Two New Phenylhydrazone Derivatives from the Pearl River Estuary Sediment-Derived Streptomyces sp. SCSIO 40020

Two new phenylhydrazone derivatives and one new alkaloid, penzonemycins A–B (1–2) and demethylmycemycin A (3), together with three known compounds including an alkaloid (4) and two sesquiterpenoids (5–6), were isolated from the Streptomyces sp. SCSIO 40020 obtained from the Pearl River Estuary sediment. Their structures and absolute configurations were assigned by 1D/2D NMR, mass spectroscopy and X-ray crystallography. Compound 1 was evaluated in four human cancer cell lines by the SRB method and displayed weak cytotoxicity in three cancer cell lines, with IC50 values that ranged from 30.44 to 61.92 µM, which were comparable to those of the positive control cisplatin. Bioinformatic analysis of the putative biosynthetic gene cluster indicated a Japp–Klingemann coupling reaction involved in the hydrazone formation of 1 and 2.


Structural Determination
Penzonemycin A (1) was obtained as yellow needles. The molecular formula of 1 was determined to be C 13 Figure S3) displayed 13 carbon signals for a 1,2,3,4-tetra-substituted benzene ring, three methyl groups, δ C 23.6 (C-10), 8.1 (C-16) and 24.2 (C-17), and 4 quaternary carbons, δ C 168.6 (C-7), 171.3 (C-9), 144.2 (C-13) and 196.3 (C-14). The COSY correlations of H-5 and H-6 and the HMBC correlations from H-5 to C-1/C-3 and from H-6 to C-2/C-4 confirmed the presence of a tetra-substituted benzene ring unit ( Figure S3). The key HMBC correlations (Figures 2 and S3) from H-6 to C-7 (δ C 168.6) indicated a carboxylic acid group linked to the tetra-substituted benzene ring at C-1. The HMBC correlations from H 3 -10 (δ H 2.21) to C-9 (δ C 171.3) and the chemical shift value of C-9 revealed an acetamide group, which was deduced to be located at C-2 of the tetra-benzene ring by the chemical shift value of C-2 (δ C 127.5). The HMBC correlations from H 3 -16 (δ H 2.00)/H 3 -17 (δ H 2.40) to C-14 (δ C 144.2)/C-15 (δ C 196.3) and from NH-12 to C-14, together with the remaining two N atoms, established a rare butanone hydrazone unit, which was linked to the benzene ring at C-4 by HMBC correlations from NH-12 to C-3/C-5. The remaining hydroxyl group (δ H 10.18, 3-OH) was placed at C-3 based on the NOESY correlations of 3-OH and NH-8 ( Figure 2). Thus, the planar structure of 1 was established. The E configuration of the double bond ∆ 13,14 in 1 was assigned by the NOESY correlation of NH-12 and H 3 -17 ( Figure 2). Fortunately, highquality crystals of 1 were obtained, allowing the successful performance of a single-crystal X-ray diffraction experiment using Cu Ka radiation (CCDC 2131907), confirming the planar structure and absolute stereochemistry (E∆ 13,14 ) of 1 ( Figure 2, Table S4).  Figure S4) were highly similar to those of 1. The difference was that 2 had a propionamide moiety instead of the acetylamino unit in 1. The presence of the propionamide group in 2 was supported by the 1 H-1 H COSY correlation of H 2 -10 (δ H 2.49) and H 3 -11 (δ H 1.17) and HMBC correlations from H 3 -11 to C-9 (δ C 174.5)/C-10 ( Figure 2). The geometry of the double bond ∆ 13,14 in 2 was determined to be trans (E) by NOESY correlations of NH-12 and H 3 -17 ( Figure 2). Compound 3 was isolated as a white solid. The molecular formula of 3 was determined to be C 14 (Table 2 and Figure S5) were highly similar to those of mycemycin A (4) [13]. Compound 3 was different from 4 by the absence of the methoxy group at C-14, which was supported by 14 mass units less compared to mycemycin A (4), as well as by a detailed analysis of 1D and 2D NMR spectra ( Figure 3 and Figure S5). The COSY correlations of H-3 (δ H 7.99)/H-4 (δ H 7.11)/H-5 (δ H 7.57)/H-6 (δ H 7.15) and the HMBC correlations from H-4 to C-2/C-6, from H-3/H-5/H-6 to C-7, and from H-3 to C-1 established the salicylic acid fragment. The COSY correlations of H-9 (δ H 8.12)/H-10 (δ H 7.58)/H-11(δ H 8.05) and the HMBC correlations from H-9/H-11 to C-13, from H-10 to C-12/C-8, and from H-11 to C-14 revealed a 3-hydroxyanthranilic acid unit. Given determined molecular formula as well as one less degree of unsaturation, two ring systems were connected by C-2-C-1-NH-C-13 and C-7-O-C-8. Thus, compound 3 was elucidated to be demethylmycemycin A.    revealed a 3-hydroxyanthranilic acid unit. Given determined molecular formula as well as one less degree of unsaturation, two ring systems were connected by C-2-C-1-NH-C-13 and C-7-O-C-8. Thus, compound 3 was elucidated to be demethylmycemycin A.  Compounds 4-6 were identified as mycemycin A (4) [13], 9(R)-ganodermanol B (5) [14], and isopterocarpolone (6) [15], respectively. Their 1 H and 13 C NMR data (Figures S6-S8) were identical to those reported in the literature. In addition, we performed a crystallographic analysis of 5. For the first time, the absolute configuration of 5 was confirmed by the way of single-crystal X-ray diffraction analysis ( Figure 4) using Cu Kα data (CCDC 2177578) with Flack parameter of 0.04(9) (Table S5).

Biosynthetic Implications
Penzonemycins A (1) and B (2) are new hydrazones harboring an −N−N=C− unit and a 3-hydroxyanthranilic acid (3-HAA) core. The whole genome of Streptomyces sp. SCSIO 40020 has been sequenced, revealing a putative gene cluster (pzm), spanning from pzm1 to pzm22 (Scheme 1, Table 3), that encodes the enzymatic machinery for the biosynthesis of 1 and 2. The 3-HAA core was proposed to be derived from a chorismate pathway, given the presence of the genes pzm9, pzm6, pzm7 and pzm8, which are predicted to encode 3deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, isochorismatase and a dehydrogenase, respectively. Pzm9 catalyzes the condensation of D-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to form DAHP, the first intermediate in the shikimate pathway leading to chorismate [16,17]. The homologous proteins of Pzm6, Pzm7 and Pzm8 in the biosynthetic pathways of calcimycin and limazepine have been shown to be responsible for converting chorismate to 3-HAA (Scheme 1) [18,19]. Next, 3-HAA is putatively acylated by the N-acetyltransferase Pzm3 to afford an acetylated (or propionylated) product ready for two rounds of oxidations, likely catalyzed by the proposed oxidases Pzm19 and Pzm21. Subsequently, an unknown enzyme-catalyzed transamination occurs, followed by a tautomerization, to generate the putative intermediate M1 (Scheme 1). The proposed oxidative transamination reactions are similar to the recent reported reactions in the formation of 8-amino-flaviolin during the biosynthesis of naphthoquinone-based meroterpenoid [20]. Notably, the three enzymes, namely, the nitrosuccinate lyase Pzm11, the monooxygenase Pzm12 and the AMP-binding protein Pzm18, are predicted to be the orthologues of CreD, CreE and CreM, respectively, which

Biosynthetic Implications
Penzonemycins A (1) and B (2) are new hydrazones harboring an −N−N=C− unit and a 3-hydroxyanthranilic acid (3-HAA) core. The whole genome of Streptomyces sp. SCSIO 40020 has been sequenced, revealing a putative gene cluster (pzm), spanning from pzm1 to pzm22 (Scheme 1, Table 3), that encodes the enzymatic machinery for the biosynthesis of 1 and 2. The 3-HAA core was proposed to be derived from a chorismate pathway, given the presence of the genes pzm9, pzm6, pzm7 and pzm8, which are predicted to encode 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, isochorismatase and a dehydrogenase, respectively. Pzm9 catalyzes the condensation of D-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to form DAHP, the first intermediate in the shikimate pathway leading to chorismate [16,17]. The homologous proteins of Pzm6, Pzm7 and Pzm8 in the biosynthetic pathways of calcimycin and limazepine have been shown to be responsible for converting chorismate to 3-HAA (Scheme 1) [18,19]. Next, 3-HAA is putatively acylated by the N-acetyltransferase Pzm3 to afford an acetylated (or propionylated) product ready for two rounds of oxidations, likely catalyzed by the proposed oxidases Pzm19 and Pzm21. Subsequently, an unknown enzyme-catalyzed transamination occurs, followed by a tautomerization, to generate the putative intermediate M1 (Scheme 1). The proposed oxidative transamination reactions are similar to the Mar. Drugs 2022, 20, 449 6 of 12 recent reported reactions in the formation of 8-amino-flaviolin during the biosynthesis of naphthoquinone-based meroterpenoid [20]. Notably, the three enzymes, namely, the nitrosuccinate lyase Pzm11, the monooxygenase Pzm12 and the AMP-binding protein Pzm18, are predicted to be the orthologues of CreD, CreE and CreM, respectively, which are responsible for the diazo formation in cremeomycin biosynthesis, utilizing L-aspartic acid as a nitrogen source [21]. Therefore, Pzm11 and Pzm12 are postulated to generate nitrite from L-aspartic acid, which is subsequently incorporated to M1 by Pzm18, leading to the diazo group in M2 after dehydration and tautomerization, similar to the biosynthesis of cremeomycin and triacsin [21,22]. Recently, an electrophilic diazo-anthranilate has been reported to undergo nonenzymatic Japp−Klingemann coupling with a β-keto aldehydecontaining precursor to furnish the hydrazone group in the biosynthesis of tasikamides [4]. Interestingly, Pzm16 encodes an acetolactate synthase and putatively catalyzes the coupling of two pyruvates to form 2-acetolactate [23], which becomes 4-hydroxy-3-methyl-3-buten-2one (M3) after two rounds of carboxyl reduction (probably catalyzed by the F420-dependent oxidoreductase Pzm4) and spontaneous dehydration (Scheme 1). Finally, the nonenzymatic Japp−Klingemann coupling reaction between M2 and M3 constructs the hydrazone moiety in 1 and 2 after subsequent hydration and decarboxylation (Scheme 1).

Strain, Screening and Culture Methods
Streptomyces sp. SCSIO 40020 (original number LW701) was isolated from a marine sediment sample (E 114.0432 • , N 22.0194 • , Pearl River Estuary in China, at the depth of 28 m), and was identified by 16S rDNA sequence analysis (GenBank accession no. MW582618). The strain SCSIO 40020 was maintained in a 40% glycerol aqueous solution at −80 • C at the Research Center for Marine Microbiology Culture Collection Center of South China Sea Institute of Oceanology, Chinese Academy of Sciences. It was found that the strain SCSIO 40020 was best maintained on 38 # -agar medium containing 3% sea salt for optimal growth and sporulation. A single colony was inoculated into 50 mL of four different media, including modifed-A1BFe+C [24]  The culture broths were extracted with an equal volume of n-butanone, and the extracts were then monitored by HPLC-DAD. HPLC analyses were carried out using the following program: solvent system (solvent A, 10% acetonitrile in water supplemented with 0.08% formic acid; solvent B, 90% acetonitrile in water); 5% B to 100% B (linear gradient, 0-18 min), 100% B (18-23 min), 100% B to 5% B (23-27 min), 5% B (27-32 min); flow rate of 1 mL/min. A single colony was inoculated into 30 mL of modified A1BFe+C medium and incubated at 28 • C for 2-3 days. Then, 20 L of fermentation culture was prepared by inoculating 30 mL of the seed culture into a 1000 mL Erlenmeyer flask containing 200 mL of the modified ISP3 medium followed by cultivation on a rotary shaker (200 rpm) at 28 • C for 7 days.

Extraction, Isolation and Purification
The 20 L of culture broth of Streptomyces sp. SCSIO 40020 was centrifuged at 3900 rpm for 15 min at 25 • C. The mycelia were extracted three times, each with 2 L of acetone. The acetone extracts were concentrated under reduced pressure to afford an aqueous residue, which was extracted four times with an equal volume of n-butanone. The supernatants were extracted four times with an equal volume of n-butanone [12,24]. The butanone extracts were combined and concentrated under reduced pressure to afford the crude extracts (7.5 g). The crude extracts were subjected to column chromatography over silica gel, eluting with a gradient of CHCl 3 /MeOH mixtures i.e., 100/0, 95/5, 90/10, 80/20, 50/50 and 0/100 (v/v). We obtained five fractions (Fr.1-Fr.5). Then Fr.2 (3.50 g) was further purified via MPLC (Medium-Pressure Preparative Liquid Chromatography) with a reverse-   Crystal data for 9(R)-ganodermanol B (5)

X-Ray Crystallographic Analysis Data of Penzonemycin A (1) and 5
Single crystals of C 13 H 15 N 3 O 5 (penzonemycin A 1) and C 15 H 26 O 2 (9(R)-ganodermanol B 5) were obtained in MeOH solution. Their data were collected on an XtaLAB AFC12 (RINC): Kappa single diffractometer. The crystals were kept at 100.00 (10) K during data collection. Using Olex2 [26], their structures were solved with the ShelXT [27] structure solution program using Intrinsic Phasing and refined with the ShelXL [28] refinement package, using Least Squares minimization. Crystallographic data for 1 and 5 were deposited at the Cambridge Crystallographic Data Center (CCDC) with the deposition numbers 2,131,907 and 2,177,578, respectively.
3.6. Bioactivity Assays 3.6.1. Cytotoxic Activity Assay The in vitro cytotoxic activities of penzonemycin A (1) were evaluated in four tumor cell lines, i.e., SF-268 (human glioma cell line), HepG-2 (human liver carcinoma cell line), MCF-7 (human breast adenocarcinoma cell line), and A549 (human lung adenocarcinoma cell) by the SRB method [24], according to a previously described protocol [29]. The cells were cultivated in RPMI 1640 medium [30]. Cells (180 µL) with a density of 3 × 10 4 cells/mL were seeded onto 96-well plates and incubated for 24 h at 37 • C, 5% CO 2 . Subsequently, 20 µL of different concentrations of penzonemycin A (1), ranging from 0 to 100 µM in dimethyl sulfoxide (DMSO), was added to each plate well. An equal volume of DMSO was used as a negative control. After further incubation for 72 h, the cell monolayers were fixed with 50% (wt/v) trichloroacetic acid (50 µL) and then stained for 30 min with 0.4% (wt/v) SRB dissolved in 1% acetic acid. The unbound dye was removed by repeatedly washing with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris-base solution (200 µL) for the determination of the optical density (OD) at 570 nm using a microplate reader. The cytotoxic compound cisplatin was used as a positive control. All data were obtained in triplicate and are presented as means ± S.D. IC 50 values were calculated with the SigmaPlot 14.0 software using the non-linear curve-fitting method.

Antifungal Activity Assay
The antifungal activity of compound 1 was measured against three phytopathogenic fungi, i.e., Colletotrichum gloeosporioides Penz, Physalospora piricola Nose, Bipolaris sorokiniana, by the broth microdilution method [31]. Compound 1 was dissolved in DMSO at the final concentration of 2.56 mg mL −1 . The indicator fungi strains were grown for 48 h on a rotary shaker at 28 • C. The cultures were diluted with sterilized medium to achieve a predetermined optical absorbance at 600 nm, then diluted 1000-fold before being added into 96-well microtiter plates. Three replicates of each compound were tested in dilution series, ranging from 128 to 0.25 µg mL −1 . The lowest concentrations that completely inhibited the visible growth of the tested strains were recorded after 48 h of cultivation from two independent experiments. Nystatin was used as a positive control against the three phytopathogenic fungi.

Antibacterial Activity Assay
The antibacterial activity of compound 1 was assayed against Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606, Staphyloccocus aureus ATCC 29213, and Methicillinresistant S. aureus (MRSA) ATCC 43300 by the broth microdilution method [31]. Compound 1 was dissolved in DMSO at the final concentration of 2.56 mg mL −1 . The indicator strain was grown for 24 h on a rotary shaker at 37 • C. The cultures were diluted with sterilized medium to achieve a predetermined optical absorbance at 600 nm, then diluted 1000-fold before being placed into 96-well microtiter plates. Three replicates of each compound were tested in dilution series, ranging from 128 to 0.25 µg mL −1 . The MIC values were recorded after 24 h of cultivation from two independent experiments. Ciprofloxacin and vancomycin were used as positive controls.

Alpha-glucosidase Inhibition Activity
A general inhibiting reaction assay contained 0.04 U/mL, 2.0 mM p-nitrophenyl-α-Dglucopyranoside solution (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) and a specific concentration of 1 and was carried out in PBS buffer (50 mM, pH 7.0), in a total volume of 100 µL. In detail, first, sample 1 was mixed with α-glucosidase and incubated at 37 • C for 10 min, and then, the reactions were initiated by adding the p-nitrophenylα-D-glucopyranoside solution and continued at 37 • C for another 15 min. After that, the absorbance at 405 nm was recorded on a microplate reader (Enspire, PerkinElmer, Singapore) within 30 min. In the assay, DMSO was used as a negative control, and acarbose (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) was used as a positive control. All treatments were performed in triplicate, and the IC 50 value was determined by regression analysis [32].

Bioinformatics Analysis
The genomic DNA of Streptomyces sp. SCSIO 40020 was isolated according to a prior study [12]. The whole genome was sequenced and assembled by Nextomics Biosciences Co., Ltd. (Wuhan, China) using Oxford Nanopore GridION and canu. The putative biosynthetic gene clusters in the genome were predicted with antiSMASH 6.0 [33]. The deduced ORFs were analyzed using online 2ndFind software (https://biosyn.nih.go.jp/2ndfind/, accessed on 13 January 2022), and their functional predictions were accomplished with an online BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 13 January 2022). The DNA sequences of the penzonemycins gene cluster were deposited in GenBank, accession number ON345781.

Conclusions
In conclusion, two new phenylhydrazone derivatives and a new alkaloid, penzonemycins A-B (1-2) and demethylmycemycin A (3), together with three known compounds (4-6) were obtained from the Pearl River Estuary sediment-derived Streptomyces sp. SCSIO 40020. Penzonemycins feature a hydrazone unit (−N−N=C−) that is rare in natural products. Biosynthetically, a Japp−Klingemann reaction is proposed as a key reaction leading to the hydrazone moiety in 1 and 2, which is worth of further investigations.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.