Isolation and Characterization of Unusual Hydrazides from

Article pubs.acs.org/jnp

Isolation and Characterization of Unusual Hydrazides from Streptomyces sp. Impact of the Cultivation Support and Extraction Procedure Géraldine Le Goff, Marie-Thérèse Martin, Bogdan I. Iorga, Emilie Adelin, Claudine Servy, Sylvie Cortial, and Jamal Ouazzani* Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette, France S Supporting Information *

ABSTRACT: Three novel hydrazides, geralcins C−E (1−3), were isolated from Streptomyces sp. LMA-545, together with MH-031 and geralcins A and B. This unusual family of compounds was isolated from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. The use of such neutral resin during the cultivation step allowed the specific adsorption of microbial secondary metabolites, avoiding any contamination of the crude extracts by the constituents of the culture medium. The trapped compounds were eluted from the resin with methanol, and their structures elucidated using 1H, 13C, and 15N NMR spectroscopic analysis and high-resolution mass spectrometry. Molecular modeling calculations were applied in order to support structural attributions. No antimicrobial, cytotoxic, or DnaG-inhibition activities were detected for geralcins D and E. Geralcin C has no antimicrobial activity but exhibited an IC50 of 0.8 μM against KB and HCT116 cancer cell lines. Furthermore, geralcin C inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC50 of 0.7 mM.

R

Antimicrobial, cytotoxic, and primase-inhibition activities were evaluated for the newly isolated compounds.

ecently, a new scaffold of natural compounds was reported consisting of unusual alkyl hydrazides.1−4 Major representatives of this family are montamine isolated from the seeds of Centaurea montana (Asteraceae),1 hydrazidomycins A, B, and C, also called elaiomycins, isolated from Streptomyces species,2,3 and recently geralcins A and B isolated from Streptomyces sp. LMA-545.4 The biological role of these compounds is still unknown, while cytotoxic activities against tumor cell lines were reported for montamine1 (IC50 of 43.9 μM), hydrazidomycins2 (IC50 of 0.37 μM), and geralcin B4 (IC50 of 5 μM). Although natural hydrazides are notably scarce, they are often found in synthetic therapeutics such as iproniazid, a monoamine oxidase inhibitor used as an antidepressant.5 The actinomycete Streptomyces sp. LMA-545 was isolated recently as an α,β-unsaturated γ-lactono-hydrazides producer leading to geralcins A and B.4 In order to widen the panel of secondary metabolites isolated from this strain, we investigated the impact of the cultivation support and the extraction procedure. We were encouraged by our recent developments in the field of agar-supported fermentation (Ag-SF)6 and solidphase extraction (SPE).4 In this paper, we report the structural characterization of three novel alkyl hydrazides produced by the bacterial strain Streptomyces sp. LMA-545, combining SPE with liquid-state fermentation (LSF) or SPE with agar-state fermentation (AgSF). The latter procedure had never been investigated by us and allowed the isolation of a novel hydrazide compound not produced from LSF. © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The bacterial strain Streptomyces sp. LMA-545 was previously reported for the production of geralcins A and B.4 The compounds being studied were produced in liquid-state fermentation and agar-supported fermentation and recovered via in situ solid-phase extraction using Amberlite XAD-16 neutral resin. Agar-supported cultivation was recently scaled-up in our laboratory in the specific device Platotex, offering 2 m2 of cultivation surface.6 The crude extracts were eluted from the XAD-16 resin by methanol and analyzed by HPLC coupled with PDA, ELS, and mass spectrometry detection. The HPLC profile obtained for the LSF extract revealed the presence of two unknown compounds (1 and 2) together with the previously reported MH-031 and geralcins A and B. The crude extract obtained from Ag-SF showed the presence of one unkown compound (3) together with MH-031 and geralcins A and B (Figure 1). The structures of the new compounds were elucidated using both 1D and 2D 1H and 13C NMR spectroscopic analysis and high-resolution mass spectrometry. 1H−15N NMR experiments were required for full structural elucidation as well as molecular Received: July 31, 2012 Published: February 6, 2013 142

dx.doi.org/10.1021/np300527p | J. Nat. Prod. 2013, 76, 142−149

Journal of Natural Products

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Figure 1. HPLC analysis of LSF extract (chromatograms A and B) and Ag-SF extract (chromatogram C).

Figure 2. Compounds isolated from Streptomyces sp. LMA-545. MH-031 and geralcins A and B were previously reported.4 Compounds 1, 2, and 3 are newly reported in this paper.

168.7 (C-16), two methine carbons at δC 123.8 (δC 126.9) (C9) and δC 118.5 (δC 128.0) (C-10) (duplication of carbon chemical shifs were recorded corresponding to major and minor rotamers), nine methylene groups, which included one oxygen-bound methylene at δC 60.7 (C-15) and one azoxybound methylene at δC 69.2 (C-3), one sp3 carbon atom at δC 63.9 (C-2), linked to the azoxy moiety, and three methyl carbons at δC 13.6 (C-8), 13.7 (C-14), and 20.2 (C-17). Duplication of the 1H and 13C NMR signals for hydrogen and carbon atoms connected to the hydrazide group was observed for compound 1. This phenomenon was due to the equilibrium between amide rotamers and was previously described for geralcin B.4 The nJ1H−13C connectivities given by HSQC and HMBC NMR, including 1H−15N correlations, are listed in Table 1. The key structural elements for compound 1 revealed by 1H NMR were the singlet at δH 11.00 (H-N, s) associated with a nitrogen atom at δN 148.0 (N-A, N) (15N-HSQC in the Supporting Information) and a broad multiplet at δH 5.18 (H-

modeling calculations. The structures of these compounds are shown in Figure 2. Compound 1 was obtained as a yellowish oil. The HRESIMS analysis gave the molecular formula C17H32N4O4. The NMR data, recorded in DMF-d7, are listed in Table 1. According to the molecular formula, four degrees of unsaturation should be present, corresponding to two carbonyl groups, two olefinic carbons (δC 118.5 and 123.8), and an azoxy group revealed by a characteristic IR band at 1506 cm−1 (NN+−O−). A characteristic IR band at 1692 cm−1 (OC−NR) suggested that the two carbonyl groups are from amides. Moreover, the 1 H−15N HMBC data revealed two nitrogen atoms at δN 148.0 (N-A, N) and δN 142.4 (N-B, NH). The correlations of H-N (δH 11.00, s, NH) to N-A supported a N−N bond corresponding of the hydrazide group. The 1H−15N HMBC spectra also showed the presence of two nitrogen atoms of an azoxy group at δN 345.1 (N-C, NO) and δN 348.0 (N-D, N N). The 13C NMR revealed the presence of 17 carbon atoms, which included two carbonyl groups at δC 168.9 (C-1) and δC 143



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Table 1. NMR Spectroscopic Dataa for Compounds 1, 2, and 3 geralcin C (1) δC

no. 1 2 3

168.9 (166.9)d 63.9 69.2

δH (mult; J in Hz)

geralcin D (2) HMBC

4.88 (lH, mb) 4.29 (2H, t, 6.9)

b

C-l, 15 C-4, 5, N-C, N-D

70.7 147.1

C-3, 5, 6

131.6

C-4, 6

20.2

C-9, 11, N-A

31.3 169.1 (169.5)d 83.0 (71.1)d 90.5 (94.2)d 157.5

C-9, 10

10.4 29.0 27.9 22.3 13.5

4

27.7

5

25.7

1.32−1.38 (2H, mb)

6 7

31.3 22.6

1.26−1.31 (2H, mb) 1.26−1.31 (2H, mb)

8

13.6

0.87 (3H, mb)

C-6, 7

9

123.8 (126.9)d 118.5 (128.0)d 26.1

6.41 (1H, bd, 9.3) 6.53 (1H, bd, 8.0)d 4.88 (1H, mb) 5.36 (1H, q, 9.0)d 2.08−2.19(2H, mb)

C-10, 11, N-A

31.8 22.6 13.7 60.7 168.7 (168.1)d 20.2 (19.9)d

1.26−1.31 (2H, mb) 1.26−1.31 (2H, mb) 0.87 (3H, mb) 3.77−3.95 (2H, mb)

C-13

2.05 (3H, s) 1.95 (3H, s)d 11.00 (1H, s) 10.53 (H, s)d 5.18 (1H, mb)

C-16, N-B

11 12 13 14 15 16 17 H-N H-O N-A N-B

148.0,c N 142.4,c NH

N-C N-D

345.1,c NO 348.0,c NN

δH (mult; J in Hz)

geralcin E (3) HMBC

174.3

1.85−1.91 (2H, m )

10

δCe

C-12, 13 C-1, 5

N-A, C-9, 10

δH (mult; J in Hz)

HMBC

172.5 4.87 (2H, mb) 7.53 (1H, mb)

C-3, 4 C-1, 2

60.9 124.5

4.16 (2H, mb) 6.31 (1H, d, 7.6)

2.53−2.58 (2H, mb)

C-l, 3, 4, 6

(124.3)d 123.6 (132.4)d 27.2

2.70, 2.90 (2H, mb)

C-4, 5, 7

32.2 23.0

6.07 (1H, d, 7.3d) 5.13 (1H, q, 7.6) 5.44 (1H, q, 7.3)d 2.07 (2H, bq, 6.7) 2.14 (2H, bq, 6.5)d 1.32−1.37 (2H, mb) 1.32−1.37 (2H, mb)

C-8

C-12

14.3

0.90 (3H, t, 6.9)

C-6, 7

C-10

169.1 2.02 (3H, s)

C-9

8.17 (1H, bs) 8.08 (1H, bs)d 4.16 (1H, mb)

N-A

4.23 3.83 6.49 6.07

(1H, mb) (1H, mb)d (1H, d, 1.86) (1H, d, 1.86)d

21.0 2.10 (3H, s) 2.07 (3H, s)d 1.22−1.45 (2H, mb) 1.22−1.45 (2H, mb) 1.22−1.45 (2H, mb) 0.85 (3H, 7.2, t) 0.86 (3H, t, 7.1)d

C-3, 6 C-3, 6

C-13, 14

12.86 (1H, bs)f (5.86)d (1H, bd)d,f −, N 241.2,c* N

C-l C-4, 5

C-10, N-B

C-14, N-A C-15

δC

C-11

137.3c NH 138.6c,d

C-3, 4 C-3

a1 H chemical shifts were recorded at 600 MHz and 13C chemical shifts at 150 MHz in DMF-d7 for compounds 1 and 2, and CD2Cl2 for compound 3. bSignals were not distinguishable. c15N chemical shift of the nitrogen atom δN in ppm. dNMR spectroscopic data recorded for the minor rotamer observed for compounds 1 to 3. eCarbon chemical shifts were deduced from HSQC and HMBC data for compound 2. fData were recorded at 233 K to observed H-O 1H NMR signal

quadruplet at δH 4.88 (5.36) (H-10) were assigned to the two methine proton signals. The coupling constant of 9.0 Hz between H-9 and H-10 of compound 1 indicated a cis configuration for the C-9/C-10 double bond. The 1H−1H COSY correlations indicated that the single proton at δH 4.88 (H-2, m) linked to the azoxy function was connected to the geminal protons H-15. The triplet at δH 4.29 (H-3, t, 6.9) was assigned to the methylene connected to the azoxy group through the nitrogen at δN 345.1 (N-C, NO) and to the methylene protons H-4, which gave a broad signal between 1.85 and 1.91 ppm. The 1H−15N HMBC correlations from H-3 and H-4 to N-C (δN 345.1, NO) and H-3 to N-D (δN 348.0, NN) corroborated this observations. A broad signal corresponding to two geminal protons H-11 at δH 2.08 and 2.19 was connected to the methine H-10. A singlet at δH 2.95 (2.05) was identified as the methyl group connected to the hydrazide function through C-16 (δC 168.7). A broad signal

O, m), linked to an oxygen atom, coupling with the two geminal protons of the methylene group, at δH 3.77−3.95 (H15, m) (Figure 3). The doublet at δH 6.41 (6.53) (H-9) and the

Figure 3. Structural assignments of compound 1, showing C−H and N−H connectivities. 144



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(C-15) and 10.4 (C-11). Duplication of the 1H and 13C NMR signals for hydrogen and carbon atoms connected to the hydrazide moiety was recorded for compound 2. The nJ1H−13C connectivities given by HSQC and HMBC NMR, including 1H−15N correlations, are listed in Table 1. The key structural elements revealed by 1H NMR were the broad and weak signal, recorded at 223 K, at δH 12.86 (5.86) (H-O, m) coupling with the proton at δH 6.49 (6.07) (H-9, d, 1.9). The multiplet at δH 7.53 (H-3, m) was assigned to the double bond of the α,β-unsatured lactone and coupled to the methylene group at δH 4.87 (H-2, m). The 1H−1H COSY correlations indicated a pair of vicinal methylene groups, giving a broad signal at δH 2.53−2.58 (H-5, m) and 2.70−2.90 (H-6, m). The multiplet at δH 4.23 (3.83) was assigned to H-8 and coupled with H-9. A set of three methylene groups, with a broad signal between 1.22 and 1.45 ppm, were attributed to the aliphatic methylene chain (C-12 to C-15). This chain was connected to the single proton H-8 and ended with a methyl group characterized by the multiplet at δH 0.86 (H-15, m). A duplicate singlet at δH 2.10 (2.07) was assigned to the methyl group attached to the quaternary carbon at δC 157.5 (C-10). The 1H−13C HMBC connectivities of H-2 to C-1/C-3/C-4 and H-3 to C-1/C-2 allowed for the construction of the α,βunsaturated γ-lactone moiety. Moreover, correlations from H-5 to C-1/C-3/C-4/C-6 and from H-6 to C-4/C-5/C-7 showed that the pair of vicinal methylene groups C-5/C-6 were connected to the α,β-unsaturated γ-lactone moiety and to the carbonyl amide-type group at δC 169.1 (C-7). According to the 1 H−1H COSY and 1H−13C HMBC, H-8 was connected to the aliphatic chain (C-12 to C-15) through C-12 and C-9. The chemical shift for C-8 (δC 83.0) and C-9 (δC 90.5) confirmed the presence of oxygen atoms in the local environment. Moreover 1H−13C HMBC together with 1H−15N HMBC data gave connectivities from H-9 to C-10 and H-11 to C-10/N-B, allowing the construction of an original heteroheptacycle as proposed in Figure 4.

between 1.85 and 1.91 ppm identified as the methylene group H-4 was connected to the methylene group H-3. A set of five methylene groups, with a broad signal between 1.26 and 1.31 ppm, was assigned to two aliphatic methylene chains (C-5 to C7 and C-12 to C-13). These chains were respectivly connected to C-4 (δC 27.7) and C-11 (δC 26.1) and ended with methyl groups at δC 13.6 (C-8) and δC 13.7 (C-14). The two methyl groups were characterized by a broad signal at δH 0.87. The 1 H−13C HMBC connectivities allowed the construction of the aliphatic chain from C-7 to C-8. The 1H−15N HMBC data showed the connection of this aliphatic chain to the azoxy function through N-C (δN 345.1, NO). The chemical shift for C-2 (δC 63.9) confirmed the presence of the azoxy group in the local environment, and 1H−13C HMBC supported the connectivities from H-15 to C-2 and from H-2 to C-1 (δC 168.9). C-1 is one of the two carbonyl groups involved in the hydrazide moiety. 1H−1H COSY and 1H−13C HMBC allowed the construction of the second aliphatic chain from C-11 to C14, which was connected to the C-10 position of the cis C-9/C10 double bond. The connectivities from H-9 to N-A deduced from the 1H−15N HMBC data attached the unsaturated carbon chain to the hydrazide function through the nitrogen N-A. The connectivities from H-17 to C-16 and to N-B, given by both 1 H−13C HMBC and 1H−15N HMBC data, allowed to connect the methyl C-17 to the second carbonyl group of the hydrazide. Finally, the 1H−15N HMBC experiments confirmed the position of the two nitrogen atoms with the observation of the connectivity from H-N (δH 11.00) to N-A (δC 148.0, N). The overall structural assignments led to compound 1, shown in Figure 2. This compound consists of a novel natural scaffold of alkyl hydrazide with the notable presence of the azoxy function. Such an azoxy group was previously reported for similar structures3 but remains scarcely reported for natural compounds. Since the discovery of macrozamin in 1951,7 a number of naturally occurring azoxy compounds have been identified, including cycasin,8 elaiomycin,9 maniwamycin I,10 azoxybacillin,11 valanimycin,12 calvatic acid,13 pyranadine A,14 and pyranadines B−G.15 Some of them exhibited antifungal or antibacterial activities. The physical properties and the small quantity obtained for compound 1 did not allow the determination of the absolute configuration for the carbon C-2. Compound 2 was obtained as a transparent oil. The HRESIMS analysis gave the molecular formula C15H22N2O6. The NMR data, recorded for 2 in DMF-d7, are listed in Table 1 and showed notable similarities to geralcins A and B,4 in particular duplication of 1H and 13C signals. According to the molecular formula, six degrees of unsaturation should be present to account for an α,β-unsatured lactone, a carbonyl group (δC 169.1), an imine group (δC 157.5), and a heterocyclic ring. A characteristic IR band at 1657 cm−1 (OC−NR) suggested that the carbonyl group could be from an amide. Moreover, the 1H−15N HMBC data revealed a nitrogen atom at δN 241.2 (N-B, N). A notable IR band at 1448 cm−1 indicated the presence of a nitogen−oxygen bond. The 13C NMR revealed the presence of 15 carbon atoms, which included two carbonyl groups at δC 174.3 (C-1) and 169.1 (C7), an imine group at δC 157.5 (C-10), two olefinic carbons at δC 147.1 (C-3) and 131.6 (C-4), six methylene groups, including one oxygen-bound methylene at δC 70.7 (C-2), two sp3 carbon atoms at δC 83.0 (71.1) (C-8) and 90.5 (94.5) (C-9) both linked to oxyen atoms, and two methyl carbons at δC 13.5

Figure 4. 1H−13C HMBC and recorded for compound 2.

1

H−15N HMBC connectivities

The characteristic IR band at 1448 cm−1 and the particular chemical shift of C-8 (δC 83.0) consolidated the existence of the nitrogen−oxygen bond between C-7 (δC 169.1) and C-8 (δC 83.0) to close the seven-membered ring. As we did not observe any 1H−15N HMBC correlations between H-8 and N-A to ensure this original heterocycle, and 145



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in the absence of any alternative structure that accounts for the NMR data, molecular modeling calculations were undertaken. Molecular modeling studies were carried out to evaluate the conformational flexibility of different conformers for compound 2 and to compare these results with the NMR data. As the relative configurations at C-8 and C-9 could not be formally assigned by NMR, both 2A and 2B diastereoisomers were included in the present study (Figure 5).

Table 2. Dihedral angles H8−C8−C9−H9 for the conformers included in this study and NMR coupling constants estimated using 4 different methods: 1 - Karplus;18 2 - Haasnoot, as implemented online;19 3 - Karplus modified, as implemented in Maestro;16 4 - DFT calculations (B3LYP/6-31+G(d,p)) using Gassian0917 2A dihedral angle (deg) estimated 3JHH (Hz, method 1) estimated 3JHH (Hz, method 2) estimated 3JHH (Hz, method 3) estimated 3JHH (Hz, method 4)

2B

a

b

c

a

b

c

−62.9 2.00

−66.5 1.59

−38.9 6.00

177.2 13.96

−84.1 0.10

−99.4 0.37

1.06

0.94

2.66

7.27

0.92

1.73

0.80

0.70

2.30

6.80

1.60

0.80

2.19

0.96

5.96

7.25

0.06

0.43

Figure 5. Cis and trans diastereomers of 2 included in the molecular modeling study.

experimental value for 3JH8−H9, which is about 1.86 Hz for both species evidenced in the NMR spectra (Table 1), and the data from Table 2 suggests that the species present in solution are 2A-a and 2A-b. Additionally, the Karplus method18 and the DFT calculations seem to give the best agreement with the experimental data. Moreover, the conformer establishing a hydrogen bond involving the hydroxyl group and C-1 could be eliminated: duplication of the 13C signal was recorded only for C-7 (δC 169.1 for the major form and δC 169.5 for the minor one). This supported the existence of hydrogen bonding between the hydroxyl group and C-7 (2A-b) in equilibrium with the 2A-a conformer, considering the NMR data. In conclusion, molecular modeling calculations allowed, by comparison with the experimental NMR data, the assignment of the relative stereochemistry at C-8 and C-9 and thus identification of the naturally occurring cis diastereomer of compound 2. This conclusion is supported by the NOE effects recorded at 223 K. Concerning the major conformer (2A-b), correlations from H-9 (δH 6.49) to H-8 (δH 4.23) were recorded, and for the minor conformer (2A-a), correlations from H-9 (δH 6.07) to H-8 (δH 3.83) were recorded. NOESY data were in accordance with molecular modeling calculations

A conformational analysis of the diastereomers 2A and 2B was carried out using MacroModel,16 to generate 53 and 44 conformers, respectively. For each diastereomer, three representative conformations were selected, presenting an extended structure (a), an intramolecular hydrogen bond between the hydroxyl group and the carbonyl group at C-7 (b), and an intramolecular hydrogen bond between the hydroxyl group and the carbonyl group at C-1 (c), respectively. The geometries of the six conformers selected in the first step were optimized using DFT calculations, with Gaussian17 at the B3LYP/6-31+G(d,p) level; then vibrational frequency calculations confirmed that these structures are local minima. The relative energies calculated for these conformers (3−10 kcal/mol) allow an easy conformer interconversion (in the case of each diastereomer) to establish a thermodynamic equilibrium. The optimized structures of these conformers are shown in Figure 6. Dihedral angles H8−C8−C9−H9 were measured for the six optimized structures presented above, and the 3JHH NMR coupling constants were estimated using four different methods (Table 2 and Supporting Information). A comparison of the

Figure 6. Conformations of the diastereomers cis 2A and trans 2B presenting an extended structure (a), an intramolecular hydrogen bond between the hydroxyl group and the carbonyl group at C-7 (b), and an intramolecular hydrogen bond between the hydroxyl group and the carbonyl group at C-1 (c). Relative energies (kcal/mol) calculated with Gaussian0917 at the B3LYP/6-31+G(d,p) level are shown in orange. 146



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to establish the cis relative stereochemistry at C-8 and C-9 for compound 2. Compound 3 was obtained as a colorless oil. The HRESIMS analysis gave the molecular formula C10H18N2O3. The NMR data, recorded in CD2Cl2, are listed in Table 1. According to the molecular formula, three degrees of unsaturation should be present to account for two carbonyl groups and two olefinic carbons (δC 123.6 and 124.5). A characteristic IR band at 1650 cm−1 (OC−NR) suggested that the two carbonyl groups are from amides. Moreover, the 1H−15N HSQC data revealed the nitrogen atom δN 137.3 (138.6) (N-B, NH), showing signal duplication corresponding to the major and the minor rotamers. The small quantity of isolated compound did not allow 1H−15N HMBC, but the very similar NMR spectra compared to those recorded for the geralcins family suggested that compound 3 was a hydrazide. The 13C NMR revealed the presence of 10 carbon atoms, which included two carbonyl groups at δC 169.1 (C-9) and 172.5 (C-1), two methine carbons at δC 123.6 (δC 132.4) (C-4) and 124.5 (δC 124.3) (C3), four methylene groups, which included one oxygen-bound methylene at δC 60.9 (C-2), and two methyl carbons at δC 14.3 (C-8) and 21.0 (C-10). As we found out for compounds 1 and 2, duplication of the 1H and 13C NMR signals for hydrogen and carbon atoms connected to the hydrazide group was recorded for compound 3. This phenomenon was due to the equilibrium between amide rotamers and was previously described for geralcin B.4 The nJ1H−13C connectivities given by HSQC and HMBC NMR, including 1H−15N correlations, are listed in Table 1. The key structural element revealed by 1H NMR was the singlet at δH 8.17 (H-N, s) associated with a nitrogen atom at δN 137.3 (138.6) (N-A, N) (15N-HSQC in the Supporting Information). The doublet at δH 6.31 (6.07) (H-3, d, 7.6) and the quadruplet at δH 5.13 (5.44) (H-4, q, 7.6) were assigned to the two methine proton signals. The coupling constant of 7.6 Hz between H-3 and H-4 of compound 3 indicated a cis configuration for the C-3/C-4 double bond. A broad and large signal at δH 4.12 was attributed to H-2 (2H, m), corresponding to the oxygen-bound methylene and to H-O. The two quadruplets, at 2.07 for the major form and 2.14 ppm for the minor one, were assigned to H-5 (2H, bq, 6.7). The 1H−1H COSY and 1H−13C HMBC correlations showed the connection from H-5 to the double bond C-3/C-4 through C-4. A singlet at δH 2.02 was assigned to the methyl group H-10 (3H, s). The broad signal between 1.32 and 1.37 ppm was attributed to the two methylene protons H-6 and H-7. The aliphatic chain ended with the methyl group at C-8, giving a triplet at δH 0.90 (3H, t, 6.9). 1H−1H COSY correlations linked the aliphatic chain C-5 to C-8 to the double bond C-3/C-4 connected to NB through C-3. 1H−13C HMBC data gave connectivities from H-10 to C-9 and from H-2 to C-1. These correlations allowed connecting the methylenoxy group C-2 (δC 60.9) to the carbonyl C-1 (δC 172.5) and the methyl group C-10 (δC 21.0) to the carbonyl C-9 (δC 169.1) (Figure 7). Antimicrobial and cytotoxic activities of compounds 1, 2, and 3 were evaluated according to our previously reported bioassays.4 Compounds were also screened for their ability to inhibit E. coli DnaG primase, a Gram-negative antimicrobial target.20−22 Geralcins D and E did not show any significant bioactivity. Geralcin C has no antimicrobial activity but exhibited an IC50 of 8 × 10−7 M against KB and HCT116 cancer cell lines (IC50 for Taxotere KB 2.5 × 10−10 M, HCT116 5 × 10−8 M). Furthermore, geralcin C inhibited the E. coli

Figure 7. 1H−13C HMBC and recorded for compound 3.

1

H−15N HMBC connectivities

Figure 8. Inhibition of E. coli DnaG primase by geralcin C (1).

DnaG primase, in a dose-dependent manner, with an IC50 of 7 × 10−4 (Figure 8). Thus, combining SPE with LSF and Ag-SF allowed the isolation of three novel hydrazides together with MH-031 and geralcins A and B.4

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EXPERIMENTAL SECTION

General Experimental Procedures. The IR spectra were obtained using a Perkin-Elmer Spectrum 100 model instrument. NMR experiments were performed using a Bruker Avance 600 MHz spectrometer equipped with a microprobe head (1.7 TXI, Bruker) for compounds 1 to 3. The spectra for compounds 1 and 2 were acquired in DMF-d7 (δH 2.75, 2.92, 8.03 ppm; δC 29.74, 34.89, 163.15 ppm) at room temperature to observe all of the correlations present. The spectra for compound 3 were obtained in methylene chloride-d2 (δH 5.32 ppm; δC 54.0 ppm) at room temperature and at 233 K. LC-MS experiments were performed using a Waters-Micromass ZQ2000 simple-stage quadrupole mass spectrometer equipped with an ESI (electrospray ionization) interface coupled to an Alliance Waters 2695 HPLC instrument with PDA and ELS detection. HRESIMS was conducted using a Waters-Micromass mass spectrometer equipped with an ESI-TOF (electrospray-time-of-flight). Biological Materials. Streptomyces sp. LMA-545 was isolated from a soil sample collected in La Réunion Island and grown on a PDB agar (potato dextrose broth, DIFCO) at 30 °C. The microorganism was examined for chemotaxonomic and morphological properties known to be useful in the systematics of Streptomyces. A phylogenetic analysis was performed using a fragment of the 16S rRNA gene amplified from the genomic DNA of Streptomyces sp. LMA-545. The 16S rRNA gene amplification and sequencing were performed, and the resulting material was compared to the corresponding sequences in the related 147



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Streptomyces using the NCBI/BLAST Web site (GenBank). The primers used for PCR amplification were 16 S F 27, AGA GTT TGA TC(AC) TGG CTC AG (Tm: 56.3 °C), and 16 S R 1492, TAC GG(CT) TAC CTT GTT ACG ACT T (Tm: 57.5 °C). The GenBank accession number for Streptomyces sp. LMA-545 sequence is BankIt1535712 8404357.seq JX025158. Fermentation. Batch fermentation of Streptomyces sp. LMA-545 was conducted in a 15 L fermentor (Chemap 20 L unit) in PDB medium (DIFCO) over 5 days at 30 °C with an aeration rate of 16 volumes of air per volume per minute and 200 rpm agitation. The initial pH of 7.2 was progressively decreased to 4.3. Amberlite XAD-16 (30 g/L) was added prior to sterilization to allow the in situ trapping of the microbial metabolites. Agar-state fermentation coupled with solid-state fermentation was performed on PDB medium (DIFCO) supplemented with 2% agar (DIFCO) over 9 days at 30 °C. Sterilizing Amberlite XAD-16 (20 g) mixed with 5 mL of inoculums was spread on each of 10 agar plates (25 cm × 25 cm). Isolation. Concerning liquid-state fermentation, the XAD-16 resin was separated from the broth culture via filtration and washed with water before being eluted with MeOH (500 mL). The eluate was concentrated to dryness in vacuo (5.4 g) and extracted with MeOH. The crude extract (4.7 g) was subjected to flash chromatography on a Combiflash Companion using a Redisep 80 g silica column, with a heptane−ethyl acetate mixture serving as the eluent. The fractions containing compounds 1 and 2 were separated as pure compounds by preparative RP-HPLC (Sunfire Prep C18 5 μm, 10 × 250 mm) eluted using a linear H2O−CH3CN gradient supplemented with 0.1% formic acid. After concentrating in vacuo, compound 1 (4 mg) was obtained as a yellowish oil, while compound 2 (0.7 mg) was obtained as a colorless oil. For agar-state fermentation, the XAD-16 resin was recovered by carefully scraping the agar plate surface. The recovered resin was washed with water to eliminate the biomass before being eluted with MeOH (500 mL). The eluate was concentrated to dryness in vacuo (2.9 g) and extracted with MeOH. The crude extract (1.2 g) was subjected to flash chromatography on a Combiflash Companion using a Redisep 24 g silica column, with a heptane−ethyl acetate mixture serving as the eluent. The fractions containing compound 3 were separated as pure compound by preparative RP-HPLC (Sunfire Prep C18 5 μm, 10 × 250 mm) eluted using a linear H2O−CH3CN gradient supplemented with 0.1% formic acid (100→0 to 0→100). After concentrating in vacuo, compound 3 (0.5 mg) was obtained as a colorless oil. Geralcin C (1): yellowish oil; IR νmax 3457, 3264, 2958, 2928, 1692, 1680, 1506, 1373, 1051 cm−1; for complete NMR data see Table 1; HRESIMS m/z [M + H]+ 357.2488 (calcd for C17H33N4O4, 357.2502). Geralcin D (2): translucent oil; IR νmax 3451, 3264, 2958, 2940, 2869, 1744, 1659, 1448, 1381, 1219, 1081, 1051 cm−1; for complete NMR data see Table 1; HRESIMS m/z [M + H]+ 327.1541 (calcd for C15H23N2O6, 327.1556). Geralcin E (3): translucent oil; IR νmax 3430, 3275, 2959, 2929, 2860, 1741, 1647, 1425, 1080, 1050 cm−1; for complete NMR data see Table 1; HRESIMS m/z [M + H]+ 215.1392 (calcd for C10H19N2O3, 215.1396). Antibacterial and Antitumor Cell Assays. The antibacterial activity was measured using the disk inhibition zone method against Bacillus subtilis ATCC.6633, Micrococcus luteus ATCC.10240, and Escherichia coli ATCC.25922. Inhibition was compared for 10 μg of gentamicin and 30 μg of chloramphenicol. Cytotoxicity Assays. A tetrazolium dye [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; MTT]-based colorimetric assay was used to measure the inhibition of proliferation of the colonic epithelial cancer cell line HCT116, the hormone-responsive breast cancer cell line MCF7, the colon adenocarcinoma cell line HT29, the naso-pharyngeal carcinoma KB cells, and the breast cancer cell line MDA231, as previously reported.4 All of the test compounds were formulated in DMSO and added to the cells such that the final DMSO concentration ranged from 1% to 3%. Cells were grown in D-MEM medium supplemented with 10% fetal calf serum (Invitrogen), in the presence of penicillin, streptomycin, and fungizone, and plated in 96-

well microplates. After 24 h of growth, cells were treated with target compounds from 100 μM to 10 nM. After 72 h, MTS reagent (Promega) was added, and the absorbance was monitored (490 nm) to measure the inhibition of cell proliferation compared to untreated cells. IC50 determination experiments were performed in separate duplicate experiments. Primase Bioassay. E. coli DnaG primase cloning, purification, and bioassay conditions are fully described in the Supporting Information. Molecular Modeling. The conformational analysis was carried out using MacroModel v9.5, as implemented in the Schrödinger Suite.16 Default values were used except the allowed energy window (42 kJ/ mol) and the number of evaluations per rotatable bond (500). The resulting conformers were clustered using a 2.0 Å cutoff. The geometries of all conformers were optimized in the gas phase using the Gaussian 09 package17 with Becke’s three-parameter hybrid exchange functional (B3LYP)23,24 and the 6-31+G(d,p) basis set. Subsequent vibrational frequency calculations confirmed that these conformations were local minima. For all calculations the IEFPCM and CPCM solvation models were also used, these results being compared with the ones obtained in the absence of solvation. However, no significant differences were observed, and only the results obtained without solvation are presented here.

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ASSOCIATED CONTENT

* Supporting Information S

Experimental section, physicochemical properties, 1D and 2D NMR spectroscopic data, IR data, and high-resolution Orbitrap-ESIMS. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33 1 69 82 30 01. Fax: +33 1 69 07 72 47. E-mail: jamal. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a scholarship grant from the Institut de Chimie des Substances Naturelles, ICSN-CNRS. REFERENCES

(1) Shoeb, M.; MacManus, S. M.; Jaspars, M.; Trevidu, J.; Nahar, L.; Kong-Thoo-Lind, P.; Sarker, S. D. Tetrahedron 2006, 62, 11172− 11177. (2) Ueberschaar, N.; Ndejouong Ble, S.; Ding, L.; Maier, A.; Fiebig, H. H.; Hertweck, C. Chem. Lett. 2011, 21, 5839−5841. (3) Helaly, S. E.; Pesic, A.; Fiedler, H. P.; Sussmuth, R. D. Org. Lett. 2011, 13, 1052−1055. (4) Le Goff, G.; Martin, M. T.; Servy, C.; Cortial, S.; Lopes, P.; Bialecki, A.; Smadja, J.; Ouazzani, J. J. Nat. Prod. 2012, 75, 915−919. (5) Gaspar, A.; Reis, J.; Fonseca, A.; Milhazes, N.; Borges, F.; Vina, D.; Uriarte, E. Bioorg. Med. Chem. Lett. 2011, 21, 707−709. (6) Adelin, E.; Slimani, N.; Cortial, S.; Schmitz-Afonso, I.; Ouazzani, J. J. Ind. Microbiol. Biotechnol. 2011, 38 (2), 299−305. (7) Langley, B. W.; Lythgoe, B.; Riggs, N. V. J. Chem. Soc. 1951, 2309−2316. (8) Wilson, J. M. B.; Khabazian, I.; Wong, M. C.; Seyedalikhani, A.; Bains, J. S.; Pasqualotto, B. A.; Williams, D. E.; Andersen, R. J.; Simpson, R. J.; Smith, R.; Craig, U.-K.; Kurland, L. T.; Shaw, C. A. NeuroMol. Med. 2002, 1, 207−221. (9) Parry, R. J.; Mueller, J. V. J. Am. Chem. Soc. 1984, 106, 5764− 5765. (10) Hasegawa, K.; Deushi, T.; Yaegashi, O.; Miyashita, Y.; Sasaki, S. Eur. J. Med. Chem. 1995, 30, 569−574. (11) Aoki, Y.; Kondoh, M.; Nakamura, M.; Fujii, T.; Yamazaki, T.; Shimada, H.; Arisawa, M. J. Antibiot. 1994, 47, 909−916.

148



Article

(12) Tao, T.; Alemany, L. B.; Parry, R. J. Org. Lett. 2003, 5, 1213− 1215. (13) Boschi, D.; Guglielmo, S.; Aiello, S.; Morace, G.; Borghi, E.; Fruttero, R. Bioorg. Med. Chem. Lett. 2011, 21, 3431−3434. (14) Kariya, Y.; Kubota, T.; Jane Fromont, J.; Kobayashi, J. Tetrahedron Lett. 2006, 47, 997−998. (15) Kariya, Y.; Kubota, T.; Fromont, J.; Kobayashi, J. Bioorg. Med. Chem. 2006, 14, 8415−8419. (16) http://www.schrodinger.com. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.2; Gaussian Inc.: Wallingford, CT, USA (http://www. gaussian.com). (18) Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870−2871. (19) http://www.stenutz.eu/conf/jhh.html. (20) Agarwal, A.; Louise-May, S.; Thanassi, J. A.; Podos, S. D.; Cheng, J.; Thoma, C.; Liu, C.; Wiles, J. A.; Nelson, D. M.; Phadke, A. S.; Bradbury, B. J.; Deshpande, M. S.; Pucci, M. J. Bioorg. Med. Chem. Lett. 2007, 17, 2807−2810. (21) Maciag, M.; Kochanowska, M.; Lyzen, R.; Wegrzyn, G.; Szalewska-Palasz, A. Plasmids 2010, 63, 61−67. (22) Frick, D. N.; Richardson, C. C. Annu. Rev. Biochem. 2001, 70, 39−80. (23) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789. (24) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652.

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Isolation and Characterization of Unusual Hydrazides from

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