Vochysiamides A and B: Two new bioactive carboxamides produced by the new species Diaporthe vochysiae

Abstract

Endophytic have been considered a rich source for bioactive secondary metabolites with novel chemical structures. A high diverse group of endophytes, isolated from different medicinal plants, belongs to the genus Diaporthe. In a previously study performed by our group the crude extract of strain LGMF1583 showed considerable antibacterial activity mainly against Gram-negative bacteria. Based on ITS phylogeny analysis, strain LGMF1583 was identified as belonging to Diaporthe genus and may represent a new species. In the present study, we described the new species Diporthe vochysiae based on multilocus phylogeny analysis and morphological characteristics, The species name refers to the host, from which strain LGMF1583 was isolated, the medicinal plant Vochysia divergens. In view of the biotechnological potential of strain LGMF1583, we have also characterized the secondary metabolites produced by D. vochysiae. Chemical assessment of the D. vochysiae LGMF1583 revealed two new carboxamides, vochysiamides A (1) and B (2), in addition to the known metabolite, 2,5-dihydroxybenzyl alcohol (3). In the biological activity analysis, vochysiamide B (2) displayed considerable antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae (KPC), a producer of carbapenemases, MIC of 80 μg/mL. Carbapenemases are considered a major antimicrobial resistance threat, and infections caused by KPC have been considered a public health problem worldwide. Thus, compound 2 may represent a new alternative to treat infections caused by Gram-negative bacteria producers of carbapenemase enzymes, and should be further investigated.

1. Introduction

Endophytic fungi comprise a heterogeneous and diverse group of species that colonize the interior of plants for part of their life without causing harm to the host [1]. Long relationships of endophytes and medicinal plants contribute to endophyte metabolic pathways, thereby influencing their natural bioactivity [2]. Thus, endophytes have been considered a rich source of bioactive secondary metabolites [3], and natural products (NPs) have been screened particularly for the discovery of new molecules with novel mechanisms-of-action to treat steadily increasing numbers of antibiotic-resistant bacteria [4].

Among the endophytic microorganisms, the genus Diaporthe has been reported as a great source of compounds with antibacterial, antifungal, antiviral and antioxidant activities [5,6, 7,8]. Diaporthe species are commonly isolated from medicinal plants, found in a range of biomes, and comprise more than 1000 names (www.indexfungorum.org) [9], however recent studies suggest that only about 220 species are supported by molecular data [5,10,11].

In a previous study performed by Noriler et al. [8], strain LGMF1583 was isolated with considerable antibacterial activity, and might be a possible new species belonging to the genus Diaporthe. The genus identification was performed based on phylogenetic analysis using ITS sequences [8]; however, for the description of new species, a polyphasic approach is required, including multilocus sequence analysis and morphological characterization [5].

Based on these data, we describe herein the new species Diaporthe vochysiae sp. nov. (LGMF1583), isolated as endophyte of the medicinal plant Vochysia divergens, based on polyphasic analysis, and performed a large-scale fermentation, purification and identification of active secondary metabolites produced by this new species.

2. Material and Methods

2.1. General Experimental Procedures

Ultraviolet-visible (UV-VIS) spectra were taken directly from analytical HPLC-PDA runs and show relative intensities. NMR spectra were measured using a Varian (Palo Alto, CA) Vnmr 400 (¹H, 400 MHz; ¹³C, 100 MHz) spectrometer, δ-values were referenced to the respective solvent signals [CD₃OD, δ_H 3.31 ppm, δ_C 49.15 ppm; CDCl₃, δ_H 7.24 ppm, δ_C 77.23 ppm], HPLC-MS analyses were accomplished using a Waters (Milford, MA) 2695 LC module (Waters Symmetry Anal C₁₈, 4.6 × 250 mm, 5 μm; solvent A: H₂O/0.1% formic acid, solvent B: CH₃CN/0.1% formic acid; flow rate: 0.5 mL min⁻¹; 0-4 min, 10% B; 4-22 min, 10-100% B; 22-27 min, 100% B; 27-29 min, 100%-10% B; 29-30 min, 10 % B). HPLC analyses were performed on an Agilent 1260 system equipped with a photodiode array detector (PDA) and a Phenomenex C₁₈ column (4.6 × 250 mm, 5 μm; Phenomenex C₁₈ column, Torrance, CA) [HPLC-conditions: solvent A: H₂O/0.1% TFA; solvent B: CH₃CN; flow rate: 1.0 mL min⁻¹; 0-30 min, 5%-100% B; 30-35 min, 100% B; 35-36 min, 100%-5% B; 36-40 min, 5 % B]. Semi-preparative HPLC was accomplished using Phenomenex (Torrance, CA) C₁₈ column (10 × 250 mm, 5 μm) on a Varian (Palo Alto, CA) ProStar Model 210 equipped with a photodiode array detector and a gradient elution profile (solvent A: H₂O, solvent B: CH₃CN; flow rate: 5.0 mL min⁻¹; 0-2 min, 25% B; 2-15 min, 25-100% B; 15-17 min, 100% B; 17-18 min, 100%-25% B; 18-19 min, 25% B). Size exclusion chromatography was performed on Sephadex LH-20 (25-100 μm; GE Healthcare, Piscataway, NJ). Column chromatography was performed on silica gel (PTLC). PC3 and A549 cells were obtained from ATCC (Manassas, VA). All solvents used were of ACS grade and purchased from the Pharmco-AAPER (Brookfield, CT).

2.2. Species identification and taxonomic description

The isolate LGMF1583 was grown on potato-dextrose agar (PDA) overlaid with sterilized cellophane for 3 days at 28 °C, and total genomic DNA was extracted according to Raeder and Broda [12]. For the multilocus sequence analysis the ITS region, and partial of the elongation factor, tubulin, histone and calmodulin genes were amplified and sequenced as described by Gomes et al. [5]. Chromatograms were visualized using the software MEGA 7 [13] and BioEdit 7.0.5.3 [14]. The phylogenetic analysis was performed using sequences of the available type strains of Diaporthe genus in the database Mycobank (http://www.mycobank.org/). Alignment of sequences was performed in the software Mafft [15] (http://mafft.cbrc.jp/alignment/server). Bayesian phylogeny analyses were performed as described by Noriler et al.[8]. Resulting trees were plotted in FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). The substitution model was selected for each gene using the MEGA software. The DNA sequences of each region were deposited in the GenBank database with the access codes: MG976391 (ITS), MK007526 (TEF1), MK007527 (TUB), MK033323 (HIS), MK007528 (CAL).

The morphological analysis of the strain LGMF1583 was performed based on macromorphological characteristics in different culture media and micromorphological analysis based on sporulation in culture and observation of asexual morphology. The colonies were cultured on 2 % tap water agar supplemented with sterile pine needles (PNA) [16], PDA, oatmeal agar (OA), and 2 % malt extract agar (MEA) (according to Crous et al. [17]), and incubated at 24 °C under a 12 h/12 h dark/light cycle to promote sporulation. The fungal structures were mounted in clear lactic acid, and 50 measurements of conidia and 30 for other structures were determinedat 1,000 x magnification and performed in microscope Axio Imager Z2 (Carl Zeiss, Jena, DE), equipped with Metafer 4/VSlide software (Metasystems, Altlussheim, DE), using differential phase interference contrast (DIC) illumination with software support, ImageJ. The 95 % confidence levels were determined, and the extremes are given in parentheses. Colony diameters were determined at 24 °C in darkness on PDA, OA and MEA. Colony colors (surface and reverse) were described after 14 days, using the color charts of Rayner [18]. Four conidiomata sections were obtained at days 15, 30, 45, and 60 after infection. The structures were fixed and stained based on Bernal et al.[19]. The slides were observed, and photomicrographs were captured using an Olympus microscope equipped with an SC30 camera. Descriptions were deposited in MycoBank (www.MycoBank.org).

2.3. Strain LGMF1583 cultivation and secondary metabolites isolation

Strain LGMF1583 was cultivated in PDA at 28 °C for 3 days, and culture was cut into 8 mm plugs. Five plugs were used to inoculate 32 Erlenmeyer flasks (500 mL) containing 250 mL MEB medium and incubated at 28 °C for 10 days on a rotary shaker (180 rpm). After this period, the culture was filtered-off on Whatman n. 4, and the supernatant was extracted with ethyl acetate (3 x v/v). The recovered organic extracts were evaporated in vacuo at 40 °C to yield 850 mg of a brown, oily crude extract (Fig. 1).

Fig. 1.

Work-up scheme of the metabolites produced by the fungus Diaporthe vochysiae LGMF1583.

As highlighted in Fig. 1, the crude extract obtained (850 mg) was subjected to a Reverse Phase C₁₈ column chromatography (20 × 2.0 cm, 250g) eluted with a gradient of H₂O- CH₃CN (100:0 - 0:100) followed by HPLC analysis to produce five fractions [FI (187 mg), FII (135 mg), FIII (191 mg), FIV (83 mg) and FV (171 mg)]. Further purification of FIII using Sephadex LH-20 (MeOH, 25.0 × 2.0 cm) followed by HPLC afforded the new compounds, vochysiamides A (1; 4.0 mg) and B (2; 7.0 mg) in pure forms as white solids. In the same manner, FI was subjected to Sephadex LH-20 (CH₂Cl₂/40% MeOH; 1 × 40 cm), followed by HPLC purification to afford 2,5-dihydroxybenzyl alcohol (3; 36.2 mg) in pure form as white solid (Fig. 1). All the remaining fractions and sub-fractions were omitted based on the HPLC analysis and/or the absence of bioactivity.

Vochysiamide A (1).

C₈H₁₃NO₄ (187); white solid, HPLC-R_t = 12.12 min (Supplementary Material, Fig. S2); UV/vis λ_max 266 nm; ¹H NMR (CD₃OD, 400 MHz) and ¹³C NMR (CD₃OD, 100 MHz), see Table 1; (−)-APCI-MS: m/z 186 [M − H]⁻; (+)-APCI-MS: m/z 188 [M + H]⁺; (−)-HRESI-MS: m/z 186.0769 [M − H]⁻ (calcd for C₈H₁₂NO₄, 186.0771).

Table 1.

¹³C (100 MHz) and ¹H (400 MHz) NMR Spectroscopic Data of Vochysiamides A (1) and B (2) [δ in ppm].

Position	Compound 1 (CD3OD)		Position	Compound 2 (CD3OD)		Compound 2 (CDC13)
	δC, type	δH (mult, J in [Hz])		δC, type	δH (mult, J in [Hz])	δC, type	δH (mult, J in [Hz])
1	175.7, C		2	173.4, C		172.5, C
2	99.2, CH	5.19 (d, 8.9)	3	125.9, CH	6.36 (s)	125.1, CH	6.28 (brs)
3	137.7, CH	7.44 (dd, 11.5, 9.1)	4	156.8, C		156.2, C
4		11.45 (brd, 9.9)	5	173.6, C		172.7, C
5	175.7, C		7	61.7, CH	3.95 (m)	62.5, CH	4.03 (m)
6	77.1, CH	4.00 (brd, 3.1)	8	60.4, CH2	3.97 (m) 3.72 (brd, 6.9)	58.7, CH2	3.94 (m) 3.88 (m)
7	33.4, CH	2.13 (m)	9	66.2, CH	4.13 (m)	67.3, CH	4.21 (m)
8	16.4, CH3	0.86 (d, 6.8)	10	21.4, CH3	1.20 (d, 6.3)	20.9, CH3	1.16 (d, 6.5)
9	19.6, CH3	1.02 (d, 6.9)	11	27.1, CH	2.81 (m)	26.1, CH	2.83 (m)
			12	21.3, CH3	1.23 (d, 6.9)	21.0, CH3	1.21 (d, 6.5)
			13	21.3, CH3	1.23 (d, 6.9)	21.0, CH3	1.21 (d, 6.5)

See Supplementary Information for NMR spectra. Assignments supported by 2D HSQC and HMBC experiments.

Vochysiamide B (2).

C₁₁H₁₇NO₄ (227); white solid, HPLC-R_t = 11.48 min (Supplementary Material, Fig. S12); UV/vis λ_max 232, 302 nm; ¹H NMR (CD₃OD, 400 MHz) and ¹³C NMR (CD₃OD, 100 MHz), see Table 1; ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz), see Table 1; (−)-APCI-MS: m/z 226 [M − H]⁻, 272 [M+HCOO ]⁻; (+)-APCI-MS: m/z 228 [M + H]⁺; (−)-HRESI-MS: m/z 226.1079 [M − H]⁻ (calcd for C₁₁H₁₆NO₄, 226.1084); (+)-HRESI-MS: m/z 228.1232 [M + H]⁺ (calcd for C₁₁H₁₈NO₄, 228.1230).

2,5-Dihydroxybenzyl alcohol (Gentisin alcohol; 3):

C₇H₈O₃ (140); white solid, HPLC-R_t = 6.03 min (Supplementary Material, Fig. S27); UV/vis λ_max 228, 294 nm; ¹H NMR (CD₃OD, 400 MHz) δ 6.76 (d, J = 2.9 Hz, 1H, 6-H), 6.61 (d, J = 8.6 Hz, 1H, 3-H), 6.54 (dd, J = 8.5, 2.8 Hz, 1H, 4-H), 4.59 (s, 2H, 1-CH₂); ¹³C NMR (CD₃OD, 100 MHz), δ 151.2 (C-5), 149.0 (C-2), 129.6 (C-1), 116.8 (CH-3), 115.9 (CH-6), 115.6 (CH-4), 61.2 (1-CH₂); (−)-APCI-MS: m/z 139 [M − H]⁻.

2.4. Minimum Inhibitory Concentration (MIC)

The MIC of compounds 1-3 against Staphylococcus aureus (MSSA), Methicillin resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carbapenemase-producing (KPC) was performed as described by Gos et al. [20]. The MIC was performed using a 96-well plate, in each well 90 μL of LB broth was added, followed by 10 μL of the crude extract and a serial dilution of compounds was used by the MIC determination. 10 μL of bacteria culture at 0.5 MacFarland scale was added to each well. The plate was incubated on rotary shaker (118 rpm) at 37 °C for 17 hours. The absence of bacterial growth was considered positive result of the analysis. Methanol was used as solvent control.

2.5. Cancer Cell Line Viability Assays

The mammalian cell line cytotoxicity [A549 (non-small cell lung) and PC3 (prostate) human cancer cell lines] assays were accomplished in triplicate following our previously reported protocols [21,22,23, 24,25, 26]. The cytotoxicity of isolated compounds 1-3 was assessed using tumor cells of human lung non-small cell carcinoma A549 and prostate adenocarcinoma PC3 cells, by monitoring the conversion of resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) to its fluorescent product resorufin. The cytototoxicity EC₅₀ values were obtained after 72 h incubation, and actinomycin D was used as positive control (Fig. 2).

Fig. 2.

Dose-response of compounds 1-3 against A549 (non-small cell lung) and PC3 (prostate) human cancer cell lines (72 h). A549: EC₅₀ for compounds 1 (>100 μM), 2 (86.4 μM) and 3 (54.8 μM). PC3: EC₅₀ for compounds 1 (>100 μM), 2 (40.25 μM) and 3 (9.45 μM).

3. Results and Discussion

3.1. Species identification and taxonomic description

The species identification was based on polyphasic analysis. The phylogenetic analysis comprised sequences from 355 ex-type strains of the Diaporthe genus, comprising 3840 characters: the partial sequence of TUB comprised 1023 bp, 310 of these characters were constant (C) and 516 parsimony informative (Pi); the sequence of CAL gene comprised 717 bp with 200 C and 410 Pi; the TEF1 comprised 731 bp with 140 C and 378 Pi; HIS comprised 703 with 329 C and 285 Pi and ITS with 665 bp with 290 C and 220 Pi.

The multilocus phylogenetic analysis included the isolate LGMF1583 in clade 1, however, this strain did not cluster with any ex-type strain, suggesting the strain LGMF1583 as a new species of the genus Diaporthe (Fig. S1). Therefore, we performed a phylogenetic tree using the 29 species belonging to clade 1 (Fig. 3). Strain LGMF1583 is closely related to the strain Diaporthe sp.1 CBS119639, forming a well-supported group (Posterior Probability: 0.9). However, despite LGMF1583 being close to the strain Diaporthe sp. 1 CBS119636, these strains differ in 9 positions in the ITS region (98% of similarity), 1 position in TEF1 (99%), 5 positions in TUB (98%), 3 positions in CAL, and 3 positions in HIS (99%). The isolate CBS 119639 was included in the reclassification of the genus Diaporthe performed by Gomes et al. [5] and together with the isolate LGMF947 was named as Diaporthe sp. 1, since both strains were sterile on culture. Thus, the impossibility of morphological comparison and the differences presented in the phylogenetic analysis do not allow us to assure that the strains CBS119639 and LGMF1583 belong to the same species. Thus, we decided to assign only the strain LGMF1583 to the new species Diaporthe vochysiae, lacking to solve the classification of the strains CBS119639 and LGMF947.

Fig. 3.

Bayesian phylogenetic analysis of the combined 5-gene sequence alignment. Isolate LGMF1583 highlighted in the grey box. Names in bold represent strains known to be ex-type strains or are authentic for the species. Bayesian posterior probabilities are shown at the nodes. The tree was rooted to Diaporthe sambucusii (strain CFCC 51986). Scale bar indicates the number of substitutions per site.

Species description

Diaporthe vochysiae Noriler, Gomes, & Glienke, sp. nov. MycoBank MB827590; Etymology: Named after the host genus from which it was collected, Vochysia.

Conidiomata pycnidial, globose to conical, semi-immersed, scattered or aggregated, brown to black, ostiolate, 328μm wide, 367μm tall, necks 90μm tall, outer surface smooth; Pycnidial wall consisting of brown, thick-walled cells of texture angular; conidial mass globose, predominantly yellow, and yellow to reddish brown. Conidiophores hyaline, smooth, 1–3-septate, subcylindrical to cylindrical, rarely branched above the septa, tapering towards the apex, 21–29 × 2.1–3.3 μm. Conidiogenous cells hyaline, subcylindrical, tapering towards the apex, collarette present and flared, with slightly periclinal thickening, (13.5−) 12.2–14.8(−15) × 3(−5) μm. Alpha conidia hyaline, oblong to ellipsoid, apex bluntly rounded, base obtuse; biguttulate, (2.8−) 3-4 (−4.2) × (1.5−) 2 μm. Beta and gamma conidia absent (Fig. 4).

Fig. 4.

Diaporthe vochysiae (LGMF1583). A) colonies on MEA at 15 days; B) conidiomata sporulating on PNA; C) conidiogenous cells (Arrow); D) colonies on OMA at 15 days; E) Transverse section through conidiomata; F) alpha conidia. Scale bars: E = 100 μm, C and F = 5 μm.

Culture characteristics:

Colonies covering dish after 2 weeks in the dark at 25 °C. On PDA raised, with an entire edge, surface mycelium dense and felty, grey olivaceous, olivaceous buff, olivaceous, greenish olivaceous, colonies reaching 49 mm diam after five days; reverse olivaceous, honey to isabelline. Colonies on MEA flat, entire edge, cottony growth of the aerial mycelium in rings, olivaceous buff, pale olivaceous, grey to olivaceous grey, 60 mm diam. Reverse saffron to pale luteous and cinnamon-buff Colonies on OA flat, entire edge, cottony growth of the aerial mycelium in rings, smoke grey, grey olivaceous and olivaceous, 45 mm diam. Reverse umber and greenish olivaceous.

Specimen examined:

Brazil, Corumba, Pantanal, Mato Grosso do Sul, S19°32’36.9”, W57°02’21.8”, endophytic species isolated from leaf of Vochysia divergens (popular name Cambara), 13 June 2016 by D.C. Savi. Holotype: UPCB 92976 (Herbarium of the Department of Botany code, University Federal of Parana), ex-type culture LGMF1583= CMRP3911 (Laboratory of Genetics of Microorganism and Rede of Microbiological Culture Collection of Paraná, University Federal of Paraná).

3.2. Biological Activities and Secondary Metabolites Identification

From 8 L fermentation of LGMF1583 using malt extract broth (MEB), 850 mg of crude extract was obtained. As highlighted in the experimental section and Fig. 1, the obtained crude extract was subjected for various chromatographic techniques [including reverse phase C₁₈-column, Sephadex LH-20 and semi-preparative HPLC purification] to afford compounds 1-3 in pure form.

3.2.1. Structure elucidation

The physicochemical properties of compounds 1-3 are summarized in the experimental section (Fig. S2–36). Compound 1 was obtained as white solid of medium polarity. The molecular formula of 1 was determined as CsFloNCri (HREI-MS), bearing 3 double-bond equivalents (DBE). The proton NMR spectrum (Table 1), showed one NH broad doublet signal at δ 11.45 (H-1) and two proton signals characteristic for an α-β-unsaturated double bond at δ 7.44 (dd, J = 11.5, 9.1, H-3) and δ 5.19 (d, J = 8.9, H-2). In addition, two proton signals were observed at δ 4.00 (brd, .7=3.1, H-6) and 2.13 (m, H-7) along with two methyl doublet signals at δ 1.02 (d, J = 6.9, CH₃-9) and 0.86 (d, J = 6.8, CH₃-8). The ¹³C NMR/HSQC spectra (Table 2) indicated the presence of two carbonyl groups (acid and/or amide) [δ 175.7 (C-5) and 171.4 (C-1)], two olefinic methines [δ 137.7 (CH-3) and 99.2 (CH-2)], two aliphatic methines [δ 77.1 (CH-6) and 33.4 (CH-7)] and two methyl carbon signals [δ 19.6 (CH₃-9) and 16.4 (CH₃-8)]. Analysis of the COSY and TOCSY spectra (Fig. 5) revealed two fragments through the connectivity of CH-2/CH-3/NH-4 and CH-6/CH-7/CH₃-8 (CH₃-9). Key HMBC correlations (Fig. 5) established 1 to be comprised of two key substructures: an α-β-un saturated double bond of acrylic acid (based on HMBC correlations from H-2/H-3 to C-1) and 2-hydroxy-3-methylbutanamide (based on HMBC correlations from CH₃-8/CH₃-9 to C-6 and C-7; from H-6 to C-5; and from CH₃-8 to CH₃-9). The crucial HMBC correlation from H-3 (δ_H 7.44) to C-5 (δ_C 175.7) served as a basis for the connection of both substructures to establish the preliminary 1 scaffold. The (Z/cis)-configuration of the double bond in 1 was established by NOESY, which highlighted H-2 and H-3 to adopt the same facial orientation (Fig. 5), which supported by the typical coupling constants (J~9 Hz), confirming the Z/cis-configuration of the double bond at C-2/C-3. Finally, the relative configuration at C-6 stereocenter was indirectly established through the analyses of NOESY correlations observed between H-6/H-7 and the detected small couling constant of H-6 (J=3.1) to adopt the same facial orientation of H-6/H-7 (Fig. S5). Thus, thorough cumulative analyses of 1D (¹H, ¹³C) and 2D (HSQC, ¹H,¹H-COSY, TOCSY, HMBC and NOESY) NMR spectra established the structure of 1 as (Z)-3-(2-hydroxy-3-methylbutanamido) acrylic acid (Fig. 5–6). Thus, the structure of 1 was established as a new natural product, and 1 was named vochysiamide A to reflect the host species name of the producing fungus strain.

Table 2.

Minimum Inhibitory Concentrations (MIC) of Secondary Metabolites Produced by Diaporthe vochysiae LGMF1583.

Microrganism	MIC (mg/mL)
	Compound 1	Compound 2	Compound 3
KPC	1.0	0.08	–
MSSA	–	1.0	–
MRSA	–	1.0	–

Note: – denotes no activity

MSSA = Methicillin-Susceptible Staphylococcus aureus; MRSA = Methicillin Resistant Staphylococcus aureus; KPC = Klebsiella pneumoniae carbapenemase-producing

Fig. 5.

A) ¹H,¹H-COSY (—) and selected HMBC (→) correlations of compounds 1-3. B) TOCSY () and selected NOESY () correlations of compounds 1-3.

Fig. 6.

Chemical structures of vochysiamides A-B (1-2) and 2,5-dihydroxybenzyl alcohol (3), produced by the fungus Diaporthe vochysiae LGMF1583.

Compound 2 was also obtained as white solid from the same fraction of 1, using various chromatographic methods (Fig. 1). The molecular weight of 2 was confirmed as 227 based on (−)- and (+)-APCI-MS. The molecular formula of 2 was established as C₁₁H₁₇NO₄(HREI-MS) and bearing 4 double-bond equivalents (DBE). The proton NMR spectrum of 2 (Table 1) showed one singlet proton at δ 6.36 (H-3) along with four proton signals at δ 4.13-3.72 (H-9, H-7 and CH₂-8). In addition, one methine and three doublet methyl proton signals were observed at δ 2.81 (m, H-11), 1.23 (d, J = 6.9 Hz, 6H, CH₃-12/CH₃-13) and 1.20 (d, J = 6.3 Hz, CH₃-10). The ¹³C/APT NMR/HSQC spectra of 2 (Table 2) displayed 11 carbon signals, including two carbonyl [δ 173.4 (C-2) and 173.6 (C-5)], two olefinic [δ 156.8 (C-4) and 125.9 (CH-3)], three methine [δ 66.2 (CH-9), 61.7 (CH-7) and 27.1 (CH-11)], one oxy-methylene [δ 60.4 (CH₂-8)], and three methyl carbon signals [δ 21.4 (CH₃-10) and 21.3 (CH₃-12/CH₃-13)]. Similarly, cumulative analyses of the ¹H,¹H-COSY/TOCSY/NOESY/HMBC spectra (Fig. 5) established the structure of compound 2 as depicted in Fig. 5. Key ³J/²J HMBC correlations [from doublet methyl signals CH₃-12/CH₃-13 (δ_H 1.23) to CH-11 (δ_C 27.1) and C-4 (δ 156.8); from H-3 (δ_H 6.36) to CH-11 (δ_C 27.1), C-2 (δ_C 173.4) and C-5 (δ_C 173.6); from doublet methyl CH₃-10 (δ_H 1.20) to CH-9 (δ_C 66.2) and CH-7 (δ_C 61.7); from CH₂-8 (δ_H 3.96, 3.72) to C-2 (δ_C 173.4) and CH-7 (δ_C 61.7); from H-7 (δ_H 3.95) to C-5 (δ_C 173.6), CH₂-8 (δ_C 60.4) and CH-9 (δ_C 66.2); Fig. 5] confirmed the structure of 2 as (Z)-7-(1-hydroxyethyl)-4-isopropyl-7,8-dihydro-2H-1,5-oxazocine-2,6(5H)-dione. The C-3/C-4 double bond configuration was established as Z/cis by NOESY, based on the correlation observed between the isopropyl group and H-3. In addition, H-7/H-9 were established to adopt the opposite orientation due to the missing NOESY correlation between H-7/H-9 which indicates their antarafacial orientation (Fig. 5). It is also important also to note that, due to the limited purified amounts of compounds 1 and 2 (which were mainly consumed in biological assays) we were unable to do the chemical derivatization at the hydroxy groups (for example by using Mosher ester analysis method) to established the absolute configurations at C-6 and C-9 stereocenters, respectively. As second new natural product isolated from the same fungus crude extract, 2 was subsequently designated as vochysiamide B.

Finally, compound 3 was established as 2,5-dihydroxybenzyl alcohol (also known as gentisin alcohol, gentisyl alcohol or salirepol) based on MS, and 1D and 2D NMR data analyses (Fig. 5), and by comparison with literature [27,28 29,30). 2,5-Dihydroxybenzyl alcohol (3) was previously reported from other fungi [including Penicillium patulum Bainier, Phyllosticta sp., marine Aspergillus varians KMM 4630, marine Penicillium terrestre, Penicillium sp. F020150, Penicillium novae-zeelandiae, Penicillium commune and Penicillium terrestre], and was shown to inhibit apoptosis of U937 human leukemia cells along with both radical-scavenging activity and oxidative mutagenicity [31, 27, 28, 29, 30,32 33].

3.2.2. Biological activities

All isolated compounds were evaluated for their antibacterial, and cancer cell line cytotoxicity assays (Table 2, Fig. 2). One of the new isolated natural products, vochysiamide B (2), showed considerable activity against the bacterium Klebsiella pneumonia (KPC), a producer of carbapenemases (Table 2, MIC of 80 μg/mL), which points out the potential of this compound to treat carbapenem-resistant Gram-negative bacteria. Carbapenemases are considered a major antimicrobial resistance threat of human pathogens belonging to the Enterobacteriaceae, further exacerbated by their broad-spectrum and additional resistance mechanisms that can cause cross-resistance to other antimicrobial classes [34], Because of this broad resistance of KPC, infections caused by K. pneumoniae (pneumonias, urinary tract infections, bacteremia and others) are no longer exclusive of immunocompromised patients but have now also reached the immunocompetent population [35], and therefore are considered a public health problem worldwide.

In the cancer cell line cytotoxicity assays, compounds 2 and 3 displayed appreciable activity against human non-small cell lung A549 and human prostate PC3 cell lines [A549: EC₅₀ for compounds 2 (86.4 μM) and 3 (54.8 μM); PC3: EC₅₀ for compounds 2 (40.25 μM) and 3 (9.45 μM)], (Fig. 2). Compound 1 was not toxic to either of the human cancer cell lines (A549, PC3) at ≤ 100 μM concentration (Fig. 2).

In summary, the new species Diaporthe vochysiae strain LGMF1583, isolated from the medicinal plant Vochysia divergens, produced two new compounds named vochysiamides A (1) and B (2), and the already known compound 2,5-dihydroxybenzyl alcohol (gentisyl alcohol). The new compound vochysiamide B (2) displayed considerable antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae (KPC), and both new compounds showed considerable cytotoxic activity against two tumor cell lines, demonstrating the biotechnological potential of Diaporthe vochysiae as a producer of new molecules with biological activity.