Note pubs.acs.org/jnp
Antibacterial Ilicicolinic Acids C and D and Ilicicolinal from Neonectria discophora SNB-CN63 Isolated from a Termite Nest Charlotte Nirma,† Véronique Eparvier,*,† and Didier Stien*,†,‡ †
CNRS−Institut de Chimie des Substances Naturelles, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France CNRS/Université Pierre et Marie Curie, LBBM, Observatoire Océanologique, 66650 Banyuls-sur-mer, France
‡
S Supporting Information *
ABSTRACT: Ilicicolinic acids A, C, and D (1−3) and ilicicolinal (4) were isolated from a fungus isolated from a Nasutitermes corniger nest in French Guiana. The structures of ilicicolinic acids C and D and ilicicolinal were elucidated using 1D and 2D NMR spectroscopic data as well as MS data. Ilicicolinic acids show antibacterial activity in vitro.
N
ew efficient and selective novel antimicrobial drugs are urgently needed. Social insect colonies may host large numbers of individuals in a constrained environment. Promiscuity, frequent interactions, and genetic homogeneity are factors that in principle favor the transmission of infectious pathogens. Social insects have developed mutually beneficial associations with microbes that may provide colonies with antimicrobial agents.1 Due to their extreme diversity and high relative proportion of unknown species,2 insect and microbial symbioses represent an alternative and attractive natural source of bioactive compounds. Antibacterial compounds have been isolated from ant symbiotic microorganisms, but there are few reports of similar studies on termite symbionts.3,4 We isolated 130 fungi and bacteria from termites and termite nests in the North Amazon (French Guiana) and evaluated the activity of extracts of these organisms against pathogenic bacteria and fungi. A fungus isolated from a Nasutitermes corniger nest and identified as Neonectria discophora SNB-CN63 yielded a strongly antibacterial ethyl acetate extract that was investigated further. Four compounds were isolated from this extract by bioactivity-guided isolation, and these were then characterized (Figure 1). The isolated compounds are ilicicolinic acids A (1),5 C (2), and D (3) and ilicicolinal (4). Compounds 2−4 have not previously been reported in the literature (Figure 1). Compound 1 was identified as ilicicolinic acid A. The spectral data were identical to those already described in the literature.5 Ilicicolinic acid C (2) was obtained as a light beige powder. A molecular formula of C23H33ClO5 was determined via highresolution ESITOFMS analysis, which showed a pseudomolecular ion at m/z 423.1933 [M − H]− (423.1938 calcd for C23H32ClO5) and corresponds to seven degrees of unsaturation. © XXXX American Chemical Society and American Society of Pharmacognosy
Figure 1. Compounds isolated from Neonectria discophora SNB-CN63.
Examining the NMR data made it clear that compound 2 was an analogue of ilicicolinic acid A (1) (Table 1). The same characteristic groups are present. The only difference was in the Received: January 24, 2014
A
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Table 1. 13C and 1H NMR Data for Compounds 1−4 (recorded in CDCl3) ilicicolinic acid A (1) atom
a
δCa, type
δHb (J in Hz)
1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′
104.8, C 160.9, C 114.1, C 154.6, C 114.2, C 137.1, C 22.4, CH2 120.7, CH 136.1, C 39.7, CH2 26.3, CH2
6′ 7′ 8′ 9′
124.4, CH 135.0, C 39.6, CH2 26.6, CH2
10′ 11′ 12′ 13′ 1-COOH 1-CHO 2-OH 4-OH 6-CH3 3′-CH3 7′-CH3 3′-OH 7′-OH 11′-OH
124.1, CH 130.7, C 25.6, CH3 17.3, CH3 174.9, C
1.69 s 1.60 s 11.70 br s
19.5, CH3 16.1, CH3 16.3, CH3
n.d. 6.32 2.69 1.82 1.59
3.45 d (7.0) 5.26 t (7.0) 2.02 t (6.6) 2.10 m 5.09 t (6.6) 1.95 m 2.03 m 5.09 t (6.6)
s s s s
ilicicolinic acid C (2) δCa, type 105.8, C 161.5, C 114.0, C 154.0, C 113.9, C 137.2, C 22.7, CH2 121.6, CH 135.9, C 40.1, CH2 22.1, CH2 41.5, 72.9, 41.4, 22.6,
CH2 C CH2 CH2
ilicolinic acid D (3)
δHb (J in Hz)
3.43 d (7.0) 5.24 t (7.0) 1.98 m 1.41 m 1.46 m 1.40 m 2.00 m
124.5, CH 131.6, C 25.6, CH3 17.6, CH3 173.7, C
5.12 t (6.9) 1.69 s 1.61 s 11.82 s
19.7, CH3 16.0, CH3 26.7, CH3
n.d. n.d. 2.67 s 1.79 s 1.16 s
δCa, type 105.7, C 161.5, C 113.9, C 154.2, C 113.9,C 137.2, C 22.7, CH2 121.5, CH 135.8, C 39.5, CH2 26.1, CH2
δHb (J in Hz)
3.42 d (6.8) 5.22 t (6.8) 2.01 t (7.2) 2.09 t (6.9)
124.2, CH2 134.6, C 39.9, CH2 22.4, CH2
5.07 t (6.6)
43.2, CH2 71.5, C 29.1, CH3 29.1, CH3 173.2, C
1.43 br s
19.7, CH3 16.1, CH3 15.9, CH3
1.93 m 1.43 br s
1.23 s 1.23 s 11.77 s n.d. n.d. 2.67 s 1.79 s 1.56 s
ilicicolinal (4) δCa, type 114.6, C 159.4, C 112.2, C 163.4, C 108.2, C 141.3, C 27.4, CH2 91.7, CH 73.6, C 36.9 21.8, CH2 124.1, CH 136.0, C 39.6, CH2 26.6, CH2 123.7, CH 131.5, C 25.6, CH3 17.7, CH3
δHb (J in Hz)
3.22 m 4.87 t (8.5) 1.55 2.17 2.09 5.15
t (8.4) m m t (6.6)
1.98 2.05 1.99 5.08
m m m t (6.4)
1.68 s 1.60 s
193.2, C
10.14 s 12.48 s
14.0, CH3 22.6, CH3 16.0, CH3
2.60 s 1.35 s 1.64 s n.d.
n.d. n.d. b
Data recorded at 125 MHz. Data recorded at 500 MHz.
farnesyl side chain, which contained only two olefinic protons. The methyl group linked to C-7′ was more shielded (δ 1.16 instead of δ 1.59), and the C-7′ quaternary carbon exhibited a signal at δ 72.9 ppm, which indicated that it carried an oxygen. Therefore, compound 2 differs from ilicicolinic acid A (1) by the presence of a hydroxy group at C-7′ in the farnesyl chain, which was hydrated across 6′−7′ according to the Markovnikov orientation. Ilicicolinic acid D (3) was obtained as a yellow powder, and its molecular formula was determined as C23H33ClO5 based on the HRESIMS experiment ([M + H]+ peak at m/z 425.2075 calcd at m/z 425.2095). The close resemblance between the NMR spectra of 1, 2, and 3 indicated 3 was another farnesylbearing benzoic acid. Its empirical formula indicated it was also a hydrate of ilicicolinic acid A (1). The COSY correlations between H-8 ′, H-9′, and H-10 ′ combined with the HMBC correlations between the methyl protons at δ 1.23 (H-12′ and H-13′) and a carbon at δ 71.5 (C-11′) placed a tertiary alcohol at C-11′ (Figure 2, Table 1). Ilicicolinal (4) was obtained as a light beige oil. The HRESIMS mass spectrum of this compound had a pseudomolecular ion peak at m/z 407.1989 [M + H]+ corresponding to the empirical formula C23H32ClO4 (calcd 407.1989). This compound (C23H31ClO4) had eight degrees of unsaturation. The 1H and 13C NMR and HSQC NMR spectra revealed similarities to ilicicolinic acids 1−3. However, there
Figure 2. Key 1H−1H COSY (bold line) and HMBC (dashed arrows) correlations of 2, 3, and 4.
B
dx.doi.org/10.1021/np500080m | J. Nat. Prod. XXXX, XXX, XXX−XXX
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was an sp3 methine bound to an oxygen atom (δH 4.87, δC 91.7) and an aldehyde at δH 10.14 and δC 193.2 (Table 1). The carbon−proton correlations clearly indicated that the aldehyde function was positioned on a carbon in the aromatic ring in place of the CO2H group of the ilicicolinic acids. COSY and HMBC correlations (Figure 2) allowed us to identify a farnesyl chain hydroxy substituted at C-3′. The presence of a single aryl hydroxy group (2-OH) and a methine at 2′ (δH 4.87, δC 91.7) as well as a H-2′−C-4 proton−carbon correlation indicated the presence of a furan ring at atoms 3 and 4 of the aromatic ring and atoms 1′ and 2′ of the farnesyl chain. All isolated and identified compounds from the Neonectria discophora strain were evaluated against several human pathogens (Table 2).6 The ilicicolinic acids were active against
High-resolution ESITOFMS measurements were performed using a Waters Acquity UPLC system with column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection equipped with an 80 g silica column. For UV-based experiments, effluents were monitored at 254 and 280 nm. TLCs were conducted on 60 A F254 Merck plates and visualized using UV and phosphomolybdic acid. Analytical and preparative HPLCs were conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector, and a prepELSII detector electrospray nebulizer. Columns used for these experiments included a Phenomenex Luna C18 5 μm 4.6 × 250 mm analytical column and Phenomenex Luna C18 5 μm 21.2 × 250 mm preparative column. The flow rate was 1 or 17 mL/min, respectively, using a linear gradient of H2O mixed with an increasing proportion of CH3CN or MeOH. Both solvents were modified with 0.1% formic acid. All solvents were HPLC grade. Potato dextrose agar (PDA) was purchased from Fluka Analytical. Isolation and Identification of Neonectria discophora SNBCN63. The strain was isolated from an N. corniger termite aerial nest sampled in Rémire-Montjoly, French Guiana, in July 2011. A piece of the nest matter was placed in suspension in sterile water, and this solution was spread on PDA medium. After 1 week of incubation at 25 °C, the first fungus hyphae were removed and transferred to another Petri dish. The colony is maintained in triplicate at −80 °C in glycerol−H2O (50/50). A sample submitted for amplification and nuclear ribosomal internal transcribed spacer region ITS4 sequencing allowed for strain identification by NCBI sequence comparison. The sequence has been registered in the NCBI GenBank database (http:// www.ncbi.nlm.nih.gov) under registry number KJ023733. Molecular analysis was performed externally by BACTUP, France. Culture, Extraction, and Isolation. The N. discophora strain was cultivated on PDA at 26 °C for 15 days, initially on a small scale and then on 150 14 cm Petri dishes. The fungus and culture medium were then transferred into a large container and macerated with EtOAc for 24 h. The insoluble residue was removed by filtration, and the organic solvent was washed with H2O in a separatory funnel and evaporated, yielding 4.56 g of extract. A portion of the extract (1.19 g) was purified by flash chromatography with a linear gradient of hexane−EtOAc followed by another gradient of EtOAc−MeOH. Eight fractions were collected based on their TLC profiles. The antifungal activity was found to be concentrated in fractions II (146.2 mg) and IV (123.1 mg). Upon further fractionation with prep-HPLC with H2O−MeOH (30/70 to 20/80 in 10 min, 20/80 during 5 min, 20/80 to 0/100 in 5 min, 0/100 during 10 min; flow rate 21 mL/min), fraction II led to the isolation of ilicicolinic acid A (1, 1.7 mg, tR = 20.56 min) and ilicicolinic acid C (2, 1.2 mg, tR = 26.18 min). Fraction IV was fractionated by prep-HPLC with H2O−ACN (70/30 to 30/70 in 15 min, 30/70 during 5 min, 30/70 to 0/100 in 10 min, 0/100 during 5 min; flow rate 21 mL/min), leading to the isolation of ilicicolinic acid D (3, 1.8 mg, tR = 31.88 min). For ilicicolinal (4), 481.0 mg of the crude extract was purified by flash chromatography with a linear gradient of hexane−EtOAc followed by another gradient of EtOAc− MeOH. Twelve fractions were collected based on their TLC profiles. The antifungal activity was found to be concentrated in fraction XI (20.0 mg). Upon further fractionation with prep-HPLC with H2O− ACN (60/40 to 50/50 in 30 min, 50/50 to 0/100 in 1 min, 0/100 during 4 min; flow rate 17 mL/min), fraction XI led to the isolation of ilicicolinal (4, 4.4 mg, tR = 29.45 min). Ilicicolinic acid C (2): light beige; [α]25D +87.5 (c 0.08, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.4), 264 (3.4), 306 (3.2) nm; NMR spectral data, see Supporting Information and Table 1; HRESITOFMS [M − H]− m/z 423.1933 (calcd for C23H32ClO5 m/z 423.1938). Ilicicolinic acid D (3): yellow powder; UV (CHCl3) λmax (log ε) 244 (3.6), 265 (3.6), 311 (3.3) nm; NMR spectral data, see Supporting Information and Table 1; HRESITOFMS [M + H]+ m/z 425.2075 (calcd for C23H34ClO5 m/z 425.2095). Ilicicolinal (4): light beige oil; [α]25D +32.3 (c 0.44, CHCl3); UV (CHCl3) λmax (log ε) 246 (3.5), 298 (3.6), 309 (3.6) nm; NMR
Table 2. Minimum Inhibitory Concentration (MIC), Antibacterial Selectivity Index (SI), and Cytotoxicity of the Isolated Compounds SIb
MIC (μg/mL) strain
1
2
3
4
pos. ctrl.a
1
2
3
C. albicans (ATCC 10231) C. parapsilosis (ATCC 22019) A. f umigatus (SNB-AF1) T. rubrum (SNB-TR1) E. coli (ATCC 25922) S. aureus (ATCC 29213)
16
32
16
128
4
0.5
0.2
1.6
16
32
16
128
4
0.5
0.2
1.6
64
32
128
>128
0.5
0.1
0.2
0.2
8
4
32
128
0.03
1.0
1.5
0.8
1
8
8
64
8
8.0
0.7
3.3
4
32
32
128
0.5
2.0
0.2
0.8
IC50 (μM) KB cells MDA435 cells MRC5 cells
1
2
3
4
pos. ctrl.c
36.6 15.1 19.7
28.4 4.9 13.7
47.3 42.4 62.1
16.5 16.4 15.1
0.0002 0.02 0.0005
a Positive control: oxacillin: S.aureus; gentamicin: E. coli; fluconazole: Candida sp.; itraconazole: T. rubrum, A. f umigatus. bSelectivity index (SI = IC50/MIC) based on IC50 measured in healthy MRC5 cells. c Positive control: taxotere: KB, MRC5; doxorubicin: MDA435.
E. coli and were weakly cytotoxic. Compound 4 was inactive against all pathogens tested. Ilicicolinic acid A was more active against E. coli than the cell line MRC5, indicating a potentially favorable selectivity (SI = 8). In conclusion, we demonstrated that N. discophora SNBCN63 produces antimicrobial metabolites. While the symbiotic nature of this strain toward the N. corniger termite species has yet to be demonstrated, it is shown that termite nests can host a diversity of microbes; this particular N. discophora strain produces previously unknown antibacterial compounds.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was measured using an Anton Paar MCP 300 polarimeter in a 100 mm long 350 μL cell. UV spectra were recorded in CHCl3 using a PerkinElmer Lambda 5 spectrophotometer. NMR spectra were recorded in CDCl3 on a Bruker 500 MHz spectrometer or a Bruker 600 MHz spectrometer equipped with a 1 mm inverse detection probe. Chemical shifts (δ) are reported as ppm based on TMS signal. C
dx.doi.org/10.1021/np500080m | J. Nat. Prod. XXXX, XXX, XXX−XXX
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spectral data, see Supporting Information and Table 1; HRESITOFMS [M + H]+ m/z 407.1989 (calcd for C23H32ClO4 m/z 407.1989). Antimicrobial Assay. The ATCC strains were purchased, and the clinical isolates were kindly provided by Prof. Philippe Loiseau, Université Paris Sud. These strains were identified by Philippe Loiseau and Christian Bories, with molecular analysis conducted by BACTUP. ITS sequences were deposited in the NCBI GenBank database under the registry numbers KC692746 (Trichophyton rubrum SNB-TR1) and KC692747 (Aspergillus f umigatus SNB-AF1). Extracts, fractions, and pure compounds were tested according to the reference protocol of the European Committee on Antimicrobial Susceptibility Testing.6 The MIC value was obtained after 18 h for yeasts, 5 days for T. rubrum, and 24 h for the other pathogens. Cytotoxicity Assays. Cytotoxicity assays were conducted with KB (nasopharyngeal epidermoid carcinoma), MDA435 (melanoma, previously described as mammary gland carcinoma), and MRC5 (normal lung tissue of a 14-week-old male Homo sapiens fetus) cell lines according to the procedure described by Tempête et al.7
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(5) Kuroda, M.; Takatsu, T.; Takahashi, H.; Hosoya, T.; Furuya, K. New compound ilicicolinic acid A or B. Jpn. Kokai Tokkyo Koho JP05255184, 1993. (6) (a) Rodrigues, A. M. S.; Theodoro, P. N. E. T.; Eparvier, V.; Basset, C.; Silva, M. R. R.; Beauchêne, J.; Espindola, L. S.; Stien, D. J. Nat. Prod. 2010, 73, 1706. (b) Casella, T. M.; Eparvier, V.; Mandavid, H.; Bendelac, A.; Odonne, G.; Dayan, L.; Duplais, C.; Espindola, L. S.; Stien, D. Phytochemistry 2013, 96, 370. (7) Tempête, C.; Werner, G.; Favre, F. Eur. J. Med. Chem. 1995, 30, 647.
ASSOCIATED CONTENT
* Supporting Information S
NMR spectra of compounds 1−4 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(V. Eparvier) Tel: +33 169 82 36 79. Fax: +33 169 07 72 47. E-mail:
[email protected]. *(D. Stien) Tel: +33 169 82 36 10. Fax: +33 169 07 72 47. Email:
[email protected]. Author Contributions
The manuscript was written through the contributions of all authors. All authors have approved the final version of this manuscript Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has benefited from an “Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-0025). The authors are very grateful to P. Loiseau for providing wild strains of pathogenic fungi.
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REFERENCES
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