Aberrarone: A Gorgonian-Derived Diterpene from Pseudopterogorgia elisabethae

Abstract

A marine metabolite based on a previously undescribed carbon skeleton, aberrarone (1), is reported as a natural product from the Caribbean sea whip, Pseudopterogorgia elisabethae. The molecular structure of the crystalline metabolite was established by spectral analysis and subsequently confirmed by X-ray crystallographic analysis. Aberrarone shows in vitro antimalarial activity against a chloroquine-resistant strain of the protozoan parasite Plasmodium falciparum.

The West Indian gorgonian octocoral Pseudopterogorgia elisabethae (Bayer, 1961) has proved to be an exceptionally rich source of diterpene glycoside natural products initially isolated and characterized by Fenical and colleagues in the 1980’s.¹ Some of these compounds display significant anti-inflammatory and wound healing properties and thus have been the focus of many investigations dealing with their isolation and structure characterization, others with synthesis, and others with their biological activity.² Specifically, the pseudopterosins are of considerable interest given their commercial market as additives in skin cream products and the successful completion of clinical trials as topical anti-inflammatory agents.³ Since the pioneering work of the Fenical group, a myriad of unusual natural products have been steadily isolated from P. elisabethae by several research groups, which are representatives of several classes of previously undescribed terpene metabolites, typically based on unusual carbon skeletons.⁴ Unlike the pseudopterosins, these metabolites lack a sugar moiety indicating an interesting departure from the structures of the previously characterized diterpenes. As of 2009, nearly 66% of all of the natural products isolated from this prolific gorgonian species (in all 109 compounds) are diterpenoids based on the amphilectane or serrulatane (“biflorane”) carbon skeletons. On the other hand, about 29% of the remaining metabolites (32 compounds) are diterpenoids based on the so called “non-amphilectane” or “rearranged amphilectane” carbon skeletons.⁴ Diterpenoids belonging to the latter group are based on over 25 distinct carbon skeletal classes, and many are endowed with intricate architectures and interesting biological properties. These unusual features render P. elisabethae as the most chemically diverse gorgonian octocoral from the West Indian region.⁵ As we near towards completion of a 12-year long chemical scrutiny of this productive marine animal, we now wish to report on the discovery of aberrarone (1),⁶ an interesting C₂₀ tetracyclic natural product based on yet another unusual carbon skeleton. When evaluated for in vitro antimalarial activity, aberrarone displayed inhibitory activity against the protozoan parasite Plasmodium falciparum.

The MeOH–CHCl₃ extract of the gorgonian (<1 kg of dry wt) was partitioned between hexane and water to yield a green thick paste, a portion of which was loaded onto a large silica gel column and separated by stepwise elution with hexane– acetone mixtures. Fractions were routinely pooled on the basis of their TLC, NMR, and biological activity profiles. Aberrarone (1, 4.6 mg) was obtained pure after one of the primary fractions obtained above was subjected to a series of purification steps consisting mainly of normal-phase silica gel column chromatography and HPLC analysis. The molecular structure of this metabolite was proposed initially on the basis of comprehensive analysis of the 1D and 2D NMR (¹³C, ¹H, ¹H–¹H COSY, HSQC, HMBC, and NOESY), IR, UV, and HRFAB-MS spectra. A single-crystal X-ray structure analysis was subsequently carried out in order to confirm the proposed molecular structure of 1.

Aberrarone (1), isolated as a UV-active, light orange crystalline solid, was assigned a molecular formula of C₂₀H₂₆O₄ based on the HRFAB-MS and NMR data, implying eight degrees of unsaturation. The presence of four ketone carbonyls (δ_C 215.3, 207.2, 202.4 and 199.0) was further corroborated by the IR absorption bands at 1768, 1753, 1724, and 1699 cm⁻¹. The UV and ¹³C NMR spectra indicated that two of the carbonyls (δ_C 202.4 and 199.0) were those of a cyclic α-diketone functionality.⁷ The carbonyl groups accounted for four of the eight degrees of unsaturation as well as all of the oxygens required by the molecular formula of 1, and the absence of any further olefinic functionalities suggested a tetracyclic skeleton for this compound. Analysis of the ¹³C and DEPT NMR spectra of 1 confirmed the presence of 20 carbons, comprising four ketones, three quaternary, five methyl, five methine, and three methylene carbons. These data suggested a diterpenoid structure, but the tetracyclic nature of 1 implied rearrangement of the typical tricyclic amphilectane (or bicyclic serrulatane) skeleton, such as in elisapterosin A (2) or colombiasin A (3).^8,9

The ¹H NMR spectrum of 1 indicated the presence of five methyl groups with three-proton singlets at δ 1.54, 1.52, and 1.23, as well as two doublets with large couplings at δ 1.02 (J = 6.5 Hz) and 0.95 (J = 7.3 Hz). It also exhibited a doublet of a doublet of doublets at δ 2.87 (J = 8.4, 6.8, 1.6 Hz) and a broad doublet of doublets with fine splitting at δ 2.28 (J = 1.6 Hz), each integrating for one proton, which subsequently were attributable to the adjoining angular methines H-9 and H-10, respectively. The rest of the ¹H NMR spectrum consisted of nine complex proton resonances between δ 0.78 and δ 2.21, suggestive of a polycyclic terpenoid structure.^8,9

Correlations deduced from extensive analyses of the 2D NMR data of 1 (¹H–¹H COSY, TOCSY, HSQC, and HMBC NMR experiments) in CDCl₃ enable initially the establishment of three partial structures (a–c) that were interconnected later on to yield the final structure of aberrarone (see Figure 1). The ¹H and ¹³C NMR data are presented in Table 1.

FIGURE 1.

Partial structures for aberrarone (1) generated from ¹H— ¹H COSY and HMBC spectral correlations.

TABLE 1.

¹H NMR (500 MHz), ¹³C NMR (125 MHz),¹H—¹H COSY, NOESY, and HMBC Spectral Data for Aberrarone (1)^a

position	δH, mult, intgt (J, Hz)	δCb	1H–1H COSY	NOESY	HMBCc
1		76.1 (C)			H-3, H-5α, H-6, H-8β, H-9, H-10
2		215.3 (C)			H-3, H-9, H3-16
3	2.07, m, 1H	44.5 (CH)	H-4αβ, H3-16	H3-16	H3-16
4α	1.77, m, 1H	28.6 (CH2)	H-3, H-4β, H-5αβ	H-4β, H3-16	H3-16
4β	1.87, m, 1H		H-3, H-4α, H-5αβ	H-4α
5α	2.21, m, 1H	27.3 (CH2)	H-4αβ, H-5β, H-6	H-5β
5β	1.07, m, 1H		H-4αβ, H-5α, H-6	H-5α
6	2.18, m, 1H	50.5 (CH)	H-5αβ, H-7	H3-17	H-4αβ, H-8β, H3-17
7	1.63, m, 1H	43.0 (CH)	H-6, H-8αβ, H3-17	H-9, H3-17	H-8α, H3-17
8α	0.78, q, 1H (12.3)	44.3 (CH2)	H-7, H-8β, H-9	H-8β, H-10, H3-17	H-10, H3-17
8β	2.16, m, 1H		H-7, H-8α, H-9	H-8α, H-9
9	2.87, ddd, 1H (8.4, 6.8, 1.6)	45.9 (CH)	H-8αβ, H-10	H-7, H-8β, H3-19	H-8α, H-10
10	2.28, br d, 1H (1.6)	55.7 (CH)	H-9	H-8α, H3-18, H3-20	H-8α, H3-18, H3-19, H3-20
11		45.0 (C)			H-10, H3-18, H3-19
12		202.4 (C)			H-10, H3-18, H3-19
13		199.0 (C)			H-10, H3-20
14		63.5 (C)			H-10, H3-20
15		207.2 (C)			H-6, H-9, H-10, H3-20
16	0.95, d, 3H (7.3)	17.6 (CH3)	H-3	H-3, H-4α
17	1.02, d, 3H (6.5)	17.9 (CH3)	H-7	H-6, H-7, H-8α
18	1.23, s, 3H	29.8 (CH3)		H-10, H3-19, H3-20	H-10, H3-19
19	1.54, s, 3H	20.9 (CH3)		H-9, H3-18	H3-18
20	1.52, s, 3H	24.2 (CH3)		H-10, H3-18	H-10

^aSpectra were recorded in CDCl3 at 25 °C. Chemical shift values are in parts per million relative to the residual CHCl₃ (7.25 ppm) or CDCl₃ (77.0 ppm) signals. Assignments were aided by 2D NMR experiments, spin-splitting patterns, number of attached protons, and chemical shift values.

^b13C NMR multiplicities were obtained from a DEPT-135 experiment.

^cProtons correlated to carbon resonances in the ¹³C column. Parameters were optimized for ^2,3J_CH = 6 and 8 Hz.

Connectivities from C-3 to C-10 (substructure a) were inferred from the ¹H–¹H COSY cross-peaks, including correlations from H-3 to H₃-16 and H-7 to H₃-17. This extended spin system, encompassing half the carbon atoms present in 1 across three rings, was quickly recognized as it is present in other compounds isolated from the same gorgonian specimen (i.e. compounds 2 and 3). Confirmation of the proton connectivity network already established from the ¹H– ¹H COSY and TOCSY experiments was obtained directly from long range ¹H–¹³C couplings (Table 1). The structure elucidation of substructures b and c, comprising the other half of carbon atoms in 1, was more difficult to achieve as they deviated substantially from those found in other rearranged diterpenes, such as 2 or 3.

The HMBC experiment showed connectivity between the C-1 carbon [δ_C 76.1 (C)] and the protons of C-3, C-5, C-6, C8, C-9 and C-10. Thus the pivotal C-1 quaternary carbon must be attached to C-2, C-6, and C-9, thereby establishing the perhydroindane substructure within 1 (substructures a and c). Furthermore, these units were linked by a strong correlation between C-15 [δ_C 207.2 (C)] and protons H-6, H-9, H-10, H₃-20, and the strong couplings between C-2 [δ_C 215.3 (C)] and protons H-3, H-9 and H₃-16. Since C-10 [δ_C 55.7 (CH)] correlated strongly with H₃-18, H₃-19, and H₃-20, and C-14 [δ_C 63.5 (C)] was strongly coupled to H-10 and H₃-20, C-14 has to be flanked by C-10, C-15 and C-20. On the other hand, substructure b was connected to partial structure c by the observation of strong HMBC correlations of C-13 [δ_C 199.0 (C)] to H-10 and H₃-20, and to unit a from the correlations of C-10 [δ_C 55.7 (CH)] to geminal methyls H₃-18 and H₃-19, and between C-11 [δ_C 45.0 (C)] with H-10. The observation of a strong HMBC correlation of C-12 [δ_C 202.4 (C)] and the protons of C-10, C-18, and C-19 showed that C-11 and C-12 must themselves be linked to one another. This strongly implied that the carbonyl carbons C-12 and C-13 (the last connecting points remaining) had to be linked to one another thus comprising the 1,2-cyclopentanedione ring system within aberrarone (1). Applying these combined 2D NMR methods resulted in the unambiguous assignment of all protons and carbons as listed in Table 1 and allowed the complete planar structure for 1 to be assigned.

Segments of the relative stereochemistry of aberrarone were readily assigned by NOE NMR spectral methods (Table 1). However, while the relative configuration about the spirocyclic carbon (C-1) in 1 could be assigned indirectly on the basis of the overall correlations observed in the NOESY spectrum, we could not confidently assigned the relative configuration at C-3 with the NMR data already at hand. Thus, confirmation of the entire structure of aberrarone by single-crystal X-ray diffraction analysis was highly desirable. Recrystallization of 1 by slow evaporation from a mixture of MeOH/H₂O gave crystals of excellent quality that were amenable to X-ray crystallographic analysis. The X-ray structure, which defines only the relative configuration, is shown in the Supporting Information. Hence, the overall relative stereochemistry for the seven stereocenters about the complex tetracarbocyclic ring system of aberrarone was assigned as 1S*, 3S*, 6R*, 7S*, 9S*, 10R*, and 14S*. The assigned configurations, which were in full agreement with the correlations observed in the NOESY spectrum of 1, were confirmed through interpretation of NMR coupling data (Table 1).¹⁰

Diterpenes with the same tetracyclic ring system found in 1 have not been previously encountered in Nature.¹¹ We therefore suggest the general name aberrarane for this class of diterpene. While the biosynthesis of this regular diterpene remains to be demonstrated, aberrarone can be considered as deriving from geranylgeranyl pyrophosphate (GGPP) via C1/C15, C3/C14, C5/C13, and C5/C10 cyclizations (Scheme 1). Nonetheless, the presence of various serrulatane-, amphilectane-, and elisabethane-based diterpenes within the same gorgonian specimen provides circumstantial support for a biosynthetic pathway in which the carbon skeleton of the latter class of metabolites, for instance, is the precursor to the aberrarane skeleton (via cleavage of the C2/C17 bond followed by concomitant C10/C15 and C11/C17 cyclizations (see Scheme 2 in the Supporting Information).^12,13

SCHEME 1.

Proposed Biogenesis of the Aberrarane Carbon Skeleton

Compounds 1–3 were tested against the most common etiologic agent of malaria, Plasmodium falciparum (W-2 chloroquine-resistant strain). The antimalarial activity was determined using a novel fluorometric method, based on the intercalation of the fluorochrome PicoGreen® in the parasite DNA, as described by Corbett et al.¹⁴ After a 24 and 48 h incubation, all P. falciparum parasites were killed by these compounds, with α-diketone 1 and colombiasin A (3) showing the highest antimalarial activity (IC₅₀ = 10 µg/mL).

Experimental Section

Biological Material

The biological specimens used for this investigation corresponded to a deep-water morphotype of Pseudopterogorgia elisabethae Bayer (order Gorgonacea, family Gorgoniidae, phylum Cnidaria) collected in May 1996 by scuba from deep reef waters (−28 m) off San Andrés island, Colombia (located at 12°33’N 81°43’W). A voucher specimen (No. PESAI-01) has been deposited at the Chemistry Department of the University of Puerto Rico.

Collection, Extraction, and Isolation

The gorgonian specimens were partially sun-dried and kept frozen prior to its extraction. The freeze-dried animal (1.0 kg) was cut into small pieces and blended with 1:1 MeOH/CHCl₃ (11 × 1 L). The combined organic extracts were filtered and then concentrated, and the green residue obtained (284 g) was suspended in water and extracted with hexane, CHCl₃ and EtOAc. A large portion (128 g) of the entire crude hexane extract (178 g) was loaded onto a column of silica gel (780 g) and separated with stepwise elution of acetone in hexane (0–100%), and then with 100% CH₃OH. Fractions were routinely pooled on the basis of their TLC, NMR, and biological activity profiles to yield 7 primary fractions, denoted I–VII. Fractions IV (83 g) was re-chromatographed over silica gel (600 g) using a step gradient eluent system composed of EtOAc–hexane mixtures leading to 16 secondary fractions (A–P). Further purification of sub-fraction F (7.0 g) by silica gel (150 g) flash column chromatography using as eluant a 1:1 mixture of hexane–CHCl₃ gave a series of smaller fractions, the most polar of which (113 mg) was dissolved in a small volume of 95:5 hexane–2-propanol, filtered, and purified by normal-phase HPLC on a 10 mm × 25 cm Magnum Partisil-10 column, 5 µm, eluted isocratically with 5% 2-propanol in hexane at 2.0 mL/min. One of the homogeneous components isolated during HPLC analysis was later identified as aberrarone (1) (4.6 mg, 6.4 × 10⁻⁴% yield) (dry gorgonian weight basis).

Biological Assay

The primary in vitro anti-microbial assay against Plasmodium falciparum was used as described before.¹⁴

Aberrarone (1)

light orange crystals; [α]_D²⁵ +2.2 (c 1.4, CHCl₃); IR (film) ν_max 2932, 2870, 1768, 1753, 1724, 1699, 1675, 1456, 1376, 1319, 1261, 1142, 1118, 1100 cm⁻¹; UV (MeOH) λ_max 203 (ε 4 700), 220 (ε 2 800), 380 (ε 30) nm; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) (see Table 1); EIMS m/z 302 [M–CO]⁺ (6), 233 (17), 231 (17), 203 (11), 215 (22), 193 (52), 175 (23), 137 (23), 109 (19), 70 (100); HRFAB-MS m/z [M + H]⁺ 331.1922 (calcd for C₂₀H₂₇O₄, 331.1909).

Single-Crystal X-Ray Structure Determination of Aberrarone (1) at 298(2) K

The X-ray data were collected at 298° K with a Bruker SMART 1 K CCD diffractometer equipped with a graphite monochromator and Mo-Kα radiation (λ = 0.71073 Å) using the SMART software. Final values of the cell parameters were obtained from least-squares refinement. The frames were processed using the SAINT software to give the hkl file corrected for Lorentz and polarization effects. No absorption correction was applied. The structures were solved by direct methods with the SHELX-90 program and refined by least-squares methods on F², SHELXTL-93, incorporated in SHELXTL, Version 5.1. The initial E-maps yielded all non-hydrogen atom positions. All non-hydrogen atoms were refined anisotropically, and the H atoms were positioned geometrically and treated as riding, with C–H distances in the range 0.93–0.98 Å and with U_iso(H) = 1.2 or 1.5 U_eq(C). The crystallographic data for 1 reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under the reference number CCDC 741374. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or deposit@ccdc.cam.ac.uk). Aberrarone (1) was recrystallized by slow evaporation from a CH₃OH/H₂O mixture to yield light orange crystals of excellent quality. Crystal Data: C₂₀H₂₆O₄, Mr = 330.41, orthorhombic, space group P2₁2₁2₁ (No. 19), a = 10.375(2), b = 11.449(3), c = 15.153(4) Å, V = 1799.9(8) ų, Z = 4, ρ_calc = 1.219 Mgm⁻³, F₀₀₀ = 712, λ(Mo Kα) = 0.71073 Å, µ = 0.084 mm⁻¹. Data collection and reduction: crystal size, 0.19 × 0.14 × 0.11 mm, θ range = 2.23–23.29°, 8732 reflections collected, 2594 independent reflections (R_int = 0.0268), final R indices (I > 2σ(I)): R₁ = 0.0341, wR₂ = 0.0785 for 222 variable parameters, GOF = 1.025.