*2.2. 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 IC50 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 IC50 = 23.10 μM (6.398 μgmL−1)]. Previously reported mansouramycins C (**4b**) and A (**5**) exhibited mean IC50 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.

**Table 3.** In vitro cytotoxic activities of mansouramycins A (**5**), C (**4b**), E (**1a**), and F (**2a**).


\* individual IC70 < 1/3 mean IC70; e.g., if mean IC70 = 2.1 μM the threshold for above average sensitivity was IC < 0.7 μM, \*\* - (% selective =<4%);+(4%>%selective>=10%);++(10%>%selective>=20%); +++(%selective>20%).

#### **3. Materials and Methods**

## *3.1. General Procedures*

NMR spectra were measured on Varian Unity 300 and Varian Inova 600 spectrometers. The spectra were referenced to the signals of partially deuterated solvents (*δ*Chl 7.270, 77.000; *δ*DMSO 2.500, 39.510). Electron spray ionization mass spectrometry (ESI HRMS): Finnigan LCQ ion trap mass spectrometer coupled with a Flux Instruments (Basel, Switzerland) quaternary pump Rheos 4000 and a HP 1100 HPLC (nucleosil column EC 125/2, 100-5, C 18) with autosampler (Jasco 851-AS, Jasco Inc., Easton, MD, USA) and a Diode Array Detector (Finnigan Surveyor LC System). High resolution mass spectra (HRMS) were recorded by ESI MS on an Apex IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker Daltonics, Billerica, MA, USA). EI mass spectra (70 eV) were recorded on a Finnigan MAT 95 spectrometer (Thermo Electron Corp., Bremen, Germany) with perfluorokerosene as reference substance for EI HRMS. IR spectra were recorded on a Perkin-Elmer 1600 Series FT-IR spectrometer from KBr pellets. UV/vis spectra were recorded on a Perkin-Elmer Lambda 15 UV/vis spectrometer. Flash chromatography was carried out on silica gel (230–400 mesh). *R*f-values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co., Düren, Germany). Size exclusion chromatography was carried out on Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany).

#### *3.2. 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].

#### *3.3. Fermentation and Working Up*

The *S.* sp. isolate B1848 was previously cultivated on M2 + 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.

#### *3.4. Isolation and Purification*

The mycelial cake extract (4.8 g) was applied to flash silica gel column chromatography (3 × 60 cm) using a CH2Cl2-CH3OH gradient. After monitoring by TLC (CHCl3/5; 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**, 8.0 mg), E (**1a**; 4.1 mg), F (**2a**; 6.0 mg), and mansouramycin G (**3a**; 4.2 mg); for the physico-chemical properties and NMR spectral data of mansouramycins E–G (**1**–**3**), see Tables 1 and 2, respectively.

Mansouramycin C (3-Carbomethoxy-7-methylaminoisoquinoline-5,8-dione; **4b**): During this investigation, we realized two errors in the previously reported 13C NMR data of mansouramycin D [10]: (CDCl3, 150 MHz): *δ* 180.6 (Cq-8), 179.8 (Cq-5), 164.3 (Cq-9),

153.3 (Cq-3), 148.8 (Cq-7), 148.0 (CH-1), 140.5 (Cq-4a), 126.0 (Cq-8a), 120.7 (CH-4), 101.5 (CH-6), 53.4 (9-OCH3), 29.3 (NCH3).

Mansouramycin F (7-Methylamino-3 *<sup>H</sup>*-pyrrolo [2,3-c]-isoquinoline-6,9-dione, **2a**): 1H NMR (CDCl3, 300 MHz): δ 10.25 (s br, 1H, NH-1), 9.12 (s, 1H, 1-H), 7.79 (t, 3*J* = 2.6 Hz, 1H, 2-H), 6.81 (t, 3*J* = 2.9 Hz, 1H, 3-H), 6.25 (s br, 1H, 9-NH), 5.69 (s, 1H, 6-H), 2.98 (d, *J* = 5.1 Hz, 3H, CH3-10).

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.
