New Napyradiomycin Analogues from Streptomyces sp. Strain CA-271078

As part of our continuing efforts to discover new bioactive compounds from microbial sources, a reinvestigation of extracts of scaled-up cultures of the marine-derived Streptomyces sp. strain CA-271078 resulted in the isolation and structural elucidation of four new napyradiomycins (1–3, 5). The known napyradiomycin SC (4), whose structural details had not been previously described in detail, and another ten related known compounds (6–15). The structures of the new napyradiomycins were characterized by HRMS and 1D- and 2D-NMR spectroscopies and their relative configurations were established through a combination of molecular modelling with nOe and coupling constants NMR analysis. The absolute configuration of each compound is also proposed based on biosynthetic arguments and the comparison of specific rotation data with those of related compounds. Among the new compounds, 1 was determined to be the first non-halogenated member of napyradiomycin A series containing a functionalized prenyl side chain, while 2–4 harbor in their structures the characteristic chloro-cyclohexane ring of the napyradiomycin B series. Remarkably, compound 5 displays an unprecedented 14-membered cyclic ether ring between the prenyl side chain and the chromophore, thus representing the first member of a new class of napyradiomycins that we have designated as napyradiomycin D1. Anti-infective and cytotoxic properties for all isolated compounds were evaluated against a set of pathogenic microorganisms and the HepG2 cell line, respectively. Among the new compounds, napyradiomycin D1 exhibited significant growth-inhibitory activity against methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis, and HepG2.


Structural Elucidation
Compound 1 was obtained as a white powder and was assigned the molecular formula C25H30O7 (11 degrees of unsaturation) by analysis of the sodium adduct present in its ESI-TOF mass spectrum (m/z 465.1878 [M + Na] + , calcd. for C25H30NaO7 + , 465.1884) ( Figure S1-A). The UV absorption pattern ( Figure S1-B) with maxima at 258, 314, and 362 nm along with the IR spectrum of 1 (broad absorption bands at around 3294 cm −1 for multiple hydroxyl groups, and at 1701 cm −1 for a conjugated carbonyl functionality) suggested that this compound possessed the dihydronaphthoquinone moiety typically present in napyradiomycin metabolites.

Structural Elucidation
Compound 1 was obtained as a white powder and was assigned the molecular formula C 25 H 30 O 7 (11 degrees of unsaturation) by analysis of the sodium adduct present in its ESI-TOF mass spectrum (m/z 465.1878 [M + Na] + , calcd. for C 25 H 30 NaO 7 + , 465.1884) ( Figure S1-A). The UV absorption pattern ( Figure S1-B) with maxima at 258, 314, and 362 nm along with the IR spectrum of 1 (broad absorption bands at around 3294 cm −1 for multiple hydroxyl groups, and at 1701 cm −1 for a conjugated carbonyl functionality) suggested that this compound possessed the dihydronaphthoquinone moiety typically present in napyradiomycin metabolites. The 1 H NMR spectrum of 1 (Table 1 and Figure S2) in DMSO-d 6 exhibited three deshielded signals attributable to two aromatic protons at δ H 6.57 ppm (1H, d, 2.1, H-7) and 6.84 ppm (1H, d, 2.1, H-9), and one downflield olefinic proton signal at δ H 6.89 ppm (1H, d, 6.4, H-4). This suggested the presence of a trisubstituted double bond attached to an electron deactivating group. Signals for one olefin methine at δ H 4.98 ppm (1H, br t, 7 Figure S3) exhibited 25 signals: two carbonyl signals at δ C 189.7 and 195.5 ppm, two phenolic carbons (δ C 164.7 and 165.7 ppm), ten sp 2 methine or quaternary carbon signals resonating between δ C 107.7 and 148.3 ppm, one sp 2 methylene at δ C 110.3 ppm, one sp 3 oxygenated quaternary carbon a at δ C 82.2 ppm, two oxygenated methine carbons at δ C 65.8 and 73.4 ppm, as well as other seven aliphatic methylene or methyl carbon signals with chemical shifts below δ C 40.4 ppm. Summing up, according to a heteronuclear single quantum coherence spectroscopy (HSQC) experiment (multiplicity edited) ( Figure S5), the 13 C NMR spectrum evidenced the presence of 4 methyl, 4 methylene, 6 methine, and 11 quaternary carbons. These NMR spectroscopic data suggested that compound 1 was structurally related to the napyradiomycin family of antibiotics, more precisely to dihydronapthoquinones with a 10-carbon monoterpenoid branched side chain (NPDs A series) [4]. A comparison of the NMR spectroscopic data of 1 with those of napyradiomycin A2a/A2b [6] revealed that 1 only differed from napyradiomycin A2a/A2b in the substitution at C-3, with the replacement of the chlorine atom in the latter compound by a hydroxy group in 1 and the presence of an additional olefinic bond at C-4/C-4a. This double bond was easily located since the signals for H 2 -4 were lost and replaced by a sp 2 methine signal at δ H 6.89 ppm. Consequently, the 13 C NMR spectrum (Table 2 and Figure S3) showed two new olefinic carbons for C-4 and C-4a at δ C 134.7 ppm (CH) and δ C 137.6 ppm (C). A comprehensive analysis of 2D NMR data ( Figure 2 and Figure S4-S7) allowed the full planar structure of 1 to be assigned, being the first member of the napyradiomycin A-series bearing a hydroxy group rather than a chlorine at position C-3 of the dihydropyran ring ( Figure 1).  The relative stereochemistry of the dihydropyran ring of compound 1 (Figure 3) was assigned by interpretation of ROESY data ( Figure S7) and the coupling constants observed in its 1 H NMR spectrum (Table 1, Figure S2) in combination with molecular modelling using Chem3D 12.0 Pro. The biosynthetic route for all napyradiomycins described to date was also considered [3,28,29]. The almost equally intense ROESY correlations observed between H-3 and both geminal methyl groups (C-20 and C-21) together with a coupling constant value of 6.4 Hz between protons H-3 and H-4 ( Figure 3), in good agreement with a dihedral angle of 28.7 • measured in the energy-minimized molecular model ( Figure S8), confirmed the relative configuration at C-3 of the dihydropyran ring in 1. Assuming an R absolute configuration at C-10a based on the common biosynthetic origin described for the napyradiomycin A series described to date, the absolute configuration at C-3 was also proposed to be The relative stereochemistry of the dihydropyran ring of compound 1 (Figure 3) was assigned by interpretation of ROESY data ( Figure S7) and the coupling constants observed in its 1 H NMR spectrum (Table 1, Figure S2) in combination with molecular modelling using Chem3D 12.0 Pro. The biosynthetic route for all napyradiomycins described to date was also considered [3,28,29]. The almost equally intense ROESY correlations observed between H-3 and both geminal methyl groups (C-20 and C-21) together with a coupling constant value of 6.4 Hz between protons H-3 and H-4 ( Figure 3), in good agreement with a dihedral angle of 28.7° measured in the energy-minimized molecular model ( Figure S8), confirmed the relative configuration at C-3 of the dihydropyran ring in 1. Assuming an R absolute configuration at C-10a based on the common biosynthetic origin described for the napyradiomycin A series described to date, the absolute configuration at C-3 was also proposed to be R. Finally, the low amount of 1 obtained prevented the determination of the absolute configuration of the chiral center at C-16 using Mosher´s approaches. . This formula requires 10 degrees of unsaturation. Their UV, IR, and NMR spectroscopic data were almost identical and shared common features. The UV absorption pattern ( Figures S9-B and S18-B) along with the IR spectrum of both (broad absorption bands at around 3294 cm −1 for multiple hydroxyl groups, and at 1701 cm −1 for a conjugated carbonyl functionality) suggested that these compounds also possessed the dihydronaphthoquinone moiety present in napyradiomycin metabolites. Comprehensive analysis of 1D and 2D NMR data of  The relative stereochemistry of the dihydropyran ring of compound 1 (Figure 3) was assigned by interpretation of ROESY data ( Figure S7) and the coupling constants observed in its 1 H NMR spectrum (Table 1, Figure S2) in combination with molecular modelling using Chem3D 12.0 Pro. The biosynthetic route for all napyradiomycins described to date was also considered [3,28,29]. The almost equally intense ROESY correlations observed between H-3 and both geminal methyl groups (C-20 and C-21) together with a coupling constant value of 6.4 Hz between protons H-3 and H-4 ( Figure 3), in good agreement with a dihedral angle of 28.7° measured in the energy-minimized molecular model ( Figure S8), confirmed the relative configuration at C-3 of the dihydropyran ring in 1. Assuming an R absolute configuration at C-10a based on the common biosynthetic origin described for the napyradiomycin A series described to date, the absolute configuration at C-3 was also proposed to be R. Finally, the low amount of 1 obtained prevented the determination of the absolute configuration of the chiral center at C-16 using Mosher´s approaches. . This formula requires 10 degrees of unsaturation. Their UV, IR, and NMR spectroscopic data were almost identical and shared common features. The UV absorption pattern ( Figures S9-B and S18-B) along with the IR spectrum of both (broad absorption bands at around 3294 cm −1 for multiple hydroxyl groups, and at 1701 cm −1 for a conjugated carbonyl functionality) suggested that these compounds also possessed the dihydronaphthoquinone moiety present in napyradiomycin metabolites. Comprehensive analysis of 1D and 2D NMR data of NMR spectroscopic data were almost identical and shared common features. The UV absorption pattern ( Figures S9-B and S18-B) along with the IR spectrum of both (broad absorption bands at around 3294 cm −1 for multiple hydroxyl groups, and at 1701 cm −1 for a conjugated carbonyl functionality) suggested that these compounds also possessed the dihydronaphthoquinone moiety present in napyradiomycin metabolites. Comprehensive analysis of 1D and 2D NMR data of compounds 2 (Tables 1 and 2; Figures S10-S16) and 3 (Tables 1 and 2; Figures S19-S25) revealed a strong similarity between them and evidenced that they were epimers at C-3 position of the dihydropyran ring. Interpretation of 2D NMR data of compound 2 allowed all the protons and carbons to be assigned and according to a HSQC experiment (multiplicity edited) ( Figure S13), we distinguished the presence of 5 methyl, 3 methylene, 6 methine, and 11 quaternary carbons. These NMR spectroscopic data suggested that compound 2 was structurally related to dihydronapthoquinones with the monoterpenoid substituent cyclized to a 6-membered ring (NPDs B series) [4]. The dihydronaphtoquinone ring was constructed based on HMBC correlations ( Figure 4 and Figure S14) from H-9 (δ H 6.88 ppm) to C-5a, Mar. Drugs 2020, 18, 22 7 of 19 C-7, C-8, and C-10, and from OH-6 (δ H 12.66 ppm) to C-5a, C-6, and C-7. In addition, correlations in the HMBC experiment between the two geminal methyl groups H 3 -18 and H 3 -19 (δ H 0.94 and δ H 1.40 ppm) and C-2 and C-3, and between the olefinic proton H-4 at δ H 6.73 ppm and C-2, C-3, C-4a, and C-10a indicated the presence of a dihydropyran ring and confirmed the position of a chlorine substituent at C-3 (δ H 4.98, s; δ C 59.9, CH) and a trisubstituted double bond at ∆ 4,4a . Analysis of the overall NMR data set (Tables 1 and 2; Figures S10-S16) for the monoterpene unit (C-11 to C-17 and C-20, C-21, and C-22) in 2 showed that it was a cyclohexane ring with a chair conformation. Based on these NMR features, the planar structure of 2 was established ( Figure 4). A similar analysis of the NMR data set (Tables 1 and 2; Figures S19-S25) for compound 3 rendered the same planar structure and evidenced the close structural similarity of 2 and 3 with the previously reported antibiotic CNQ525.510A, which is additionally methylated at C-7 [3]. dihydropyran ring.
Interpretation of 2D NMR data of compound 2 allowed all the protons and carbons to be assigned and according to a HSQC experiment (multiplicity edited) ( Figure S13), we distinguished the presence of 5 methyl, 3 methylene, 6 methine, and 11 quaternary carbons. These NMR spectroscopic data suggested that compound 2 was structurally related to dihydronapthoquinones with the monoterpenoid substituent cyclized to a 6-membered ring (NPDs B series) [4]. The dihydronaphtoquinone ring was constructed based on HMBC correlations (Figures 4 and S14) from H-9 (δΗ 6.88 ppm) to C-5a, C-7, C-8, and C-10, and from OH-6 (δΗ 12.66 ppm) to C-5a, C-6, and C-7. In addition, correlations in the HMBC experiment between the two geminal methyl groups H3-18 and H3-19 (δΗ 0.94 and δΗ 1.40 ppm) and C-2 and C-3, and between the olefinic proton H-4 at δΗ 6.73 ppm and C-2, C-3, C-4a, and C-10a indicated the presence of a dihydropyran ring and confirmed the position of a chlorine substituent at C-3 (δH 4.98, s; δC 59.9, CH) and a trisubstituted double bond at Δ 4,4a . Analysis of the overall NMR data set (Tables 1 and 2; Figures S10-S16) for the monoterpene unit (C-11 to C-17 and C-20, C-21, and C-22) in 2 showed that it was a cyclohexane ring with a chair conformation. Based on these NMR features, the planar structure of 2 was established ( Figure 4). A similar analysis of the NMR data set (Tables 1 and 2; Figures S19-S25) for compound 3 rendered the same planar structure and evidenced the close structural similarity of 2 and 3 with the previously reported antibiotic CNQ525.510A, which is additionally methylated at C-7 [3]. The relative stereochemistry of 2 and 3 was assigned by analysis of NOESY and ROESY NMR data (Figures S15, S16, S24, and S25) and the coupling constants observed in their 1 H NMR spectra. NOESY correlations between the H3-21 methyl protons and the methine protons H-12 and H-16 showed these protons were on the same face of the cyclohexane ring in both compounds ( Figure 5  The relative stereochemistry of 2 and 3 was assigned by analysis of NOESY and ROESY NMR data (Figures S15, S16, S24, and S25) and the coupling constants observed in their 1 H NMR spectra. NOESY correlations between the H 3 -21 methyl protons and the methine protons H-12 and H-16 showed these protons were on the same face of the cyclohexane ring in both compounds ( Regarding the dihydropyran ring, we used the vicinal 3 J HH spin-spin coupling constants and key NOESY correlations to establish the relative configuration of this moiety. The coupling constant between the olefinic proton at H-3 and its vicinal proton H-4 in 2 had a value of 1.6 Hz (Figure 6), which is in good agreement with a dihedral angle of 91.4 • measured in the energy-minimized molecular model ( Figure S17). Furthermore, only one NOESY correlation is observed between H-3 and one of the geminal methyl groups (H 3 -19), due to the antiperiplanar position of the H 3 -18 methyl with respect to H-3 ( Figure 6 and Figure S15). Thus, the relative configuration on the dihydropyran ring for compound 2 was confirmed as depicted in Figure 1. Conversely, the almost equally intense and strong nOe correlations observed between H-3 and both geminal methyl groups (C-18 and C-19) in the spectra of 3 ( Figure 6 and Figure S24), together with a coupling constant value of 6.9 Hz between protons H-3 and H-4 in accordance with a dihedral angle of 25.2 • measured in the energy-minimized molecular model ( Figure S26), confirmed the opposite relative configuration at C-3 for compound 3. Regarding the dihydropyran ring, we used the vicinal 3 JHH spin-spin coupling constants and key NOESY correlations to establish the relative configuration of this moiety. The coupling constant between the olefinic proton at H-3 and its vicinal proton H-4 in 2 had a value of 1.6 Hz (Figure 6), which is in good agreement with a dihedral angle of 91.4° measured in the energy-minimized molecular model ( Figure S17). Furthermore, only one NOESY correlation is observed between H-3 and one of the geminal methyl groups (H3-19), due to the antiperiplanar position of the H3-18 methyl with respect to H-3 (Figures 6 and S15). Thus, the relative configuration on the dihydropyran ring for compound 2 was confirmed as depicted in Figure 1. Conversely, the almost equally intense and strong nOe correlations observed between H-3 and both geminal methyl groups (C-18 and C-19) in the spectra of 3 (Figures 6 and S24), together with a coupling constant value of 6.9 Hz between protons H-3 and H-4 in accordance with a dihedral angle of 25.2° measured in the energy-minimized molecular model ( Figure S26), confirmed the opposite relative configuration at C-3 for compound 3. The absolute configurations of 2 and 3 were assumed to be the same as those reported for all the napyradiomycins in the B series. Apart from the common biosynthetic origin, this proposal was additionally supported by the comparison of specific rotation data. The sign of the specific rotation for compound 2 is negative ([α] 25 D -41.0°, c 0.4, MeOH), as is that reported for CNQ525.510A [3], which is a C-7 methylated version of 2. On the contrary, the sign of [α] 25 D value for the epimeric compound 3 is positive ([α] 25 D +32.0°, c 0.13, MeOH). The previously reported B-type napyradiomycin MDN-0170 [23] displays the same relative configuration at C-3 (but bearing a hydroxy group instead of a chlorine  Regarding the dihydropyran ring, we used the vicinal 3 JHH spin-spin coupling constants and key NOESY correlations to establish the relative configuration of this moiety. The coupling constant between the olefinic proton at H-3 and its vicinal proton H-4 in 2 had a value of 1.6 Hz (Figure 6), which is in good agreement with a dihedral angle of 91.4° measured in the energy-minimized molecular model ( Figure S17). Furthermore, only one NOESY correlation is observed between H-3 and one of the geminal methyl groups (H3-19), due to the antiperiplanar position of the H3-18 methyl with respect to H-3 (Figures 6 and S15). Thus, the relative configuration on the dihydropyran ring for compound 2 was confirmed as depicted in Figure 1. Conversely, the almost equally intense and strong nOe correlations observed between H-3 and both geminal methyl groups (C-18 and C-19) in the spectra of 3 (Figures 6 and S24), together with a coupling constant value of 6.9 Hz between protons H-3 and H-4 in accordance with a dihedral angle of 25.2° measured in the energy-minimized molecular model ( Figure S26), confirmed the opposite relative configuration at C-3 for compound 3. The absolute configurations of 2 and 3 were assumed to be the same as those reported for all the napyradiomycins in the B series. Apart from the common biosynthetic origin, this proposal was additionally supported by the comparison of specific rotation data. The sign of the specific rotation for compound 2 is negative ([α] 25 D -41.0°, c 0.4, MeOH), as is that reported for CNQ525.510A [3], which is a C-7 methylated version of 2. On the contrary, the sign of [α] 25 D value for the epimeric compound 3 is positive ([α] 25 D +32.0°, c 0.13, MeOH). The previously reported B-type napyradiomycin MDN-0170 [23] displays the same relative configuration at C-3 (but bearing a hydroxy group instead of a chlorine corroborates the presence of this tetrahydropyran ring. Comprehensive NMR analyses allowed all protons and carbons to be assigned, and compound 4 was confirmed to be napyradiomycin SC, whose structure was incompletely reported by Kamimura and co-workers in a Japanese patent in 1997 [27]. Details on how the structure was assigned, spectroscopic data, and the absolute stereochemistry of compound 4 were never reported. The relative stereochemistry of 4 was assigned by analysis of NOESY data (Figure 7 and Figure S33). Correlations of the protons on the cyclohexane ring were identical to those observed in the NOESY/ROESY experiments for 2 and 3. Key NOESY correlations between the exchangeable OH signal at C-4a with both H-11 protons and the methine H-12 indicated that the tetrahydropyran ring was cis-fused to the dihydroquinone, as observed for other napyradiomycins. A correlation observed between the OH-4a hydroxy group and the methine H-3 indicated that these were both oriented on the bottom face of the tetrahydropyran ring. The typical axial-equatorial coupling constants measured between H-3 (J = 11.8, 4.4 Hz) and the methylene proton pair H 2 -11 are in good agreement with dihedral angles of -172.6 • and -55.3 • measured in the energy-minimized molecular model ( Figure S34) and confirm that the tetrahydropyran ring was in a chair form, identical to the configuration of this ring in the crystal structure of napyradiomycin B4 [8], a methylated version of compound 4 bearing a chlorine instead of a hydroxy group at C-4a. The full stereostructure of napyradiomycin B4 was assigned by X-ray diffraction methods [8] and hence, the absolute setereochemistry is assumed identical to that of napyradiomycin B4 based on comparable specific rotation values.   (Tables 1 and 2; Figures S28-S33) showed signals similar to those of napyradiomycin metabolites. The presence of a tetrahydropyran ring fused to the dihydronaphthoquinone moiety in 4 was clearly evidenced from the absence of the olefinic proton signal at C-4 present in compounds 1-3, now replaced by two new diastereotopic protons at δΗ 2.12 and 2.19 at that position. Furthermore, the presence of a hydroxy substituent at C-4a, easily assigned based on HMBC correlations ( Figure S32) of that OH-4a at δΗ 6.74 with C-4, C-4a, C10, and C-10a that corroborates the presence of this tetrahydropyran ring. Comprehensive NMR analyses allowed all protons and carbons to be assigned, and compound 4 was confirmed to be napyradiomycin SC, whose structure was incompletely reported by Kamimura and co-workers in a Japanese patent in 1997 [27]. Details on how the structure was assigned, spectroscopic data, and the absolute stereochemistry of compound 4 were never reported.
The relative stereochemistry of 4 was assigned by analysis of NOESY data (Figures 7 and S33). Correlations of the protons on the cyclohexane ring were identical to those observed in the NOESY/ROESY experiments for 2 and 3. Key NOESY correlations between the exchangeable OH signal at C-4a with both H-11 protons and the methine H-12 indicated that the tetrahydropyran ring was cis-fused to the dihydroquinone, as observed for other napyradiomycins. A correlation observed between the OH-4a hydroxy group and the methine H-3 indicated that these were both oriented on the bottom face of the tetrahydropyran ring. The typical axial-equatorial coupling constants measured between H-3 (J = 11.8, 4.4 Hz) and the methylene proton pair H2-11 are in good agreement with dihedral angles of -172.6° and -55.3° measured in the energy-minimized molecular model ( Figure S34) and confirm that the tetrahydropyran ring was in a chair form, identical to the configuration of this ring in the crystal structure of napyradiomycin B4 [8], a methylated version of compound 4 bearing a chlorine instead of a hydroxy group at C-4a. The full stereostructure of napyradiomycin B4 was assigned by X-ray diffraction methods [8] and hence, the absolute setereochemistry is assumed identical to that of napyradiomycin B4 based on comparable specific rotation values.   Compound 5 was obtained as a white powder and was assigned the molecular formula C 25 H 28 Cl 2 O 5 by evaluation of HRMS data ( Figure S35-A)  Interestingly, compound 5 lacks the characteristic deshielded singlet signal of OH-6. HMBC correlations ( Figure S40-A) from H-3 (δ H 4.40 ppm) to C-19/C-20, C-2, and C-4a, and from H 2 -4 (δ H 2.52, 2.25 ppm) to C-2, C-3, C-4a, C-5, and C-10a defined the presence of the archetypal tetrahydropyran ring with two chlorine substituents at C-3 and C-4a positions reported for other napyradiomycins such as 18-hydroxynapyradiomycin A1 (10) [6], napyradiomycin A2a (11) [6], napyradiomycin A2b (12) [6], or napyradiomycin B4 (13) [8]. COSY NMR spectroscopic data ( Figure S38) allowed the identification of three key proton spin systems within the monoterpenoid moiety: H 2 -11 / H-12, H 2 -14/-H 2 -15/H-16, and H 2 -18 ( Figure 8). These three proton sequences were connected as a linear monoterpenoid side chain by interpretation of HMBC correlations (Figure 8 and Figure S40) from H 3 -21 to C-12, C-13, and C-14, and from H 3 -22 to C-16, C-17, and C-18. As expected, additional HMBC correlations from both H 2 -11 and H-12 clearly established the attachment of this side chain to C-10a of the dihydronaphthoquinone moiety ( Figure 8). All these assignments accounted for ten of the eleven degrees of unsaturation and indicated that 5 was composed of a tetracyclic ring system, evidencing that 5 is closely related to napyradiomycin C1 and other compounds within the C series [1,26]. The chemical shift of the methylene carbon signal C-18 (δ C 76.3 ppm) strongly supported the presence of an oxygen substituent at this position. Moreover, the deshielding of this carbon signal in comparison with that found for 18-hydroxynapyradiomycin A1 (10) (δ C 66.6 ppm) suggested the etherification of the 18-hydroxy groups in 5. Finally, the strong key HMBC correlation from H 2 -18 to C-6 (δ C 162.7 ppm; Figure S40b) allowed us to establish unambiguously the existence of such ether link between C-6 and C-18, which results in a 14-membered bridging macrocycle between C-6 and C-10a ( Figure 8). This kind of O-linked cyclization has no precedent within the napyradiomycin metabolites, and therefore compound 5 represents the first member of a new subfamily of napyradiomycins, the D series. Thus, we propose the name napyradiomycin D1 for compound 5.
The chemical shift of the methylene carbon signal C-18 (δC 76.3 ppm) strongly supported the presence of an oxygen substituent at this position. Moreover, the deshielding of this carbon signal in comparison with that found for 18-hydroxynapyradiomycin A1 (10) (δC 66.6 ppm) suggested the etherification of the 18-hydroxy groups in 5. Finally, the strong key HMBC correlation from H2-18 to C-6 (δC 162.7 ppm; Figure S40b) allowed us to establish unambiguously the existence of such ether link between C-6 and C-18, which results in a 14-membered bridging macrocycle between C-6 and C-10a (Figure 8). This kind of O-linked cyclization has no precedent within the napyradiomycin metabolites, and therefore compound 5 represents the first member of a new subfamily of napyradiomycins, the D series. Thus, we propose the name napyradiomycin D1 for compound 5. The relative configuration of 5 was determined by inspection of the NOESY correlations and multiplet analysis for some key proton signals ( Figure 9). As in napyradiomycin SC (4), the values of the coupling constants between H-3 and the pair H2-4 (J = 11.8, 4.4 Hz) confirmed a chair conformation The relative configuration of 5 was determined by inspection of the NOESY correlations and multiplet analysis for some key proton signals ( Figure 9). As in napyradiomycin SC (4), the values of the coupling constants between H-3 and the pair H 2 -4 (J = 11.8, 4.4 Hz) confirmed a chair conformation of the tetrahydropyran ring and the axial orientation of H-3. Although the substitution at C-4a with a chlorine atom in 5 prevents establishing a relative configuration with respect to C-10a, it was assumed to be cis, as for all tetrahydropyran-containing napyradiomycins described to date. The geometry of the two double bonds in 5 was assigned as E based on the existence of NOESY cross-peaks between H 2 -11/H 3 -21/H 2 -14 and H-15/H 3 -22/H 2 -18, as well as the absence of correlations between H-12/ H 3 -21 and H-16/H 3 -22 (Figure 9a). The absolute configuration of 5 is assumed to be the same as for napyradiomycin C1 considering the comparable specific rotation values and the common biosynthetic origin [1]. of the tetrahydropyran ring and the axial orientation of H-3. Although the substitution at C-4a with a chlorine atom in 5 prevents establishing a relative configuration with respect to C-10a, it was assumed to be cis, as for all tetrahydropyran-containing napyradiomycins described to date. The geometry of the two double bonds in 5 was assigned as E based on the existence of NOESY crosspeaks between H2-11/H3-21/H2-14 and H-15/H3-22/H2-18, as well as the absence of correlations between H-12/ H3-21 and H-16/H3-22 (Figure 9a). The absolute configuration of 5 is assumed to be the same as for napyradiomycin C1 considering the comparable specific rotation values and the common biosynthetic origin [1]. Apart from the unambiguous NOESY correlations described above, other sets of cross-peaks were observed for proton signals within the linear monoterpenoid chain ( Figure S41). The slight broadening for these signals in the 1 H NMR spectrum and the presence of such different sets of nOe correlations clearly points to a fast (in NMR time-scale) conformational equilibrium in solution for 5. Not surprisingly, this interconversion might be assisted by the flexibility of the terpenoid chain due to the presence of seven rotatable bonds within it. To better illustrate this, a conformational search using ConfBuster Web Server, a recently delivered open-source tool for the conformational analysis of macrocycles was launched [30]. As a result, up to four close-energy conformers were obtained ( Figure  S42), differing to each other in about 1.5 kcal/mol, which supports the rapid interconversion between them. Interestingly, the minimum-energy conformer proved to be consistent with the most abundant one, as evidenced by the more intense set of NOESY cross-peaks between H2-18 (4.67 ppm) and the aromatic proton at H-7 (7.04 ppm), and between the olefinic proton H-16 (4.91 ppm) and H2-14 (1.42 ppm) (Figure 9b).

Evaluation of Antimicrobial Activity
Antibacterial, Antifungal, and Cytotoxic Activities Compounds 1-15 were evaluated for their antibacterial and antifungal properties against a clinical isolate of methicillin resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, Escherichia coli, Acinetobacter baumannii, and Aspergillus fumigatus (Table 3). Napyradiomycins 2, 5, 7, 12, 13, 14, and 15 showed antibacterial activities against MRSA with MIC values ranging from 3 to 48 μg/mL. The new napyradiomycin D1 (5) was one of the most active compounds and displayed activities comparable to those of napyradiomycin B4 (13) and napyradiomycin B5 (15). Napyradiomycin A2b (12) and napyradiomycin B2 (14) exhibited the best antibacterial activities (MIC values of 3-6 μg/mL) among these fifteen napyradiomycins. Except compounds 3, 6, 8, and 10, the Apart from the unambiguous NOESY correlations described above, other sets of cross-peaks were observed for proton signals within the linear monoterpenoid chain ( Figure S41). The slight broadening for these signals in the 1 H NMR spectrum and the presence of such different sets of nOe correlations clearly points to a fast (in NMR time-scale) conformational equilibrium in solution for 5. Not surprisingly, this interconversion might be assisted by the flexibility of the terpenoid chain due to the presence of seven rotatable bonds within it. To better illustrate this, a conformational search using ConfBuster Web Server, a recently delivered open-source tool for the conformational analysis of macrocycles was launched [30]. As a result, up to four close-energy conformers were obtained ( Figure S42), differing to each other in about 1.5 kcal/mol, which supports the rapid interconversion between them. Interestingly, the minimum-energy conformer proved to be consistent with the most abundant one, as evidenced by the more intense set of NOESY cross-peaks between H 2 -18 (4.67 ppm) and the aromatic proton at H-7 (7.04 ppm), and between the olefinic proton H-16 (4.91 ppm) and H 2 -14 (1.42 ppm) (Figure 9b).
Compound 1 is to the best of our knowledge the first example of a napyradiomycin in the A series bearing a hydroxy group instead a chlorine atom at position C-3 of the dihydropyran ring. This substitution pattern has been previously reported for napyradiomycins of types B (MDN-0170) [23] and C [1,26], further displaying the same relative configuration as 1 at this chiral center. The presence of the C-3 hydroxy group with this absolute configuration can be explained considering the precursor of 1 might be the corresponding chlorinated compound at the same position, and that a non-enzymatic S N 2 nucleophilic substitution with water on the C-3 chloride would result in the production of 1.
Mar. Drugs 2020, 18, 22 13 of 19 Compounds 2 and 3 are epimers at C-3 of their dihydropyran ring. The relative configuration at this chlorinated position for compound 3 is reported herein for the first time in the napyradiomycin series, since all the natural products of this family found in the literature or databases have the opposite configuration at the same chiral center (i.e., that found for compound 2) when it has a chlorine substituent. A first tentative explanation for this variant arises from the proposed mechanism of oxidative halogenation and subsequent halonium-induced cyclization in meroterpenoids [28]. Although the vanadium-dependent chloroperoxidase (VCPO) NapH1 has been shown to act in a stereoselective fashion when introducing chlorine atoms in napyradiomycin biosynthetic intermediates, it was also found that the same enzyme catalyzed a non-stereoselective bromination of the same substrate [31]. This result was explained because of the production of a diffusible hypobromous acid, which would depart the active site of the enzyme and then would brominate the substrate in a nonspecific manner. Indeed, the involvement of a hypohalous acid (HOX species) in this mechanism is widely accepted for vanadium-dependent haloperoxidases (VHPOs) from algae and fungi, which do not exhibit specificity [32], while for VCPOs from Streptomycetes it has been postulated that an enzyme-bound chlorine species would make possible the stereoselective halogenation [33,34].
The isolation herein of both diastereomeric versions of the same chlorinated product (compounds 2 and 3) could suggest the participation of a hypochlorous acid mediated chlorination along with the enzyme-assisted mechanism. However, the fact that this nonspecific chlorination has not been observed for any other napyradiomycin derivatives isolated in this work, i.e. from the same culture and therefore under the same conditions, did not support this overall hypothesis.
The other possibility is that the chlorine atom at C-3 in compound 3 may come from a S N 2 displacement, therefore with inversion of configuration. The only reasonable way for that to occur is that a chloride ion is displacing the C-3 chlorine in compound 2. Although it is known that chloride is a poor nucleophile (and a fair leaving group), the activation of this allylic position within the dihydropyran ring could provide enough driving force for the reaction to proceed. Despite this epimerization has not been observed for any other dihydropyran ring-containing napyradiomycin (isolated in this work or not), we consider that the nucleophilic substitution to produce compound 3 may be the most plausible hypothesis.
Finally, compound 5 presents in its structure a 16-membered macrocycle because of a bridging ether link between C-6 of the dihydronaphtoquinone unit and C-18 of the monoterpenoid chain. This kind of ether link is unprecedented within the napyradionmycin family, and therefore compound 5 represents the first member reported of a new series of napyradiomycins that we have designated as napyradiomycin D1. Remarkably, although a similar 15-membered cyclic ether ring has been previously reported for merochlorin C-a compound belonging to another family of meroterpenoid metabolites [35]-the O-linked bridging observed in 5 still represents, to the best of our knowledge, one of the largest ether cyclization events observed for a natural product. Regarding its biosynthesis, since NapH1 has been proved responsible for the halogenation and the formation of a 6-membered cyclic ether ring in napyradiomycins [31], it is tempting to think that the same enzyme could also catalyze this macrocyclization. However, the attachment in napyradiomycin D1 of the ether linkage to a former methyl group without the installation of any chiral chlorinated center vicinal to the linking position is inconsistent with a chloronium-induced cyclization catalyzed by NapH1. The whole genome sequencing of the producer strain CA-271078 and the inspection of its napyradiomycin BGC may further provide valuable data about this and other biosynthetic questions raised in this work.
Although the specific mechanism of action for this family of meroterpenoids is not totally clear [5,14,15], previous studies about the structure activity relationship (SAR) have shown that structural variations among the napyradiomycin metabolites scaffold known so far, such as the different halogenation patterns or the presence or absence of the methyl group at C-7 among others, can attenuate or enhance their biological activities [4,14].
Some of the compounds isolated displayed antibacterial activity against MRSA. Notably, the activity of compound 5, the first in the napiradiomycin D series, was comparable to that of other known compounds isolated in this work. In line with previous results, none of the compounds isolated were found to be active against Gram-negative bacteria or fungi. Additionally, our cytotoxicity data illustrate that, for the napyradiomycin B series, the C-3 chlorinated derivatives (compound 2 and 3) exhibit a higher cytotoxic activity compared to the hydroxylated analogous at that stereocenter (compound 6). This cytotoxic is in turn significantly influenced by the absolute stereochemistry of the chlorine group at that position (compound 2 vs. 3). On the other hand, there is a clear correlation between rising levels of cytotoxicity when the substitution pattern at C-4a varies between hydroxy group (compound 4), hydrogen (compound 9), or chlorine (compound 13).

Antibacterial and Antifungal Assays Cytotoxic Activities
Compounds 1-15 were tested in antimicrobial assays against the growth of Gram-negative (E. coli ATCC 25922 and A. baumannii MB5973) and Gram-positive bacteria (M. tuberculosis H37Ra and methicillin-resistant S. aureus (MRSA) MB5393), and fungi (A. fumigatus ATCC46645) and in a cytotoxicity assay against the human liver adenocarcinoma cell line (HepG2) following previously described methodologies [36][37][38]. Briefly, each compound was serially diluted in DMSO with a dilution factor of 2 to provide 10 concentrations starting at 96 µg/mL for all the antimicrobial assays except for compounds 3 and 8 which started at 64 µg/mL. For the adenocarcinoma cell line each compound was serially diluted in DMSO with a dilution factor of 2 to provide 10 concentrations starting at 150 µg/mL (compounds 2, 4, 6, 7, and 9-15), 100 µg/mL (compounds 3 and 8) or 30.0 µg/mL (compounds 1 and 5). 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 [39]. In all experiments performed in this work the RZ' factor obtained was between 0.87 and 0.98.

Conclusions
In this work, we report the isolation and structural characterization of four new napyradiomycins (1-3, 5) and the known napyradiomycin SC (4), whose structural details had not been previously described. Additionally, another ten known napyradiomycins or related compounds (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were also isolated from the same culture broth of the marine-derived Streptomyces sp. CA-271078 from MEDINA's microbial collection. The antibacterial, antifungal and cytotoxic properties of all the compounds isolated were tested. Napyradiomycins B2 (14), B4 (13), and B5 (15) and the new napyradiomycin D1 (5) were the most active compounds, exhibiting similar antibacterial activities against MRSA and M. tuberculosis H37Ra, as well as comparable cytotoxic activities against the HepG2 tumoral cell line. On the contrary, none of the compound tested showed significant activity against E. coli, A. baumannii, or A. fumigatus.
The four new compounds isolated displayed remarkable structural features. Thus, compound 1 is the first member of napyradiomycins in the A series bearing a hydroxy group rather than a chlorine atom at C-3. The isolation of the new B-type napyradiomycin 3 represents to our knowledge the first example reported with a different relative configuration at the C-3 chlorinated position in napyradiomycins and raises biosynthetic and mechanistic questions to be considered. Compound 5 harbors in its structure an unprecedented 16-membered macrocyclic ether ring between the naphthoquinone moiety and the monoterpenoid chain, thus inaugurating a new class of napyradiomycins, the D series.
The results reported here highlight the wide range of structural possibilities for napyradiomycin metabolites. The further whole genome sequencing of the producer strain CA-271078 and the analysis of the corresponding napyradiomycins gene cluster could bring new data about the biosynthesis of these fascinating natural products.