Mansouramycins E–G, Cytotoxic Isoquinolinequinones from Marine Streptomycetes

Chemical investigation of the ethyl acetate extract from the marine-derived Streptomyces sp. isolate B1848 resulted in three new isoquinolinequinone derivatives, the mansouramycins E–G (1a–3a), in addition to the previously reported mansouramycins A (5) and D (6). Their structures were elucidated by computer-assisted interpretation of 1D and 2D NMR spectra, high-resolution mass spectrometry, and by comparison with related compounds. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated a significant cytotoxicity and good tumor selectivity for mansouramycin F (2a), while the activity profile of E (1a) was less attractive.

While the marine-derived Streptomyces sp. isolate B1848 was previously noted as a producer of 6-hydroxy-isatine and several other known compounds [12][13][14], further fermentations led now to the isolation and characterization of three unusual mansouramycins E-G (1a-3a) along with mansouramycins A (5), D (6) ( Figure 1) and 13 known metabolites [12][13][14]. The chemical structures of 1a-3a were elucidated by NMR (1D, 2D) and HRMS, by comparison with related compounds and by computer-assisted methods. The cytotoxic activity of the isolated isoquinolinequinones was determined.

Results and Discussion
With a malt extract medium with 50% synthetic seawater (M2 + medium), the marinederived Streptomyces sp. isolate B1848 produced only traces of mansouramycins A (5) and D (6), along with the zizaene derivative albaflavenol [9], 6-hydroxy-isatine [13,14], 2′deoxythymidine, 2′-deoxuridine, 2′-deoxyadenonsine, anthranilic acid, tyrosol, indolyl-3acetic acid, phenyl acetamide, indolyl-3-carboxylic acid, N ß -acetyltryptamine, Nacetyltyramine, and p-hydroxybenzoic acid [12]. Better yields of the mansouramycins and further red pigments were obtained now on a meat extract medium in a fermentation with a 50 L shaker culture. After extraction and chromatographic separation, the strain B1748 afforded under these conditions the mansouramycins A (5) and D (6) and three new congeners, the mansouramycins E-G (1a-3a) as dark red solids. The isoquinolinequinones gave brown-red zones on TLC, with UV absorptions in solution similar as of perihydroxyquinones. Their reversible color change with sodium dithionite from orange to nearly colorless confirmed quinones; peri-hydroxyquinones were excluded, however, by the missing bathochromic shift with sodium hydroxide. Unlike the orange-red phenoxazinone chromophore of actinomycins and related pigments, which are becoming red with concentrated sulfuric acid, the isoquinolinequinones turned yellow. Further physicochemical properties of compounds 1a−3a are summarized in Table 1.

Results and Discussion
With a malt extract medium with 50% synthetic seawater (M 2 + medium), the marinederived Streptomyces sp. isolate B1848 produced only traces of mansouramycins A (5) and D (6), along with the zizaene derivative albaflavenol [9], 6-hydroxy-isatine [13,14], 2 -deoxythymidine, 2 -deoxuridine, 2 -deoxyadenonsine, anthranilic acid, tyrosol, indolyl-3acetic acid, phenyl acetamide, indolyl-3-carboxylic acid, N ß -acetyltryptamine, N-acetyltyramine, and p-hydroxybenzoic acid [12]. Better yields of the mansouramycins and further red pigments were obtained now on a meat extract medium in a fermentation with a 50 L shaker culture. After extraction and chromatographic separation, the strain B1748 afforded under these conditions the mansouramycins A (5) and D (6) and three new congeners, the mansouramycins E-G (1a-3a) as dark red solids. The isoquinolinequinones gave brown-red zones on TLC, with UV absorptions in solution similar as of peri-hydroxyquinones. Their reversible color change with sodium dithionite from orange to nearly colorless confirmed quinones; peri-hydroxyquinones were excluded, however, by the missing bathochromic shift with sodium hydroxide. Unlike the orange-red phenoxazinone chromophore of actinomycins and related pigments, which are becoming red with concentrated sulfuric acid, the isoquinolinequinones turned yellow. Further physicochemical properties of compounds 1a−3a are summarized in Table 1.

Structure Elucidation
Compound 1a was obtained as red powder of moderate polarity. The molecular formula was determined as C 16 H 11 N 3 O 2 by EI-HRMS, indicating 13 double bond equivalents (DBE). The color change to yellow with concentrated sulfuric acid and the characteristic UV curve with a flat absorption at λ max 509 nm as for 5, 6 pointed to an isoquinolinequinone moiety as well [10] ( Table 1). The 13 C NMR spectrum ( Table 2) showed six aromatic/olefinic methines and one methyl signal. Furthermore, signals of nine non-protonated carbon atoms were observed, of which two at δ 182.8 and 181.4 pointed to carbonyl groups of a quinone. In the proton NMR spectrum (Table 2), the CH singlets at δ 9.01 (H-1) and 5.71 (H-6), in addition to a broadened NH signal at δ 7.83 and a methyl doublet at δ 2.85 of the CH 3 NH fragment were typical for mansouramycins.
A 1,2-disubstituted benzene ring was deduced from the typical signal pattern of four o,m-coupled protons at δ 8.23 (d), 7.79 (d), 7.61 (td), and 7.31 (td) ppm and from the expected HMBC correlations ( Figure 2). A further broadened NH signal was seen at δ 11.98, which formed with the remaining atoms an aniline residue. With respect to the two open valencies in both fragments, the isoquinoline and the aniline unit can be merged only in two ways under formation of structures 1a or 1b. The more in-depth analysis of the NMR data by means of the structure elucidation program COCON [15] confirmed isomers 1a and 1b as allowed structures, but delivered >7600 additional alternatives! Most of them were highly strained (cyclobutenes, non-linear allenes, or bridged aromatic systems) and therefore excluded. the NMR data by means of the structure elucidation program COCON [15] confirmed isomers 1a and 1b as allowed structures, but delivered >7600 additional alternatives! Most of them were highly strained (cyclobutenes, non-linear allenes, or bridged aromatic systems) and therefore excluded.
Amongst 24 plausible indoloquinoline-and indoloisoquinoline-quinones, only 6 (1a, 1b, S1c, S1e, S1f, S1h) * were found by COCON and therefore only these are in agreement with the COSY and HMBC correlations. For mansouramycin E, the isomer 1a showed the best agreement of experimental NMR data with shifts calculated by SPARTAN'20 [16] using ab initio methods on a high level of theory. This structure was therefore assumed for mansouramycin E (see Supplementary Materials). Further applications of this technique have been described previously [17]. * Formula numbers with a leading bold letter "S" are refering to structures in Supplementary Materials. For the dark red mansouramycin F (2a) the molecular formula C12H9N3O2 (ESI-HRMS) was determined, which entails 10 DBE. The 1 H and 13 C NMR shifts (Table 2), as well as the HMBC couplings, confirmed again an N-methyl-isoquinolinequinone substructure as in all other mansouramycins (Figure 1). According to the chemical shifts and 2D correlations, the unassigned residual atoms C2H3N were belonging to an annulated pyrrole ring, which was confirmed by the 1H triplet at δH 7.93 and the dd signal at 6.70 with the expected small coupling constants (~5 Hz). The pyrrole ring can be fused with the isoquinolinequinone core in three different ways, yielding structures 2a, 2b, S2m, and the respective isomers with the N-methyl group at C-6 instead at C-7 (see Supplementary Materials). With COCON using atom types, 19 isomers were found. Four of them were quinones (2a, 2b, S2c, S2d). The other structures were azepin-2-ones or highly strained bridged systems. Isomers of type S2m were excluded by COCON as well, and also o-quinones were not predicted for mansouramycin F.
H-3′ in 2b should show a 3 J correlation with C-4a, which is missing in the experimental spectrum and therefore better fitting on 2a. In S2c/S2d, the quinonoid proton H-7 (δ ~5.6) should show a 3 J correlation with C-8a at δ ~147. However, this was also not observed, so that only structure 2a was left. For further confirmation, we compared the experimental with calculated shifts of all possible pyrrolo-quinoline-and pyrroloisoquinoline-5,8-quinones. The results (Table S2) pointed again clearly to structure 2a for mansouramycin F. This conclusion was further confirmed by comparison with similarly fused pyrrolo-pyridine skeletons [18,19]. Amongst 24 plausible indoloquinoline-and indoloisoquinoline-quinones, only 6 (1a, 1b, S1c, S1e, S1f, S1h) * were found by COCON and therefore only these are in agreement with the COSY and HMBC correlations. For mansouramycin E, the isomer 1a showed the best agreement of experimental NMR data with shifts calculated by SPARTAN'20 [16] using ab initio methods on a high level of theory. This structure was therefore assumed for mansouramycin E (see Supplementary Materials). Further applications of this technique have been described previously [17]. * Formula numbers with a leading bold letter "S" are refering to structures in Supplementary Materials.
For the dark red mansouramycin F (2a) the molecular formula C 12 H 9 N 3 O 2 (ESI-HRMS) was determined, which entails 10 DBE. The 1 H and 13 C NMR shifts (Table 2), as well as the HMBC couplings, confirmed again an N-methyl-isoquinolinequinone substructure as in all other mansouramycins (Figure 1). According to the chemical shifts and 2D correlations, the unassigned residual atoms C 2 H 3 N were belonging to an annulated pyrrole ring, which was confirmed by the 1H triplet at δ H 7.93 and the dd signal at 6.70 with the expected small coupling constants (~5 Hz). The pyrrole ring can be fused with the isoquinolinequinone core in three different ways, yielding structures 2a, 2b, S2m, and the respective isomers with the N-methyl group at C-6 instead at C-7 (see Supplementary Materials).
With COCON using atom types, 19 isomers were found. Four of them were quinones (2a,  2b, S2c, S2d). The other structures were azepin-2-ones or highly strained bridged systems. Isomers of type S2m were excluded by COCON as well, and also o-quinones were not predicted for mansouramycin F.
H-3 in 2b should show a 3 J correlation with C-4a, which is missing in the experimental spectrum and therefore better fitting on 2a. In S2c/S2d, the quinonoid proton H-7 (δ~5.6) should show a 3 J correlation with C-8a at δ~147. However, this was also not observed, so that only structure 2a was left. For further confirmation, we compared the experimental with calculated shifts of all possible pyrrolo-quinoline-and pyrroloisoquinoline-5,8quinones. The results (Table S2) pointed again clearly to structure 2a for mansouramycin F. This conclusion was further confirmed by comparison with similarly fused pyrrolo-pyridine skeletons [18,19].
Compound 3a was obtained as a red solid as well, which displayed isoquinolinequinone-like UV/vis and other physicochemical properties. The molecular formula of 3a was established as C 15 H 11 N 3 O 4 by ESI-HRMS and 1 H and 13 C NMR analysis, entailing 12 DBE. The 1 H and 13 C NMR spectra confirmed a further isoquinoline-quinone, which showed, however, remarkable differences compared with 1a and 2a. Instead of one N-methyl residue (7-NHCH 3 ) and one quinonoid proton (6-H), as in the other mansouramycins, the 1 H NMR spectrum showed each two of these signals. In addition, the 13 C NMR spectrum displayed four carbonyl groups (δ C 178.2, 179.8, 177.6, and 180.9) instead of two carbonyls as in 1a and 2a ( Table 2). Interpretation of the HMBC spectrum of 3a (Figure 2) revealed an isoquinoline-quinone and an N-methylaminobenzoquinone substructure, which can be connected in two different ways only, resulting in 3a or 3b, respectively (Figures 1 and 2). The alternative 3b was excluded, however, based on the significant 3 J HMBC correlations of H-6 (δ H 5.75) and NH-9 (δ H 7.84) with CO-8 (δ C 179.8) and not with CO-5 (δ C 178.2); the position of the second N-methyl group at C-3 was determined in a similar way. All the remaining HMBC and COSY correlations ( Figure 2) were in full agreements with structure 3a, a novel azaphenanthrene diquinone, which we named mansouramycin G (see also Supplementary Materials).

Biological Activities
Isolated compounds were evaluated in cytotoxicity assays against the same 36 cancer cell lines as published before [10]. Consistent with results previously reported herein for other members of the group, cytotoxicity profiling of the new mansouramycin 2a revealed good anti-tumor activity in vitro with a mean IC 50 value of 7.92 µM (1.797 µgmL −1 ). Furthermore, 2a showed good tumor selectivity across the panel of 36 cell lines. Mansouramycin E (1a) was less active and selective [mean IC 50 = 23.10 µM (6.398 µgmL −1 )]. Previously reported mansouramycins C (4b) and A (5) exhibited mean IC 50 values of 0.089 µM (0.022 µgmL −1 ) and 13.44 µM (2.902 µgmL −1 ), respectively (Table 3). Mansouramycin G (3a) was not tested, due to a lack of material. In the agar diffusion test, crude extracts of S. isolate B1848 exhibited high bioactivity against Mucor miehei (Tü 284) and Candida albicans, and moderate activity against Escherichia coli and the alga Chlorella vulgaris. The samples of 1-6 were nearly consumed in the cytotoxicity assays and therefore not tested for their antimicrobial activity.

Isolation and Taxonomy of the Producing Strain
The marine Streptomyces sp. strain B1848 was isolated and deposited in the Actinomycetes culture collection of the Alfred-Wegner Institute for Polar-und Marine Research, Am Handelshafen, Bremen, Germany. The taxonomy of the strain has been described previously [12].

Fermentation and Working Up
The S. sp. isolate B1848 was previously cultivated on M 2 + medium with 50% seawater in a 25 L jar fermenter (72 h at 28 • C) [12,13]. Optimization of the culture conditions has been performed now using six different media [14] at two pH values (6.5, 7.8), temperatures (28, 35 • C), and shaking rates (110, 95 rpm) for four days. TLC analysis and antimicrobial screenings indicated that medium C (meat extract medium: 10 g glucose, 2 g peptone, 1 yeast, 1 g meat extract, pH 7.8) gave the best yield of mansouramycins.
A 50-L jar fermenter with C-medium was inoculated with strain B1848 and stirred for 4 days at 28 • C with 120 rpm. The resulting pale yellow culture broth was mixed with diatomaceous earth (Celite, ca. 1.8 kg), and filtered-off under pressure. The mycelial cake was extracted with ethyl acetate (3×), and then with acetone (2×). The acetone extract was concentrated under reduced pressure, and the aqueous residue was extracted once more with ethyl acetate. The combined organic phases were concentrated in vacuo, yielding 4.8 g of reddish-orange residue. None of the compounds of interest were detected in the aqueous phases, and therefore they were discarded.

Isolation and Purification
The mycelial cake extract (4.8 g) was applied to flash silica gel column chromatography (3 × 60 cm) using a CH 2 Cl 2 -CH 3 OH gradient. After monitoring by TLC (CHCl 3 /5; 10% MeOH), four fractions were obtained. Purification of fractions II-IV, using PTLC and Sephadex LH 20, led to isolation of five dark red compounds: mansouramycin A (5; 3.0 mg), D (6, 8. Data of mansouramycins E-G (1a-3a) are listed in Tables 1-3, and spectra are depicted in the Supplementary Information. The working up and isolation of mansouramycins E-G (1a-3a) was carried out in August 2004. Spectral measurements, structural interpretation, and biological activity testing of 1a-3a were achieved in the beginning of 2005.

Cytotoxicity Assays
A modified propidium iodide assay was used to examine the antiproliferative activity of the compounds against human tumor cell lines. Cell lines tested were derived from patient tumors engrafted as a subcutaneously growing tumor in NMRI nu/nu mice or obtained from American Type Culture Collection, Rockville, MD, USA, National Cancer Institute, Bethesda, MD, USA, or Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany, and details of the test procedure have been described previously [20][21][22]. For the results, see Table 3.

DFT-Calculations
The calculation of NMR shifts was performed in a sequence of six calculation steps implemented in SPARTAN'20 [16]: (1) for all molecules of interest, the least energy conformers were determined using the "systematic approach" of the Merck Molecular Force Field program (MMFF). Up to 500 MMFF conformers within 40 kJ/mol above the global minimum were kept; in step (2), geometries were further optimized with a Hartry-Fock calculation (HF/3-21G); up to 200 conformers with <40 kJ/mol above the global minimum the energies were kept and (3) optimized (energies) with the DFT functional ωB97X-D and the 6-31G* basis set; (4) for up to 100 conformers within a window of 15 kJ/mol, the geometries were calculated now with the same functional and basis set; up to 50 conformers with <10 kJ/mol were kept for step (5); for the remaining conformers, energies and Boltzmann factors (300 K) were calculated with ωB97X-V/6-311+G(2df,2p) [6-311G*]; (6) for up to 30 resulting conformers with <10 kJ/mol the NMR data were calculated with ωB97X-D/6-31G* using the geometries from step (4). The conformer shifts were averaged with the Boltzmann factors from step five.

Conclusions
Isoquinoline-quinones from marine invertebrates and associated streptomycetes attracted scientific attention due to their strong anticancer activities [1][2][3][4][5][6]. From marinederived Streptomyces spp., we isolated recently five isoquinoline-quinone derivatives, the mansouramycins A-D and 3-methyl-7-(methylamino)-5,8-isoquinolinedione (4a), which showed significant cytotoxicity in a panel of up to 36 tumor cell lines, with pronounced selectivity for non-small cell lung cancer, breast cancer, melanoma, and prostate cancer cells [10]. After a culture optimization, we succeeded now to isolate three further mansouramycins E-G (1a-3a) from the same marine Streptomyces sp. strain B1848, used optimized culture conditions. Their structures were elucidated by computer-assisted interpretation of 1D and 2D NMR spectra, high resolution mass spectrometry, by comparison with ab initio-calculated NMR data and by comparison with related compounds. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated only a moderate cytotoxicity and tumor selectivity for the new quinones E (1a) and F (2a). The novel azaphenanthrene-diquinone mansouramycin G (3a) was not tested due to insufficient material.