Phenopyrrolizins A and B, Two Novel Pyrrolizine Alkaloids from Marine-Derived Actinomycetes Micromonospora sp. HU138

Two previously undescribed pyrrolizine alkaloids, named phenopyrrolizins A and B (1 and 2), were obtained from the fermentation broth of marine-derived Micromonospora sp. HU138. Their structures were established by extensive spectroscopic analysis, including 1D and 2D NMR spectra as well as HRESIMS data. The structure of 1 was confirmed by single-crystal diffraction analysis and its racemization mechanism was proposed. The antifungal activity assay showed that 2 could inhibit the mycelial growth of Botrytis cinerea with the inhibitory rates of 18.9% and 35.9% at 20 μg/disc and 40 μg/disc, respectively.


Introduction
Micromonospora, one of the most important actinomycetes genera, is a valuable repository of natural bioactive ingredients [1].Micromonospora-derived secondary metabolites include a large and structurally diverse class of metabolites such as macrolides [2,3], peptides [4,5], aminoglycosides [6,7], macrolactams [8][9][10], alkaloids [11,12], quinones [13][14][15], oligosaccharides [16,17], and miscellaneous compounds [18,19], some of which have developed as important products of the pharmaceutical industry, for example, gentamicin, erythromycin B, and sisomicin [1,20,21].However, the rate of discovery of new secondary metabolites from Micromonospora has been rapidly decreasing in the past two decades, and the discovery of new pharmaceutical compounds from Micromonospora has become more difficult [1].Therefore, more efforts should be taken to investigate these microbes from unexplored habitats, making them a promising storehouse of drug leads.Although a number of secondary metabolites with diverse bioactivity have been isolated from marine Micromonospora [22], they are still revealed to be rather untapped and potential sources of chemically diverse and unique bioactive natural products.
In our previous research, some bioactive secondary metabolites were isolated and identified from marine-derived Micromonospora.For example, three new isoflavonoid glycosides with moderate cytotoxic activity to the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116 were obtained from Micromonospora aurantiaca 110B [18].A new derivative of diterpenoid with strong cytotoxicity against HCT-116 cells and moderate cytotoxicity against HepG2 cells and A549 cells was isolated from Micromonospora zhangzhouensis sp.nov.HM134 [23].In the continuous efforts to discover bioactive chemical entities from marine Micromonospora, a marine Micromonospora sp.HU138 demonstrated antifungal activity against Botrytis cinerea.Further research on this strain, two previously undescribed pyrrolizine alkaloids, named phenopyrrolizin A (1) and phenopyrrolizin B (2) (Figure 1), were isolated from its cultures.Their isolation, structure characterization, and bioactivity are described in this paper.
In our previous research, some bioactive secondary metabolites were isolated and identified from marine-derived Micromonospora.For example, three new isoflavonoid glycosides with moderate cytotoxic activity to the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116 were obtained from Micromonospora aurantiaca 110B [18].A new derivative of diterpenoid with strong cytotoxicity against HCT-116 cells and moderate cytotoxicity against HepG2 cells and A549 cells was isolated from Micromonospora zhangzhouensis sp.nov.HM134 [23].In the continuous efforts to discover bioactive chemical entities from marine Micromonospora, a marine Micromonospora sp.HU138 demonstrated antifungal activity against Botrytis cinerea.Further research on this strain, two previously undescribed pyrrolizine alkaloids, named phenopyrrolizin A (1) and phenopyrrolizin B (2) (Figure 1), were isolated from its cultures.Their isolation, structure characterization, and bioactivity are described in this paper.

Identification of Strain
The 16S rRNA genes of the strain HU138 were sequenced and the sequence (NCBI GenBank accession No.: OR708545) was blasted against the sequences available in EzBio-Cloud data base.The matching result suggested that strain HU138 belonged to the genus Micromonospora and showed the highest sequence similarity to Micromonospora fluminis A38 T (99.01%).The phylogenetic analysis based on a neighbor-joining tree (Figure 2) revealed that the strain HU138 fell within the cluster of the genus Micromonospora and formed a coherent clade with Micromonospora fluminis A38 T .The clade had firm bootstrap support and represented an independent lineage.Similar results were obtained by using the maximum-parsimony and maximum-likelihood trees (see Figures S1 and S2 in Supplementary Materials).

Identification of Strain
The 16S rRNA genes of the strain HU138 were sequenced and the sequence (NCBI GenBank accession No.: OR708545) was blasted against the sequences available in EzBio-Cloud data base.The matching result suggested that strain HU138 belonged to the genus Micromonospora and showed the highest sequence similarity to Micromonospora fluminis A38 T (99.01%).The phylogenetic analysis based on a neighbor-joining tree (Figure 2) revealed that the strain HU138 fell within the cluster of the genus Micromonospora and formed a coherent clade with Micromonospora fluminis A38 T .The clade had firm bootstrap support and represented an independent lineage.Similar results were obtained by using the maximum-parsimony and maximum-likelihood trees (see Figures S1 and S2 in Supplementary Materials).Bootstrap values are expressed as a percentage of 1000 replicates; only those higher than 50% are given at the branch points.Bar, 0.002 substitutions per nucleotide position.

Structural Elucidation of Compounds 1 and 2
Compound 1 was isolated as a colorless prism.The molecular formula of 1 was established as C14H15NO3 on the basis of HRESIMS ion peak at m/z 246.1124 [M + H] + (calculated for C14H16NO3, 246.1125) (see Figure S3 in Supplementary Materials) and NMR data (Table 1, Figures S4-S9 in Supplementary Materials).Its IR spectrum showed absorption Bootstrap values are expressed as a percentage of 1000 replicates; only those higher than 50% are given at the branch points.Bar, 0.002 substitutions per nucleotide position.

Structural Elucidation of Compounds 1 and 2
Compound 1 was isolated as a colorless prism.The molecular formula of 1 was established as C 14 H 15 NO 3 on the basis of HRESIMS ion peak at m/z 246.1124 [M + H] + (calculated for C 14 H 16 NO 3 , 246.1125) (see Figure S3 in Supplementary Materials) and NMR data (Table 1, Figures S4-S9 in Supplementary Materials).Its IR spectrum showed absorption bands at 3281 cm −1 (OH) and 1599 cm −1 (C=O) in the functional group region.The structure of 1 was elucidated and named phenopyrrolizin A (Figure 1).The above assignment was further confirmed by the single-crystal X-ray diffraction analysis of 1.We obtained the eutectics of 1 with MeOH by slow evaporation of MeOH at 5 • C, which was suitable for an X-ray analysis.However, as shown in Figures 3 and 4, the X-ray experiment demonstrated that 1 is a racemate.The specific optical rotation ([α] 25  D 0) of 1 also supported the conclusion.In order to obtain optically pure enantiomers for further research, compound 1 was analyzed by chiral-phase HPLC.Chromatographic analysis of 1 by a Chiralcel OD-H column using n-hexane/isopropanol = 80:20 (v/v) as the eluent suggested that 1 is a racemic mixture (1a and 1b) with an enantiomeric ratio of 1:1 (Figure 5A).However, efforts to obtain the enantiomers were not successful as the racemization occurred spontaneously in the isolation procedure (Figure 5B).We speculate that racemization of 1 takes place through a key tautomer (1c), as shown in Figure 6.In order to obtain optically pure enantiomers for further research, compound 1 was analyzed by chiral-phase HPLC.Chromatographic analysis of 1 by a Chiralcel OD-H col umn using n-hexane/isopropanol = 80:20 (v/v) as the eluent suggested that 1 is a racemic mixture (1a and 1b) with an enantiomeric ratio of 1:1 (Figure 5A).However, efforts to obtain the enantiomers were not successful as the racemization occurred spontaneously in the isolation procedure (Figure 5B).We speculate that racemization of 1 takes place through a key tautomer (1c), as shown in Figure 6.Compound 2 was obtained as a colorless powder.Its molecular formula C14H15NO2 (8 degrees of unsaturation) was deduced from the HRESIMS at m/z 230.1181 [M + H] + (see Figure S10 in Supplementary Materials) and was supported by the NMR data (Table 1, Figures S11-S16 in Supplementary Materials), which was only one oxygen less than that of 1.The presence of a mono-substituted benzene ring (δH 7.02 (1H, t, J = 7.9 Hz), 7.26 (2H, t, J = 7.9 Hz), and 7.66 (2H, d, J = 7.9 Hz)) was observed in the 1 H NMR spectrum of 1. Careful comparison of the 1 H NMR and 13 C NMR data (Table 1) of 2 with those of 1 revealed that 2 possessed the identical structure features to those found in 1, except that the para-hydroxy phenyl moiety in 1 was replaced by a mono-substituted benzene ring in 2. Based on the optical rotation value, 2 was also a pair of enantiomers similar to 1. Therefore, the chemical structure of 2 was established and named phenopyrrolizin B. Compound 2 was obtained as a colorless powder.Its molecular formula C 14 H 15 NO 2 (8 degrees of unsaturation) was deduced from the HRESIMS at m/z 230.1181 [M + H] + (see Figure S10 in Supplementary Materials) and was supported by the NMR data (Table 1, Figures S11-S16 in Supplementary Materials), which was only one oxygen less than that of 1.The presence of a mono-substituted benzene ring (δ H 7.02 (1H, t, J = 7.9 Hz), 7.26 (2H, t, J = 7.9 Hz), and 7.66 (2H, d, J = 7.9 Hz)) was observed in the 1 H NMR spectrum of 1. Careful comparison of the 1 H NMR and 13 C NMR data (Table 1) of 2 with those of 1 revealed that 2 possessed the identical structure features to those found in 1, except that the para-hydroxy phenyl moiety in 1 was replaced by a mono-substituted benzene ring in 2. Based on the optical rotation value, 2 was also a pair of enantiomers similar to 1. Therefore, the chemical structure of 2 was established and named phenopyrrolizin B.

Bioactivity of Compounds 1 and 2
The antibacterial and antifungal activity assay showed that these two compounds exhibited no obvious inhibitory activity against the tested bacteria (Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Salmonella typhimurium, Pseudomonas aeruginosa, Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas syringae, and Staphylococcus aureus) and fungi (Fusarium oxysporum, Ustilago maydis, Cucumber fusarium, Fusarium moniliforme, Helminthosporium maydis, Alternaria alternata, Phoma foveate, and Magnaporthe oryzae).However, compound 2 could inhibit the mycelial growth of Botrytis cinerea, and demonstrated the inhibitory rates of 18.9% and 35.9% at 20 µg/disc and 40 µg/disc, respectively (Figure 7).Notably, compound 1 showed no antifungal activity against B. cinerea, suggesting that hydroxylation at C12 has a negative effect on the bioactivity.The cytotoxicity activity assay for compounds 1 and 2 showed that these two compounds exhibited no cytotoxicity activity to A549, HepG2, and HCT116 cells.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 10 The antibacterial and antifungal activity assay showed that these two compounds exhibited no obvious inhibitory activity against the tested bacteria (Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Salmonella typhimurium, Pseudomonas aeruginosa, Ralstonia solanacearum, Xanthomonas oryzae, Pseudomonas syringae, and Staphylococcus aureus) and fungi (Fusarium oxysporum, Ustilago maydis, Cucumber fusarium, Fusarium moniliforme, Helminthosporium maydis, Alternaria alternata, Phoma foveate, and Magnaporthe oryzae).However, compound 2 could inhibit the mycelial growth of Botrytis cinerea, and demonstrated the inhibitory rates of 18.9% and 35.9% at 20 µg/disc and 40 µg/disc, respectively (Figure 7).Notably, compound 1 showed no antifungal activity against B. cinerea, suggesting that hydroxylation at C12 has a negative effect on the bioactivity.The cytotoxicity activity assay for compounds 1 and 2 showed that these two compounds exhibited no cytotoxicity activity to A549, HepG2, and HCT116 cells.

General Experimental Procedures
Preparative HPLC was conducted on a Shimadzu LC-8A device with an SIL-10AP autosampler, an SPD-20A detector and an FRC-10A fraction collector.Optical rotations were recorded in EtOH on a PerkinElmer 341 polarimeter at 25 °C.UV spectra were ob-

General Experimental Procedures
Preparative HPLC was conducted on a Shimadzu LC-8A device with an SIL-10AP autosampler, an SPD-20A detector and an FRC-10A fraction collector.Optical rotations were recorded in EtOH on a PerkinElmer 341 polarimeter at 25 • C. UV spectra were obtained on a CARY 300 BIO spectrophotometer (Varian, CA, USA); IR spectra were recorded on an FT-IR 750 spectrometer with ν max in cm −1 (Nicolet Magna, WI, USA); 1 H and 13 C NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1 H and 100 MHz for 13 C) spectrometer (Bruker, Rheinstetten, Germany).Chemical shifts are reported in parts per million (δ), using residual DMSO-d 6 signal (δ H 2.49 ppm; δ C 39.5) as an internal standard, with coupling constants (J) in Hz. 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HMQC, and HMBC experiments.The HRESIMS spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Waters, MA, USA).The analytical HPLC was conducted on an Agilent 1100 series (Agilent, CA, USA).Commercial silica gel (100-200 mesh, Qing Dao Hai Yang Chemical Group Co., Ltd., Qingdao, China) was used for column chromatography.

Strain Isolation and Identification
Strain HU138 was isolated from a mangrove soil sample collected from Huaniao island, in Zhoushan city, Zhejiang province, China.The samples were placed into 50 mL sterile plastic centrifuge tubes and stored at 4 • C prior to isolation.Samples were 10-fold diluted and spread on the four selective isolation media by the traditional dilution plating method: HV (humic acid-vitamin agar medium), GS (Gauze's modified medium No. 1), ISP2 (ISP medium No. 2), and MA (marine agar 2216 medium).The selective isolation media were supplemented with 50 mg/L of nalidixic acid and 80 mg/L of cycloheximide.After 6 days of incubation at 28 • C, the actinomycetes colonies were picked from the plate and pure-cultured.Strain HU138 was picked from an ISP2 plate.All of the actinomycetes strains were preserved at −80 • C as suspension with 25% glycerol after pure clone was obtained.

Fermentation, Extraction, and Isolation
The fermentation for the Micromonospora sp.HU138 was carried out in a 50 L fermenter containing 30 L of production medium that consisted of malt extract 0.5%, yeast extract 0.5%, cottonseed meal 1.0%, soluble starch 2.0%, maltodextrin 2.0%, and CaCO 3 0.2% at pH 7.2.The seed medium consisted of glucose 0.4%, malt extract 1%, and yeast extract 0.4% at pH 7.0.A total of 2 L seed was incubated by 1 L flasks containing 250 mL of the seed medium at 28 • C for 48 h, shaken at 250 r.p.m.The fermentation was carried out at 28 • C, stirred at 150~280 r.p.m. (control based on dissolved oxygen) with the aeration rate of 1000~1800 L (control based on dissolved oxygen) of air per hour, dissolved oxygen control ≥ 15%, and tank pressure control at 0.05 MPa.After 7 days of fermentation, a total of 26 L of fermentation broth was obtained.

Bioactivity Assay
The antibacterial and antifungal activities of the obtained compounds were evaluated by the disc diffusion method [30,31].The disc containing 20 µg or 40 µg of the compounds was used, the diameters of the inhibition zone of bacterial or fungal growth around the disc were measured, and inhibition rate was calculated.The cytotoxicity of the obtained compounds was assayed in vitro against HepG2, A549, and HCT116 cells by the CCK8 colorimetric method [32].

Conclusions
Pyrrolizine alkaloids usually exist in hundreds of plant species and herbs [33]; however, they are seldomly produced by microbes.To date, only few pyrrolizine alkaloids have been reported to be produced by Streptomyces and entomopathogenic bacteria [34].To the best of

Figure 1 .
Figure 1.Structures and key 1 H-1 H COSY and HMBC correlations of compounds 1 and 2.

Figure 1 .
Figure 1.Structures and key 1 H-1 H COSY and HMBC correlations of compounds 1 and 2.

Figure 2 .
Figure 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain HU138 and the type strains of the highest 16S rRNA sequence similarity.Bootstrap values are expressed as a percentage of 1000 replicates; only those higher than 50% are given at the branch points.Bar, 0.002 substitutions per nucleotide position.

Figure 2 .
Figure 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain HU138 and the type strains of the highest 16S rRNA sequence similarity.Bootstrap values are expressed as a percentage of 1000 replicates; only those higher than 50% are given at the branch points.Bar, 0.002 substitutions per nucleotide position.

The 1 H
NMR spectrum of 1 displayed a set of proton signals at δ H 6.69 (2H, d, J = 8.6 Hz) and 7.45 (2H, d, J = 8.6 Hz), suggesting the existence of a para-substituted benzene ring moiety.The 13 C NMR and DEPT135 spectra of 1, in conjunction with HMQC spectrum, showed the presence of 14 carbon resonances that included one methyl (δ C 21.5), three aliphatic methylenes (δ C 23.1, 29.0, and 42.9), one oxygenated tertiary carbon (δ C 85.7), one ketone carbonyl (δ C 199.8), two aromatic methine carbons, each accounting for two carbon resonances (δ C 115.4, 126.1), an aromatic quaternary carbon (δ C 125.1), and one phenolic carbon (δ C 154.4), in addition to two quaternary carbon signals (δ C 100.8, 179.0).The presence of three consecutive methylene connectivity was evidenced by the correlations of H 2 -5/H 2 -6/H 2 -7 in the 1 H-1 H COSY spectrum (Figure 1).The observed HMBC correlated signals from H 3 -8 (δ H 1.24) and 1-OH (δ H 6.26) to C-1 (δ C 85.7) and C-2 (δ C 199.8) established the connection of C-2, C-1, and C-8, as shown in Figure 1.The remaining two carbon signals at δ C 100.8, 179.0, together with the ketone carbon at δ C 199.8, suggested the presence of an α, β-unsaturated ketone unit.The long-range correlation in the HMBC spectrum from H-10 (14) to α-carbon (δ C 100.8) of the α, β-unsaturated ketone unit indicated that the connectivity of the para-hydroxy phenyl moiety was located at C-3.The HMBC correlation of H 2 -5 and H 2 -6 with δ C 179.0 established the connection of C-4 and C-5.Considering the molecular formula of 1, the linkage of C-1, C-4, and C-7 via a nitrogen atom established the pyrrolizine skeleton in 1.
Molecules 2023, 28, x FOR PEER REVIEW 4 of X-ray experiment demonstrated that 1 is a racemate.The specific optical rotation ([α] 25 Dof 1 also supported the conclusion.

Figure 3 .
Figure 3.The ORTEP view of the crystal structure of compound 1.

Figure 3 .
Figure 3.The ORTEP view of the crystal structure of compound 1.

Figure 3 .
Figure 3.The ORTEP view of the crystal structure of compound 1.

Figure 5 .
Figure 5.The enantiomeric analysis of compounds 1 (A) and 1a (B) by a Chiralcel OD-H column.

Figure 5 .
Figure 5.The enantiomeric analysis of compounds 1 (A) and 1a (B) by a Chiralcel OD-H column.

Figure 5 .
Figure 5.The enantiomeric analysis of compounds 1 (A) and 1a (B) by a Chiralcel OD-H column.

Figure 7 .
Figure 7.The antifungal activity of compound 2 against B. cinerea.

Figure 7 .
Figure 7.The antifungal activity of compound 2 against B. cinerea.