4 Terrestrial Streptomyces sp
4.2 Terrestrial Streptomyces sp. TN58
4.4.1 Optimization of the Terrestrial Streptomyces sp. bl 2/5831
The prolific productivity of pyrrol-2-carboxylic acid from these three terrestrial Streptomyces spp. isolates bl 2/5831, b1 4/5844 and bl 10/5742, prompted us to se-lect the isolate bl 2/5831 for optimization, in an effort to find further interesting bio-active metabolites. Therefore, the strain bl 2/5831 was cultivated (6 days) using five culture media: Meat extract (C-medium) containing 50% sea water, C-medium with tap water, soja-mannite (SM-medium), fish powder (F-medium) and M2 (A-Medium), at different pH (7.8 and 6.5), temperature (28 and 35 °C), and shaking rate (95, 110 rpm); (Table 51). TLC screening of the extracts obtained (Figure 34) indi-cated that the C-medium with 50% sea water (pH 7.8, 28 °C and 95 rpm) afforded a different main component in a yield of 130 mg/l, while the previously reported pyr-rol-2-carboxylic acid was not present.
bl 10/5742 bl 10/5742 bl 2/5831 bl 2/5831
bl 4/5844
Figure 34: TLC of optimization after 10 days in 4 medium: A = C-medium with 50% sea water; B = C-medium; C = SM-medium; D = F-medium A 4 L culture was harvested after 6 days, the culture broth was extracted with ethyl acetate and the crude extract (4.20 g) was purified by silica gel column chroma-tography to afford 106 as orange solid (520 mg, i.e. 130 mg/L) (Figure 35).
bl 2/5831 (4 l shaker)
Biomass Filtrate
mixing with cilite and filtered by filterpress
3 x with EtOAC i.vac 4 x with EtOAC
Crude extract (4.2 g)
chrom. on silica gel (CH2Cl2-MeOH ) 6 days
IB-00208 (520 mg)
Figure 35: Work-up procedure of the terrestrial Streptomyces sp. bl 2/5831 using meat extract medium with 50% sea water
4.4.2 IB-00208
Compound 106 was obtained as an UV absorbing orange powder, which showed no colour change after treatment with sulphuric acid or spraying with anisalde-hyde/sulphuric acid reagent. The colour was changed, however, to light red by treat-ment with dil. sodium hydroxide, which is indicative of peri-hydroxyquinones. The
A B C D IB-00208
A B C D
molecular weight was deduced by (+)-ESI MS as 690 Dalton, and the corresponding molecular formula was C36H34O14, containing ten double bond equivalents.
The IR spectrum of 106 indicated the presence of hydroxyl (3444 cm-1) and con-jugated carbonyl (1697, 1650 cm–1) functionalities. The UV spectrum exhibited sig-nals (λmax 390, 321, 251 and 225 nm), characteristic for a polycyclic xanthone moi-ety[143].
The 1H NMR spectrum displayed four 1H signals in the sp2 region, representing two o-coupled benzenoide AB systems. The first set was visible at δ 8.58 and 8.28 (J
= 8.8 Hz), while the second one was found at δ 7.46 and δ 7.38 (d, J = 9.2 Hz), in addition to a singlet of a chelated hydroxyl group at δ 12.71. In the aliphatic region, six oxy-methines, one methylene and five singlet oxy-methyl signals were detected.
Additionally, two methyl doublets were observed at δ 1.56 and 1.15; (Table 7).
Figure 36: 1H NMR spectrum (CDCl3, 300 MHz) of IB-00208 (106).
The 13C NMR/ APT spectra and HMQC experiments of compound 106 revealed 36 carbon signals (Table 7), i.e. 18 quaternary sp2 carbon atoms (δ 178~104) includ-ing four carbonyl signals (δ 181.5~170.1), and four sp2 methines (δ 127~114.4). Ad-ditionally, six sp3 oxy-methines (δ 105~71) were displayed, among which one signal (δ 105.0) was assigned as an acetal H. Moreover, five methyl ether signals (δ
62.0~56.8), one methylene (δ 30.2) together with two methyl signals (δ 20.9 and 17.5) were visible (Table 7).
Figure 37: 13C NMR spectrum (CDCl3, 150 MHz) of IB-00208 (106).
Compound 106 was established to bear a rhamno-pyranosyl moiety based on the H,H COSY and the coupling constants of the attached protons (J= 8~9 Hz). Accord-ingly, the anomeric methine C-1' (δH 4.51; δC 105.0) was of β−configuration (J~8 Hz). The three hydroxy groups of the sugar at C-2’, C-3’ and C-4’ were replaced by three methoxy groups, at where protons of the three methoxy groups (δ 3.81, 3.69 and 3.56) showed a 3J correlation in HMBC at the corresponding carbons (C-2’:
61.4, C-3’: 61.3, C-4’: 61.0), respectively. Subsequently, the double methyl 5’-CH3
(1.15) of trimethoxy-rhamnose was recognized as its directed 2J correlation towards C-5’ (71.7). The whole structure of sugar system was further deduced by H, H COSY giving the partial structure of A. As rather confirmation, the ESI MS2 exhibited a peak fragment at m/z 525.2 due to the splitting of the glycoside moiety (A) from the whole molecule (m/z 713.2, [M+Na]+) (Figure 40). The long-range coupling from H-1' to C-5 (δ 139.7) shown by HMBC confirmed the attachment of the sugar at C-5 of ring B.
O O O O
O C H3
C H3
C H3 C
H3 3' 1'
5'
O O OH C
H3
1 3
5 16
A B
Additionally, the methylene protons of H2-4 (δ 3.74, 2.80) exhibited five cross-sections in HMBC towards C-3 (δ 75.9, 2J), 3-CH3 (δ 20.9, 3J), C-4a (δ 131.5, 2J), C-5 (δ 139.7, 3J) and C-16a (δ 104.5, 3J). On other hand, the 3-CH3 (δ 1.57) directed
two important correlations at C-3 (2J), and the lactonic carbonyl C-1 (δ 170.1, 4J) along with those at C-4 (3J). This lead to construction of a lactone moiety fused with an aromatic residue (fragment B).
O OH
5
7 16
O O
O O
O OH C
H3
A
B
C D
O O
O CH
3CH
3O
1114
C D
The singlet chelated OH signal (δ 12.71, 16-OH) displayed three significant connectivities versus C-15b (119.9, 3J), C-16 (160.3, 2J), and C-16a (104.8, 3J) as indicative of its joining at C-16 (ring B) in peri-position to the lactonic carbonyl (C-1, ring A). Alternatively, the two ortho-coupled protons (J~8.8 Hz) of H-6 (δ 8.57) and H-7 (8.26) showed relevant cross-sections against 5 (139.9), 5a (138.6), C-7a (129.8), C-15a (136.9), C-15b (119.9) as well as to those of the quinone carbon-yls, C-8 (178.8) and C-15 (181.8). Hence, the three rings (B, C and D) allowed the construction of the phenanthrone moiety fused with the lactone ring of A.
The remaining molecular formula (C9H8O4) was constructed by HMBC correla-tions as follows: the residual ortho-coupled protons, H-11 (δ 7.40) and H-12 (δ 7.47) showed interchangeable correlations at C-9a (δ 149.5), C-10 (δ 148.5), C-11 (δ 119.5), C-12 (δ 114.4), and C-13 (δ 151.2), C-13a beside to a γ-lactonic carbonyl (C-14, δ 173.1). In addition, the residual methyl-ether groups at δ 4.04 and 3.96 dis-played connectivities at C-10 and C-13, respectively, confirming their location at the assigned carbons. This supported the assignment of F ring (fragment D). The 4J cou-pling from H-12 at C-14 gave an evidence for the location of the carbonyl of γ -pyrone system. Alternatively, this F fragment was connected to the ring C of phenan-throne moiety via the pyrone ring E. Hence two alternatives were suggested: 16- hydroxy-10,13-dimethoxy-3-methyl-5-(3,4,5-trimethoxy-6-methyl-tetrahydro-pyran-2-yloxy)-3,4-dihydro-2,9-dioxa-hexaphene-1,8,14,15-tetraone (106) and
16-hydroxy-
Figure 38: H,H COSY ( , ) and selected HMBC ( ) connectivities of IB-00208 (106).
Based on the similarity of NMR data of the two isomers, the final structure could not be determined. Also, additional spectroscopic techniques (e.g. NOE, NO-ESY and/or CD experiments), could not give a final confirmation of the structure.
Therefore, the compound could be subjected to some chemical reactions in order to determine the correct isomer. Since this compound isolated, searching in all
data-bases resulted no hits, indicating the novelty of 106. However one month later, searching in AntiBase using the deduced substructures (Figure 39) identified the compound 106 as IB-00208[144,145], which is coincided with the reported data (Table 7) but still may be not the correct structure.
Table 7: 1H and 13C NMR assignments for IB-00208 (106) in comparison with literature data in CDCl3.
Position Litrature[144,145] IB-00208 (106)
δCa δH (J in[Hz]) b δCc δH (J in [Hz])d
1 170.4 - 170.1 -
3 76.2 4.68 (m) 75.9 4.70 (m)
3-CH3 21.1 1.56 (d, 6.4) 20.9 1.57 (d, 6.3) 4 30.5 2.80 (dd, 16.2, 11.2)
3.74 (dd, 16.4, 3.0) 30.2 2.82 (dd, 16.6, 11.3) 3.75 (dd, 16.7, 2.9)
4a 131.8 - 131.5 -
5 139.9 - 139.7 -
5a 138.6 - 138.4 -
6 127.6 8.56 (d, 8.8) 127.4 8.57 (d, 8.8)
7 124.8 8.26 (d, 8.8) 124.6 8.26 (d, 8.8)
7a 129.8 - 129.6 -
8 178.8 - 178.6 -
8a 153.2 - 153.0 -
9a 149.8 - 149.5 -
10 148.7 - 148.5 -
10-OCH3 62.3 4.03 (s) 62.0 4.04 (s)
11 119.7 7.38 (d, 9.2) 119.5 7.40 (d, 9.2)
12 114.6 7.46 (d, 9.2) 114.4 7.47 (d, 9.2)
13 151.5 - 151.2 -
13-OCH3 57.0 3.95 (s) 56.8 3.96 (s)
13a 121.1 - 120.8 -
14 173.3 - 173.1 -
14a 121.3 - 121.1 -
15 181.8 - 181.5 -
15a 136.9 - 136.7 -
15b 119.9 - 119.7 -
16 160.3 - 160.0 -
16-OH - 12.71 (s) - 12.71 (s)
16a 104.8 - 104.5 -
1' 105.2 4.49 (d, 8.0) 105.0 4.51 (d, 7.9)
2' 84.7 3.34 (dd, 9.1, 8.0) 84.4 3.36 (dd, 9.2, 7.9)
2'-OCH3 61.4 3.81 (s) 61.1 3.82 (s)
3' 86.7 3.17 (t, 9.0) 86.5 3.19 (t, 8.9)
3'-OCH3 61.3 3.69 (s) 61.0 3.70 (s)
4' 85.4 2.84 (t, 9.0) 85.1 2.85 (t, 9.0)
4'-OCH3 61.0 3.56 (s) 60.7 3.57 (s)
5' 71.7 3.02 (dd, 9.2, 6.0) 71.4 3.05 (m) 5'-CH3 17.7 1.15 (d, 6.0) 17.5 1.17 (d, 6.0)
a (100 MHz); b(400 MHz); c(150 MHz); b(300 MHz)
C(H0)
Figure 39: AntiBase search of IB-00208 (106).
O O
m/z = 525.2 [M-sugar(A)+Na]+ A
Figure 40: (+)-ESI-MS/MS of IB-00208 (106).
IB-00208 (106), a polycyclic compound possessing a xanthone unit, exhibit po-tent cytotoxic and antibiotic activity. This agrees well with our own results, deter-mined in a monolayer cell proliferation assay using a panel of 36 human tumor cell lines, with a mean IC50 of 0.002 µM (mean IC70 = 0.007 µM), Table 53. Compound 106 is structurally related to a small family of polycyclic antibiotics including cerv-inomycins[146], citreamicins[148], simaomycins[149] and the highly cytotoxic polycyclic xanthones actinoplanones A~G; which have a lactam with partial reduction at ring F
[150]. They were isolated from terrestrial Micromonospora, Streptomyces, Actino-madura and Actinoplanes strains, respectively.