Anteaglonialides AâF and Palmarumycins CE1 ... - ACS Publications

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Anteaglonialides A−F and Palmarumycins CE1−CE3 from Anteaglonium sp. FL0768, a Fungal Endophyte of the Spikemoss Selaginella arenicola Ya-ming Xu,† Jair Mafezoli,†,‡ Maria C. F. Oliveira,†,‡ Jana M. U’Ren,§ A. Elizabeth Arnold,§,⊥ and A. A. Leslie Gunatilaka*,† †

Southwest Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United States ‡ Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Campus do Pici, Caixa Postal 6044, Fortaleza-CE 60455-970, Brazil § Division of Plant Pathology and Microbiology, School of Plant Sciences, College of Agriculture and Life Sciences, and ⊥Department of Ecology and Evolutionary Biology, College of Sciences, University of Arizona, Tucson, Arizona 85721, United States S Supporting Information *

ABSTRACT: Anteaglonialides A−F (1−6), bearing a spiro[6-(tetrahydro-7-furanyl)cyclohexane-1,2′-naphtho[1,8-de][1,3]dioxin]-10-one skeleton, three new spirobisnaphthalenes, palmarumycins CE1−CE3 (7−9), nine known palmarumycin analogues, palmarumycins CP5 (10), CP4a (11), CP3 (12), CP17 (13), CP2 (14), and CP1 (15), CJ-12,371 (16), 4-O-methyl CJ12,371 (17), and CP4 (18), together with a possible artifact, 4a(5)-anhydropalmarumycin CE2 (8a), and four known metabolites, O-methylherbarin (19), herbarin (20), herbaridine B (21), and hyalopyrone (22), were encountered in a cytotoxic extract of a potato dextrose agar culture of Anteaglonium sp. FL0768, an endophytic fungus of the sand spikemoss, Selaginella arenicola. The planar structures and relative configurations of the new metabolites 1−9 were elucidated by analysis of extensive spectroscopic data, and the absolute configuration of 1 was determined by the modified Mosher’s ester method. Application of the modified Mosher’s ester method combined with the NOESY data resulted in revision of the absolute configuration previously proposed for 10. Co-occurrence of 1−6 and 7−18 in this fungus led to the proposal that the anteagloniolides may be biogenetically derived from palmarumycins. Among the metabolites encountered, anteaglonialide F (6) and known palmarumycins CP3 (12) and CP1 (15) exhibited strong cytotoxic activity against the human Ewing’s sarcoma cell line CHP-100, with IC50 values of 1.4, 0.5, and 1.6 μM, respectively.

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spikemoss (Selaginella arenicola; Selaginellaceae). Fractionation of this extract led to the isolation of six metabolites bearing a spiro[6-(tetrahydro-7-furanyl)cyclohexane-1,2′-naphtho[1,8de][1,3]-dioxin]-10-one carbon skeleton,5 named anteaglonialides A−F (1−6), three new palmarumycin analogues, palmarumycins CE1−CE3 (7−9), and nine known spirobisnaphthalenes, palmarumycins CP5 (10),6 CP4a (11),6 CP3 (12),7,8 CP17 (13),9 CP2 (14),8 and CP1 (15),8 CJ-12,371 (16),10 4-O-methyl CJ-12,371 (17),11 and palmarumycin CP4

lant-associated fungi such as endophytes represent a large and relatively untapped resource of small-molecule natural products.1 Although first observed over a century ago, endophytes have drawn increasing interest in recent years due to their ecological relevance2 and their potential to yield metabolites with diverse structures and interesting biological activities.3 In our continuing search for bioactive and/or novel metabolites from endophytic and endolichenic fungi,4 we have investigated a cytotoxic EtOAc extract of a potato dextrose agar (PDA) culture of Anteaglonium sp. FL0768 (Anteagloniaceae, Pleosporales, Pezizomycotina, Ascomycota), an endophytic fungus isolated from the living photosynthetic tissue of sand © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 12, 2015

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DOI: 10.1021/acs.jnatprod.5b00717 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of metabolites 1−22 from Anteaglonium sp. FL0768.

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of Metabolites 1−3 in CDCl3 1 position

δC, type

1 2

102.1, C 29.6, CH2

3

30.2, CH2

4 5

68.8, CH 35.2, CH2

6 7 8

41.7, CH 84.3, CH 69.1, CH

9

37.9, CH2

10 1′a 2′b 3′c 4′d 4a′ 5′d 6′c 7′b 8′a 8a′ a−d

175.1, 145.8, 109.2, 127.4, 121.1, 134.2, 121.4, 127.7, 110.0, 145.9, 113.3,

C C CH CH CH C CH CH CH C C

δH, mult. (J in Hz) α. 1.57, m β. 2.24, dd (3.2, 10.4) α. 1.57, m β. 1.78, m 3.89, tt (4.8, 10.4) β. 1.97, dt (11.2, 12.8) α. 2.51, m 2.71, dt (4.0, 10.4) 4.63, dd (3.2, 10.4) 4.52, dd (3.2, 5.2) β. 2.55, d (17.6) α. 2.69, dd (5.2, 17.6)

6.86, dd (0.8, 7.6) 7.40, t (7.6) 7.50, dd (0.8, 7.6) 7.50, dd (0.8, 7.6) 7.44, t (7.6) 6.96, dd (0.8, 7.6)

2 δC, type

δH, mult. (J in Hz)

101.7, C 29.7, CH2

α. 1.49, m β. 2.20, m α. 1.56, m β. 1.76, m 3.84, m β. 1.95, dt (10.8,12.4) α. 2.38, m 2.29, m 4.86, m β. 2.27, m α. 2.43, m 2.44−2.52, m

30.4, CH2 69.0, CH 34.2, CH2 47.8, CH 80.4, CH 29.2, CH2 29.3, CH2 177.0, 146.5, 108.9, 127.3, 120.4, 134.2, 120.6, 127.4, 109.5, 146.9, 113.4,


6.81, dd (0.8, 7.6) 7.37, t (7.6) 7.43, dd (0.8, 7.6) 7.45, dd (0.8, 7.6) 7.40, t (7.6) 6.92, dd (0.8, 7.6)

3 δC, type 102.4, C 26.1, CH2 28.7, CH2 64.6, CH 32.1, CH2 43.7, CH 80.3, CH 29.3, CH2 29.3, CH2 177.2, 146.9, 109.0, 127.3, 120.3, 134.2, 120.5, 127.4, 109.3, 147.0, 113.5,


δH, mult. (J in Hz) α. 1.92, dt (4.0, 12.4) β. 1.98, m α. 1.62, m β. 1.74, ddt (2.8, 4.8, 12.4) 4.24, quint (3.2) β. 2.09, ddd (2.8, 12.0, 14.8) α. 2.22, m 2.66, ddd (4.4, 7.2, 12.0) 4.90, m β. 2.28, m α. 2.43, m 2.45−2.52, m

6.84, dd (0.8, 7.6) 7.38, t (7.6) 7.43, dd (0.8, 7.6) 7.44, dd (0.8, 7.6) 7.39, t (7.6) 6.88, dd (0.8, 7.6)

Assignments for positions with identical superscripts are interchangeable.

B



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Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations for metabolites 1−9.

(18),8 together with a possible artifact, 4a(5)-anhydropalmarumycin CE2 (8a), formed from 8, and four other known metabolites, O-methylherbarin (19),12 herbarin (20),13 herbaridine B (21),14 and hyalopyrone (22).15 Palmarumycins constitute a subclass of spirobisnaphthalenes bearing a cis-decalin moiety attached to a naphthalene moiety through a spiro-ketal linkage with two oxygen bridges.16 Analogues of palmarumycins with the same carbon skeleton, but containing a trans-decalin moiety instead of the cis-decalin, a β-OH group at C-8a, and epoxy substituent(s) in the decalin moiety are also known, and these are referred to as decaspirones,17 cladospirones,18 and diepoxins,19 respectively. Herein we report the isolation and identification of metabolites 1−22 (Figure 1), the cytotoxic activity of 6, 12, and 15, and a proposed biosynthetic pathway to anteaglonialides from palmarumycins. Although the genus Anteaglonium was proposed only recently,20 thus far no reports have appeared describing secondary metabolites of this genus. Thus, this constitutes the first report of the occurrence of secondary metabolites in the fungal genus Anteaglonium.

Hz), 4.52 (1H, dd, J = 3.2, 5.2 Hz), and 4.63 (1H, dd, J = 3.2, 10.4 Hz). The 13C NMR spectrum of 1 (Table 1) displayed 20 signals consisting of four methylenes, 10 methines, of which 6 were aromatic/olefinic, and six nonprotonated, of which one each was a ketal carbon (δC 102.1) and a carbonyl carbon and four were aromatic/olefinic carbons, all of which were assigned by the analysis of its phase-sensitive HSQC spectrum. These data suggested that 1 contained a naphtho-1,8-dioxin moiety, as in the spirobisnaphthalenes6−10 that were also found to cooccur in this fungus (see below), but the presence of a signal due to a lactone/ester carbonyl at δC 175.1 in its 13C NMR spectrum indicated that the upper non-naphthalene portion of the molecule was somewhat different. The presence of the partial structure CH2−CH2−CH(O)−CH2−CH−CH(O)− CH(O)−CH2 in this part of the molecule was confirmed by its 1H−1H COSY spectrum (Figure 2), complete assignment of which was made with the help of the HSQC data. The HMBC correlations (Figure 2) of the signal at δC 175.1 (C-10) with δH 2.55/2.69 (CH2-9) and δH 4.52 (CH-8) and those of δC 102.1 (C-1) with δH 1.57/2.24 (CH2-2), δH 1.97/2.51 (CH2-5), and δH 4.63 (CH-7) suggested that this portion of the molecule consisted of an oxygenated cyclohexane ring bearing an oxygenated 5-oxotetrahydrofuran moiety. NOESY correlations of H-4/H-6 and H-7/H-8 suggested that H-4 and H-6 were on the same face of the cyclohexane ring and H-7 and H-8 were on the same face of the 5-oxotetrahydrofuran ring (Figure 2). The absolute configuration of 1 was elucidated by application of the modified Mosher’s ester method.21 Analysis of the 1H NMR data for (S)-MTPA and (R)-MTPA diesters of 1 confirmed the absolute configuration of C-4 and C-8 as R and S, respectively

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RESULTS AND DISCUSSION Anteaglonialide A (1) was obtained as an off-white, amorphous solid that analyzed for C20H20O6 by a combination of HRESIMS and NMR data, which indicated 11 degrees of unsaturation. The 1H NMR spectrum of 1 (Table 1) exhibited signals at δH 6.86 (1H, dd, J = 0.8, 7.6 Hz), 6.96 (1H, dd, J = 0.8, 7.6 Hz), 7.40 (1H, t, J = 7.6 Hz), 7.44 (1H, t, J = 7.6 Hz), and 7.50 (2H, dd, J = 0.8, 7.6 Hz), together with three downfield aliphatic methines at δH 3.89 (1H, tt, J = 4.8, 10.4 C



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oxotetrahydrofuran moiety in 1 was replaced with a CH2 in both 2 and 3. Anteaglonialides B (2) and C (3) differed from each other in their 1H and 13C chemical shift data for the hydroxylated cyclohexane moiety, and this difference was suspected to be due to the orientation of the OH group at C-4. This was confirmed by NOESY data. A correlation between H4 and H-6 in the NOESY data for 2 suggested that these protons were on the same side of the cyclohexane ring. However, no such NOESY correlation between these two protons was observed for 3, suggesting that H-4 and H-6 in 3 were on opposite sides of the cyclohexane moiety. On the basis of their common biosynthetic origin (see below), the absolute configurations at C-6 and C-7 of 2 and 3 were presumed to be the same as those of 1. Thus, the structures of anteaglonialides B and C were established as spiro[4R,6S-((7R)-tetrahydro-7furanyl)-4-hydroxycyclohexane-1,2′-naphtho[1,8-de][1,3]-dioxin]-10-one (2) and spiro[4S,6S-((7R)-tetrahydro-7-furanyl)-4hydroxycyclohexane-1,2′-naphtho[1,8-de][1,3]-dioxin]-10-one (3), respectively (Figure 1). On the basis of their HRESIMS and NMR data, anteaglonialides D (4) and E (5) were determined to have the same molecular formula, C20H18O5, suggesting 12 units of unsaturation. Anteaglonialide F (6) analyzed for C20H16O5 by a combination of HRESIMS and NMR data and indicated 13 degrees of unsaturation. These data suggested that 4 and 5 were probably monounsaturated analogues of anteaglonialides B (2) and C (3), while compound 6 was a diunsaturated analogue. The assignments of 1H and 13C NMR data of 4−6 (Table 2) by 2D NMR experiments (Figure 2) confirmed that they contained the same carbon skeleton as 2 and 3. The NMR data also indicated the presence of two olefinic methines in 4 in place of the two methylenes at C-2 and C-3 in 2. Analysis of the

(Figure 3). Thus, the structure of anteaglonialide A was established as spiro[4R,6S-((7S,8S)-8-hydroxytetrahydro-7-fur-

Figure 3. Δδ values [Δδ values (in ppm) = δS − δR] obtained for (S)and (R)-MTPA esters of 1 and 10.

anyl)-4-hydroxycyclohexane-1,2′-naphtho[1,8-de][1,3]-dioxin]10-one (1; Figure 1). Anteaglonialides B (2) and C (3) were determined to have the same molecular formula, C20H20O5, on the basis of their HRESIMS and NMR data, indicating 11 units of unsaturation and suggesting that 2 and 3 may be deoxygenated analogues of 1. The 1H and 13C NMR data of 2 and 3 (Table 1) analyzed with the help of HSQC showed that both contained five methylenes, nine methines, of which six were aromatic/olefinic, and six nonprotonated carbons consisting of a lactone carbonyl, a ketal carbon, and four aromatic/olefinic carbons. The 1H and 13 C NMR spectra of 2 and 3 analyzed with the help of 1H−1H COSY and HMBC data (Figure 2) revealed that these were almost the same as for 1 except that the CHOH of the 5-

Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data of Metabolites 4−6 in CDCl3 4 position

5


δC, type

1 2

98.8, C 124.1, CH

5.73, dd (2.0, 10.0)

100.9, C 30.0, CH2

3

136.6, CH

5.91, dt (10.0, 1.6)

36.1, CH

4.41, m β. 2.00, m α. 2.61, m 2.39, m 4.85, dt (6.4, 8.4) 2.30−2.45, m

207.0, C 38.0, CH2

2.47−2.54, m

28.7, 176.1, 146.3, 109.4, 127.5, 120.9, 134.2, 121.1, 127.5, 109.5, 146.4, 113.4,

4 5

67.0, CH 32.7, CH2

6 7 8

46.8, CH 80.4, CH 28.6, CH2

9 10 1′a 2′b 3′c 4′d 4a′ 5′d 6′c 7′b 8′a 8a′ a−d

δC, type

29.7, 176.9, 146.7, 109.4, 127.4, 120.7, 134.2, 120.7, 127.5, 109.5, 146.9, 113.3,

CH2 C C CH CH CH C CH CH CH C C

6.86, d (7.6) 7.40, t (7.6) 7.46, d (7.6) 7.47, d (7.6) 7.40, t (7.6) 6.88, d (7.6)

6

δH, mult. (J in Hz) 2.10, 2.47, 2.42, 2.52,

m m m m

2.73, dd (8.4, 15.0) 2.86, ddd (1.2, 6.0, 15.0) 2.64, m 5.02, ddd (4.8, 6.4, 9.6) 2.11, m 2.37, m 2.47−2.54, m

47.4, CH 78.3, CH 27.7, CH2 CH2 C C CH CH CH C CH CH CH C C

6.91, d (7.6) 7.42, t (7.6) 7.49, dd (0.8, 7.6) 7.50, d (7.6) 7.43, t (7.6) 6.96, d (7.6)

δC, type


97.5, C 141.8, CH

6.73, d (10.0)

131.4, CH

6.03, d (10.0)

196.2, C 36.4, CH2

2.82−3.50, m

47.8, CH 79.0, CH 28.5, CH2 29.0, 176.3, 145.9, 109.7, 127.6, 121.4, 134.2, 121.5, 127.6, 110.1, 146.4, 113.3,

CH2 C C CH CH CH C CH CH CH C C

2.87, m 4.97, m 2.32, m 2.48, m 2.47−2.55, m

6.93, d (7.6) 7.42, t (7.6) 7.52, d (7.6) 7.54, d (7.6) 7.45, t (7.6) 6.97, d (7.6)

Assignments for positions with identical superscripts are interchangeable. D



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Table 3. 1H (400 MHz) and 13C (100 MHz) NMR Data of 7, 8, 8a, and 9 in CDCl3 7 position

δC, type


8 δC, type

1 2

97.9, C 139.8, CH

6.69, dd (2.0, 10.4)

102.6, C 32.6, CH2

3

134.1, CH

6.19, d (10.4)

37.9, CH2

4 4a 5

201.1, C 45.3, CH 71.7, CH

3.39, t (4.8) 3.54, dt (12.4, 4.8)

212.9, C 47.3, CH 71.8, CH

6

25.8, CH2

7

31.2, CH2

8 8a 1′a 2′b 3′c 4′d 4a′ 5′d 6′c 7′b 8′a 8a′ 5-OH a−d

63.6, CH 50.3, CH 146.3, C 109.6, CH 127.5, CH 121.1, CH 134.2, C 121.4, CH 127.6, CH 109.6, CH 146.5, C 113.2, C

α. 1.78, m β. 2.01, ddt (3.2, 12.4, 13.2) α. 1.38, ddt (2.8, 4.0, 14.4) β. 1.88, ddd (3.2, 6.8, 14.4) 4.43, dd (2.2, 5.8) 2.66, dt (2.2, 5.0) 6.93, d (7.6) 7.43, t (7.6) 7.51, d (7.6) 7.53, d (7.6) 7.44, t (7.6) 6.97, d (7.6)

8a


δC, type 102.7, C 28.2, CH2

α. 2.28, m β. 3.06, dt (6.4, 13.2) β. 2.30, m α. 2.70, m

34.3, CH2

196.1, C 129.9, C 141.7, CH

3.15, t (5.2) 3.31, m;

27.1, CH2

α. 1.55, m β. 2.13, m

21.9, CH2

32.8, CH2

α. 1.30, m

27.5, CH2

β. 1.80, m 63.6, CH 49.1, CH 147.7, C 110.0, CH 128.6, CH 121.4, CH 135.3, C 121.5, CH 128.7, CH 110.4, CH 148.3, C 114.6, C

δH, mult. (J in Hz) α. 1.95, m β. 2.40, m

9 δC, type


105.6, C 76.3, CH

4.21, d (5.2)

7.27, ddd (1.0, 4.6, 2.6) 2.34, m 2.66, m

71.1, CH 42.3, CH 66.1, CH

α. 2.07, dd (9.6, 13.2) β. 2.31, ddd (4.8, 8.0, 13.2) 4.44, dt (9.6, 8.0) 2.57, ddd (3.6, 4.4, 8.0) 4.39, ddd (1.6, 3.6, 4.4)

135.0, CH

5.88, dt (10.0, 1.6)

α. 1.67, m

125.1, CH

5.94, ddd (1.6, 4.4, 10,0)


4.59, t (4.4) 2.37, t (3.6)

2.40, m 2.53, m

35.9, CH2

β. 2.08, m

4.26, brs 2.42, m


7.09, dd (0.8, 7.2) 7.47, dd (8.0, 7.6) 7.55, d (8.0) 7.56, d (8.0) 7.52, dd (8.0, 7.6) 6.96, dd (0.8, 7.6)

4.91, dt (4.4, 2.0) 3.15, m 6.94, dd (0.7, 7.6) 7.41, dd (7.6, 8.4) 7.50, d (8.4) 7.53, d (8.4) 7.44, dd (7.6, 8.4) 6.96, dd (0.8, 7.6)

6.96, dd (0.8, 7.6) 7.40, t (7.6) 7.48, dd (0.8, 7.6) 7.50, dd (0.8, 7.6) 7.42, t (7.6) 7.04, dd (0.8, 7.6)

3.92, d (11.6)

Assignments for positions with identical superscripts are interchangeable.

Palmarumycin CE1 (7) was determined to have the molecular formula C20H18O5 by analysis of its HRESIMS and NMR data, indicating 12 degrees of unsaturation. The 1H NMR data of 7 (Table 3) showed the presence of two olefinic protons and two oxygenated methines in addition to signals typical of the 1,8-di-O-naphthelene moiety. The 13C NMR spectrum (Table 3) showed the presence of 20 signals, which were assigned with the help of HSQC data to two methylenes, 12 methines (of which six were aromatic and two were olefinic), and six nonprotonated carbons consisting of a ketone carbonyl, a ketal, and four aromatic/olefinic carbons. Analysis of its 1 H−1H COSY spectrum revealed the presence of spin systems assignable to −CHCH− and −CH−CHOH−CH2−CH2− CHOH−CH− units in addition to the 1,8-di-O-naphthelene moiety and suggested that 7 contained the palmarumycin carbon skeleton.6−10 The HMBC spectrum of 7 showed correlations of H-3 to C-1 and H-2 to C-4 (Figure 2), suggesting the presence of a 2(3)-en-4-one moiety in 7 and reflecting its structural resemblance to decaspirone C.17 However, careful comparison of the NMR data of 7 with those reported for decaspirone C (which bears a trans-fused decalin ring system) revealed that they are isomeric with each other and that 7 contained a cis-fused decalin ring system. 1D NOESY data confirmed that the protons H-4a, H-5, H-8, and H-8a were on the same face of the ring based on correlations of H-8 and H-8a, H-8a and H-5, H-8a and H-4a, and H-5 and H4a. The above data established the structure of palmarumycin

1D NOESY data of 4 (Figure 2) showed a strong correlation between H-4 and H-6, suggesting the cis orientation of these two protons (i.e., β-orientation of 4-OH). On the basis of biosynthetic considerations, the configurations at C-6 and C-7 of 4 were presumed to be S and R, respectively. Thus, the structure of anteaglonialide D was established as spiro[4S,6S((7R)-tetrahydro-7-furanyl)-4-hydroxycyclohex-2-ene-1,2′naphtho[1,8-de][1,3]-dioxin]-10-one (4; Figure 1). Comparison of the NMR data of anteaglonialide E (5) (Table 2) with those of 2 and 3 (Table 1) indicated that the CHOH at C-4 was replaced with a ketone carbonyl (δC 207.0) in 5. The presence of this carbonyl group at C-4 was confirmed by the presence of strong HMBC correlations of CH2-2 and CH-6 to this carbonyl carbon (Figure 2). The foregoing data and its presumed biosynthetic relationship to 1 established the structure of anteaglonialide E as spiro[6S-((7R)-tetrahydro-7furanyl)cyclohexane-1,2′-naphtho[1,8-de][1,3]-dioxin]-4,10dione (5; Figure 1). Analysis of the 1H and 13C NMR data of anteaglonialide F (6) (Table 2) with the help of 1H−1H COSY and HMBC data (Figure 2) suggested that its structure was closely related to that of 5, but had an additional double bond at the 2(3)position conjugated to the carbonyl at C-4 (δC 196.2). Thus, the structure of anteaglonialide F was established as spiro[6S((7R)-tetrahydro-7-furanyl)cyclohex-2-ene-1,2′-naphtho[1,8de][1,3]-dioxin]-4,10-dione (6; Figure 1). E



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Figure 4. Proposed biosynthetic pathway to anteaglonialide F (6) from palmarumycin CP4 (18).

CE1 as (4aα,5β,8β,8aα)-4a,5,6,7,8,8a-hexahydro-5,8-dihydroxyspiro[naphthalene-1(4H)-2′-naphtho[1,8-de][1,3]-dioxin]-4one (7, Figure 1). Attempted isolation of palmarumycin CE2 (8) in its pure form failed, as it was consistently found to be contaminated with ca. 30% of its dehydration product 8a, as indicated by the 1 H NMR spectrum of the mixture. Thus, 8a was considered to be an artifact formed from 8. The 13C NMR spectrum of the mixture contained 20 signals corresponding to the major constituent, palmarumycin CE2 (8). Analysis of the NMR data for 8 with the help of an HSQC experiment suggested that it contained a ketone, two oxymethines, a ketal, two other methines, and four methylene groups, in addition to the 1,8-diO-naphthalene moiety typical of palmarumycins. The presence of a carbonyl at C-4 and OH groups at C-5 and C-8 in 8 was supported by the 1H−1H COSY correlations (Figure 2) and HMBC correlations of C-4 with H-5 and H-4a, C-1 with H-8a, C-5 with H-4a, C-8 with H-4a, and C-4a with H-8. Strong NOESY correlations observed between H-8/H-8a, H-8/H-4a, H-4a/H-8a, and H-8a/H-5 suggested that H-4a, H-5, H-8, and H-8a were all on the same face of the decalin ring as those of palmarumycin CE1 (7). Thus, the structure of palmarumycin CE2 was established as (4aα,5β,8β,8aα)-2,3,4a,5,6,7,8,8aoctahydro-5,8-dihydroxyspiro[naphthalene-1(4H)-2′-naphtho[1,8-de][1,3]-dioxin]-4-one (8, Figure 1). HPLC purification of the above mixture afforded 8a, the molecular formula of which was determined to be C20H18O4 by analysis of its HRESIMS and NMR data, indicating 13 degrees of unsaturation. The 1H NMR spectrum of 8a (Table 3) showed the presence of an olefinic proton, an oxygenated methine, and an aliphatic methine besides signals typical of a palmarumycin-type 1,8-di-O-naphthalene bearing a decalin moiety. The 13C NMR spectrum of 8a (Table 2) showed 20 signals characteristic of an α,β-unsaturated ketone, a ketal, two aliphatic methines, and four methylenes in addition to the 10 signals typical of the 1,8-di-O-naphthalene moiety. The 1H−1H COSY and HMBC data (Figure 2) confirmed the presence of a ketone carbonyl at C-4, a double bond at C-4a(C-5), and an OH group at C-8. 1D NOESY data indicated that H-8 and H8a were on the same face of the decalin ring (Figure 2), confirming the structure of 8a as 4a(5)-anhydropalmarumycin CE2 (Figure 1). It is noteworthy that palmarumycin CE1 (7), palmarumycin CP5 (10), and palmarumycin CP4a (11), which were found to co-occur in this fungus, also contain a 5βhydroxy-4-oxo moiety amenable to dehydration, but no corresponding dehydration products were detected. A possible explanation for this is the restricted conformational mobility of their structures due to the presence of a 2(3)-double bond (in 7) or a 3,8-ether linkage (in 10 and 11), which do not favor the trans-diaxial orientation of 4α-H and 5β-OH groups that would presumably be required for elimination.22

Palmarumycin CE3 (9) was determined to have the molecular formula C20H18O5 by analysis of its HRESIMS and NMR data. The 1H and 13C NMR data (Table 3) revealed that 9 contained two adjoining olefinic methines, four oxygenated methines, two aliphatic methines, one methylene, and a ketal in addition to signals characteristic of a 1,8-di-O-naphthalene moiety. The 1H−1H COSY and HMBC data of 9 (Figure 2) confirmed that it had a palmarumycin core structure and suggested that C-2, C-4, C-5, and C-8 of the decalin moiety were oxygenated. The presence of the C-2−C-8 ether linkage was confirmed by an HMBC correlation between H-2 and C-8. NOESY correlations observed for H-4/H-3β, H-4a/H-3α, H4a/H-8a, H-8a/H-8, and H-8a/H-5 (Figure 2) indicated that H-4a, H-5, H-8, and H-8a were α-oriented and H-4 was βoriented, suggesting relative configurations of α-H-4a, 5β-OH, α-H-8a, 4α-OH, and 2β,8β for the C-2−C-8 ether linkage. These data established the structure of palmarumycin CE3 as (2β,4α,4aα,5β,8β,8aα)-2,3,4,4a,5,8,8a-heptahydrospiro[2,8-epoxynaphthalene-1(4H)-2′-naphtho[1,8-de][1,3]-dioxin]-4,5diol (9, Figure 1). Comparison of spectroscopic and specific rotation data of the metabolite 10 with those reported for palmarumycin CP56 suggested that they are identical with each other. The absolute configuration 2R,4aS,5R,8S,8aR previously proposed for palmarumycin CP5 involved comparison of its experimental CD spectrum with that predicted using calculated conformational behavior of the two possible enantiomers.6 We were intrigued by the fact that the proposed absolute configuration for palmarumycin CP5 was the opposite of palmarumycin CP4a (11) and palmarumycin CP3 (12) reported by these workers from the same fungus.6 It was therefore of interest to determine the absolute configuration of palmarumycin CP5 (10) by the application of other techniques such as the modified Mosher’s ester method21 combined with NOESY data. Thus, the esterification of 10 with (S)- and (R)-MTP acids afforded its (R)- and (S)-MTPA esters, respectively. Analysis of the 1H NMR data for these MTPA esters confirmed the Sconfiguration for C-5 of 10 (Figure 3), which is the opposite of that proposed previously.6 The absolute configurations of the remaining chiral carbons were determined to be 2S,4aR,8R,8aS by analysis of the NOESY data for 10 (Supporting Information, Figures S53 and S54). On the basis of these data, the structure of palmarumycin CP5 should be revised to (2S,4aR,5S,8R,8aS)2,3,4a,5,6,7,8,8a-octahydro-5,8-dihydroxyspiro[2,8-epoxynaphthalene-1(5H)-2′-naphtho[1,8-de][1,3]-dioxin]-4-one (10; Figure 1). Co-occurrence of anteaglonialides with palmarumycins in this fungal strain prompted us to postulate a biosynthetic pathway from palmarumycins to anteaglonialides involving several simple transformations. A proposed biosynthetic pathway to anteaglonialide F (6) from palmarumycin CP4 (18) involving the reversible addition of 8β-OH to the F



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Corp.). The resulting sequence has been deposited in GenBank (accession JQ760428.1). Because the isolate did not produce diagnostic fruiting structures in culture, two methods were used to tentatively identify isolate FL0768 using molecular sequence data. First, the LSU rDNA portion of the ̈ Bayesian classifier for fungi28 sequence was evaluated using the naive available through the Ribosomal Database Project (http://rdp.cme. msu.edu/). The Bayesian classifier estimated placement within the Dothideomycetes with high support, but placement at finer taxonomic levels was not possible. Therefore, the entire sequence was compared against the GenBank database using BLAST.29 The top BLAST matches were primarily to unidentified Dothideomycetes, Lophiostoma spp., Phoma spp., and diverse Pleosporales (e.g., Massarina spp.). To clarify the phylogenetic placement of the strain, the top 100 BLAST matches were downloaded from GenBank. The query sequence and the 100 top matches were aligned automatically using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) with default parameters. The alignment was trimmed to consistent starting/ending points and adjusted manually in MacClade30 prior to analysis using maximum likelihood in GARLI31 using the GTR+I+G model of evolution as determined by ModelTest,32 followed by a bootstrap analysis with 100 replicates. The resulting analysis suggested placement of FL0768 within Anteaglonium (data not shown). To confirm this placement, a second phylogenetic analysis was conducted using narrower taxon sampling (12 isolates of Anteaglonium, the query sequence, and Lophiostoma cyanoides as the outgroup taxon). Analyses as described above focused on 563 base pairs of the partial LSU rDNA and placed FL0768 within Anteaglonium. These analyses did not provide evidence that FL0768 is identical to any previously sequenced species within this genus (for phylogenetic tree, see Supporting Information Figure S55). Comparison of the sequence of FL0768 with the aligned LSU rDNA sequence of any closely related species of Anteaglonium revealed that it has distinctive nucleotides in seven of 563 positions (data not shown). On the basis of these analyses we designated the fungal isolate as Anteaglonium sp. FL0768, pending morphological description. Culturing and Isolation of Metabolites. A culture of Anteaglonium sp. FL0768 grown on PDA for 2 weeks was used for extraction. The culture media and mycelium from 13 T-flasks (800 mL), each containing 135 mL of PDA coated on five sides of the flasks, maximizing the surface area for fungal growth (total surface area/flask ca. 460 cm2), were extracted with MeOH (3.2 L) in an ultrasonic bath for 1 h. After filtration, the aqueous MeOH extract was concentrated to about one-third of its volume under reduced pressure, and the resulting solution was extracted with EtOAc (3 × 500 mL). The EtOAc extract was concentrated under reduced pressure to afford the crude EtOAc extract (1.03 g), which showed cytotoxicity activity. This extract was fractionated by preparative RP HPLC using a gradient solvent system from 60% MeOH(aq) to 100% MeOH in 30 min. Ten fractions (I−X) were collected according to the HPLC trace at retention times of around 7.6, 9.3, 12.5, 13.7, 17.5, 20.1, 22.4, 23.8, 26.0, and 30.6 min. Fraction X (50.0 mg) was separated by preparative TLC (SiO2, eluent: CHCl3−hexanes, 1:1) to afford 14 (19.0 mg, Rf = 0.6) and 15 (10.1 mg, Rf = 0.7). Separation of fraction IX (55.0 mg) by preparative TLC (SiO2, eluent: CHCl3) gave 13 (4.0 mg, Rf = 0.8) and 17 (13.0 mg, Rf = 0.6). Fraction VIII (58.0 mg) on purification by preparative TLC (SiO2, eluent: CHCl3−MeOH, 99:3) afforded 16 (35.8 mg, Rf = 0.5). Metabolite 18 (9.0 mg, Rf = 0.8) was isolated from fraction VII (35.0 mg) by preparative TLC (SiO2, CHCl3−MeOH, 99:1). Fraction VI (90.0 mg) was chromatographed on a column of Si gel (20 g), which on elution with CHCl3, CHCl3−MeOH (99:1), and CHCl3−MeOH (98:2) afforded 12 (31.5 mg) (Rf = 0.9, CHCl3− MeOH, 99:1), 3 (6.0 mg) (Rf = 0.4, EtOAc−hexanes, 1:1), 9 (9.5 mg) (Rf = 0.4, EtOAc−hexanes, 3:2), and two crude fractions, VI-1 (25.0 mg) and VI-2 (8.4 mg). Further purification of fraction VI-1 by column chromatography on Si gel (1:2 EtOAc−hexanes) followed by RP preparative TLC (C-18; 75% MeOH(aq)) gave 11 (11.2 mg) (Rf = 0.5, 1:1 EtOAc−hexanes) and 19 (0.7 mg) (Rf = 0.3, 1:1 EtOAc− hexanes). Fraction VI-2 on purification by RP HPLC (65% MeOH(aq)) and preparative TLC (SiO2, CHCl3−MeOH, 95:5) afforded 8a (2.2 mg) (Rf = 0.8, CHCl3−MeOH, 95:5) and 8

carbonyl at C-5 to form a hemiketal, which would then undergo a retro aldol-type reaction generating the 5-oxotetrahydrofuran moiety characteristic of anteagloniolides, is depicted in Figure 4. It is noteworthy that a similar biosynthetic pathway has recently been proposed by Bunyapaiboonsri et al. for the formation of palmarumycin P1 from palmarumycin CP3.5 These authors found that palmarumycin CP3, the major metabolite of BCC 25093 (Pleosporales, Dothideomycetes), on treatment with methanolic K 2 CO 3 afforded a trace quantity of palmarumycin P1, a minor metabolite of this fungus, suggesting that palmarumycin P1 may be an artifact formed from palmarumycin CP3 during their isolation process. Although the possibility of anteaglonialides 1−6 being artifacts cannot be ruled out with certainty, the absence of spirobisnaphthalenes bearing structures corresponding to anteaglonialides 1−5 and our inability to find palmarumycin P1 in Anteaglonium sp. FL0768, despite the presence of palmarumycin CP3 (12) as a major metabolite in this fungus, suggested that 1−6 encountered in this study were genuine fungal metabolites. All isolated compounds were evaluated for their potential cytotoxic activity against the human Ewing’s sarcoma cell line CHP-100. Of these, anteaglonialide F (6), palmarumycin CP3 (12), and palmarumycin CP1 (15) showed cytotoxic activity with IC50’s of 1.4, 0.4, and 1.6 μM, respectively. It is noteworthy that all three active compounds contained an α,β-unsaturated ketone (enone) moiety characteristic of many cytotoxic agents.23

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Jasco Dip-370 polarimeter using MeOH as solvent. UV spectra were recorded with a Shimadzu UV 2601 spectrophotometer. 1D and 2D NMR spectra were recorded in CDCl3, unless otherwise stated, using residual solvents as internal standards on a Bruker Avance III 400 spectrometer at 400 MHz for 1H NMR and 100 MHz for 13C NMR, respectively. Chemical shift values (δ) are given in parts per million (ppm), and the coupling constants are in hertz (Hz). Low-resolution and high-resolution MS were recorded on Shimadzu LCMS-DQ8000α and Shimadzu LCMS-IT-TOF (225-07100-34) spectrometers, respectively. Preparative HPLC purifications were carried out on a 10 × 250 mm Phenomenex Luna 5 μm C18 column with a Waters Delta Prep system consisting of a PDA 996 detector. The solvent flow rate was 3 mL/min, and detection wavelength was 230 nm. Repeated injections were used for large-scale purifications. When necessary, MM2 energy minimizations of all possible conformers were carried out using Chembio3D Ultra 14.0 of CambridgeSoft Corp. Fungal Isolation and Identification. Endophyte FL0768 was isolated from healthy, surface-sterilized photosynthetic tissue of a freshly collected stem of the sand spikemoss (Selaginella arenicola) obtained from a pine-dominated forest in central Florida, USA. The host was identified as S. arenicola by one of the authors (A.E.A.), and the tissue vouchers of it are maintained with living fungal cultures in the fungal collection, which is housed at the Robert L. Gilbertson Herbarium of the University of Arizona, with the same accession number as the fungal strain (FL0768). For details of fungal isolation, see U’Ren et al.24 The strain was accessioned as a living mycelial voucher at the Robert L. Gilbertson Mycological Herbarium (MYCOARIZ, FL0768). Total genomic DNA was isolated from fresh mycelium, and the nuclear ribosomal internal transcribed spacers and 5.8s gene (ITS rDNA; ca. 600 base pairs [bp]) and the adjacent portion of the nuclear ribosomal large subunit (LSU rDNA) were amplified as a single fragment by PCR. The positive amplicon was cleaned, normalized, and sequenced as described previously.24 Basecalls were made by phred25 and phrap26 with orchestration by Mesquite,27 followed by manual editing in Sequencher (Gene Codes G



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(containing ca. 30% of 8a, 5.1 mg) (Rf = 0.4, CHCl3−MeOH, 95:5). Fraction V (72.0 mg) on separation by Si gel (30 g) column chromatography and elution with CHCl3 and CHCl3−MeOH (99:1) gave 5 (14.0 mg) (Rf = 0.9, EtOAc−hexanes, 2:1), 6 (9.8 mg) (Rf = 0.8, EtOAc−hexanes, 2:1), 2 (20.5 mg) (Rf = 0.6, EtOAc−hexanes, 2:1), and three crude fractions containing 7, 4, 12, and 21 as major constituents. Each of these crude fractions was further purified by preparative TLC (SiO2, eluent: EtOAc−hexanes, 1:1) and RP HPLC (C-18, eluent: 80% MeOH(aq)) to afford 7 (0.9 mg) (Rf = 0.6, EtOAc−hexanes, 1:1), 4 (1.8 mg) (Rf = 0.5, EtOAc−hexanes, 1:1), 12 (1.1 mg) (Rf = 0.6, EtOAc−hexanes, 2:1), and 21 (1.8 mg) (Rf = 0.3, EtOAc−hexanes, 2:1). The combined fractions III and IV (58.0 mg) were subjected to Si gel (8 g) column chromatography and elution with CHCl3, CHCl3−MeOH (98:2), CHCl3−MeOH (95:5), and CHCl3−MeOH (90:10) to afford four crude fractions, each of which on further purification by RP HPLC gave 10 (4.5 mg) (tR = 12.8, 60% MeOH(aq)), 20 (3.0 mg) (tR = 13.7, 60% MeOH(aq)), 22 (2.5 mg) (tR = 13.8, 65% MeOH(aq)), and 1 (2.3 mg) (tR = 11.2, 65% MeOH(aq)), respectively. Anteaglonialide A (1): off-white, amorphous solid; [α]25 D −39 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 227 (4.58), 299 (3.73), 314 (3.60), 328 (3.50) nm; 1H and 13C NMR data, see Table 1; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/ z 379.1191 (calcd for C20H20O6Na, 379.1152). Anteaglonialide B (2): off-white, amorphous solid; [α]25 D −41 (c 1.56, MeOH); UV (MeOH) λmax (log ε) 227 (4.68), 300 (3.82), 314 (3.60), 328 (3.60) nm; 1H and 13C NMR data, see Table 1; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/ z 363.1252 (calcd for C20H20O5Na, 363.1203). Anteaglonialide C (3): off-white, amorphous solid; [α]25 D −39 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 227 (4.68), 300 (3.82), 328 (3.57) nm; 1H and 13C NMR data, see Table 1; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 363.1246 (calcd for C20H20O5Na, 363.1203). Anteaglonialide D (4): off-white, amorphous solid; [α]25 D +26 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 227 (4.42), 301 (3.64), 315 (3.53), 329 (3.42) nm; 1H and 13C NMR data, see Table 2; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/ z 361.1081 (calcd for C20H18O5Na, 361.1046). Anteaglonialide E (5): off-white, amorphous solid; [α]25 D +136 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 225 (4.66), 297 (3.75), 328 (3.50) nm; 1H and 13C NMR data, see Table 2; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 361.1068 (calcd for C20H18O5Na, 361.1046). Anteaglonialide F (6): off-white, amorphous solid; [α]25 D −48 (c 0.45, MeOH); UV (MeOH) λmax (log ε) 226 (4.69), 299 (3.83), 328 (358) nm; 1H and 13C NMR data, see Table 2; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 359.0938 (calcd for C20H16O5Na, 359.0890). Palmarumycin CE1 (7): off-white, amorphous solid; [α]25 D −64 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 226 (4.41), 300 (3.53), 328 (3.31) nm; 1H and 13C NMR data, see Table 3; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 361.1089 (calcd for C20H18O5Na, 361.1046). Palmarumycin CE2 (8): off-white, amorphous solid; 1H and 13C NMR data, see Table 3; For key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 363.1229 (calcd for C20H20O5Na, 363.1203). 4(5a)-Anhydropalmarumycin CE2 (8a): off-white, amorphous solid; [α]25 D +11 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 267 (4.64), 299 (3.77), 313 (3.64), 328 (3.51) nm; 1H NMR and 13C NMR data, see Table 3; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/z 345.1101 (calcd for C20H18O4Na, 345.1097). Palmarumycin CE3 (9): off-white, amorphous solid; [α]25 D +24 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 228 (4.64), 299 (3.76), 313 (3.62), 328 (3.52) nm; 1H and 13C NMR data, see Table 3; for key COSY, HMBC, and NOESY data, see Figure 2; positive HRESIMS m/ z 361.1062 (calcd for C20H18O5Na, 361.1046).

Palmarumycin CP5 (10): off-white, amorphous solid; [α]25 D +43 (c 0.13, CH2Cl2) (lit.6 + 45.45); 1H NMR (400 MHz, CDCl3): δ 7.58 (1H, d, J = 8.4 Hz, H-5′ or H-4′), 7.57 (1H, d, J = 8.4 Hz, H-5′ or H4′), 7.47 (1H, dd, J = 8.4, 7.6 Hz, H-3′ or H-6′), 7.43 (1H, dd, J = 8.4, 7.6 Hz, H-3′ or H-6′), 7.04 (1H, d, J = 7.6 Hz, H-2′ or H-7′), 6.89 (1H, d, J = 7.6 Hz, H-2′ or H-7′), 4.60 (1H, m, H-2), 3.68 (1H, dt, J = 12.4, 4.0 Hz, H-5), 3.00 ((1H, t, J = 4.8 Hz, H-4a), 2.86 (1H, brd, J = 19.6 Hz, H-3), 2.73 (1H, d, J = 6.0 Hz, H-8a), 2.67 (1H, brd, J = 19.6 Hz, H-3), 2.19 (1H, brdd, J = 4.8, 12.4 Hz, H-7), 1.89 (2H, m, H-7 and H-6), 1.46 (1H, m, H-6); 1H NMR (400 MHz, pyridine-d5) δ 7.60 (2H, d, J = 8.0 Hz, H-5′ or H-4′), 7.46 (1H, dd, J = 8.0, 7.6 Hz, H-3′ or H-6′), 7.40 (1H, dd, J = 8.0, 7.6 Hz, H-3′ or H-6′), 7.26 (1H, d, J = 7.6 Hz, H-2′ or H-7′), 7.03 (1H, s, 8-OH), 6.88 (1H, d, J = 7.6 Hz, H2′ or H-7′), 5.56 (1H, 5-OH), 4.84 (1H, brs, H-2), 3.93 (1H, m, H-5), 3.21 (1H, brt, J = 4.8 Hz, H-4a), 3.12 (1H, ddd, J = 1.2, 2.8, 18.8 Hz, H-3), 2.93 (1H, dd, J = 2.0, 6.4 Hz, H-8a), 2.88 (1H, ddd, J = 1.2, 2.0, 18.8 Hz, H-3), 2.43 (1H, ddd, J = 2.8, 4.0, 14.4 Hz, H-7), 2.12 (1H, dt, J = 2.8, 13.6 Hz, H-7), 1.95 (1H, m, H-6), 1.75 (1H, ddt, J = 4.0, 13.2, 12.8 Hz, H-6); 13C NMR (100 MHz, CDCl3) δ 213.2 (C, C-4), 146.4 (C, C-8′ or C-1′), 145.9 (C, C-8′ or C-1′), 134.6 (C, C-4a′), 127.7 (CH, C-3′ or C-6′), 127.6 (CH, C-3′ or C-6′), 122.2 (CH, C-4′ or C5′), 122.1 (CH, C-4′ or C-5′), 113.7 (C, C-8a′), 110.0 (CH, C-2′ or C-7′), 109.7 (CH, C-2′ or C-7′), 107.8 (C, C-1), 103.4 (C, C-8). 75.6 (CH, C-2), 71.1 (CH, C-5), 50.0 (CH, C-4a), 47.9 (CH, C-8a), 46.6 (CH2, C-3), 33.0 (CH2, C-7), 28.8 (CH2, C-6); 13C NMR (100 MHz, pyridine-d5) δ 211.2 (C, C-4), 148.24 (C, C-8′ or C-1′), 148.20 (C, C8′ or C-1′), 135.3 (C, C-4a′), 128.6 (CH, C-3′ or C-6′), 128.3 (CH, C-3′ or C-6′), 122.4 (CH, C-4′ or C-5′), 121.9 (CH, C-4′ or C-5′), 115.2 (C, C-8a′), 111.2 (CH, C-2′ or C-7′), 110.0 (CH, C-2′ or C-7′), 109.2 (C, C-1), 104.6 (C, C-8). 76.9 (CH, C-2), 71.4 (CH, C-5), 52.2 (CH, C-4a), 48.4 (CH2, C-3), 48.3 (CH, C-8a), 36.0 (CH2, C-7), 30.0 (CH2, C-6). Preparation of the (R)- and (S)-MTPA Ester Derivatives (1a and 1b) of 1 by a Convenient Mosher Ester Procedure.21 Anteaglonialide A (1; 1.0 mg) was dissolved in pyridine-d5 (0.5 mL), and the solution was transferred into a clean and dry NMR tube. (S)(−)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride (5 μL) was added to the NMR tube immediately under a stream of N2, and the tube was shaken carefully to mix the sample and the MTPA chloride. The NMR tube was allowed to stand at room temperature for 1 h to afford the (R)-MTPA ester derivative (1a) of 1: 1H NMR data of 1a (400 MHz, pyridine-d5) δ 6.20 (1H, dd, J = 3.2, 4.8 Hz, H-8), 5.26 (1H, dd, J = 3.2, 8.8 Hz, H-7), 5.14 (1H, m, H-4), 3.53 (1H, overlapped, H-9), 3.11 (1H, d, J = 18.0 Hz, H-9), 2.69 (1H, m, H-6), 2.52 (1H, brd, J = 12.8 Hz, H-5), 2.30 (1H, t, J = 12.8 Hz, H-5), 2.03 (1H, dt, J = 14.0, 4.0 Hz, H-2), 1.88 (1H, m, H-3), 1.61 (1H, m, H-3), 1.39 (1H, m, H-2). In the manner described for 1a, another portion of 1 (0.9 mg) was reacted in a second NMR tube with (R)-(+)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride (5 μL) at room temperature for 2 h in pyridine-d5 to afford the (S)-MTPA ester derivative (1b): 1H NMR data of 1b (400 MHz, pyridine-d5) δ 6.22 (1H, dd, J = 3.2, 4.8 Hz, H-8), 5.41 (1H, m, H-4), 5.23 (1H, dd, J = 3.2, 9.2 Hz, H-7), 3.46 (1H, overlapped, H-9), 3.08 (1H, m, H-6), 2.85 (1H, d, J = 18.0 Hz, H-9),, 2.57 (1H, brd, J = 13.2 Hz, H-5), 2.24 (1H, t, J = 12.8 Hz, H-5), 2.20 (1H, brd, J = 14.0 Hz, H-2), 1.97 (1H, m, H3), 1.83 (1H, m, H-3), 1.64 (1H, m, H-2). Preparation of the (R)- and (S)-MTPA Ester Derivatives (10a and 10b) of 10 by a Convenient Mosher Ester Procedure.21 The same procedure for the preparation of 1a and 1b was used to prepare the (R)-MTPA ester derivative (10a) and the (S)-MTPA ester derivative (10b) of 10: 1H NMR data of 10a (400 MHz, pyridine-d5) δ 5.32 (1H, dt, J = 11.2, 4.8 Hz, H-5), 4.78 (1H, brs, H-2),, 3.70 (1H, overlapped, H-4a), 3.14 (1H, dd, J = 1.2, 6.4 Hz, H-8a), 2.95 (1H, ddd, J = 1.2, 3.6, 19.2 Hz, H-3), 2.83 (1H, brd, J = 19.2 Hz, H-3), 2.51 (1H, dt, J = 14.0, 3.2 Hz, H-7), 2.26 (1H, m, H-7), 1.99 (1H, m, H-6), 1.94 (1H, m, H-6); 1H NMR data of 10b (400 MHz, pyridine-d5) δ 5.34 (1H, dt, J = 12.4, 4.8 Hz, H-5), 4.78 (1H, brs, H-2),, 3.71 (1H, overlapped, H-4a), 3.15 (1H, dd, J = 1.2, 6.4 Hz, H-8a), 2.98 (1H, ddd, J = 1.2, 3.2, 18.8 Hz, H-3), 2.83 (1H, brd, J = 18.8 Hz, H-3), 2.45 (1H, dt, J = 14.0, 3.2 Hz, H-7), 2.22 (1H, m, H-7), 1.87 (1H, m, H-6), 1.83 H



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(1H, m, H-6); 13C NMR (100 MHz, pyridine-d5) δ 205.8 (C, C-4), 115.9 (C, C-8a′), 112.1 (CH, C-2′ or C-7′), 110.7 (CH, C-2′ or C-7′), 109.9 (C, C-1), 104.9 (C, C-8), 77.7(CH, C-2), 76.6 (CH, C-5), 49.8 (CH, C-4a), 49.2 (CH, C-8a), 49.1 (CH2, C-3), 36.3 (CH2, C-7), 26.9 (CH2, C-6). Cytotoxicity Assay. Ewing’s sarcoma CHP-100 cells were cultured at 37 °C under 6% CO2 in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS). Cultures were passaged twice weekly, and cells in exponential growth were used for experiments. Stock solutions of compounds were formulated in DMSO and maintained at −20 °C protected from light. To measure acute toxicity, CHP-100 cells were seeded in flat-bottom 96-well plates (7500 cells/well) and allowed to adhere overnight. Serial dilutions of compounds or DMSO vehicle control (not exceeding 0.2%) were added, and relative viable cell number was determined 24 h later by dye reduction assay using the substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) as previously described.33 Doxorubicin and DMSO were used as positive and negative controls, respectively.

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Ramakrishnan, R.; Faeth, S. H.; Ahmad, N.; Gunatilaka, A. A. L. Bioorg. Med. Chem. 2014, 22, 6112−6116. (e) Wijeratne, E. M. K.; EspinosaArtiles, P.; Gruener, R.; Gunatilaka, A. A. L. J. Nat. Prod. 2014, 77, 1467−1472. (f) Wijeratne, E. M. K.; Xu, Y.; Arnold, A. E.; Gunatilaka, A. A. L. Nat. Prod. Commun. 2015, 10, 107−111. (5) After preparation of this article a paper describing the isolation of a minor metabolite with the same carbon skeleton as anteaglonialides, but called a palmarumycin (palmarumycin P1), from an unidentified fungus belonging to Dothideomyces (Pleosporales) has recently appeared in the literature; see: Bunyapaiboonsri, T.; Yoiprommarat, S.; Nopgason, R.; Intereya, K.; Suvannakad, R.; Sakayaroj, J. Tetrahedron 2015, 71, 5572−5578. (6) Krohn, K.; Beckmann, K.; Floerke, U.; Aust, H.-J.; Draeger, S.; Schulz, B.; Busemann, S.; Bringmann, G. Tetrahedron 1997, 53, 3101− 3110. (7) Bringmann, G.; Busemann, S.; Krohn, K.; Beckmann, K. Tetrahedron 1997, 53, 1655−1664. (8) Krohn, K.; Michel, A.; Floerke, U.; Aust, H.-J.; Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 11, 1093−1097. (9) Martinez-Luis, S.; Della-Togna, G.; Coley, P. D.; Kursar; Thomas, A.; Gerwick, W. H.; Cubilla-Rios, L. J. Nat. Prod. 2008, 71, 2011−2014. (10) Sakemi, S.; Inagaki, T.; Kaneda, K.; Hirai, H.; Iwata, E.; Sakakibara, T.; Yamauchi, Y.; Norcia, M.; Wondrack, L. M.; Sutcliffe, J. A.; Kojima, N. J. Antibiot. 1995, 48, 134−142. (11) Ohishi, H.; Chiba, N.; Mikawa, T.; Sakaki, T.; Miyaji, S.; Sezaki, M. Jpn. Pat. 01294686, Nov 28, 1989. (12) Narasimhachari, N.; Gopalkrishnan, K. S. J. Antibiot. 1974, 27, 283−287. (13) (a) Nagarajan, R.; Narasimhachari, N.; Kadkol, M. V.; Gopalkrishnan, K. S. J. Antibiot. 1971, 24, 249−252. (b) Paranagama, P. A.; Wijeratne, E. M. K.; Burns, A. M.; Marron, M. T.; Gunatilaka, M. K.; Arnold, A. E.; Gunatilaka, A. A. L. J. Nat. Prod. 2007, 70, 1700− 1705. (14) Schueffler, A.; Liermann, J. C.; Kolshorn, H.; Opatz, T.; Anke, H. Z. Naturforsch., C: J. Biosci. 2009, 64, 25−31. (15) Venkatasubbaiah, P.; Chilton, W. S. J. Nat. Prod. 1992, 55, 461− 467. (16) Cai, Y.-S.; Guo, Y.-W.; Krohn, K. Nat. Prod. Rep. 2010, 27, 1840−1870. (17) Jiao, P.; Swenson, D. C.; Gloer, J. B.; Campbell, J.; Shearer, C. A. J. Nat. Prod. 2006, 69, 1667−1671. (18) Bode, H. B.; Walker, M.; Zeeck, A. Eur. J. Org. Chem. 2000, 2000, 3185−3193. (19) Tian, J.; Liu, X. C.; Liu, Z. L.; Lai, D.; Zhou, L. Pest Manage. Sci. 2015, DOI: 10.1002/ps.4075. (20) Mugambi, G. K.; Huhndorf, S. M. Syst. Biodiversity 2009, 7, 453−464. (21) (a) Su, B. N.; Park, E. J.; Mbwambo, Z. H.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 1278−1282. (b) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2004, 104, 17−117. (c) Gao, S.; Xu, Y.; Valeriote, F. A.; Gunatilaka, A. A. L. J. Nat. Prod. 2011, 74, 852−856. (22) Anglea, T. A.; Pinder, A. R. Tetrahedron 1987, 43, 5537−5543. (23) Gersch, M.; Kreuzer, J.; Sieber, S. A. Nat. Prod. Rep. 2012, 29, 659−682. (24) U’Ren, J. M.; Lutzoni, F.; Miadlikowska, J.; Laetsch, A. D.; Arnold, A. E. Am. J. Bot. 2012, 99, 898−914. (25) Ewing, B.; Green, P. Genome Res. 1998, 8, 186−194. (26) Ewing, B.; Hillier, L.; Wendl, M. C.; Green, P. Genome Res. 1998, 8, 175−185. (27) Maddison, W. P.; Maddison, D. R. Mesquite, 2011. www. mesquiteproject.org. (28) Liu, K. L.; Porras-Alfaro, A.; Kuske, C. R.; Eichorst, S. A.; Xie, G. Appl. Environ. Microbiol. 2012, 78, 1523−1533. (29) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. J. Mol. Biol. 1990, 215, 403−410. (30) Maddison, D. R.; Maddison, W. P. MacClade v. 4.08a; 2005, http://macclade.org.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00717. 1D and 2D NMR spectra of compounds 1−9, 1D NOESY spectrum and key NOESY correlations for compound 10, and the majority-rule consensus tree indicating placement of FL0768 within Anteaglonium (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (520) 621-9932. Fax: (520) 621-8378. E-mail: leslieg1@ email.arizona.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this work was provided by the Grant R01 CA090265 funded by the National Cancer Institute (NCI), Grant P41 GM094060 funded by the National Institute of General Medical Sciences (NIGMS), and Grant DEB-0640996 funded by the National Science Foundation. We are also thankful to Dr. L. Whitesell (Whitehead Institute) for cytotoxicity assays, Dr. M. Carlos de Mattos (Federal University of Fortaleza, Ceara) for his valuable suggestions, and CNPq, Brazil, for award of fellowships to J.M. (Process: 236451/2012-0) and MCFO (Process: 233171/2012-6).

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