT level by use of the SCRF/PCM method (Cammi and Tomasi, 1995).

Cytotoxic Assays
The cytotoxic activities of compounds 1 and (±)-2 were detected by CellTiter-Glo R (CTG) assay (He et al., 2019)  penicillin-streptomycin solution under a humidified atmosphere of 95% air and 5% CO 2 at 37 • C. Ninety microliter of culture solution (containing fetal bovine serum) and 100 µl of cell suspension at a density of 2 × 10 3 cell/ml was plated in 96-well microtiter plates, allowed to attach overnight, and then exposed to 10 µl of drugs for 72 h within the final concentrations of 0.032, 0.16, 0.8, 4, 20, and 100 µM, respectively. Hundred microliter of the CTG solution was then added to each well and incubated for 10 min, and the absorbance was read at 500 nm on a Spectra Max Plus plate reader. Adriamycin was used as the positive control.
The cytotoxicities of compounds 3-12 against Jurkat, K562, MCF-7, and P6C cell lines were assayed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Mosmann, 1983). MCF-7 and P6C cell lines were grown in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) under a humidified atmosphere of 5% CO 2 and 95% air at 37 • C, respectively. Cell suspension, 200 µl, at a density of 5 × 10 4 cell/ml was plated in 96-well microtiter plates and incubated for 24 h. Then, the samples to be tested (final concentration, 10 µM) were added to each well and further incubated for 72 h. Twenty microliter of MTT solution (5 mg/ml in IPMI-1640) was then added to each well and incubated for 4 h. Old medium containing MTT (150 µl) was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. Jurkat and K562 cell lines were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin under a humidified atmosphere of 5% CO 2 at 37 • C. The cells in the logarithmic growth phase were seeded in 96-well plates at 8,000 cells/well (180 ml/well). After 24 h at 37 • C and 5% CO 2 , the samples to be tested were added (final concentration, 10 µM) and three replicate wells were set for each concentration. The solvent control Dimethyl Sulfoxide (DMSO) was used in an amount of 0.1% of the maximum dose used in the test group. After 72 h of drug treatment at 37 • C and 5% CO 2 , 20 µl of MTT (5 mg/ml) was added and incubated for another 4 h. Then, 100 µl mixture of 10% SDS, 5% isopropanol and 12 mM HCl was added and incubated for 12-20 h. The optical density of each well at 570 nm was read by a microplate reader.
The cytotoxicities of compounds 3-12 against HT29, HCT-116, and BXPC-3 cell lines were assayed by SRB method (Skehan et al., 1990). They were cultured as K562 and Jurkat cell lines described above. The cells in the logarithmic growth phase were seeded in 96-well plates at 8,000 cells/well (180 ml/well). After 24 h in 5% CO 2 and 37 • C, the samples to be tested were added (final concentration, 10 µM) and three replicate wells were set for each concentration. The solvent control DMSO was used in an amount of 0.1% of the maximum dose used in the test group. After 72 h of drug treatment at 37 • C and 5% CO 2 , 50% (m/v) ice-cold trichloroacetic acid was added to each well to fix the cells. After SRB staining, 150 µl/well of Tris solution was added and the optical density of each well at 540 nm was read in a microplate reader.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.