The CH₂Cl₂/MeOH (v/v, 1:1) extract of the gorgonian was subjected to chromatography using Sephadex LH-20 followed by silica gel and HPLC separations to yield compounds 1–6.

Compound 1, a colorless oil, exhibited a molecular formula of C₂₆H₃₆N₄O₂ as determined by HRESIMS ([M + Na]⁺, 459.2721, calcd. 459.2736), implying 11 double bond equivalents (DBE). The IR absorption bands at 1709 and 1610 cm⁻¹ indicated the presence of two carbonyls. The ¹H NMR spectrum of 1 (Table 1) showed signals for two aromatic singlets [δ_H 8.26 (H-2) and 7.69 (H-8)], four olefinic protons [δ_H 6.07 (H-14′), 5.45 (H-2′), 5.19 (H-10′), and 5.05 (H-6′)], five vinylic methyls [δ_H 2.10 (H-17′), 1.84 (H-16′), 1.77 (H-20′), 1.58 (H-18′), and 1.56 (H-19′)], one heteroatom-functionalized methyl [δ_H 3.86 (H-10)], and a series of aliphatic methylene multiplets. The ¹³C NMR spectrum of 1 (Table 2) resolved 26 resonances attributable to five double bonds (δ_C 155.5 C, 122.9 CH; 147.3 C, 115.0 C; 143.4 C, 117.6 CH; 135.5 C, 123.5 CH; and 129.6 C, 129.0 CH), two carbonyls (δ_C 199.3 and 162.0), two imines (δ_C 147.7 and 140.3), five vinylic methyls (δ_C 27.7, 20.6, 16.5, 16.4, and 16.0), a N-methyl (δ_C 35.0), and six sp³ methylenes (δ_C 55.3, 44.5, 39.4, 39.3, 26.7, and 26.1). As nine of the eleven DBE were accounted for by abovementioned unsaturated functional groups, the remaining two DBE required that 1 was bicyclic. The collective spectroscopic information pointed clearly to a fused diterpene-N-methylhypoxanthine structure, which bore a high similarity to that of malonganenone D [4]. The N-methylhypoxanthine moiety of 1 was readily identified by comparison of its NMR data with that of malonganenone D, which gave almost identical ¹³C NMR data regarding to this portion. The tetraprenyl side-chain of 1 was deduced from detailed analysis of COSY and HMBC data (Figure 2). ¹H–¹H COSY correlations revealed four spin systems: (a) H-1′/H-2′/H-3′/H₃-20′; (b) H-4′/H-5′/H-6′/H-7′/H₃-19′; (c) H-8′/H-9′/H-10′/H-11′/H₃-18′; and (d) H-14′/H-15′/H-16′ or H-17′. The connections from a to c were achieved by HMBC correlations of H-20′/C-4′ and H-19′/C-8′. The fragments c and d were linked via a methylene and a ketone (C-13) by HMBC correlations of H-18′/C-12′, H-12′/C-13′, and H-14′/C-13′. The geometries of the three olefins in the tetraprenyl side-chain were established from analysis of both ¹³C chemical shift and NOE data. The ¹³C chemical shifts of the vinylic methyls of C-18′, C-19′, and C-20′ (δ_C 16.4, 16.0, and 16.5, respectively) suggested the E geometries for ∆^10′, ∆^6′, and ∆^2′ as the vinyl methyl corresponding to Z geometry are known to resonate at around 25 ppm [4]. This was further supported by NOE correlations (Figure 2) of H-9′/H-18′ and H-10′/H-12′, H-5′/H-19′ and H-6′/H-8′, and H-1′/H-20′ and H-2′/H-4′, respectively. Finally, the tetraprenyl side-chain was attached to N-7 by HMBC correlations from H-1′ to C-5 and C-8. Thus, compound 1 was determined as depicted and given the trivial name malonganenone L.

Compound 2 exhibited the same molecular formula of C₂₆H₃₆N₄O₂ as 1 on the basis of the HRESIMS data ([M + Na]⁺, 459.2725, calcd. 459.2736). The NMR spectroscopic data of 2 (Table 1 and Table 2) was very similar to that of 1. In comparison with 1, the ¹³C NMR spectroscopic data for 2 differed significantly about ∆^10′ moiety, with the upfield-shifted carbon at C-12′ and the downfield-shifted vinyl methyl at C-18′ (δ_C 55.3 and 16.3 in 1; δ_C 47.9 and 24.1 in 2, respectively). This indicated that ∆^10′ in 2 adopts a Z configuration. Similar ¹³C NMR changes were also reported in malonganenone I [5], which possessed the same Z configuration of ∆^10′ as 2. Thus, compound 2 was determined as depicted and named malonganenone M.

Compound 3 had a molecular formula of C₂₆H₃₆N₄O₂ as established by HRESIMS data. The ¹H and ¹³C NMR data of 3 (Table 1 and Table 2) showed high similarity to those of 1 except that the ∆^10′ double bond in 1 was migrated to ∆^11′, forming a conjugated system with the C-13′ carbonyl. This was suggested by the significant downfield-shifted carbon at C-11′ and the upfield-shifted carbon at C-13′ as compared with those of 1 (δ_C 129.6 and 199.3 in 1; δ_C 157.8 and 191.7 in 3, respectively), and by the presence of a singlet olefinic signal (δ_H 6.00, H-12′) in the ¹H NMR spectra of 3 instead of a triplet olefinic signal (δ_H 5.19, t, J = 6.4 Hz, H-10′) in 1. The configuration of ∆^11′ in 3 was established to be E by the characteristic chemical shift of the vinylic methyl at C-18′ (δ_C 19.1) and by comparison of its NMR data with those of reported. Therefore, the structure of compound 3 was determined as depicted and given the trivial name malonganenone N.

Compound 4 exhibited an [M − H]⁻ ion at m/z 467.3021 (calcd. for C₂₇H₃₉N₄O₃, 467.3022), suggesting the molecular formula C₂₇H₄₀N₄O₃ (ten DBE). The ¹H and ¹³C NMR spectra of 4 (Table 1 and Table 2) bore a resemblance to those of 1, with the notable differences occurring in the hypoxanthine core. The NMR spectra of 4 showed the presence of an N-methylamide (δ_H 2.84, H-11; δ_C 26.1, C-11) and an N-methylformamide (δ_H 8.23, H-2 and 3.13, H-10; δ_C 163.3, C-2 and 31.9, C-10) groups, which were identical to those previously reported in malonganenones B, F, and G, indicating that 4 possessed the same trisubstituted imidazole ring. This was further supported by HMBC correlations (Figure 2) of H₃-11/C-6, H₃-10/C-4, and H-2/C-10. Thus, the structure of compound 4 was determined as depicted and given the trivial name malonganenone O.

Compound 5 had a molecular formula C₂₇H₄₀N₄O₃ by analysis of the HRESIMS data. Comparing the NMR data (Table 1 and Table 2) of 5 and 4, it appeared that the former had a Z configuration of ∆^10′ instead of an E configuration of ∆^10′ in 4. This was suggested by the upfield-shifted carbon at C-12′ and the downfield-shifted vinyl methyl at C-18′ (δ_C 48.3 and 24.4 in 5; δ_C 55.8 and 16.5 in 4, respectively). Thus, the structure of compound 5 was determined as depicted and given the trivial name malonganenone P.

The molecular formula of compound 6 was established as C₂₇H₄₀N₄O₃ by HRESIMS. The NMR data of 6 (Table 1 and Table 2) showed high similarity to that of 4 except that the ∆^10′ double bond in 6 was migrated to ∆^11′, forming a conjugated system with the C-13′ carbonyl. This was further suggested by the significant downfield-shifted carbon at C-11′ and the upfield-shifted carbon at C-13′ as compared with those of 4 (δ_C 130.7 and 198.8 in 4; δ_C 158.6 and 191.0 in 6, respectively), and by the presence of a singlet olefinic signal (δ_H 6.08, H-12′) in the ¹H NMR spectra of 6 instead of a triplet olefinic signal (δ_H 5.26, t, J = 6.8 Hz, H-10′) in 4. The characteristic chemical shift of the vinylic methyl at C-18′ (δ_C 25.5) indicated the Z configuration of ∆^11′ in 6. Thus, the structure of 6 was determined as depicted and given the trivial name malonganenone Q.

Compounds 1–6 were screened for their inhibitory activities against PDE4D, PDE5A, and PDE9A by using our previously reported methods [20,21,22,23]. As shown in Table 3, all compounds exhibited inhibition at 50 µM against PDE4D with degree of inhibition from 72% to 85%, while displaying weaker activities against PDE5A and PDE9A. The two most active compounds, 1 and 6, were selected to test for the half maximal inhibitory concentration (IC₅₀), which gave IC₅₀ values of 8.5 and 20.3 µM, respectively (Figure 3).

UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer with KBr disks. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS and HRESIMS were carried out on a Finnigan LC Q^DECA instrument. A Shimadzu LC-20 AT equipped with an SPD-M20A PDA detector was used for HPLC, a YMC-pack ODS-A column (250 × 10 mm, 5 µm, 12 nm) and a chiral column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 µm) was used for semipreparative HPLC separation. Silica gel (300–400 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, Shandong, China), C₁₈ reversed-phase (Rp-C₁₈) silica gel (12 nm, 50 µm, YMC Co., Ltd., Kyoto, Japan), Sephadex LH-20 gel (Amersham Biosciences, Piscataway, NJ, USA), used for column chromatography (CC). All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd., Guangzhou, Guangdong, China).

The gorgonian E. pseudosassapo was collected at a depth of 18–25 m in 29 July 2012 in Daya Bay of Guangdong Province, China and frozen immediately after collection, and were identified by one of the authors (Cheng-Qi Fan). A voucher specimen (accession number: LSH201207) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University, China.

Specimens of E. pseudosassapo (550 g, wet weight) were extracted with CH₂Cl₂/MeOH (1:1, 3 × 1 L) at room temperature (rt) to give 13.7 g of crude extract. The crude extract was subjected to silica gel column chromatography eluted with a CH₂Cl₂/MeOH gradient (9:1→1:9) to afford five fractions (Fr. I–V). Fr. III (1.4 g) was chromatographed over Sephadex LH-20 (CH₂Cl₂/MeOH, v/v, 1:1), followed by Rp-C₁₈ silica gel eluted with a CH₃CN/H₂O gradient (5:5→10:0) to obtain four sub-fractions (Fr. IIIa–IIId). Fr. IIIb was further separated by HPLC equipped with a chiral column (CH₃CN, 3 mL/min) to afford 1 (17 mg, t_R 17 min) and 2 (4.9 mg, t_R 20 min). Fr. IIId was purified by repeating the HPLC conditions described above to yield 4 (11 mg, t_R 23 min) and 5 (3.7 mg, t_R 27 min). Fr. IIIc was chromatographed by HPLC equipped with an ODS-A column (CH₃CN/H₂O, 90:10, 3 mL/min) to afford 3 (5.2 mg, t_R 15 min) and 6 (5.1 mg, t_R 20min).

Malonganenone L (1): colorless oil; UV (MeOH) λ_max (log ε) 211 (4.42), 225 (4.38), 253 (4.31) nm; IR ν_max 3145, 1709, 1610, 1464, 1250, 1128, 1060 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M + Na]⁺ m/z 459.2721 (calcd. for C₂₆H₃₆N₄O₂Na, 459.2736).

Malonganenone M (2): colorless oil; UV (MeOH) λ_max (log ε) 209 (4.22), 225 (4.17), 254 (4.06) nm; IR ν_max 3011, 1714, 1607, 1458, 1240, 1123, 1046 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M + Na]⁺ m/z 459.2725 (calcd. for C₂₆H₃₆N₄O₂Na, 459.2736).

Malonganenone N (3): colorless oil; UV (MeOH) λ_max (log ε) 208 (3.68), 223 (3.60), 254 (3.16) nm; IR ν_max 2937, 1732, 1627, 1439, 1379, 1215, 1136, 1039 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M + Na]⁺ m/z 459.2725 (calcd. for C₂₆H₃₆N₄O₂Na, 459.2736).

Malonganenone O (4): colorless oil; UV (MeOH) λ_max (log ε) 211 (4.39), 249 (4.22) nm; IR ν_max 2932, 1744, 1654, 1514, 1445, 1218, 1127, 1033 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M − H]⁻ m/z 467.3021 (calcd. for C₂₇H₃₉N₄O₃, 467.3022).

Malonganenone P (5): colorless oil; UV (MeOH) λ_max (log ε) 210 (4.27), 254 (4.02) nm; IR ν_max 2933, 1674, 1621, 1437, 1220, 1128, 1018 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M + Na]⁺ m/z 491.2994 (calcd. for C₂₇H₄₀N₄O₃Na, 491.2998).

Malonganenone Q (6): colorless oil; UV (MeOH) λ_max (log ε) 212 (4.52), 257 (4.46) nm; IR ν_max 2932, 1664, 1623, 1532, 1443, 1224, 1057 cm⁻¹; ¹H and ¹³C NMR, see Table 1 and Table 2; HRESIMS [M + Na]⁺ m/z 491.2994 (calcd. for C₂₇H₄₀N₄O₃Na, 491.2998).