4.7 Marine Streptomyces sp. B7936
4.7.2 Actinomycin D analogue
Compound 131 was isolated along with compound 130 as a red solid pheno-xazinone derivative, where the 1H NMR spectrum showed a high similarity to that of
A search in AntiBase gave three possible structures: Azetomycin I (131), auran-tin III[177] (132) and actinomycin D0[178] (133). The first two actinomycins were pre-viously isolated from Streptomyces antibioticus and Streptomyces aurantiacus, re-spectively, while the third one was found in Actinomyces olivobrunneus. However, azetomycin I (131) required a medium containing L-azetidine-2-carboxylic acid (134) or its derivatives, and was excluded therefore. Aurantin III (132) showed a replacement of the methyl group at 6-position in phenoxazinone chromophore (129) by a hydrogen, compared with actinomycin D, while the N-methyl group of valin (β -ring) was replaced by H in actinomycin D0. Therefore, aurantin III[177] (132) or actin-omycin D0 (133) is supposed. Due to the small amount, no further attempts were made to differentiate between both structures.
N
In fraction III, a middle polar yellow zone of compound 135 was found which turned to brown by treatment with conc. sulphuric acid. It exhibited additionally a yellowish-green colour with Ehrlich’s reagent, however, no colour change with so-dium hydroxide or anisaldehyde/sulphuric acid was observed. PTLC and Sephadex LH-20 delivered a yellow solid. This pointed to compound 135 possibly as peri-hydroxy xanthone moiety, present in linear or angular form (A, B).
O O
OH OH
O
OHO OH
A B
The 1H NMR spectrum showed three peri-hydroxyl protons at δ 12.37, 11.98 and 11.88. In the aromatic region, it displayed two doublets each of 1H at δ 7.56 (d,
3J = 9.1 Hz), and 6.96 (d, 3J = 9.1 Hz) of a 1,2,3,4-tetrasubstituted benzene ring. An additional aromatic singlet of 1H was observed at δ 7.09 (H-5). In the sugar and ali-phatic region, it showed three anomeric protons at δ 5.14 (dd, J = 9.7, 2.0 Hz), 4.51 (m) and 4.50 (dd, J = 9.8, 1.8 Hz), pointing to three sugar moieties in compound 135.
Between δ 4.8-3.0, it exhibited 12 oxygenated methine protons, two singlets of meth-oxy protons at δ 3.41 and 3.40, as well as two singlets of 2 OH at δ 4.54 and 4.49 which disappeared in the presence of TFA. Furthermore, it showed multiplets and twofold doublet signals between δ 2.60-1.60 of 10 protons, which could be assigned as 5 methylene groups. In addition, two other multiplets of 2 CH2 of an open chain between δ 1.91- 1.45 are exhibited. Three 3H doublets of methyl groups at δ 1.36, 1.34, and 1.30 were pointing again to the existence of three sugar units in 135. Fi-nally, a terminal methyl triplet at δ 1.00 was observed.
The 13C/APT NMR spectra displayed 44∼45 carbon signals which were classi-fied into several categories i.e. two α,β-unsaturated carbonyl carbons at δ 186.2 and 170.3, fifteen sp2 quaternary carbons, three sp2 methine carbons, fifteen oxygenated sp3 methine carbons of which three were assigned as anomeric carbons, two methoxy carbons, five sp3 methylene carbons and finally four methyl carbon signals.
Figure 49: 1H NMR spectrum (CDCl3, 300 MHz) of FD-594 (135).
Figure 50: APT NMR spectrum (CDCl3, 150 MHz) of FD-594 (135)
The ESI mass spectra confirmed the molecular weight of 135 as 940 Dalton due to the existence of two quasi-molecular ions at m/z 963 ([M + Na]+) and 941 ([M + H]+) in the (+)-mode, and 939 ([M-H]- in (-)-ESI mode.
Applying the above spectral data and the molecular weight of 135 to AntiBase resulted in FD-594 (135). The compound was confirmed by direct comparison of the data with the literature[179,180]. Due to the high interest of the compound 135 and its high antimicrobial activity, this drew our attention to confirm it furthermore by 2 D correlations (see the experimental part). Detailed analysis of the spin-coupling data and H,H COSY spectra as well as HMBC experiments revealed that the trisaccharide moiety of the compound (135) consisted of one 2,6-dideoxy-3-O-methyl-β -arabino-hexopyranoside (oleandrose) unit and two 2,6-dideoxy-β-arabino-hexopyranosides (olivose) units. The positions of the glycoside linkages between these hexo-pyranose
were established by HMBC spectra. The HMBC correlations of 1'''-H ( 4.51) with the C-4'' ( 87.9), and 1''-H (4.50) with C-4' (88.7) clearly indicated the connectivities of the pyranose units.
OO
The antitumor antibiotic FD-594 (135) is produced by Streptomyces sp. TA-0256[179,180]. Compound 135 was reported to be closely related to BE-13973X and MS 901809, isolated from other Streptomyces sp.[181,182].
FD-594 (135) shows high activity against tumor cell lines, comparative to that of adriamycin[183] (136), as well as antibacterial activity against some Gram-positive bacteria.
OHOH O
Figure 51: Plausible biosynthetic pathway of FD-594 (135).
The biosynthetic pathway[180] of 135 appears to proceed via a polyacetate equivalent. In an early stage, the benzo[α]naphthacene quinone chromophore may first be derived from 14 acetate units. Thereafter, the Baeyer-Villiger type oxidation occurs at a quinone carbonyl group, followed by additional oxidation and decarboxy-lation to yield an open intermediate. Recyclization via path A leads to 135, an alter-native cyclization via path B may afford the structurally related MS 901809 (137).
4.7.4 Fungichromin (14-Hydroxyfilipin III)
Fraction IV showed a highly polar yellowish-green UV fluorescent band (366 nm), which turned to blue by anisaldehyde/sulphuric acid. Compound 138a was iso-lated as a yellow solid by applying the fraction to PTLC and Sephadex LH-20.
The UV spectrum of 138a showed three sharp absorption peaks (λmax 300-400 nm) which are characteristic for a polyene system without conjugation with a car-bonyl group. Comparison of the UV pattern of 138a with AntiBase data pointed to five conjugated double bonds of a pentaene system.
The 1H NMR spectrum displayed no signals in the aromatic region. However, it showed 9H of five olefinic double bonds between δ 6.5-5.9, the first of which was
that of a terminal one. In addition, between δ 5.29-4.36 many singlets with an inten-sity of 10H appeared, of which 9 disappeared by addition of TFA, to assign them as OH signals, and the residual one, a triplet at δ 4.67, is assigned as an oxygenated methine group. In addition, several multiplets with an intensity of 10H between δ 4.03-3.15 were observed which could be assigned to oxygenated methine protons.
Furthermore, a twofold doublet of 1H at δ 2.50 (3J = 8.1, 7.1 Hz) of a methine proton attached to sp2 carbon (e.g. CO), a singlet of a methyl group at δ 1.73 (perhaps at an olefinic sp2 carbon), multiplets of 18H at δ 1.54-1.23 of 9 methylene protons, a dou-blet of a methyl at δ 1.22 (3J = 7.1 Hz), and a terminal methyl triplet at δ 0.90 ( 3J = 7.1 Hz) were shown.
Figure 52: 1H NMR spectrum ([D6]DMSO, 300 MHz) of fungichromin (138a).
The 13C/APT NMR spectra showed a CO signal at δ 171.0, in addition to an-other quaternary sp2 carbon at δ 138.7, and 9 sp2 olefinic methine carbons pointing to the presence of 5 conjugated double bonds, one of which being of the CH=Cq bond type. The spectra revealed also 11 oxygenated sp3 methine carbons as well as one CH at δ 58.6 which could be assigned to a methine carbon attached to CO or to a hetero atom. In addition, 9 sp3 carbon signals were displayed, which assigned as methylene groups, three methyl carbon signals at δ 17.7, 13.8 and 11.6, of which the first could be attached to sp2 carbon of an olefinic double bond.
Figure 53: 13C NMR spectrum ([D6]DMSO, 125 MHz) of fungichromin (138a).
The molecular weight of the 138a was established as 670 Dalton by ESI mass spectra. A search in AntiBase resulted in fungichromin (138a) which was further confirmed by direct comparison with the literature[184,48].
Fungichromin (138a), isolated first from a Streptomyces sp.[185,186],and the re-lated analogue filipin 138b belong to the class of macrocyclic polyene antibiotics, a group of over 200 compounds, produced mainly by Streptomyces sp., that possess antifungal and antiprotozoal activity[184]. Despite of their toxicity and the develop-ment of other classes of antifungal antibiotics, especially the polyene amphotericin B (139) and nystatin (140) remain the gold standard for treatment of many fungal infec-tions in humans. The antihypercholesterolemic, antitumor, and antiviral activities of such steroid-binding polyenes also attracted considerable interest[184]. Biosyntheti-cally, the macrocyclic ring of the polyenes derived from acetate and propionate[,184]. The compounds FD-594 (135) and fungichromin (138a) were subjected to cyctotox-city measurements against nematode, and showed a weak activity (13%, 1mg/l) by 135, and high activity (95 %, 1 mg/l) by 138a.
CH3 OH
OH OH
OH OH
R OH O H
OH
O O
CH3 OH
CH3
1 3 2 13
14
16 2627 28
29 15
1' 2'
4'
6'
138a: R = OH, 138b: R = H
O 4.8 Terrestrial Streptomyces sp. GW2/577
Terrestrial Streptomyces strains are a prolific source of many diverse metabolic compounds that might represent useful leads in the development of new pharmaceu-tical agents. During the systematic search for new secondary metabolites from bacte-ria, the ethyl acetate extract of a terrestrial Streptomyces sp. isolate GW 2/577 was found to produce four new compounds designated as cPHB (318), p-hydroxyphenethyl propionamide (144), 3-hydroxy-N-phenethyl-butyramide (145) and 5-methyl-1H-quinazoline-2,4-dione (148), in addition to some other known compounds (Figure 54). Moreover, the crude extract possessed high activity against Escherichia coli, and moderate activity against Bacillus subtilis.
The strain was fermented in M2 medium as 27-liter fermenter for 3 days at 28
°C. The obtained crude extract was applied to defatting and the resulting methanol extract was chromatographed on Sephadex LH-20 and eluted with methanol to give four fractions. Purification of the resulting fractions led to the described compounds.
Ssephadex LH-20 (MeOH) Fraction I Fraction II Fraction III Fraction IV
Chrom on Ssephadex LH-20 (MeOH)
Sephadex LH-20 (MeOH) PRv HPLC (STM2) Defatting with Cyclohexane
adenine
Mycelium Water phase
mixing with celite and filtered by filterpress
3 x with EtOAc and 1x with
acetone 5 x with EtOAc
i.vac
Sephadex LH-20 (MeOH) HPLC (STM2)
Figure 54: working-up procedure of strain GW2/577.
4.8.1 N-(2-Phenylethyl)-propionamide
Compound 141 was isolated as colourless solid from fraction II by applying the fraction to Sephadex LH-20 and HPLC, was UV absorbing, and turned to violet and pink when sprayed with anisaldehyde/sulphuric acid and Ehrlich’s reagent, respec-tively.
The 1H NMR spectrum displayed a multiplet of a phenyl group at δ 7.24, in ad-dition to a broad singlet of 1H at δ 5.5, was indicative for an amide (NH). Further-more, an ethandiyl group was observed due to the existence of two aliphatic signals each of 2H at δ 3.40 (q) which changed to triplet after H/D exchanging, and the other one was a triplet at δ 2.77. An additional ethyl group was observed due to the pres-ence of a quartet methylene group at δ 2.13, and the adjacent methyl group was found as triplet at δ 1.04.
The molecular weight of compound 141 was established as 177 Dalton by EI MS. The molecular ion exhibited a base fragment at m/z 104, which is attributed to an expulsion of acetamide group [M – (HN-COCH3)]+.. A search in AntiBase re-sulted in N-(2-phenylethyl)-propionamide (141), which was further confirmed by its direct comparison with the literature. This type of compounds possesses a moderate antimicroalgal activity and can be used as herbicides[187].
N