Polycyclic polyprenylated acylphloroglucinols: natural phosphodiesterase-4 inhibitors from Hypericum sampsonii

Abstract

Chemical investigation of the aerial parts of Hypericum sampsonii led to the isolation of seven new polycyclic polyprenylated acylphloroglucinols, hypersampsonones A–G (1–7), together with 23 known analogs (8–30). Their structures including the absolute configurations were elucidated by combined spectroscopic analysis, quantum chemical ECD calculations, and chemical methods. Compound 1 represents an unprecedented cyclocitral monoterpene-coupled bicyclo[3.3.1]nonane skeleton, while 2 features an unusual hexahydrofuro[2,3-b]furan-diepoxy ring system fused in a tricyclo[4.3.1.1^5,7]undecane skeleton. All the compounds were screened by using tritium-labeled adenosine 3′,5′-cyclic monophosphate ([³H]-cAMP) as substrate for their inhibitory activity against phosphodiesterase-4 (PDE4), which is a drug target for the treatment of asthma and chronic obstructive pulmonary disease. Compounds 1, 18–19, 21, and 25–30 exhibited inhibition with IC₅₀ values less than 10 μM, in which compound 19 represented the most active compound (IC₅₀ = 0.64 μM), being comparable to the positive control, rolipram (IC₅₀ = 0.62 μM).

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Introduction

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a class of hybrid natural products characterized by a highly oxygenated and densely substituted bicyclo[3.3.1]nonane-2,4,9-trione or other related core structures. PPAPs are biosynthetically generated through a “mixed” mevalonate/methylerythritol phosphate and polyketide pathway and are usually decorated with prenyl or geranyl side chains.^1,2 Over the past decades, more than 300 structurally diverse PPAPs have been reported,^1,2 and their fascinating highly rigid “diamond-like” architectures as well as important biological activities have attracted broad interest from both natural products and synthetic chemists.^3–6 Hyperforin, for instance, has been reported to possess a wide range of biological effects including antibiotic, anticancer, antidepressant, and procognitive activities, and has been totally synthesized by five research groups within the last five years.^7,8 So far, natural PPAPs have been reported exclusively from plants of the Guttiferae family, and nearly half of these PPAPs were isolated from the genus Hypericum.^1,2 Particularly, Hypericum sampsonii, an evergreen shrub used in traditional Chinese medicine for removing blood stasis and relieving swelling,⁹ has been proved to be a rich source of complex caged PPAPs with a wide variety of biological properties, such as antimicrobial,¹⁰ cytotoxic,⁵ and anti-HIV¹¹ activities.

Phosphodiesterase-4 (PDE4), which specifically catalyzes the hydrolysis of adenosine 3′,5′-cyclic monophosphate (cAMP), is a promising drug target of high interest for central nervous system (CNS), inflammatory, and respiratory diseases.¹² In our continuing search for PDE4 inhibitors from medicinal plants,^13,14 seven new PPAPs, hypersampsonones A–H (1–7), and 23 analogues were isolated from the aerial parts of H. sampsonii. Compound 1 represents an unprecedented carbon skeleton formed from coupling of a cyclocitral monoterpene and a bicyclo[3.3.1]nonane, while 2 features an unusual hexahydrofuro[2,3-b]furan-diepoxy ring system fused in a tricyclo[4.3.1.1^5,7]undecane skeleton. All of the isolates were screened for their inhibitory activity against PDE4, and ten compounds were identified as PDE4 inhibitors with IC₅₀ values less than 10 μM. Herein, the details of the isolation, structure elucidation, and inhibitory activity of these compounds are described.

Results and discussion

The air-dried powder of aerial parts of H. sampsonii was extracted with 95% EtOH at room temperature (rt) to give a crude extract, which was suspended in H₂O and successively partitioned with petroleum ether (PE) and EtOAc. Various column chromatographic separations of the PE extract afforded compounds 1–30 (Fig. 1).

Fig. 1 Structures of compounds 1–30.

Compound 1, a colorless oil, had the molecular formula C₃₈H₅₀O₅ as determined by the HRESIMS ion at m/z 585.3561 (calcd for [M − H]⁻, 585.3585), indicating 14 degrees of unsaturation (DOUs). The IR spectrum exhibited absorption bands for hydroxyl (3505 cm⁻¹) and carbonyl (1721 and 1694 cm⁻¹) functionalities. The ¹H and ¹³C NMR data (Tables 1 and 2) and HSQC spectrum showed signals for a benzoyl [δ_H 7.22 (2H, brt, J = 7.5 Hz), 7.37 (1H, tt, J = 7.5, 1.2 Hz), and 7.48 (2H, brd, J = 7.5 Hz); δ_C 127.8 × 2, 128.2 × 2, 131.7, 137.0, and 193.8], an α,β-unsaturated ketone (δ_C 123.3, 167.6, and 192.6), a ketone (δ_C 206.2), and two prenyl groups [δ_H 1.54 (3H, s), 1.59 (3H, s), 1.61 (3H, s), 1.68 (3H, s), 2.13 (2H, m), 2.96 (1H, dd, J = 13.5, 7.1 Hz), 3.08 (1H, dd, J = 13.5, 7.7 Hz), 4.87 (1H, dd, J = 7.2, 6.0 Hz), 4.99 (1H, dd, J = 7.7, 7.1 Hz); δ_C 17.8, 18.0, 22.4, 25.8, 25.9, 29.0, 120.4, 124.8, 132.1, 132.4], suggesting that 1 was a PPAP derivative.¹⁰ The characteristic carbon signals for three sp³ quaternary carbons at δ_C 76.7 (C-1), δ_C 52.4 (C-5), and δ_C 48.2 (C-8), together with a sp³ methine at δ_C 47.7 (C-7) indicated that 1 possessed a bicyclo[3.3.1]nonane core, similar to that of propolone D.¹⁵ Comparison of the NMR data of 1 with those of propolone D indicated that their major differences arose from the C₁₀ unit linked at C-5 of the bicyclo[3.3.1] core. As 12 of the 14 DOUs were accounted for by a benzoyl group, two keto groups, three double bonds, and a bicycle core, the remaining DOUs required two additional rings in the C₁₀ unit.

Table 1 ¹H NMR spectroscopic data of 1–7 in CDCl₃ (400 MHz, J in Hz, δ in ppm)

	1	2	3	4	5	6	7
6	a 2.09, m; b 2.41, d (14.4)	a 2.23, m; b 2.35, m	a 2.27, m; b 2.45, m	2.26, m	1.99, m	a 1.89, m; b 2.51, m	a 1.84, m; b 2.51, m
7	1.46, m	1.78, m	2.03, m	1.73, m	2.00, m	2.11, m	2.14, m
12	7.48, brd (7.5)	7.57, a (7.5)	7.58, brt (7.4)	7.44, brd (7.5)	7.11, brd (7.1)	7.10, brd (7.4)	7.11, brd (7.4)
13	7.22, brt (7.5)	7.36, brt (7.5)	7.26, brt (7.4)	7.27, brt (7.5)	7.31, brt (7.1)	7.28, brt (7.4)	7.26, brt (7.4)
14	7.37, tt (7.5, 1.2)	7.39, brt (7.5)	7.38, brt (7.4)	7.35, brt (7.5)	7.42, brt (7.1)	7.39, brt (7.4)	7.39, brt (7.4)
15	7.22, brt (7.5)	7.36, brt (7.5)	7.26, brt (7.4)	7.27, brt (7.5)	7.31, brt (7.1)	7.28, brt (7.4)	7.26, brt (7.4)
16	7.48, brd (7.5)	7.57, brd (7.5)	7.58, brt (7.4)	7.44, brd (7.5)	7.11, brd (7.1)	7.10, brd (7.4)	7.11, brd (7.4)
17	a 2.96, dd (13.5, 7.1); b 3.08, dd (13.5, 7.7)	a 2.45, m; b 2.96, m	5.08, brs	6.44, d (16.5)	a 2.01, m; b 3.04, m	a 2.17, m; b 2.49, m	a 2.43, dd (9.5, 13.5); b 2.91, dd (11.4, 13.5)
18	4.99, dd (7.7, 7.1)	5.86, m	4.00, brs	5.90, d (16.5)	4.30, dd (7.2, 7.2)	2.84, dd (7.9, 12.3)	1.95, m
20	1.61, s		1.28, s	1.17, s	1.24, s	1.35, s	1.30, s
21	1.59, s		1.33, s	1.21, s	1.16, s	1.39, s	1.38, s
22	α 1.19, m; β 3.15, d (11.3)	a 2.45, m; b 2.76, dd (14.4, 7.2)	a 2.53, m; b 2.85, m	a 2.45, m; b 2.70, dd (6.9, 6.9)	2.59, d (7.2)	2.56, d (6.8)	2.54, m
23	1.14, m	5.39, dd (7.2, 7.2)	5.48, m	5.52, dd (6.9, 6.9)	5.23, dd (7.2, 7.2)	5.13, dd (6.8, 6.8)	5.01, m
25	α 2.12, m; β 1.67, m	2.04, m	2.10, m	2.04, m	2.03, m	1.98, m	1.95, m
26	1.50, s	1.65, s	1.73, s	1.68, s	1.65, s	1.65, s	1.65, s
27	α 1.89, m; β 1.58, m	2.03, m	2.11, m	2.05, m	1.61, m	1.99, m; 2.05, m	1.96, m; 2.04, m
28	3.37, dd (11.2, 4.3)	5.07, dd (6.8, 6.8)	5.06, m	5.07, brs	5.04, dd (7.2, 7.2)	5.05, m	5.04, m
30	1.08, s	1.58, s	1.61, s	1.59, s	1.57, s	1.58, s	1.58, s
31	0.82, s	1.63, s	1.69, s	1.68, s	1.65, s	1.65, s	1.66, s
32	2.13, m	a 1.26, m; b 1.95, m	2.07, m	2.05, m	2.10, m	1.62, m	1.49, m
33	4.87, dd (7.2, 6.0)	2.46, m	2.50, m	2.62, m	4.21, dd (8.3, 8.3)	2.05, m	2.04, m
35	1.68, s	1.58, s	1.35, s	1.51, s		0.92, s	1.08, s
36	1.54, s	1.47, s	1.35, s	1.44, s		1.17, s	0.98, s
37	1.35, s	1.22, s	1.48, s	1.22, s	1.40, s	1.39, s	1.44, s
38	1.44, s	1.42, s; 18-OH: 3.41, d (3.9)	1.39, s; 4-OH: 4.35, s	1.41, s; 19-OMe: 2.79, s	1.42, s	1.46, s	1.50, s

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Table 2 ¹³C NMR spectroscopic data of 1–7 in CDCl₃ (100 MHz, δ in ppm)

	1	2	3	4	5	6	7
1	76.7	81.4	82.3	81.0	81.1	80.8	81.3
2	192.6	207.2	203.4	202.4	201.3	203.6	205.7
3	123.3	67.5	77.2	67.6	76.5	73.4	73.1
4	167.6	115.2	106.6	108.8	200.7	206.4	205.6
5	52.4	56.4	57.0	59.2	67.7	69.0	68.4
6	39.8	31.2	41.2	33.6	35.5	42.0	44.9
7	47.7	43.7	44.7	43.5	41.5	43.8	43.4
8	48.2	47.9	52.1	48.1	47.8	50.8	51.6
9	206.2	208.1	207.5	209.0	204.1	204.7	205.0
10	193.8	194.4	194.4	194.3	192.4	192.5	192.8
11	137.0	135.8	136.7	135.9	134.8	134.9	135.1
12	128.2	129.3	129.6	129.1	128.6	128.9	129.1
13	127.8	128.3	127.7	127.9	128.3	128.1	127.9
14	131.7	132.1	131.8	131.8	132.4	132.1	132.0
15	127.8	128.3	127.7	127.9	128.3	128.1	127.9
16	128.2	129.3	129.6	129.1	128.6	128.9	129.1
17	22.4	45.8	83.1	127.2	32.7	33.1	31.0
18	120.4	98.6	89.4	141.3	87.6	54.4	59.5
19	132.1		69.8	75.2	72.5	85.6	73.0
20	25.8		27.3	24.4	26.7	24.7	31.0
21	18.0		26.4	26.5	24.7	23.0	30.2
22	22.9	27.3	28.5	28.8	28.9	29.3	29.9
23	48.7	119.8	120.0	120.2	118.4	119.0	119.0
24	82.7	137.9	140.4	139.3	139.3	138.4	138.4
25	38.9	40.0	40.0	40.0	40.0	40.0	39.9
26	21.8	16.2	16.3	16.2	16.3	16.4	16.4
27	28.5	26.7	26.3	25.8	26.5	26.6	26.6
28	77.4	124.3	123.8	124.0	124.0	124.1	124.1
29	38.8	131.4	131.9	131.7	131.5	131.4	131.3
30	27.3	17.7	17.7	17.7	17.7	17.7	17.7
31	14.3	25.7	25.8	25.7	25.7	25.7	25.7
32	29.0	25.3	28.9	26.5	31.3	28.6	28.3
33	124.8	49.4	50.1	52.7	81.6	57.8	58.4
34	132.4	84.2	86.8	84.7		44.5	47.2
35	25.9	30.4	32.0	33.2		28.0	30.3
36	17.8	27.9	24.1	27.0		27.5	15.7
37	26.8	22.4	22.9	22.1	22.7	25.2	25.9
38	22.1	25.0	26.9	25.0	24.5	22.8	22.7
19-OMe				50.6

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Detailed 2D NMR analysis regarding this unit was thus unfolded. In ¹H–¹H COSY spectrum two structural fragments (C-22–C-23 and C-25–C-27–C-28) were first established by the correlations observed (Fig. 2). The connectivity of the two fragments, quaternary carbons, and three tertiary methyls was achieved by analysis of the HMBC correlations. The HMBC correlations from two tertiary methyls (CH₃-30 and CH₃-31) to C-23, C-28, and C-29 allowed the connections of C-23, C-28, C-30, and C-31 to the quaternary carbon C-29. Moreover, HMBC correlations from H₃-26 to C-23, C-24, and C-25 linked C-23, C-25, and C-26 to C-24. Thus, a cyclocitral monoterpenoid substructure was constructed for the C₁₀ unit. The cyclocitral unit was further linked to C-5 by HMBC correlations from H₂-22 to C-4, C-5, C-6, and C-9. Although no direct HMBC correlations were available to construct the remaining ring, the still “loose end” of an oxygenated quaternary carbon (δ_C 82.7, C-24) and the severely downfield-shifted sp² carbon at C-4 (δ_C 167.6) suggested that an oxygen bridge was located between C-24 and C-4 to form a tetrahydropyran ring. Thus, the gross structure of 1 was established, which represented an unprecedented carbon skeleton formed from coupling of a cyclocitral monoterpene and a bicyclo[3.3.1]nonane.

Fig. 2 Selected ¹H–¹H COSY () and HMBC () correlations of 1 and 2.

The relative stereochemistry of 1 was established on the basis of a NOESY experiment. NOESY correlation from H₃-38 to H-6a indicated that CH₃-38 and H-6a adopted the axial bonds of the chair conformational D-ring and designed as β-orientation (Fig. 3). Consequently, NOESY correlations of H-6a/H-7 indicated that H-7 adopted the equatorial bond, and was assigned as β-orientation. The α-orientation of the prenyl chain at C-7 was further supported by the NOESY correlation of H-32a/H-18. In addition, NOESY correlations of H-23/H₂-6, H-23/H-28, and H-28/H-25β indicated that these protons resided in the axial position of the chair conformational rings A and B, and were designated as β-oriented. Thus, the NOESY correlation of H₃-31/H₃-26 and H-22α indicated that CH₃-26 was axially-oriented and was assigned as α-orientation.

Fig. 3 Key NOE () correlations of 1 and 2.

The absolute configuration of 1 was determined by quantum chemical TDDFT calculations of its theoretical ECD spectrum. Firstly, conformational analysis of the 1S,5R,7S,23R,24R,28R stereoisomer was carried out via Monte Carlo searching using molecular mechanism with MMFF94 force field in the Spartan 08 program, which resulted in 14 conformers with relative energy within 2.0 kcal mol⁻¹.¹⁶ These conformers showed conformational possibilities on the directions of the O–H bond at C-28 (three directions) and the prenyl side chains at C-7 (two directions) and C-3 (three directions). The conformers with different directions of the O–H bond were ignored because the far distance between the OH and the chromophore had little influence on the carbon skeleton and ECD spectrum. Then six conformers (1a–1f) with different directions of the two prenyl side chains (ESI Fig. S1†) were selected for further calculation. In addition, a simplified model with the absence of two prenyl side chains (1g, ESI Fig. S1†) was also designed and calculated. As shown in Fig. 4, in the 200–400 nm region, the experimental first positive Cotton effect around 275 nm with a shoulder at 320 nm was simulated by a positive Cotton effect peak at 272 nm with a shoulder at 345 nm. The experimental negative Cotton effects around 205 nm and 242 nm were simulated correspondingly by a broad negative theoretical peak at 219 nm. Therefore, qualitative analysis of the calculated and experimental ECD spectra allowed the assignment of the absolute configuration of 1. In addition, the model conformer without the two prenyl side chains also showed similar theoretical ECD spectrum, indicating that the two side chains also had little influence on the ECD spectrum of compound 1. Thus, the absolute configurations of 1 were assigned as 1S,5R,7S,23R,24R,28R, and 1 was given a trivial name hypersampsonone A.

Fig. 4 Experimental and B3LYP/6-311++G(2d,2p)//B3LYP/6-31G(d) calculated ECD spectra of 1 and the model conformer.

Compound 2, a colorless oil, had the molecular formula C₃₅H₄₄O₆, as determined by HRESIMS at m/z 559.3065 (calcd for [M − H]⁻, 559.3065), corresponding to 14 DOUs. The NMR data (Tables 1 and 2) and HSQC spectrum showed, in addition to a number of aliphatic methylene multiplets, signals for a benzoyl group [δ_H 7.36 (2H, brt, J = 7.5 Hz), 7.39 (1H, brt, J = 7.5 Hz), and 7.57 (2H, brd, J = 7.5 Hz); δ_C 128.3 × 2, 129.3 × 2, 132.1, 135.8, and 194.4], seven methyls [δ_H 1.22 (3H, s), 1.42 (3H, s), 1.47 (3H, s), 1.58 (3H, s), 1.58 (3H, s), 1.63 (3H, s), and 1.65 (3H, s); δ_C 16.2, 17.7, 22.4, 25.0, 25.7, 27.9, and 30.4], two keto groups [δ_C 207.2 and 208.1], and two trisubstituted double bonds [δ_H 5.07 (1H, dd, J = 6.8, 6.8 Hz) and 5.39 (1H, dd, J = 7.2, 7.2 Hz); δ_C 119.8, 124.3, 131.4, and 137.9]. The above-mentioned information suggested that 2 was also a PPAP derivative. Observations of characteristic signals for three sp³ quaternary carbons [δ_C 56.4 (C-5), δ_C 67.5 (C-3), and δ_C 115.2 (C-4)], two sp³ methines [δ_C 43.7 (C-7) and δ_C 49.4 (C-33)], two sp³ methylenes [δ_C 25.3 (C-32) and 31.2 (C-6)], two geminal methyls [δ_C 22.4 (C-37) and δ_C 25.0 (C-38)], and the two keto groups indicated that 2 was a homoadamantyl type PPAP with a tricyclo[4.3.1.1]undecane core.² The geranyl and benzoyl groups were readily attached to C-5 and C-1 of the core, respectively, by HMBC correlation (Fig. 2) and ¹³C chemical shift analysis of C-5 and C-1.² As 12 of the 14 DOUs were accounted for by a benzoyl group, two keto groups, two double bonds, two carbonyl groups, and the tricyclo core, the remaining two DOUs required that the rest five carbons and three oxygen atoms were constructed to give two additional rings in 2. The HMBC correlations from two tertiary methyls (CH₃-35 and CH₃-36) to an oxygenated quaternary carbon (δ_C 84.2, C-34) and C-33 indicated that a 2-oxyisopropyl was linked to C-33. The HMBC correlations from a hydroxyl proton to a hemiacetal carbon (δ_C 98.6) and a methylene (δ_C 45.8) together with those from H-17 to C-3, C-4, and C-33 linked a 2-hydroxyethyl moiety to C-3. Taking the ketal carbon of C-4 (δ_C 115.2) and the still “loose end” carbons of C-18 (δ_C 98.6) and C-34 (δ_C 84.2) into account, the two additional rings could only be formed, via oxygen bridges, between C-4 and C-18 and between C-4 and C-34, respectively. Thus the gross structure of 2 was figured out, which represented an unusual hexahydrofuro[2,3-b]furan-fused tricyclo[4.3.1.1^5,7]undecane skeleton.

The relative configuration of 2 was assigned by analyzing its NOESY data and by comparison of its 1D NMR data with those of the compounds sharing the same structural cores.² In particular, the configurations of the two additional furan rings were readily fixed by the rigid “diamond-like” tricyclo[4.3.1.1^5,7] core, while the NOESY correlations of H-18/H₃-35 indicated that the OH-18 at one of the furan rings was α-oriented. Compound 2 was given the trivial name hypersampsonone B.

Compound 3 had a molecular formula of C₃₈H₅₀O₇, as established by HRESIMS. The ¹H and ¹³C NMR spectra of 3 (Table 1 and 2) showed high similarity to those of dioxasampsone B, a known PPAP with a unique tetrahydrofuro[3,4-b]furan-fused tricyclo[4.3.1.1^5,7]undecane core.¹⁷ Extensive comparison of NMR data of 3 with those of dioxasampsone B revealed that their only difference arose from the side chain at C-5, where the prenyl linked to C-5 in dioxasampsone B was replaced by a geranyl in 3 to give an extended carbon skeleton for this type of compounds. Detailed 2D NMR analyses (¹H–¹H COSY, HSQC, and HMBC) permitted the establishment of the gross structure of 3 (ESI S25†). The relative configuration of 3 was assigned to be the same as that of dioxasampsone B by comparison of its 1D NMR and NOESY data with those of dioxasampsone B. In particular, NOE correlations of H-17/H-33 and H₃-21 indicated that these protons were cofacial and designated as α-oriented. Accordingly, compound 3 was established and named hypersampsonone C.

Compound 4 displayed the HRESIMS ion at m/z 639.3683 [M + Na]⁺, consistent with a molecular formula of C₃₉H₅₂O₆, 14 mass units more than that of hyphenrone N (8),² a co-isolated metabolite in the current study. The ¹H and ¹³C NMR data of 4 (Tables 1 and 2) were very similar to those of 8 except for the presence of an additional methoxyl (δ_H 2.79) and the downfield-shifted carbon signal at C-19 [δ_C 75.2 in 4, δ_C 71.4 in 8], indicating that 4 was a 19-O-methyl derivative of 8. This was further confirmed by HMBC correlation from the methoxyl to C-19. The relative configuration of 4 was assigned to be the same as that of 8 by comparing their 1D NMR data and by analyzing its NOESY data. Thus, compound 4 was deduced as depicted and was given the trivial name hypersampsonone D.

Compound 5, a colorless oil, had the molecular formula C₃₅H₄₄O₆ as determined by HRESIMS at m/z 561.3225 (calcd 561.3211). The 1D NMR data of 5 was very similar to that of hypersampsone N (9),¹⁸ except for the replacement of the prenyl group in 9 by a geranyl group in 5. Detailed 2D NMR analyses (¹H–¹H COSY, HSQC, and HMBC) permitted the establishment of the gross structure of 5 as depicted. The relative configuration of 5 was assigned to be the same as that of 9 by NOESY experiment and by comparison of their NMR data. Thus, compound 5 was deduced as shown and was given the trivial name hypersampsonone E.

The HRESIMS of 6 gave a pseudo-molecular ion at m/z 601.3511 [M − H]⁻ (calcd 601.3535), corresponding to the molecular formula of C₃₈H₅₀O₆, with one more O-atom than that of sampsonione C (10).¹⁹ The ¹³C NMR data of 6 was very similar to that of 10, with the differences scattering around C-19. In comparison with 10, the carbon resonances of C-18, C-19, C-20, and C-21 in 6 were shifted by Δδ_C −3.1, +12.2, −5.3, −7.1 ppm, respectively, indicating that 6 possessed a hydroperoxy group at C-19.²⁰ The structure of 6 was further confirmed by the conversion of 6 to 10 in acidic CDCl₃ under several days' storage. Accordingly, compound 6 was established as depicted and was named hypersampsonone F.

Compound 7, a colorless oil, had the molecular formula C₃₈H₅₀O₅ as determined by HRESIMS at m/z 587.3758 [M + H]⁺ (calcd 587.3731), indicating that it was an isomer of sampsonione C (10).¹⁹ Comprehensive analysis of 1D and 2D NMR spectra revealed that 7 shared the same planar structure with 10. The only structural difference between 7 and 10 was occurring at C-18, as implied by the slightly shifted carbon resonances around this stereocenter (ΔC-17, ΔC-18, and ΔC-34 within 2 ppm), indicating that 7 was a 18-epimer of 10. This was further supported by the NOE correlations of H₃-35/H-18 and H-33. Thus, compound 7 was assigned as depicted and was named hypersampsonone G.

The absolute stereochemistry of 2–7 were assigned by CD analysis. Based on the CD benchmark summarized by Zhang et al.,¹¹ the R configuration for C-1 of benzoyl substituted adamantyl and homoadamantyl PPAPs would generate a negative Cotton effect around 333 nm. Thus, compounds 2–5 (homoadamantyl type) and 6–7 (adamantane type) displaying the negative cotton effects around 330 nm (ESI S3†) were assigned the R configuration at C-1. This assignment was consistent with the biosynthetic origin of the PPAPs from the genus Hypericum.^11,17 Hence, The absolute configuration of 2–7 was assigned as depicted.

The known compounds hyphenrone N (8),² hypersampsone N (9),¹⁸ sampsonione C (10),¹⁹ hypersampsone Q (11),¹⁸ plukenetione B (12),²¹ hyperattenin I (13),²² sampsonione F (14),¹⁹ attenuatumione D (15),²³ sampsonione G (16),¹⁹ hypercohone A (17),²¹ otogirinin D (18),²⁴ hyperattenin C (19),²² hyperibone A (20),²⁵ hyperattenin G (21),²² cowabenzophenone B (22),²⁶ sampsonione H (23),¹⁹ hypersampsone P (24),¹⁸ sampsonione A (25),²⁷ sampsonione B (26),²⁷ hypersampsone N (27),¹⁸ sampsonione L (28),²⁸ hyperisampsin I (29),³ and hyperisampsin J (30)³ were identified by comparison of their NMR and MS data with those in the literature.

Most of the structurally related PPAPs isolated from the genus Hypericum are believed to be biosynthesized from the biogenetically acceptable 2,4,6-trihydroxybenzophenone i.^2,17,28 It is further decorated with dimethylallyl diphosphates (DMAPP) to give intermediate ii, which is the common precursor of diverse PPAPs.^2,17,28 A plausible biosynthetic pathway of 1–30 starting from ii through a series of reactions including epoxidation, intramolecular cyclization, oxidation cleavage, dehydration, reduction, and cationic polycyclization^17,28,29 was proposed in Scheme 1.

Scheme 1 Plausible biosynthetic pathway for 1–30.

Compounds 1–30 were tested for their inhibitory activity against PED4D2 at an initial concentration of 10 μM by using tritium-labeled adenosine 3′,5′-cyclic monophosphate ([³H]-cAMP) as substrate (ESI S1†). Rolipram, a well-known PDE4 inhibitor, was used as the reference compound (IC₅₀ = 0.62 μM), comparable to the reported value of 1.0 μM.³⁰ Compounds with inhibition greater than 50% at 10 μM were further analyzed to determine their corresponding IC₅₀ values. As shown in Table 3, compounds 1, 18–19, 21 and 25–30 had significant inhibitory activity against PDE4D2, with IC₅₀ values less than 10 μM, and 19 turned out to be the most active compound with an IC₅₀ value of 0.64 μM, being comparable to that of rolipram. The inhibitory curves of compounds 19, 27, 30, and rolipram are represented in Fig. 5.

Table 3 IC₅₀ values of the active compounds against PDE4D2

Compound	IC50 (μM)	Compound	IC50 (μM)
a Positive control.
1	8.08 ± 0.34	18	2.73 ± 0.13
19	0.64 ± 0.02	21	3.47 ± 0.13
25	3.05 ± 0.14	26	2.66 ± 0.09
27	1.59 ± 0.11	28	4.68 ± 0.27
29	3.08 ± 0.12	30	1.91 ± 0.07
		Rolipram^a	0.62 ± 0.03

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Fig. 5 Inhibitory curves of compounds 19, 27, 30 and rolipram (positive control) against PDE4D2.

Conclusions

In summary, seven new polycyclic polyprenylated acylphloroglucinols (1–7) and 23 known analogs (8–30) were isolated from aerial parts of Hypericum sampsonii. Compound 1 represents an unprecedented cyclocitral monoterpene-coupled bicyclo[3.3.1]nonane skeleton, while 2 features an unusual hexahydrofuro[2,3-b]furan-diepoxy ring system fused in a tricyclo[4.3.1.1^5,7]undecane skeleton. Ten compounds exhibited inhibitory activity against PDE4D2 with IC₅₀ values less than 10 μM, and the most active compound, 19 (IC₅₀ = 0.64 μM), was comparable to that of rolipram (IC₅₀ = 0.62 μM).

Natural PDE4 inhibitors are rare, and the current study revealed a new group of PDE4 inhibitors from Hypericum sampsonii, which not only expand the structural diversity of PPAPs categories, but also explain the anti-inflammatory efficacy of this plant in traditional Chinese medicine. Studies toward their selectivity versus other PDE members are in progress.

Experimental section

General experimental procedures

Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 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 LCQ Deca instrument. A Shimadzu LC-20AT equipped with a SPD-M20A PDA detector was used for HPLC, and a YMC-pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) was used for semipreparative HPLC separation. A chiral column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 μm) was used for chiral separation. Silica gel (300–400 mesh, Qingdao Haiyang Chemical Co. Ltd.), reversed-phase C₁₈ (RP-C₁₈) silica gel (12 nm, S-50 μm, YMC Co. Ltd.), and MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.) were used for column chromatography (CC). All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.).

Plant material

The aerial parts of H. sampsonii were collected in August from Jiangsu province, and were identified by Dr Gui-Hua Tang of Sun Yat-sen University. A voucher specimen (accession number: YBC201411) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

Extraction and isolation

The air-dried powder of the aerial parts of H. sampsonii (5 kg) was extracted with 95% EtOH (3 × 5 L) at room temperature to give 450 g of crude extract. The extract was suspended in H₂O (3 L) and successively partitioned with petroleum ether (PE, 3 × 3 L) and EtOAc (3 × 3 L). The petroleum ether extract (181 g) was subjected to MCI gel column chromatography (CC) eluted with a MeOH/H₂O gradient (3 : 7 → 10 : 0) to afford five fractions (I–V). Fraction II (22.6 g) was chromatographed over a silica gel CC (n-hexane/EtOAc, 40 : 1) to give four fractions (Fr. IIa–IId). Fr. IIb (2.9 g) was subjected to C₁₈ reversed-phase (RP-C₁₈) silica gel CC eluted with MeOH/H₂O (8 : 2 → 10 : 0) to afford four fractions (Fr. IIb1–IIb4). Fr. IIb1 (162 mg) was further purified on RP-C₁₈ HPLC using MeOH/H₂O (90 : 10, 3 mL min⁻¹) as mobile phase to give 19 (35 mg, t_R 18.5 min), 18 (15 mg, t_R 19.5 min), and 9 (8 mg, t_R 22 min), Fr. IIb2 (40 mg) further purified on RP-C₁₈ HPLC using MeCN/H₂O (80 : 20, 3 mL min⁻¹) as mobile phase to afford 1 (11 mg, t_R 8.5 min), 28 (7 mg, t_R 12.5 min) and 5 (3.4 mg, t_R 15.5 min). Fr. IIb3 (362 mg) was further purified on RP-C₁₈ HPLC using MeOH/H₂O (90 : 10, 3 mL min⁻¹) as mobile phase to give 7 (50 mg, t_R 18 min), 14 (15 mg, t_R 18.5 min), 10 (25 mg, t_R 19.5 min), and 15 (16 mg, t_R 21 min). Fr. IIb4 (145 mg) was further purified on RP-C₁₈ HPLC using MeCN/H₂O (80 : 20, 3 mL min⁻¹) as mobile phase to give 17 (24 mg, t_R 14 min), 11 (15 mg, t_R 16.5 min), 16 (11 mg, t_R 17.5 min), and 12 (16 mg, t_R 21 min). Fraction III (12.6 g) was chromatographed over a silica gel CC (n-hexane/EtOAc, 50 : 1) to give five fractions (Fr. IIIa–IIIe). Fr. IIIb (1.9 g) was subjected to C₁₈ reversed-phase (RP-C₁₈) silica gel CC eluted with MeOH/H₂O (6 : 4 → 10 : 0) and further purified on RP-C₁₈ HPLC using MeOH/H₂O (90 : 10, 3 mL min⁻¹) as mobile phase to give 4 (8.4 mg, t_R 21 min), 29 (4 mg, t_R 24 min) and 30 (6.4 mg, t_R 24.5 min). Fr. IIIc (45 mg) was further purified on RP-C₁₈ HPLC using MeCN/H₂O (85 : 15, 3 mL min⁻¹) as mobile phase to give 13 (8 mg, t_R 14 min) and 6 (6 mg, t_R 16.5 min). Fr. IIId (370 mg) was chromatographed over a Sephadex LH-20 eluted with MeOH, and further purified on RP-C₁₈ HPLC using MeOH/H₂O (90 : 10, 3 mL min⁻¹) as mobile phase to afford 2 (5.6 mg, t_R 13 min), 25 (12.6 mg, t_R 16 min), and 26 (3.8 mg, t_R 18 min). Fr. IIIe (1.9 g) was chromatographed over a Sephadex LH-20 eluted with MeOH to give two fractions (Fr. IIIe1–IIIe2). Fr. IIIe1 (36 mg) was further purified on RP-C₁₈ HPLC using MeCN/H₂O (80 : 20, 3 mL min⁻¹) as mobile phase to give 22 (14 mg, t_R 12.4 min) and 20 (6 mg, t_R 16.5 min), while Fr. IIIe2 (900 mg) followed by a C₁₈ reversed-phase (RP-C₁₈) silica gel CC eluted with MeOH/H₂O (8 : 2 → 10 : 0) to afford 3 (6.2 mg) and 8 (8.8 mg). Fraction IV (8 g) was chromatographed over a Sephadex LH-20 (CH₂Cl₂/MeOH, v/v, 1 : 1), to give three fractions (Fr. IVa–IVc). Fr. IVa was further purified on RP-C₁₈ HPLC using MeCN/H₂O (90 : 10, 3 mL min⁻¹) as mobile phase to give 23 (6 mg, t_R 14 min), 24 (5 mg, t_R 16 min), 21 (9 mg, t_R 21 min) and 27 (6 mg, t_R 21.5 min).

Hypersampsonone A (1). Colorless oil; [α]²⁰_D +46.1 (c 0.59, CHCl₃); UV (MeOH) λ_max (log ε) 206.5 (7.10), 247 (6.89), 286 (6.85) nm; IR (KBr) ν_max 3505, 2925, 1721, 1694, 1633, 1585, 1221, 745 cm⁻¹; CD (c 2.85 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 205 (−16.31), 242 (−5.06), 275 (+9.04), 318 (+4.02) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 585.3561 (calcd for C₃₈H₄₉O₅⁻ [M − H]⁻, 585.3585).

Hypersampsonone B (2). Colorless oil; [α]²⁰_D +14.9 (c 0.35, CHCl₃); UV (MeOH) λ_max (log ε) 208.5 (7.51), 247.5 (7.38) nm; IR (KBr) ν_max 3442, 2927, 1725, 1691, 1450, 1224 cm⁻¹; CD (c 6.21 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 216 (+1.86), 243 (+7.20), 258 (+1.45), 297 (−7.09) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 559.3065 (calcd for C₃₅H₄₃O₆⁻ [M − H]⁻, 559.3065).

Hypersampsonone C (3). Colorless oil; [α]²⁰_D +6.7 (c 0.15, CHCl₃); UV (MeOH) λ_max (log ε) 207 (7.14), 246.5 (6.83) nm; IR (KBr) ν_max 3443, 2927, 1724, 1693, 1227 cm⁻¹; CD (c 4.09 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 213 (+15.06), 243 (−6.57), 259 (−6.58), 283 (+3.30), 314 (−2.31) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 641.3480 (calcd for C₃₈H₅₀O₇Na⁺ [M + Na]⁺, 641.3449).

Hypersampsonone D (4). Colorless oil; [α]²⁰_D −66.2 (c 0.52, CHCl₃); UV (MeOH) λ_max (log ε) 208.5 (7.27), 245.5 (7.07) nm; IR (KBr) ν_max 2979, 1694, 1223, 764, 749 cm⁻¹; CD (c 2.33 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 243 (+3.46), 297 (−3.34) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 639.3683 (calcd for C₃₉H₅₂O₆Na⁺ [M + Na]⁺, 639.3656).

Hypersampsonone E (5). Colorless oil; [α]²⁰_D +3.21 (c 1.06, CHCl₃); UV (MeOH) λ_max (log ε) 205 (3.64), 244 (3.18) nm; IR (KBr) ν_max 2927, 1738, 1703, 1597, 1236 cm⁻¹; CD (c 2.33 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 206 (−15.51), 211 (+5.49), 251 (+4.57), 284 (−1.36), 322 (−2.06) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 561.3225 (calcd for C₃₅H₄₅O₆⁺ [M + H]⁺, 561.3211).

Hypersampsonone F (6). Colorless oil; [α]²⁰_D +6.73 (c 1.04, CHCl₃); UV (MeOH) λ_max (log ε) 207.5 (7.32), 245.5 (7.12) nm; IR (KBr) ν_max 2979, 1694, 1223, 764, 749 cm⁻¹; CD (c 2.45 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 208 (+9.21), 238 (−6.11), 258 (+1.45), 311 (−3.02), 324 (−2.96) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 601.3511 (calcd for C₃₈H₄₉O₆⁻ [M − H]⁻, 601.3535).

Hypersampsonone G (7). Colorless oil; [α]²⁰_D +4.22 (c 0.9, CHCl₃); UV (MeOH) λ_max (log ε) 208 (4.49), 245 (4.31) nm; IR (KBr) ν_max 2971, 2935, 1734, 1696, 1222 cm⁻¹; CD (c 4.55 × 10⁻⁴ M, CH₃CN) λ_max (Δε) 218 (+4.15), 241 (−7.37), 285 (+2.11), 324 (−2.61) nm; ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 587.3758 (calcd for C₃₈H₅₁O₅⁺ [M + H]⁺, 587.3731).

Acknowledgements

The authors thank the National High Technology Research and Development Program of China (863 Project, No. 2015AA020928), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306047), and the National Natural Science Foundation of China (No. 81573302 and No. 81402813) for providing financial support to this work.

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Footnote

† Electronic supplementary information (ESI) available: HRESIMS, 1D and 2D NMR spectra of 1–7. See DOI: 10.1039/c6ra08805h

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