Nannozinones and Sorazinones, Unprecedented Pyrazinones from

Article pubs.acs.org/jnp

Nannozinones and Sorazinones, Unprecedented Pyrazinones from Myxobacteria Rolf Jansen,†,‡ Sakshi Sood,†,‡ Kathrin I. Mohr,†,‡ Brigitte Kunze,‡,§ Herbert Irschik,†,‡ Marc Stadler,†,‡ and Rolf Müller*,†,‡,⊥ †

Department of Microbial Drugs, §Research Group Microbial Communication, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany ‡ German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany ⊥ Helmholtz Institute for Pharmaceutical Research and Department of Pharmaceutical Biotechnology, Saarland University, P.O. Box 15115, 66041 Saarbrücken, Germany S Supporting Information *

ABSTRACT: Nannozinones A (1) and B (2) were discovered as metabolites of the recently isolated Nannocystis pusilla strain MNa10913 belonging to the poorly studied myxobacterial family Nannocystaceae. In contrast, the structurally related sorazinones A (5) and B (6) were isolated from Sorangium cellulosum strain Soce895, which was known as the producer of the antibiotic thuggacin A. The extract also contained methyl indole-3-carboxylate (4). HRESIMS and 1H, 13C, and 15N NMR spectroscopy revealed the structures of nannozinones A (1) and B (2) as unusual dihydropyrrolo- and pyrrolopyrazinone derivatives, while sorazinone A (5) was characterized as an aromatic diketopiperazine and sorazinone B (6) as a dibenzyl 2(1H)-pyrazinone derivative. While the dihydropyrrolo derivative nannozinone A (1) showed weak antibacterial and antifungal activity, nannozinone B (2) inhibited the growth of cell cultures with IC50 values between 2.44 and 16.9 μM. The nannochelin A iron complex (3), which was isolated besides 1 and 2, was even more active, with IC50 values between 0.05 and 1.95 μM. On the other hand, the indole 4 and sorazinones 5 and 6 did not show any significant cytotoxicity and only weak activity against the Gram-positive Nocardia sp.

M

were identified in the extracts from strain MNa10913 by RPHPLC-UV-HRESIMS, and the remarkable cytotoxic activity of nannochelin A (3) as an iron complex was revealed. Strain MNa10913, an isolate from a Majorcan soil sample (Spain), was cultivated in 10 L of a protein-containing medium in the presence of 1% Amberlite XAD-16 adsorber resin at 30 °C. After 7 days the resin was recovered by sieving, and bound compounds were eluted with methanol. The crude extract was subjected to partitioning between methanol and n-heptane to eliminate lipophilic byproducts to give 0.6 g of an enriched crude extract, which was fractionated by RP-MPLC to give three fractions. These were purified by preparative RP-HPLC using ammonium acetate-buffered aqueous methanol gradients to yield pure nannozinones A (1) (3 mg) and B (2) (2 mg) as well as nannochelin A as an iron complex (3) (3 mg). The elemental formula C15H16N2O of nannozinone A (1) was suggested by the molecular ion cluster [M + H]+ at m/z 241.1326 in the high-resolution ESIMS (HRESIMS). All signals of the 16 protons were correlated in the 1H,13C HSQC-DEPT spectrum in CDCl3 with their corresponding carbon atoms (Table 1). The 1H,1H COSY spectrum showed the presence of a phenyl ring connected by a long-range correlation with a

yxobacteria are an intriguing group of bacteria with unique features such as gliding on solid surfaces and formation of complex fruiting bodies. In addition, their ability to produce a variety of natural products reflects their rich secondary metabolism that has established Myxobacteria as valuable source of potential drugs and drug leads. These in turn frequently display novel modes of action.1 Further advances in this research area rely largely on isolation of new and phylogenetically diverse strains or genera that might contribute to the pipeline of novel therapeutic molecules.2 The genus Nannocystis belongs to the family Nannocystaceae within Myxococcales and is closely related to marine myxobacteria such as Enhygromyxa, Pseudenhygromyxa, and Plesiocystis. Compared to other genera such as Sorangium and Myxococcus, there have been limited investigations on the secondary metabolite potential of Nannocystis spp. They have been reported to produce a variety of compounds such as phenylnannolones,3 germacran,4 geosmin,5 and the siderophores of the nannochelin type.6 Most recently, we described phenazine derivatives and the pyrronazols, a new class of secondary metabolites, from Nannocystis sp.7 Herein, we report two novel secondary metabolites, the cytotoxic nannozinones A (1) and B (2), from a recently isolated N. pusilla, strain MNa10913. Their structures were elucidated by HRESIMS and comprehensive 1H, 13C, and 15N NMR data analysis. Additionally, nannochelins A (3) and B © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 7, 2014

A

dx.doi.org/10.1021/np500632c | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Selected 2D NMR correlations and numbering of nannozinone A (1) (bold bonds: 1H,1H COSY-derived structural parts; green arrows: 1H,13C HMBC correlations; blue arrows: 1H,15N HMBC; red lines: 1H,1H ROESY).

The 1H,13C HSQC NMR spectrum of nannozinone B (2) in DMSO-d6 (Table 2) provided the correlations of 13 protons with their respective carbon atoms, thus leaving one heteroatom-bound proton to be connected, which was visible as a broad singlet signal at δ 11.07 in the 1H NMR spectrum. Similar to 1 the 1H,1H COSY NMR spectrum revealed the presence of a phenyl ring; however, in 2 it was connected to only one methylene group, C-10, having a conspicuous 1H NMR shift of δ 3.97. A second structural element derived from the COSY NMR spectrum was composed of three unsaturated methines (δH 6.62 to 7.71). According to the direct coupling constant 1JHC 192 Hz of methine C-2, they were part of a heterocyclic ring. The latter was recognized as a five-membered pyrrole from the small vicinal coupling constants between 2.8 and 4.1 Hz. Additionally, the 1H NMR spectrum presented only a singlet of a methyl group at δH 2.51, which was connected to a carbonyl carbon, C-17 (δC 193.82), by a correlation in the 1H,13C HMBC NMR spectrum to give an acetyl residue. As presented in Figure 2, the remaining structure elements were connected to give a pyrazinone ring based on unequivocal 1H,13C HMBC correlations. Again, the 1H,15N HMBC spectrum strongly supported the assignment in 2 with three correlations within the pyrrole and the only correlation of N-7 to the methylene protons of C-10. Finally, the only free position at N-7 was filled with the heteroatom-bound proton (δH 11.07). The observation of nannozinones in an extract of Nannocystis sp. inspired us to analyze additional myxobacteria for the presence of similar small aromatic metabolites. Sorangiineae

short chain of two aliphatic methylene groups (C-10 and C11), which appeared as typical triplets (J10,11 = 7.3 Hz) in the 1 H NMR spectrum. Further, the COSY spectrum presented a set of three correlated methylene groups (C-2 to C-3) in a ring, which were observed as a double doublet, quintet, and triplet in the 1H NMR spectrum, respectively, as well as a singlet of an unsaturated methine, C-8 (δH 8.06, δC 145.03), with a direct coupling of JH,C 189 Hz, suggesting a heterocyclic conjugated sp2 methine. All three structural parts were connected with the remaining three quaternary carbon and two nitrogen atoms following the correlations indicated in the 1H,13C HMBC and the especially useful correlations in the 1H,15N HMBC spectra (Table 1) to unambiguously provide the dihydropyrrolopyrazinone core of nannozinone A (1) as illustrated in Figure 1. The position of the phenylethyl side chain was also suggested by the nuclear Overhauser effect (NOE) between methylene groups C-10 and C-4 observed in the 1H,1H ROESY spectrum. A second congener, nannozinone B (2), appeared at 11.4 min in our RP HPLC system with a characteristic UV spectrum of three bands at 208, 259, and 288 nm. Compound 2 was assigned to have the elemental formula C16H14N2O2 from the molecular ion clusters [M + H]+ and [2 M + Na]+ at m/z 267.1118 and 555.1994 in the HRESIMS, respectively. Table 1. NMR Data of Nannozinone A (1) in CDCl3

a1

pos.

δC (δN)a

m

1 2 3 4 5 6 7 8 9 10 11 12 13, 17 14, 16 15

(186.1) 48.79 20.92 29.32 139.40 130.27 (343.4) 145.03 155.50 34.94 35.16 141.12 128.66 128.37 126.11

N CH2 CH2 CH2 C C N CH C CH2 CH2 C CH CH CH

δH

m (J [Hz])

ROESY

4.06 2.01 2.58

dd (8.0, 6.8) quin (7.6) t (7.7)

3>4 2 10 >11

8.06

s br.

2.81 2.96

t (7.3) t (7.3)

4, 13/17 13/17 > 4

7.07 7.25 7.20

dd br (7.1, 1.3) t br (7.1) tt (7.1, 1.5)

11, 10

H in HMBC 8, 3, 4, 2 3, 4 > 8 4, 2 3, 2 4, 10 > 3, 2 > 8 10, 11 > 4, 8 8, 10 >4 (JC,H 189 Hz) 8≫2 11 10, 13/17 10, 11, 14/16 11, 15, 17/13 16/14 13/17

H, 13C, and 15N: 700.44, 176.14, and 70.99 MHz; for numbering see Figure 1. B



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Table 2. NMR Data of Nannozinone B (2) in DMSO-d6

a1

pos.

δC (δN)a

m

1 2 3 4 5 6 7 8 9 10 11 12, 16 13, 15 14 17 18

(176.0) 120.89 112.32 109.81 123.20 155.36 (147.3) 131.47 118.42 34.54 137.62 128.14 128.53 126.60 193.82 30.69

N CH CH CH C C N C C CH2 C CH CH CH C CH3

δH

m (J [Hz])

7.71 6.62 6.99

dd (2.8, 1.5) dd (3.9, 2.8) dd (4.1, 1.5)

11.07

NOESY 18, 3 4, 2 3

br s

3.97

s

12/16 > 18

7.28 7.32 7.23

br d (6.7) br t (7.1) tt (7.3, 1.3)

10 > 18

2.51

s

10, 2 > 12/16

H in HMBCb 2, 4, 3 3, 4 (192 Hz) 2 > 4 (173 Hz) 2 > 3 (177 Hz) 2 > 3, 4 10 > 2 10 10 10 > 18 > 2 12/16 10, 13/15 10, 14, 13/15 15/13 12/16 18 ≫ 10

H, 13C, and 15N: 700.44, 176.14, and 70.99 MHz; for numbering see Figure 2. b(1JH,C).

Figure 2. Selected 2D NMR correlations and numbering of nannozinone B (2) (bold bonds: 1H,1H COSY-derived structural parts; green arrows: 1H,13C HMBC correlations; blue arrows: 1H,15N HMBC; red lines: 1H,1H ROESY).

NMR data in CDCl3, which agreed with reported data.12 In extracts and fractions, indole 4 can easily be recognized by analytical TLC detected with vanillin-sulfuric acid spray reagent because it develops an intense bright orange color upon heating to 120 °C. The second UV peak of the RP-MPLC was purified by preparative TLC. The elemental formula C11H8N2O2 was assigned to sorazinone A (5) from the [M + H]+ and [M + Na]+ ion clusters m/z 201.0666 and 223.0485, respectively, observed in the HRESIMS. The high number of nine ring or double-bond equivalents of the small molecule was reflected in the band-rich UV spectrum with absorptions at 235, 275, 308, and 319 nm. In the 1H NMR spectrum all eight protons were present as one aromatic methyl group (δH 2.45) and five aromatic methine signals between δH 7.75 and 6.44 ppm (Table 3). They were correlated to their respective carbon atoms in the 1 13 H, C HSQC NMR spectrum. This left five quaternary carbon atoms between 152.4 and 121.0 ppm. Similar to nannozinone B (2), the 1H,1H COSY spectrum indicated the presence of a three-proton spin system of a pyrrole ring with vicinal coupling constants between 3.1 and 3.7 Hz. Additionally, a two-proton aromatic spin system was observed with a similar small coupling constant (J 3.2 Hz). A weak correlation of a long-range coupling connected these protons with the aromatic methyl group. The carbon skeleton of 5 resulted from a detailed analysis of the correlations in the 1H,13C HMBC NMR spectrum. The most significant correlations are presented in Figure 3. Furthermore, the positions of the two nitrogen atoms were

have been established as a prolific source of diverse structures and biological activities, such as the antibiotic sorangicin A,8 the antifungal soraphen A,9 and the antitumor lead compound epothilon A.10 Sorangium cellulosum strain Soce895 was found to produce the macrolides thuggacins,11 active as antibiotics against Mycobacterium tuberculosis. Some side fractions of the isolation procedure of thuggacins had been tagged as containing putative aromatic compounds and were examined in detail recently. For thuggacin production, S. cellulosum strain Soce895 was cultivated in the presence of Amberlite XAD 16 adsorbing resin. Since the strain additionally produces ambruticins as byproducts, the isolation procedure includes a liquid−liquid extraction with 1% sodium carbonate to furnish an ambruticinfree raw product for chromatographic purification by preparative RP medium-pressure liquid chromatography (RP MPLC). The early eluting UV peaks of unknown byproducts of the RP-MPLC were collected and found to contain aromatic compounds. The first fraction was identified as methyl indole3-carboxylate (4). The second fraction was purified further by preparative thin-layer chromatography (TLC) to give sorazinone A (5), and the third fraction provided a first portion of 3 mg of sorazinone B (6) as fine needles from acetone and further material. By HPLC-UV-HRESIMS compound 4 provided the elemental formula C10H9NO2. It was assigned as methyl indole-3-carboxylate from the NMR data in acetone-d6 (Table S3 and Figure S17). The assignment was supported by the C



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Table 3. NMR Data of Sorazinone A (5) in Acetone-d6 pos.

δC (δN)a

type

1 2 3 4 5 6 7 8 9 10 11 12 13

151.5 125.5 122.6 115.2 123.9 (192.7) 152.4 139.4 121.0 118.0 123.4 (189.7) 12.9

C C CH CH CH N C C C CH CH N CH3

δHa

m (J [Hz])

ROESY

7.31 6.56 7.75

dd (3.7, 1.5) t (3.4) dd (3.2, 1.6)

4 3, 5 4

6.44 7.66

d (3.2) d (3.2)

11, 13 10

2.49

s

10

H in HMBC >3 4, 3, 5 5 3, 5 4>3 4 > 3, 5 13 > 11, 5 13 > 10, 11 13, 10, 11 13 > 11 13, 10 10 > 11 >10

Figure 4. Selected 2D NMR correlations and numbering of sorazinone B (6) (bold bonds: 1H,1H COSY-derived structural parts; green arrows: 1H,13C HMBC correlations; blue arrows: 1H,15N HMBC; red lines: 1H,1H ROESY).

NMR spectrum did not provide a link between both molecule parts. Since two nitrogen atoms from the elemental formula remained, this connection was expected to be revealed by a 1 15 H, N HMBC NMR spectrum. However, only one nitrogen atom, N-4 at δN 344.2, showed correlations with the protons of methylene group C-7 and methyl group C-14 and was thus inserted as a bridge between C-3 and C-5. Finally, the remaining oxygen atom was placed at C-2 in order to account for the chemical shift δC 156.6, and the last nitrogen and the amine proton at δH 11.06 were used to close the pyrazinone ring, thus consuming the last ring equivalent. The signal broadening of the quaternary carbons C-3, C-5, and C-6, which are not visible in the 13C NMR spectrum while their correlations appear in the inverse-detected 1H,13C HMBC spectrum, indicated a slow dynamic process of the pyrazinone ring.13 A similar finding was reported for dragmacidin E (11), a heptacyclic pyrazinone-containing metabolite of a deep-water marine sponge, Spongosorites sp. In the case of 11 a ROESY correlation with an exchangeable OH proton was observed, indicating the existence of a pyrazine tautomer in an equilibrium.14 The dynamic tautomerism also accounts for the missing correlation signals of N-1 in the 1H,15N HMBC spectrum of 6. Due to the coupling with the amide proton, this signal a priori was expected as a doublet with half signal intensity. Nannozinones A (1) and B (2) and the nannochelin A−iron complex (3) were analyzed for their antimicrobial activity against bacteria, yeasts, and filamentous fungi. All three compounds showed very weak biological activity. While 1 was active against a number of Gram-positive bacteria such as Nocardia sp., Staphylococcus aureus, and Mycobacterium diernhoferi, 2 was active only against Nocardia sp., and 3 showed no antibacterial activity. In addition, 1 and 3 showed some antifungal activity with a MIC value of 33.3 μg/mL (Table 5). When 1−6 were tested for their cytotoxicity against growing primary and cancer cell lines, 1 did not show any significant activity, while 2 and 3 were remarkably active against most cell lines (Table 6). The strongest cytotoxicity for 2 was observed against human ovarian carcinoma cell line SKOV-3 and for 3 against cervix carcinoma cell line KB3-1 and HUVEC with IC50 values as low as 0.05 μM. Although antimicrobial activity of nannochelins has been described previously,6 this is the first report of the high cytotoxic activity of nannochelin A as an iron complex (3). Methyl indole-3-carboxylate (4) and sorazinones A (5) and B (6) on the other hand did not show any significant cytotoxicity and only weak activity against the Gram-positive Nocardia sp. Using the pyrazinone cores of nannozinones A (1) and B (2) as partial structures, a search in the Dictionary of Natural Products (DNP) revealed that they belong to a rare compound

a1

H, 13C, and 15N: 700.44, 176.14, and 70.99 MHz; for numbering see Figure 3.

Figure 3. Selected 2D NMR correlations and numbering of sorazinone A (5) (bold bonds: 1H,1H COSY-derived structural parts; green arrows: 1H,13C HMBC correlations; blue arrows: 1H,15N HMBC; red line: 1H,1H ROESY).

independently indicated by their correlations in the 1H,15N HMBC NMR spectrum (Figure 3). The structure elucidation of sorazinone A (5) was completed by the observation of the only possible nonvicinal 1H,1H ROESY correlation between the methyl group C-13 and methine H-10. RP-MPLC fraction 3 yielded 3 mg of sorazinone B (6). Compound 6 always crystallized as very fine needles from acetone and various other solvents. Sorazinone B (6) showed [M + H]+ and [M + Na]+ ion clusters in the HRESIMS at m/z 291.1492 and 313.1314, respectively, which were used to calculate the elemental formula C19H18N2O and 12 ring or double-bond equivalents. The 1H NMR spectrum presented signals of a broad NH proton at δH 11.06 ppm, 10 aromatic protons of two phenyl groups between δH 7.35 and 7.15 ppm, two singlets of methylene groups (δH 3.99 and 3.94), and an unsaturated methyl group (δH 2.26). Besides the direct correlations within both phenyl groups, the 1H,1H COSY NMR spectrum revealed only weak long-range correlations connecting the methylene groups with their respective phenyl residues to give two benzyl groups and a further long-range correlation indicating the vicinity of the methyl group C-14 and methylene group C-15. The 13C NMR spectrum showed signals of only 16 of the 19 carbon atoms, and those bearing protons were easily assigned using the 1H,13C HSQC-DEPT NMR spectrum. However, at this stage three quaternary carbon atoms of the elemental formula remained hidden. C-3 was detected at δC 154.7 in the 1 13 H, C HMBC NMR spectrum due to a correlation with methylene group C-7, while the 13C shift of C-5 and C-6 was revealed at δC 130.5 and 136.1, respectively, from their HMBC correlations to methylene group C-15 and methyl group C-14 (Figure 4). Although thoroughly examined, the 1H,13C HMBC D



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unsaturated ring. These were isolated from the Caribbean sponges Agelas longissima and A. dispar, respectively.17 Compound 8 showed moderate antibacterial activity.

Table 4. NMR Data of Sorazinone B (6) in Acetone-d6 pos.

δC(N)a

type

1 1a 2 3 4 5 6 7 8 9, 13 10, 12 11 14 15 16 19 20, 18 21, 17

(130.1)c

N H C Cb N Cb Cb CH2 C CH CH CH CH3 CH2 C CH CH CH

a1

13

156.6 154.7 (344.2) 130.5 136.1 39.7 139.6 130.1 129.1 127.0 19.1 37.0 138.4 127.6 129.6 129.4

δH

m (J [Hz])

NOESY

H in HMBC (1a)

11.06

br s

3.99

s

9/13

7.35 7.24 7.15 2.26 3.94

d (7.3) m m s s

7

15, 17/21 14, 17/21

7.22 7.29 7.26

m m m

15, 14

7 7 7, 14 14, 15 14, 15 9/13 7, 10/12 7, 11 9/13 17/21 15, 18/20 17/21 19 15, 18/20

15

H, C, and N: 700.44, 176.14, and 70.99 MHz; for numbering see Figure 4. b13C shift from HMBC. cCalculated value; not visible in the 1 15 H, N HMBC spectrum due to signal broadening and direct coupling to an NH proton.

class. The similarity search provided only one closely related hit with unsaturated pyrazine and pyrrole rings comparable to 2, i.e., the insect deterrent and antifeedant peramine (7) that was isolated from perennial ryegrass herbage and seed, Lolium perenne, infected with the symbiont Acremonium lolii,15 an endophytic fungus, which was renamed Neotyphodium lolii in 1996.16

Previously the Sorangium metabolite 4, methyl indole-3carboxylate, had been isolated from red algae,18 marine-derived bacteria,12 and the sponge Spongosorites sp.19 and was isolated from fungi as a weakly active cytotoxin.20 As most closely related to 4 the well-known phytohormone indole-3-acetic acid might be considered, which also was reported to act as a signaling molecule in bacteria,21 or indole, a common bacterial decomposition product but also an exogenous effector of antibiotic resistance of Pseudomonas putida.22 The DNP search indicated the desmethyl derivative of sorazinone A (5), pyrocoll (10), which is a moderate antibiotic, antiparasitic, and antitumor compound isolated from the alkaliphilic Streptomyces strain AK 40923 and from the mangrove endophytic fungus Xylaria sp. (#2508).24 Pyrocoll (10) also had been isolated from cigarette smoke.25 However,

Further, the similarity search indicated the dibromopyrrole metabolites longamides A (8) and B (9), featuring only one

Table 5. Antimicrobial Minimum Inhibitory Concentrations (MIC) of Compounds 1−6 MIC (μg/mL) test organisms/DSM number

d

Nocardia sp./43069 Staphylococcus aureus/346 Mycobacterium diernhoferi/43542 Paenibacillus polymyxa/36 Escherichia coli/1116 Chromobacterium violaceum/30191 Pseudomonas aeruginosa/50071 Saccharomyces cerevisiae/70449 Candida albicans/1665 Wickerhamomyces anomalus/6766 Trichosporon oleaginous/11815 Mucor hiemalis/2656 a

1

2

3

4

5

6

ref

66.6 66.6 33.3 66.6 66.6 66.6 66.6 66.6 33.3 >66.6 66.6 33.3

66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6

>66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 33.3 >66.6 66.6 >66.6

33.3 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6

16.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 66.6 >66.6 >66.6

67.0 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6 >66.6

41.5 >41.5 >41.5 >41.5 >41.5 >41.5

>37.6 15.5 8.45 16.9 5.3 14.1 8.7 2.4

1.82 0.05 0.10 0.19 0.05 0.22 0.12 1.95

>56.8

>50.0

>34.5

0.0077

>56.8 >56.8 >56.8 >56.8 >56.8

>50.0 >50.0 >50.0 >50.0 >50.0

>34.5 >34.5 >34.5 >34.5 >34.5

0.0024 0.0096 0.0022 0.0013

Epothilone A.

10 was named pyrocoll in 1811 due to its first characterization after dry distillation of animal glue (gelatin).26 While 5 is a diketopiperazine derivative, sorazinone B (6) contains a rare trisubstituted 2(1H)-pyrazinone moiety. Other trisubstituted metabolites include the highly conjugated heteroaromates dragmacidin E (11), a metabolite of a Spongosorites sp.,14 maremycin E (12), which was found in a terrestrial Streptomyces sp. (strain GT 051237)27 by chemical screening, and JBIR-56 (13), featuring a peptide side chain, which was isolated from a marine sponge-derived Streptomyces sp. (SpD081030SC-03).28 From the sponge Suberea sp. disubstituted 2(1H)-pyrazinone derivatives with less conjugation, the ma’edamines, such as 14, were isolated as cytotoxic metabolites.29

formation under starvation conditions for the preservation of spores. These processes require intracellular and extracellular intraspecies signaling, for example, by excretion of signaling molecules such as 8-hydroxy-2,5,8-trimethyl-4-nonanone (stigmolone) by Stigmatella aurantiaca38 or the family of yellow DKxanthenes by Myxococcus xanthus,39 or by transient fusion recently described as outer-membrane exchange,40 a process involving the transfer of proteins and lipids. Compounds 1, 2, 4, 5, and 6 do not exert antimicrobial effects that may be beneficial for the producers as observed with antibiotics such as thuggacin A. However, they are more easily and economically biosynthesized than larger signaling peptides, and thus they may also have a function in the very complex myxobacterial intra- and extraspecies cell−cell interaction.

Interestingly, the most related disubstituted 2(1H)-pyrazinone derivatives are tyrvalin (aureusimine A) (15) and phevalin (aureusimine B) (16), which were isolated as putative virulence factors from a pathogenic Staphylococcus aureus strain.30 Their corresponding nonribosomal peptide synthetase (NRPS), which also produces leuvalin (17), was found in pathogenic strains of S. aureus, S. epidermidis, S. capitis, and S. lugdunensis31 but not in other staphylococci.32 From the domains present, the NRPS was predicted as producing a dipeptide with a Cterminal aldehyde, which cyclizes with the N-terminal amino group upon release and spontaneously undergoes oxidation to give the 2(1H)-pyrazinones. In the case of sorazinone B (6) the final NRPS product could be a methyl ketone or a β-keto acid intermediate for cyclization, followed by oxidation or oxidation and decarboxylation, respectively. Although participation in the virulence was excluded for aureusimines,33 their overexpression in Staph. aureus biofilms34 suggests they might play a role in intra- or interspecies signaling, because previous to its detection in Staph. aureus, aureusimine B (16) had been isolated as phevalin (16) from a Streptomyces sp. in the course of a screening for calpain (cysteine protease) inhibitors in 1995.35 Further, two biologically inactive 2(1H)-pyrazinones, arglecin (18)36 and argvalin (19),37 were detected in several diverse Streptomyces sp. strains by chemical screening. Nannocystis pusilla and Sorangium cellulosum represent two distinct species of Myxobacteria, which as a common survival strategy developed swarming, predation, and fruiting body

General Experimental Procedures. UV data were recorded on a Shimadzu UV/vis-2450 spectrophotometer in methanol (Uvasol, Merck); 1H, 13C, and 15N NMR spectra were recorded on Bruker AVANCE III HD 700 MHz with Cryo Platform or Bruker Avance III 500 MHz spectrometers, locked to the deuterium signal of the solvent. Data acquisition, processing, and spectral analysis were performed with standard Bruker software and ACD/Spectrus Processor (ACD/Labs, Toronto, Canada). Chemical shifts are given in parts per million (ppm), and coupling constants in hertz (Hz). HRESIMS data were recorded on a Maxis ESI TOF mass spectrometer (Bruker Daltonics), and molecular formulas were calculated including the isotopic pattern (Smart Formula algorithm). Analytical RP HPLC was carried out with an Agilent 1260 HPLC system equipped with a diode-array UV detector and a Corona Ultra detector (Dionex) or a Maxis ESI TOF mass spectrometer (Bruker Daltonics). HPLC conditions: Waters Acquity C18 column 50 × 2 mm, 1.7 μm; solvent A: H2O, 0.1% HCOOH; solvent B: acetonitrile, 0.1% HCOOH; gradient system: 5% B for 1 min, increasing to 95% B in 20 min; flow rate 0.6 mL/min; 40 °C. Myxobacterial Strain. Nannocystis pusilla strain MNa10913 was isolated in 2012 from a soil sample that was collected from Majorca, Spain, in April 2011. Strain MNa10913 showed the highest similarity (99.9%) to the type strain of Nannocystis pusilla (DSM 14622T) in the 16S rRNA gene sequence analysis. The sequence of strain MNa10913 was deposited at the National Center for Biotechnology Information (NCBI) under the number KP057225. Cultivation of MNa10913. The strain had been stored at −80 °C. It was reactivated in 100 mL of CY/H medium consisting of 0.15% casitone, 0.15% yeast extract, 0.1% soymeal extract, 0.1% glucose, 0.4% starch (Cerestar), 0.05% MgSO4 × 7H2O, 0.1% CaCl2 × 2H2O, 50 mM HEPES, and 4 mg/L Fe-EDTA at pH 7.3. The strain was then subcultured into 400 mL of P-medium [0.2% peptone (Marcor), 0.8% starch, 0.4% probion (single-cell protein, Hoechst), 0.2% yeast extract, 0.1% CaCl2 × 2H2O, 0.1% MgSO4 × 7H2O, 100 mM HEPES, 8 mg/L Fe-EDTA] supplemented with 400 μL of vitamin solution and used as a preculture. Ten liters of the same medium in flasks supplemented with 1% Amberlite XAD-16 adsorber resin (Rohm & Haas) was inoculated with the preculture and incubated at 30 °C under stirring

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with 160 rpm for 7 days. The XAD resin was harvested from the culture broth at the end of fermentation by sieving. Extraction and Isolation of Nannozinones. The adsorber resin XAD-16 was eluted in a glass column with 600 mL of methanol, and the eluate was evaporated to an aqueous mixture. It was diluted with water and extracted three times with dichloromethane. The organic layer was evaporated to yield 841 mg of extract. This extract was dissolved in methanol and subjected to solvent−solvent partitioning between methanol and n-heptane to eliminate lipophilic compounds. The partitioning was repeated three times to give 636 mg of an enriched crude methanol extract. This extract was dissolved in methanol (15 mL) and filtered through Whatman filter paper to collect the undissolved residue. The residue on the filter paper was again washed with dichloromethane. Filtered extract was used as a sample for RP-MPLC chromatography [column 480 × 30 mm (Kronlab), ODS-AQ C18 (YMC), S 16 μm; solvent A: H2O− methanol, 1:1; solvent B: methanol; gradient: 20% B to 60% B in 90 min; flow rate 30 mL/min; UV detection at 300 nm]. Methanol from each fraction collected was evaporated, and the remaining aqueous solution was extracted with ethyl acetate to obtain three compounds in three fractions. The fraction containing 3 was dissolved in methanol and purified subsequently with preparative RP-HPLC [column 250 × 21 mm, Nucleodur 100−10 C18 EC, S 10 μm; solvent A: H2O−methanol (1:1), 50 mmol of NH4Ac, 0.04 mL of CH3COOH; solvent B: methanol, 50 mmol of NH4Ac, 0.04 mL of CH3COOH; gradient: 10% B to 30% B in 33 min; flow rate 20 mL/min; UV detection at 310 nm]. Methanol was evaporated from the collected fraction, and the remaining aqueous layer was extracted with ethyl acetate. The ethyl acetate was also evaporated to yield 3 mg of nannochelin A iron complex (3). Fractions containing 2 and 3 were also purified subsequently by preparative RP-HPLC chromatography [column 250 × 21 mm, Nucleodur 100−10 C18 EC, S 10 μm; H2O−methanol (1:1), 50 mmol of NH4Ac, 0.04 mL of CH3COOH; solvent B: methanol, 50 mmol of NH4Ac, 0.04 mL of CH3COOH; gradient: 0% B to 10% B in 35 min; flow rate 20 mL/min; UV detection at 350 and 210 nm]. Methanol was evaporated from fractions obtained from both runs, and aqueous layers were extracted with ethyl acetate to finally yield pure nannozinone B (2) (3 mg) and nannozinone A (1) (2 mg). Nannozinone A (1): 1-(2-phenylethyl)-7,8-dihydropyrrolo[1,2-a]pyrazin-4(6H)-one, C15H16N2O, M = 240.30; HPLC system A tR 7.6 min; UV (MeOH) λmax (log ε) 232 (4.019), 333 (3.743) nm; NMR data, see Table 1; HRESIMS m/z 241.1326 [M + H]+ (calcd for C15H17N2O, 241.1335), m/z 503.2426 [2 M + Na]+ (calcd for C30H32N4O2 + Na, 503.2417). Nannozinone B (2): 4-acetyl-3-benzylpyrrolo[1,2-a]pyrazin-1(2H)one, C16H14N2O2, M = 266.29; HPLC system A tR 8.2 min; UV (MeOH) λmax (log ε) 208 (4.208), 224 (sh), 259 (4.008), 288 (3.752), 298, 332 (sh) nm; NMR data, see Table 2; HRESIMS m/z 267.1118 [M + H]+ (calcd for C16H15N2O2, 267.1128), m/z 555.1994 [2 M + Na]+ (calcd for C32H28N4O4Na, 555.2003). Extraction and Isolation of Sorazinones. Amberlite XAD-16 (0.5 kg) was recovered from 70 L of fermentation broth of Sorangium cellulosum strain Soce895 by sieving. Residual cell material was removed by floating with water before the XAD was eluted in a column with 50% methanol (3 column volumes) followed by 100% acetone (3 column volumes). In order to stabilize thuggacin A, 1 mL of acetic acid was added to the acetone eluate and the solvent was evaporated. The remaining water−oil mixture was extracted with dichloromethane to give 8 g of raw extract, which was subjected to methanol−heptane partition. Thus, 2.4 g of lipophilic byproducts was removed with the heptane layer. The remaining material (5.9 g) were dissolved in ethyl acetate (150 mL), cooled to 0 °C in an ice bath, and extracted with three portions of cold 1% sodium carbonate solution (each about 120 mL). After evaporation the organic layer provided 2.4 g of thuggacin-enriched raw extract, while the ethyl acetate extract of the acidified water layers contained 0.94 g of raw ambruticins. The thuggacin fraction was separated in two portions by RP-MPLC [column 460 × 30 mm, ODS-AQ 120 Å, 16 μ (YMC); solvent

gradient 65% to 80% methanol with 0.1% acetic acid for 3 h, 80% methanol with 0.1% acetic acid for 1 h; detection UV at 226 nm; flow 30 mL/min]. Besides the expected thuggacin-containing fractions the early eluting fractions of unknown UV peaks showed signals of aromatic metabolites in the 1H NMR spectra.11 Fraction 1 (tR ∼18 min; 19 mg) was identified as methyl indolecarboxylate (4) by HPLC-HRESI-UV-MS and NMR spectroscopy. Fraction 2 (tR ∼31 min; 18 mg) was purified further by preparative PSC [KG 60 F254 (Merk), 20 × 20 cm; solvent petroleum benzine−ethyl acetate, 6:1] to give sorazinone A (5) (7 mg). Fraction 3 (tR ∼36.5 min; 30 mg) provided 3 mg of crystalline (fine needles) sorazinone B (6) from acetone. Methyl 1H-indole-3-carboxylate (4): C10H9NO2, M = 175.18; HPLC system A tR 7.4 min; UV (MeOH) λmax (log ε) 211 (4.481), 225, 246, 272 (sh), 280 (3.936) nm (acidic: 214, 228, 245 (sh), 282 nm); 1H NMR (700 MHz, CDCl3) δ ppm 3.94 (s, 3 H) 7.27−7.31 (m, 2 H) 7.41−7.45 (m, 1 H) 7.94 (d, J = 2.8 Hz, 1 H) 8.18−8.22 (m, 1 H) 8.72 (br s, 1 H); 13C NMR (CDCl3) δ ppm 165.7, 136.0, 131.0, 125.8, 123.2, 122.1, 121.5, 111.5, 108.8, 51.1; HRESIMS m/z 176.0716 [M + H]+ (calcd for C10H10NO2, 176.0706), m/z 198.0532 [M + Na]+ (calcd for C10H9NO2 + Na, 198.0525). Since we observed small deviations between our 1H NMR data in CDCl3 and those given by Zheng et al.,11 which possibly arise due to different pH of the sample, we also measured a complete NMR data set in acetone-d6 and analyzed the 2D NMR correlations (Table S3 and Figure S17). Sorazinone A (5): 1-methyl-5H,10H-dipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-dione, C11H8N2O2, M = 200.19; HPLC system A tR 8.7 min; UV (MeOH) λmax (log ε) 235 (4.389), 275 (4.185), 308 (4.116), 319 (4.148) nm; NMR data see Table 3; HRESIMS m/z 201.0666 [M + H]+ (calcd for C11H9N2O2, 201.0658), m/z 223.0485 [M + Na]+ (calcd for C11H8N2O2 + Na, 223.0478). Sorazinone B (6): 3,6-dibenzyl-5-methylpyrazin-2(1H)-one, C19H18N2O, M = 290.36; mp 178 °C; HPLC system A tR 10.4 min; UV (MeOH) λmax (log ε) 202 (4.343), 231 (3.957), 338 (3.966) nm; NMR data see Table 4; HRESIMS m/z 291.1492 [M + H]+ (calcd for C19H19N2O, 291.1492), m/z 313.1314 [M + Na]+ (calcd for C19H18N2O + Na, 313.1311). Antimicrobial Testing. Aliquots of 2 and 20 μL (conc 1 mg/mL) of compounds 1−6 and reference drugs were tested against five bacteria, four yeasts, and three filamentous fungi (Table 6). The MIC values were determined in 96-well microtiter plates by 1:1 serial dilution in 0.15 mL EBS medium (0.5% casein peptone, 0.5% protease peptone, 0.1% meat extract, 0.1% yeast extract, pH 7.0) for bacteria and 0.15 mL MYC medium (1.0% glucose, 1.0% phytone peptone, 50 mM HEPES [11.9 g/L], pH 7.0) for yeasts and filamentous fungi, as previously described.7 The test organisms were cultivated under shaking at 160 rpm at 30 °C for 24−48 h. The lowest concentration of the drug preventing visible growth of the pathogen was taken as the MIC. Cytotoxicity Assay. In vitro cytotoxicity (IC50) was determined for compounds 1−6 against a number of mammalian cell lines (Table 5) including the mouse fibroblast cell line L929, the cervix carcinoma cell line KB-3-1, nontransformed human umbilical vein endothelial cell line HUVEC, epidermoid carcinoma cell line A-431, prostatic adenocarcinoma cell line PC-3, breast cancer cell line MCF-7, human ovarian carcinoma cell line SKOV-3, and adenocarcinomic human alveolar basal epithelial cell line A549. Lines KB-3-1, A431, A549, and L929 were cultured in DMEM (Lonza), PC-3 was cultured in F12K (Gibco), MCF-7 was cultured in RPMI (Gibco), HUVECs were cultured in EBM-2 (Lonza), and SKOV-3 was cultured in McCoy’s (Lonza) media, all supplemented with 10% of fetal bovine serum (Gibco) and incubated under 10% CO2 at 37 °C. The MTT (2(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method was used for the cytotoxicity assay on 96-well microplates41 as described previously.7 Methanol was used as the negative and epothilone A as positive control. G



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ASSOCIATED CONTENT

S Supporting Information *

Tables of complete 1D and 2D NMR data and figures of all NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-681-30270201. Fax: +49-681-30270202. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.S. is highly indebted to Erasmus Mundus External Cooperation Window for a Ph.D. scholarship and all of their support. We thank C. Kakoschke for measuring the NMR spectra, W. Kessler and his team for large-scale fermentation, and K. Schober, A. Teichmann, D. Telkemeyer, and W. Collisi for technical assistance.

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Nannozinones and Sorazinones, Unprecedented Pyrazinones from

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