Scheme 4: proposed biosynthesis of 79 and 80
6.5.3 N-Methyl-L-valyl-tryptophanol (valindolmycin)
Compound 81 was isolated as a colourless solid. The 1H NMR spectrum showed in the aromatic region the characteristic fingerprint of a 3-monosubstituted indole.
The aliphatic region showed signals between 4.40 and 0.98. The search in Anti-Base gave valindolmycin.
NH
NH NH
O O H
81
N-Methyl-L-val-tryptophanol is the precursor in the biosynthesis of indolactam V [131], and is also the precursor in the biosynthesis of teleocidin [132] and this may explain its presence in this strain (Scheme 4).
6.5.4 2-Hydroxy-6-methyl-cinnamic acid
Compound 82a was obtained as a white solid, which showed on TLC an orange colour after spraying with anisaldehyde/sulphuric acid. The 1H NMR spectrum of 82a in MeOH-d4 exhibited two doublets at 7.89 and 6.82 typical for a trans double bond. A triplet at 7.08 and two doublets at 6.87 and 6.83 were attributed to a 1,2,3-trisubstituted aromatic ring system, which was confirmed by the H,H COSY couplings. In the aliphatic region, only a 3H singlet at 2.40 characteristic of an aromatic methyl group was observed. The EI mass spectrum showed a molecular ion at m/z 178, and the HRESIMS delivered the molecular formula C10H10O2.
The 13C NMR spectrum exhibited 10 signals including a carbonyl at 172.1, three quaternary carbons, 5 sp2 methines and a methyl carbon. Compound 82a was identified as 2-hydroxy-6-methylphenylcinnamic acid and the structure was con-firmed by HMBC correlations; this compound is reported here for the first time.
Terrestrial Streptomyces sp. AdM02 105
R2
R1 O
82
R1 R2
82a OH OH
82b OCH3 OH
82c OCH3 NH2
82d H NH2
R2
R1 O
1 4 3
7 5 9
Figure 81: Selected HMBC ( ) and COSY ( ) correlations in compounds 82b-c 6.5.5 2-Methoxy-6-methyl-cinnamic acid
The EI mass spectrum of compound 82b gave a molecular ion at m/z 192, which de-livered the molecular formula C11H12O3 by HREIMS. The 1H NMR spectra of 82b showed a trans double bond signal, the trisubstituted aromatic system and the aro-matic methyl and was very similar to those of 82a, the major difference being the presence of a methoxy group at 3.86 in 82b.
Figure 82: 1H NMR spectrum (300 MHz) of 2-methoxy-6-methylcinnamic acid (82b) in MeOH-d4
The 13C NMR spectrum confirmed the presence of eleven carbon atoms as de-manded by high resolution MS.
Figure 83: 13C NMR spectrum (75 MHz) of 2-methoxy-6-methylphenylcinnamic acid (82b) in MeOH-d4
The HMBC spectrum indicated a correlation of the methoxy group to the qua-ternary carbon at 160.6, confirming the methoxy group in position C-9.
Figure 84: HMBC spectrum (300 MHz) of 2-methoxy-6-methylphenylcinnamic acid (82b) in MeOH-d4
Further HMBC correlations (Figure 81) confirmed the structure of the new compound as 2-methoxy-6-methyl-cinnamic acid (82b). The related trans-2,6-dimethoxycinnamic acid was previously isolated from the plant Taxus floridana.[133]
Terrestrial Streptomyces sp. AdM02 107
6.5.6 2-Methoxy-6-methylcinnamide
Compound 82c was obtained as white solid with a molecular ion at m/z 191 in the EI mass spectrum. The molecular formula C11H12NO2 was derived by HREI MS.
The 1H and 13C NMR data of 82c (Table 10) were very similar to those of 82a and 82b. The major difference was the replacement of an oxygen by a nitrogen atom in 82c, which indicated the presence of an amide group instead of an carboxylic acid in 82c. Compound U77863 (82d) is closely related to 82c and was isolated from Strep-tomyces griseoluteus by Herper et al.[134] It showed antiinvasive and antimetastatic activities versus K-1735-M2 and B16-F10 murine melonomas.[135] Compound 82c is also a new natural product.
Table 10: 1H (300 MHz); (J, Hz) and 13C NMR (75 MHz) data of 82a, b and c in MeOH-d4.
1H 13C
No 82a 82b 82c 82a 82b 82c
1 - - - 172.1 171.5 172.2
2 6.82 (d,16.2) 6.62 (d, 16.2) 6.82 (d, 16.0) 121.3 123.0 123.5 3 7.89 (d,16.2) 7.89 (d, 16.2) 7.78 (d, 16.2) 140.8 140.4 136.9
4 - - - 122.4 122.9 125.2
5 - - - 140.8 140.8 140.6
5- CH3 2.39 (s) 2.40 (s) 2.41 (s) 20.9 20.9 21.0
6 6.87 (d, 8.3) 6.88 (d, 8.3) 6.87 (d, 8.0) 114.9 110.1 110.1 7 7.06 (t, 7.9) 7.21 (t, 7.9) 7. 18 (t, 8.0) 131.4 131.6 131.0 8 6.83 (d, 7.5) 6.83 (d, 7.5) 6.83 (d, 8.0) 122.9 124.2 124.2
9 - - - 159.2 160.8 160.6
9-OCH3 - 3.86 (s) 3.86 (s) - 55.9 55.9
6.5.7 Heramide
Compound 83a was obtained as a yellowish solid with a blue fluorescence under UV at 366 nm; it gave a yellow colour reaction with anisaldehyde/sulphuric acid.
The molecular ion peak of 83a was deduced from (+)-ESI HR mass spectrum to be m/z 253.15467, corresponding to the molecular formula C13H20N2O3. The 1H NMR spectrum of 83a in MeOH-d4 indicated two doublets of doublets of intensity six at 1.00 and one methine signal at 2.14, typical for an isopropyl fragment, the singlet
of a methyl group connected to a double bond at 1.57, and in addition signals of four methylene groups at 2, 35 (12-H2), 1.94 and 1.64 (3-H2), 2.35 and 2.13 (4-H2) and 3.41, 3.05 (5-H2). The NMR spectrum in DMSO-d6 indicated the presence of two H/D exchangeable protons by signals at 6.00 and 10.14.
Figure 85: 1H NMR spectrum (300 MHz) of heramide (83a) in DMSO-d6
The 13C NMR spectrum of indicated 13 carbon signals including three sp2 car-bon signals at 201.7, 172.9 and 169.5 which may be attributed to a conjugated ketone, acid or amide, respectively. The spectrum showed two quaternary carbons at 99.2 and 97.6, and signals due to four methylenes ( 48.2, 46.5, 33.8, 27.7), one methine ( 27.2) and three methyls ( 22.7, 22.6, 7.1).
Figure 86: 13C NMR Spectrum (75 MHz) of heramide (83a) in DMSO-d6
Terrestrial Streptomyces sp. AdM02 109
A search in AntiBase[68] and the Dictionary of Natural Products (DNP)[69] with the 1H and 13C NMR data, the molecular mass and the molecular formula delivered no answer, indicating 83a to be probably a new natural product. The interpretation of the H,H COSY spectrum confirmed the presence of an isopropyl group connected to a methylene group as well as a three methylene fragment as the only sub-structures.
N
13 3
5
In the HMBC spectrum, the methylene protons at 1.94 and 1.64, as well as the methine 27.2 (C-13) and the methylene 2.35 (C-12) protons of the isobutyl fragment showed a correlation with the carbonyl at 172.9 (C-9). In addition, corre-lations of both methyls of the isobutyl group with the methylene ( 2.35, 12-H2) were visible. The methylene and the methyl at 1.64 (3-H2) and 1.58 (8-CH3), respec-tively, showed correlations to the carbonyl at 201.7 (C-11). Further HMBC correla-tions were observed between the methylene signals at 3.41/3.03 (5-H2) and the methyl at 1.58 (8-CH3) to the carbon at 169.5 (C-7). Finally, the 8-CH3 exhibited a correlation with the quaternary carbon at 99.2 (C-8), and of 3-H2 with the signal at 97.2 (C-2). Careful interpretation of these HMBC correlations delivered the sub-structure I.
Figure 87: HMBC ( ) correlation of the substructures I and II in 83a
The substructure I (Figure 87) contains already C13H19N2O3 and three double bond equivalences. With respect to the molecular formula of 83a, only one acidic proton, one oxygen, one nitrogen and two double bond equivalences were remaining.
In respect of the chemical shift of 5-H2 (3.03/3.41), this methylene group cannot be bound to oxygen, and due to the correlation of the 5-H2 to the carbon signal at
169.5 (C-7) substructure II (Figure 87) was found. The chemical shift at 97.2 (C-2) for a quaternary sp3 carbon was explained by a cyclisation via oxygen. The possibil-ity of structure 83b was consequently excluded and the structure 83a was finally con-firmed by the fact that in the 13C NMR spectrum no signal for an oxymethylene group was visible. Compound 83a was named heramide.
N the only natural products containing the 9-oxa-2-aza-bicyclo[4.2.1]nonane ring (84), however, similar ring systems were observed in synthetic products.[138]
N O
84
To further support structure 83a, the mass-spectrometric fragmentation pattern was investigated. The EI MS afforded a base peak at m/z 139 and several other peaks (167). The ESI MS/MS presented the fragments at m/z 169, 151, whose fragments formulas were identified through HRESI MS/MS (Figure 89).
Terrestrial Streptomyces sp. AdM02 111
EI-fragmentation HRESI MS/MS [M+H]+: m/z 253.3
[M+H]+: m/z 169.097 for C8H13N2O2
-H2O
[M+H]+: m/z 151.086 for C8H11N2O
Figure 89: Proposed EI and HRESI MS/MS fragmentations of compound 83a Retrosynthetically, heramide (83a) can be deduced from propionic acid and or-nithine. Both acids could afford the keto-diamino acid intermediate by means of an ester condensation. Similar compounds are reported in the literature [139] as precursor in the enzymatic synthesis of the roast smelling 2-propionyl-1-pyrroline (R = CH3) (Figure 90[139]) Subsequent lactam formation should yield an ozacinone derivative, which under oxidative conditions might form heramide (83a).
N
Figure 90: Retrosynthesis of heramide (83a) i: Retrosynthesis from aminodiketo acid derivative [139], ii: ester condensation; iii: lactamization; iv: aminal
Terrestrial Streptomyces sp. AdM02 113
6.5.8 3-Hydroxy-5-hydroxyaminisochroman-1-one
Compound 85a was obtained as a colourless solid, which showed on TLC a blue fluorescent under UV 366 nm and pink colour after spraying with anisalde-hyde/sulphuric acid. The 1H NMR spectrum of 85a in MeOH-d4 exhibited two dou-blets of doudou-blets at 7.45 (J = 8.1, 1.5 Hz) and 6.84 (J = 7.8, 1.5 Hz) and one triplet at 6.49 (J = 7.9 Hz), attributed to a 1,2,3-trisubstituted aromatic ring system and confirmed by the H,H COSY couplings. The doublet of doublet of an oxymethine signal at 5.40 (J = 2.4, 4.3 Hz) indicated a correlation to the methylene group at 3.49 and 3.25 (J = 2.4, 11.9 Hz). The proton NMR spectrum in MeOH-d4 indicated only six protons, suggesting three further acidic protons.
The 13C NMR spectrum exhibited 9 signals including a carbonyl at 172.0, three quaternary carbons, three sp2 methines, one methylene carbon and a methine carbon. The interpretation of the HMBC spectrum showed the correlations of a pro-ton at 7.45 (8-H) and the methylene group at 3.25/3.49 (4-H2) to the quaternary carbon at 139.0 (C-4a). The methylene group gave also the correlation to the he-miacetal carbon at 90.5 (C-3). This methine (3-H) showed a H,H COSY correlation to the methylene group. The three aromatic protons (6-, 7- and 8-H) showed tions to the quaternary carbons C-5, C-4a and C-8a. Finally, 8-H exhibited a correla-tion to the carbonyl carbon at 172 (C-1), which is the only correlacorrela-tion to this sig-nal. The information from all the NMR data (1D, 2D) delivered the substructure de-picted in Figure 91.
The CI mass spectrum showed a quasimolecular ion at m/z 196 ([M+H]+), and HREI MS delivered the molecular formula C9H9NO4. Compound 85a contained 6 double bond equivalences. From the substructure, one oxygen, one nitrogen, 2 hy-dogen atoms and one double bond equivalence are missing. Comparison of the 1H data from compounds 85a and 85c showed similarity, so that C-5 seems to be substi-tuted with an amino group. The missing oxygen can only be assigned to the amine group to form an oxime. So the structure of compound 85a was finally elucidated as 3-hydroxy-5-hydroxyaminisochroman-1-one (Figure 92).
O
Compounds possessing the isochroman chromophore were isolated mainly from fungi and rarely from bacteria. They possessed moderate antifungal activity. [140] The new compound 85a may be derived from 85b by reduction of the nitro group, 85b and 85c are reported as intermediates in the synthesis of 5-nitroisocoumarins and methylindoles. [141,142]
O Figure 92: HMBC ( ) correlations in the structure 85
Terrestrial Streptomyces sp. AdM02 115
Table 13:1H NMR data comparison (J in Hz) of 85a in MeOH-d4 and 85c in CDCl3.
No 85a 85c[141]
3 5.40 (dd, 2.4, 4.3) 5.36 (t, 4)
3-CH3 - 3.53 (s)
4 3.49 (dd, 2.4, 11.9) 3.25 (dd, 4.3, 11.9)
3.05 (dd, 4.0, 15.0) 2.73 (dd, 4.0, 15.0)
4a - -
5 - -
6 6.84 (dd, 7.8, 1.5) 6.80 (dd, 8.0, 2.0) 7 6.49 ( t, 7.8) 7.10 ( t, 8.0) 8 7.45 (dd, 8.1, 1.5) 7.48 (dd, 8.0, 2.0)
8a - -
6.5.9 N-(2-methoxyphenyl)-benzamide
Purification of fraction 3 by PTLC followed by HPLC delivered compound 86 as colourless crystals; the molecular mass was deduced to be m/z 227. The 1H NMR spectrum exhibited only nine aromatic protons as four doublets ( 8.08, 7.98, 7.97, 7.60) and five triplets, respectively, at 7.58, 7.54, 7.20, 7.10 and 6.98 suggesting the presence of two spins system with four consecutives protons for the first one (a aromatic ring) and five consecutives protons for the second spin system, in addition a methoxy group was visible ( 3.90).
Figure 93: 1H NMR spectrum (300 MHz) 86 in MeOH-d4
The 13C NMR spectrum indicated only fourteen carbon signals including a car-bonyl of an amide at 168.1 (C-7), three quaternary carbons at 152.0, 136.1 and
128.0, nine methines at 133.0-121.6 and a methoxy carbon at 56.4. The H,H COSY spectrum confirmed the presence of two spin systems as derived from the 1H NMR spectrum. The HMBC spectrum indicated the correlations of the methoxy sig-nal together with the doublet at 8.08 (3-H) to the carbon at 152.0 (C-2), indicat-ing that the methoxy group should be located in the disubstituted aromatic system.
From these interpretations, two different aromatic rings are resulting: a 1,2-disubstituted (I) and a mono substituted benzene rings (II).
O
4 2 56.4
I II
Figure 94: Fragments I and II from the COSY and HMBC spectra of 86
The HMBC spectrum showed no correlation to the carbonyl group at 168.1 so that including fragments I and II, two plausible structures can be derived: N-(2-methoxyphenyl)-benzamide (86a) und 2-(methoxy-Nphenyl)-benzamide (86b).
O NH O
NH O O
86a 86b
Both structures are known from the synthesis. [143,144]
The preparation of N- (2-methoxyphenyl)-benzamide was repeated (86b) according to the literature [144] by reacting benzoyl chloride with 2-methoxyaniline. Comparison of the proton spectra identified compound 86 as N- (2-methoxyphenyl)-benzamide, which is described here for the first time as natural product.
NH O O
4 2
6 7
9 11
13
86
Terrestrial Streptomyces sp. AdM02 117
6.5.10 Azomycin
Compound 87 was isolated as a yellow powder. The 1H NMR spectrum of 87 in DMSO-d6 exhibited one singlet at 7.22 with the intensity of 2 and one broad singlet for an exchangeable proton at 14.60. EI MS gave a molecular ion at 113 Dalton. By searching in the MS database NIST, compound 87 was identified as azomycin, which was already isolated in 1954. Azomycin (87) is an antimicrobial antibiotic firstly isolated from a strain of Nocardia mesenterica.[145]
N N
N+ O O 87 6.5.11 Trivial compounds
Vanillic acid
Compound 88 was isolated as white powder. The UV active compound showed on TLC a light blue colour reaction which anisaldehyde. Its proton NMR spectrum showed only four signals: at 7.58 (d, J = 1.5 Hz), 7.40 (dd, J = 8.6, 1.5 Hz), 6.88 (d, J = 8.6 Hz) for a 1,3,4-trisubstituted benzene ring and a singlet at 3.92 attributed to a methoxy group. The search in AntiBase gave 4-hydroxy-5-methoxy-benzoic acid as a plausible structure. Comparison of the 1H NMR data with the spectra in our spectra collection confirmed the assignment.
OH
OH O O
88