Cyclized 9,11-secosterol enol-ethers from the gorgonian Pseudopterogorgia americana

Accepted Manuscript Cyclized 9,11-Secosterol Enol-Ethers from the Gorgonian Pseudopterogorgia americana Yang-Qing He, Stacee Lee Caplan, Paul Scesa, Lyndon M. West PII: DOI: Reference:

S0039-128X(17)30101-0 http://dx.doi.org/10.1016/j.steroids.2017.06.008 STE 8119

To appear in:

Steroids

Received Date: Revised Date: Accepted Date:

8 March 2017 14 June 2017 17 June 2017

Please cite this article as: He, Y-Q., Lee Caplan, S., Scesa, P., West, L.M., Cyclized 9,11-Secosterol Enol-Ethers from the Gorgonian Pseudopterogorgia americana, Steroids (2017), doi: http://dx.doi.org/10.1016/j.steroids. 2017.06.008

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Cyclized 9,11-Secosterol Enol-Ethers from the Gorgonian Pseudopterogorgia americana Yang-Qing He a,b, Stacee Lee Caplan b , Paul Scesa b, Lyndon M. West b,* a

Department of Applied Chemistry, Xi’an University of Technology, Xi’an 710048 China

b

Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431, United

States

∗ Corresponding author. Tel.: +1-561-297-0939; Fax: +1-561-297-2759 E-mail address: [email protected].

1

ABSTRACT Chemical investigation of the MeOH extract from the gorgonian Pseudopterogorgia americana afforded two rare sterols, ameristerenol A (1) and B (2), both 9,11-secosterols possesses a seven-membered cyclic enol-ether in ring C, and ameristerol A (3) is the first example of a naturally occurring 9,11-secosterol containing a gorgosterol side chain with a C-24(28) double bond. Ameristerenol A (1) was converted to the sterol derivatives 4−6 to provide additional chemical diversity and comparison for biological screening. The structures of compounds 1−6, along with three related known analogues 7−9, were determined on the basis of extensive spectroscopic analysis and by comparison with literature data. Compound 6 exhibited slight cytotoxicity activity against human breast cancer cell line MDA-MB-231.

Keywords: Sterols; 9,11-secosterol enol-ethers; Gorgonian corals; Pseudopterogorgia americana

2

1. Introduction Pseudopterogorgia americana, is a widely distributed marine gorgonian on the reefs in Florida and the Caribbean Sea and have been subjected to numerous chemical investigations [1]. It has been found to produce sesquiterpenoids [2-6], phospholipid fatty acids [7], an imidazole betaine [8] and the 9,11-secosterols [9], which are characterized by a cleaved ring C of the steroid tetracyclic skeleton. There have been at least nine 9,11-secosterols reported from P. americana, which includes the first example of a 9,11-secosterol (22R,23R,24R)-22,23-methylene-23,24-dimethyl-9,11-secocholest-5-en-3,11-diol

that

was

isolated

by

Spraggins in 1970 [10]. All of the reported 9,11-secosterols have a ketone at C-9, a hydroxyl group at C-11 and differ in the degree of unsaturation, hydroxylation and side chain moieties. Two have also been reported with an epoxide at the C-5/C-6 position [11]. The 9,11-secosterols have been reported to exhibit diverse biological activities including anti-histaminic, anti-proliferative, anti-inflammatory, cytotoxicity and protein kinase C (PKC) inhibition [9]. It has also been suggested that the secosterols provide P. americana with a chemical defense against fish predation [12]. Continuing the search for new bioactive polar metabolites from marine organisms, chemical constituents of the gorgonian P. americana collected off the south west coast of Great Abaco Island, Bahamas were investigated. A large-scale extraction yielded two new cyclized 9,11-secosterol enol-ethers, ameristerenol A (1) and B (2), a new 9,11-secosterol, ameristerol A (3), together with three known compounds, 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (7) [13], 3β,11,24-trihydroxy-9,11-secogorgost-5-en-9-one (8) [13] and 3β,11-dihydroxy-5,6-epoxy-9,11-secogorgost-9-one (9) [11] (Fig. 1). Ameristerenols A (1) and B (2) are the first reported cyclized 9,11-secosterols from P. americana. The structures of the compounds were elucidated based on extensive spectroscopic analysis and comparison with literature data. Herein the details of the isolation and structural elucidation of 1−3 and conversion of compounds 1 to the 9,11-secosterols 4−6 are

3

reported. 2. Experimental 2.1. General methods Optical rotations were measured on a JASCO P-2000 polarimeter (c g/100 mL) equipped with a halogen lamp (589 nm) and a 1 mL microcell. IR spectra were recorded on a Thermo Electronic Corporation Nicolet IR-100 spectrophotometer. All NMR spectra were acquired with a Varian MercuryPlus 400 spectrometer using solvent signals (CDCl3: 1H, δH 7.26 ppm;

13

C, δC 77.0 ppm) as references. Short-and long-range 1H-13C

correlations were determined with gradient-enhanced inverse-detection HSQC and HMBC experiments, respectively. NOE correlations were detected with NOESY experiments with a 0.5 s mixing time. The HRESIMS were obtained using an Agilent 6220 series TOF mass spectrometer. HPLC was performed on a Shimadzu LC-20AT instrument with a Shimadzu SPD-M20A UV/vis photodiode array detector and a Shimadzu ELSD-LTII detector. 2.2. Animal material The gorgonian Pseudopterogorgia americana was collected by hand using SCUBA at a depth of 6 m off the south west coast of Great Abaco Island in the Bahamas in 2007. The specimen was immediately frozen and kept at ‒20 °C until extraction. A voucher specimen has been deposited in Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton (BA07-075). 2.3. Extraction and isolation The soft coral P. americana (270 g wet wt.) was minced and extracted with MeOH (3 × 1000 mL). The combined extracts were concentrated in vacuo to give a crude residue (70.0 g). The crude residue was then chromatographed on HP-20 (6.0 × 65 cm) with 1000 mL fractions of (1) H2O, (2) 20% Me2CO/H2O, (3) 40% Me2CO/H2O, (4) 60% Me2CO/H2O, (5) 80% Me2CO/H2O and (6) Me2CO to yield six fractions (A−F). Fraction

4

D (8.0 g) was separated on a silica gel column (3.0 × 50 cm) using a stepwise gradient of hexanes/EtOAc (50:50, 40:60, 30:70 10:90 and 0:100, v/v, each 500 mL) and EtOAc/MeOH mixtures (80:20, 60:40, 40:60, 20:80 and 0:100, v/v, each 500 mL) to afford ten sub-fractions (Fr. D1−D10). Fraction D3 (800 mg) was subjected to gel permeation Sephadex LH-20 column chromatography to afford 6 fractions (Fr. D3a−D3f) of MeOH (20 mL each). Fraction D3c (510 mg) was chromatographed on normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 10 × 250 mm; 5−35% hexanes/EtOAc over 85 min, 7 mL/min) to yield ameristerenol A (1, 110.0 mg, tR 76.0 min). Fraction D4 (2.0 g) was subjected to Sephadex LH-20 size-exclusion chromatography to afford 7 fractions (Fr. D4a−D4g) of 50 mL MeOH each. Fraction D4d (350 mg) was subjected to silica gel column chromatography (3.0 × 50 cm) using CH2Cl2/MeOH (100:0 to 0:100, v/v) to afford ten fractions (Fr. D4d-1 to Fr. D4d-10). Fraction D4d-4 (30 mg) was further purified on a normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 × 250 mm; 5−35% hexanes/EtOAc over 70 min, 7 mL/min) to yield a major fraction (tR 60.6 min), which was further separated on a normal phase column using a different gradient (Phenomenex Luna Silica; 5 µm; 20 × 250 mm; 5−25% hexanes/EtOAc over 70 min, 7 mL/min) to yield ameristerol A (3, 3.0 mg, tR 39.9 min) and 3β,11,24-trihydroxy-9,11-secogorgost-5-en-9-one (8, 4.0 mg, tR 51.2 min). Fraction D4d-5 (70 mg) was purified on a polymeric HPLC column (Hamilton PRP-1; 5 µm; 20 × 250 mm; 10−40% CH3CN/H2O over 50 min, 7 mL/min) to yield a major fraction with tR 65.3 min. This fraction (25 mg) was chromatographed on a normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 × 250 mm; 5−25% hexanes/EtOAc over 60 min, 7 mL/min) to yield 3β,11-dihydroxy-5,6-epoxy-9,11-secogorgost-9-one (9, 5.0 mg, tR 53.5 min). Fraction D4d-10 (18 mg) was further purified by normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 × 250 mm; 10−35% hexanes/EtOAc over 70 min, 7 mL/min) to afford ameristerenol B (2, 8.0 mg, tR 13.1 min). Finally, fraction E (300 mg) was subjected to a flash chromatography on a silica gel column (3.0 × 50 cm) eluting with hexanes EtOAc (from 100:0 to 0:100 gradient) to afford 10

5

fractions of 100 mL each. Sub-fraction E3 (82 mg) was purified on a normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 × 250 mm; 5−25% hexanes/EtOAc over 70 min, 7 mL/min) to yield 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (7, 30 mg, tR 48.7 min). 2.3.1. Ameristerenol A (9,11-epoxy-9,11-secogorgosta-5,8(9)-dien-3β-ol) (1) 18

Colorless gum; [α] 1

-1

D

+80 (c 0.94, MeOH); IR (KBr) νmax 3392, 3080, 2957, 2876, 1455, 1101, 1056 cm ;

13

+

H and C NMR (400 MHz, CDCl3), see Table 1; HRESIMS m/z 441.3727 [M + H] (calcd for C30H49O2,

441.3727). 2.3.2. Ameristerenol B (9,11-epoxy-9,11-secogorgosta-5,8(9)-dien-3β-yl acetate) (2) 18

Colorless gum; [α] 13

-1

D

1

+140 (c 0.45, MeOH); IR (KBr) νmax 3070, 2957, 2876, 1734, 1460, 1033 cm ; H and +

C NMR (400 MHz, CDCl3), see Table 1; HRESIMS m/z 483.3831 [M + H] (calcd for C32H51O3, 483.3833).

2.3.3. Ameristerol A (3β,11-dihydroxy-9,11-secogorgost-5,24(28)-dien-9-one) (3) Colorless gum; [α]18D +72 (c 0.54, MeOH); IR (KBr) νmax 3410, 3030, 2959, 1705, 1462, 1054 cm-1; 1H and 13

+

C NMR (400 MHz, CDCl3), see Table 1; HRESIMS m/z 457.3668 [M + H] (calcd for C30H49O3, 457.3676).

2.4. Preparation of 3β,11-acetoxy-9,11-secogorgost-5-en-9-one (4) Ameristerenol (1) (20.0 mg, 0.045 mmol) was dissolved in pyridine (1 mL) and Ac2O (1 mL). The reaction mixture was stirred at rt for 24 h, and then quenched with H2O (1 mL), and neutralized with 10% NaHCO3. The mixture was extracted with CH2Cl2 (3 × 20 mL) and the combined extracts were concentrated in vacuo. The brown gum was purified on a normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 x 250 mm; 5−20% hexanes/EtOAc over 60 min, 7 mL/min) to yield 15.2 mg of 4 (62.3%, tR 32.1 min). Colorless gum; 18

[α]

-1

D

1

13

+43.3 (c 1.38, MeOH); IR (KBr) νmax 3070, 2957, 2876, 1736, 1712, 1465, 1237, 1030 cm ; H and C +

NMR (400 MHz, CDCl3), see Table 2; HRESIMS m/z 543.4045 [M + H] (calcd for C34H54O5, 543.4044). 2.5. Preparation of 3,9-dioxo-9,11-secogorgost-5-en-11-al (5)

6

To a stirred solution of oxalyl chloride (40 µL, 0.38 mmol) in CH2Cl2 (1 mL) at –78 °C, was added a solution of DMSO (65 µL, 1.0 mmol). After 3 min a solution of 1 (30 mg, 0.068 mmol) in CH2Cl2 (1.0 mL) was added dropwise. After 15 min, Et3N (0.50 mL, 3.59 mmol) was added dropwise. The solution was stirred 30 min at –78 °C and then slowly warmed to rt. The mixture was concentrated in vacuo and dissolved in water (20 mL) and extracted with Et2O (3 × 20 mL). The combined organic extracts were dried (Na2SO4) and concentrated in vacuo to give a yellow gum, which was subjected to separation on a normal phase HPLC column (Phenomenex Luna Silica; 5 µm; 20 x 250 mm; 10−30% hexanes/EtOAc over 60 min, 7 mL/min) to yield 5 (12.0 mg, 38.8% 18

yield, tR 44.8 min). Colorless gum; [α] -1

1

D

+26.8 (c 0.35, MeOH); IR (KBr) νmax 3070, 2957, 2876, 1736, 1712,

13

+

1465, 1237, 1030 cm ; H and C NMR (400 MHz, CDCl3), see Table 2; HRESIMS m/z 543.4045 [M + H] (calcd for C34H54O5, 543.4044). 2.6. Preparation of 3β-hydroxy-9-oxo-9,11-secogorgost-5-en-11-al (6)

To a stirring solution of 1 (25 mg, 0.0568 mmol) and DMP (36.1 mg, 0.0852 mmol) in CH2Cl2 (2 mL) was added dropwise a solution of CH2Cl2 (2 mL) and H2O (2 µL) over 30 min. Et2O (10 mL) was added, the resultant solution concentrated in vacuo. The crude product was dissolved in Et2O (20 mL) and washed with 1:1 10% Na2S2O3 and saturated aqueous saturated NaHCO3, aqueous saturated NaHCO3 (15 mL), followed by H2O (10 mL) and brine (10 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo to give a white gum, which was subjected to separation on a normal phase HPLC column (Phenomenex Luna Silica; 5

µm; 20 x 250 mm; 10−40% hexanes/EtOAc over 70 min, 7 mL/min) to yield 6 (3.9 mg, 15.0%, tR 57.9 min). 18

Colorless gum; [α] and

13

-1

D

1

+28.4 (c 0.35, MeOH); IR (KBr) νmax 3389, 3060, 2961, 2876, 1712, 1460, 1049 cm ; H +

C NMR (400 MHz, CDCl3), see Table 2; HRESIMS m/z 457.3678 [M + H] (calcd for C30H49O3,

457.3676). 3. Results and discussion

7

Ameristerenol A (1) was isolated as a colorless gum. Its molecular formula C30H48O2, that was determined from the HRESIMS by the [M + H]+ ion at m/z 441.3727, required seven degrees of unsaturation. A preliminary analysis of the NMR data (Table 1) revealed two double bonds (δC 156.6, 138.2, 118.6, 115.0), the presence of a doublet of doublet signal at δH 0.48 (dd, J = 9.2, 4.4 Hz), a triplet signal at −0.13 (t, J = 5.6 Hz), and a resonance integrating for two hydrogens at δH 0.23 that could be assigned to C-22 and C-24, which were characteristic of a cyclopropyl group substituted at C-22 and C-23 of the side chain of gorgosterols [11]. These data accounted for three of the seven double bond equivalents, required by the molecular formula and indicated that ameristerenol (1) was tetracyclic. One of the rings could be assigned to a seven-membered cyclic enol-ether from the presence of the signals for an oxymethylene group [δH 4.01 (1H, dt, J = 12.4, 3.6 Hz, H-11α), δH 3.62 (1H, dt, J = 12.1, 1.6 Hz H-11β); 1

1

δC 68.0 (C-11)] and two olefinic quaternary carbons at δC 115.0 (C-8) and δC 156.6 (C-9). H– H COSY correlations between H2-11 and H2-12 and HMBC correlations from H-11 to C-9, C-12, and C-13, together with HMBC correlations from H-14 to C-8, C-9, C-12 and C-13 confirmed this assignment (Fig. 2).

This

suggested ameristerenol A (1) was a 9,11-secosterol containing a seven-membered cyclic enol-ether in ring C. Examination of the NMR data revealed near identical chemical shift values in the tetracyclic skeleton to the cyclized 9,11-secosterol 9,11-epoxy-9,11-secocholest-5,8(9)-dien-3β-ol (10), that was obtained as an artifact from the decomposition of 3β,11-dihydroxy-9,11-secocholest-5-en-9-one in CDCl3 during acquisition of the NMR data [14]. This suggested that the tetracyclic skeleton of ameristerenol A (1) was identical to 9,11-epoxy-9,11-secocholest-5,8(9)-dien-3β-ol except it contained the gorgosterol side chain. 1

1

A detailed analysis of the HSQC, H− H COSY and HMBC permitted assignment of the signals in ring A, B and D and the connection of the cyclopropane containing side chain to the tetracyclic skeleton. HMBC correlations from H3-19 to C-10, C-1 and the two double bond carbons at C-9 and C-5 established the

8

connection of C-1, C-5, C-9 and C-19 to C-10. Additionally, HMBC correlations from the trisubstituted double bond signal at δH 5.33 (H-6) to C-4, C-7, C-8 and C-10 together with 1H−1H COSY correlations between the H-6 and H2-7 signals, and the oxygenated methane signal at δH 3.55 (H-3) to both H2-2 and H2-4 and between the H2-2 to H2-1 signals completed the assignment and closure of ring A and B and allowed the connection of ring B to C. Similarly, HMBC correlations from H3-18 to C-12, C-13, C-14, and C-17 and from signal of H2-15 1

1

to C-8, C-14 and C-17 combined with H− H COSY correlations between the H2-15 and H2-16 signals allowed the closure and assignment of ring D. Finally, HMBC correlations from the signal of H3-21 to C-17, C-20 and C-22 and from H-19 to C-17 allowed the connection of the cyclopropyl containing side chain to the tetracyclic skeleton. 1

The relative configuration of ameristerenol A (1) was determined from coupling patterns in the H NMR and NOE correlations observed in a NOESY spectrum (Fig. 3), as well as comparison with reported data. The relative configuration of C-3, C-10, C-13, C-14, and C-17 of the tetracyclic skeleton and at C-20, C-21, C-22, and

C-23

of

the

gorgosterol

side

chain

were

proven

to

be

the

same 1

as 13

9,11-epoxy-9,11-secocholest-5,8(9)-dien-3β-ol [14] and 7 [13], respectively from near identical H and C NMR data. NOE correlations from Me-19 to H-1β, H-2β, and H-4β, together with correlations from H-3 to H-2α, H-6 established the axial orientation of the Me-19 and the equatorial geometry of the hydroxyl group at C-3. The presence of a large coupling constant (J = 12.6 Hz) between H-3 and H-4β was consistent with the anti-periplanar relationship of these two protons and suggested that ring A was in a chair conformation. NOE correlations from Me-18 to H-11β and H-20, together with correlations H-14 to H-12α and H-17 established the trans-fused relationship between ring C and D. Finally, NOE correlations observed between protons on the gorgosterol side chain were consistent with those observed for 9 [11] (Fig. 3), therefore the structure of ameristerenol (1) was established as 9,11-epoxy-9,11-secogorgosta-5,8(9)-dien-3β-ol.

9

Ameristerenol B (2) was isolated as a colorless gum. Its molecular formula C32H50O3, that was determined from the HRESIMS of the [M + H]+ ion at m/z 483.3831, is 42 mass units higher than that of 1. The 1H NMR data of 2 was similar to that of ameristerenol B (1), except that H-3 [δH 4.62, tt (11.2, 4.8)] was shifted 13

downfield by 1.07 ppm as compared with that of 1. In the C NMR spectrum, the resonance of C-3 (δC 74.0) was shifted downfield by 2.1 ppm and those of C-2 (δC 27.3) and C-4 (δC 37.0) were both shifted upfield by 4.0 ppm in comparison with those of 1. This suggested that the 3-hydroxy group of 1, was replaced by an acetate group at C-3 in 2. The presence of the acetate group was confirmed by the NMR data [δH 2.03 (3H, s), δC 21.4 (q), 170.2 (CO)]. An HMBC correlation observed from H-3 to the ester carbonyl carbon at 170.2 confirmed the 1 placement of the acetate group at the C-3 position. The similarity of proton−proton coupling constants and H

13

and C chemical shifts together with a NOESY spectrum of 2 showed the same relative configuration as that of ameristerenol

A

(1).

Therefore,

the

structure

of

ameristerenol

B

(2)

was

established

as

9,11-epoxy-9,11-secogorgosta-5,8(9)-dien-3β-yl acetate. Ameristerol A (3) was isolated as a colorless gum. Its molecular formula C30H48O3, that was determined from +

the HRESIMS of the [M + H] ion at m/z 457.3668, required seven degrees of unsaturation. Initial analysis of the NMR data (Table 1) suggested that 3 had the same carbon skeleton as the co-isolated compound 7 [13] except for the absence of the signals for the methyl group at C-24, and the addition of signals for a 1,1-disubstituted double bond [δH 4.81 (1H, s), δH 4.77 (1H, s), δC 161.4 (q), 105.3 (t)]. This suggested that a C-24(28) double bond replaced the methyl group at C-24 of the gorgosterol side chain in 3. HMBC correlation observed from H-25 to both C-24 and C-28 and from double bond methylene proton signals H2-28 to C-23, C-24 and C-25 confirmed this assignment. The relative configuration of 3 was assumed to be the same as that 1

13

of 7 due to the similarity of proton−proton coupling constants and H and C chemical shifts. Therefore, the structure of 3 was determined to be 3β,11-dihydroxy-9,11-secogorgost-5,24(28)-dien-9-one and is the first

10

example of a marine sterol containing a gorgosterol side chain containing an C24(28) double bond. Compound 3 could either be the dehydration product of the co-isolated 3β,11,24-trihydroxy-9,11-secogorgost-5-en-9-one (8) that contains a hydroxyl group at C-24 or from an enzymatic dehydrogenation of 7. To increase chemical diversity for biological screening, the sterol derivatives 4−6 were prepared from 1 (Fig. 4), which was isolated in relatively large quantities (110 mg). Acylation of 1 with acetic anhydride in pyridine interestingly afforded the diacetylated ring-opened 9,11-secosterol 4. The Swern oxidation of 1 with oxalyl chloride and DMSO yielded the ring-opened oxidation product 5. In an attempt to maintain the enol-ether linkage, a Dess-Martin periodinane (DMP) oxidation was attempted; however, this also produced a ring-opened product, compound 6 [15]. The structures of compounds 4−6 were elucidated on the basis of extensive spectroscopic analysis and by comparison of their NMR data with those of the starting materials. Compounds 1−6 and the three known compounds were evaluated for cell growth inhibitory activities against human breast cancer cell lines (MCF-7 and MDA-MB-231). No cytotoxicity was observed for any of the compounds at 100 µM except for slight cytotoxicity being observed for compound 6 against the MDA-MB-231 cell line. To date only two 9,11-secosterol containing a seven-membered cyclic enol−ether in ring C have been reported. This includes the artificially produced 9,11-epoxy-9,11-secocholest-5,8(9)-dien-3β-ol (10), formed overnight

in

CDCl3

from

the

acid-catalyzed

intramolecular

cyclization

and

dehydration

of

3β,11-dihydroxy-9,11-secocholest-5-en-9-one that was isolated from the Formosan soft coral Sinularia leptoclados [14] and stellattasterenol from the Australian sponge Euryspongia arenaria [16]. It was speculated that stellattasterenol could be an artifact of isolation; however, experiments performed on the ring-opened 9,11-secosterol stellattasterol in the presence of silica gel and acid showed no conversion to stellattasterenol. Similarly, all the 9,11-secosterols isolated in this study were stable on silica gel and in CDCl3, including 7

11

which is the ring-opened relative of ameristerenol A (1). In addition, no other previous reports of the isolation of hydroxyl ketone 7 resulted in the isolation of an enol-ether product [10-14]. Two cyclic enol-ether natural products have been reported and have been found to exist in equilibrium with their ring-opened hydroxyl ketones, erythromycin A enol ether and the dihydropyran derivative of the prokaryotic pheromone stigmolone [17,18]. Erythromycin A enol ether, once thought to be an acid-catalyzed degradation product, was found to convert to erythromycin upon incubation under mild acidic conditions and similarly, stigmolone and its dihydropyran derivatives were also found to exist in an equilibrium that is both solvent- and pH-dependent. Purification procedures of the later has shown to change the equilibrium, where it slows in organic solvents or neutral conditions (yielding the dihydropyran) and is more rapid at low pH (yielding the ring-opened hydroxyl ketone). This could suggest a possible equilibrium may exists between the ameristerenols and their

ring-opened hydroxyl ketones,

and could explain the conversion of

3β,11-dihydroxy-9,11-secocholest-5-en-9-one to 10 in CDCl3 [14]. It is interesting that if ameristerenols A (1) and B (2) are artifacts of isolation, then it is surprising that 9,11-secosterol enol-ethers are not encountered more often. Given the large number of 9,11-secosterol isolated over the last 40 years using similar methods as reported herein, it would be expected that the related enol-ethers should be routinely isolated. Since this is not the case, it lends merit to further review of these compounds, their interconversion, and potential role in the biogenesis of 9,11-secosterols from polyhydroxylated steroids. Acknowledgements We thank Dr. Howard Lasker at the University at Buffalo for allowing Dr. Maia Mukherjee to participate on a research cruise to the Bahamas aboard the University of Miami’s R/V F.G. Walton Smith for collection and identification of the Pseudopterogorgia americana. The authors thank Drs. A. Robbins and T. Schulz at Viacyte Inc. for cytotoxicity testing. This research was supported by National Institutes of Health grants

12

(P41GM079597 and P01GM085354), the Science and Technology Project of Shaanxi Province in China (2015SF087), the Scientific Research Program funded by Shaanxi Provincial Education Department (15JK1500), and the Program for Scientific Activities of Selected Returned Overseas Professionals in Shaanxi Province (302-253081601).

References [1] F. Berrue, R.G. Kerr, Diterpenes from gorgonian corals, Nat. Prod. Rep. 26 (2009) 681−710. [2] A.J. Weinheimer, P.H. Washecheck, D. van der Helm, M.B. Hossain, The sesquiterpene hydrocarbons of the gorgonian, Pseudopterogorgia americana, the nonisoprenoid β-gorgonene, Chem. Commun. 18 (1968) 1070−1071. [3] S.L. Miller, W.F. Tinto, S. McLean, W.F. Reynolds, M. Yu, Bisabolane sesquiterpenes from Barbadian Pseudopterogorgi spp., J. Nat. Prod. 58 (1995) 1116−1119. [4] A.D. Rodriguez, A. Boulanger, New guaiane metabolites from the Caribbean gorgonian coral, Pseudopterogorgia americana, J. Nat. Prod. 60 (1997) 207−211. [5] A.D. Rodriguez, A. Boulanger, J.R. Martinez, S.D. Huang, Sesquiterpene lactones from the Caribbean sea plume Pseudopterogorgia americana, J. Nat. Prod. 61 (1998) 451−455. [6] W.R. Chan, W.F. Tinto, R. Moore, New gemacrane derivatives from gorgonian octocorals of the genus Pseudopterogorgia, Tetrahedron 46 (1990) 1499−1502. [7] N.M. Carballeira, A. Sostre, A.D. Rodriguez, Phospholipid fatty acid composition of gorgonians of the genus Pseudopterogorgia: Identification of tetracosapolyenoic acids, Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 113 (1996) 781−783. [8] A. Weinheimer, E. Metzner, M. Mole, A new marine betaine, norzooanemonin, in the gorgonian Pseudopterogorgia americana, Tetrahedron 29 (1973) 3135−3136.

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[9] D. Sica, D. Musumeci, Secosteroids of marine origin, Steroids 69 (2004) 743−756. [10] E.L. Enwall, D. van der Helm, I.N. Hsu, T. Pattabhiraman, F.J. Schmitz, R.L. Spraggins, A.J. Weinheimer, Crystal structure and absolute configuration of two cyclopropane containing marine steroids, J. Chem. Soc. Chem. Commun. 4 (1972) 215−216. [11] S. Naz, R.G. Kerr, R. Narayanan, New antiproliferative epoxysecosterols from Pseudopterogorgia americana, Tetrahedron Lett. 41 (2000) 6035−6040. [12] R.A. Epifanio, L.F. Maia, J.R. Pawlik, W. Fenical, Antipredatory secosterols from the octocoral Pseudopterogorgia americana, Mar. Ecol. Prog. Ser. 329 (2007) 307−310. [13] H. He, P. Kulanthaivel, B.J. Baker, K. Kalter, J. Darges, D. Cofield, L. Wolff, L. Adams, New antiproliferative and antiinflammatory 9,11-secosterols from the gorgonian Pseudopterogorgia sp, Tetrahedron 51 (1995) 51−58. [14] S.Y. Cheng, H.P. Chen, S.K. Wang, C.Y. Duh, Three new 9,11-secosterols from the formosan soft coral Sinularia leptoclados, Bull. Chem. Soc. Jpn. 84 (2011) 648−652. [15] S.D. Meyer, S.L. Schreiber, Acceleration of the Dess-Martin Oxidation by Water, J. Org. Chem. 59 (1994) 7549−7552. [16] I.A. Van Altena, A.J. Butler, S.J. Dunne, A new cyclized 9,11-secosterol enol−ether from the Australian sponge Euryspongia arenaria, J. Nat. Prod. 62 (1999) 1154−1157. [17] A. Hassanzadeh, J. Barber, G.A. Morris, P. A. Gorry, Mechanism for the Degradation of Erythromycin A and Erythromycin A 2'-Ethyl Succinate in Acidic Aqueous Solution, J. Phys. Chem. A,111 (2007) 10098–10104 [18] W.E. Hull, A. Berkessel, W. Plaga, Structure elucidation and chemical synthesis of stigmolone, a novel type of prokaryotic pheromone, Proc. Natl. Acad. Sci. USA. 95 (1998) 11268–11273.

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Fig. 1. Structures of compounds 1−3 1-10and 7−10. Fig. 2. Key 1H−1 H COSY and HMBC correlations for compounds 1 and 3. Fig. 3. Selected NOESY correlations observed for 1. Fig. 4. Conversion of 1 to sterol derivatives 4−6.

15

30 21 11 12 18 1

13 14

9 10

3

HO

22

26

20 23 19 O

28

H

17

29

24

H

25

O

H

27

H

H

5

HO

RO

3

1R=H 2 R = Ac

HO

OH HO H

O

H

H

O

H

H H

HO HO

7

8

HO H O

O

H

H H

H HO

HO O

10

9

16

17

18

19

Table 1. NMR Spectroscopic Data for Compounds 1 – 3 in CDCl3a 1 2 position δ δ (J in Hz) δ δ (J in Hz) δ 1α 34.0 1.34, dd (12.4, 3.2) 33.9 1.37, m 31.0 1β 1.97, dt (12.8, 3.2) 1.96, m 2α 31.2 1.87, m 27.3 1.88, d (13.6) 30.8 2β 1.56, m 1.63 3 71.9 3.55, tt (11.2, 4.8) 74.0 4.62, m 71.5 4α 41.0 2.33, ddd (12.4, 4.8, 1.8) 37.0 2.36, ddd (12.4, 5.2, 1.6) 40.6 4β 2.25, brt (12.6) 2.29, brt (12.4) 5 138.2 137.1 140.1 6 118.6 5.33, br s 119.6 5.36, br s 121.5 7α 30.3 2.68, brd (21.0) 30.3 2.67, brd (23.0) 32.8 7β 2.59, brd (21.0) 2.60, brd (23.0) 8 115.0 115.0 43.5 9 156.6 156.4 217.6 10 39.3 39.4 48.3 11α 68.0 4.01, dt (12.4, 3.6) 68.0 4.03, dt (12.4, 3.6) 59.1 11β 3.62, dt (12.0, 1.6) 3.63, dt (12.4, 1.6) 12α 46.4 1.78, m 46.4 1.76, dt (14.4, 4.0) 40.5 12β 2.00, m 2.00, m 13 42.8 42.9 45.4 14 50.4 2.86, t (10.4) 50.7 2.90, t (10.4) 41.6 15α 24.4 1.62, m 24.7 1.61, m 24.3 15β 1.62, m 1.61, m 16α 27.9 2.02, m 27.9 2.00 26.8 16β 1.42, m 1.40, m 17 58.4 1.45, m 58.5 1.44, m 50.7 18 12.2 0.74, s 12.2 0.72, s 17.3 19 21.5 1.29, s 21.5 1.29, s 22.9 20 35.4 1.02, m 35.4 1.01, m 33.8 21 21.7 1.01, br s 21.7 1.00, br s 20.5 22 32.0 0.21, m 32.3 0.21, m 31.4 23 25.9 26.2 26.5 24 50.7 0.25, m 50.8 0.25, m 161.4 25 32.1 1.56, m 32.2 1.55, m 29.4 26 21.5 0.85, d (6.4) 21.5 0.84, d (6.4) 24.2 27 22.2 0.95, d (5.2) 22.2 0.92, d (5.2) 24.2 28 15.3 0.93, d (4.4) 15.4 0.94, d (4.4) 105.3 29 14.2 0.90, s 14.2 0.89, s 20.4 30a 21.4 0.48, dd (9.2, 4.4) 21.4 0.46, dd (8.8, 4.4) 17.8 30b -0.13, t (5.6) -0.14, t (4.8) 21.4 2.03, s 3-OAc b a1 13 H NMR measured at 400 MHz, C NMR measured at 100 MHz, b3-OAc C=O (δC 170.2).

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3

δ (J in Hz) 1.85, m 1.55, m 1.95, m 1.52, m 3.50, m 2.40, m 2.26, dt (10.4, 2.0) 5.49, br d (5.6) 2.42, m 2.03, m 3.04, dt (12.0, 6.8)

3.90, m 3.75, m 1.73, m 1.39, m 2.69, m 1.56, m 1.34, m 1.97, m 1.32, m 1.74, m 0.70, s 1.36, s 1.20 1.08, d (6.4) 0.82, s

2.17, m 1.05, d (6.8) 1.05, d (6.8) 4.81, s; 4.77, s 1.17, s 0.72 -0.01, dd (6.4, 4.4)

Table 2. NMR Spectroscopic Data for Compounds 4 – 6 in CDCl3a 4b position δ δ (J in Hz) 1α 31.2 1.88, t (3.6) 1β 1.52, m 2α 26.9 1.92, m 2β 1.61, m 3 73.7 4.56, m 4α 36.9 2.45, dd (12.8, 4.8) 4β 2.30, m 5 139.0 6 122.5 5.53, br d (5.6) 7α 32.2 2.37, m 7β 2.08, m 8 42.2 2.97, dt (12.0, 5.2) 9 215.0 10 47.9 11α 61.8 4.22, dt (9.2, 6.0) 11β 4.16, dt (9.6, 6.0) 12α 35.9 1.80, dt (8.8, 6.0) 12β 1.46, m 13 45.7 14 41.7 2.54, m 15α 24.0 1.54, m 15β 1.30, m 16α 27.7 2.04, m 16β 1.30, m 17 50.8 1.68, m 18 16.9 0.68, s 19 23.5 1.36, s 20 35.3 0.99, m 21 21.7 1.04, s 22 32.1 0.21, m 23 25.8 24 50.4 0.25, m 25 32.6 1.53, m 26 21.6 0.84, d (6.8) 27 22.2 0.94, d (6.8) 28 15.1 0.91, d (7.2) 29 14.1 0.87, s 30a 21.2 0.46, dd (8.8, 4.0) 30b -0.15, t (4.8) a1 H NMR measured at 400 MHz, 13C NMR

5 δ 29.6

δ (J in Hz) 2.04, dd (4.8, 2.2) 2.01, t (4.4) 2.46, dd (7.6, 3.6) 2.44, d (5.2)

33.6 198.6 32.5

2.83, dd (7.2, 3.6) 2.51, dd (6.4, 3.2)

166.4 125.6 28.6

5.81, d (2.0) 2.12, m 1.46c 2.89, dd (13.2, 4.4)

44.8 211.3 51.0 203.7

9.91, s

51.4

2.55, dd (16.0, 3.0) 2.27, dd (16.0, 3.6)

46.2 43.5 23.0

2.80, m 1.55c 1.39c 2.09, m 1.39c 1.86, m 0.83, s 1.49, s 1.08c 1.08c 0.21, m

27.5 52.8 16.3 23.2 35.1 21.6 31.8 26.0 50.6 32.0 21.5 22.2 15.4 14.2 21.4

6 δ 31.0 30.7 71.4 40.4 140.5 121.2 32.7 43.0 215.8 48.1 204.2 50.4 45.7 42.6 24.2 27.3 52.6 16.2 23.0 34.9 21.5 31.5 26.0 50.7 32.0 21.7 22.1 15.3 14.3 21.4

δ (J in Hz) 1.82, t (3.2) 1.80, t (3.2) 1.89, m 1.47, m 3.50, tt (11.2, 4.4) 2.39, dd (5.6, 2.4) 2.24, m 5.48, br d (6.0) 2.43, m 2.05, m 3.02, dt (13.4, 7.2)

9.97, s 2.45, dd (16.8, 3.6) 2.17, dd (17.2, 1.6) 2.84, dt (10.4, 8.4) 1.60, m 1.34c 2.05, m 1.34c 1.93, m 0.72, s 1.32, s 1.00c 1.00c 0.20, s

0.26, m 0.22, m 1.56, m 1.53, m 0.85, d (6.8) 0.84, d (6.4) 0.95, d (6.8) 0.93, d (6.8) 0.92, d (6.8) 0.91, d (7.2) 0.90, s 0.88, s 0.51, dd (8.8, 4.4) 0.47, dd (8.8, 4.4) -0.11, dd (5.6, 4.8) -0.14, dd (5.6, 4.4) measured at 100 MHz; bAcO: δH 2.03 (3H, s); δC 170.5, 22.1

and for AcO-11: δH 1.99 (3H, s); δC 171.3, 21.5; cOverlapped signals.

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Three new 9,11-secosterols 1-3 were isolated from Pseudopterogorgia americana. Compounds 1 and 2 are sterols bearing a seven-membered cyclic enol−ether in ring C. Compounds 3 is the first 9,11-secosterol containing a C-24(28) double bond. The results presented can be used for further synthetic and pharmacological studies.

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