Actinoranone, A Cytotoxic Meroterpenoid of Unprecedented Structure from a Marine Adapted Streptomyces sp

Abstract

The isolation and structure elucidation of a new meroterpenoid, actinoranone (1), produced by a marine bacterium closely related to the genus Streptomyces is reported. Actinoranone is composed of an unprecedented dihydronaphthalenone polyketide linked to a bicyclic diterpenoid. The stereochemistry of 1 was defined by application of the advanced Mosher's method and by interpretation of spectroscopic data. Actinoranone (1) is significantly cytotoxic to HCT-116 human colon cancer cells with an LD₅₀ = 2.0 μg/mL.

The variability of environmental chemical and physical conditions in the oceans has clearly played a major role in the evolution of a great diversity of microorganisms.¹ Our evidence suggests that obligate marine microorganisms have, in part, adapted to life in the sea through the production of unique secondary metabolites that distinguish them from their terrestrial counterparts.² Thus, investigations of the chemistry and biology of these unique molecules could lead to the discovery of a significant number of new drug leads.³

The chemically-prolific genus Streptomyces, which is abundant and well-known in terrestrial habitats, also appears to be represented in marine ecosystems. In recent years, expanding studies of marine-derived and obligate marine actinomycetes have yielded bioactive molecules with diverse chemical structures. ⁴ Phylogenetic classification, by analysis of 16S rRNA gene sequence data, has indicated that new taxa at both the genus and species levels are present.⁵

As part of our continuing program to explore the biomedical potential of marine actinomycetes, we have previously isolated and reported the structures of several highly modified peptides, actinoramides A-C, from our marine actinomycete strain CNQ-027 (GenBank accession number EU214912).⁶ This seawater-requiring strain shares only 97.6% 16S rRNA gene sequence identity with an obligate marine Streptomyces species, S. marinus, suggesting it may represent a new Streptomyces species adapted to live in the sea. ⁷

A more extensive study of the secondary metabolite composition of this strain, has now yielded a new meroterpenoid, actinoranone (1), which possesses a new carbon skeleton and significant cancer cell cytotoxicity. Details of the isolation, characterization, and biological activity of actinoranone are presented here.

The molecular formula of actinoranone (1)⁸ was defined as C₃₂H₄₆O4, based on analysis of HRESIMS data (a pseudomolecular ion peak at m/z 495.3450 [M+H]⁺), and on interpretation of ¹³C NMR data. The ¹H NMR spectrum of 1 displayed meta-coupled aromatic protons [δ 6.56 (d, J = 2.0 Hz), 6.50 (d, J = 2.0 Hz)], two olefinic protons [δ 5.38 (br s), 5.25 (d, J = 8.5 Hz)], and one downfield methine protons [δ 4.47 (dd, J = 8.5, 8.5 Hz)]. The ¹H NMR spectrum also showed two methoxyl groups [δ 3.87, 3.83], and five methyl singlets [δ 1.71, 1.63, 0.90, 0.86, 0.78](Table 1). Analysis of ¹³C NMR and gHSQC spectral data revealed seven methyl, eight methylene, eight methine, and nine fully-substituted carbons. Analysis of the gCOSY spectroscopic data for 1 revealed the connectivity of four partial structures; substructure a (C-1 to C-3), substructure b (C-5 to C-7), substructure c (C-9 to C-11 and C-11 to C-12), and substructured (C-15 to C-8′, and C-8′ to C-10′), as shown in Figure 1. Substructures (a and b) and gHMBC correlations from gem-dimethyl singlets (H-16 and H-17) to carbons C-3, C-4, and C-5, and from a methyl singlet H-18 to carbons C-1, C-5, C-9, and C-10, and from a methyl singlet H-19 to carbons C-7, C-8, C-9 allowed the terpenoid bicyclic ring to be defined. The dihydronaphthalenone moiety was also constructed by interpretation of gCOSY and gHMBC spectroscopic data. The presence of meta-coupled aromatic protons [δ 6.56 (d, J = 2.0 Hz), 6.50 (d, J = 2.0 Hz)] indicated a 1, 2, 3, 5-tetrasubstituted benzene ring. The composition of substructure d and gHMBC correlations from H-9′ to carbons C-1′, C-7′, and from H-8′ to carbons C-2′, C-6′, and C-7′, and from H-11′ to C-3′, and from H-12′ to C-5′, and from H-4′ to C-2′, C-6′ confidently established the dihydronaphthalenone ring. Last, gHMBC correlations from the H-20 methyl singlet to carbons C-12, C-13, and C-14 permitted the connectivity of C-14 and C-15, thus completing the assignment of the planar structure of 1 (Figure 1).

Table 1. NMR Data for Actinoranone 1 (methanol-d₄)^a.

no	δC, #H.b	δH (mult, J (Hz))	HMBC
1	39.3, CH2	1.85* 0.94 dt (13.5, 3.6)	3, 18
2	25.4, CH2	1.55, m 1.29, m	4, 10
3	42.3, CH2	1.42, m 1.16*	5
4	32.7, C
5	50.4, CH	1.16, m	7
6	23.7, CH2	1.97, m 1.86, m	7, 8
7	122.2, CH	5.38, br s	6, 8, 19
8	135.0, C
9	54.2, CH	1.61, m	11, 12
10	36.7, C
11	25.4, CH2	1.55, m 0.78, m	8, 13
12	42.1, CH2	2.24, m 2.01, m	9, 13, 14
13	139.5, C
14	126.8, CH	5.25, d (8.5)	15, 20
15	70.4, CH	4.47, dd (8.5, 8.5)	8′
16	32.5, CH3	0.86, s	3, 4, 5, 17
17	21.0, CH3	0.90, s	3, 4, 5, 16
18	12.8, CH3	0.78, s	1, 5, 9, 10
19	21.5, CH3	1.71, s	7, 8, 9
20	15.6, CH3	1.63, s	12, 13, 14
1′	197.3, C
2′	115.5, C
3′	162.7, C
4′	97.2, CH	6.50, d (2.0)	2′, 5′, 6′
5′	164.4, C
6′	107.5, CH	6.56, d (2.0)	2′, 4′, 5′, 7′, 8′
7′	150.4, C
8′	45.3, CH	2.97, m	15, 2′, 6′, 7′, 9′, 10′
9′	23.8, CH2	2.12, m	1′, 7′
10′	36.3, CH2	2.44, ddd (18.0, 11.0, 8.0) 2.59, dt (18.0, 4.5)	1′, 2′, 8′, 9′
11′	54.9, CH3	3.83, s	3′
12′	55.0, CH3	3.87, s	5′

^a500 MHz for ¹H NMR and 75 MHz for ¹³C NMR.

^bThe number of attached protons was determined by analysis of 2D spectroscopic data.

^*The coupling constant was not determined because of overlapping signals.

Figure 1. gCOSY and key gHMBC correlations assisting in the structure elucidation of actinoranone (1).

The relative configuration of the bicyclic terpenoid ring in 1 was assigned by interpretation of ROESY NMR correlations. Correlations between the H-18 methyl singlet and the H-11 methylene protons, and between H-5 and H-9, allowed the relative configurations to be assigned as 5R*, 9R*, and 10R*. The 7Z and 13E doublebond geometries were also assigned by the observation of ROESY NMR correlations between the H-7 olefinic proton and the H-19 methyl singlet, and between the H-12 methylene protons and the H-14 olefinic proton, respectively.

Next, application of the advanced Mosher's method allowed the absolute configuration at C-15 to be defined.⁹ Treatment of 1 with (R)- and (S)-MTPA-Cl [α-methoxy-α-(trifluoromethyl) phenylacetyl chloride] yielded the (S)- and (R)-MTPA ester derivatives, respectively. Calculation of the ¹H NMR Δδ_S-R values for the mono-MTPA esters of 1 established the absolute configuration at C-15 as R (Figure 2a). HETLOC ¹⁰ and HSQMBC ¹¹ NMR experiments were undertaken to determine the configuration of C-8′, but we were unable to measure the coupling constants due to the complexity of signals (data not shown). As an alternative approach, we carefully analyzed the ROESY NMR data hoping to assign the absolute configuration of C-8′. There were only two possible stereoisomers, (15R, 8′ R) or (15R, 8′S), for 1. The median value of the coupling constant (³J_H15-H8′ = 8.0 Hz) and ROESY correlations from H-8′ and H-6′ to H-15′, and from H-8′ and H-9′ to H-14 supported our assignment of an 8′R configuration (Figure 2b).

Figure 2.

a) Mosher's ester analysis of actinoranone (1), Δδ_S-R of ¹H for S- and R-mono-MTPA esters of 1, and b) A Newman projection of the C-8′ — C-15 bond illustrating the assigned C-8′ relative configuration.

In an attempt to determine the absolute configurations for C-5, C-9, and C-10, we performed a series of synthetic modifications. We protected the hydroxyl group at C-15 of 1 by acetylation and then we treated the 15-acetoxyl derivative 2 with OsO₄ to afford a mixture of 7, 8, 13, 14-tetra-ol products, which were then subjected to HPLC purification to obtain a major stereoisomer 3. ¹² The relative configuration of the bicyclic ring system of this stereoisomer was suggested as 5R*, 7S*, 8R*, 9S*, and 10R* based upon ROESY correlations between H-7 and H-19, between H-19 and H-11, and between H-11 and H-18 (see supporting information). Treatment of this stereoisomer with (R)- and (S)-MTPA-Cl produced crude bis-(S)- and bis-(R)-MTPA ester derivatives, respectively. However, on HPLC purification these ester derivatives spontaneously decomposed under all conditions explored. Next, we attempted chemical reactions to eliminate the stereocenters at C-15 and C-8′ in an effort to compare the optical rotations of derivatives of 1 with the known (+) isozonarol^13a and (-) isozonarol.^13b Actinoranone A was treated with p-toluenesulfonic acid (TSA) hoping to obtain the Δ^13,14,15,8′-diene.^14a However, we obtained the undesired dehydration product possessing the Δ^11,12,13,14-diene. We also attempted a base dehydration of actinoranone A using p-toluenesulfonyl chloride (TsCl),^14b but this yielded the same result. In addition, attempted aromatization of these unexpected products with Pd/C^14c did not produce any isolable products. Then, actinoranone A was treated with Dess-Martin periodinane (DMP) ^14d to oxidize the secondary alcohol to ketone 5, and then further oxidized by using 2-iodoxybenzoic acid (IBX)^14e hoping to obtain the C-8′-C-9′ α,β-unsaturated ketone. The reaction did not yield identifiable products.

All attempts defined above to eliminate the stereocenters at C-15 and C-8′ were unsuccessful (see supporting information for synthetic details). Thus, only the relative stereochemistry of 1 could be defined as 5R*, 9R*, 10R*, along with the absolute configurations 15R and 8′R.

Traditionally, actinomycetes are not considered producers of terpenoids. However, strains of a largely marine lineage we have classified as MAR4 consistently produce diverse meroterpenoids. ¹⁵ Three classes of meroterpenoids, exempified by azamerone, ¹⁶ marinone and neomarinone, ¹⁷ and the many members of the napyradiomycin family,¹⁸ have been isolated from these organisms. They commonly share a 2,3-dihydronaphthalene-1,4-dione or naphthalene-1,4-dione derived from the polyketide precursor 1,3,6,8-tetrahydroxynaphthalene (THN) and terpenoid substituents in the molecules. Given these comparisons, it is surprising that strain CNQ-027 does not belong to the MAR4 group of marine actinomycetes.

To the best of our knowledge, this is the first observation of the common diterpenoid “labdane” bicyclic ring system in microbial natural products. This is also the first report of a meroterpenoid isolated from a marine-derived actinomycete possessing the dihydronaphthalenone moiety. Actinoranone (1) showed in vitro cytotoxicity against HCT-116 human colon carcinoma with an LD₅₀ = 2.0 μg/mL. Testing in a broader number of diverse cancer cell lines is in progress.