Anthracycline derivatives from a marine-derived New Zealand Streptomycete

Four new anthracycline derivatives, (7S*9R*10R*)- pyrromycin 1 , (7R*9R*10R*)- pyrromycin 2 , 1-hydroxyauramycin T 3 , 1-hydroxysulfurmycin T 4 , and the previously reported 1-hydroxyaclacinomycin B 5 were isolated from a New Zealand marine-derived Streptomycete . All five compounds were cytotoxic against the P388 murine leukaemia cell line.


Introduction
Actinomycetes are well known as producers of biologically active compounds with members of the genus Streptomyces, in particular, being prolific producers of metabolites of a diverse range of biological activities. 1 To date, a large number of anthracyclines have been isolated from a wide variety of Streptomycetes. 1 Some of these anthracyclines, such as adriamycin and daunomycin, have found uses in medicine for the treatment of some cancers. 2,3

Results and Discussion
In continuing studies on bioactive natural products from New Zealand micro-organisms one isolate, a Streptomyces sp.(CANU Fox 21-2-6), was of interest.The crude EtOAc extract from the fermentation showed strong cytotoxicity against the murine P388 leukaemia cell line.Furthermore, dereplication of this extract using an in-house approach (HPLC, MS and UV profiles) indicated good potential for novel chemistry.Further investigation led to the isolation of four new anthracycline derivatives, (7S*9S*10R*)-pyrromycin 1, (7R*9R*10R*)-pyrromycin 2, 1-hydroxyauramycin T 3, 1-hydroxysulfurmycin T 4, as well as the previously reported 1hydroxyaclacinomycin B 5.
The Streptomycete, (CANU Fox 21-2-6a), was isolated from well-weathered driftwood collected below the low-tide mark at the mouth of the Fox River on the West Coast of New Zealand.After 18 days fermentation in starch-casein broth under static conditions at 26°C, the EtOAc extract prepared from the fermentation broth was fractionated using flash reverse-phase (RP) chromatography.Repeated chromatography on DIOL of selected fractions from the RP column yielded 5. Further purification of selected DIOL fractions by HPLC yielded 1, 2, 3 and 4.
The molecular formula of 1, a red solid, was deduced as C 30 H 35 NO 11 (fourteen double bond equivalents) by HRESIMS and from 13 C NMR data.The 1 H NMR spectrum of 1 in CDCl 3 showed three signals above 12 ppm, interpreted as hydrogen-bonded phenolic groups.The 13 C NMR experiment confirmed thirty carbon signals, five CH 3 , including one OCH 3 and two NCH 3 , three CH 2 , nine CH, and thirteen quaternary carbon signals.
The UV-visible spectrum of 1, maxima at 202, 234, 258, 290 and 492 nm, was characteristic of a quinone. 4That spectral data, coupled with the 1 H and 13 C NMR chemical shifts, indicated the presence of an anthraquinone moiety containing three phenolic groups.In addition to the fourteen signals that could be assigned to the anthraquinone system, a single anomeric signal at δ C 101.9 suggested that 1 was also a mono-glycoside.
The partial connectivities from COSY and HSQC NMR experiments established four subunits, a, b, c, and d as shown in Figure 1.The assembly of these partial structures followed from a long range 1 H-13 C correlation NMR (CIGAR) experiment. 5Correlations were observed from the NCH 3 signals to a carbon in subunit b.The anomeric proton (δ H 5.56) showed strong correlations to two oxygenated CH's (δ C 72.9 and 68.2) thus linking subunits b and c and closing the glycosidic ring.A correlation from H7 (δ H 5.10) to an oxygenated aromatic carbon (δ C 163.6) connected the subunits a and c.The H11 aromatic proton (δ H 7.51) correlated to C10 (δ C 58.5).H8, H10 and H16 (δ H 2.28, 2.56, 4.07 and 1.11) all correlated to an oxygenated quaternary carbon (δ C 72.2) connecting subunit d.The OCH 3 group and H10 both correlated to a carbonyl (δ C 172.8) completing the assignment of the planar structure of 1 as a pyrromycin derivative. 6xamination of the coupling constants and a 2D NOE experiment enabled assignment of the relative stereochemistry of the anthraquinone moiety and the sugar residue in 1.The starting point was the H7 proton (δ H 5.10) which showed a single 3 J HH of 5 Hz to H8a (δ H 2.56) enabling placement of H8a in a pseudo-axial position and H8b in a pseudo-equatorial position.NOE correlations were observed from H15a (δ H 1.56) to H8b (δ H 2.28) and from H15b (δ H 1.76) to H10 (δ H 4.07) indicating that the ethyl side chain and the carboxyl groups were both pseudoaxial to give the relative stereochemistry of 1 as (7S*9S*10R*)-pyrromycin.The parent pyrromycin is the (7S9R10R)-stereoisomer. 6 This stereoisomer also allows for the formation of the observed hydrogen-bond between the D-ring hydroxyl and carboxyl groups and completed the stereochemical assignment of the anthraquinone.
Based on the NOESY data the sugar could be identified as rhodosamine.Particularly important was the NOESY correlation seen from H5' (δ H 4.26) to H3' (δ H 3.52) which placed these two protons in axial positions.If one or both of these protons had been in an equatorial position then NOESY correlations would not be observed.H3' also showed NOESY correlations to H4' (δ H 3.96) and H2' (δ H 2.08) as expected for vicinal axial/equatorial orientations.The final stereocentre in the rhodosamine sugar residue was at the anomeric center.The anomeric proton (H1'; δ H 5.55) showed no NOESY correlations to either axial proton at H3' and H5' and so could be assigned to an equatorial orientation in keeping with the only observed 3 J HH coupling to the H2' protons of 2.5 Hz.This asssigned stereochemistry confirmed the sugar residue as rhodosamine ( NMR spectrum of 2 were attributed to a change in the stereochemistry of the D-ring.The change in the multiplicity seen for H7 indicated that the D-ring was in a different conformation allowing both couplings to the H8 protons to be observed.The slight increase in polarity and upfield shift of the ethyl protons suggested that the potential for a hydrogen-bond between the Dring hydroxyl and carboxyl groups was removed thereby placing both groups in pseudo-axial positions and limiting the possible stereoisomers to just two.Energy minimisation using the MM2 parameters in Chem3D ® (CambridgeSoft ® ) showed dihedral angles (H7-C7-C8-H8a and H7-C7-C8-H8b) of 55° and 170° and, 33° and 145° respectively for the possible stereoisomers.Application of the Karplus equation 7  1-Hydroxyauramycin T 3, C 29 H 33 NO 11 from HRESIMS, also displayed very similar spectral properties to 1.However, for 3, the signals arising from the ethyl group in the 1 H NMR spectrum of 1 were replaced with a singlet methyl resonating at δ H 1.43.This, when coupled with the necessary decrease in mass and change in molecular formula allowed 3 to be assigned as the methyl derivative of 1.The 1 H NMR signals for the H7 and H8 protons showed minor variation from those observed for 1 suggesting that the relative stereochemistry is identical to that established for 1.The anthracycline pyrromycin core is well established in a range of mono-, di-and triglycosides, but the auramycins and sulfurmycins have previously only been reported as triglycosides. 9,10This is the first report of mono-glycosylated auramycins and sulfurmycins.
The bioactivity evaluation showed that all four compounds displayed very good cytotoxicity against P388 cultured cells with ID 50 values ranging from 0.4 -0.06 µg/mL.

Experimental Section
General Procedures.UV spectra were recorded with a Hewlett Packard 8452 diode array spectrometer.Optical rotation values were obtained on a Perkin Elmer 341 polarimeter. 1 H, 13 C-APT and 2D NMR ( 1 H-1 H COSY, 1 H-13 C HSQC, 1 H-13 C CIGAR) spectra were recorded on a Varian INOVA 500 MHz spectrometer.ESI mass spectra were acquired using a Micromass TOF LCT mass spectrometer.Column chromatography used 40 µM Prep LC Bakerbond Octadecyl (C 18 ) and 40 µM Prep LC Bakerbond Diol.Solvents for extraction and chromatography were distilled prior to use.HPLC was carried out using a Shimadzu LC-10ADvp equipped with an SPD-M10Avp photodiode array detector.

Figure 1 .
Figure 1.Substructures a-d and important CIGAR correlations for 1.

spectrum of 1 .
In the1 H NMR spectrum of 4, C 31 H 35 NO 12 from HRESIMS, a methyl ketone (δ H 2.25) and a pair of isolated doublets (δ H 2.66, 3.04) replaced the ethyl group signals seen in the 1 H NMR Issue in Honor of Prof.This methyl group showed a long range TOCSY correlation to the isolated doublets and a CIGAR correlation to a carbonyl group (δ C 210.6) to establish the structure and relative stereochemistry as 1-hydroxysulfurmycin T 4.1-Hydroxyaclacinomycin B (5) was identified by comparison of NMR data to those in the literature.