Ring D-Modified and Highly Reduced Angucyclinones From Marine Sediment-Derived Streptomyces sp.

Angucyclines and angucyclinones represent the largest family of type II PKS-engineered natural products. Chemical analysis of a marine Streptomyces sp. KCB-132 yielded three new members, actetrophenone A (1) and actetrophenols A–B (2–3). Their structures were elucidated by NMR spectroscopy, X-ray crystallography and CD calculations. Actetrophenone A (1) is the first representative of a novel-type angucyclinone bearing a nonaromatic D-ring. Actetrophenol A (2) features a highly reduced and aromatized four-ring system, which is unprecedented for natural products. While (R a )- and (S a )-actetrophenol B (3) bear an unprecedented N-acetyltryptamine-substituted tetraphene core skeleton, this is the first report of a pair of atropisomeric isomers in the angucyclinone family. Actetrophenol A (2) exhibits remarkable antibiotic activity, notably including potent activity to multiple resistant Staphylococcus aureus and Enterococcus faecium with MIC values of 4 μg/ml, in contrast, the positive control antimicrobial agent penicillin was inactive up to 32 μg/ml.


INTRODUCTION
Angucyclines and angucyclinones, characterized by an unsymmetrically assembled benz [a] anthraquinone frame, represent by far the largest family of type II PKS-engineered natural products (Kharel et al., 2012). This family of antibiotics has attracted much attention due to their broad biological activities and remarkable structural diversity that is mainly derived from oxidation, hydroxylation and glycosylation at various positions (Bringmann et al., 2005;Shaaban et al., 2012;Xie et al., 2016), and additionally, rearrangement of A-, B-and C-ring (Ma et al., 2015;Liu et al., 2019;Wu et al., 2019). However, an aromatic D-ring and a high number of oxygen functions on the tetracyclic backbone remain the common features of their structures, and neither ring D-modified nor highly reduced angucyclin(on)es have been reported as yet (Cabrera-Afonso et al., 2018).
As part of our research on the discovery of new-type angucyclinones from marine sedimentderived bacteria, we reported several angucyclinones featuring C-ring cleavage and expansion, produced by Streptomyces pratensis KCB-132 (Zhang et al., 2017;Zhang et al., 2019). Recently, a comprehensive LC-MS-based analysis of this strain identified a group of metabolites possessing previously unreported UV chromophores. The subsequent purification of the fraction containing these components led to the discovery of three further unique angucyclinone-derived polyketides, with ring D-modified (1) and highly reduced (2 and 3) structural characteristics ( Figure 1). We describe herein the isolation and structural elucidation of these new metabolites, named actetrophenone A (1) and actetrophenols A-B (2-3). In addition, the atropisomerism of 3 is discussed.

RESULTS AND DISCUSSION
Actetrophenone A (1) was isolated as a light brown powder. Its molecular formula was predicted to be C 19 H 16 O 4 with twelve degrees of unsaturation, based on high-resolution electrospray ionization (HR-ESI) mass spectrometry ([M + H] + at m/z 309.1122, calculated 309.1127). The 1 H NMR spectrum of 1 in DMSO-d 6 showed a methyl singlet at δ H 2.43, an oxygenated methine signal at δ H 4.90, two pairs of geminal protons at δ H 2.92, 2.78 and at δ H 2.27, 2.09, as well as five aromatic protons at δ H 9. 38, 8.11, 7.68, 7.26 and 7.01 (Table 1). The 13 C NMR spectrum revealed the expected 19 carbon signals, classified as a methyl, an oxymethine (δ C 67.3), two methylenes, five aromatic methines (δ C 115.2-127.1), nine aromatic tertiary carbons (δ C 110.7-157.5), and a carbonyl carbon (δ C 205.2) with the aid of a DEPT-135 measurement, accounting for eight degrees of unsaturation, which required four rings in the scaffold of 1.
Inspection of 1 H-1 H COSY NMR data led to identification of two isolated spin systems ( Figure 2). The first spin system showed correlations from H 2 -9 to 11-OH, which was extended to include two tertiary carbons C-7a/C-11a and a carbonyl carbon C-8 based on HMBC correlations from H 2 -10 to C-8/C-11a and from H-11 to C-7a to construct a 4-hydroxy-2-cyclohexen-1-one ring. The second spin system containing two aromatic protons, H-5 and H-6, showed HMBC couplings from H-5 to C-6a/C-12b and from H-6 to C-4a/C-12a to form a benzene unit and was further expanded by HMBC correlations from 1-OH to C-1/C-2/ C-12b, from 3-CH 3 to C-2/C-3/C-4 and from H-4 to C-4a/C-12b, revealing the presence of a 3-methylnaphthalen-1-ol ring. Additional HMBC signals with key correlations from 7-OH to C-6a/C-7/C-7a and from H-12 to C-11a/C-12a fused the two ring systems together and resulted in the planar structure of 1.
Since ring D-modified angucycline was unprecedented in natural products, and since ring C-aromatized representative was very rare in the angucycline family, crystals of 1 were subjected to X-ray crystallographic analysis (CCDC 2045687). Slow evaporation of a concentrated solution containing 1 in a MeOH/CH 2 Cl 2 mixture furnished X-ray quality crystals. The X-ray crystal data corroborated the structure assignment of 1 and provided a centrosymmetric space group P-1, supporting a racemic mixture ( Figure 3). Interestingly enough, the crystal structure of 1 belongs to the relatively rare case of "solid solution" with both enantiomers appearing in the asymmetric unit with disordered chiral centre (Rekis, 2020).
Actetrophenol A (2) was purified as a light brown powder. Its molecular formula was assigned as C 20 H 16 O 2 based on HR-ESI mass spectrometry ([M + H] + at m/z 289.1200, calculated 289.1228). The NMR data of 2 ( Table 1) were quite similar to those of 1 and showed main changes in ring D ( Figure 2). In 2, the nonaromatic D-ring previously seen in 1 was replaced with a conventional 1,2,3-trisubstituted benzene ring, as evidenced by COSY cross-peaks between H-10 and H-9/H-11 alongside HMBC contacts from H-9 to C-7a, from H-10 to C-8/C-11a and from 8-OCH 3 to C-8. Furthermore, an additional change in the NMR data of 2 was observed in C-7, whose chemical shift was significantly downfield-shifted from δ C 159.8 to 120.1, which indicated the lack of an oxygen atom in line with the molecular formula, and thus completed the planar structure of 2. Frontiers in Chemistry | www.frontiersin.org October 2021 | Volume 9 | Article 756962 2 Actetrophenol B (3) was also obtained as a light brown powder. Its molecular formula was determined to be C 32 H 28 N 2 O 3 based on HR-ESI mass spectrometry ([M + Na] + at m/z 511.1991, calculated 511.1998). Careful comparison of the 1 H and 13 C NMR spectra of 2 and 3 showed superimposable resonances for the tetraphene part as subunit A ( Figure 2; Table 1). Further analysis of 2D NMR data of 3 delineated the presence of a N-acetyltryptamine motif as subunit B. In particular, COSY NMR data quickly established the H-4'/H-5'/H-6'/H-7' spin system. Combined HMBC correlations from H-4' to C-8', from H-7' to C-3' and from 9'-NH to C-1'/C-2'/C-3'/C-8', allowed the construction of an indole ring. A further COSY sequence of signals from H-10' to 12'-NH along with HMBC correlations from H-10' to C-2' and from both H-11' and 13'-CH 3 to the amide carbonyl carbon C-13' (δ C 169.7) permitted the attachment of an N-ethylacetamide side chain in C-2' of the indole ring.
At this point of the structure elucidation two aromatic tertiary carbons, C-7 in subunit A and C-1' in subunit B, were still loose ends. Considering the molecular formula of 3, subunits A and B had to be linked between C-1' and C-7, unequivocally, although no obvious correlation was observed between 9'-NH and C-7 in the HMBC spectrum as it might have been expected. However, this assignment was supported by X-ray diffraction experiment (CCDC 2045689). The X-ray analysis confirmed the structure assigned for 3 but also indicated its racemic nature from a centrosymmetric space group P121/c1 ( Figures 4C,D). Consequently, resolution of    single atropisomers was achieved by HPLC equipped with a chiral column (CHIRALCEL OD-H). The interconversion (racemization) barrier between the two atropisomers was estimated by DFT calculations at ωB97X-D/6-31+G(d) level and amounted to ≈ 20 kcal mol −1 ( Figure 5). Finally, the absolute configurations of the two enantiomers were determined by electronic circular dichroism (ECD) calculations run with TDDFT method at CAM-B3LYP/def2-TZVP//ωB97X-D/6-311+G(d,p)/PCM level (see Supporting Information for details) (Pescitelli and Bruhn, 2016;Superchi et al., 2018). The ECD spectra of these two isomers recorded in acetonitrile exhibited a perfect mirror-image relationship and were in good agreement with the TDDFT calculated spectra of the possible atropisomers with S a and R a configurations, respectively ( Figures 4A,B).

EXPERIMENTAL SECTION General Experimental Procedures
Opitical rotations were measured on an Autopol VI (Serial #91058) manufactured by Rudolph Research Analytical, Hackettstown, NJ, United States CD spectra were recorded using a JASCO J-810 spectropolarimeter. NMR spectra were measured by a Bruker AVANCE IIITM 600 spectrometer. ESI-

Cultivation and Culture Extraction
Streptomyces sp. strain KCB-132 was isolated from a sediment sample collected off Kiaochow Bay, China, as described previously. The sequence is deposited in GenBank under accession no. KX033803. The strain KCB-132 was cultured in seawater-based ISP2 medium (5 g of malt extract, 4 g of yeast extract, 4 g of glucose, 500 ml of deionized water and 500 ml of seawater, pH 7.8) with the addition of 50 μM lanthanum chloride, at a total volume of 21.6 L (72 × 0.3 L), for 10 days at 28°C. The culture broth was filtered to provide filtrate and mycelium. The filtrate was absorbed onto XAD-16 amberlite resin, and the resin was eluted with methanol, then dry out methanol under reduced pressure, the resulting aqueous layer was extracted with ethyl acetate, while the mycelium was extracted by ethyl acetate under ultrasonic radiation directly, both ethyl acetate phases were combined to yield 7.2 g crude extract.

Computational Section
MMFF and DFT calculations were run with Spartan'18 (Wavefunction, Inc., Irvine CA, 2019), with standard parameters and convergence criteria. TDDFT calculations were run with Gaussian'16 (Rev. C.01, Gaussian, Inc., Wallingford CT, 2013), with default grids and convergence criteria. Conformational searches were run on Three with the Monte Carlo algorithm implemented in Spartan'18 using Merck molecular force field (MMFF). All structures thus obtained were first optimized with DFT method using B97X-D functional and 6-31G(d) basis set in vacuo, and then re-optimized using B97X-D functional and 6-311+G(d,p) basis set including SMD solvent model for acetonitrile. Torsional energy scans were run on a simplified model of Three with the substituent at C-2' replaced by a methyl group, by varying the dihedral angle relative to the biaryl axis by 10 deg steps; calculations were run at B97X-D/6-31+G(d) level in vacuo. TDDFT calculations were run using various functionals (B3LYP, CAM-B3LYP, M06-2X) and def2-TZVP basis set, including 32 excited states and the IEF-PCM solvent model for acetonitrile. CAM-B3LYP and M06-2X functionals led to consistent results, while B3LYP results were in poorer agreement with the experiment. ECD spectra were generated using the program SpecDis (v. 1.71, Bruhn T., Schaumlöffel A., Hemberger Y., Pescitelli G., Berlin, Germany, 2017, https://specdis-software.jimdo.com/), by applying a Gaussian band shape with 0.26 eV exponential half-width, from dipole-length rotational strengths. The calculated spectra were red shifted by 18 nm and scaled by a factor 10 for better comparison with the experimental spectra. Boltzmann populations were estimated at 300K from internal energies calculated at B97X-D/6-311+G(d,p)/SMD level. The conformers with Boltzmann population >2% at 300K were included in the calculations, which amounted to 4 conformers for 3. The structures and relative energies are shown in Supplementary Figure S1. All stable conformers had an intramolecular hydrogen bond between the amide N-H and 8-OMe and a similar value of the aryl-aryl dihedral angle C2'-C1'-C7-C6a ≈ 71-74°.

Cytotoxicity Bioassay
The THP-1 (acute monocytic leukemia) cells were plated at a density of 5,000 cells/well in 100 μl DMEM medium. All cell lines were incubated overnight then treated with various concentrations of purified compounds in triplicate. After cultured for 72 h, 20 μl/well of MTT solution (5 mg/ml, Sigma-Aldrich, United States) was added to each well, plate was cultured for 4 h at 37°C in a 5% CO 2 atmosphere, which was followed by adding 150 μl DMSO to dissolve the formazan crystals, and FIGURE 5 | Torsional energy scan of a simplified model of 3 run at ωB97X-D/6-31+G(d) level in vacuo. In the truncated model trunc-3, the substituent at C2' in 3 (CH 2 CH 2 NHCOCH 3 ) was replaced by a methyl group (see Figure). The substituent CH 2 CH 2 NHCOCH 3 is capable of stabilizing the energy minima by intramolecular hydrogen bonding (see Supplementary Figure S1); on the contrary, it hardly affects the energy of the transition state because it folds away from the benz[a]anthracene ring. Therefore, the energy barrier of trunc-3 offers a lower estimate of the racemization barrier in 3. The plot was obtained by running two independent torsional energy scans varying the C2'-C1'-C7-C6a dihedral angle in steps of 10°and considering for each value of the dihedral angle the lowest energy value.
Frontiers in Chemistry | www.frontiersin.org October 2021 | Volume 9 | Article 756962 6 shaking for 5 min. The absorbance was recorded at 570 nm by a microplate Reader. IC 50 value was taken using Graph pad Prism 5 software.

CONCLUSION
Common structural features of angucyclin(on)es are an angular tetracyclic benz[a]anthracene backbone with an aromatic D-ring and a high number of oxygen functionalities, mainly at C-1, C-7, C-8, and C-12 positions (Metsä-Ketelä et al., 2003). To the best of our knowledge, actetrophenone A (1) is the first representative of a novel-type angucyclinone bearing an nonaromatic D-ring. Actetrophenol A (2) featured a highly conjugated and aromatized tetraphene ring system, which is unprecedented for natural products. From known angucyclinones, it differs in the unusually high degree of reduction and, hence, only two oxygens in molecule. This finding provided clear proof that the oxygen atoms initially originated from molecular oxygen during biosynthesis of the benz[a]anthracene frame, can be removed later by the post-PKS tailoring enzymes, except for the two oxygens at C-1 and C-8 originating from acetate (Weber et al., 1994). While angucyclines are common with various sugar residues at diverse attachment positions, such as the single saccharide chains of 2, five or six sugar units attached at O-8 in landomycins , or the C-glycosidic sugar D-olivose located at C-9 in urdamycins and saquayamycins (Shaaban et al., 2011), actetrophenol B (3) is the first example of the family to contain a non-glycosylated side chain linked at the unusual C-7 position of the angucyclinone chromophore. This results in a unique N-acetyltryptamine-substituted tetraphene skeleton and offers new opportunities to enrich the existing structural diversity of angucyclines. Another striking difference between 3 and other angucyclines is the dynamic type of axial chirality in that atropisomers interconversion can occur spontaneously, thanks to relatively little hindrance to rotation about the unusual chiral axis as present only in actetrophenol B (3). More strikingly, actetrophenol A (2) exhibits remarkable antibiotic activity, notably including potent activity to multiple resistant S. aureus and E. faecium, consequently, actetrophenol A may be a promising new candidate to combat ESKAPE pathogens in the future.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
LG performed the experiments and analyzed the data. QL and GW performed the experiments. ML and XP analyzed the data. GP calculated the data and revised the manuscript. SZ and ZX designed the study and wrote the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was funded by the National Natural Science Foundation of China (81503040), the Natural Science