Hormonemate Derivatives from Dothiora sp., an Endophytic Fungus

Article pubs.acs.org/jnp

Hormonemate Derivatives from Dothiora sp., an Endophytic Fungus Mercedes Pérez-Bonilla,*,† Víctor González-Menéndez,† Ignacio Pérez-Victoria,† Nuria de Pedro,† Jesús Martín,† Joaquín Molero-Mesa,‡ Manuel Casares-Porcel,‡ María Reyes González-Tejero,‡ Francisca Vicente,† Olga Genilloud,† José R. Tormo,† and Fernando Reyes*,† †

Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico Ciencias de la Salud, Avda. del Conocimiento 34, 18016 Armilla, Granada, Spain ‡ Departamento de Botánica, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain S Supporting Information *

ABSTRACT: A search for cytotoxic agents from cultures of the endophytic fungus Dothiora sp., isolated from the endemic plant Launaea arborescens, led to the isolation of six new compounds structurally related to hormonemate, with moderate cytotoxic activity against different cancer cell lines. By using a bioassay-guided fractionation approach, hormonemates A−D (1−4), hormonemate (5), and hormonemates E (6) and F (7) were obtained from the acetone extract of this fungus. Their structures were determined using a combination of HRMS, ESI-qTOF-MS/MS, 1D and 2D NMR experiments, and chemical degradation. The cytotoxic activities of these compounds were evaluated by microdilution colorimetric assays against human breast adenocarcinoma (MCF-7), human liver cancer cells (HepG2), and pancreatic cancer cells (MiaPaca_2). Most of the compounds displayed cytotoxic activity against this panel.

C

Endophytes are relatively underexplored as potential sources of novel natural products for exploitation in medicine, agriculture, or food. Of the approximately 300,000 higher plant species that exist on the earth, each individual plant is host to one or more endophytes.6,7 Consequently, the opportunity to find new and interesting endophytic microorganisms among myriads of plants in different settings and ecosystems is very wide. In addition, if endophytes can produce the same rare and important bioactive compounds as their host plant, this would not only reduce the need to harvest slowgrowing and possibly rare plants but also help to preserve the world’s ever diminishing biodiversity. Thus, more background information on a given plant species and its microorganism biology would be exceedingly helpful in directing the search for bioactive products.8 During the period 1990−2010, one hundred anticancer compounds belonging to 19 different chemical classes, with activity against 45 different cell lines, were isolated from over 50 different fungal species belonging to six different endophytic fungal groups. Of the total compounds isolated from endophytic fungi, 57% were novel compounds or analogues of known compounds.9 The identification and isolation of important plant-derived anticancer drugs, such as Taxol, camptothecin, podophyllotoxin, vinblastine, and vincristine, from endophytic fungi isolated from the producing plants and

ancer is one of the top ten leading causes of death globally, and the second after cardiovascular diseases in middle income countries according to the World Health Organization, and a big effort has been directed to identify new pathways, biomarkers, and agents that are likely to be effective against the treatment of this disease.1,2 The search for novel drugs is a priority in cancer chemotherapy due to the rapid development of resistance to multiple chemotherapeutic drugs, the high toxicity usually associated with this type of treatment, and their undesirable side effects. The discovery of novel natural drugs with reduced side-effects, high selectivity for cancer cell lines, low toxicity to normal cells, and efficient killing of cancer cells is desirable.3 Arid zones in Andalucı ́a have special edaphological and climatic conditions, such as low rainfall, high sunshine levels, and specific lithology, where loamy materials and evaporites abound. Among them, the Tabernas desert in Almerı ́a is designated as one of the Mediterranean’s biodiversity hot spots, with one of the richest steppic to subdesertic floras in Europe.4 Native plants from these ecosystems possess distinctive survival characteristics for these conditions, which has led to the existence of a high degree of endemic plants with highly adapted endophyte ecosystems, previously poorly studied. It is precisely this singularity which turns them into a valuable potential source for the isolation of new and unique hostspecific endophytes.4,5 Investigation of the secondary metabolites of microorganisms from unusual or specialized niches may increase the chances of finding novel compounds. © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 21, 2016 Published: March 9, 2017 845

DOI: 10.1021/acs.jnatprod.6b00680 J. Nat. Prod. 2017, 80, 845−853

Journal of Natural Products

Article

the identification of the gene/gene products controlling the production of these drugs could provide alternative sources for the production of key bioactive natural products.10 In fact, it is becoming increasingly clear that host specificity is a bona fide phenomenon in endophyte relationships with higher plants. Knowledge of such interactions can provide guidance as to which endophytes might be selected in the search for novel medicinal natural products.11 In our search for bioactive compounds from endophytes of endemic plants of the Tabernas desert, we observed cytotoxicity against the human cell lines MCF-7 and HepG2 in an acetone extract of the fungus Dothiora sp., isolated from Launaea arborescens. Additionally, the LC-HRMS profile of the extract revealed the presence of diverse secondary metabolites, which prompted a more detailed investigation of the extract. Extraction of the culture broth followed by a bioassay-guided fractionation of the extract led to the isolation of six new compounds structurally related to hormonemate. Despite the fact that the isolation of hormonemate had been reported previously,12 no spectroscopic data have been so far disclosed for this molecule. Structure elucidation of the new compounds and hormonemate itself is reported herein for the first time.

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RESULTS AND DISCUSSION Cultures of Dothiora sp. in YES medium were extracted with acetone and proved to be active against the MCF-7 and HepG2 human cancer cell lines. These extracts were fractionated using a bioassay-guided approach, by column chromatography and semipreparative reversed phase HPLC, to yield compounds 1− 7. Compound 1 was obtained as a colorless syrup. A molecular formula of C35H62O15 was assigned after analysis by ESI-TOF. The 1H NMR data of 1 indicated the presence of five doublets at 4.20, 5.09, 5.10, 5.11, and 5.15 ppm, corresponding to five oxygenated methines. In addition, three hydroxylated methines at 3.52−3.90 ppm, together with two oxygenated methylenes at 3.63 (2H), and 4.22 and 4.47 ppm were observed, suggesting the presence of a glycol moiety. Five methines at 1.88−2.10 ppm appeared in the aliphatic region, along with ten overlapped methylene protons at 1.35 and 1.53 ppm and ten overlapped aliphatic methyl groups at 0.93−1.02 ppm (Table 1). Although the 13C NMR spectrum displayed several overlapped signals, 35 carbons were identified based on multiplicity edited HSQC and HMBC experiments. The 13C NMR data exhibited different sets of signals corresponding to five ester carbonyl carbons appearing at 170.6 (×2), 170.8, 171.2, and 175.7 ppm; eight oxygenated methine carbons at 70.5, 71.6, 72.0, 74.2, 76.1, 76.3, 76.5, and 76.7 ppm; two oxygenated methylene carbons at 64.8 and 68.7 ppm; five aliphatic methine carbons at 37.8, 38.0, 38.1 (×2), and 40.1 ppm; five aliphatic methylene carbons at 26.9 (×2) and 27.0 (×3); and ten methyl carbons at 12.0 (×3), 12.1, 12.3, 14.0, 14.7 (×2), and 14.8 (×2). The 1D and 2D NMR data established five identical partial substructures of 2-hydroxy-3-methylpentanoyl moieties and one alditol of five carbons. The C-2 position of all but one of the 2-hydroxy-3-methylpentanoyl units was acylated, as confirmed by the downfield chemical shift of proton H-2 (δH 5.09−5.15). The remaining unit showed an upfielded chemical shift for H-2 (δH 4.20 ppm), indicating its terminal, nonacylated, position. HMBC correlations were used to establish the points of attachment between the different moieties. Longrange correlation of the methylene protons at 4.22 and 4.47 ppm to the carbonyl carbon at 171.2 (C-1) revealed that the

alditol moiety is attached to C-1 via an ester linkage (Figure 1). Methine protons at 5.09−5.15 ppm exhibited long-range correlations to the carbonyl carbons at 170.6−175.7 ppm (C1), suggesting that the five 2-hydroxy-3-methylpentanoyl moieties are connected to each other through ester linkages between the 1- and 2- positions (Figure 1), similar to that in liamocins and the antibiotic substance exophilin A.13,14 As further evidence, the saponification reaction of compound 1 with NaOH led to the cleavage of the ester bond and released the alditol and the acid salt. Subsequent acidification of the alkoxide ion with hydrochloric acid produced the carboxylic acid. NMR analysis of both units and their comparison with standards verified that the sugar alcohol was arabitol and the carboxylic acid was 2-hydroxy-3-methylpentanoic acid or isoleucic acid. As a result, the structure of compound 1 consists of a single arabitol headgroup 1-O-acylated with a chain of five 2-hydroxy-3-methylpentanoic ester units that was named hormonemate A. Compound 2 was also obtained as a colorless syrup. Its ESITOF spectrum had an ammonium adduct at m/z 710.4331 corresponding to a molecular formula of C34H60O14. The proton and carbon chemical shifts of compound 2 were similar 846



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Table 1. NMR Spectroscopic Data (500 Mz, CD3OD) for Hormonemate A (1) and Hormonemate B (2) hormonemate A (1) position

δC, type

hormonemate B (2)

δH (J in Hz)

HMBC

L-arabitol

1′a 68.7, CH2 1′b 2′ 70.5, CH 3′ 72.0, CH 4′a 71.6, CH 4′b 5′ 64.8, CH2 D-allo-isoleucic acid A 1 171.2, C 2 76.7, CH 3 37.8, CH 4a 27.0, CH2 4b 5 12.0, CH3 6 14.8, CH3 D-allo-isoleucic acid B 1 170.6, C 2 76.5, CH 3 38.1, CH 4a 27.0, CH2 4b 5 12.1, CH3 6 14.8, CH3 D-allo-isoleucic acid C 1 170.6, C 2 76.3, CH 3 38.1, CH 4a 27.0, CH2 4b 5 12.0, CH3 6 14.7, CH3 D-allo-isoleucic acid D 1 170.8, C 2 76.1, CH 3 38.0, CH 4a 26.9, CH2 4b 5 12.0, CH3 6 14.7, CH3 D-allo-isoleucic acid E 1 175.7, C 2 74.2, CH 3 40.1, CH 4a 26.9, CH2 4b 5 12.3, CH3 6 14.0, CH3

4.22, 4.47, 3.90, 3.52, 3.90,

dd (11.4, 6.6) dd (11.4, 2.6) m dd (8.8, 1.7) m

δH (J in Hz)

HMBC

erythritol 2′, 3′, 3′, 1′, 2′,

3′, 1A 1A 5′ 2′, 5′ 3′, 5′

3.63, d (6.3)

3′

5.15, 2.10, 1.35, 1.53, 0.96, 1.02,

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.11a, d (3.6) 2.10, m 1.35, m 1.53, m 0.96, m 1.02, m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.10a, d (3.8) 2.10, m 1.35, m 1.53, m 0.96, m 1.02, m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.09, 2.10, 1.35, 1.53, 0.96, 1.02,

d (3.3) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

4.20, 1.88, 1.35, 1.53, 0.96, 0.93,

d (3.5) m m m m m

3, 1, 2, 2, 4 2,

4, 4, 3, 3,

d (3.2) m m m m m

δC, type

4, 6, 1B 6 5, 6 5, 6

68.2, CH2 71.3, CH 73.5, CH 64.6, CH2

171.0, 76.6, 37.9, 27.1,

C CH CH CH2

12.0, CH3 14.9, CH3

3, 4

4, 6, 1C 6 5, 6 5, 6

170.6, 76.3, 38.1, 27.0,

C CH CH CH2

12.1, CH3 14.8, CH3

3, 4

4, 6, 1D 6 5, 6 5, 6

170.6, 76.6, 38.1, 27.0,

C CH CH CH2

12.1, CH3 14.7, CH3

3, 4

4, 6, 1E 6 5, 6 5, 6

170.8, 76.1, 38.1, 27.0,

C CH CH CH2

12.1, CH3 14.7, CH3

3, 4

6 5, 6 5, 6 5, 6

175.7, 74.2, 40.1, 26.9,

C CH CH CH2

12.3, CH3 14.1, CH3

3, 4

4.21, 4.41, 3.75, 3.56, 3.61, 3.75,

dd (11.6, 6.6) dd (11.6, 3.0) m m dd (11.2, 5.9) m

2′, 3′, 4′ 1′ 2′, 2′,

3′, 1A 1A

5.14, 2.10, 1.35, 1.53, 0.96, 1.01,

d (3.2) m m m m m

1, 1, 2, 2, 4 2,

3, 4, 3, 3,

5.11b, d (3.2) 2.10, m 1.35, m 1.53, m 0.96, m 1.01, m

1, 1, 2, 2, 4 2,

3, 4, 3, 3,

5.10b, d (3.6) 2.10, m 1.35, m 1.53, m 0.96, m 1.01, m

1, 1, 2, 2, 4 2,

3, 4, 3, 3,

5.09, 2.10, 1.35, 1.53, 0.96, 1.01,

d (3.2) m m m m m

3, 1, 2, 2, 4 2,

4, 4, 3, 3,

4.20, 1.88, 1.35, 1.53, 0.96, 1.01,

d (3.2) m m m m m

3, 1, 2, 2, 4 2,

4, 4, 3, 3,

3′ 3′

4, 5, 5, 5,

6, 1B 6 6 6

3, 4

4, 5, 5, 5,

6, 1C 6 6 6

3, 4

4, 5, 5, 5,

6, 1D 6 6 6

3, 4

6, 5, 5, 5,

1E 6 6 6

3, 4

6 5, 6 5, 6 5, 6

3, 4

a,b

Interchangeable signals.

to those of compound 1 for the polyester chain of five isoleucic acids. The major difference between the 1H NMR data of both compounds was the presence of only two hydroxylated methines at 3.56−3.75 ppm, together with two oxygenated methylenes (3.61 and 3.75, and 4.21 and 4.41 ppm), suggesting the presence of a glycol moiety having four carbon atoms instead of five. The 13C NMR spectrum showed 34 carbons with only seven oxygenated methine carbons at 71.3, 73.5, 74.2, 76.1, 76.3, and 76.6 (×2) ppm and two oxygenated methylene

carbons at 64.6 and 68.2 ppm (Table 1). A cross peak between the methylene protons H-1′ at 4.21 and 4.41 ppm and the carbonyl carbon at 171.0 (C-1) indicated that the alditol moiety is attached to the polyester chain of isoleucic acids via an ester linkage. Saponification followed by acidification and spectroscopic analysis of the products obtained, secured that the sugar alcohol was erythritol. As a consequence, the structure of compound 2 consists of a single erythritol headgroup 1-O-acylated with a 847



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and 4.23 and 4.50 ppm, suggesting that the alditol was connected to the chain of isoleucic acids by a terminal position. The downfield shift of the methylene carbon at 68.9 ppm in the 13 C NMR spectrum (C-1′) and the presence of a cross-peak in the HMBC spectrum between the methylene protons H-1′ at 4.23 and 4.50 ppm and the carbon at 171.1 ppm (C-1) confirmed the evidence of an ester linkage between position 1′ of the alditol and position 1 of the polyester chain of isoleucic acids, confirming the structure previously described as hormonemate.12 The HRMS spectrum of compound 6 presented an ammonium adduct at m/z 740.4437 corresponding to a molecular formula of C35H62O15. The proton and carbon chemical shifts of compound 6 were similar to those of compound 1. The main difference between the 1H and 13C NMR spectra of both compounds was the chemical shift of the oxygenated protons of the alditol. An oxygenated methine at 5.03 ppm appeared in the 1H NMR spectrum together with a downfield shifted signal at 76.8 ppm in the carbon spectrum. In addition, an upfield shifted resonance was observed for the methylene protons at 3.80 and 3.90 ppm along with the corresponding upfield shift of their carbon at 61.6 ppm. The long-range correlation in the HMBC spectrum between the methine proton at 5.03 ppm and the carbon at 170.5 ppm (C1) supported the evidence of an ester linkage between position 2′ of the alditol and position 1 of the polyester chain, to give the structure that we named hormonemate E. Compound 7 was assigned a molecular formula of C29H52O13, based on its HRMS analysis. The chemical shifts of the protons and carbons of compound 7 were similar to those of compound 1. The difference between the 1H and 13C NMR data of both compounds was the absence of signals corresponding to one unit of D-allo-isoleucic acid. Compound 7 consisted therefore in a single arabitol headgroup O-acylated with polyester tails containing four isoleucic ester groups, and was called hormonemate F. Figure 2 displays the overlapped HSQCs spectrum of hormonemate A (1) together with the standard L-arabitol. The zoom shot of the alditol region shows the shift of the methylene protons (H-1a and H-1b) from 3.66/3.83 ppm to 4.22/4.47 ppm due to the esterification of this position. As expected, the methine proton H-2 is also affected, with a slight shift downfield. These findings indicate that the O-linkage between the backbone of the arabitol to the 2-hydroxy-3methylpentanoyl polyester tail is through position 1 of the arabitol moiety. Similarly, Figure S1 (Supporting Information) points out the 1-O-linkage between erythritol and the 2hydroxy-3-methylpentanoyl polyester chain, due to the remarkable chemical downshift of the methylene protons H1a/H-1b, as well as the downshift of the methine proton H-2. The aforementioned evidence was also applicable to esterified mannitol as shown in Figure S2 (Supporting Information). Protons H-1′ and carbon C-1′ of the mannitol unit are highly affected by the esterification with the polyester chain (3.69/ 3.84 ppm in the mannitol to 4.23/4.50 in the esterified mannitol). The methine proton at position C-2′ is likewise being affected with a slight downfield shift from 3.74 ppm to 3.90. At the same time, the remaining protons and carbons were assigned from the multiplicity edited HSQC and HMBC data and were slightly affected. Further evidence of the structures proposed was also obtained through analysis of compounds 1, 2, and 5 by quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS/

Figure 1. Key HMBC correlations observed for compound 1.

chain of five isoleucic ester groups which was named hormonemate B. Compound 3, obtained as a colorless syrup, was assigned a molecular formula of C36H64O16 based on the ESI-TOF MS of the ammonium adduct. The NMR data of this compound were very similar to those of compounds 1 and 2, with the major difference being the presence of four oxygenated methines at 3.40, 3.92, 3.96, and 5.22, and two oxygenated methylenes at 3.50 and 3.66 ppm and 3.62 and 3.79 ppm, suggesting the presence of an alditol with six carbon atoms. The 13C NMR data displayed 36 carbons, accounting for the presence of five 2hydroxy-3-methylpentanoyl units, and four oxygenated methine carbons at 70.8, 72.0, 72.4, and 74.8 ppm, and two oxygenated methylene carbons at 64.7 and 65.2 ppm corresponding to the alditol moiety (Table 2). The downfield shifted resonance of proton H-3′ at 5.22 ppm in the 1H NMR data along with the downfield shift of the corresponding carbon C-3′ at 74.8 ppm in the 13C NMR data, and the long-range HMBC correlation between this proton and the carbon at 170.9 ppm (C-1) were consistent with the existence of an ester linkage between the 3′position of the alditol and position 1 of the chain of five isoleucic acids. COSY and HMBC correlations (Table 2) to other protons and carbons of the alditol confirmed this structural proposal, and compound 3 was named hormonemate C. Hormonemate D (4) was obtained as a colorless syrup. The HRESIMS data showed an ammonium adduct with the molecular formula C36H64O16. Its NMR data were similar to those of compound 3, with the major difference being in the chemical shift of the four oxygenated methines at 3.68, 3.46, 4.11, and 5.03, and two oxygenated methylenes at 3.60 and 3.79 ppm and 3.81 and 3.91 ppm, suggesting that the alditol was connected to the chain of isoleucic acids at a different position. The 13C NMR data also displayed 36 carbons with nine oxygenated methine carbons at 69.1, 71.6, 72.8, 74.2, 76.0, 76.3, 76.5, 76.6, and 76.8 ppm, together with two oxygenated methylene carbons at 61.8 and 65.2 ppm (Table 2). In this case, the deshielded proton at 5.03 ppm in the 1H NMR spectrum corresponded to H-2′ according to COSY and HMBC measurements. Its downshifted carbon at 76.8 ppm in the 13 C NMR data and the cross-peak in the HMBC spectrum between this proton and the carbon at 170.5 ppm (C-1) provided further evidence of an ester linkage between position C-2′ of the alditol and position C-1 of the chain of five isoleucic acids. Compound 5 was assigned a molecular formula of C36H64O16 based on its HRESIMS analysis. Its NMR data were similar to those of compounds 3 and 4, with the major difference being in the chemical shift of the four oxygenated methines at 3.68−3.90 ppm, and the two oxygenated methylenes at 3.64 and 3.81 ppm 848



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Table 2. NMR Spectroscopic Data (500 Mz, CD3OD) for Hormonemate C (3), Hormonemate D (4), and Hormonemate (5) hormonemate C (3) position

δC, type

D-mannitol 1′a 64.7, CH2 1′b 2′ 72.0, CH 3′ 74.8, CH 4′ 70.8, CH 5′ 72.4, CH 6′a 65.2, CH2 6′b D-allo-isoleucic acid A 1 170.9, C 2 76.7, CHa 3 37.7, CH 4a 27.5, CH2 4b 5 12.0, CH3 6 14.8, CH3 D-allo-isoleucic acid B 1 170. 8, Cb 2 76.3, CHa 3 38.1, CH 4a 27.0, CH2 4b 5 12.1, CH3 6 14.7, CH3 D-allo-isoleucic acid C 1 170.6, Cb 2 76.6, CHa

3 38.1, CH 4a 26.9, CH2 4b 5 12.1, CH3 6 14.7, CH3 D-allo-isoleucic acid D 1 170.7, C 2 76.1, CHa 3 38.1, CH 4a 27.1, CH2 4b 5 12.1, CH3 6 14.7, CH3 D-allo-isoleucic acid E 1 175.7, C 2 74.2, CH 3 40.1, CH 4a 27.1, CH2 4b 5 12.3, CH3 6 14.1, CH3 a−j

hormonemate D (4)

δH (J in Hz) 3.50, 3.66, 3.92, 5.22, 3.96, 3.40, 3.62, 3.79,

dd dd m dd dd m dd dd

(11.7, 6.9) (11.7, 3.5)

5.13, 2.12, 1.36, 1.53, 0.96, 1.02,

d (2.8) m m m m m

(7.8, 1.4) (9.4, 1.4) (11.4, 5.6) (11.4, 2.7)

HMBC 2′, 3′ 3′ 1′, 3′, 4′ 4′, 4′

3′

61.8

2′, 1A 5′, 6′ 5′

1, 3, 4, 6, 1B 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

5.12c, d (3.3) 2.12, m 1.36, m 1.53, m 0.96, m 1.02, m

1, 3, 4, 6, 1C 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

5.10c, d (3.4) 2.12, 1.36, 1.53, 0.96, 1.02,

m m m m m

1, 3, 4, 1D 1, 4, 5, 2, 3, 5, 2, 3, 5, 4 2, 3, 4

5.09, 2.12, 1.36, 1.53, 0.96, 1.02,

(3.3) m m m m m

1, 3, 4, 6, 1E 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

4.20, 1.88, 1.36, 1.53, 0.96, 0.92,

d (3.2) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

6, 6 6 6

4, 6 6 5, 6 5, 6

3, 4

δC

76.8 69.1 71.6d 72.8d 65.2

170.5 76.6e 37.7 27.2 12.0 14.9 170. 5 76.5e 38.1 26.9

hormonemate (5)

δH (J in Hz) 3.81, dd (12.3, 3.91, dd (12.3, 5.03, m 4.11, d (8.3) 3.46f, d (8.3) 3.68f, m 3.60, dd (11.1, 3.79, dd (11.1,

5.20, 2.10, 1.36, 1.54, 0.96, 1.02,

d (3.0) m m m m m

3.4) 3.4)

6.0) 4.6)

HMBC 2′, 3′ 3′, 1′, 5′, 3′, 4′, 4′,

3′

68.9

4′, 1A 2′ 6′ 4′, 6′ 5′ 5′

70.4 71.1 71.1 73.0 65.2

1, 3, 4, 6, 1B 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

12.1 14.8

5.12g, d (3.5) 2.10, m 1.36, m 1.54, m 0.96, m 1.02, m

1, 3, 4, 6, 1C 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

170.6 76.3e

5.11g, d (3.7)

1, 3, 4, 6, 1D

2.10, 1.36, 1.54, 0.96, 1.02,

m m m m m

1, 2, 2, 4 2,

5.09, 2.10, 1.36, 1.54, 0.96, 1.02,

d (3.4) m m m m m

1, 3, 4, 6, 1E 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

4.20, 1.88, 1.36, 1.54, 0.96, 0.93,

d (3.4) m m m m m

1, 1, 2, 2, 4 2,

38.1 27.0 12.1 14.7 170.7 76.0 38.0 27.0 12.0 14.7 175.7 74.2 40.1 27.0 12.3 14.0

δC

4, 5, 6 3, 5, 6 3, 5, 6

3, 4, 3, 3,

4, 5, 5, 5,

3, 4

6 6 6 6

HMBC

4.23, 4.50, 3.90, 3.78, 3.78, 3.68, 3.64, 3.81,

dd (11.4, 6.7) dd (11.4, 2.4) m m m m m m

2′, 3′, 4′ 1′, 2′, 3′ 5′ 5′

5.16, 2.10, 1.36, 1.53, 0.95, 1.01,

d (3.2) m m m m m

1, 3, 4, 6, 1B 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

12.1 14.8

5.11j, d (3.2) 2.10, m 1.36, m 1.53, m 0.95, m 1.01, m

1, 3, 4, 6, 1C 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

170.5h 76.3i

5.10j, d (3.4)

171.1 76.6 37.8 27.1 12.0 14.9 170.6h 76.5i 38.1 27.0

38.1 27.0 12.1 14.7

3, 4

δH (J in Hz)

170.7 76.0 38.0 26.9 12.1 14.7 175.6 74.2 40.1 26.9 12.3 14.0

1A 1A 5′ 6′

2.10, 1.36, 1.53, 0.95, 1.01,

m m m m m

1, 3, 4, 1D 1, 4, 5, 2, 3, 5, 2, 3, 5, 4 2, 3, 4

5.09, 2.10, 1.36, 1.53, 0.95, 1.01,

d (3.2) m m m m m

1, 3, 4, 6, 1E 1, 4, 5, 6 2, 3, 5, 6 2, 3, 5, 6 4 2, 3, 4

4.20, 1.88, 1.36, 1.53, 0.95, 0.92,

d (3.4) m m m m m

1, 1, 2, 2, 4 2,

3, 4, 3, 3,

4, 5, 5, 5,

6, 6 6 6

6 6 6 6

3, 4


MS). Data shown in the Supporting Information (Figures S53, S54, and S55) confirmed the connectivity of the monomers, and were also consistent with the structures proposed. To determine the absolute configuration of the 2-hydroxy-3methylpentanoic acid moiety present in all the compounds of the series, its NMR and optical rotation data after alkaline hydrolysis were compared with those published,15 demonstrat-

ing this was the D-allo (2R,3S) form of the acid based on the negative value of its specific optical rotation [α]25 D = −30.4° (c 0.01, MeOH).16 NMR and optical rotation data from the alditol residues were compared with those obtained for the authentic L-arabitol, erythritol, and D-mannitol (Figures S45−S50). In the case of arabitol, its negative specific optical rotation [α]25 D = −17.8° (c 0.06, MeOH) was consistent with that of standard L849



Article

Figure 2. Overlapped multiplicity edited HSQC spectra of hormonemate A (1) and L-arabitol, showing the “alditol” region. Red and blue signals correspond to hormonemate A whereas green and pink signals are those from L-arabitol.

arabitol, [α]25 D = −9.2° (c 0.08, MeOH). Erythritol is the meso form of an alditol with four carbon atoms, so the specific optical rotation measured was zero. Measurement of the optical rotation of mannitol in the presence of 10% ammonium molybdate and 1 N sulfuric acid gave a positive value of [α]25 D = +103.1°, confirming the presence of D-mannitol in compounds 3−5. Recently, a family of similar structures has been isolated from the polymorphic fungus Aureobasidium pullulans, a microorganism isolated from tiger lily flowers. A. pullulans, often considered to be a black yeast because cultures produce melanin, is an ascomycetous yeast belonging to the Dothideomycetes class, order Dothideales: the same as Dothiora sp. Apart from polysaccharide pullulans, A. pullulans produces extracellular heavy oils; these oils are 3,5-dihydroxydecanoyl and 5-hydroxy-2-decenoyl esters of mannitol, arabitol, or glycerol, named as liamocins.13,14 Interestingly, these alditol lipids exerted an antiproliferative effect on the human lung cancer cell line A54914,17 as well as in two human breast cancer cell lines (T47D and SK-BR3) and a human cervical cell line (HeLa).18 In addition, liamocins also showed antibacterial activity19 and act as biosurfactants with important biotechnological applications in the food industry, medicine, and cosmetics due to their specific modes of action and low toxicity.20 Similarly, hormonemates could also act as antibacterial and/or biosurfactants, expanding their potential applications.19,20 The cytotoxic activity of the hormonemates was tested against three tumor cell lines. Table 3 shows the IC50 values obtained for the pure compounds. No clear structure−activity relationship could be established from the values obtained. Overall, human breast cancer cells (MCF-7) exhibited the greatest sensitivity to hormonemates, mainly to hormonemate B (IC50 = 11.1 μM) and hormonemate A (IC50 = 15.6 μM). Additionally, an IC50 around 30 μM was measured for most of the compounds against human hepatic cancer cells (HepG2). Except hormonemate E (IC50 = 36.4 μM), none of them were active against the pancreatic cell line (MiaPaca_2) at the highest concentration tested.

Table 3. Cytotoxic Activity of the Isolated Compounds Expressed as Inhibitory Concentration (IC50) HepG2 hormonemate (1) hormonemate (2) hormonemate (3) hormonemate (4) hormonemate (5) hormonemate (6) hormonemate (7) doxorubicin

■

MCF-7

MiaPaca_2

μg/mL

μM

μg/mL

μM

μg/mL

μM

A

20.9

29.0

11.3

15.6

>40.0

>55.4

B

19.9

28.7

7.7

11.1

>40.0

>57.8

C

>40.0

>53.2

20.9

27.8

>40.0

>53.2

D

27.3

36.2

13.8

18.3

>40.0

>53.2

>40.0

>53.2

18.2

24.3

>40.0

>53.2

E

18.7

26.2

13.9

19.0

27.4

36.4

F

>40.0

>65.7

>40.0

>65.7

>40.0

>65.7

0.1

0.2

0.8

1.4

1.0

1.9

compounds

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Jasco P-2000 polarimeter. UV spectra were obtained with an Agilent 1100 DAD. IR spectra were recorded on a JASCO FT/ IR-4100 spectrometer equipped with a PIKE MIRacle single reflection ATR accessory. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively) equipped with a 1.7 mm TCI MicroCryoProbe (Bruker Biospin). Chemical shifts were reported in ppm using the signals of the residual solvent as internal reference (δH 3.31 and δC 49.15 for CD3OD, δH 4.75 for deuterium oxide). LC-MS and LC-HRMS analysis were performed as described previously.21 Experimental Biological Material. Cell culture medium, fetal bovine serum, L-glutamine, sodium pyruvate, MEM nonessential amino acids, penicillin-streptomycin, triple express, PBS, and bovine insulin were purchased from Life Technologies (Gigco, Invitrogen Corporation, CA, USA). Actinomycin D, doxorubicin, MTT (3-(4,5dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium bromide), and MMS (methylmethanesulfonate) were purchased from Sigma Chemical Co. (St. Louis MO. USA). Cells were maintained and plated with a cell culture robotic system, SelecT (TAP Biosystems). All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Medium, serum, and complements were purchased from Invitrogen. HepG2 cells (human liver carcinoma, CCL-8065) were grown in ATCC-formulated Eagle’s M essential medium (MEM) with 10% qualified FBS, 2 mM L-glutamine, 1 mM 850



Article

Table 4. NMR Spectroscopic Data (500 Mz, CD3OD) for Hormonemate E (6) and Hormonemate F (7) hormonemate E (6) position

δC, type

L-arabitol 1′a 61.6, CH2 1′b 2′ 76.8, CH 3′ 70.0, CH 4′ 71.8, CH 5′ 64.5, CH2 D-allo-isoleucic acid A 1 170.5, C 2 76.5, CHa 3 37.9, CH 4a 27.3, CH2 4b 5 12.0, CH3 6 14.9, CH3 D-allo-isoleucic acid B 1 170. 6, Cb 2 76.4, CHa 3 38.2, CH 4a 26.9, CH2 4b 5 12.1, CH3 6 14.8, CH3 D-allo-isoleucic acid C 1 170.7, Cb 2 76.7, CHa 3 38.1, CH 4a 27.0, CH2 4b 5 12.1, CH3 6 14.8, CH3 D-allo-isoleucic acid D 1 170.8, C 2 76.1, CHa 3 38.1, CH 4a 27.0, CH2 4b 5 12.0, CH3 6 14.7, CH3 D-allo-isoleucic acid E 1 175.7, C 2 74.2, CH 3 40.1, CH 4a 27.0, CH2 4b 5 12.3, CH3 6 14.1, CH3

a−d

hormonemate F (7)

δH (J in Hz) 3.80, 3.90, 5.03, 3.87, 3.61, 3.61,

dd (12.3, 3.3) dd (12.3, 4.5) m d (8.4) m m

3′ 2′, 3′, 1′, 5′ 3′,

5.21, 2.10, 1.35, 1.53, 0.98, 1.02,

d (3.1) m m m m m

1, 4, 2, 2, 4 2,

c

5.12 , d (3.2) 2.10, m 1.35, m 1.53, m 0.98, m 1.02, m

c

δC, type

HMBC

1, 4, 2, 2, 4 2,

68.7, CH2 3′ 4′, 1A 2′ 4′

3, 5, 3, 3,

4, 6, 1B 6 5, 6 5, 6

4, 6, 1C 6 5, 6 5, 6

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.09, 2.10, 1.35, 1.53, 0.98, 1.02,

d (3.4) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

4.20, 1.88, 1.35, 1.53, 0.98, 0.93,

d (3.4) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

171.2, 76.7, 37.9, 27.1,

C CH CH CH2

170.9, 76.5, 37.1, 27.0,

Cd CH CH CH2

12.1, CH3 14.8, CH3

3, 4

5.10 , d (3.4) 2.10, m 1.35, m 1.53, m 0.98, m 1.02, m

CH CH CH CH2

12.0, CH3 14.9, CH3

3, 4

3, 5, 3, 3,

70.5, 72.1, 71.7, 64.8,

4, 6, 1D 6 5, 6 5, 6

170.7, 76.1, 38.2, 27.0,

Cd CH CH CH2

12.1, CH3 14.7, CH3

3, 4

4, 6, 1E 6 5, 6 5, 6

170.7, 74.2, 40.1, 27.0,

C CH CH CH2

12.3, CH3 14.1, CH3

3, 4

δH (J in Hz)

HMBC

4.23, 4.47, 3.90, 3.53, 3.89, 3.63,

dd (11.4, 6.5) dd (11.4, 2.7) m dd (8.7, 1.8) dd (6.2, 1.8) d (6.2)

2′, 3′, 5′ 1′, 3′ 3′,

3′, 1A 1A

5.15, 2.09, 1.36, 1.53, 0.97, 1.02,

d (3.3) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.10, 2.09, 1.36, 1.53, 0.97, 1.02,

d (3.4) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

5.09, 2.09, 1.36, 1.53, 0.97, 1.02,

d (3.4) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

4.20, 1.88, 1.36, 1.53, 0.97, 0.93,

d (3.5) m m m m m

1, 4, 2, 2, 4 2,

3, 5, 3, 3,

2′, 5′ 4′

4, 6, 1B 6 5, 6 5, 6

3, 4

4, 6, 1C 6 5, 6 5, 6

3, 4

4, 6, 1D 6 5, 6 5, 6

3, 4

4, 6 6 5, 6 5, 6

3, 4

4, 6 6 5, 6 5, 6

3, 4


sodium pyruvate, and 100 μM MEM nonessential amino acids. MCF-7 cells (human breast HTB-22) were maintained in the previous medium supplemented with 0.01 mg/mL bovine insulin. MiaPaca_2 cell line (ATCC CRL-1420), a fibroblast primary pancreatic adenocarcinoma cell line which has mutated K-RAS, P16 y P53 genes, was grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 2.5% fetal horse serum, 1% L-glutamine, and 1% penicillin/streptomycin. Cell cultures were maintained in a humidified incubator at 37 °C with 5% CO2. Plant Material. Aerial parts of L. arborescens were collected from Tabernas desert (Almerı ́a, Spain), in May 2014. The taxonomical

identification was made by one of us, J. Molero-Mesa from the Deparment of Botany, Faculty of Pharmacy, University of Granada, where a voucher specimen has been deposited. Producing Fungus, Fungal Dimorphic Growth, and Cultivation. The producing organism (CF-285353) was isolated from aerial parts of the plant by using a method for isolating fungal endophytes described previously.5 Its ITS1-5.8S-ITS228S region or independent ITS and 28S rDNA was completely sequenced and compared with GenBank (KU295575) and the NITE Biological Resource Center (http://www.nbrc.nite.go.jp/) databases by using the BLAST application. Frozen stock cultures in 10% glycerol (−80 °C) 851



Article 13

C NMR data see Table 1; (+)-HRESIMS m/z 740.4437 [M+NH4]+ (calcd for C35H66NO15, 740.4427). Hormonemate B (2). colorless syrup; [α]D25 +27.4° (c 0.25, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3374, 2965, 2937, 2879, 1749, 1462, 1385, 1185, 1127, 1105, 1046, 1023 cm−1; 1H and 13C NMR data see Table 1; (+)-HRESIMS m/z 710.4331 [M +NH4]+ (calcd for C34H64NO14, 710.4321). Hormonemate C (3). colorless syrup; [α]25 D +5.25° (c 0.08, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3363, 2965, 2937, 2879, 1750, 1642, 1385, 1185, 1127, 1105, 1021 cm−1; 1H and 13 C NMR data see Table 2; (+)-HRESIMS m/z 770.4547 [M+NH4]+ (calcd for C36H68NO16, 770.4533). Hormonemate D (4). colorless syrup; [α]25 D +11.3° (c 0.17, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3363, 2965, 2937, 2879, 1750, 1642, 1385, 1185, 1127, 1105, 1021 cm−1; 1H and 13 C NMR data see Table 2; (+)-HRESIMS m/z 770.4546 [M+NH4]+ (calcd for C36H68NO16, 770.4533). Hormonemate (5). colorless syrup; [α]25 D +39.2° (c 1.11, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3363, 2965, 2937, 2879, 1750, 1642, 1385, 1185, 1127, 1105, 1021 cm−1; 1H and 13C NMR data see Table 2; (+)-HRESIMS m/z 770.4547 [M+NH4]+ (calcd for C36H68NO16, 770.4533). Hormonemate E (6). colorless syrup; [α]D25 +32.5° (c 0.27, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3387, 2965, 2937, 2879, 1751, 1462, 1185, 1126, 1046, 1023 cm−1; 1H and 13C NMR data see Table 4; (+)-HRESIMS m/z 740.4437 [M+NH4]+ (calcd for C35H66NO15, 740.4427). Hormonemate F (7). colorless syrup; [α]25 D + 0.6° (c 0.03, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3387, 2965, 2937, 2879, 1751, 1462, 1385, 1185, 1126, 1046, 1023 cm−1; 1H and 13C NMR data see Table 4; (+)-HRESIMS m/z 626.3750 [M+NH4]+ (calcd for C29H56NO13, 626.3746). Alkaline Hydrolysis of 1, 2, and 5. To solutions in THF (0.4 mL) of compounds 1 (3.1 mg), 2 (3.1 mg), and 5 (2.6 mg) was added NaOH 2 N (0.2 mL), and the resulting solution was stirred for 15 min at room temperature. After hydrolysis was completed, reaction mixtures were neutralized with HCl 1 N and evaporated to dryness. The residues were subjected to semipreparative reversed-phase HPLC (Zorbax SB-C18, 9.4 × 250 mm, 5 μm, 3.6 mL/min, detection at 210 nm) using a linear gradient of CH3CN in water (5−10% CH3CN over 20 min after a 10 min period at 5% CH3CN) for elution. This afforded the alditols, L-arabitol (0.4 mg, 4.0 min), erythritol (0.3 mg, 4.0 min), and D-mannitol (0.4 mg, 4.0 min), together with D-allo-isoleucic acid (1.9, 2.1, and 1.6 mg respectively, 6.0 min). 25 D-allo-Isoleucic acid. white amorphous powder; [α]D −30.4° (c 0.01, MeOH); 1H NMR (500 MHz, D2O) δ 4.00 (1H, d, J = 2.80 Hz, H-2), 1.79−1.74 (1H, dddq, J = 2.80, 4.20, 7.04, 9.88 Hz, H-3), 1.44− 1.35 (1H, m, H-4b), 1.31−1.22 (1H, m, H-4a), 0.91 (3H, t, J = 7.41 Hz, H-5), 0.77 (3H, d, J = 7.04 Hz, H-6); 13C NMR (D2O) (obtained from HSQC) δ 75.32 (C-2), 38.76 (C-3), 26.63 (C-4), 13.67 (C-6) and 11.99 (C-5). 25 L-Arabitol. white amorphous powder; [α]D −17.8° (c 0.06, 1 MeOH); H NMR (500 MHz, D2O) δ 3.95 (1H, td, J = 2.06, 7.18 Hz, H-4), 3.84 (1H, dd, J = 2.96, 11.71 Hz, H-1b), 3.77 (1H, td, J = 2.96, 8.10 Hz, H-2), 3.67−3.69 (3H, m, H-1a, H-5) and 3.60 (1H, dd, J = 2.06, 8.10 Hz, H-3). 1 Erythritol. white amorphous powder; [α]25 D 0° (c 0.95, MeOH); H NMR (500 MHz, D2O) δ 3.76 (2H, d, J = 11.46 Hz, H-1b, H-4b), 3.69−3.70 (2H, m, H-2, H-3) and 3.62 (2H, dd, J = 6.18, 11.46 Hz, H1a, H-4a). 25 D-Mannitol. white amorphous powder [α]D 0° (c 1.15, MeOH); 1 [α]25 D + 103.1° (c 0.11, 10% (NH4)2MoO4, 1 N H2SO4); H NMR (500 MHz, D2O) δ 3.86 (2H, dd, J = 2.69, 11.76 Hz, H-1b, H-6b), 3.81 (2H, d, J = 8.47 Hz, H-3, H-4), 3.74−3.78 (1H, dd, J = 2.69, 8.47 Hz, H-2), 3.74−3.78 (1H, dd, J = 2.69, 6.03 Hz, H-5) and 3.67−3.69 (2H, dd, J = 6.03, 11.76 Hz, H-1a, H-6a). MTT Cytotoxicity Assay. The MTT reduction rate is an indicator of the functional integrity of the mitochondria and, hence, of cellular viability.22,23 Two different cell concentrations were dependent on the assay time: 20000 cells/well concentration for HepG2 and MiaPaca_2,

were maintained in the collection of Fundación MEDINA (www. medinadiscovery.com). Different morphologies of this strain have recently been reported as dependent on the number of subcultures performed.5 Changes in the fermentation morphology (pigmentation and conidia/hyphae conversion) during the growth of the fungus affected the production of the secondary metabolites, and hormonemates were only present when Dothiora sp. (Dothideales) was grown as a yeast, featuring a black thick broth with high density of conidia. However, when a subculturing step was added, it resulted in a yellowish broth containing hyphae and a few conidia. Similar findings were observed for Hormonema dematioides (Dothideales), producing hormonemate at its mature state, in a melanized culture with a dark-green appearance12 and the polymorphic fungus Aureobasidium pullulans (Dothideales), producing liamocins.13 Hence, hormonemates could be identified as biomarkers for the conidia morphology of the fungus Dothiora sp. To scale up the fermentation to 600 mL, 10 mycelial discs of fungal strain grown on YM agar plates at 22 °C for 7 days were used to inoculate 50 mL of SMYA (Difco neopeptone 10 g, maltose 40 g, Difco yeast extract 10 g, agar 4 g, distilled H2O 1 L). After 7 days incubation at 22 °C and 220 rpm, 3 mL aliquots of this culture were used to inoculate YES medium (Difco yeast extract 20 g, sucrose 150 g, MgSO4·7H2O 0.5 g, trace elements 1 mL (ZnSO4·7H2O 1 g/100 mL and CuSO4·5H2O 0.5 g/100 mL) and distilled H2O 1 L) distributed among 6 × 100 mL in 500 mL Erlenmeyer flasks. The flasks were incubated statically at 22 °C, 70% relative humidity for 14 days. Isolation and Identification of Metabolites. The conidia culture and fermentation broth (600 mL) were extracted by adding acetone (600 mL) and shaking at 220 rpm for 2 h. After filtration, the acetone extract was concentrated under reduced pressure to a final volume of 600 mL (100% water). The aqueous residue was loaded onto a SP207ss resin column (65 g, 32 × 100 mm) and eluted with an acetone−H2O stepped gradient (10/90 for 6 min, 20/80 for 6 min, 40/60 for 6 min, 60/40 for 6 min, 80/20 for 6 min, and 100/0 for 12 min, 10 mL/min, 20 mL/fraction) to give 19 fractions. Bioactive fractions 15 to 18 were pooled and subjected to preparative reversedphase HPLC (Zorbax SB-C18 PrepHT, 21.2 × 250 mm, 7 μm, 16 mL/ min, UV detection at 210 and 280 nm, 10 mL/fraction) using H2O + 0.1% TFA (solvent A) and CH3CN + 0.1% TFA (solvent B). Elution was carried out using isocratic conditions of 5% B for 5 min and then a linear gradient from 5% to 100% B in 43 min, yielding 79 fractions that were tested against MCF-7 and HepG2 cells. Subfractions 53−57 and 60 were active against MCF-7 and fractions 53 and 60 against HepG2 cells. Subfractions 57 and 60 of this chromatography yielded compounds 1 (tR 40 min, 54.0 mg) and 2 (tR 42 min, 18.3 mg), respectively. Subfraction 53 was further purified by semipreparative reversedphase HPLC (Zorbax SB-C18 SemiPrep column, 9.4 × 250 mm, 5 μm, 3.6 mL/min, UV detection at 210 and 280 nm, 1.8 mL/fraction) using H2O + 0.1% TFA (solvent A) and CH3CN + 0.1% TFA (solvent B). Elution was carried out using isocratic conditions of 50% B for 65 min to yield compounds 3 (tR 38 min, 1.0 mg), 4 (tR 40 min, 4.8 mg), and 5 (tR 49 min, 5.4 mg). Subfraction 55 was further purified by preparative reversed-phase HPLC (Zorbax SB-C18 PrepHT, 21.2 × 250 mm, 7 μm, 16 mL/min, UV detection at 210 and 280 nm, 10 mL/fraction) eluting with isocratic conditions of 50% CH3CN in H2O for 75 min to yield compounds 5 (tR 39 min, 3.2 mg), 6 (tR 46 min, 7.4 mg), and 1 (tR 52 min, 15.8 mg). Subfraction 56 was further purified by preparative reversed-phase HPLC (Waters XBridge Prep C18, 10 × 250 mm, 5 μm, 16 mL/min, UV detection at 210 and 280 nm, 10 mL/fraction) eluting with isocratic conditions of 55% CH3CN in H2O for 75 min to yield compounds 7 (tR 16 min, 1.5 mg), 6 (tR 44 min, 27.1 mg), and 1 (tR 48 min, 75.6 mg). Hormonemate A (1). colorless syrup; [α]D25 +26.2° (c 1.18, MeOH); UV (DAD) end absorption; IR (ATR) νmax 3387, 2965, 2937, 2879, 1751, 1462, 1385, 1185, 1126, 1046, 1023 cm−1; 1H and 852



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(10) Cragg, G. M.; Newman, D. J. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 3670−3695. (11) Sekar, S.; Kandavel, D. J. Phytol 2010, 2, 91−100. (12) Filip, P.; Weber, R. W. S.; Sterner, O.; Anke, T. Z. Naturforsch. 2003, 58c, 547−552. (13) Leathers, T. D.; Price, N. P. J.; Bischoff, K. M.; Manitchotpisit, P.; Skory, C. D. Biotechnol. Lett. 2015, 37, 2075−2081. (14) Price, N. P. J.; Manitchotpisit, P.; Vermillion, K. E.; Bowman, M. J. Carbohydr. Res. 2013, 370, 24−32. (15) Feifel, S. C.; Schmiederer, T.; Hornbogen, T.; Berg, H.; Süssmuth, R. D.; Zocher, R. ChemBioChem 2007, 8, 1767−1770. (16) Kallmerten, J.; Gould, T. J. J. Org. Chem. 1986, 51, 1152−1155. (17) Manitchotpisit, P.; Price, N. P. J.; Leathers, T. D.; Punnapayak, H. Biotechnol. Lett. 2011, 33, 1151−1157. (18) Manitchotpisit, P.; Watanapoksin, R.; Price, N. P. J.; Bischoff, K. M.; Tayeh, M.; Teeraworawit, S.; Kriwong, S.; Leathers, T. D. World J. Microbiol. Biotechnol. 2014, 30, 2199−2204. (19) Bischoff, K. M.; Leathers, T. D.; Price, N. P. J.; Manitchorpisit, P. J. Antibiot. 2015, 68, 642−645. (20) Kim, J. S.; Lee, I. K.; Yun, B. S. PLoS One 2015, 10, e0122917. (21) Martín, J.; Crespo, G.; González-Menéndez, V.; Pérez-Moreno, G.; Sánchez-Carrasco, P.; Pérez-Victoria, I.; Ruiz-Pérez, L. M.; González-Pacanowska, D.; Vicente, F.; Genilloud, O.; Bills, G. F.; Reyes, F. J. Nat. Prod. 2014, 77, 2118−2123. (22) Verhulst, C.; Coiffard, C.; Coiffard, L. J. M.; Rivalland, P.; De Roeck-Holtzhauer, Y. J. Pharmacol. Toxicol. Methods 1998, 39, 143− 146. (23) Liu, Y.; Nair, M. G. J. Nat. Prod. 2010, 73, 1193−1195. (24) Audoin, C.; Bonhomme, D.; Ivanisevic, J.; de la Cruz, M.; Cautain, B.; Monteiro, M. C.; Reyes, F.; Rios, L.; Pérez, T.; Thomas, O. P. Mar. Drugs 2013, 11, 1477−1489.

and 30000 cells/well for MCF-7 in the 24 h assay, and 10000 cell/well for HepG2 and MiaPaca_2 and 15000 cells/well for MCF-7 in the 72 h assay. Extract and fractions were tested at a dilution 1/40 (5 μL of extract/fractions into 195 μL of fresh medium), whereas pure compounds were analyzed at 1/200 dilution (3 μL of pure compound into 597 μL of fresh medium). MMS was used as positive control, and 0.5% DMSO was used as negative control. The maximum concentration of DMSO was 0.5% to minimize any solvent toxicity background. Pure compounds were tested in triplicate at 10 concentrations using 2-fold serial dilutions starting at a maximum concentration of 40 μg/mL following the method previously described.24

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00680. 1D and 2D NMR spectra of hormonemates and degradation products, and MS/MS fragmentation data of selected compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E- mail: [email protected] (M.P.-B.). Tel.: +34 958993965. *E-mail: [email protected] (F.R.). Tel.: +34 958993965, ext. 7006. ORCID

Ignacio Pérez-Victoria: 0000-0002-4556-688X Fernando Reyes: 0000-0003-1607-5106 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Junta de Andalucı ́a through Project RNM-7987 is gratefully acknowledged. The polarimeter; HPLC, IR, and NMR equipment; and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovación [Grants No. PCT-010000-2010-4 (NMR), INP2011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR), and PCT-01000-ACT7, 2011-13 (plate reader)].

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REFERENCES

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