Dimeric Pimprinine Alkaloids From Soil-Derived Streptomyces sp. NEAU-C99

Six new pimprinine alkaloids (1–6), including four dimers, dipimprinines A–D (1–4), and two monomers, (±)-Pimprinol D (5), and pimprinone A (6), along with six known congeners (7–12), were isolated from a soil-derived actinomycete Streptomyces sp. NEAU-C99. Structures of the new compounds were elucidated by extensive spectroscopic analyses, single-crystal X-ray diffractions, and ECD calculations. Dipimprinines A–D (1–4) showed weak cytotoxic activities against five tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW-480, with IC50 values ranging from 12.7 to 30.7 μM.


INTRODUCTION
Natural products, in particular secondary metabolites derived from actinomycetes, Gram-positive bacteria (Hoshino et al., 2018;Yang et al., 2018), such as antibiotics, enzymes, enzyme inhibitors, and other pharmacologically active agents (Sripreechasak et al., 2013), have contributed substantially to modern medical care (Onaka, 2017). These microbial natural products are still an attractive and indispensable resources for drug discovery due to their potential productivity of unique core skeletons, such as the antiparasitic drug ivermectin (Cragg and Newman, 2013) and the anticancer agent eribulin (Yu et al., 2011). Pimprinine is an indole alkaloid, which was first isolated from the filtrates of Streptomyces pimprina cultures in 1963 (Joshi et al., 1963). Members of this family display a range of biological activities, such as antiepileptic (Naik et al., 2001;Roy et al., 2006), platelet-aggregation-inhibitory (Miao et al., 2004), antitumor (Pettit et al., 2002), fungicidal (Zhang et al., 2012), and anti-plant-viral activities (Liu et al., 2019).

General Experimental Procedures
NMR spectra were recorded in methanol-d 4 or CDCl 3 using a Bruker AVANCE III-600 or AVANCE III-400 spectrometer (Bruker Corp., Switzerland), and tetramethylsilane (TMS) was used as internal standard. HRESIMS data were obtained using an Agilent G6230 Q-TOF mass instrument (Agilent Corp., USA) or a Shimadzu UPLC-IT-TOF mass instrument (Shimadzu Corp., Japan). Optical rotation data were determined in MeOH on an Autopol VI S2&Plus polarimeter (Rudolph Research Analytical, Hackettstown, USA). CD spectra were recorded on an Applied Photophysics digital circular dichroism chiroptical spectrometer (Applied Photophysics Limited, Surrey, United Kingdom). IR spectra were measured on a Nicolet TM iS TM 10 FT-IR spectrometer with KBr disks (Thermo Fisher Scientific, Waltham, USA). X-ray crystallographic analysis was carried out with a Bruker APEX DUO single crystal X-ray diffractometer (Bruker Corp., Switzerland). Thin-layer chromatography (TLC) was performed using precoated silica gel GF254 plates (0.25 mm in thickness, Qingdao Marine Chemical Inc., China), and spots were visualized by UV light (254 nm) and colored by spraying heated silica gel plates with 10% H 2 SO 4 in ethanol. Semipreparative HPLC was conducted on a HITACHI Chromaster system (Hitachi Corp., Japan) equipped with a DAD detector, an YMC-Hydrosphere C 18 column (250 × 10 mm i.d., 5 µm) at a flow rate of 3.0 mL/min and a column temperature of 25 • C.

Bacterial Strains
The strain Streptomyces sp. NEAU-C99 was isolated from a soil sample collected in Mount Song, Henan Province, China, in 2016. It was identified as Streptomyces sp. on the basis of the morphological characteristics and 16S rRNA gene sequence (GenBank: MN647558) with closest homology to that of Streptomyces netropsis strain SXYM16 (100% similarity, GenBank: JN999913.1).

Cytotoxicity Assay
Five tested human tumor cell lines, human leukemia (HL-60), hepatocellular carcinoma (SMMC-7721), lung cancer (A-549), breast adenocarcinoma (MCF-7), and colon carcinoma (SW-480), were purchased from ATCC (Manassas, VA, USA). Each of these cell lines was incubated in medium DMEM or RPMI-1640 containing 10% fetal bovine serum at 37 • C under humidified atmosphere with 5% CO 2 . Cytotoxicity of the isolates toward these tumor cell lines was assessed via the 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4sulfopheny)-2H tetrazolium (MTS) (Promega, Madison, WI, USA) method (Cory et al., 1991), and cisplatin (Sigma) was used as a positive control. The cell lines were inoculated into each well of the normal 96-well plates and incubated for 12 h before addition of the test isolates. Different concentrations of each compound were added and exposed to the cells for a continuous cultivation of 48 h. The isolates with inhibition rates ≥50% against the cell lines were further assessed in triplicate at different concentrations (0.064, 0.32, 1.6, 8, and 40 µM). The IC 50 values were measured based on Reed and Muench's method (Reed and Muench, 1938). All the experiments were carried out in triplicate. . The 13 C and DEPT spectra of 1 suggested the presence of 26 carbons, which were classified into two methyls, two methylenes, 11 aromatic nonprotonated carbons, and 11 aromatic methine carbons (Table 1, Figure S4). These signals appeared in pairs in the 13 C NMR spectrum, which were very similar to those of pimprinethine (Pettit et al., 2002). The aforementioned spectroscopic evidences suggested that compound 1 was likely a dimeric pimprinine alkaloid.
Dipimprinine C (3) shared the same molecular formula C 28 H 26 N 4 O 2 with 2 as determined by the HRESIMS ion peak at m/z 435.1840 [M-H] − (calcd for 435.1826) (Figure S24), suggesting 3 is an isomer of 2. Indeed, the 1 H and 13 C-NMR chemical shifts of 3 were almost the same as those of 2 (Table 1, Figures S19, S20), but differed in the 1 H-NMR splitting pattern of the proton signals at δ H 2.99 and δ H 2.81, the signal at δ H 2.99 was a quartet in 2 but a triplet in 3, while the other signal at δ H 2.81 was a triplet in 2 but a quartet in 3. Based on the in-depth  interpretation of its 1D NMR data ( Table 1) and 2D NMR data (Figures S21-S23), particularly the 1 H-1 H COSY and HMBC correlations, 3 was further revealed as a structural analog of 2 with the obvious HMBC correlations from H 2 -13 to C-14 and from H 2 -13 ′ to C-14 ′ /C-15 ′ and the 1 H-1 H COSY cross-peaks of H 2 -13/H 3 -14 and H 2 -13 ′ /H 2 -14 ′ /H 3 -15 ′ (Figure S2). Therefore, the structure of compound 3 was identified as shown in Figure 1.
As mentioned above, the stereochemistry of rings in the monomeric pimprinines turns out to be planar according to the result of X-ray crystallographic analysis (Figure 3). To investigate the possible potential axial chirality in dimeric pimprinine molecules, CD spectra for dipimprinines A-D (1-4) were acquired (Figures S10, S18, S26, S34). Unlike the reported natural dimeric atropisomers (Wang et al., 2013;Tshitenge et al., 2019), no Cotton effects can be found in any CD spectra of dipimprinines A-D (1-4). Consequently, either dipimprinines A-D (1-4) have no atropisomeric stereochemistry (that's to say a plane structure) or they were all racemates. Actually, it's more likely that compounds 1-4 have no atropisomeric stereochemistry. The carbon-nitrogen bond (N-1-C-11 ′ ) in compounds 1-4 can rotate in a circle without any steric hindrance for the reason that no substituents can be found at neither C-2 nor N-10 ′ .
All the new compounds were evaluated for their cytotoxic activities against five human tumor cell lines, human leukemia (HL-60), hepatocellular carcinoma (SMMC-7721), lung cancer (A-549), breast adenocarcinoma (MCF-7), and colon carcinoma (SW-480), and cisplatin was used as a positive control. As shown in Table 3, compounds 1, 2, and 4 showed antiproliferative activity against breast adenocarcinoma cell line MCF-7 with IC 50 values ranging from 13.8 to 18.2 µM, while the same treatment on cisplatin turned out to be an IC 50 value of 26.8 µM. Compound 3 showed weak inhibitory activity against hepatocellular carcinoma cell line SMMC-7721 with an IC 50 value of 25.2 µM.

CONCLUSIONS
In summary, this work describes the isolation and characterization of six new pimprinine alkaloids (1-6) from a soil-derived actinomycete Streptomyces sp. NEAU-C99. Their structures including absolute configurations were determined by extensive spectroscopic data, single-crystal X-ray diffraction analysis, and ECD calculations. Cytotoxicity assays showed that compounds 1, 2, and 4 displayed moderate antitumor activity against breast adenocarcinoma MCF-7. Compounds 1-4 were represented as the first examples of dimeric pimprinine alkaloids, which could further enrich the structure diversities of pimprinine alkaloids.

DATA AVAILABILITY STATEMENT
All datasets for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
ZY performed the experiments, identified the structures, and prepared the original manuscript. HJ isolated and identified the strain, and conducted the cytotoxicity assay. LW collected the spectrographic data, assisted with the structure elucidation and manuscript revision. F-XY, J-PH, CL and XG revised the manuscript. WX and S-XH designed and supervised the research and revised the manuscript.