Anandins A and B, Two Rare Steroidal Alkaloids from a Marine Streptomyces anandii H41-59

Abstract

Anandins A (1) and B (2), two rare steroidal alkaloids, were isolated from the fermentative broth of a marine actinobacteria Streptomyces anandii H41-59. The gross structures of the two alkaloids were elucidated by spectroscopic methods including HR-ESI-MS, and NMR. Their absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of their experimental and calculated electronic circular dichroism spectra, respectively. Anandin A exhibited a moderate inhibitory effect against three human cancer cell lines MCF-7, SF-268, and NCI-H460 with IC₅₀ values of 7.5, 7.9, 7.8 μg/mL, respectively.

1. Introduction

Because of the increasing difficulty to discover new bioactive compounds from terrestrial sources and the structure diversity of marine metabolites, many researchers have great interest in investigating secondary metabolites from marine-derived organisms [1]. Marine organisms are regarded as a prolific resource of novel bioactive metabolites, including a vast array of macrolide, cyclic peptides, pigments, polyketides, terpenes, steroids and alkaloids, but only a few steroidal alkaloids [2]. Steroidal alkaloids are a class of alkaloids with the basic steroidal skeleton containing a nitrogen atom, either in a ring or in a side chain. Structurally, these alkaloids can be classified into three major groups according to their carbon skeleton, namely, pregnane alkaloids, cholestane alkaloids and C-nor-D-homosteroidal alkaloids. Accumulated evidence in previous studieshas demonstrated that steroidal alkaloids and their glycosides have a wide range of bioactivities, such as antimicrobial, cytotoxic, anti-HIV, anti-inflammation, and antinociceptive activities [3,4,5].

Steroidal alkaloids are well-known metabolites of certain terrestrial plants, mainly from Solanaceae, Liliaceae, Apocynaceae and Buxaceae families [6,7,8]. The first steroidal alkaloid was isolated from the berries of Solanum nigrum almost 200 years ago [9]. However, steroidal alkaloid was not found from marine organisms until 1984 when two antimicrobial plakinamines were isolated from sponge Plakina sp. [10]. Later, four novel cortistatins exhibiting highly selective anti-proliferative activity were isolated from Corticium sp. [11], and cytotoxic cephalostatins were discovered from Cephalodiscus gilchristi [12]. Compared to those from plants, the marine steroidal alkaloids were relatively few, but with diverse chemistry structures.

Streptomyces are known for their ability to produce novel structural and bioactive metabolites [13,14,15]. While searching for bioactive compounds from marine actinomycete, we encountered a strain of actinomycete Streptomyces anandii H41-59, the crude extract of which showed strong antifungal activity against Candida albicans. In aprevious report, we have isolated three new ergosterols and ten known ones from culture broth of the strain H41-59 [16]. Further investigation on the broth of the same marine actinomycete led to the isolation of two novel ergostane-type steroidal alkaloids, named anandins A (1) and B (2). In addition, the antibioticand cytotoxic activities of the two steroidal alkaloids were tested in the present study. Herein, details of the isolation, structure elucidation, configuration assignment, and bioactivities of the new metabolites are described.

2. Results and Discussion

2.1. Elucidation of New Compounds

Actinomycete Streptomyces anandii H41-59 was isolated from a sea sediment sample from the mangrove zone in the South China Sea. An ethyl acetate partition of the ethanol extract was subjected to silica gel and LH-20 column chromatography, followed by RP-HPLC purification to yield two rare steroidal alkaloids (Figure 1).

Compound 1 was obtained as a colorless plate crystal and given a molecular formula of C₂₃H₃₅NO₂. Its seven degrees of unsaturation were determined on the basis of HR-ESI-MS [M + H]⁺ ion at m/z 358.2752 (C₂₃H₃₆NO₂, calcd. 358.2741) (see Figure S1-8). The ¹H NMR spectrum (see Figure S1) of 1 measured in acetone-d₆ (

Table 1. ¹H and ¹³C NMR data of 1 and 2 (δ in ppm, J in Hz).

Position	1 a		2 b
	δH	δC	δH	δC
2	-	171.1	-	171.1
3	5.63 (br.s)	115.3	5.52 (d, 1.9)	116.5
4	-	152.0	-	164.9
5	-	141.5	-	89.3
6	5.60 (m)	107.4	1.65 (m), 1.92 (m)	36.0
7	2.63 (m), 2.34 (dd, 2.5, 17.5)	40.8	2.23 (m), 1.60 (m)	35.7
8	-	47.0	-	49.1
9	2.68 (m)	49.4	2.65 (m)	50.1
10	1.48 (m), 1.93 (m)	29.7	1.46 (m), 1.91 (m)	29.6
11	1.51 (m), 1.94 (m)	23.1	1.58 (m), 1.65 (m)	22.0
12	1.59 (m)	55.6	1.53 (m)	56.3
13	0.71 (s)	12.4	0.60 (s)	12.1
14	2.13 (m)	41.1	2.11 (br. s)	41.2
15	1.08 (d, 6.9)	21.5	1.08 (d, 6.9)	21.5
16	5.27 (dd, 7.7, 15.1)	136.4	5.28 (dd, 7.6, 14.6)	136.4
17	5.30 (dd, 7.2, 15.1)	133.1	5.30 (dd, 7.2, 14.6)	133.1
18	1.90 (m)	43.8	1.90 (m)	43.8
19	1.51 (m)	33.9	1.50 (m)	33.9
20	0.84 (d, 6.9)	20.1	0.85 (d, 6.9)	20.1
21	0.87 (d, 6.9)	20.4	0.88 (d, 6.9)	20.4
22	0.95 (d, 6.8)	18.2	0.96 (d, 6.9)	18.2
1′	3.64 (m)	42.8	3.32 (m), 3.60 (m)	42.5
2′	3.63 (m)	61.1	3.66 (m)	62.3
5-OH			5.12 (br.s)	-
2′-OH	3.87 (br.s)	-	4.36 (br.s)	-

^a Measured in CD₃COCD₃ at 300 MHz for ¹H and 75 MHz for ¹³C NMR; ^b Measured in CD₃COCD₃ at 600 MHz for ¹H and 125 MHz for ¹³C NMR.

) revealed the presence of five characteristic steroid methyl groups including a methyl singlet at δ_H 0.71 (Me-13) and four methyl doublets at δ_H 1.08 (Me-15), 0.84 (Me-20), 0.87 (Me-21) and 0.95 (Me-22). The ¹³C NMR and DEPT spectra indicated the presence of 23 carbon signals including a carbonyl carbon at δ_C 171.1 (C-2), six olefinic carbons at δ_C 115.3 (C-3), 152.0 (C-4), 141.5 (C-5), 107.4 (C-6), 136.4 (C-16), and 133.1 (C-17), a quaternary carbon at δ_C 47.0 (C-8), five methine carbons at δ_C 49.4 (C-9), 55.6 (C-12), 41.1 (C-14), 43.8 (C-18) and 33.9 (C-19), one oxygenated methylene at δ_C 61.6 (C-2′), four methylenes at δ_C 40.8 (C-7), 29.7 (C-10), 23.1 (C-11), 20.8 (C-1′), and five methyl carbons at δ_C 12.4 (C-13), 21.5 (C-15), 20.1 (C-20), 20.4 (C-21) and 18.2 (C-22). Further analyses of the 1D NMR and HSQC data suggested that compound 1 was more likely to be a highly degraded sterol.

The H-H COSY spectrum of 1 gave several spin systems which belong to substructures as shown in Figure 2 with bold lines. The HMBC correlations from a sp²methine proton at δ_H 5.27 (1H, dd, H-16) to carbons at δ_C 21.5 (C-15, ³J) and 43.8 (C-18, ³J), and from a sp²methine proton at δ_H 5.30 (1H, dd, H-17) to carbon at δ_C 41.1 (C-14, ³J), confirmed the presence of a characteristic side chain of ergosterol [16]. A methyl proton at δ_H 0.71 (3H, s, Me-13) displayed four HMBC correlations to carbons at δ_C 40.8 (C-7, ³J), 47.7 (C-8, ²J), 49.4 (C-9, ³J) and 55.6 (C-12, ³J), interpreted as direct attachment to the quaternary C-8. The HMBC correlations from the proton at δ_H 1.93 (1H, m, H-10) to carbons at δ_C 47.7 (C-8, ³J) and 55.6 (C-12, ³J), from the proton at δ_H 5.60 (1H, m, H-6) to carbons at δ_C 47.7 (C-8, ³J) and 152.0 (C-4, ³J), from protons at δ_H 2.63 (2H, m, H-7)/2.34 (dd, 2.5, 17.5) to carbons at δ_C 141.5 (C-5, ³J), 49.4 (C-9, ³J) and 55.6 (C-12, ³J), confirmed the substructure of the B ring and C ring of ergosterol. The HMBC correlations from the proton at δ_H 5.63 (1H, br.s, H-3) to carbons at δ_C 171.1 (C-2, ²J), 141.5 (C-5, ³J) and 49.4 (C-9, ³J), from protons at δ_H 3.64 (2H, m, H-1′) to carbons at 171.1 (C-2, ³J) and 141.5 (C-5, ³J), and from protons at δ_H 3.63 (2H, m, H-2′) to carbon at δ_H 171.1 (C-2, ⁴J), confirmed the presence of an α,β-unsaturated γ-lactam and two methylene carbons linked to γ-lactam and the hydroxyl group.

The relative configuration of 1 was determined on the basis of the analysis of ¹H-¹H coupling constants and the NOESY information (Figure 3). Large coupling constants of 15.1 Hz between H-16 and H-17, and the NOESY correlations between H-17 and H-14, between H-17 and H-20/H-21, between H-16 and H-12, and between H-16 and H-18 indicated the E configuration of the double bond in the side chain. A correlation was also observed between H-13β and H-14, confirming the β-orientation of H-14. The absolute configuration of compound 1 was unequivocally defined as (16E,8R,12R,14S,18R) by single crystal X-ray diffraction analysis (see Table S1) using Cu Ka radiation with Flack and Hooft parameters of 0.1 (2) and 0.15 (6), respectively. Compound 1 is a new steroidal alkaloid, named as anandin A.

Compound 2 was isolated as white powder. Its molecular formula was determined as C₂₃H₃₇NO₃ with six degrees of unsaturation, by the HR-ESI-MS [M + H]⁺ ion at m/z 376.2860 (C₂₃H₃₈NO₃, calcd. 376.2853) (see Figure S2-8). The ¹³C NMR and DEPT (see Figure S2) spectroscopic data of 2 measured in acetone-d₆ (Table 1) indicated the presence of 23 carbon signals for a carbonyl carbon at δ_C 171.1 (C-2), four olefinic carbons at δ_C 116.5 (C-3), 164.9 (C-4), 136.4 (C-16), 133.1 (C-17), one quaternary signal at δ_C 49.1 (C-8), one oxygenated methylene at δ_C 62.3 (C-2′), five methine carbons at δ_C 50.1 (C-9), 56.3 (C-12), 41.2 (C-14), 43.8 (C-18) and 33.9 (C-19), five methyl groups at δ_C 12.1 (C-13), 21.5 (C-15), 20.1 (C-20), 20.4 (C-21) and 18.2 (C-22). The ¹H and ¹³C NMR spectroscopic data of 2 were comparable to those of 1, suggesting that 2 is an analogue of 1 with an additional oxygenated quaternary carbon and the absence of a double bond. The gross structure of 2 was further elucidated by analysis of COSY and HMBC spectrum data (Figure 4). The substructure of γ-hydroxy-α,β-unsaturated γ-lactam was deduced by the HMBC correlations from H-3 (δ_H 5.52, br.s, 1H) to C-2 (δ_C 171.1, ²J) and C-5 (δ_C 89.3, ³J), from H-1′ (δ_H 3.60, 3.32) to C-2 (δ_C 171.1, ³J) and C-5 (δ_C 89.3, ³J), from H-2′ (δ_H 3.66, m, 2H) to C-2 (δ_C 171.1, ⁴J), and from H-7 (δ_H 2.23/1.60, m, 2H) to C-5 (δ_C 89.3, ³J). The HMBC correlation from H-9 (δ_H 2.65, m, 1H) to C-5 (δ_C 89.3, ³J), C-3 (δ_C 116.5, ³J) and C-12 (δ_C 56.3, ³J), from H-11 (δ_H 1.65/1.58, m, 2H) to C-9 (δ_C 50.1, ³J), from H-9 (δ_H 2.65, m, 1H) to C-5 (δ_C 89.3, ³J) and C-3 (δ_C 116.5, ³J), confirmed the substructure of the B ring and the saturated C ring of ergosterol. Other HMBC correlations permitted further confirmation of the carbon skeleton of 2.

In the NOESY spectrum, NOE correlations (Figure 5) of H-13 with H-14, and H-9 with H-12, confirmed the β-orientation of the side chain and the α-orientation of H-12, respectively. A large coupling constant (14.7 Hz) between H-16 and H-17 indicated the E configuration of the double bond between C-16 and C-17. The relative configuration at C-14 and C-18 was tentatively assigned to be identical to those in 1 based on similar NMR spectrum data and biogenetic consideration. The orientation of 5-OH (δ_H 5.12) was tentatively defined to be α, because the comparison of the experimental and calculated CD spectra (Figure 6) facilitated assignment of the absolute configuration of 2 as 16E,5R,8R,12R,14S,18R. Compound 2 is named as anandin B.

Anandins A (1) and B (2) are two rare steroidal alkaloids possessing a unique highly degraded ergosterol skeleton and a conjugated γ-lactam moiety. Why do two novel conjunction-type steroidal alkaloids exist in the ethanol extract of the mycelium material of Streptomyces anandii H41-59? With further investigation, we found that a cholesterol oxidase (Cho), usually observed among Gram-positive G+C-rich actinobacteria, is able to oxidize and degrade steroids [17]. In a previous study, we discovered a series of ergosterols including three new ones [16]. Thus, we considered that anandins A (1) and B (2) were probably derived from a highly degraded ergosterol [18,19].

2.2. Bioactivities of 1 and 2

Anandins A (1) and B (2) were evaluated in a wide panel of biological assays, including antimicrobial activity against Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus sp. and Dickey azeae, and anti-proliferative activity against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268 and human lung cancer cell line NCI-H460 by the methods described below. As shown in

Table 2. Cytotoxicities of 1 and 2 (IC₅₀: μg/mL).

Cell Line	1	2	cis-Dichlorodiamine Platinum
MCF-7	7.5	>50	4.0
SF-268	7.9	>50	41.0
NCI-H460	7.8	>50	25.1

Concentration range: 1.56–100 μg/mL; IC₅₀: half maximal inhibitory concentration.

, both compounds 1 and 2 were shown to be active against three cancer cell lines. As a result, compound 1 exhibited moderate cytotoxicity against MCF-7, SF-268 and NCI-H460 with IC₅₀ values of 7.5, 7.9, 7.8 μg/mL, respectively. However, both the compounds were inactive against the tested strains at the concentration of 20 μg/mL, even if the crude extract displayed moderate antimicrobial activity (inhibiting the zone of 16 mm against C. albicans with 6 mm paper discs at the concentration of 20 μg/mL).

3. Experimental Section

3.1. General

Melting points were measured on an X-5 micro-MP apparatus (Huayan Corporation, Shanghai, China), uncorrected. Optical rotations were measured with a JASCO digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were measured on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). IR data were recorded with a Nicolet Impact 410-FTIR instrument (Thermo, San Jose, CA, USA) in KBr pellets. HR-ESI-MS were acquired on an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were measured on a Bruker AV-300 and AV-600 spectrometer (Bruker Instrument, Inc., Zurich, Switzerland). Chemical shifts were expressed in δ (ppm) and referenced to the NMR solvent used. X-ray crystallographic analysis was performed on an X calibur, sapphires, Gemini ultra diffractometer (Oxford Diffraction Ltd., Tokyo, Japan). The crystal was kept at 173.00(10) K during data collection. Using Olex2, the structure was solved with the ShelXS structure solution program using Direct Methods and refined with the ShelXL refinement package using Least Squares minimisation. HPLC was performed on an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array detector, using a column A (Ultimate XB-C18, 5 μm, 4.6 × 250 mm, Welch, Potamac, MA, USA) for analysis and a semi-preparative HPLC column B (Ultimate XB-C18, 5 μm, 10 × 250 mm, Welch, Potamac, MA, USA) for purification. Open column chromatography was performed on silica gel (300–400 mesh, Qingdao Haiyang Chemical Group Corporation, Qingdao, China). Sephadex LH-20 (25–100 mm) was purchased from Pharmacia (Uppsala, Sweden). HSGF254 silica gel TLC plates (0.2 mm thickness, 200 × 200 mm, Qingdao Marine Chemicals Co., Qingdao, China) were used for routine analysis of fractions. Strains Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus sp., and Dickey azeae were from the Institute of New Drug Research (Guangzhou, China) in our college.

3.2. Strain Isolation and Identification

The isolation and activation of actinomycete strain H41-59; the identification of morphological characteristics, physiological and chemical properties; the molecular genetic analysis and information on storing the strain are mentioned in our previous report [16].

3.3. Fermentation and Isolation

Fermentation of strain H41-59 including condition and media compositions were reported in our earlier paper [16]. The extract and partition procedure from fermentation material for the EtOAc extract were introduced in the same paper. The ethyl acetate-soluble extract (50 g) was dissolved in chloroform and loaded on the silica gel column (1.5 kg, 300–400 mesh, Qingdao, China) after filtration. A stepwise gradient elution of petroleum ether-EtOAc (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 2:8 and 0:10 (v/v)) was used, and 20 fractions (Fr-1 to Fr-20) were obtained through TLC analysis and combination of fractions with same TLC pattern. Fr-6 was separated on Sephadex LH-20 (2 × 200 cm, CH₂Cl₂-MeOH, 1:1) and purified via semi-preparative HPLC (MeOH-H₂O, 90:10) to yield two new natural products, namely, 1 (10 mg) and 2 (3 mg).

Compound 1 (anandin A): colorless plate crystal; M.P. = 133–135 °C; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −13.5° (c 0.25, CHCl₃); UV (MeOH) λ_max (logε): 204.6 (3.88), 263.8 (3.73) nm; IR (KBr) ν_max: 3382, 2958, 2930, 2871, 1666, 1650, 1372, 1300, 1191, 1111, 1015, 981, 620 cm⁻¹; ¹H NMR (CD₃COCD₃, 300 MHz) and ¹³C NMR (CD₃COCD₃, 75 MHz), see Table 1; HR-TOF-ESI-MS (positive) m/z 358.2752 [M + H]⁺ (calcd. for C₂₃H₃₆NO₂, 358.2741).

Compound 2 (anandin B): white powder; M.P. = 129–131 °C; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −15.1° (c 0.25, CHCl₃); UV (MeOH) λ_max (logε): 208.2 (3.78) nm; IR (KBr) ν_max: 3423, 2929, 2853, 1650, 1542, 1461, 1381, 1190, 1110, 620 cm⁻¹; ¹H NMR (CD₃COCD₃, 600 MHz) and ¹³C NMR (CD₃COCD₃, 150 MHz), see Table 1; HR-ESI-MS: m/z 376.2860 [M + H]⁺ (calcd. for C₂₃H₃₈NO₃, 376.2846).

3.4. Single-Crystal X-ray Data for Anandin A (1)

Crystal data (CCDC No. 1544792) for 1: C₂₃H₃₅NO₂, M = 357.52, T = 100(2) K, monoclinic, space group C2, a = 21.7844 (3) Å, b = 7.2393 (11) Å, c = 13.1475 (20) Å, α = 90.00°, β = 93.2128 (13)°, γ = 90.00°, V = 2070.15 (5) ų, Z = 4, μ (Cu Kα) = 0.555 mm⁻¹, 16,181 reflections measured, 3274 independent reflections (R_int = 0.0282). The final R₁ values were 0.0286 (I > 2σ (I)). The final wR (F²) values were 0.0747 (I > 2σ (I)). The final R₁ values were 0.0290 (all data). The final wR (F²) values were 0.0750 (all data). The goodness of fit on F² was 1.058. Flack parameter = 0.1 (2). Hooft parameter = 0.15 (6).

3.5. ECD Calculation

ECD calculations of Anandium B (2): The systematic random conformational analysis of two possible stereoisomers (5R and 5S) of 2 was performed in the SYBYL 8.1 program by using a MMFF94s molecular force field, which afforded 40 and 42 conformers for 5R and 5S respectively, with an energy cutoff of 10 kcal mol⁻¹ to the global minima. All the obtained conformers were further optimized using DFT at the B3LYP/6-31+G (d) level in gas phase by using Gaussian09 software (Gaussian, Inc., Wallingford, CT, USA), and eight conformers of each stereoisomer were selected. All of the optimized stable conformers were used for TDDFT (cam-B3LYP/6-31+G (d)) computation of the excited states at the same levels, with consideration of the first 50 excitations. The overall ECD curves were weighted by the Boltzmann distribution of each conformer (with a half-band width of 0.25 eV), with a UV correction of 10 nm. The calculated ECD spectra of 5R and 5S of 2 were subsequently compared with the experimental spectra, respectively. The ECD spectra were produced by SpecDis 1.6 software (T. Bruhn, Y. Hemberger, A. Schaumlöffel, G. Bringmann, SpecDis version 1.6, University of Wuerzburg, Wuerzburg, Germany).

3.6. Biological Activities

3.6.1. Antimicrobial Activity

Antimicrobial evaluation of compounds 1 and 2 was performed following the same method and procedure as in a previous paper [16].

3.6.2. Cytotoxicity Assay

Three cancer cell lines MCF-7, SF-268, and NCI-H460 were used for measuring the cytotoxicities of compounds 1 and 2. For details on the procedure, we refer readers to our previous paper [16]. The 50% inhibition concentrations (IC₅₀) of compounds 1 and 2 against the test cells were calculated using Origin 8 software (OriginLab, Northampton, MA, USA).

4. Conclusions

Anandins A (1) and B (2) were obtained from the fermented mycelia of Sterptomyces anandii H41-59. They have the same basic skeleton, and belong to a rare type of steroidal alkaloids. The structures of 1 and 2 were elucidated on the basis of extensive spectroscopic data including HR-ESI-MS, NMR, and X-ray crystallography for 1, and calculation chemistry for 2. To the best of our knowledge, 1 and 2 are the second reported compounds and second and third compounds with this type of structure. Anandin A (1) exhibited moderate in vitro inhibitory activity against MCF-7, SF-268, and NCI-H460 cell lines by the MTT method. Neither 1 nor 2 showed any antibiotic activities against the test pathogenic microorganisms.