Prostaglandin Derivatives: Nonaromatic ... - American Chemical Society

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Prostaglandin Derivatives: Nonaromatic Phosphodiesterase‑4 Inhibitors from the Soft Coral Sarcophyton ehrenbergi Zhong-Bin Cheng,†,§ Ya-Lin Deng,†,§ Cheng-Qi Fan,‡ Qing-Hua Han,† Shu-Ling Lin,† Gui-Hua Tang,† Hai-Bin Luo,*,† and Sheng Yin*,† †

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Ten new prostaglandin derivatives (PGs), sarcoehrendins A−J (1−10), together with five known analogues (11−15) were isolated from the soft coral Sarcophyton ehrenbergi. Compounds 4−8 represented the first examples of PGs featuring an 18-ketone group. The structures including the absolute configurations were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates and six synthetic analogues (3a, 3b, 4a, and 11a−11c) were screened for inhibitory activity against phosphodiesterase-4 (PDE4), which is a drug target for the treatment of asthma and chronic obstructive pulmonary disease. Compounds 2, 10, 11a, 11b, and 13−15 exhibited inhibition with IC50 values less than 10 μM, and compound 15 (IC50 = 1.4 μM) showed comparable activity to the positive control rolipram (IC50 = 0.60 μM). The active natural PGs (2, 10, and 13−15) represent the first examples of PDE4 inhibitors without an aromatic moiety, and a preliminary structure−activity relationship is also proposed.

T

drug target of high interest for central nervous system (CNS), inflammatory, and respiratory diseases.14 At present, roflumilast, the sole PDE4 inhibitor launched in the market as a therapy for chronic obstructive pulmonary disease, has shown some side effects, such as nausea, diarrhea, weight loss, and headaches.15 Therefore, the search for natural products to serve as PDE inhibitors with more efficacy and reduced side effects has attracted considerable interest. In our continuing search for a PDE4 inhibitor from natural resources,16−19 a fraction of the CH2Cl2/MeOH extract of the soft coral S. ehrenbergi showed inhibitory activity of 44.3% at a concentration of 10 μg/mL toward PDE4. Subsequent fractionation led to the isolation of 10 new PGs (1−10) and five known analogues (11−15). All of the isolates together with synthetic derivatives were tested for inhibition against PDE4. Seven compounds exhibited inhibition, with IC50 values less than 10 μM. Herein, the details of the isolation, structure elucidation, and the PDE4 inhibitory activity as well as a preliminary structure−activity relationship (SAR) of these compounds are described.

he prostaglandins (PGs) are a group of hormone-like intracellular second messengers that regulate a broad range of physiological activities, including blood circulation, digestion, and reproduction.1,2 Structurally, they are derived enzymatically from arachidonic acid and feature a 20-carbon skeleton containing a five-membered ring and two side chains. The biological activities and complex molecular architectures have made PGs popular targets for synthetic chemists and natural product chemists in the past several decades.3 Prostaglandin analogues are widely used as pharmaceuticals, and to date, there have been 14 PGs or analogues approved by the U.S. Food and Drug Administration (FDA) for the treatment of various diseases.4 Some of them, such as latanoprost, which is used to treat glaucoma, have become billion-dollar drugs.3,4 So far, nearly 120 naturally occurring PGs have been reported, the majority of which come from marine organisms, especially from soft corals.5 Soft corals of the genus Sarcophyton are chemically prolific and are represented by about 52 species.6 The species Sarcophyton ehrenbergi is often encountered in the South China Sea. Previous chemical investigation of this species revealed six PGs,7 a ceramide and five cerebrosides,8 and several cembranoids,9−12 some of which showed significant anti-inflammatory and antiviral activities. The phosphodiesterases (PDEs) represent a family of enzymes with 11 members that catalyze the hydrolysis of the second messengers, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP).13 Phosphodiesterase4 (PDE4), which specifically catalyzes the hydrolysis of adenosine 3′,5′-cyclic monophosphate (cAMP), is a promising © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The frozen sample was chopped and exhaustively extracted with CH2Cl2/MeOH at room temperature (rt). After removal of solvent in vacuo, the residue was suspended in H2O and then partitioned sequentially with petroleum ether (PE), EtOAc, and Received: May 8, 2014 Published: July 30, 2014 1928

dx.doi.org/10.1021/np500394d | J. Nat. Prod. 2014, 77, 1928−1936

Journal of Natural Products

Article

Figure 1. ΔδH values of (S)- and (R)-MTPA esters of 3 and 11, respectively.

Scheme 1. Chemical Correlations of Compounds 1−8, 11, and 12

J = 10.4, 5.5 Hz)]. The 13C NMR spectrum, in combination with DEPT experiments, resolved 27 carbon resonances attributable to four carbonyl groups (δC 174.2, 170.9, 170.8, and 170.1), two disubstituted double bonds (δC 130.0, 128.6, 133.3, and 130.4), five methyls (including a methoxy group at δC 51.5), six sp3 methines (four bearing oxygen atoms), and eight sp3 methylenes. As six of the seven degrees of unsaturation were consumed by four carbonyls and two double bonds, the remaining degree of unsaturation required that 1 was monocyclic. The above-mentioned information was quite similar to that of the co-isolated known PG, methyl 11α,l8diacetoxy-9α,l5(S)-dihydroxy-5-cis-13-trans-prostadienoate (11), isolated from the soft coral Lobophyton depressum

n-BuOH. Various column chromatographic separations of the EtOAc extract afforded compounds 1−15. Compound 1, a colorless oil, had the molecular formula C27H42O9, as established by HRESIMS, corresponding to seven degrees of unsaturation. The strong IR absorption band at 1736 cm−1 indicated the existence of carbonyl groups. The 1H NMR and HSQC spectra exhibited signals for five methyl groups [δH 2.04 (3H × 2, s), 2.03 (3H, s), 3.66 (3H, s), and 0.86 (3H, t, J = 7.4 Hz)], four oxymethine protons [δH 5.20 (1H, dd, J = 12.4, 6.2 Hz), 4.87 (1H, ddd, J = 8.8, 7.3, 3.7 Hz), 4.78 (1H, quint, J = 6.1 Hz), and 4.15 (1H, t, J = 5.2 Hz)], and four olefinic protons [δH 5.56 (1H, dd, J = 15.4, 7.9 Hz), 5.47 (1H, dd, J = 15.4, 6.2 Hz), 5.41 (1H, dd, J = 10.4, 6.5 Hz), and 5.38 (1H, dd, 1929



Article

collected in the Gulf of Eilat,20 except for the presence of an additional acetyl group [δH 2.04 (3H, s); δC 21.2, 170.1] in 1, indicating that 1 was an acetylated derivative of 11. The location of the acetyl group was assigned at OH-15 by an HMBC correlation from H-15 to the carbonyl group at δC 170.1. This was supported by the downfield-shifted H-15 signal in 1 with respect to that in 11 [δH 5.20 in 1; δH 4.02 in 11]. In addition, the introduction of an acetyl group at OH-15 resulted in a γ-gauche shielding effect on C-14 and C-16 of 1,21 which shifted these carbons upfield approximately 5.1 and 2.6 ppm, respectively, as compared to those in 11. The relative configuration of 1 was established on the basis of a NOESY experiment and analyses of its 1H−1H coupling constants. In particular, the coupling constants of H-5/H-6 (J = 10.4 Hz) and H-13/H-14 (J = 15.4 Hz) are typical values for the Δ5 Z and Δ13 E, respectively. The coupling constants of H-9/H-10 (J = 5.2 Hz) and H-10/H-11 (J = 8.8 Hz) are diagnostic for the H-9β and H-11β, respectively.22 The NOESY interactions of H8/H-9, H-11, and H-13 and H-12/H-7 further assigned the β and α configuration of H-8 and H-12, respectively. The specific rotation of 1 was nearly zero, implying the possibility of a racemic nature. However, the chiral-phase HPLC analysis and the enlarged specific rotation value of the acetylation product of 1 (2, [α]25D +9.1) demonstrated that 1 was enantiopure. To assign the absolute configuration (AC) of 1, the ACs at C-9 and C-15 in 11 were first determined as 9S and 15S by the modified Mosher’s method23 in the current study (Figure 1). As the alkaline hydrolysis of 1 and 11 generated the same product 11c (Scheme 1), which was verified by comparison of their Rf, NMR data, and specific rotations, the ACs of 1 were assigned as 8R, 9S, 11R, 12R, 15S. The AC at C-18 in 1 remained unresolved after the failure of several efforts including attempts to apply Mosher’s method (Supporting Information S100). Compound 1 was given the trivial name sarcoehrendin A. Compound 2 had a molecular formula of C29H44O10, as determined by HRESIMS and 13C NMR data. The 1D NMR spectra of 2 were very similar to those of 1 except for the presence of an additional acetyl group [δH 2.02 (s); δC 21.1, 170.2], indicating 2 was an acetylated derivative of 1. The acetyl group was located at 9-OH by HMBC correlation from H-9 to the carbonyl group at δC 170.2, which was supported by the severely downfield-shifted H-9 signal in 2 with respect to that in 1 (δH 5.06 in 2; δH 4.15 in 1). The structure of 2 was further confirmed by chemical correlation of 1 to 2 via acetylation. Compound 2 was named sarcoehrendin B. Compound 3 had the same molecular formula as that of 1, implying that they were structural isomers. The NMR data of 3 were very similar to those of 2 except for the absence of one acetyl group and an upfield-shifted H-15 signal [δH 4.09 (1H, dd, J = 11.6, 5.9 Hz)], indicating that 3 was the 15-deacetylated derivative of 2. The planar structure of 3 was further secured by detailed analyses of its 2D NMR data. The AC of C-15 was assigned as S by the modified Mosher’s method (Figure 1), and the ACs of other sites, except C-18, were assigned by the chemical conversion of 3 to 11c (Scheme 1). Compound 3 was given the trivial name sarcoehrendin C. Compound 4 had a molecular formula of C27H40O9, as established by HRESIMS, indicating eight degrees of unsaturation. The 1D NMR spectra of 4 exhibited most of the structural features found in 2, with major difference being the presence of a ketone group (δC 210.2) in 4 instead of the 18-acetoxy group in 2. HMBC correlations from the terminal methyl [δH 1.04 (3H, t, J = 7.4 Hz)] and H2-19 [δH 2.41 (2H,

q, J = 7.4 Hz)] to the keto-carbon situated the ketone group at C-18. This was supported by the downfield-shifted C-17 and C19 signals in 4 (∼ 7 ppm) as compared to those bearing an 18OAc. The planar structure of 4 was further established by detailed interpretation of its 2D NMR data. The AC of 4 was determined to be the same as that of 2 by chemical transformation of 4 to 4c (Scheme 1), which was confirmed to contain 2 by chiral-phase HPLC analysis (S101). Compound 4 represents the first example of a PG featuring an 18-ketone group and was named sarcoehrendin D. Compound 5, a colorless oil, had the molecular formula C23H36O7, as determined by HRESIMS and 13C NMR data. The NMR data of 5 bore a resemblance to those of 11 except for the absence of the 18-acetoxy group and the presence of a ketone group (δC 212.1). Detailed 2D NMR analyses of 5 located the ketone group at C-18. The structure of 5 was further confirmed by alkaline hydrolysis of 5, which yielded the same hydrolysis product (4a) as that of 4 (Scheme 1). Compound 5 was given the trivial name sarcoehrendin E. Compound 6 had the molecular formula C25H38O8, as established by HRESIMS. The 1D NMR spectra of 6 showed high similarity to those of 5 except for the presence of an additional acetyl group [δH 2.04 (3H, s); δC 21.2, 170.1], implying that 6 was an acetylated derivative of 5. The additional acetyl group was located at OH-15 by the HMBC correlation from H-15 [δH 5.24 (dd, J = 12.6, 6.0 Hz)] to the carbonyl group at δC 170.1, which was further supported by the downfield-shifted H-15 signal in 6 with respect to that in 5 (δH 5.24 in 6; δH 4.12 in 5). The structure was secured by chemical correlation of 6 to 4a via alkaline hydrolysis (Scheme 1). Compound 6 was given the trivial name sarcoehrendin F. The HRESIMS data for compound 7 indicated a molecular formula of C22H34O7. The 1H and 13C NMR spectra of 7 resembled those of 5 with the notable differences being the absence of the methoxy group and the downfield-shifted C-1 signal (δC 177.0 in 7, δC 174.3 in 5), indicating that 7 was the free acid of 5. The structure was confirmed by alkaline hydrolysis of 7 to afford 4a (Scheme 1). Compound 7 was given the trivial name sarcoehrendin G. The presence of both free acid and methyl ester prostaglandin derivatives raises the possibility that the methyl esters could be artifacts formed by reaction with MeOH during the extraction process. This possibility was not investigated further. Compound 8 had the molecular formula C24H36O8. The NMR spectra of 8 were very similar to those of 7 except for the presence of an additional acetyl group [δH 2.03 (3H, s); δC 21.2, 170.8], indicating that 8 was an acetylated derivative of 7. An HMBC correlation from H-15 to the carbonyl group at δC 170.8 positioned the acetyl group at OH-15, which was upheld by the severely downfield-shifted H-15 signal in 8 with respect to that in 7 (δH 5.25 in 8; δH 4.17 in 7). The structure was confirmed using the same chemical correlation described above (Scheme 1). Compound 8 was given the trivial name sarcoehrendin H. Compound 9 had the molecular formula C25H42O7, two mass units more than that of the co-isolated known compound (5Z,9α,11α,13E,15S)-11,15-bis(acetyloxy)-9-hydroxyprosta5,13-dien-1-oic acid methyl ester (14).24 The NMR spectra of 9 revealed similar structural features to those found in 14, except for the presence of the two additional methylenes (δC 29.5 and 27.6) rather than the olefinic carbons at δC 130.0 and 128.7 in 14, indicating that 9 was the Δ5-hydrogenated derivative of 14. The planar structure of 9 was further established by detailed 1930


1931

5.47, dd (15.4, 6.2)

5.20, dd (12.4, 6.2) 1.60, m

1.54, m 4.78, quint (6.1) 1.54, m

0.86, t (7.4) 3.66, s

14

15 16

17 18 19

20 −COOCH3 9-OAc 11-OAc 15-OAc 18-OAc

13

12

11

2.04, s 2.04, s 2.03, s

4.15, t (5.2) 1.68, m 2.40, ddd (15.0, 8.8, 5.2) 4.87, ddd (8.8, 7.3, 3.7) 2.55, ddd (11.7, 7.3, 7.9) 5.56, dd (15.4, 7.9)

9 10α 10β

8

5 6 7

2.32, td (7.3, 2.3) 1.68, m a2.08, m b2.13, m 5.38, dd (10.4, 5.5) 5.41, dd (10.4, 6.5) a2.26, m b2.04, m 1.53, m

1

2 3 4

position

0.84, 3.63, 2.02, 1.98, 2.01, 2.01,

t (7.4) s s s s s

1.52, m 4.76, quint (6.0) 1.52, m

5.18, dd (11.7, 5.9) 1.57, m

5.49, dd (15.5, 5.9)

5.52, dd (15.5, 7.4)

4.85, ddd (8.8, 7.5, 4.4) 2.53, m

5.06, t (4.6) 1.63, m 2.49, m

1.65, m

5.34, dd (11.4, 6.1) 5.28, dd (11.4, 5.6) 2.05, m

2.27, t (7.5) 1.64, m 2.00, m

2

t (7.1) s s s

2.05, s

0.87, 3.66, 2.01, 2.03,

a1.66, m, b1.53, m 4.80, quint (5.8) 1.55, m

4.09, dd (11.6, 5.9) 1.52, m

5.57, dd (15.8, 5.9)

5.54, dd (15.8, 7.8)

4.89, ddd (7.5, 7.5, 4.6) 2.54, m

5.08, t (4.9) 1.66, m 2.52, m

1.66, m

2.29, t (7.5) 1.65, m a2.06, m b1.99, m 5.35, dd (10.9, 5.5) 5.32, dd (10.9, 5.9) 2.10, m

3

1.04, 3.66, 2.05, 2.01, 2.04,

t (7.4) s s s s 2.03, s

2.44, q (7.2) 1.04, t (7.2) 3.66, s

2.41, q (7.4)

2.42, t (7.4)

4.12, m a1.87, m b1.77, m 2.54, t (6.7)

5.54, m

5.54, m

2.55, m

4.89, m

4.15, m 1.68, m 2.38, m

5.37, m 5.41, m a2.24, m b2.06, m 1.51, m

2.31, t (7.2) 1.68, m 2.09, m

5

5.24, dd (12.3, 5.5) 1.88, m

5.48, dd (15.7, 5.5)

5.54, dd (15.7, 7.4)

4.88, ddd (8.1, 8.1, 4.6) 2.54, m

5.07, t (5.0) 1.66, m 2.50, m

5.36, dd (11.3, 6.8) 5.30, (11.3, 6.8) a2.13, m b2.08, m 1.67, m

2.29, (7.5) 1.66, m 1.99, m

4

6

2.03, s 2.04, s

1.04, t (7.2) 3.66, s

2.41, q (7.2)

2.42, t (7.2)

5.24, dd (12.6, 6.0) 1.90, m

5.47, dd (15.5, 6.0)

4.88, ddd (8.7, 7.4, 3.8) 2.54, ddd (11.4, 7.9, 7.4) 5.56, dd (15.5, 7.9)

5.39, m 5.38, m a2.26, m b2.03, m 1.52, ddd (15.1, 11.4, 4.3) 4.15, t (4.3) 1.68, m 2.40, m

2.32, td (7.3, 2.5) 1.69, m 2.09, m

Table 1. 1H NMR Data of Sarcoehrendins A−J (1−10) in CDCl3 (400 MHz, δ in ppm, J in Hz)

2.00, s

2.44, q (7.3) 1.02, t (7.3)

2.54, t (6.7)

4.17, m 1.78, m

5.52, m

5.52, m

2.50, m

4.87, m

4.18, t (4.2) 1.68, m 2.36, m

5.37, m 5.47, m a2.18, m b2.11, m 1.46, m

2.31, t (7.2) 1.63, m 2.10, m

7

2.06, s 2.03, s

1.05, t (7.3)

2.43, m

2.44, m

5.58, dd (15.6, 8.3) 5.48, dd (15.6, 6.0) 5.25, m 1.91, m

2.54, m

4.88, m

4.18, t (4.3) 1.69, m 2.40, m

1.53, m

5.40, m 5.45, m 2.15, m

2.35, m 1.70, m 2.12, m

8

2.03, s 2.04, s

0.88, t (6.6) 3.66, s

1.28, m 1.28, m 1.28, m

5.21, (12.9, 6.4) 1.57, m

5.46, dd (15.4, 6.4)

5.53, dd (15.4,7.9)

4.19, t (4.0) 1.69, m 2.37, ddd (14.4, 8.6,5.2) 4.87, ddd (8.6, 6.6, 3.4) 2.49, m

1.45, m

1.30, m 1.28, m 1.41, m

2.30, t (7.5) 1.61, m 1.28, m

9

2.04, s 2.04, s

0.88, t (7.2) 3.67, s

1.28, m 1.28, m 1.29, m

5.19, m 1.60, m

5.49, m

5.49, m

2.90, m

3.99, m 1.64, m 2.63, td (15.0, 7.4) 4.93, m

2.10, m

2.08, m 5.43, m 5.43, m

2.32, t (7.3) 1.70, m 1.98, m

10

Journal of Natural Products Article



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Table 2. 13C NMR Data of Sarcoehrendins A−J (1−10) (100 MHz, δ in ppm) position

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OCH3 9-OAc

174.2 33.3 24.7 26.6 130.0 128.6 25.0 49.3 71.7 40.8 78.8 51.0 133.3 130.4 73.8 30.1 29.1 74.9 26.8 9.5 51.5

173.9 33.4 24.7 26.5 130.0 127.8 24.9 47.4 74.2 38.8 77.7 51.9 132.5 130.7 73.0 28.1 37.6 210.2 35.9 7.74 51.4 21.1 170.3 21.0 170.5 21.1 170.0

174.2 33.4 24.8 26.6 130.1 128.6 25.0 49.3 71.7 40.8 78.7 51.1 133.2 130.2 73.2 28.2 37.7 210.3 36.0 7.80 51.6

177.0 32.8 24.4 26.2 129.9 128.5 24.5 50.0 71.6 40.9 78.9 51.4 130.8 134.2 71.2 30.3 38.0 213.2 36.0 7.8

176.5 32.8 24.5 26.4 130.1 128.4 25.0 49.5 71.8 40.9 78.8 51.3 133.3 130.2 73.4 28.3 37.8 211.3 36.1 7.8

174.3 34.0 24.8 29.0 29.5 27.6 27.1 49.4 72.1 41.1 79.2 51.6 133.3 130.7 74.3 34.4 24.8 31.5 22.5 13.9 51.4

174.0 33.4 24.8 26.5 26.7 130.2 128.7 50.5 75.8 40.2 77.5 49.8 131.7 129.8 74.4 34.42 24.8 31.5 22.5 13.9 51.5

21.2 170.8 21.2 170.1 21.2 170.9

174.0 33.3 24.8 26.5 129.9 128.0 24.8 47.6 74.2 38.8 77.9 52.0 130.7 135.8 72.1 32.7 29.5 75.3 27.0 9.5 51.5 21.2a 170.4 21.1a 170.7

174.3 33.3 24.7 26.6 129.9 128.7 24.9 49.5 71.7 40.8 79.0 51.3 131.1 135.1 71.5 30.7 38.1 212.1 36.0 7.8 51.6

11-OAc

173.8 33.3 24.7 26.5 129.9 127.8 24.9 47.4 74.2 38.8 77.6 51.8 132.6 130.8 73.5 30.0 29.0 74.9 26.8 9.4 51.3 21.1a 170.2 20.9a 170.4 21.1a 170.0 21.1a 170.7

21.2 171.0

21.2 170.8 21.2 170.1

21.2 171.0

21.2 170.6 21.2 170.8

21.3 170.7 21.2 170.3

21.2a 170.6 21.3a 170.2

15-OAc 18-OAc a

21.2a 171.0

May be interchanged in each column.

dien-1-oic acid methyl ester (14),24 and 9,11,15-triacetoxy PGF2α methyl ester (15)29,30 have been previously reported without NMR information, of which 13−15 were synthetic intermediates. Thus, the full NMR assignments and specific rotations of 12−15 are first reported in the current study (S102). All of the isolates (1−15), the Mosher’s esters (3a, 3b, 11a, and 11b), and the hydrolysis products (4a and 11c) were tested for their inhibitory activity against PED4D2 at an initial concentration of 10 μM by using our previously reported methods.16−19 Rolipram, a well-known PDE4 inhibitor, was used as the reference compound (IC50 = 0.6 μM), comparable to the reported value of 1.0 μM.31 Compounds with inhibition greater than 50% (S1, Supporting Information) were further analyzed to determine their corresponding IC50 values (Table 3). The bioassay results showed that compounds 2, 10, 11a, 11b, and 13−15 had strong activity, with IC50 values less than

interpretation of its 2D NMR data. The AC of 9 was proposed to be the same as that of 14 based on their identical 13C chemical shifts regarding the C-7 to C-20 part and on biosynthetic considerations. Compound 9 was given the trivial name sarcoehrendin I. Compound 10, a colorless oil, had the molecular formula C25H40O7 as determined by HRESIMS. The 1H and 13C NMR spectra of 10 were similar to those of 9 except for the presence of an additional disubstituted double bond [δH 5.43 (2H, m); δC 130.2, 128.7] in 10. The COSY correlations from the double-bond protons to H-5 and H-8 located the double bond at C-6 and C-7. This was supported by the downfield-shifted H8 signal (δH 2.10) in 10 as compared to its Δ5 analogues (δH ∼1.5). The configuration of Δ6 was assigned as Z by comparing its 13C chemical shifts with those of methyl cymatherol A,25 bearing a similar Δ6 moiety, and was further supported by the chemical shift of the methylene carbon C-5 at δC 26.7 (Z alkenes, δC < 27; E alkenes, δC > 30).26 The AC of 10 was proposed to be the same as that of 14 and other analogues based on analyses of their NMR data and on biosynthetic considerations. Compound 10 was given the trivial name sarcoehrendin J. Compound 11 was identified to be methyl 11α,l8-diacetoxy9α,15(S)-dihydroxy-5-cis-13-trans-prostadienoate by comparison of its NMR data with those in the literature. 20 (5Z,9α,11α,13E,15S)-11,18-Bis(acetoxy)-9,15-dihydroxyprosta5,13-dien-1-oic acid (12),20 9α,15α-diacetoxy-11α-hydroxy5Z,13E-prostadienoic acid methyl ester (13), 2 7 , 2 8 (5Z,9α,11α,13E,15S)-11,15-bis(acetoxy)-9-hydroxyprosta-5,13-

Table 3. IC50 Values of the Active PGs against PDE4D2

a

1932

compound

IC50 (μM)

compound

2 4 6 8 10 roliprama

3.7 10.6 12.1 16.9 7.2 0.6

± ± ± ± ± ±

11a 11b 13 14 15

0.3 0.7 0.5 0.8 0.8 0.04

IC50 (μM) 5.9 3.6 4.7 5.5 1.4

± ± ± ± ±

0.4 0.1 0.6 0.6 0.1

Positive control. dx.doi.org/10.1021/np500394d | J. Nat. Prod. 2014, 77, 1928−1936


Article

10 μM toward PDE4D2 (Table 3), and 15 turned out to be a potent inhibitor, with an IC50 value of 1.4 μM, comparable to that of rolipram. The inhibitory curves of 15 and rolipram are represented in Figure 2.

PDE4 inhibitors without an aromatic moiety. Moreover, the active natural PGs (2, 10, and 13−15) may serve as a novel structural motif for the design of PDE4 inhibitors in the future. The mechanism of inhibition against PDE4 of these PGs requires further investigation.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS spectra were obtained on a Finnigan LCQ Deca instrument, and HRESIMS was performed on a Waters Micromass Q-TOF spectrometer. A Shimadzu LC-20 AT equipped with an SPDM20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) and a chiral-phase column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 μm) were used for semipreparative HPLC separation. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S50 μm, YMC Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography (CC). All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Expression and purification of PDE4D were carried out by using a Hielscher UP200S ultrasonic cell disruption processor, a Sigma 6K15 centrifugal machine, an Eppendorf BioPhotomer spectrophotometer, and a Qiagen nickel-nitriloacetic acid column. The radioactivity of the samples was measured on a PerkinElmer Tricarb 2910 liquid scintillation counter. The yeast extract and tryptone prepared for the LB medium were purchased from Oxoid Ltd., and the substrate [3H]cAMP was from Waukesha GE Healthcare. Rolipram was purchased from Sigma. Animal Material. Specimens of the soft coral S. ehrenbergi were collected from Yang Meikeng, Guangdong Province, P.R. China, in October 2012, at a water depth of 4−5 m. The biological material was frozen immediately until used and was identified by one of the authors (C.-Q.F.). A voucher specimen (accession number XPHM201210) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation. The frozen sample (0.8 kg, wet weight) was chopped and exhaustively extracted with CH2Cl2/MeOH (1:1) (1 L × 3) at rt. After removal of solvent in vacuo, the residue (8 g) was suspended in H2O (120 mL) and then partitioned sequentially with petroleum ether (3 × 200 mL), EtOAc (3 × 200 mL), and n-BuOH (3 × 200 mL). Separation of the EtOAc extract (3 g) by Sephadex LH-20 eluted with EtOH led to three fractions (fr. I−III). Fr. I (1.2 g) was chromatographed over silica gel CC (PE/acetone, 20:1 → 3:1) to give eight fractions (fr. Ia−Ih). Fr. Ib (97 mg) was purified on a semipreparative reversed-phase (RP) HPLC system equipped with a YMC column (MeCN/H2O, 90:10, 3 mL/min) to give 14 (8 mg, tR 6.7 min), 4 (6 mg, tR 7.5 min), 2 (10 mg, tR 8.9 min), and 15 (5 mg, tR 10.6 min). Fr. Id (126 mg) was subjected to RP-HPLC (MeCN/H2O, 70:30, 3 mL/min) to afford 1 (12 mg, tR 10.8 min), 9 (6 mg, tR 13.2 min), and a mixture (tR 17.8 min). Further purification of the mixture (13 mg) on HPLC equipped with a chiral-phase column (Phenomenex Lux, cellulose-2, MeOH/H2O, 85:15, 3 mL/min) yielded 10 (2.5 mg, tR 11.0 min) and 13 (3 mg, tR 12.6 min). Fr. Ie (68 mg) was subjected to further purification by RP-HPLC (MeCN/H2O, 80:20, 3 mL/min) to give 3 (6 mg, tR 10.2 min) and fr. Iea (tR 13.8 min). The latter (12 mg) was successively purified on RP-HPLC (MeCN/H2O, 85:15, 3 mL/min) equipped with a chiral-phase column to afford 6 (6 mg, tR 12.3 min). Fr. If (36 mg) was chromatographed over RP-HPLC (MeCN/H2O, 65:35, 3 mL/min) to give 11 (11 mg, tR 9.2 min) and 5 (4 mg, tR 10.5 min). Fr. Ig (43 mg) was separated by RP-HPLC (MeCN/H2O, 55:45, 3 mL/min) to obtain 7 (5 mg, tR 7.5 min), 12 (6 mg, tR 9.3 min), and 8 (4 mg, tR 11.3 min). Sarcoehrendin A (1): colorless oil; [α]25D −0.9 (c 0.2, CHCl3); IR (KBr) νmax 3675, 2953, 2926, 2871, 2855, 1736, 1457, 1437, 1374, 1245, 1023, 968 cm−1; 1H and 13C NMR data, see Tables 1 and 2;

Figure 2. Inhibition curves of compounds 15 and rolipram (positive control) against PDE4D2.

The structural variability present within the small PG library and their inhibitory activity toward PDE4 in this study clearly define some SAR (Figure 3): (a) The esterification of OH-15

Figure 3. Schematic view of the SAR.

seems to be essential for the enhanced activity, as the inhibition by all of the compounds possessing a free OH-15 was less than 30% at the concentration of 10 μM (S1, Supporting Information). (b) The acetylation of OH-9 or OH-11 produces a small increase of the activity (∼2-fold), as compounds 2, 3, and 15, with a 9-acetoxy group, showed greater activity than their OH-9 counterparts 1, 11, and 14, respectively, and 15 is 2fold more active than its OH-11 counterpart 13 (Table 3 and S1). (c) Esterification of C-1 does not affect the activity, as 5 and 6 showed similar activity to their corresponding free acids 7 and 8, respectively (Table 3 and S1). (d) Oxidation at C-18 led to a small decrease in activity (∼3-fold), as compounds 1/6 and 2/4, with an acetoxy or ketone group at C-18, showed weaker activity than their C-18 saturated counterparts, 14 and 15, respectively. (e) The Δ5 or Δ6 may contribute to good activity, as 14 and 10 displayed much better inhibition than their Δ5- or Δ6-hydrogenated counterpart 9 (Table 3 and S1). Thus, 9,11,15-triacetoxy PGF2α methyl ester (15) with all of the above-mentioned favorable features was the most active compound. In summary, this work describes the isolation and identification of 15 PGs including 10 new ones from the soft coral S. ehrenbergi. The active PGs account for the inhibitory activity of the extract toward PDE4. Compound 15, the most active compound in the current study, is comparable to the positive control rolipram. Notably, all of the previously reported PDE4 inhibitors are aromatic-containing compounds,16−19,32,33 and we present here the first examples of 1933



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ESIMS m/z 528.3 [M + NH4]+; HRESIMS m/z 533.2731 [M + Na]+ (calcd for C27H42O9Na, 533.2727). Sarcoehrendin B (2): colorless oil; [α]25D +7.8 (c 0.2, CHCl3); IR (KBr) νmax 3683, 2953, 2926, 2870, 2854, 1738, 1462, 1441, 1434, 1371, 1237, 1174, 1020, 966 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 570.3 [M + NH4]+; HRESIMS m/z 575.2849 [M + Na]+ (calcd for C29H44O10Na, 575.2832). Sarcoehrendin C (3): colorless oil; [α]25D +17 (c 0.1, CHCl3); IR (KBr) νmax 3482, 2936, 2871, 1736, 1456, 1437, 1373, 1244, 1025, 971 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 528.2 [M + NH4]+; HRESIMS m/z 533.2715 [M + Na]+ (calcd for C27H42O9Na, 533.2727). Sarcoehrendin D (4): colorless oil; [α]25D +14 (c 0.1, CHCl3); IR (KBr) νmax 3663, 2953, 2925, 2870, 2853, 1737, 1454, 1435, 1372, 1236, 1170, 1019, 968 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 526.4 [M + NH4]+; HRESIMS m/z 531.2562 [M + Na]+ (calcd for C27H40O9Na, 531.2570). Sarcoehrendin E (5): colorless oil; [α]25D +19 (c 0.1, CHCl3); IR (KBr) νmax 3587, 2954, 2927, 2856, 1734, 1717, 1457, 1437, 1374, 1245, 1171, 1028, 969 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 447.1 [M + Na]+; HRESIMS m/z 447.2358 [M + Na]+ (calcd for C23H36O7Na, 447.2359). Sarcoehrendin F (6): colorless oil; [α]25D +6.1 (c 0.1, CHCl3); IR (KBr) νmax 3675, 2953, 2926, 2870, 2854, 1734, 1717, 1457, 1437, 1374, 1243, 1171, 1154, 1020, 969 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 484.3 [M + NH4]+; HRESIMS m/z 489.2462 [M + Na]+ (calcd for C25H38O8Na, 489.2464). Sarcoehrendin H (7): colorless oil; [α]25D +20 (c 0.1, CHCl3); IR (KBr) νmax 3565, 2938, 2870, 1715, 1640, 1556, 1536, 1395, 1363, 1265, 1104, 1078, 1063, 978 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 393.2 [M − H2O + H]+; ESIMS m/z 409.3 [M − H]−; HRESIMS m/z 393.2273 [M − H2O + H]+ (calcd for C22H33O6, 393.2277). Sarcoehrendin G (8): colorless oil; [α]25D +7.3 (c 0.1, CHCl3); IR (KBr) νmax 3690, 2954, 2923, 2852, 1735, 1718, 1458, 1376, 1243, 1149, 1022, 965 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 475.2 [M + Na]+; ESIMS m/z 451.3 [M − H]−; HRESIMS m/z 475.2308 [M + Na]+ (calcd for C24H36O8Na, 475.2308). Sarcoehrendin I (9): colorless oil; [α]25D −4.2 (c 0.1, CHCl3); IR (KBr) νmax 3588, 2928, 2859, 1763, 1663, 1439, 1403, 1384, 1290, 1246, 1140, 966 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 477.2 [M + Na]+; HRESIMS m/z 477.2830 [M + Na]+ (calcd for C25H42O7Na, 477.2828). Sarcoehrendin J (10): colorless oil; [α]25D −3.7 (c 0.05, CHCl3); IR (KBr) νmax 3447, 2955, 2925, 2854, 1735, 1718, 1458, 1376, 1264, 1243, 1068, 1022, 976 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 470.3 [M + NH4]+; HRESIMS m/z 475.2668 [M + Na]+ (calcd for C25H42O7Na, 475.2672). Preparation of (R)- and (S)-MTPA Esters of 3 and 11. Each PG (2 mg) was dissolved in freshly distilled anhydrous pyridine (250 μL). The mixture was stirred for 10 min at rt. Treatment with (R)-(−)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl) at 50 °C yielded the (S)-MTPA ester after 12 h. The reaction was quenched with 1 mL of MeOH. After removal of solvent under vacuum, the residue was purified on a flash silica gel eluted with CH2Cl2 to afford the (S)-MTPA ester. The (R)-MTPA ester was prepared with (S)-MTPA-Cl in the same manner. (S)-MTPA ester of 3 (3a): colorless oil; 1H NMR (CDCl3, 400 MHz) δH 5.69 (1H, m, H-13), 5.59 (1H, m, H-14), 5.44 (1H, m, H15), 5.34 (1H, m, H-5), 5.32 (1H, m, H-6), 5.09 (1H, t, J = 4.9 Hz, H9), 4.87 (1H, m, H-11), 4.74 (1H, m, H-18), 3.67 (3H, s, COOCH3), 2.55 (1H, m, H-12), 2.54 (1H, m, H-10β), 2.30 (2H, t, J = 7.5 Hz, H2), 2.15 (1H, m, H-7a), 2.02 (1H, m, H-7b), 2.01 (2H, m, H-4), 1.68 (1H, m, H-8), 1.68 (1H, m, H-10α), 1.67 (2H, m, H-16), 1.66 (2H, m, H-3), 1.49 (2H, m, H-19), 1.46 (2H, m, H-17), 0.82 (3H, t, J = 7.3 Hz, H-20); 13C NMR (CDCl3, 100 MHz) δC 134.9 (CH, C-13), 130.1 (CH, C-5), 129.6 (CH, C-14), 127.4 (CH, C-6), 77.6 (CH, C-11), 76.5 (CH, C-15), 75.1 (CH, C-18), 74.4 (CH, C-9), 51.9 (CH, C-12), 51.5 (COOCH3), 47.2 (CH, C-8), 38.8 (CH2, C-10), 33.3 (CH2, C-

2), 30.0 (CH2, C-16), 28.7 (CH2, C-17), 27.1 (CH2, C-19), 26.5 (CH2, C-4), 24.8 (CH2, C-7), 24.6 (CH2, C-3), 9.3 (CH3, C-20); ESIMS m/z 744.3 [M + NH4]+, 749.3 [M + Na]+. (R)-MTPA ester of 3 (3b): 1H NMR (CDCl3, 400 MHz) δH 5.57 (H, dd, J = 14.9, 8.1 Hz, H-13), 5.55 (1H, dd, J = 14.9, 6.7 Hz, H-14), 5.41 (1H, m, H-15), 5.34 (1H, m, H-5), 5.31 (1H, m, H-6), 5.07 (1H, t, J = 4.9 Hz, H-9), 4.81 (1H, m, H-11), 4.78 (1H, m, H-18), 3.66 (3H, s, COOCH3), 2.55 (1H, m, H-12), 2.54 (1H, m, H-10β), 2.30 (2H, t, J = 7.5 Hz, H-2), 2.08 (2H, m, H-7), 1.99 (2H, m, H-4), 1.71 (2H, m, H16), 1.65 (2H, m, H-3), 1.65 (1H, m, H-10α), 1.63 (1H, m, H-8), 1.56 (2H, m, H-17), 1.54 (2H, m, H-19), 0.86 (3H, t, J = 7.4 Hz, H-20); 13 C NMR (CDCl3, 100 MHz) δC 134.4 (CH, C-13), 130.0 (CH, C-5), 129.6 (CH, C-14), 127.5 (CH, C-6), 77.6 (CH, C-11), 76.6 (CH, C15), 74.8 (CH, C-18), 74.2 (CH, C-9), 51.9 (CH, C-12), 51.5 (COOCH3), 47.2 (CH, C-8), 38.9 (CH2, C-10), 33.4 (CH2, C-2), 30.0 (CH2, C-16), 28.8 (CH2, C-17), 26.9 (CH2, C-19), 26.6 (CH2, C4), 24.7 (CH2, C-7), 24.5 (CH2, C-3), 9.4 (CH3, C-20); ESIMS m/z 744.3 [M + NH4]+, 749.3 [M + Na]+. (S)-MTPA ester of 4 (11a): colorless oil; 1H NMR (CDCl3, 400 MHz) δH 5.69 (H, dd, J = 15.3, 8.4 Hz, H-13), 5.55 (1H, dd, J = 15.3, 7.2 Hz, H-14), 5.42 (1H, dd, J = 14.2, 7.2 Hz, H-15), 5.37 (1H, m, H5), 5.33 (1H, m, H-9), 5.31 (1H, m, H-6), 4.85 (1H, m, H-11), 4.73 (1H, quint, J = 6.1 Hz, H-18), 3.66 (3H, s, COOCH3), 2.53 (1H, m, H-10β), 2.50 (1H, m, H-12), 2.25 (2H, t, J = 7.4 Hz, H-2), 2.11 (1H, m, H-7a), 2.03 (3H, s, 18-OAc), 2.0 (1H, m, H-7b), 1.94 (2H, m, H4), 1.85 (3H, s, 11-OAc), 1.79 (1H, m, H-10α), 1.73 (1H, m, H-8), 1.65 (2H, m, H-16), 1.63 (2H, m, H-3), 1.47 (2H, m, H-19), 1.44 (2H, m, H-17), 0.81 (3H, t, J = 7.4 Hz, H-20); 13C NMR (CDCl3, 100 MHz) δC 174.0 (C, C-1), 171.1 (18-OCOCH3), 170.7 (11OCOCH3), 134.7 (CH, C-13), 130.8 (CH, C-5), 129.9 (CH, C-14), 127.1 (CH, C-6), 77.8 (CH, C-9), 77.7 (CH, C-11), 76.6 (CH, C-15), 74.9 (CH, C-18), 52.1 (CH, C-12), 51.5 (COOCH3), 47.9 (CH, C-8), 38.9 (CH2, C-10), 33.3 (CH2, C-2), 30.1 (CH2, C-16), 28.8 (CH2, C17), 26.9 (CH2, C-19), 26.6 (CH2, C-4), 24.9 (CH2, C-7), 24.6 (CH2, C-3), 21.1 (18-OCOCH3), 20.7 (11-OCOCH3), 9.4 (CH3, C-20); ESIMS m/z 918.4 [M + NH4]+, 923.3 [M + Na]+; ESIMS m/z 935.3 [M + Cl]−. (R)-MTPA ester of 4 (11b): 1H NMR (CDCl3, 400 MHz) δH 5.59 (H, dd, J = 14.9, 8.4 Hz, H-13), 5.47 (1H, dd, J = 14.9, 6.5 Hz, H-14), 5.42 (1H, dd, J = 12.9, 6.5 Hz, H-15), 5.34 (1H, m, H-9), 5.30 (1H, m, H-5), 5.24 (1H, m, H-6), 4.86 (1H, ddd, J = 9.1, 7.0, 3.3 Hz, H-11), 4.78 (1H, quint, J = 6.1 Hz, H-18), 3.66 (3H, s, COOCH3), 2.55 (1H, m, H-10β), 2.52 (1H, m, H-12), 2.25 (2H, t, J = 7.6 Hz, H-2), 2.04 (3H, s, 18-OCOCH3), 2.02 (1H, m, H-7a), 1.94 (3H, s, 11OCOCH3), 1.89 (1H, m, H-7b), 1.88 (2H, m, H-4), 1.83 (1H, m, H-10α), 1.70 (2H, m, H-16), 1.67 (1H, m, H-8), 1.61 (2H, m, H-3), 1.55 (2H, m, H-17), 1.53 (2H, m, H-19), 0.85 (3H, t, J = 7.4 Hz, H20); 13C NMR (CDCl3, 100 MHz) δC 174.0 (C, C-1), 171.1 (18OCOCH3), 170.6 (11-OCOCH3), 134.1 (CH, C-13), 130.5 (CH, C5), 129.8 (CH, C-14), 127.1 (CH, C-6), 77.7 (CH, C-11), 77.6 (CH, C-9), 76.4 (CH, C-15), 75.1 (CH, C-18), 52.2 (CH, C-12), 51.5 (COOCH3), 48.0 (CH, C-8), 38.9 (CH2, C-10), 33.3 (CH2, C-2), 30.0 (CH2, C-16), 28.9 (CH2, C-17), 26.9 (CH2, C-19), 26.4 (CH2, C4), 24.6 (CH2, C-3), 24.6 (CH2, C-7), 21.1 (18-OCOCH3), 20.8 (11OCOCH3), 9.4 (CH3, C-20); ESIMS m/z 918.4 [M + NH4]+, 923.3 [M + Na]+; ESIMS m/z 935.3 [M + Cl]−. Chemical Transformation of 1 to 2. Acetic anhydride (200 μL) was added to a stirred solution of compound 1 (2 mg) in freshly distilled pyridine (1 mL). The reaction was stirred at rt for 12 h and quenched by adding 0.2 mL of H2O. After removal of solvent under vacuum, the residue was purified on a flash silica gel column eluting with CH2Cl2 to afford 2 (1.95 mg), which was identified by the 1H NMR data, Rf, and [α]20D. Chemical Transformation of 4 to 4c. NaBH4 (1 mg) was added to a stirred solution of 4 (2 mg) in MeOH (0.5 mL), and the reaction was stirred for 15 min at rt. The mixture was then purified on Sephadex LH-20 using EtOH as eluent to afford 4b, which was further acetylated to obtain 4c (Scheme 1) following the same procedure as mentioned above. Compound 4c was identified by the 1H NMR spectrum, Rf, and MS data. 1934



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Alkaline Hydrolysis of 1−8, 11, and 12. Each PG (1 mg) was stirred with 1 mL of 0.1 M NaOH (MeOH/H2O, 3:1) for 2 h at rt; then the mixture was subjected to Sephadex LH-20 using EtOH as eluent to afford the pure hydrolysis product, which was identified by the 1H NMR spectrum, Rf, and [α]20D. Hydrolysis product of 11 (11c): colorless oil; [α]25D +27.3 (c 0.41, MeOH); IR (KBr) νmax 3664, 2928, 2872, 1569, 1142, 1026, 964 cm−1; 1H NMR (methanol-d4, 400 MHz) δH 5.55 (H, dd, J = 15.4, 6.5 Hz, H-13), 5.49 (1H, dd, J = 15.4, 8.1 Hz, H-14), 5.45 (1H, dd, J = 10.9, 7.2 Hz, H-6), 5.37 (1H, dd, J = 10.9, 7.2 Hz, H-5), 4.10 (1H, m, H-9), 4.04 (1H, m, H-15), 3.85 (1H, m, H-11), 3.46 (1H, m, H-18), 2.32 (1H, m, H-10β), 2.27 (1H, m, H-12), 2.21 (2H, m, H-7), 2.18 (2H, t, J = 7.6 Hz, H-2), 2.09 (2H, m, H-4), 1.64 (2H, m, H-3), 1.62 (2H, m, H-16), 1.61 (1H, m, H-10α), 1.47 (2H, m, H-19), 1.47 (1H, m, H-8), 1.42 (2H, m, H-17), 0.93 (3H, t, J = 7.4 Hz, H-20); 13C NMR (methanol-d4, 100 MHz) δC 183.7 (C, C-1), 136.3 (CH, C-13), 134.2 (CH, C-14), 131.0 (CH, C-5), 129.8 (CH, C-6), 77.9 (CH, C11), 73.9 (CH, C-18), 73.8 (CH, C-15), 72.3 (CH, C-9), 56.1 (CH, C12), 51.0 (CH, C-8), 44.2 (CH2, C-10), 38.3 (CH2, C-2), 34.6 (CH2, C-16), 33.8 (CH2, C-17), 31.1 (CH2, C-19), 28.2 (CH2, C-4), 27.6 (CH2, C-3), 26.3 (CH2, C-7), 10.4 (CH3, C-20); ESIMS m/z 353.3 [M(COOH) − H2O + H]+, ESIMS m/z 369.1 [M(COOH) − H]−. Hydrolysis product of 4 (4a): colorless oil; [α]25D +19.4 (c 0.10, MeOH); IR (KBr) νmax 3385, 2936, 2860, 1709, 1560, 1407, 1120, 1024, 971 cm−1; 1H NMR (methanol-d4, 400 MHz) δH 5.52 (1H, m, H-14), 5.51 (H, m, H-13), 5.45 (1H, dd, J = 11.5, 5.1 Hz, H-6), 5.37 (1H, dd, J = 11.5, 7.0 Hz, H-5), 4.10 (1H, m, H-9), 4.04 (1H, dd, J = 11.6, 6.2 Hz, H-15), 3.84 (1H, ddd, J = 8.0, 7.6, 5.4 Hz, H-11), 2.54 (2H, t, J = 7.3 Hz, H-17), 2.48 (2H, q, J = 7.3 Hz, H-19), 2.33 (1H, J = 14.5, 8.0, 6.0 Hz, H-10β), 2.27 (1H, m, H-12), 2.23 (1H, m, H-7a), 2.17 (2H, t, J = 7.6 Hz, H-2), 2.09 (2H, m, H-4), 2.09 (1H, m, H-7b), 1.76 (2H, m, H-16), 1.65 (2H, m, H-3), 1.60 (1H, m, H-10α), 1.49 (1H, m, H-8), 1.02 (3H, t, J = 7.3 Hz, H-20); 13C NMR (methanol-d4, 100 MHz) δC 214.2 (C, C-18), 182.6 (C, C-1), 135.7 (CH, C-13), 134.3 (CH, C-14), 131.1 (CH, C-5), 129.8 (CH, C-6), 77.9 (CH, C11), 72.8 (CH, C-15), 72.3 (CH, C-9), 56.1 (CH, C-12), 51.0 (CH, C8), 44.2 (CH2, C-10), 39.1 (CH2, C-17), 38.6 (CH2, C-2), 36.7 (CH2, C-19), 32.2 (CH2, C-16), 28.3 (CH2, C-4), 27.7 (CH2, C-3), 26.3 (CH2, C-7), 8.2 (CH3, C-20); ESIMS m/z 351.2 [M(COOH) − H2O + H]+; ESIMS m/z 367.1 [M(COOH) − H]−; HRESIMS m/z 391.2098 [M(COOH) + Na]+ (calcd for C20H32O6Na, 391.2091). PDE4D Inhibitory Screening Assays. The protocols for expression, purification, and enzymatic assays of PDE4D2 were similar to those we described previously.16−19

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and the Fundamental Research Funds for the Central Universities (11ykzd05) for providing financial support to this work. We cordially thank Prof. H. Ke in the Department of Biochemistry and Biophysics at University of North Carolina, Chapel Hill, for his help with the expression, purification, and enzymatic assay of PDE4.

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of 1−15, 4a, 11c, and the Mosher’s esters of 3 and 11 are available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(H.-B. Luo) E-mail: [email protected]. *(S. Yin) Tel: +86-20-39943090 or +86-20-39943031. Fax: +86-20-39943090. E-mail: [email protected]. Author Contributions §

Z.-B. Cheng and Y.-L. Deng contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Nos. 81102339, 21103234, and 81373258), the Guangdong Natural Science Foundation (Nos. S2011040002429, S2011030003190, and S2013010014867), 1935



Article

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