Introduction

Human African trypanosomiasis, also known as sleeping sickness, is a potentially fatal parasitic disease transmitted by the bite of the tsetse fly that plagues many regions of Africa. Although the number of people being infected with the disease has declined due to better diagnosis and treatment, there were still more than 7000 new cases recorded in 2010.1 Without treatment, sleeping sickness is fatal. Morever current therapies often have unpleasant side effects.

During the course of our screening program for compounds that treat the disease, which is caused by the parasite T. brucei, we have reported on various natural products, such as microbial metabolites and plant products that exhibit potent antitrypanosomal properties.2, 3, 4, 5, 6, 7, 8 This time we focused on metabolites from cultures of Cordyceps that showed antitrypanosomal activity. Cordyceps is a fungal genus which is endopathogenic with arthropods, principally insects. This genus is proving to be a rich source of novel secondary metabolites including important compounds for pharmaceutical discovery.9, 10, 11, 12, 13, 14

In 2004, Professor Joseph W. Spatafora reported on C. cardinalis, a pathogen of lepidopteran larvae, a new species from the southern Appalachian Mountains of the eastern United States and from southeastern Japan.15 C. cardinalis NBRC 103832 was isolated from single ascospores of a fruiting body occurring on a lepidopteran larva from Japan (Takatsuki, Osaka). The single stroma is ochraceous orange to red and less than 30 mm. The anamorphic stage on PDA is Mariannaea-like, producing imbricate conidia chains. Sequence analysis of this strain based on rDNA ITS1-5.8S-ITS2 showed 99% similarity with C. cardinalis BCMU CC01 (AB237660).

Here, we report the isolation, structural determination and antitrypanosomal activity of three depsipeptides, cardinalisamides A–C (1–3).

Results

Extraction and isolation

Laboratory cultures of C. cardinalis NBRC 103832 were grown as lawns on solid agar. The mycelia were harvested and extracted exhaustively with MeOH. The mycelial extract was evaporated to an aqueous concentrate and successively extracted with EtOAc. The extracted material (7.1 g) was chromatographed on silica gel by n-hexane and an increasing ratio of CHCl3 and by preparative HPLC using 70–75% MeOH to afford three new compounds, cardinalisamides A–C (1–3) (Figure 1).

Figure 1
figure 1

Structures of cardinalisamides A–C (1–3). A full color version of this figure is available at The Journal of Antibiotics journal online.

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Structure elucidation

Cardinalisamide A (1) was obtained as an oil, for which analysis based on HRFABMS data (m/z 687.3373 for [M+Na]+) gave the molecular formula C36H48N4O8. The 1H and COSY NMR spectra (Table 1) showed typical features for peptides, with an amide proton signal (δH 7.49), an N-methyl group (δH 3.27) and four α protons (δH 5.60, 5.23, 4.43, 3.18). The 13C NMR and HSQC spectra and a DEPT experiment indicated three carbonyl carbons, six olefinic carbons, three methines, three methylenes, one downfield methyl at 37.7 ppm and two upfield methyl signals. Based on the carbon numbers in 13C NMR and the molecular formula, 1 possesses C2 symmetry. Interpretation of COSY, HSQC and HMBC spectra allowed three distinct fragments to be constructed (Figure 2). These included two phenyllactic acid (Pla) moieties and amino acid moieties, such as two N-methylglycines (N-MeGly) and two leucines (Leu). The sequence of 1 was assigned by HMBC analysis using correlations from the α-protons or the N-methyl protons to the carbonyl carbon resonances. The linkage of N-MeGly and Pla was established by HMBC correlations from the α-proton of Pla (δH 5.60) to the N-MeGly carbonyl (δC 166.7). Connection of Leu and Pla was determined on the basis of the HMBC correlation from the α-proton of Leu (δH 5.23) to the Pla carbonyl (δC 168.4). Further, an HMBC correlation from the N-methyl singlet proton (H-12, δH 5.23) to the Leu carbonyl (δC 173.9) revealed the linkage of N-MeGly to Leu. Intense NOESY correlations from the 14-NH to H-2′ and from the N-methyl protons to H-14 strongly suggested an alternate linkage of two amide bonds. Finally, the closure of the ring allowed the planar structure of cardinalisamide A (1) to be assigned.

Table 1 NMR data for cardinalisamides A (1)–C (3) in CDCl3
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Figure 2
figure 2

COSY and selected HMBC correlations for 1, indicating the sequence of six residues. A full color version of this figure is available at The Journal of Antibiotics journal online.

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The absolute configuration of Pla in 1 was determined as D by chiral HPLC analyses using direct comparison with authentic specimens. The absolute configuration of the amino acid (Leu) in 1 was assigned by application of Marfey’s method.16, 17, 18 Acid hydrolysis of the hydrogenated product of 1 yielded the expected free amino acids, which were derivatized using FDAA (Marfey’s reagent, 1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide) and analyzed by LC/MS by comparing the chromatograms with those derivatives of authentic specimens. Retention times (in min) of the FDAA derivatives of Leu were L-Leu (16.6) and D-Leu (17.8). The amino acids (Leu × 2) in 1, which had a retention time of 17.4, thus have the D-configuration.

The same extract afforded cardinalisamide B (2) as an oil, which was assigned the molecular formula C37H50N4O8 based on HRFABMS data (m/z 701.3534 for [M+Na]+). This indicated that 2 differed from 1 by the presence of a CH2 unit. The 1H and 13C NMR spectra of 2 (Table 1) were similar to those of 1 except for C2 symmetry in 1. COSY, HSQC and HMBC spectral data allowed the assignment of one N-MeGly, one N-methylalanine (N-MeAla), two Leu and two Pla. The sequence of 2 was also determined by HMBC spectra and NOE correlations between H-12 and H-14, 14-NH and H-20, H-30 and H-32 and 32NH and H-2. The absolute configuration of the amino acids and the Pla was deduced as follows. Acid hydrolysis and FDAA derivatization of Leu (16.7) revealed L-Leu by LC-MS analysis. The absolute configuration of N-MeAla was determined using the advanced Marfey method (using an FDLA derivative) because the retention times of the FDAA derivatives of D- and L-N-MeAla were very similar. FDLA derivatization of N-MeAla (9.2) revealed L-N-MeAla by LC-MS analysis. The configuration of the Pla was determined to be D, based on comparison of the retention time of the Pla from the hydrolysate with those of authentic specimens of D- and L- Pla on a chiral HPLC column.

Cardinalisamide C (3) was obtained as an oil that showed an [M+Na]+ peak at m/z 715.3712, for a molecular formula of C38H52N4O8 by HRFABMS, which differs from that of 2 by the presence of a CH2 unit. The 1H and 13C NMR spectra of 3 (Table 1) were similar to those of 2. 3 possessed C2 symmetry based on its carbon numbers in the 13C NMR and the molecular formula. The difference in 3 was the replacement of an N-MeGly unit in 2 with an N-MeAla unit. Analysis of 1H NMR, 13C NMR, HSQC, COSY and HMBC spectra revealed the presence of two N-MeAla, two Leu and two Pla moieties. Finally, the sequence of 3 was established by HMBC correlations and supported by observed NOE enhancements. The absolute configurations of the amino acid units of 3 were determined in an identical fashion to that used for 2. Acid hydrolysis and L-FDAA derivatization of Leu (16.6) and L-FDLA derivatization of N-MeAla (9.2) revealed L-N-MeAla and L-Leu by LC-MS analysis, respectively. Finally, chiral-phase HPLC revealed L-Pla, completing the absolute configurational assignment of cardinalisamide C (3).

Biological activity

Cardinalisamides A–C (1–3) showed in vitro antitrypanosomal activity against T. brucei brucei with IC50 values of 8.56, 8.65 and 8.63 μg ml−1, and cytotoxicity against normal human diploid fibroblasts (MRC-5 cells) with IC50 values of 18.48, 14.00 and 23.84 μg ml−1, respectively.

Experimental section

General experimental procedures

Optical rotations were recorded on a JASCO P-1030 polarimeter. IR spectra were measured on a Shimazu FTIR-8400S instrument. NMR spectra were obtained on a Varian UNITY 600 NMR spectrometer. The chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. HRMS spectra were obtained on a JEOL JMS-700 instrument. Kieselgel 60 (230–400 mesh, Merk) was used for column chromatography, and silica gel 60 F-254 (Merk) for TLC. HPLC was performed on a JASCO-PU 1580 instrument with a COSMOSIL C18 P-MS II (250 × 20 mm).

Fungal material, extraction and isolation

The fungus C. cardinalis NBRC 103832 was identified by Dr Ban of the Nite Biological Resource Center (NBRC). A voucher specimen (C. cardinalis NBRC 103832) is deposited at the NBRC ulture Collection. C. cardinalis NBRC 103832 was cultured for 2 weeks at 25°C in potato sucrose medium (10 l culture). Mycelia were separated from the broth by filtration and subsequently extracted twice with MeOH to yield 16.3 g of a crude extract in an ultrasonic bath for 60 min. The extract was concentrated and the resulting residue was extracted with EtOAc (500 ml × 3). The EtOAc-soluble portion (7.1 g) was repeatedly subjected to silica gel flash column chromatography (using increasing concentrations of CHCl3 and MeOH in n-hexane as eluent). Further purification was carried out by repeated preparative HPLC on an ODS C-18 column, using MeOH and H2O as a solvent system to afford 1 (12.6 mg), 2 (53.6 mg) and 3 (34.9 mg).

Cardinalisamide A (1): pale yellow oil; [α]D23−46.6° (c 0.33, MeOH); FT-IR (film) cm−1: 3299, 1751, 1672, 1636, 1626; HRFABMS m/z: [M+Na]+ 687.3373, calcd for C36H48N4O8Na 687.3370; COSY and HMBC data see Figure 1.; Selected NOESY (H/H) 12/14, 14-NH/20.

Cardinalisamide B (2): pale yellow oil; [α]D23−86.8° (c 0.59, MeOH); FT-IR (film) cm−1: 3279, 1753, 1672, 1636, 1628; HRFABMS m/z: [M+Na]+ 701.3534, calcd for C37H50N4O8Na 701.3526; COSY (H/H) 2/3, 5/6, 6/7, 7/8, 8/9, 11/11′, 14NH/14, 14/15, 15/16, 16/17, 16/18, 20/21, 23/24, 24/25, 25/26, 26/27, 32NH/32, 32/33, 33/34, 34/35, 34/36; HMBC (H/C) 2/1, 2/10, 3/5, 3/4, 3/9, 5/3, 9/3, 11/10, 11/12, 11/13, 12/11, 12/13, 13/12, 14/13, 14/19, 20/19, 20/28, 21/22, 21/23, 21/27, 23/21, 27/21, 29/28, 29/30, 29/31, 30/29, 30/31, 32/31, 32/1, 32NH/1; Selected NOESY (H/H) 12/14, 14-NH/20, 30/32, 32-NH/2.

Cardinalisamide C (3): pale yellow oil; [α]D23−105.4° (c 0.84, MeOH); FT-IR (film) cm−1: 3275, 1751, 1672, 1659, 1629; HRFABMS m/z: [M+Na]+ 715.3712, calcd for C38H52N4O8Na 715.3683; COSY (H/H) 2/3, 5/6, 6/7, 7/8, 8/9, 11/11′, 14NH/14, 14/15, 15/16, 16/17, 16/18; HMBC (H/C) 2/1, 2/4, 3/1, 3/4, 3/5, 5/6, 5/7, 7/5, 11/10, 11/12, 11/13, 11′/10, 12/11, 12/13, 14/13, 14/1, 15/13; Selected NOESY (H/H) 5 or 9/11, 12/14, 14-NH/2′.

Acid hydrolysis of 1–3

A tube containing 1–3 (1.2 mg) and 6 M HCl (1 ml) was kept at 110 °C for 21 h. This solution was evaporated to dryness under vacuum to give the hydrolysate of 1–3, respectively.

Absolute configuration of Pla in 1–3

The hydrolysate was dissolved in 1 ml of water, with 5 μl of this solution used for one analysis. HPLC analysis was performed on DAICEL OD-RH chiral column (250 × 4.6, 5 μm; solvents: (A) pH 2 formic acid+water, (B) 20% MeCN; 0.5 ml per min; 25 °C). The configuration was determined by co-injection with L-Pla (tR 8.8) and D-Pla (tR 8.5).

Preparation and analysis of Marfey derivatives

1–3 (0.2 mg) was hydrolyzed by heating in HCl (6 M; 1 ml) at 110 °C for 14 h. After cooling, the solution was lyophilized and redissolved in 0.1 N HCl (100 μl). To the acid hydrolysate solution (or to 50 μl of a 50 mM solution of the respective amino acid) was added a 1% (w/v) solution (100 μl) of FDAA9 or FDLA10 in acetone. After addition of NaHCO3 solution (1 M; 20 μl) the mixture was incubated for 1 h at 40 °C. The reaction was stopped by addition of HCl (1 M; 20 μl), the solvents were evaporated to dryness, and the residue was redissolved in acetonitrile (100 μl). An aliquot of this solution (20 μl) was analyzed by HPLC (COSMOSIL 5C18 MS-II, 150 × 4.6, 5 μm; solvents: (A) 0.05% TFA water, (B) 0.05% TFA MeCN; linear gradient of from 10% MeCN to 70% MeCN in 15 min and isocratic at 70% MeCN for the next 10 min.; 0.5 ml min−1; 25 °C). Retention times (min) of the FDAA derivatives of Leu were as follows: L-Leu (16.6) and D-Leu (17.8).

(COSMOSIL 5C18 MS-II, 150 × 4.6, 5 μm; solvents: (A) water+pH 2 formic acid, (B) MeCN; 40% B; 0.5 ml min−1; 25 °C). Retention times (min) of the FDLA derivatives of N-MeAla were as follows: L-N-MeAla (9.2) and D-N-MeAla (11.7).

Trypanosome

The bloodstream forms of T. b. brucei strain GUTat 3.1 parasites were used for experimentation as described previously.19

In vitro assay

The in vitro antitrypanosomal assays using T b brucei strain GUTat 3.1 have been described previously.19 In brief, 95 μl of parasite suspension was incubated with 5 μl of drug solution for 72 h and Alamar Blue was used for parasite survival determination to calculate IC50 values.

Cytotoxicity assay against human diploid embryonic cell line MRC-5 was carried out as previously described.20