Article pubs.acs.org/jnp
Indothiazinone, an Indolyl Thiazolyl Ketone from a Novel Myxobacterium Belonging to the Sorangiineae Rolf Jansen,†,§ Kathrin I. Mohr,†,§ Steffen Bernecker,†,§ Marc Stadler,†,§ and Rolf Müller*,†,‡,§ †
Department Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany Centre for Infection Research and Department of Pharmaceutical Biotechnology, Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University, P.O. Box 151150, 66041 Saarbrücken, Germany § German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany ‡
S Supporting Information *
ABSTRACT: Indothiazinone (1), an indolyl thiazolyl ketone, was discovered in cultures of novel myxobacterial strain 706, recently isolated from compost in Germany. Molecular phylogenetic studies based on 16S rRNA gene sequences revealed strain 706 to be a representative of a new family of the Sorangiineae. A screening of the culture broth for antimicrobial metabolites followed by isolation and characterization of these compounds revealed six indole derivatives and a 1,4-naphthoquinone derivative. The structures were determined to be indothiazinone (1; 1H-indol-3-yl(1,3-thiazol-2-yl)methanone) and three 3methylbuta-1,3-dien-1-yl-substituted indoles, indolyl ethanol 2 and the E- and Z-isomers of indolyl ethylidenehydroxylamine 4 and 5 by MS and NMR spectroscopic analyses. In the indolyl ethanol derivative 3 the unsaturated methylene group of the butadienyl residue was replaced by an oxygen atom to give the keto group of the butanone side chain. Further 1H-indol-3ylacetonitrile (6) was identified, which was already known as a myxobacterial metabolite. 2-Hydroxyethyl-3-methyl-1,4naphthoquinone (7) was recognized during dereplication as an antibiotic previously isolated from Actinoplanes capillaceus. Whereas 1, 4, 5, and 7 showed weak activity against yeasts and filamentous fungi, isomers 4 and 5 were weakly active against Gram-positive bacteria and mouse fibroblasts. Compound 6 is volatile, and 2 and 3 showed no activity in a number of assays.
I
acids by testing analytical-scale fractions of the raw extract, other less active fractions were disclosed by HPLC-UV-MS to contain unknown metabolites. Consequently, five indole derivatives (Figure 1) were isolated using solvent partitioning, LH-20 gel chromatography, and preparative RP-HPLC. A comparison of the 16S rRNA gene sequences of strain 706 (Acc.-No. JF719608) with sequences of a public database (BLAST, Basic Local Alignment Search Tool) provided by the National Center for Biotechnology Information (NCBI) showed the most similar sequence to be surprisingly different, with only 91.7% identity on the DNA level to Polyangium spumosum (Acc.-No. GU207881).6 Thus, strain 706 belongs to the Sorangiineae but seems to be a member of a novel family of myxobacteria. Myxobacteria are well-known producers of structurally diverse secondary metabolites.7,8 In the past more
n our ongoing screening for biologically active secondary metabolites, myxobacteria and other often uncharacterized gliding bacteria are being explored as novel sources of chemical diversity. In the past it could be demonstrated that representatives of new families, genera, and new species were promising sources for new metabolites. For example, the 3formylindol derivatives indiacens A and B were isolated from a representative of a new myxobacterial family,1 and the aetheramides A and B, potent HIV-inhibitory depsipeptides, were isolated from a myxobacterium of the new genus Aetherobacter,2 which is also a novel source of polyunsaturated fatty acids.3 From the novel Ohtaekwangia kribbensis4 belonging to the Bacteroidetes, unique pyrroloquinolines, the marinoquinolines A−F, were isolated.5 In the present study, culture broth extracts of strain 706 were screened for the presence of antimicrobial secondary metabolites using broad biological testing, as well as HPLC-UV-HRESIMS, with database comparison to identify known and novel metabolites. Although the main biological activity could be traced back to some fatty © 2014 American Chemical Society and American Society of Pharmacognosy
Received: February 15, 2014 Published: April 3, 2014 1054
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three methines C-2, C-12, and C-13 belong to heterocyclic rings in accord with 1JH,C values of 190, 188, and 188 Hz, respectively.11 Detailed analysis of the 1H,13C HMBC and NOESY correlations (Figure 1) initially connected structural parts A and C and the carbonyl C-10 to give the indolyl ketone residue that was also proposed by the sulfur-free fragment ion m/z 144.0423 (C9H6NO, calcd 144.0443) in the HRESIMS. The final bis-heteroaromatic ketone indothiazinone (1) was the only structure that was consistent with the elemental analysis and the NMR data (Table 1). Figure 1. Selected correlations in the 2D NMR spectra and the main MS fragment ion of indothiazinone (1) (gray zones 1H,1H COSY; blue arrows 1H,13C HMBC; red arrows 1H,1H NOESY).
Table 1. NMR Data of Indothiazinone (1) in CDCl3a (700.4 MHz; 176.1 MHz)
than 120 new basic structures with about 600 variants exhibiting numerous biological activities have been reported from these soil-dwelling bacteria.9,10
pos.
δC
type
δH
1 2 3 4 5 6 7 8 9 10 11 12 13
136.9 114.1 122.5 123.0 123.9 111.6 136.1 126.8 178.3 170.3 144.0 124.7
NH CH C CH CH CH CH C C C C CH CH
9.72 9.12
brs d (3.0)
8.54 7.35 7.31 7.46
dd (7.7, 0.6) ddd (8.2, 7.1, 1.3) ddd (8.1, 7.1, 1.3) dt (8.0, 0.8)
NH
8.01 7.64
d (3.2) d (3.0)
13 12
■
RESULTS AND DISCUSSION Amberlite XAD 16 adsorber resin was recovered from a 70 L fermentation of strain 706 and eluted with methanol. After methanol−heptane partitioning the polar part of raw extract was separated by Sephadex LH-20 chromatography in methanol. Preparative RP HPLC of the LH-20 fractions provided six indole derivatives, 1−6, and the 1,4-naphthoquinone derivative 7. a
m (J in Hz)
NOESY
H in HMBC
7, 2 NH 4, 2 6, 5 7 4 5, 4 6, 4, 2 5, 7, 2 2 13, 12 13 12
Trace of CD3OD.
Compound 2 was isolated by RP-HPLC from another fraction of an LH-20 chromatography of the raw extract, which additionally provided 2-(1H-indol-3-yl)acetonitrile (6). HRESIMS of the molecular ion clusters [M + H]+, [M + H]+, and [M − H]− suggested the elemental composition C15H17NO with eight double-bond equivalents. The high degree of unsaturation was represented in the UV spectrum with bands at 264, 276, and 306 nm. With the exception of two H/Dexchanged protons the 1H NMR signals in CD3OD were correlated to their corresponding carbon signals (Table 2) in the 1H,13C HSQC NMR spectrum. The 1H,1H COSY spectrum of 2 contained two broad singlets for three aromatic protons in CD3OD, which were resolved in CDCl3 as an aromatic structural part A of two vicinal coupling protons (J = 8.6 Hz) H-6 and H-7 and a proton H-9 in ortho position with a coupling of 1.5 Hz (Table 2). The COSY spectrum further allowed the assignment of a 3-methyl-1,3-butadienyl residue B based on 1 1 H, H long-range couplings and the correlations in the 1H,13C HMBC NMR spectrum (Figure 2). According to the large coupling constant of 16.5 Hz, the Δ12,13 double bond of the side chain was trans, as was the methyl-substituted Δ14,16 double bond, as indicated by the ROESY correlations between H-13 and H-16a and between H-16b and the methyl group C-15. Another ROESY correlation between H-12 and the aromatic methine H-9 together with appropriate HMBC correlations (Figure 2) suggested the attachment of the butadienyl residue as a side chain to the aromatic residue A. The COSY spectrum further showed the presence of the methylene group C-10 (δH/C 2.98/29.89) connected to an oyxmethylene group (δH/C 3.82/63.85), which formed the primary alcohol C-11 in structural part C. This side chain was combined with the remaining NH atom of the molecular
The elemental formula C12H8N2OS of indothiazinone (1) was indicated by the molecular ion clusters [M + H]+, [M + Na]+, and [M − H]− in the HRESIMS. Ten calculated doublebond equivalents were the basis for a band-rich UV spectrum (λmax 207, 261, 272, 278, and 349 nm), suggesting a heterocyclic aromatic molecule. Seven of the eight protons were assigned to their corresponding sp2 carbons from correlations in the 1H,13C HSQC NMR spectrum in CDCl3. The 1H,1H COSY NMR spectrum presented three structural parts (A−C) (Figure 1), an NH proton (δ 9.72) coupling with the aromatic methine proton H-2 (δ 9.12) (part A), a pair of aromatic protons (H-12 and H-13) with doublets at 8.01 and 7.64 ppm (part B), and four sequential aromatic protons (H-4 to H-7) in structural part C. While the direct 1H,13C coupling constants observed in the HMBC NMR spectrum in part C (1JH,C 159−166 Hz) indicated their position in a carbocyclic ring, the remaining 1055
dx.doi.org/10.1021/np500144t | J. Nat. Prod. 2014, 77, 1054−1060
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Table 2. NMR Data of 3-Methyl-1,3-butadienylindolylethanol (2) and (3E)-4-[3-(2-Hydroxyethyl)-1H-indol-5-yl]but-3-en-2one (3) 2a m (J in Hz)
2b
δ Cd
type
δH
1 2 3
124.37 113.44
NH CH C
7.06
4 5 6 7
129.40 138.08 112.58 121.06
C C CH CH
7.28e 7.28e
brs brs
8 9
129.72 118.38
C CH
7.59
brs
10
29.89
CH2
2.98
t (7.1)
11 12 13 14 15 16a 16b
63.85 131.87 129.47 144.05 19.07 115.63
CH2 CH CH C CH3 CH2
3.82 6.68 6.86
t (7.1) d (16.5) d (16.5)
9 16a
1.99 5.05 4.97
s brs brs
16b 13 15
no.
NOESY
brs
H in HMBC 10 10, 11, 2>9 10, 6 2, 7, 9
12
m (J in Hz)
δH 8.03 7.09
brs brd (2.5)
12, 9
7.33 7.38
d (8.6) dd (8.6, 1.5)
6 12, 7
7.66
brs
11
3.05
10 7, 9, >13 8, 15, 16a 15, 12, >13 13, >16a 15, 13
3.93 6.69 6.89
td (6.3, 1.0) brm d (16.3) d (16.3)
2.01 5.10 5.03
s brm brm
3c m (J in Hz)
δC
m
δH
125.24 114.49
CH C
7.14
s
10, 11
129.55 139.94 113.11 122.25
C C CH CH
7.37 7.46
d (8.5) dd (8.5, 1.7)
7 13, 6, 12
126.60 122.52
C CH
7.86
d (1.7)
29.73
CH2
3.00
63.74 149.09 124.46 201.85 27.19
CH2 CH CH C CH3
3.83 7.82 6.76
td (7.1, 0.8) t (7.1) d (16.2) d (16.2)
10, 11, 13 2, 9
2.39
s
NOESY
2, 9 15, 7 15, 7, 9 13, 12
H in HMBC 10 10, 11, >2, 9 10, 2, 6, >9 7, 9, >2 >9 12, 9 13, 6, >12 7, 12 11, >2 10 7, 9, >15 15, 12 15, 12, >13 13
a
In CD3OD, 1H/13C 300/75 MHz. bIn CDCl3, 400 MHz. cIn CD3OD, 1H/13C 500.3/125.8 MHz. dNarrow signals require two decimals. e1H signal overlap was resolved in CDCl3.
4 and 5 showed identical UV spectra and molecular ion clusters [M + H]+, [2M + H]+, and [M − H]− in the HPLC-UVHRESIMS, suggesting the elemental formula C15H16N2O for both compounds. Their NMR spectra in CDCl3 (Table 3) were very similar to those of 2, and they indicated two very similar compounds with only small NMR shift differences for the major structural parts of 4 and 5 from C-3 to C-16. A methylene group (C-10) at δC 21.38 or 25.84 ppm and a methine group (C-11) at δH/δC 6.97/151.64 or 7.63/150.82 ppm discriminated 4 and 5 and suggested their structures as Eand Z-aldoximes, respectively. The oximes 4 and 5 underwent isomerization during NMR spectroscopy. Characteristically, the E-oxime 4 was the isomer with the shorter retention time in the RP18 chromatography12 and showed a distinctive low-field shift of the oxime methine proton C-11′ (ΔδH 0.7 ppm) compared to the Z-oxime 5 (C-11).13 The LH-20 fraction 8 was purified by RP-HPLC to yield 1.7 mg of the antibiotic 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (7), which has been previously described as a metabolite of the rare actinomycete Actinoplanes capillaceus.14 Compound 7 was recognized during dereplication by searching the DNP database15 using the elemental composition C13H12O4 obtained from the HRESIMS data, together with the hydroxyethyl group detected by the 1H NMR spectrum, similar to the indoles 2 and 3. Comparison of 1H and 13C NMR data (in CDCl3) and of the UV data (in methanol) confirmed the identification of 7.16 Biological activity was evaluated using different Grampositive and Gram-negative bacteria, yeasts, fungi, and eukaryotic cells. Indothiazinone (1) and oximes 4 and 5 exhibited weak antimicrobial activity against certain yeasts and filamentous fungi (Table 4; MIC between 67 and 33.3 μg/mL) compared to the reference nystatin. Additionally, isomers 4 and 5 are weakly active against Gram-positive bacteria (Nocardioides simplex and Nocardia sp.). The hydroxyethylnaphthoquinone 7
Figure 2. Selected 2D NMR correlations of 3-methyl-1,3-butadienylindolylethanol (2) (gray zones 1H,1H COSY; blue arrows 1H,13C HMBC; red arrows 1H,1H NOESY).
formula and the aromatic methine C-2 (δH/C 7.06/124.37) and three quaternary carbons (113.44, 129.40, 138.08) using the interconnections (Figure 2) indicated in the HMBC NMR spectrum to assign the remaining part of the 3-methyl-1,3butadienylindolylethanol (2). Repeated preparative RP-HPLC of LH-20 fraction 10 provided another indolyl-ethanol derivative, 3, with the elemental composition C14H15NO2, which compared with 2 contained one additional oxygen atom and the loss of a methylene unit. The UV spectrum of 3 presented three strong bands at 267, 281, and 335 nm due to the effect of the oxygen atom. The NMR spectra suggested the same indolyl-ethanol skeleton for 3 and indicated that the unsaturated side chain still contained the trans double bond, although their 1H doublets of 16.2 Hz now were about 1 ppm apart, at 7.82 and 6.76 ppm. The unusual butenone side chain then was established from HMBC correlations (Table 2) of the new keto group C-14 (δC 201.8 ppm) and the methyl group C-15 (δH 2.39, δC 27.1 ppm). Compounds 4 and 5 were isolated by RP-HPLC from the same late eluting LH-20 fraction containing indothiazinone (1). 1056
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Table 3. NMR Data of Oximes 4 and 5 in CDCl3 [1H 600 MHz; 13C 100.6/150.9 MHz] pos. 1 2 2′ 3 3′ 4, 4′ 5, 5′ 5, 5′ 6, 6′ 6, 6′ 7, 7′ 7, 7′ 8, 8′ 9 9′ 10 10′ 11 11′ 12,12′ 12, 12′ 13, 13′ 14, 14′ 15, 15′ 16, 16a′ 16, 16b′ a
δC
type
δH
122.65 122.71 111.29 111.02 127.57 136.01 135.93 111.40 111.40 121.01 121.01 129.51 117.31 117.45 21.38 25.84 151.64 150.82 129.86 129.89 129.44 142.41 18.72 115.86 115.89
NH CH CH C C C C C CH CH CH CH C CHa CHa CH2 CH2 CH CH CH CH CH C CH3 CH2 CH2
8.02 7.07 7.05
brs brd (2.2) brd (2.6)
2′, 2, >6/6′ NH, >10 NH, >10′
7.34 7.33 7.40 7.39
d (8.4) d (8.1) dd (8.4, 1.5) dd (8.6, 1.7)
NH NH 13/13′, >12/12′ 13/13′, >12/12′
7.63b 7.64b 3.87 3.68 6.97 7.63b 6.69
brs brs brd (5.1) brd (6.2) t (5.1) t (6.2) d (16.1)
12/12′, 13/13′ 12/12′, 13/13′ >2 >2′
6.89
d (16.1)
16/16′a, 7/7′, >9,9′
2.01 5.10 5.03
s brd (1.1) quin (1.5)
16/16′b, 12/12′ 13/13′ 15/15′
m (J in Hz)
NOESY
H in HMBC 10 10′ 10, >2, 9 10′, >2′, 9′ 10′, 10, 2′, 2, 6/6′ 7/7′, 9/9′, >2′/2 7/7′, 9/9′, >2′/2
15/15′, 9′, 9, >7/7′
12/12′, 9/9′ 12/12′, 9/9′ 13/13′, 6/6′, 9/9′ 12/12′, 7/7′ 12/12′, 7/7′ 11 11′ 10 10′ 13/13′, 9/9′, >7/7′ 13/13′, 9/9′, >7/7′ 15/15′, 16/16′a,b 15/15′, 12/12′, >13/13′ 16/16′a,b, 13/13′ 15/15′, 13/13′ 15/15′, 13/13′
C/H correlation interchangeable. bOverlapping signals.
Table 4. Minimum Inhibitory Concentration (MIC) in μg/mL of All Compounds and Positive Controlsa
Gram +
Gram −
yeasts
fungi
a
mouse fibroblasts, L929 LC50 Bacillus subtilis DSM 10 Nocardioides simplex DSM 20130 Nocardia sp. DSM 43069 Staphylococcus aureus DSM 346 Mycobacterium diernhoferi DSM 43524 Micrococcus luteus DSM 20030 Paenibacillus polymyxa DSM 36 Pseudomonas aeruginosa DSM 50071 Chromobacterium violaceum DSM 30191 Escherichia coli DSM 1116 Schizosaccharomyces pombe DSM 70572 Rhodotorula glutinis DSM 10134 Candida albicans DSM 1665 Pichia anomala DSM 6766 Nematospora coryli DSM 6981 Trichosporon oleaginosus DSM 11815 Saccharomyces cerevisiae BTc-3A Pichia membranifaciens DSM 21959 Mucor hiemalis DSM 2656 Aspergillus clavatus DSM 816 Hormoconis resinae DSM 1203 Penicillium capsulatum DSM 2210 Debaryomyces hansenii DSM 3428
1
2
>10
>10
3
4 and 5
n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i. n.i.
refb,c,d
33.3
8.3b 16.6b 8.3b 0.21b
