MDN-0170, a New Napyradiomycin from Streptomyces Sp. Strain CA-271078

marine drugs

Article MDN-0170, a New Napyradiomycin from Streptomyces sp. Strain CA-271078

Rodney Lacret *, Ignacio Pérez-Victoria, Daniel Oves-Costales, Mercedes de la Cruz, Elizabeth Domingo, Jesús Martín, Caridad Díaz, Francisca Vicente, Olga Genilloud and Fernando Reyes * Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Avda. del Conocimiento 34, 18016 Armilla (Granada), Spain; [email protected] (I.P.-V.); [email protected] (D.O.-C.); [email protected] (M.d.l.C.); [email protected] (E.D.); [email protected] (J.M.); [email protected] (C.D.); [email protected] (F.V.); [email protected] (O.G.) * Correspondence: [email protected] (R.L.); [email protected] (F.R.); Tel.: +34-958-993-965 (F.R.)

Academic Editor: Miguel O. Mitchell Received: 11 August 2016; Accepted: 13 October 2016; Published: 18 October 2016 Abstract: A new napyradiomycin, MDN-0170 (1), was isolated from the culture broth of the marine-derived actinomycete strain CA-271078, together with three known related compounds identified as 4-dehydro-4a-dechloronapyradiomycin A1 (2), napyradiomycin A1 (3) and 3-chloro- 6,8-dihydroxy-8-α-lapachone (4). The structure of the new compound was determined using a combination of spectroscopic techniques, including 1D and 2D NMR and electrospray-time of flight mass spectrometry (ESI-TOF MS). The relative configuration of compound 1, which contains two independent stereoclusters, has been established by molecular modelling in combination with nOe and coupling constant analyses. Biosynthetic arguments also allowed us to propose its absolute stereochemistry. The antimicrobial properties of the compounds isolated were evaluated against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Aspergillus fumigatus, and Candida albicans. The potent bioactivity previously reported for compounds 2 and 3 against methicillin-sensitive S. aureus has been extended to methicillin-resistant strains in this report.

Keywords: Streptomyces; napyradiomycin; structural elucidation; antimicrobial activity

1. Introduction The napyradiomycins (NPDs) constitute an interesting family of halogenated natural compounds mainly produced by bacteria of the family Streptomycetaceae, which were first discovered from cultures of the actinomycete Chainia rubra [1,2], later reclassified as Streptomyces ruber [3]. This class of secondary metabolites consists of a naphthoquinone core, a prenyl unit attached at C-4a, a monoterpenoid substituent at C-10a, and some congeners have a methyl group at C-7 [1,4–6]. At present, nearly 47 different NPDs have been described [1,2,4–12]. Compounds belonging to this structural class possess significant antibacterial activity against pathogenic bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA) and inhibit the growth of several tumor cell lines [13–15]. In the course of our continuous search for new bioactive natural products from marine actinomycetes, over 400 marine-derived strains were grown in carefully selected media and their fermentations extracts were assayed against clinically relevant pathogenic microbial strains [16]. Growth inhibition of MRSA was observed in the acetone crude extract from fermentation broths of strain CA-271078, which upon 16S rRNA sequencing was found to be closely related to Streptomyces aculeolatus NBRC 14824(T). A bioassay-guided fractionation of the ethyl acetate extract of this microorganism and a dereplication by LC/MS of the bioactive fractions was carried out in order

Mar. Drugs 2016, 14, 188; doi:10.3390/md14100188 www.mdpi.com/journal/marinedrugs Mar.Mar. Drugs Drugs2016 2016, 14, 14, 188, 188 2 of2 of 12 12

this microorganism and a dereplication by LC/MS of the bioactive fractions was carried out in order toto isolate isolate and and identify identify the the new new chemicalchemical constituents that that were were responsible responsible for for the the activities activities observed. observed. HereinHerein we we report report the the isolation isolation of MDN-0170of MDN‐0170 (1), a(1 new), a new napyradiomycin napyradiomycin alongside alongside three relatedthree related known compoundsknown compounds (2–4). The ( structural2–4). The elucidationstructural elucidation of MDN-0170 of wasMDN accomplished‐0170 was accomplished using a combination using a of spectroscopiccombination of techniques, spectroscopic including techniques, HRMS including and extensive HRMS and 1D andextensive 2D NMR 1D and analyses 2D NMR in combination analyses in withcombination molecular with modelling. molecular modelling.

2.2. Results Results andand DiscussionDiscussion

2.1.2.1. Isolation Isolation and and Taxonomy Taxonomy of of the the Producing Producing Microorganism Microorganism TheThe producing producing strain, strain, CA-271078, CA‐271078, was was isolatedisolated from an ascidian collected collected at at the the sea sea shore shore in in Baía Baía AnaAna Chaves, Chaves, Sao Sao Tome Tome (Sao (Sao Tome Tome and and Principe). Principe). A BLASTNA BLASTN search search employing employing the the PCR-ampliﬁed PCR‐amplified 16S rRNA16S rRNA sequence sequence (1359 bp)(1359 indicated bp) indicated that the that strain the wasstrain related was related to Streptomyces to Streptomyces aculeolatus aculeolatusNBRC 14824(T)NBRC (99.34%14824(T) similarity) (99.34% similarity) [17]. A phylogenetic [17]. A phylogenetic tree was tree constructed was constructed using the using neighbor-joining the neighbor‐joining method correctedmethod corrected with the with Jukes-Cantor the Jukes‐Cantor algorithm algorithm [18,19 [18,19]] (Figure (Figure1) showing1) showing the the relatedness relatedness with StreptomycesStreptomyces aculeolatus aculeolatusNBRC NBRC 14824(T) 14824(T) (99.34% (99.34% similarity)similarity) and Streptomyces synnematoformans synnematoformans S155(T)S155(T) (98.73%(98.73% similarity). similarity). TheThe remainingremaining closestclosest members of of the the genus genus StreptomycesStreptomyces exhibitedexhibited sequence sequence similaritiessimilarities below below 98%. These These data data strongly strongly indicate indicate that thatstrain strain CA‐271078 CA-271078 is a member is a member of the genus of the genusStreptomycesStreptomyces. .

S. javensis NBRC 100777T (AB249940)

69 S. violaceusniger NBRC 13459T (AB184420)

81 S. yogyakartensis NBRC 100779T (AB249942)

S. albiflaviniger NRRL B-1356T (AJ391812) 60 S. demainii NRRL B-1478T (DQ334782)

S. hygroscopicus NRRL 2387T (AB231803)

94 S. endus NRRL 2339T (AY999911)

S. sporocinereus NBRC 100766T (AB249933)

S. castelarensis DSM 40830T (AY508511) 68 S. mordarskii NRRL B-1346T (EF408735) 53 96 S. melanosporofaciens NBRC 13061T (AB184283)

S. antimycoticus NBRC 12839T (AB184185) 64 S. sporoclivatus NBRC 100767T (AB249934)

62 S. rapamycinicus NRRL 5491T (EF408733)

S. morookaense LMG 20074T (AJ781349) 98 S. lacticiproducens GIMN4.001T (GQ184344)

S. albospinus NBRC 13846T (AB184527) 66 63 S. angustmyceticus NBRC 3934T (AB184817)

80 S. ramulosus NRRL B-2714T (DQ026662)

69 S. caniferus NBRC 15389T (AB184640)

S. cacaoi NBRC 12748T (AB184115)

S. ruber NBRC 14600T (AB184604)

S. synnematoformans S155T (EF121313)

99 Streptomyces sp. strain CA-271078

55 S. aculeolatus NBRC 14824T (AB184624)

Micromonospora auratinigra TT1-11T (AB159779)

0.01 FigureFigure 1. 1. NeighborNeighbor-joining‐joining (NJ) (NJ) tree built tree with built MEGA with MEGA6.06 based 6.06 on nearly based‐complete on nearly-complete 16S rRNA gene 16S rRNAsequences gene of sequences CA‐271078 of and CA-271078 the closest and type the strains closest of typethe genus strains Streptomyces of the genus. MicromonosporaStreptomyces . Micromonosporaauratinigra TT1 auratinigra‐11(T) wasTT1-11(T) employed was as employedout‐group. asThe out-group. numbers The at the numbers nodes at indicate the nodes bootstrap indicate bootstrapsupport (%) support based (%) on basedNJ analysis on NJ of analysis 1000 replicates; of 1000 replicates;only values only higher values that higher 50% are that shown. 50% areThe shown. scale Thebar scale indicates bar indicates 0.01 substitutions 0.01 substitutions per site. per site.

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2.2. Extraction, Dereplication and Bioassay-GuidedBioassay‐Guided Isolation The producing strain CA CA-271078‐271078 was fermented at at 28 28 °C◦C in in 1 1 L L of of R358 medium for for six six days. days. Extraction withwith an an equal equal volume volume of acetoneof acetone and and evaporation evaporation of the of organic the organic solvent solvent afforded afforded an acetone an crudeacetone extract, crude whichextract, was which subsequently was subsequently subjected subjected to liquid-liquid to liquid extraction‐liquid extraction with ethyl with acetate ethyl (EtOAc). acetate This(EtOAc). organic This extract organic showed extract showed antibacterial antibacterial activity activity against against MRSA. MRSA. LC-UV-MS LC‐UV analysis‐MS analysis of the of ethyl the acetateethyl acetate extract extract revealed revealed the presencethe presence of some of some compounds compounds that that were were not not included included in in our our in-housein‐house microbial natural products library [20] [20] nor in the the Chapman & Hall dictionary of of Natural Natural Products Products [21]. [21]. The ethyl ethyl acetate acetate extract extract was was chromatographed chromatographed on Sephadex on Sephadex LH‐20 LH-20using dichloromethane/methanol using dichloromethane/ methanolto afford ten to afford fractions: ten fractions:A–J. Fractions A–J. FractionsC, D, F, G, C, and D, F, H G, were and the H were most the active most in active a test inagainst a test MRSA. against MRSA.LC‐DAD LC-DAD-HRMS‐HRMS analysis analysis allowed allowed us to detect us to possible detect possiblemilbemycin milbemycin-like‐like antibiotics antibiotics in fractions in fractions C and D C[4,22]. and DThis [4 ,22analysis]. This also analysis showed also that showed fractions that F, fractions G, and F,H G, contained and H contained possible known possible and known bioactive and bioactiveNDPs bearing NDPs chlorine bearing chlorine(according (according to their to isotopic their isotopic pattern), pattern), such suchas napyradiomycin as napyradiomycin A1 A1and and 4‐ 4-dehydro-4a-dechloronapyradiomycindehydro‐4a‐dechloronapyradiomycin A1 A1 [8,12]. [8,12]. Additionally, Additionally, these these fractions fractions also also contained minor amounts of of related related NPDs NPDs whose whose molecular molecular formulae formulae suggested suggested their their novelty novelty as natural as natural products products since sincethey were they not were found not foundin the Dictionary in the Dictionary of Natural of Natural Products Products [21]. According [21]. According to the abundance, to the abundance, chemical chemicalprofile and proﬁle our andinterest our in interest new inNPDs, new NPDs,we decided we decided to continue to continue the bioassay the bioassay-guided‐guided isolation isolation with withfractions fractions F, G, F,and G, H. and Further H. Further chromatographic chromatographic separation separation of bioactive of bioactive fractions fractions (F, G, (F, and G, and H) on H) onSephadex Sephadex LH‐ LH-2020 and and on reversed on reversed phase phase C8 HPLC C8 HPLC using using a gradient a gradient of CH of3CN/H CH3CN/H2O, allowed2O, allowed us to usisolate to isolate four compounds four compounds (Figure (Figure 2). MDN2).‐ MDN-01700170 (1) was ( 1identified) was identiﬁed as a new as compound a new compound on the basis on the of basisESI‐TOF of ESI-TOF and NMR and analysis. NMR analysis. The spectroscopic The spectroscopic data of 2– data4 were of 2identical–4 were identicalto those previously to those previously reported reportedfor 4‐dehydro for 4-dehydro-4a-dechloronapyradiomycin‐4a‐dechloronapyradiomycin A1 (2) [12], A1 napyradiomycin (2)[12], napyradiomycin A1 (3) [1,12] A1 and (3)[ 13,‐12chloro] and‐ 3-chloro-6,8-dihydroxy-8-6,8‐dihydroxy‐8‐α‐lapachoneα-lapachone (4) [12]. (4)[12].

OH O OH O 5a 4a 3 7 OH Cl 8 10a 2 HO 9a O 18 HO O O 19 11 H O 21 17 13 OH 22 16 20 Cl (1) (2)

OH O Cl OH O Cl Cl

HO O HO O O O

(3) (4)

Figure 2. Compounds isolated from culture broths of Streptomyces sp. CA-271078.CA‐271078.

Mar. Drugs 2016, 14, 188 4 of 12

2.3. Structural Determination of MDN-0170 (1)

Compound 1 was isolated as a yellow oil. Its molecular formula was determined as C25H31ClO7 + 35 + on the basis of ESI-TOF measurements (m/z 479.1818 [M + H] , calcd. for C25H32 ClO7 , 479.1831) accounting for ten degrees of unsaturation. The UV absorption pattern, with maxima at 258, 310 and 362 nm of 1 suggested that this compound possessed the dihydronaphthoquinone moiety typically observed in NPDs [6,8,12]. The 1H NMR spectrum of 1 (Table1 and Figure S2) displayed two aromatic protons at δ 6.60 (1H, d, 2.0, H-7) and 6.96 (1H, d, 2.0, H-6), one downfield olefinic proton at δ 7.10 (1H, d, 6.9, H-3) that suggested the presence of a trisubstituted double bond attached to an electron withdrawing group, and two methine protons at δ 3.70 (1H, dd, 12.2, 4.0, H-16) and 3.88 (1H, d, 6.8, H-3). On the other hand, five methyl groups in the aliphatic region at 0.56 (3H, s, H-21), 0.71 (3H, s, H-22), 1.04 (3H, s, H-18), 1.17 (3H, s, H-20), and 1.44 (3H, s, H-19) could also be distinguished. The 13C NMR spectrum exhibited 25 signals: 5 methyl, 3 methylene, 6 methine, and 11 quaternary carbons, according to an heteronuclear single quantum coherence spectroscopy (HSQC) experiment (multiplicity edited). Eight of these quaternary carbons were two carbonyl groups (δ 190.6 and 196.3), two phenolic hydroxyl groups (δ 167.3 and 166.3), and four sp3 carbons (δ 41.8, 72.1, 78.8, and 83.4). The NMR data confirmed that compound 1 was structurally related to dihydronaphthoquinones with two terpenoid substituents (NPDs B series) [8,12].

1 13 Table 1. H and C NMR (500 and 125 MHz in CD3OD) data for compound 1.

Position δ 1H (mult, J, Hz) δ 13C Position δ 1H (mult, J, Hz) δ 13C 1.96, dd, 15.7, 1.4 2 —- 78.8, s 11 40.9, t 2.22, dd, 15.7, 7.8 3 3.88, d, 6.9 67.0, d 12 1.55, d, 7.8 52.6, d 4 7.10, d, 6.9 134.2, d 13 —- 72.1, s 1.55, m 4a —- 140.2, s 14 42.2, t 1.77, m 1.82, m 5 —- 190.6, s 15 31.6, t 1.91, dd, 13.7, 4.0 5a —- 111.7, s 16 3.70, dd, 12.2, 4.0 72.2, d 6 —- 166.3, s 17 —- 41.8, s 7 6.60, d, 2.0 109.7, d 18 1.04, s 25.6, q 8 —- 167.3, s 19 1.44, s 24.7, q 9 6.96, d, 2.0 109.5, d 20 1.17, s 24.4, q 9a —- 137.8, s 21 0.56, s 29.2, q 10 —- 196.3, s 22 0.71, s 16.3, q 10a —- 83.4, s

The COSY spectrum of 1 showed the presence of the fragment –CH2–CH2–CHX– (C-14 to C-16). Thus, the methylene that resonated at δ 1.82 (H-15 ax) and 1.91 (H-15 eq) showed a coupling with the signals at δ 3.70 (H-16), 1.55 (H-14a), and 1.77 (H-14b). The signal at δ 3.88 (1H, d, 6.9) was assigned to H-3 whose carbon signal appears at δ 67.0 and suggests that an oxygenated function is located at this position. All these proposals were further supported by the similarity of the spectroscopic data of 1 with those reported for other analogous compounds [5,8]. Correlations in the HMBC experiment (Figure3) between the oleﬁnic proton at δH 7.10 and the signals at δC 78.8 (C-2), 67.0 (C-3), 140.2 (C-4a), 190.6 (C-5), and 83.4 (C-10a) conﬁrmed the position of the trisusbtituted double bond at ∆4 and the hydroxyl group at C-3. This NMR analysis thus allowed establishing the connectivity of Compound 1 as 3-hydroxy-10a-(3-chloro-6-hydroxy-2,2,6-trimethylcyclohexylmethyl)-6,8-dihydroxy-2,2-dimethyl- 3,10a-dihydro-2H-benzo[g]chromene-5,10-dione, which was given the name MDN-0170 according to our proprietary compound library. Mar. Drugs 2016, 14, 188 5 of 12 Mar. Drugs 2016, 14, 188 5 of 12

Mar. Drugs 2016, 14, 188 OH O 5 of 12 OH OH O HO O OH O HO O OH O Cl OH Cl COSY HMBC COSY FigureFigure 3. Key 3. COSYKey COSY and and HMBC HMBC correlations correlations observed observed in inthe the spectra spectra of compound of compound 1. 1. HMBC The relative stereochemistry of MDN‐0170 (1) was determined by analysis of key NOESY Thecorrelations relativeFigure stereochemistry(Figure 3. Key 4) andCOSY the and coupling of HMBC MDN-0170 constantscorrelations observed (1 observed) was in determined in its the 1H spectra NMR spectrumof by compound analysis in combination 1. of key NOESY correlationswith molecular (Figure4 )modelling. and the coupling The biogenetic constants origin described observed for in the its napyradiomycin1H NMR spectrum B series in was combination also with moleculartakenThe intorelative modelling. account stereochemistry [5,23]. The The biogenetic cyclohexane of MDN origin ring‐0170 displays described (1) was the expecteddetermined for the chair napyradiomycin by conformation analysis of in Bkey which series NOESY H was‐ also 16 is located in axial disposition since it appears as a double doublet1 with large (12.2 Hz) axial‐axial takencorrelations into account (Figure [5,23 4)]. and The the cyclohexane coupling constants ring displays observed the in its expected H NMR chair spectrum conformation in combination in which withand molecular medium (4.0modelling. Hz) axial The‐equatorial biogenetic coupling origin constants described to foreach the of napyradiomycinits vicinal methylene B series protons. was On also H-16 is located in axial disposition since it appears as a double doublet with large (12.2 Hz) axial-axial takenthe intobasis account of the [5,23].strong The NOESY cyclohexane correlation ring observed displays thebetween expected H‐16 chair and conformation H‐12, their 1 ‐in3 whichdiaxial H ‐ and medium16 isrelationship located (4.0 in Hz)on axial the axial-equatorial dispositionsame face of since the ring couplingit appears was also as constants clear. a double Additionally, todoublet each the with of C its‐ 21large vicinal methyl (12.2 group methylene Hz) axialshowed‐axial protons. On theandkey basis medium NOESY of the (4.0 correlations strongHz) axial NOESY ‐withequatorial both correlation H coupling‐16 and H ‐constants observed12, indicating to between each its equatorial of its H-16 vicinal display and methylene H-12, sharing their protons. the same 1-3 On diaxial relationshiptheface basis of on theof the cyclohexanethe same strong face NOESYring. of On the thecorrelation ring other was hand, also observed the clear. strong Additionally,between NOESY Hcorrelation‐16 theand C-21 Hbetween‐12, methyl their the C1 group‐‐320 diaxial and showed C‐22 methyl groups indicated that they are axially displayed on the same face of the ring (opposite key NOESYrelationship correlations on the same with face both of the H-16 ring and was H-12, also clear. indicating Additionally, its equatorial the C‐21 displaymethyl group sharing showed the same to the face where H‐12 is located). The relative configuration on the cyclohexane ring was, therefore, face ofkey the NOESY cyclohexane correlations ring. with On both the otherH‐16 and hand, H‐12, the indicating strong NOESY its equatorial correlation display between sharing the the same C-20 and identical to that reported for related compounds [2,4–6,24]. Thus, it is expected that the absolute face of the cyclohexane ring. On the other hand, the strong NOESY correlation between the C‐20 and C-22 methylconfiguration groups of indicated the chiral centers that they on this are cyclohexane axially displayed ring is also on the the same same since face MDN of‐ the0170 ring (1) and (opposite C‐22 methyl groups indicated that they are axially displayed on the same face of the ring (opposite to the facethe wherepreviously H-12 mentioned is located). compounds The relative must configurationshare a common on biosynthetic the cyclohexane route [21]. ring This was, same therefore, identicalto thebiogenetic to face that where argument reported H‐12 promptedis for located). related us The to compounds assume relative that configuration the [2 relative,4–6,24 ].stereochemicalon Thus, the cyclohexane it is relationship expected ring was, thatbetween therefore, the C absolute‐ identical to that reported for related compounds [2,4–6,24]. Thus, it is expected that the absolute configuration10a and of C‐ the12, which chiral connects centers the on stereochemistry this cyclohexane of the ring two is independent also the same stereoclusters since MDN-0170 (cyclohexane (1) and the configurationring and dihydropyran of the chiral ring), centers must on be this the cyclohexane same as that ringfound is foralso CNQ525.510A the same since [5,6] MDN (Figure‐0170 5). (1) and previouslythe previously mentioned mentioned compounds compounds must share must a share common a common biosynthetic biosynthetic route [route21]. This [21]. same This biogeneticsame argumentbiogenetic prompted argument us prompted to assume us that to assume the relative that the stereochemical relative stereochemical relationship relationship between between C-10a C‐ and H H C-12,10a which and connectsC‐12, which the connects stereochemistry the stereochemistry of the two of independentthe two independentO stereoclusters stereoclusters (cyclohexane (cyclohexane6.9 Hz ring and HO 18 12 H 14 dihydropyranring and dihydropyran ring), must bering), the must same be as the that same found as that for found CNQ525.510A for CNQ525.510AH3C [5,6]H (Figure [5,6] (Figure5). 5). 11 4 16 H H C H 3 H 3H H 21 20 Cl 15 O O 6.9 Hz CH3 O 18 HO CH 12 CH H 14 H 11 H C 3 11 3 3 19 HOH 22 4 16 H H C H 3 H Figure 34. Key NOESY correlations (dashed lines) and coupling constant which determine the relative 21 20 Cl 15 O O configuration of eachCH stereocluster3 in MDN‐0170 (1). CH CH H 11 3 3 19 OH Establishing the22 relative stereochemistry of the dihydropyran ring required an evaluation of the energy‐minimized molecular models of the two possible epimers of compound 1 at C‐3 for their FigurecompatibilityFigure 4. Key 4. Key NOESY with NOESY the correlations experimentalcorrelations (dashed (dashed NMR lines) datalines) (observed and coupling coupling nOes constant constant and coupling which which determine constant determine thebetween relative the relative H ‐3 configurationandconfiguration H‐4). One of eachof of the each stereocluster epimers stereocluster (epimer in in MDN-0170 MDNa) has‐ 0170the hydroxyl ((11).). group with the same relative configuration as the chlorine atom at C‐3 found in CNQ525.510A [5,6] while the other epimer (epimer b) has the oppositeEstablishing configuration the relative for thisstereochemistry hydroxyl group. of the Starting dihydropyran from the ringreported required crystal an structure evaluation of ofthe the Establishing the relative stereochemistry of the dihydropyran ring required an evaluation of energyrelated‐minimized antibiotic molecularA80915C [24] models (Figure of 5),the the two molecular possible models epimers of theof compoundtwo possible 1 epimers at C‐3 forat C their‐3 the energy-minimizedcompatibility with the molecular experimental models NMR of data the two(observed possible nOes epimers and coupling of compound constant between1 at C-3 H for‐3 their compatibilityand H‐4). One with of the the experimental epimers (epimer NMR a) has data the hydroxyl (observed group nOes with and the coupling same relative constant configuration between H-3 and H-4).as the One chlorine of the atom epimers at C‐3 (epimer found ina CNQ525.510A) has the hydroxyl [5,6] while group the with other the epimer same (epimer relative b configuration) has the as theopposite chlorine configuration atom at C-3 for found this hydroxyl in CNQ525.510A group. Starting [5,6] from while the the reported other epimercrystal structure (epimer ofb) the has the oppositerelated configuration antibiotic A80915C for this [24] hydroxyl (Figure 5), group. the molecular Starting models from theof the reported two possible crystal epimers structure at C‐3 of the related antibiotic A80915C [24] (Figure5), the molecular models of the two possible epimers at C-3 were created and energy-minimized using Chem3D 12.0 Pro software (see Figure S8). The coupling Mar. Drugs 2016, 14, 188 6 of 12

Mar. Drugs 2016, 14, 188 6 of 12 constantwere between created and H-3 energy and‐minimized H-4 has a using value Chem3D of 6.9 Hz12.0 (FigurePro software4) which (see Figure perfectly S8). The agrees coupling with the ◦ correspondingconstant between dihedral H‐3 angle and H (23.1‐4 has) a observed value of 6.9 in theHz (Figure molecular 4) which model perfectly of epimer agreesb, with and itthe is not ◦ compatiblecorresponding with the dihedral dihedral angle angle (23.1°) (−97.5 observed) observed in the in molecular the molecular model model of epimer of epimer b, anda. Interestingly,it is not the correspondingcompatible with coupling the dihedral constant angle reported (−97.5°) for observed the equivalent in the protons molecular in CNQ525.510A,model of epimer which a. has the relativeInterestingly, configuration the corresponding displayed bycoupling epimer constanta, has a valuereported of 1.8for Hz the [5 ]equivalent that would protons be compatible in withCNQ525.510A, the dihedral angle which of has− 97.5the relative◦ measured configuration in the molecular displayed by model epimer of epimera, has a valuea. On of the 1.8 other Hz [5] hand, the distancethat would measured be compatible between with protons the dihedral H-3 and angle H-4 equalsof −97.5° 2.83 measured Å and 2.41 in the Å inmolecular the molecular model modelsof epimer a. On the other hand, the distance measured between protons H‐3 and H‐4 equals 2.83 Å and of epimers a and b, respectively. A very strong NOESY correlation is observed between these two 2.41 Å in the molecular models of epimers a and b, respectively. A very strong NOESY correlation is protons in compound 1, which would be more compatible with the distance measured in the model of observed between these two protons in compound 1, which would be more compatible with the epimerdistanceb, although measured the in distance the model measured of epimer in b, the although model the of distance epimer ameasuredwould likely in the result model in of a epimer significant NOESYa would correlation. likely result In fact, in a suchsignificant a correlation NOESY correlation. is observed In for fact, the such equivalent a correlation protons is observed in CNQ525.510A for the (whichequivalent has the protons relative in configuration CNQ525.510A (which displayed has the by relative epimer configurationa)[5]. The definite displayed evidence by epimer confirming a) [5]. that theThe dihydropyran definite evidence ring confirming has the relative that the dihydropyran configuration ring displayed has the relative by epimer configurationb came from displayed the almost equallyby epimer intensely b came strong from NOESY the almost correlations equally intensely observed strong between NOESY H-3 correlations and both observed the C-18 between and C-19 Mar. Drugs 2016, 14, 188 7 of 12 geminalH‐3 methyland both groups the C‐18 (Figure and C4‐).19 In geminal the model methyl of groups epimer (Figureb, the 4). distances In the model between of epimer H-3 and b, the each of the geminaldistances methyl between carbons H‐3 and are each 2.75 of Åthe and geminal 2.78 Åmethyl explaining carbons the are equally 2.75 Å and intense 2.78 Å NOESY explaining correlation the protonsequally while intense the small NOESY coupling correlation is the of Hvecinal‐3 with couplingboth groups. with However, H‐12. These in the valuesmodel ofindicate epimer a, dihedraljust of H-3 with both groups. However, in the model of epimer a, just one strong correlation would be angleone between strong Hcorrelation‐12 and H11awould ( Jbe = observed1.4 Hz) closesince tothe − distances74° while between the dihedral H‐3 and angle each betweenof the geminal H‐12 and Hobserved‐11bmethyl (J = since7.8 carbons Hz) the would are distances 2.77 be Åclose and between to3.54 141°. Å H-3 (this Interestingly, and methyl each is ofantiperiplanar in the the geminal energy with‐minimized methyl respect carbons to model H‐3). are ofThus, MDN 2.77 the Å‐ 0170 and (3.541) the Ådihydropyran (thisdihedral methyl angles ring is antiperiplanar in measuredMDN‐0170 (are1 with) has −74.2° respectthe relative and to 170° H-3). configuration respectively, Thus, the displayed dihydropyran in good by epimer agreement ring b (Figure in MDN-0170 with 6). the (observed1) hasThe the relativecoupling relative configuration configuration constants, bearingof displayed H‐3 inin mindMDN by epimer that‐0170 the bis(Figure torsiontherefore6 ).around Theopposite relative the toC configuration ‐11–Cthat ‐reported12 bond of foris H-3 more in flexibleMDN-0170CNQ525.510A than is the therefore rigid [5]. rings opposite of the to molecule. that reported Regarding for CNQ525.510A the NOESY correlations, [5]. the axial C‐22 methyl group showed correlations with H‐11a (medium) and H‐11b (weak) while the equatorial C‐21 methyl OH O OH O showed a strong correlation with H‐11a but no correlation with H‐Cl11b. These key correlations are in Cl Cl agreement with the corresponding distances observed in the energy‐minimized model of MDN‐0170. On the other hand, althoughHO very weak,O a clear genuineHO NOESY correlationO is observed between the aromatic H‐9 proton and the equatorialO C‐21 methyl group. ThisO correlation is in agreement with the molecular model of MDN‐0170 (1) in whichH the corresponding distanceH equals 3.7 Å and definitely validates the relative stereochemistry of theOH whole molecule, provingOH that the stereochemical relationship between C‐10a and C‐12, which relates the two independent stereoclusters (cyclohexane Cl Cl ring and dihydropyran ring), is the same as that found in CNQ525.510A [5,6]. As already mentioned, it is expected that the absolute configuration of all the chiral centers except C‐3 in MDN‐0170 (1) is CNQ525.510A A80915C the same as that reported for CNQ525.510A [5,6] since both compounds must share a common biosynthetic route [21]. FigureFigure 5. 5.Structures Structures ofof CNQ525.510ACNQ525.510A and and A80915C. A80915C. It could be speculated that the precursor of MDN‐0170 (1) might be the corresponding chlorinated compound at C‐3 with the same configuration found for CNQ525.510A [5] and that an enzymatic hydroxylation via a nucleophilic substitution (with chloride ion as the leaving group) at this position, proceeding with inversion of configuration, would render the relative stereochemistry found for compound 1. In fact, the presence of a hydroxyl substitution at C‐3 on the dihydropyran ring has been previously reported in a napyradiomycin of the “C type” [8]. Not surprisingly, that napyradiomycin and MDN‐0170 (1) share the same relative configuration for the chiral centers on the dihydropyran ring suggesting a common enzymatic mechanism for the incorporation of the hydroxyl group at C‐3. MDN‐0170 (1) represents the first napyradiomycin of the “B type” bearing a hydroxyl group rather than a chlorine at this position. Finally, NMR also provided evidence of the stereochemical relationship between C‐10a and C‐ 12. Analysis of the coupling constants involving the geminal H‐11 protons showed H‐11a as a broad doublet (15.7 Hz and 1.4 Hz, the small coupling measured after resolution enhancement using a gaussian window function) and H‐11b as a double doublet (15.7 Hz and 7.8 Hz). In each multiplet, the large 15.7 Hz coupling constant corresponds to the geminal coupling between the H‐11 methylene FigureFigure 6. 6. EnergyEnergy-minimized‐minimized molecular molecular model model of MDN MDN-0170‐0170 ( 1)) showing showing its its relative stereochemistry.

2.4. EvaluationIt could be of speculated Antimicrobial that Activity the precursor of MDN-0170 (1) might be the corresponding chlorinated compound at C-3 with the same conﬁguration found for CNQ525.510A [5] and that an enzymatic Antibacterial and Antifungal Activities Compounds 1–4 were evaluated for their antibacterial and antifungal properties against a clinical isolate of MRSA, E. coli, A. fumigatus, and C. albicans (Table 2). Compounds 2 and 3 showed outstanding antibacterial activity inhibiting the growth of MRSA with minimum inhibitory concentration (MIC) values between 0.5 and 8 μg/mL, while compounds 1 and 4 did not inhibit the growth of MRSA when tested at 64 μg/mL. Otherwise, none of the compounds displayed activity against E. coli, A. fumigatus, and C. albicans when tested at 64 μg/mL.

Table 2. Antimicrobial activity (μg/mL) of compounds 1–4.

MIC (μg/mL) Microbial Strain Strain Number (1) (2) (3) (4) V R A MRSA MB5393 >64 4–8 0.5–1 >64 2–4 E. coli MB2884 >64 >64 >64 >64 6.5–12.5 A. fumigatus ATCC46645 >64 >64 >64 >64 4 C. albicans MY1055 >64 >64 >64 >64 2–4 V (vancomycin), R (rifampicin), A (amphotericin B).

Mar. Drugs 2016, 14, 188 7 of 12 hydroxylation via a nucleophilic substitution (with chloride ion as the leaving group) at this position, proceeding with inversion of configuration, would render the relative stereochemistry found for compound 1. In fact, the presence of a hydroxyl substitution at C-3 on the dihydropyran ring has been previously reported in a napyradiomycin of the “C type” [8]. Not surprisingly, that napyradiomycin and MDN-0170 (1) share the same relative configuration for the chiral centers on the dihydropyran ring suggesting a common enzymatic mechanism for the incorporation of the hydroxyl group at C-3. MDN-0170 (1) represents the first napyradiomycin of the “B type” bearing a hydroxyl group rather than a chlorine at this position. Finally, NMR also provided evidence of the stereochemical relationship between C-10a and C-12. Analysis of the coupling constants involving the geminal H-11 protons showed H-11a as a broad doublet (15.7 Hz and 1.4 Hz, the small coupling measured after resolution enhancement using a gaussian window function) and H-11b as a double doublet (15.7 Hz and 7.8 Hz). In each multiplet, the large 15.7 Hz coupling constant corresponds to the geminal coupling between the H-11 methylene protons while the small coupling is the vecinal coupling with H-12. These values indicate a dihedral angle between H-12 and H11a (J = 1.4 Hz) close to −74◦ while the dihedral angle between H-12 and H-11b (J = 7.8 Hz) would be close to 141◦. Interestingly, in the energy-minimized model of MDN-0170 (1) the dihedral angles measured are −74.2◦ and 170◦ respectively, in good agreement with the observed coupling constants, bearing in mind that the torsion around the C-11–C-12 bond is more flexible than the rigid rings of the molecule. Regarding the NOESY correlations, the axial C-22 methyl group showed correlations with H-11a (medium) and H-11b (weak) while the equatorial C-21 methyl showed a strong correlation with H-11a but no correlation with H-11b. These key correlations are in agreement with the corresponding distances observed in the energy-minimized model of MDN-0170. On the other hand, although very weak, a clear genuine NOESY correlation is observed between the aromatic H-9 proton and the equatorial C-21 methyl group. This correlation is in agreement with the molecular model of MDN-0170 (1) in which the corresponding distance equals 3.7 Å and definitely validates the relative stereochemistry of the whole molecule, proving that the stereochemical relationship between C-10a and C-12, which relates the two independent stereoclusters (cyclohexane ring and dihydropyran ring), is the same as that found in CNQ525.510A [5,6]. As already mentioned, it is expected that the absolute configuration of all the chiral centers except C-3 in MDN-0170 (1) is the same as that reported for CNQ525.510A [5,6] since both compounds must share a common biosynthetic route [21].

2.4. Evaluation of Antimicrobial Activity

Antibacterial and Antifungal Activities Compounds 1–4 were evaluated for their antibacterial and antifungal properties against a clinical isolate of MRSA, E. coli, A. fumigatus, and C. albicans (Table2). Compounds 2 and 3 showed outstanding antibacterial activity inhibiting the growth of MRSA with minimum inhibitory concentration (MIC) values between 0.5 and 8 µg/mL, while compounds 1 and 4 did not inhibit the growth of MRSA when tested at 64 µg/mL. Otherwise, none of the compounds displayed activity against E. coli, A. fumigatus, and C. albicans when tested at 64 µg/mL.

Table 2. Antimicrobial activity (µg/mL) of compounds 1–4.

MIC (µg/mL) Microbial Strain Strain Number (1) (2) (3) (4) V R A MRSA MB5393 >64 4–8 0.5–1 >64 2–4 E. coli MB2884 >64 >64 >64 >64 6.5–12.5 A. fumigatus ATCC46645 >64 >64 >64 >64 4 C. albicans MY1055 >64 >64 >64 >64 2–4 V (vancomycin), R (rifampicin), A (amphotericin B). Mar. Drugs 2016, 14, 188 8 of 12

3. Materials and Methods

3.1. General Experimental Procedures Optical rotation was measured with a Jasco P-2000 polarimeter (JASCO Corporation, Tokyo, Japan). Infrared spectra were measured with a JASCO FT/IR-4100 spectrometer (JASCO Corporation) equipped with a PIKE MIRacle™ single reflection ATR accessory. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively) equipped with a 1.7 mm TCI MicroCryoProbe™ (Bruker Biospin, Fällanden, Switzerland). Chemical shifts were reported in ppm using the signals of the residual solvents as internal reference (δH 3.31 and δC 49.0 for CD3OD). LC-UV-MS analysis was performed on an Agilent 1100 (Agilent Tehcnologies, Santa Clara, CA, USA) single quadrupole LC-MS system as previously described [25]. ESI-TOF and MS/MS spectra were acquired using a Bruker maXis QTOF (Bruker Daltonik GmbH, Bremen, Germany) mass spectrometer coupled to an Agilent 1200 LC (Agilent Technologies, Waldbronn, Germany). Acetone used for extraction was analytical grade. Solvents employed for isolation were HPLC grade. Molecular models were generated using Chem3D Pro 12.0 (CambridgeSoft, PerkinElmer Informatics, Waltham, MA, USA). The structures were energy-minimized by molecular mechanics with the MM2 force field using an RMS value of 0.001 as gradient convergence criteria. Molecular modelling figures were generated with PyMol (W. L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific LLC, Palo Alto, CA, USA, 2002).

3.2. Taxonomical Identification of the Producing Microorganism Genomic DNA from CA-271078 was isolated employing the following protocol. Strain CA-271078 was grown on ATCC-2-M medium (soluble starch 20 g/L, glucose 10 g/L, NZ Amine Type E 5 g/L, meat extract 3 g/L, peptone 5 g/L, yeast extract 5 g/L, sea salts 30 g/L, calcium carbonate 1 g/L, pH 7) for about 96 h. The broth (1.5 mL) was centrifuged for 15 min at 13,000 rpm and 4 ◦C in an eppendorf tube. The supernatant was discarded and the pellet was re-suspended in 800 µL of extraction buffer (0.2% SDS, 50 mM EDTA, pH 8.5) and heated at 70 ◦C for 30 min. The resulting mixture was then centrifuged for 15 min at 13,000 rpm and 4 ◦C. The supernatant was then transferred to an eppendorf tube containing 60 µL of sodium acetate (3 M, pH 5.2). The mixture was incubated at 4 ◦C for 2 h and then centrifuged for 15 min at 13,000 rpm and 4 ◦C. Five hundred microliters of the supernatant were transferred to an eppendorf tube containing 1 mL of i PrOH and the mixture was incubated at 4 ◦C overnight. The next day the content was centrifuged (15 min, 13,000 rpm, 4 ◦C), the supernatant was discarded, and the pellet was washed with 200 µL of 70% ethanol. The washed pellet was centrifuged in the same conditions (15 min, 13,000 rpm, 4 ◦C), the supernatant was discarded, and the pellet was dried for several hours at room temperature. The pellet (genomic DNA) was then resuspended in 100 µL of sterile water. The 16S rRNA gene was PCR-amplified employing the universal eubacterial primers fD1 (50-AGAGTTTGATCCTGGCTCAG-30) and rP2 (50-ACGGCTACCTTGTTACGACTT-30). PCR mixtures contained 5 µL of PCR buffer (10×), 4 µL of dNTPs (2.5 mM each), 0.5 µL of each of the primers (100 µM), 2 µL of a 1/50 dilution of the genomic DNA, and 0.4 µL of Taq Polymerase (5 U/µL) in a total volume of 50 µL. The PCR product was purified and sequenced at Secugen S. L. (Madrid, Spain) employing the above primers and the internal primers 926F (50-AAACTYAAAKGAATTGACGG-30) and 1100R (50-GGGTTGCGCTCGTTG-30). The resulting DNA sequence lectures were aligned and visually inspected with Bionumerics 6.6 to obtain a nearly complete (1359 nt) sequence (Text S9).

3.3. Fermentation of the Producing Microorganism A 1 L fermentation of strain CA-271078 was generated as follows: a seed culture of the strain was obtained by inoculating two 25 × 150 mm tubes containing 16 mL of ATCC-2-M medium (soluble starch 20 g/L, glucose 10 g/L, NZ Amine Type E 5 g/L, meat extract 3 g/L, peptone 5 g/L, yeast extract 5 g/L, sea salts 30 g/L, calcium carbonate 1 g/L, pH 7) with 0.8 mL of a freshly thawed inoculum stock Mar. Drugs 2016, 14, 188 9 of 12 of the producing strain. The tubes were incubated in a rotary shaker at 28 ◦C, 70% relative humidity, and 220 rpm for about 96 h. The fresh inoculum thus generated was mixed and employed to inoculate twenty 250 mL ﬂasks, each containing 50 mL of R358 medium (2.5% v/v) (soluble starch 10 g/L, yeast extract 4 g/L, peptone 2 g/L, KBr stock solution 5 mL/L (stock containing 8 g/L), FeSO4·7H2O stock solution 5 mL/L (stock containing 8 g/L), sea salts 30 g/L, pH adjusted to 7.0). The inoculated ﬂasks were incubated in a rotary shaker at 28 ◦C, 70% relative humidity, and 220 rpm for six days before harvesting.

3.4. Extraction and Bioassay Guided Isolation The fermentation broth (1 L) was extracted with acetone (1 L) under continuous shaking at 220 rpm for 2 h. The mycelium was separated by filtration and the supernatant (ca. 2L) was concentrated to 1 L under reduced pressure. The aqueous crude extract was extracted with ethyl acetate to afford an ethyl acetate extract (0.226 g). After confirming bioactivity against MRSA, this ethyl acetate extract was chromatographed on Sephadex LH-20 (30 × 500 mm) using methanol/dichloromethane (2:1) to afford 24 fractions of 15 mL. They were combined into ten fractions according to LC-UV-MS chemical profiles: fractions A–J. Fractions C (0.029 g), D (0.030 g), F (0.060 g), G (0.010 g), and H (0.058 g) were the most active against MRSA. Fraction F (0.060 g) was chromatographed on Sephadex LH-20 using mixtures of chloroform/ methanol (2:1) affording five fractions grouped according to LC-UV-MS profiles. The fifth fraction was purified by reverse-phase semipreparative HPLC (column Agilent Zorbax RX-C8, 9.4 × 250 mm, 7 µm; 3 mL/min, UV detection at 210 and 260 nm) with a linear gradient of CH3CN/H2O with 0.1% trifluoroacetic acid, from 60% to 75% CH3CN over 50 min yielding 1 (0.3 mg, tR 30 min). Fraction G (0.015 g) was subjected to reversed-phase semipreparative HPLC (column Agilent Zorbax RX-C8, 9.4 × 250 mm, 7 µm; 3 mL/min, UV detection at 210 and 260 nm) with a linear gradient of CH3CN/ H2O with 0.1% trifluoroacetic acid, from 50% to 80% CH3CN over 50 min, yielding 1 (0.5 mg, tR 31 min). Fraction H (0.058 g) was chromatographed on Sephadex LH-20 (30 × 250 mm) using mixtures of chloroform/methanol (2:1), affording 16 fractions of 5 mL. They were reduced to four final fractions according to LC-UV-MS chemical profiles. Fraction 2 was subjected to reversed-phase semipreparative HPLC (column Agilent Zorbax RX-C8, 9.4 × 250 mm, 7 µm; 3 mL/min, UV detection at 210 and 260 nm) with a linear gradient of CH3CN/H2O with 0.1% trifluoroacetic acid, from 50% to 80% CH3CN over 50 min, yielding 2 (1.2 mg, tR 40 min) and 3 (1.5 mg, tR 42 min). Fraction 4 was subjected to reversed-phase semipreparative HPLC (column Agilent Zorbax RX-C8, 9.4 × 250 mm, 7 µm; 3 mL/min, UV detection at 210 and 260 nm) with a linear gradient of CH3CN/ H2O with 0.1% trifluoroacetic acid, from 50% to 80% CH3CN over 50 min, yielding 4 (2.2 mg, tR 32 min). 25 ◦ −1 MDN-0170 (1): yellow oil; [α]D +6.3 (c 0.04, MeOH); IR (ATR) cm : 3350, 2955, 1712, 1680, + 35 + 1618, 1265, 1082, 1017, 877, 779; (+)-ESI-TOFMS m/z 496.2080 [M + NH4] (calcd. for C25H35 ClNO7 , + 35 + + 496.2097), 479.1818 [M + H] (calcd. for C25H32 ClO7 , 479.1831), 461.1721 [M + H − H2O] (calcd. 35 + + 35 + 1 for C25H30 ClO6 , 461.1725), 443.1614 [M + H − 2H2O] (calcd. for C25H28 ClO5 , 443.1620); H and 13C NMR data see Table1.

3.5. Antibacterial and Antifungal Assays Compounds 1–4 were tested for their ability to inhibit the growth of Gram negative and Gram positive bacteria (E. coli MB2884 and methicillin-resistant S. aureus MRSA, MB5393), fungi (A. fumigatus ATCC46645), and yeast (C. albicans MY1055) following previously described methodologies [26–28]. Brieﬂy, each compound was serially diluted in DMSO with a dilution factor of two to provide ten concentrations starting at 64 µg/mL for all the assays. The MIC was deﬁned as the lowest concentration of compound that inhibited ≥95% of the growth of a microorganism after overnight incubation. The Genedata Screener software (Genedata, Inc., Basel, Switzerland) was used to process and analyze the data and also to calculate the RZ’ factor, which predicts the robustness of an assay [29]. In all experiments performed in this work, the RZ’ factor obtained was between 0.87 and 0.98. Mar. Drugs 2016, 14, 188 10 of 12

4. Conclusions Compounds 1–4 have been identified from the culture of Streptomyces sp. strain CA-271078, a Streptomyces strain related to Streptomyces aculeolatus NBRC 14824(T) which was isolated from an ascidian collected at the seaside in Baía Ana Chaves, Sao Tome (Sao Tome and Principe). Compounds 2 and 3 were able to inhibit strongly the growth of MRSA, one of the most common causes of hospital-acquired infections, with MIC values in the micromolar range. The activity found for compounds 2 and 3 against a clinical isolate of MRSA expands the panel of activity previously reported for these compounds against methicillin-sensitive S. aureus [12] to methicillin-resistant strains. Additionally, in view of the bioactivity displayed by some members of the family against Gram-positive bacteria and cancer cell lines, some conclusions about structure-activity relationships of these naphtoquinones can be established. A prenyl chain attached to C-10a or at least a double bond at position ∆13,20 seem to be essential for the biological activity of this structural class [4–6,12]. Using molecular modelling in combination with nOe and coupling constants analyses, the relative configuration of compound 1, containing two independent stereoclusters, has been established. Biosynthetic arguments for the compound also allowed us to propose its absolute stereochemistry. This article constitutes the first report on the chemical composition of extracts from a marine derived Streptomyces strain related to S. aculeolatus, and has identified this strain as a new source of milbemycins and NPDs related compounds. The research described herein also confirms that marine derived actinomycetes continue to be a rich and underexploited source of new small molecules that could lead to the discovery of new antibiotics.

Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/10/188/s1, Figure S1: Electrospray-time of flight (ESI-TOF) (A) and UV (B) spectra for compound 1; Figure S2: 1H NMR 13 spectrum (CD3OD, 500 MHz) of compound 1; Figure S3: C NMR spectrum (CD3OD, 125 MHz) of compound 1; Figure S4: COSY spectrum of compound 1; Figure S5: HSQC spectrum of compound 1; Figure S6: HMBC spectrum of compound 1; Figure S7: NOESY spectrum of compound 1; Figure S8: Energy-minimized models of the two possible epimers at C-3 of compound 1; Text S9: 16S rRNA gene sequence from CA-271078. Acknowledgments: The authors acknowledge the assistance of M.E. in the fermentation of the strain, E.P. and I.S. for assistance in the preparation of extracts, and M.A.M. and P.S. for the assistance in the performance of antimicrobial assays. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 312184. The polarimeter, HPLC, IR, and NMR equipment used in this work were acquired with two grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovación [Grants No. PCT-010000-2010-4 (NMR) and INP-2011-0016-PCT-010000-ACT6 (Polarimeter, HPLC and IR)]. Author Contributions: R.L. and E.D. performed the extraction of the culture broths and the isolation of the compounds. D.O.-C. carried out the fermentation and the taxonomic identification of the microbial strain. C.D., M.d.l.C. and F.V. performed the antibacterial and antifungal assays. R.L. and I.P.-V. analyzed the spectroscopic data, performed the molecular modelling, and elucidated the structure of the new molecule. J.M. performed the LC/MS analyses and analyzed and interpreted the ESI-TOF spectra. F.R., O.G. and F.V. conceived the research and supervised the work. R.L., I.P.-V., D.O.-C., O.G. and F.R. wrote the paper, which was revised and approved by all the authors. Conflicts of Interest: The authors declare no conflict of interest.

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