Sorazolons, Carbazole Alkaloids from Sorangium cellulosum Strain

Article pubs.acs.org/jnp

Sorazolons, Carbazole Alkaloids from Sorangium cellulosum Strain Soce375 Sabrina Karwehl,†,‡ Rolf Jansen,†,‡ Volker Huch,§ and Marc Stadler*,†,‡ †

Department of Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany § Department of Inorganic Chemistry, Saarland University, Building C 4.1, 66123 Saarbrücken, Germany ‡

S Supporting Information *

ABSTRACT: Sorazolons A (1) to E2 (9) were isolated from Sorangium cellulosum strain Soce375. Their molecular structures were elucidated using extensive HRESIMS and NMR analysis. The absolute configuration of sorazolon A (1) was determined by comparison of the experimental CD spectrum with quantum chemical calculated spectra for both enantiomers. Sorazolons D2 (7), E (8), and E2 (9) exhibit a moderate cytotoxic activity against mouse fibroblast cell line L929 with IC50 values between 5.0 μM and 0.09 mM.

Na]+ (m/z 268.09) and [M − H2O + H]+ (m/z 228.10) revealed the molecular formula C14H15NO3 with eight doublebond equivalents. The UV spectrum of 1 showed a maximum at 282 nm, indicating the presence of an aromatic ring. All protons and carbons were visible in the 1D NMR spectra in acetone-d6, and those directly connected to each other were correlated by a 1 13 H, C HMQC spectrum revealing the singlets at δH = 10.17, 4.55, and 4.35 ppm as NH and OH signals, respectively. 13 C and 13C DEPT NMR data indicated the presence of one carbonyl, two aromatic quaternary (C-2a and C-3a), two tertiary alcohol (C-8 and C-9), and two tertiary amine (C-6a and C-8a) carbons as well as four aromatic methine (C-3 to C6), one methylene (C-2), and two methyl (C-10 and C-11) carbons. The 1H,1H COSY NMR data showed the aromatic ring was ortho-disubstituted with a series of correlations from H-3 to H6 and compatible coupling constants in the 1H NMR spectrum. The aromatic ring was completed with carbons C-3a and C6a from correlations in the 1H,13C HMBC spectrum with metapositioned protons H-4 and H-6 or H-3 and H-5, respectively (Figure 2). Since the only correlation of the aromatic structure element was detected between H-3 and C-2a, the latter was positioned as a substituent at C-3a. Carbons C-2a and C-3a were correlated with the protons of methylene group C-2. These appeared as a well-separated AB system of two doublets (J = 19.8 Hz) in the 1H NMR spectrum and thus suggested they were part of another ring. Strong 1H,13C HMBC

M

yxobacteria are appreciated by natural-product chemists all over the world for their large reservoir of biologically active secondary metabolites.1,2 The most famous of these myxobacterial secondary metabolites are the fungicidal soraphens,3 the antibacterial sorangicins,4,5 and the antitumor agent ixabepilone, which is a derivative of epothilon B.6 The importance of myxobacteria is not justified only by these past achievements; even after 30 years of screening for biologically active metabolites from myxobacteria and other gliding bacteria, these sources still yield new biologically active secondary metabolites with unique scaffolds and modes of action. These include the highly active topoisomerase-inhibiting cystobactamides7 from Cystobacter sp., the antibacterial disciformycins8 from Pyxidicoccus fallax, and the antifungal pinensins, the first lantibiotics from the Gram-negative bacteria Chitinophaga pinensis.9 Given new screening methods, cultivation conditions, and isolation of organisms from previously unexplored habitats, the future perspective on finding novel secondary metabolites from myxobacteria is promising.10 In our ongoing metabolome screening11 for new antibiotics from myxobacteria, Sorangium cellulosum strain Soce375 was found to produce an entirely new family of metabolites, the sorazolons. Herein, we report the isolation and structure elucidation of sorazolons 1 to 9 using extensive HRESIMS, NMR, and CD spectroscopy and X-ray.

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RESULTS AND DISCUSSION The main compound of this family, sorazolon A (1), was obtained as white crystals by preparative RP-HPLC and preparative TLC with an overall yield of 30.5 mg/L. HRESIMS and isotopic pattern analysis of the molecular ion clusters [M + © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 9, 2015

A

DOI: 10.1021/acs.jnatprod.5b00997 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

comparison between calculated and measured CD spectra (Figure 3) proved the absolute configuration of sorazolon A (1).

Figure 1. Sorazolons isolated from Sorangium cellulosum Soce375.

Figure 3. Assignment of the absolute configuration of sorazolon A (1) by comparison of the experimental CD spectrum with the calculated spectra for R,S-sorazolon A (blue line) and S,R-sorazolon A (red line).

HRESIMS and isotopic pattern analysis of the molecular ion clusters [M + H]+ (m/z 489.22) and [2M + Na]+ (m/z 999.42) of sorazolon A2 (2) revealed the molecular formula C28H28N2O6 of a dimeric form of 1 with 16 double-bond equivalents. 1D and 2D NMR spectra of 2 displayed only the signals of one-half of the molecular structure, thus indicating a completely symmetric dimer with high similarity to sorazolon A (1). However, the methylene group C-2 of sorazolon A (1) was replaced by a methine appearing as a singlet at δH 5.28 ppm, while the signals of the aromatic protons H-3 and H-6, which were overlapping in sorazolon A (1), were well separated (Δδ = 0.29 ppm) in dimer 2 mainly due to a low-field shift of H-3 (Δδ = 0.39 ppm) induced by the anisotropy of the ketone double bond. Consequently, the dimer 2 contains two sorazolon A (1) residues, which are directly connected at C-2 with one-half of the molecule skewed against the other completely symmetrically. Since a 1H,1H ROESY signal was observed between H-2 and the methyl-group C-11, the absolute configuration of C-2 was determined as R. For sorazolon A3 (3) HRESIMS and isotopic pattern analysis of the molecular ion clusters [M + Na]+ (m/z 282.11) and [2M + Na]+ (m/z 541.14) revealed the molecular formula C15H17NO2 with eight double-bond equivalents. 1D and 2D NMR data showed a carbon skeleton similar to sorazolon A (1). The only difference was one additional methyl ether at δH/C 3.38/52.25 ppm in the 1H and 13C NMR spectra. The position of the methyl ether in 3 was indicated in the 1 13 H, C HMBC spectrum by a strong correlation signal of the methyl ether with C-8 and a weak signal with methyl group C10. From the HRESIMS and isotopic pattern analysis of the molecular ion clusters [M]+ (m/z 229.11), [M + H]+ (m/z 230.12), [M + Na]+ (m/z 252.10), and [M − H2O + H]+ (m/z 212.11) the molecular formula C14H15NO2 with eight doublebond equivalents was determined for sorazolon B (4). The

Figure 2. Selected correlations in the 2D NMR spectra of sorazolons A (1), A2 (2), and A3 (3) (bold lines 1H,1H COSY; blue arrows 1 13 H, C HMBC; red arrows 1H,1H ROESY) and crystal structure (the displayed numbering reflects the NMR description) of sorazolon A (1).

correlation of both methylene protons with carbons C-2a, C-8a, and carbonyl C-1 and of Hb-2 with tertiary alcohol carbon C-9 defined the next neighbors, while a weak correlation with the remaining tertiary alcohol carbon C-8 completed the ring (Figure 2). Both methyl groups C-10 and C-11 had 1H,13C HMBC correlations with C-8 and C-9; however, C-10 could be connected to C-8 due to a 1H,13C HMBC correlation with C8a, and C-11 similarly could be connected to C-9 due to a correlation with C-1. Further, the HMBC spectrum allowed assigning the OH signals at δH 4.55 and 4.35 ppm to positions 12 and 13 from their correlations with C-8a and methyl group C-10 as well as C-1 and methyl group C-11, respectively. Finally, the NH group remained filling the gap between C-6a and C-8a. This was supported by correlations in the 1H,1H ROESY spectrum between the NH proton, methine H-6, and methyl group C-10.12 The resulting structure, a tetrahydro carbazole derivative, is shown in Figure 2 with blue arrows indicating 1H,13C HMBC correlations and red arrows 1H,1H ROESY correlations. The result of an X-ray crystallography of 1 (Figure 2) showed that the two methyl groups were in a trans relative configuration. A B



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NMR data. The position of the methyl ether in 7 was indicated in the 1H,13C HMBC spectrum by a strong correlation signal of the methyl ether with C-8. The resulting structure of sorazolon D2 (7) is shown in Figure 1. For sorazolon E (8) HRESIMS and isotopic pattern analysis of the molecular ion clusters [M − MeOH + H]+ (m/z 180.08), [M]+ (m/z 211.10), [M + H]+ (m/z 212.11), and [2M + H]+ (m/z 421.19) provided the molecular formula C14H13NO with nine double-bond equivalents. 1D and 2D NMR spectra analysis furnished the same carbazole moiety as in 5. 1H and 13C NMR data showed the second aromatic ring was substituted by two methyl groups and one hydroxy group. The positions of the methyl groups were determined by strong 1 1 H, H ROESY correlations of H-7 with H-10 and H-10 with H11. The 1H,13C HMBC NMR spectrum showed a strong correlation between H-11 and C-1 connecting the hydroxy group to C-1. The resulting structure of sorazolon E (8) is shown in Figure 1. Eighteen double-bond equivalents for a dimeric sorazolon E2 (9) resulted from HRESIMS and isotopic pattern analysis of the molecular ion clusters [M]+ (m/z 420.18), [M + H]+ (m/z 421.19), [M + Na]+ (m/z 443.17), [M + K]+ (m/z 459.15), [2M + H]+ (m/z 841.37), [2M + Na]+ (m/z 863.36), and [2M + K]+ (m/z 879.33), corresponding to the molecular formula C28H24N2O2. Similar to dimer 2, 1D and 2D NMR spectra of 9 displayed signals of one-half of the structure, thus indicating a completely symmetrical dimer with high similarity to sorazolon E (8). However, the hydrogen at C-2 of 8 was replaced by a C− C bond between C-2 and C-2′ directly connecting the two sorazolon E (8) residues with one-half of the molecule skewed against the other, resulting in a completely symmetrical, axially chiral dimer. Noteworthy, the signals of the aromatic proton H3 and of the phenol OH group were shifted considerably to higher field in 9 when compared to 8 with shift differences of Δδ = 1.21 and 1.29 ppm, respectively. In a model (Figure 4)

difference in mass compared to 1 resulted from a missing oxygen. The NMR spectra of 4 displayed the same tetrahydro carbazole moiety as described for 1 with deviations in the substitution of the nonaromatic, six-membered ring. The 13C NMR data indicated the tertiary alcohol C-8 of 1 was replaced in 4 by a double bond with C-10, which was present as an exocyclic methylene group (δH 5.44 ppm, δC 103.9 ppm). Its position was indicated by strong correlation signals of CH2-10 with carbons C-8a and C-9 in the NMR data. Further, weaker 1 13 H, C HMBC correlations were present with C-8 (δC 145.6 ppm) and methyl group C-11. Instead of the carbonyl group in 1 a secondary alcohol (δH/C 3.90/75.1 ppm) was detected in 4. The H-1 proton showed vicinal couplings in the 1H NMR spectrum with methylene group (C-1), and the three protons appeared as doublets of doublets. Since strong 1H,1H ROESY signals were observed between H-1 and Hb-2 and between Hb-2 and H-11, respectively, the absolute configuration of C-1 was determined to be R. The resulting structure of sorazolon B (4) is shown in Figure 1. For the very lipophilic sorazolon C (5) HRESIMS and isotopic pattern analysis of the molecular ion clusters [M]+ (m/ z 209.08), [M + H]+ (m/z 210.09), [M + Na]+ (m/z 232.07), [M + K]+ (m/z 248.05), [2M + Na]+ (m/z 441.16), and [2M + K]+ (m/z 457.13) revealed the molecular formula C14H11NO with 10 double-bond equivalents. The NMR spectra showed the presence of the typical sorazolon indole moiety. Although the aliphatic ring was replaced by an aromatic ring, the sorazolons’ numbering scheme was retained. The new aromatic methine C-2 was assigned from a 1H,1H ROESY signal between H-3 and H-2, while a strong 1H,1H COSY correlation between H-2 and H-1 defined the next neighbor in the aromatic ring. The only methyl group CH3-11 was placed at C-9 based on a strong 1H,1H ROESY correlation between H-1 and H-11, while another 1H,1H ROESY correlation between the methyl group and H-10 connected the aldehyde group C-10 (δH/C 10.69/ 192.6 ppm) to C-8. The resulting structure of sorazolon C (5) is shown in Figure 1. For the dione sorazolon D (6) HRESIMS and isotopic pattern analysis of the molecular ion clusters [M + H]+ (m/z 260.09), [M + Na]+ (m/z 282.07), [M + K]+ (m/z 298.05), [M − H2O + H]+ (m/z 242.08), [2M + H]+ (m/z 519.18), [2M + Na]+ (m/z 541.16), and [2M + K]+ (m/z 557.13) revealed the molecular formula C 14 H 13 NO4 with nine double-bond equivalents. The difference in mass resulted from an additional oxygen and two missing hydrogen atoms. The NMR spectra confirmed the same tetrahydro carbazole moiety as described for 1; however, the methylene group C-2 was replaced by a second carbonyl group at δC 177.4 ppm. C-2 was indirectly assigned since the rest of the molecule could be determined from 1H,13C HMBC and 1H,1H ROESY correlations as described for 5, leaving only the carbonyl C-2 to complete the aliphatic six-membered ring. The resulting structure of sorazolon D (6) is shown in Figure 1. For sorazolon D2 (7) HRESIMS and isotopic pattern analysis of the molecular ion clusters [M − MeOH + H]+ (m/z 242.08), [M − H2O + H]+ (m/z 256.10), [M + H]+ (m/z 274.11), [M + Na]+ (m/z 296.09), [2M + H]+ (m/z 547.21), [M + K]+ (m/z 312.06), [2M + Na]+ (m/z 569.19), and [2M + K]+ (m/z 585.16) revealed the molecular formula C15H15NO4 with nine double-bond equivalents. 1D and 2D NMR data analysis showed the same tetrahydro carbazole moiety as in sorazolon D (6). The only difference was an additional methyl ether (δH/C 3.46/52.76 ppm), as shown by the 1H and 13C

Figure 4. Calculated 3D model of sorazolon E2 (9) (HyperChem, pm3 method).

calculated with the pm3 method, a semiempirical method to calculate 3D models of molecular structure, in HyperChem (Ver. 8.0.10), methine proton H-3 and the OH group are directly positioned above and under the substituted aromatic ring of the opposite halves, thus explaining the strong 1H deepfield shifts as an effect of the anisotropy of the aromatic rings. By analogy to the results attained with sorazolon A (1) we reason the absolute configurations of compounds 2, 3, 4, 6, and 7 at C-8 and C-9 are similar because the basic core of the sorazolons can be assumed to be synthesized from the same gene cluster and by the same basic biosynthetic pathway. The sorazolons are structurally related to carbazomycins A− D (10−13) from Streptoverticillium ehimensis strain H1051 MY C



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spectral analysis were performed with standard Bruker software and ACD/NMRSpectrus. 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: XBridge C18 column 100 × 2.1 mm (Waters), 3.5 μm, solvent A [H2O−acetonitrile (95/5), 5 mmol NH4Ac, 0.04 mL/L CH3COOH]; solvent B [H2O− acetonitrile (5/95), 5 mmol NH4Ac, 0.04 mL/L CH3COOH]; gradient system, 10% B increasing to 100% B in 30 min; flow rate 0.3 mL/min; 40 °C. CD spectra were recorded on a Jasco J-815 spectropolarimeter, and optical rotations on a PerkinElmer 241 MC polarimeter (using the sodium D line and a quartz cuvette with 10 cm path length and 1 mL volume). Cultivation of Sorangium cellulosum Soce375. The strain was isolated in 1989 from a soil sample from Nairobi, Kenya, and stored at −80 °C. It was reactivated in 20 mL of liquid medium consisting of 0.5% asparagine, 0.05% CaCl2, 0.05% MgSO4·7H2O, 10 mg/L Na− Fe−EDTA, 23.8 g/L HEPES, 0.1% glucose, and 1 mg/L ZnSO4· 7H2O. The culture was scaled up to 1 L and used as inoculum for a fermentation of strain Soce375 that was performed in the same medium as above but supplemented with 2% Amberlite XAD-16 resin in a 70 L bioreactor. The bioreactor was kept at 30 °C, aerated at 0.07 vvm per minute, and agitated with a flat blade turbine stirrer at 100 rpm for 250 h while the pH was regulated at 7.35−7.45. At the end of fermentation the XAD resin (1.48 kg) was recovered from the culture broth by sieving. Isolation and Purification of Sorazolons. The XAD adsorber resin was extracted in a glass column with 6 L each of methanol and acetone. The methanol extract was evaporated to an aqueous mixture, diluted with water, and extracted with ethyl acetate (four times with 400 mL). To improve the separation of the layers, 100 mL of saturated NaCl solution was added. The combined ethyl acetate extract was dried with Na2SO4. It was evaporated to give 46.3 g of crude extract. Isolation of Sorazolon A (1). Two grams of crude extract were separated by RP-HPLC [column 480 × 30 mm (Kronlab), ODS-AQ C18, 15 μm; solvent A: H2O−acetonitrile (95/5), 50 mmol NH4Ac; solvent B: H2O−acetonitrile (5/95), 50 mmol of NH4Ac; gradient system: 0% B increasing to 60% B in 60 min, holding at 60% B for 20 min; flow rate 30 mL/min, UV detection at 250 nm]. Two fractions with enriched sorazolon A (1) (722 mg) were obtained after evaporation of the organic solvent. A 21 mg amount of enriched 1 was dissolved in dichloromethane−methanol (8/2) and further purified by preparative TLC, yielding pure sorazolon A (1) (17.7 mg). Isolation of Sorazolons A3 (3), C (5), D2 (7), E (8), and E2 (9). The crude extract in amounts of 0.93 and 3.61 g was fractionated by Sephadex LH-20 chromatography in methanol. Two fractions containing a crude mixture of sorazolons A3 (3), C (5), D2 (7), E (8), and E2 (9) (3.07 g) were obtained after evaporation of the organic solvent. The sorazolons were further purified by Si-flash chromatography [column: Reveleris Silica 120 g; solvent A: dichloromethane; solvent B: acetone; gradient system: holding at 0% B for 5 min, increasing to 15% B in 40 min, increasing to 100% B in 30 min; flow rate 64 mL/min, UV detection at 254 nm, 280 and 360 nm] to give pure sorazolon C (5) (15.86 mg), one fraction (28.23 mg) containing sorazolons D2 (7), E (8), and E2 (9), and another fraction containing crude sorazolon A3 (3) (65.92 mg). The fraction containing sorazolons D2 (7), E (8), and E2 (9) was dissolved in petroleum ether−ethyl acetate (2/1) and further purified by preparative TLC, yielding pure sorazolons D2 (7) (1.21 mg), E (8) (7.82 mg), and E2 (9) (6.67 mg). The fraction containing crude sorazolon A3 (3) was dissolved in petroleum ether−ethyl acetate (2/ 1) and further purified by preparative TLC, yielding pure sorazolon A3 (3) (55.17 mg). Isolation of Sorazolon A2 (2). The crude extract in amounts of 0.93, 3.61, and 4.22 g was separated by LH-20 chromatography. Three fractions containing crude sorazolon A2 (2) were obtained after

10 and Streptomyces ehimensis strain JB201, carbazomycin dimers (14 and 15) from Streptomyces sp. strain BCC26924,13 pimprinine (16) from Streptomyces pimprina, Streptoverticillium griseocrameum, and Streptoverticillium olivoreticuli,14 and compound 17 from Streptomyces ehimensis.15 Carbazomycins B (11) and C (12) were active against malaria, and carbazomycins C (12) and D (13), the dimer 15, and pimprinine (16) showed antituberculosis activity. Furthermore, carbazomycin B (11) and (1R,2R,3R)-3-hydroxy-1,2-dimethyl-1,2,3,9-tetrahydro-4Hcarbazol-4-one (17) were active against Candida albicans and carbazomycin A (10) was active against cancer cell lines (KB, MCF-7, NCI-H187) and noncancer cells (Vero).13

Figure 5. Molecular structures of carbazomycins A−D (10−13), carbazomycin dimers (14 and 15), pimprinine (16), and (1S,2S,3S)-3hydroxy-1,2-dimethyl-1,2,3,9-tetrahydro-4H-carbazol-4-one (17).

Some synthetic carbazole derivatives such as 6-methoxy1,2,3,4-tetrahydrocarbazol (18) and 1,2,3,4-tetrahydrocarbazol (19) prohibit hemolysis and protect DNA against oxidative stress.16

Figure 6. Molecular structures of 6-methoxy-2,3,4,9-tetrahydro-1Hcarbazole (18) and 2,3,4,9-tetrahydro-1H-carbazole (19).

Comparing the sorazolons to the most similar compound 10, 11, 17, or 19 shows their potential for biological activity. To date, the sorazolons were tested against a set of microorganisms, revealing moderate activities of 7, 8, and 9 against B. subtilis, S. aureus, M. luteus, and E. coli with MIC values of 8.3− 33.3 μg/mL. Furthermore, these three sorazolons showed moderate cytotoxic activities against the mouse fibroblast cell line L929 with IC50 values of 76.8, 85.2, and 4.99 μM, respectively (see Table 2). Consequently, our future aim is to study the sorazolons in as many activity assays as possible to access their potential activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on a Büchi-510 melting point apparatus; UV data were recorded on a Shimadzu UV/vis-2450 spectrophotometer in methanol (UVASOL, Merck); IR data were recorded on a Bruker Tensor 27 IR spectrophotometer. 1H NMR and 13C NMR spectra were recorded on Bruker Ascend 700 and Ascend 500 NMR spectrometers, locked to the deuterium signal of the solvent. Data acquisition, processing, and D



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Table 1. NMR Data of Sorazolons A (1) to E2 (9) in Acetone-d6 1a position 1 2a

δC, mult

34.6, CH2

a1

3b

δH, mult (J in Hz)

δC, mult 211.2, C

3.64, d (19.6)

δC, mult

4b

δH, mult (J in Hz)

210.2, C

δC, mult 75.1, CH

45.7, CH

5.28, s

34.2, CH2

105.9, C 119.5, CH

7.82, m

104.1, C 119.3, CH

126.8, C 120.2, CH

7.06, t (7.4)

127.2, C 120.0, CH

122.2, CH

7.15, m

122.7, CH

113.0, CH

7.53, d (8.0)

112.4, CH

3.75, d (3.8)

28.4, CH2

3.88, d (19.8)

2a 3

102.4, C 119.1, CH

3a 4

127.4, C 120.0, CH

5

122.5, CH

6

112.5, CH

6a 7 8 8a 9 10 11 12 13

137.7, C

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

δH, mult (J in Hz)

211.9, C

2b

position

2a

7.43, m

7.03, ddd (8.0, 7.1, 1.1) 7.12, ddd (8.2, 7.1, 1.5) 7.44, m

137.9, C

C C C CH3 CH3

δC, mult

1.38, 1.41, 4.55, 4.35, 6b

s s br s s

C CH C CH CH CH C

75.5, 159.1, 86.2, 28.1, 21.7,

C C C CH3 CH3

1.70, 1.38, 4.55, 4.11,

79.9, 137.9, 82.5, 19.2, 52.3, 20.4,

s s s s

δH, mult (J in Hz)

δC, mult

C C C CH3 CH3 CH3

7.31, dt (7.3, 1.4) 7.35, dt (7.5, 1.4) 7.62, dt (8.1, 0.9)

113.5, 122.5, 125.4, 124.1, 125.6, 113.5, 137.4,

C CH C CH CH CH C

79.6, 156.1, 85.0, 19.5, 20.3, 52.8,

C C C CH3 CH3 CH3

11.69, br s

1.63, 1.42, 4.91, 5.61,

s s br s br s

δH, mult (J in Hz)

H/13C at 700.4/176.1 MHz.

b1

111.9, CH

3.90, br dd (10.0, 5.9) 2.58, dd (15.7, 10.2) 3.16, dd (15.6, 5.7)

122.7, CH

7.46, ddd (7.8, 2.0, 1.1)

1.51, 3.38, 1.41, 4.60,

s s s s

127.6, CH

122.9, C 120.8, CH

8.12, m

124.1, C 121.0, CH

6.99, ddd (7.9, 7.1, 0.9) 7.11, ddd (8.2, 7.1, 1.1) 7.31, dt (8.1, 0.8)

C C C CH2 CH3

δC, mult

8.15, ddd (7.6, 1.6, 1.0) 7.32, m 7.35, dt (7.3, 1.5) 7.59, m

121.3, 120.5, 124.5, 118.9, 125.5, 111.7, 141.4,

C CH C CH CH CH C

120.0, 135.2, 122.7, 14.1, 12.5,

C C C CH3 CH3

10.03, s

127.0, CH 112.9, CH

5.44, 1.29, 3.93, 4.01,

δH, mult (J in Hz) 7.38, s

7.89, dt (7.8, 0.9) 7.05, m 7.27, ddd (8.2, 7.0, 1.1) 7.41, m

2.48, s 2.32, m 7.79, br s

7.25, ddd (7.9, 7.1, 1.0) 7.43, ddd (8.2, 7.1, 1.1) 7.77, dt (8.2, 0.8)

141.6, C 11.18, br s 118.4, 141.0, 140.0, 192.6, 18.6,

d (1.7) s br s br s

C C C CH CH3

10.69, s 2.89, s

9b δC, mult

δH, mult (J in Hz)

148.0, C 111.8, C 123.2, 122.1, 124.7, 118.7, 125.2, 111.3, 141.5,

C CH C CH CH CH C

120.4, 135.6, 120.2, 14.4, 13.2,

C C C CH3 CH3

9.85, s

s s br s s

δH, mult (J in Hz) 7.13, br dd (7.8, 0.6) 8.30, d (7.8)

10.19, br s 145.6, 133.4, 75.9, 103.9, 21.7,

150.1, C 102.9, CH

1.74, 1.46, 3.46, 5.12,

123.4, CH

δC, mult

8b

199.7, C 177.5, C

8.13, dq (7.8, 1.0)

128.3, C 119.9, CH

10.20, br s

7b

201.9, C 177.4, C 113.7, 122.4, 125.4, 124.0, 125.5, 113.5, 137.7,

C C C CH3 CH3

110.3, C 119.5, CH

δH, mult (J in Hz)

138.9, C

10.34, br s 76.3, 140.0, 83.4, 25.9, 22.6,

7.04, ddd (7.9, 7.0, 1.1) 7.13, ddd (8.2, 7.0, 1.1) 7.42, m

137.6, C

10.20, br s 75.8, 140.1, 83.2, 26.6, 21.5,

7.45, dq (7.9, 0.9)

5b

6.64, m 6.53, ddd (8.0, 7.1, 1.0) 7.06, ddd (8.1, 7.0, 1.2) 7.34, m 10.04, s

2.66, s 2.45, s 6.50, s

H/13C at 500.3/125.8 MHz. in 85 min, holding at 100% B for 30 min; flow rate 30 mL/min, UV detection at 220 nm] to give one fraction containing sorazolon B (4). This fraction was further purified by RP-HPLC [column 125 × 40 mm, Nucleodur C18, 7 μm; solvent A: H2O, 0.5% acetic acid; solvent B: acetonitrile, 0.5% acetic acid; gradient system: 25% B increasing to 40% B in 90 min, increasing to 100% B in 1 min, holding at 100% B for 30 min; flow rate 20 mL/min, UV detection at 254 nm, 210 nm], yielding pure sorazolon B (4) (32.00 mg). Isolation of Sorazolon D (6). A 6.41 g amount of crude extract was separated by Si-flash chromatography [column: Reveleris Silica 120 g; solvent A: dichloromethane; solvent B: acetone; gradient: holding 5% B for 8 min, increasing to 15% B in 8 min, holding at 15% B for 5 min, increasing to 30% B in 5 min, holding at 30% B for 5 min, increasing to 100% B in 0.5 min, holding at 100% B for 15 min; flow rate 30 mL/ min, UV detection at 250 nm]. The fraction containing sorazolon D

evaporation of the organic solvent. 2 was further purified by RP-MPLC [column: Kronlab ODS-AQ C18, 480 × 30 mm, 15 μm; solvent A: H2O−methanol (8/2); solvent B: methanol; gradient: 10% B increasing to 80% B in 260 min, increasing to 100% B in 1 min, holding at 100% B for 60 min; flow rate 30 mL/min, UV detection at 210 nm]. The fraction containing crude dimer 2 was dissolved in dichloromethane−methanol (9/1) and used for further purification by preparative TLC, yielding pure sorazolon A2 (2) (1.13 mg). Isolation of Sorazolon B (4). The crude extract in amounts of 3.61 and 4.22 g was separated by LH-20 chromatography. Six fractions containing crude sorazolon B (4) (316.78 mg) were obtained after evaporation of the organic solvent. Sorazolon B (4) was further purified by RP-MPLC [column 480 × 30 mm (Kronlab), ODS-AQ C18, 15 μm; solvent A: H2O−methanol (8/2); solvent B: methanol; gradient: 10% B increasing to 60% B in 260 min, increasing to 100% B E



Article

Table 2. MIC [in μg/mL] and IC50 [in μM] Values of Sorazolons A (1) to E, Dimer 9, and Methanol As Negative Control Gram +

Gram −

yeasts fungi cytotoxicity a

Staphylococcus aureus Newmanb Micrococcus luteusc DSM1790 Mycobacterium smegmatisd ATCC700084 Bacillus subtilise DSM10 Escherichia colib DSM1116 Escherichia coli TolCb Pseudomonas aeruginosa PA14b Wickerhamomyces anomalaf DSM6766 Candida albicansf DSM1665 Mucor hiemalisf DSM2656 L929g

1

2

3

4

5

6

7

8

9

MeOH

n.i.a 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.





16.6 16.6 n.i. 8.3 n.i. n.i. n.i. n.i. n.i. n.i. 76.8

33.3 33.3 n.i. 16.6 n.i. 67 n.i. n.i. n.i. n.i. 85.2

16.6 16.6 n.i. 16.6 n.i. 33.3 n.i. n.i. n.i. 67 4.99


n.i. = no inhibition. bGentamycin. cAmpicillin. dKanamycin. eOxytetracyclin hydrochloride. fNystatin. gEpothilone A.

(6) (63.14 mg) was further purified by RP-HPLC [column 250 × 21 mm, Nucleodur C18, 10 μm; solvent A: H2O, 0.5% formic acid; solvent B: acetonitrile, 0.5% formic acid; gradient: 20% B increasing to 35% B in 50 min, increasing to 100% B in 1 min, holding at 100% B for 10 min; flow rate 20 mL/min, UV detection at 220 nm, 254 nm, 300 nm, 360 nm], yielding pure sorazolon D (6) (17.37 mg). Sorazolon A (1): C14H15NO3, M = 245.28; colorless crystal; mp 89 °C; [α]25D = +133 (c 5.4 mg/mL, MeOH); analytical HPLC system A tR = 9.0 min; UV (MeOH) λmax (log ε) 220 (4.53), 282 (2.90), 290 (3.82) nm; IR (KBr) νmax 3408 (br, s), 3055 (w), 2980 (w), 2932 (w), 2868 (w), 1718 (s), 1645 (w), 1454 (m), 1410 (w), 1369 (m), 1334 (m), 1228 (w), 1176 (s), 1120 (m), 1102 (s), 1068 (m), 1048 (m), 954 (m), 901 (w), 833 (w), 745 (s), 702 (w), 514 (w) cm−1; CD (MeOH) Δε204 13.10, Δε206 13.10, Δε208 12.15, Δε219 −14.23, Δε222 −3.87, Δε224 −17.11, Δε260 18.46, Δε285 4.89, Δε293 8.24, Δε318 0.83; NMR data, see Table 1; HRESIMS m/z 228.1015 [M − H2O + H]+ (calcd for C14H14NO2+, 228.0995), m/z 268.0944 [M + Na]+ (calcd for C14H15NaNO3+, 268.0944). Sorazolon A2 (2): C28H28N2O6, M = 488.54; [α]25D = +23 (c 10 mg/mL, MeOH); analytical HPLC system tR = 12.21 min; UV (MeOH) λmax (log ε) 203 (4.50), 223 (4.70), 284 (4.05), 290 (4.00) nm; NMR data, see Table 1; HRESIMS m/z 489.2219 [M + H]+ (calcd for C28H29N2O6+, 489.1947), m/z 999.4207 [2M + Na]+ (calcd for C56H56NaN4O12+, 999.3792). Sorazolon A3 (3): C15H17NO3, M = 259.31; analytical HPLC system A tR = 13.5 min; UV (MeOH) λmax (log ε) 217 (4.36), 272 (3.85), 278 (3.84), 346 (3.34) nm; NMR data, see Table 1; HRESIMS m/z 282.110 441 [M + Na]+ (calcd for C15H17NaNO3+, 282.110 064), m/z 541.142 606 [2M + Na]+ (calcd for C30H34 NaN2O6+, 541.231 46). Sorazolon B (4): C14H15NO2, M = 229.28; [α]25D = +34 (c 10.7 mg/mL, MeOH); analytical HPLC system tR = 8.96 min; UV (MeOH) λmax (log ε) 205 (4.26), 307 (4.17) nm; NMR data, see Table 1; HRESIMS m/z 212.106 695 [M − H2O + H]+ (calcd for C14H14NO+, 212.106 990), m/z 229.109264 [M]+ (calcd for C14H15NO2+, 229.109730), m/z 230.117 074 [M + H]+ (calcd for C14H16NO2+, 230.117 555), m/z 252.099 205 [M + Na]+ (calcd for C14H15NaNO2+, 252.099 499). Sorazolon C (5): C14H11NO, M = 209.25; analytical HPLC system tR = 18.35 min; UV (MeOH) λmax (log ε) 294 (4.10), 335 (3.50), 386 (3.81) nm; NMR data, see Table 1; HRESIMS m/z 209.083 196 [M]+ (calcd for C14H11NO+, 209.083 515), m/z 210.091 605 [M + H]+ (calcd for C14H12NO+, 210.091 340), m/z 232.073 361 [M + Na]+ (calcd for C14H11NaNO+, 232.073 285), m/z 248.048 434 [M + K]+ (calcd for C14H11KNO+, 248.047 222), m/z 441.155 804 [2M + Na]+ (calcd for C28H22NaN2O2+, 441.157 349), m/z 457.129 431 [2M + K]+ (calcd for C28H22KN2O2+, 457.131 286). Sorazolon D (6): C14H13NO4, M = 259.26; [α]25D = +240 (c 12.8 mg/mL, MeOH); analytical HPLC system tR = 8.53 min; UV (MeOH) λmax (log ε) 209 (4.44), 245 (9105.61/3.96), 270 (4.01), 278 (3.96), 314 (3.69), 346 (3.73), 361 (3.71) nm; NMR data, see Table 1; HRESIMS m/z 242.081 439 [M − H2O + H]+ (calcd for

C14H12NO3+, 242.081 170), m/z 260.092 439 [M + H]+ (calcd for C14H14NO4+, 260.091 734), m/z 282.073 475 [M + Na]+ (calcd for C14H13NaNO4+, 282.073 679), m/z 298.047 430 [M + K]+ (calcd for C14H13KNO4+, 298.047 616), m/z 519.175 947 [2M + H]+ (calcd for C28H27N2O8+, 519.176 192), m/z 541.158 368 [2M + Na]+ (calcd for C28H26NaN2O8+, 541.158 137), m/z 557.129 252 [2M + K]+ (calcd for C28H26KN2O8+, 557.132 074). Sorazolon D2 (7): C15H15NO4, M = 273.28; [α]25D = +41 (c 1.2 mg/mL, MeOH); analytical HPLC system tR = 9.62 min; UV (MeOH) λmax (log ε) 208 (4.31), 243 (3.81), 271 (3.92), 278 (3.86), 301 (3.50), 347 (3.65) nm; NMR data, see Table 1; HRESIMS m/z 242.081 686 [M − MeOH + H] + (calcd for C 14 H 12 NO 3 + , 242.081 170), m/z 256.096 856 [M − H2O + H]+ (calcd for C15H14NO3+, 256.096 820), m/z 274.107 464 [M + H]+ (calcd for C15H16NO4+, 274.107 384), m/z 296.089 446 [M + Na]+ (calcd for C15H15NaNO4+, 296.089 329), m/z 312.063 470 [M + K]+ (calcd for C15H15KNO4+, 312.063 266), m/z 547.208 989 [2M + H]+ (calcd for C30H31N2O8+, 547.207 492), m/z 569.189 801 [2M + Na]+ (calcd for C30H30NaN2O8+, 569.189 437), m/z 585.162 715 [2M + K]+ (calcd for C30H30KN2O8+, 585.163 374). Sorazolon E (8): C14H13NO, M = 211.26; analytical HPLC system tR = 14.22 min; UV (MeOH) λmax (log ε) 217 (4.45), 233 (4.36), 253 (4.12), 265 (4.02), 302 (4.11), 341 (3.48) nm; NMR data, see Table 1; HRESIMS m/z 211.098 217 [M]+ (calcd for C14H13NO+, 211.099 165), m/z 212.107 951 [M + H]+ (calcd for C14H14NO+, 212.106 990). Sorazolon E2 (9): C28H24N2O2, M = 420.51; analytical HPLC system tR = 19.64 min; UV (MeOH) λmax (log ε) 221 (4.92), 234 (4.90), 237 (4.90), 302 (4.64), 354 (4.15) nm; NMR data, see Table 1; HRESIMS m/z 420.181 550 [M]+ (calcd for C28H24N2O2+, 420.183 229), m/z 421.191 157 [M + H]+ (calcd for C28H25N2O2+, 421.191 054), m/z 443.173 206 [M + Na] + (calcd for C28H24NaN2O2+, 443.172 999), m/z 459.146 925 [M + K]+ (calcd for C28H24KN2O2+, 459.146 936), m/z 841.374 336 [2M + H]+ (calcd for C56H49N4O4+, 841.374 832), m/z 863.356 644 [2M + Na]+ (calcd for C56H48N4NaO4+, 863.356 777), m/z 879.330 083 [2M + K]+ (calcd for C56H48KN4O4+, 879.330 714). X-ray Crystallographic Analysis of 1. A colorless flat crystal of 1 was obtained via very slow vapor diffusion with petroleum ether as precipitant and diethyl ether as solvent. The data were collected at 152 K on a BrukerAXS X8Apex CCD diffractometer operating with graphite-monochromated Mo Kα radiation. Frames of 0.5° oscillation were exposed, deriving data in the θ range of 1.372° to 26.371° with a completeness of 97.6%. Structure solution and full least-squares refinement with anisotropic thermal parameters of all non-hydrogen atoms and free refinement of the hydrogen were performed using SHELX.17 Crystallographic data of 1: C14H15NO3, M = 245.27 g/mol, monoclinic, a = 17.851(3) Å, b = 15.920(3) Å, c = 18.784(3) Å, V = 5063.7(16) Å3, space group I2, Z = 16, Dcalc = 1.287 mg/m3, 23 818 reflections measured in the range 1.37° ≤ θ ≤ 26.37°, 8543 independent reflections, Rint = 0.0753, 666 parameters, 637 restraints. F



Article

The final refinement resulted in R1 = 0.113, wR2 = 0.284 (I > 2σ(I)). The goodness of fit on F2 was 1.069. Antimicrobial Testing. Twenty microliter aliquots (concentration 1 mg/mL) of compounds 1 to 9 were tested against four different Gram-positive (Staphylococcus aureus, Micrococcus luteus, Mycobacterium smegmatis, Bacillus subtilis) and three Gram-negative (Escherichia coli, Escherichia coli TolC, Pseudomonas aeruginosa) bacteria and three yeasts (Wickerhamomyces anomala, Candida albicans, Mucor hiemalis) with methanol as negative control (see Table 2). The MIC values were determined by serial dilution (150 μL) in 96-well plates for tissue cultures (TPP). In addition a total of 3.0 μL of each sorazolon was tested against eukaryotic cells (mouse fibroblast cell line; L929). MIC values were determined by serial dilution (60 μL) in 96-well plates for tissue cultures (Falcon).18 Computational Details. A conformational analysis of 1 was done with B3LYP-D3/def2-TZVP19−21 in combination with the chain-ofspheres approximation22 utilizing the software package ORCA,23,24 and only one relevantly populated conformation was found. The coupled-cluster method SCS-RICC2/def2-SVP21,25 was performed with Turbomole.26,27 For the evaluation of the CD and UV spectra calculations (length formalism) and for the comparisons of the computed curves with the experimental measurements SpecDis28 was used. A σ value of 0.3 eV and a UV shift29 of 37 nm have been applied.

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(9) Mohr, K. I.; Jansen, R.; Wray, V.; Hoffmann, J.; Bernecker, S.; Wink, J.; Gerth, K.; Stadler, M.; Müller, R. Angew. Chem., Int. Ed. 2015, 54, 11254−11258. (10) Müller, R.; Wink. Int. J. Med. Microbiol. 2014, 304, 3−13. (11) Krug, D.; Müller, R. Nat. Prod. Rep. 2014, 31, 768−783. (12) Probably due to the low intensity of the broad NH signal, no correlations were observed in the 1H,13C HMBC spectrum. (13) Intaraudom, C.; Rachtawee, P.; Suvannakad, R.; Pittayakhajonwut, P. Tetrahedron 2011, 67, 7593−7597. (14) Wei, Y.; Fang, W.; Wan, Z.; Wang, K.; Yang, Q.; Cai, X.; Shi, L.; Yang, Z. Virol. J. 2014, 11, 195−209. (15) Hwang, B. S.; Kim, H.-J.; Jeong, G. S.; Oh, J. S.; Rho, J.-R. Bull. Korean Chem. Soc. 2010, 31, 3457−3459. (16) Zhao, F.; Liu, Z.-Q. J. Biochem. Mol. Toxicol. 2009, 23, 273−9. (17) The crystallographic data of sorazolon A (1) were deposited in the Cambridge Crystallographic Data Centre with the deposition number CCDC 1421680. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). (18) Steinmetz, H.; Mohr, K. I.; Zander, W.; Jansen, R.; Gerth, K.; Müller, R. J. Nat. Prod. 2012, 75, 1803−1805. (19) Grimme, S.; Ehrlich, S.; Goerigk, L. J. Comput. Chem. 2011, 32, 1456−1465. (20) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. J. Chem. Phys. 2010, 132, 154104−154119. (21) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005, 7, 3297−3305. (22) Petrenko, T.; Kossmann, S.; Neese, F. J. Chem. Phys. 2011, 134, 054116−14. (23) Neese, F.; Wenmohs, F.; Becker, U.; Bykov, D.; Ganyushin, D.; Hansen, A.; Izsák, R.; Liakos, D. G.; Kollmar, C.; Kossmann, S.; Pantazis, D. A.; Petrenko, T.; Reimann, C.; Riplinger, C.; Roemelt, M.; Sandhöfer, B.; Schapiro, I.; Sivalingam, K.; Wezisla, B. ORCA−an ab initio, density functional and semiempirical program package, version 3.0.3; Max Planck Institute for Chemical Energy Conversion: Germany, 2014. (24) Neese, F. WIREs Comput. Mol. Sci. 2012, 2, 73−78. (25) Hellweg, A.; Grun, S. A.; Hattig, C. Phys. Chem. Chem. Phys. 2008, 10, 4119−4127. (26) Ahlrichs, R.; Armbruster, M. K.; Bachorz, R. A.; Bär, M.; Baron, H.-P.; Bauernschmitt, R.; Bischoff, F. A.; Böcker, S.; Crawford, N.; Deglmann, P.; Della Sala, F.; Diedenhofen, M.; Ehrig, M.; Eichkorn, K.; Elliott, S.; Friese, D.; Furche, F.; Glöß, A.; Haase, F.; Häser, M.; Hättig, C.; Hellweg, A.; Höfener, S.; Horn, H.; Huber, C.; Huniar, U.; Kattanek, M.; Klopper, W.; Köhn, A.; Kölmel, C.; Kollwitz, M.; May, K.; Nava, P.; Ochsenfeld, C.; Ö hm, H.; Pabst, M.; Patzelt, H.; Rappoport, D.; Rubner, O.; Schäfer, A.; Schneider, U.; Sierka, M.; Tew, D. P.; Treutler, O.; Unterreiner, B.; von Arnim, M.; Weigend, F.; Weis, P.; Weiss, H.; Winter, N. TURBOMOLE, version 6.6; TURBOMOLE GmbH: Karlsruhe, Germany, 2014. (27) Furche, F.; Ahlrichs, R.; Hättig, C.; Klopper, W.; Sierka, M.; Weigend, F. WIREs Comput. Mol. Sci. 2014, 4, 91−100. (28) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249. (29) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2009, 2717−2727.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00997. Tables of 1D and 2D NMR data and figures of all NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-531-61814240. Fax: +49-531-61819499. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. T. Bruhn for the calculated CD spectra, W. Collisi and K. Schober for excellent technical assistance, C. Kakoschke for recording the NMR spectra, A. Gollasch for the HRESIMS measurements, and S. Bernecker and co-workers for large-scale cultivation.

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REFERENCES

(1) Bode, H. B.; Müller, R. Angew. Chem., Int. Ed. 2005, 44, 6828− 6846. (2) Weissman, K. J.; Müller, R. Bioorg. Med. Chem. 2009, 17, 2121− 2136. (3) Gerth, K.; Bedorf, N.; Irschik, H.; Höfle, G.; Reichenbach, H. J. Antibiot. (Tokyo) 1986, 47, 23−31. (4) Irschik, H.; Jansen, R.; Gerth, K.; Höfle, G.; Reichenbach, H. J. Antibiot. 1987, 40, 7−13. (5) Campbell, E. A.; Pavlova, O.; Zenkin, N.; Leon, F.; Irschik, H.; Jansen, R.; Severinov, K.; Darst, S. A. EMBO J. 2005, 24, 674−682. (6) Gerth, K.; Bedorf, N.; Höfle, G.; Irschik, H.; Reichenbach, H. J. Antibiot. 1996, 49, 560−563. (7) Baumann, S.; Herrmann, J.; Raju, R.; Steinmetz, H.; Mohr, K. I.; Hüttel, S.; Hamrolfs, K.; Stadler, M.; Müller, R. Angew. Chem. 2014, 126, 14835−14839. (8) Surup, F.; Viehrig, K.; Mohr, K. I.; Herrmann, J.; Jansen, R.; Müller, R. Angew. Chem., Int. Ed. 2014, 53, 13588−13591. G


Sorazolons, Carbazole Alkaloids from Sorangium cellulosum Strain

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