Antiangiogenic Polyketides from Peperomia dindygulensis Miq.

Abstract

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxy- phenyl)tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[(3,4- methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.

1. Introduction

Peperomia dindygulensis Miq. (Piperaceae), a widespread herb in the south of China, is used in the folk medicine to treat cough, asthma, phthisis, and stomach, lung, mammary and liver cancers [1]. The common constituents of Peperomia genus include secolignans, tetrahydrofuran lignans, chromones and acylcyclohexane-1,3-diones [2,3,4,5,6,7], which possess various bioactivities, such as antitumor and anti-HIV activities [8]. We found that the chloroform extract prepared from P. dindygulensis showed significant suppression acitivity against the proliferation of primary human umbilical vein endothelial cells (HUVEC). In our previous research, we isolated some antiangiogenic secolignans including two new secolignans from P. dindygulensis [9]. Further fractionation and purification of the CHCl₃ extract led to the isolation and characterization of two new polyketides: 2-(heptadec-12-enyl)-4-hydroxy-3,4,7,8- tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen-4- one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxyphenyl)tridecanoyl] cyclohexane-1,3-dione (3) [10], oleiferinone (4) [11], 4-hydroxy-2-[(3,4-methylenedioxyphenyl) undecanoyl]cyclohexane-1,3-dione (5) [10], 4-hydroxy-2-[(11-phenylundecanoyl)cyclohexane-1,3-dione (6) [12], proctorione C (7) [13], surinone C (8) [14], 5-hydroxy-7,8,4'-trimethoxyflavone (9) [15], 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10) [16], 5-hydroxy-7,3',4'-trimethoxyflavone (11) [17], 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) [15] and cepharanone B (13) [18] (Figure 1).

Figure 1. Structures of compounds 1–13.

Figure 1. Structures of compounds 1–13.

2. Results and Discussion

Compound 1 was assigned the molecular formula C₂₆H₄₄O₃ from the HRESI-MS m/z 427.3185 [M+Na]⁺ peak (calcd for C₂₆H₄₄O₃Na, 427.3188). The IR spectrum exhibited a hydroxyl absorption at 3,466 cm⁻¹. A long alkyl chain was indicated from the multiple-proton signal in the range δ_H 1.26–1.37 in the ¹H-NMR spectrum (Table 1). Two olefinic signals at δ_C 129.8 and 129.9 in the ¹³C-NMR spectrum (Table 1) suggested the presence of a double bond, and its position at C-12' was further confirmed by EI-MS (Figure 2), HMBC and ¹H-¹H COSY spectra (Figure 3). The cis-geometry of a double bond could be deduced from chemical shifts of C-11' (δ_C 27.2) and C-14' (δ_C 26.9) [19]. Two oxymethine [δ_H 4.00 (1H, dddd, 1.7, 5.4, 7.3,11.2, H-2) and δ_H 4.75 (1H, dd, 7.0, 9.3, H-4)] and five methylenes [δ_H 2.22 (1H, ddd, 2.0, 6.7, 13.5, H-3a) and 1.70 (1H, m, H-3b), δ_H 2.33 (1H, dd, 9.6, 16.8, H-6a) and 2.39 (1H, m, H-6b), δ_H 1.97 (1H, dd, J = 6.3, 12.9, H-7a) and 1.96 (1H, dd, J = 6.1, 12.9, H-7b), δ_H 2.40 (2H, m, H-8), δ_H 1.62 (1H, m, H-1'a) and 1.73 (1H, m, H-1'b)] were found to be connected to two moieties, ⁶CH₂–⁷CH₂–⁸CH₂ and O–⁴CH–³CH₂–²CH(O)–^1'CH₂ using the ¹H–¹H COSY spectrum. The above two moieties were linked by the two quaternary olefinic carbons δ_C 173.4 (C-8a) and 114.7 (C-4a) which showed HMBC cross-peaks with H-4, H-7, H-8, and with H-3, H-8, respectively. The location of carbonyl carbon δ_C 200.6 (C-5) could be confirmed by the HMBC correlations with H-6 and H-7. Thus, the moiety were determined as O=⁵C–⁶CH₂–⁷CH₂–⁸CH₂–^8aC=^4aC–⁴CH(O)–³CH₂–²CH(O)–^1'CH₂ and further confirmed by the homoallylic coupling between H-4 and H-8 in the ¹H-¹H COSY spectrum. The downfield chemical shift of C-8a and the remaining two degrees of unsaturation suggested that the oxygen at C-2 connected to C-8a, and C-5 to C-4a. The 5-hydroxytetrahydrochromen moiety was also confirmed by the mass spectrum which showed a fragment at m/z 167 (Figure 2). Thus, the structure of 1 was elucidated as 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one. The NOE correlation between H-2 and H-4 indicated their cis-configuration.

Table 1. ¹H- (400 MHz) and ¹³C-NMR (100 MHz) data of compound 1 (in CDCl₃; δ in ppm, J in Hz).

Position	δC	δH
2	77.1	4.00 (1H, dddd, 1.7, 5.4, 7.3,11.2)
3	35.2	2.22 (1H, ddd,2.0, 6.7, 13.5); 1.70 (1H, m)
4	62.1	4.75 (1H, dd, 7.0, 9.3)
4a	114.7
5	200.6
6	36.7	2.33 (1H, dd, 9.6, 16.8); 2.39 (1H, m)
7	20.5	1.97 (1H, dd, 6.3, 12.9); 1.96 (1H, dd, 6.1, 12.9)
8	28.4	2.40 (2H, m)
8a	173.4
1'	34.7	1.62 (1H, m); 1.73 (1H, m)
2'	25.0	1.46 (2H, m)
3'-10'	29.3–29.8	1.26–1.37 (16H, m)
11'	27.2	2.00 (2H, m)
12', 13'	129.8, 129.9	5.35 (2H, m)
14'	26.9	2.01 (2H, m)
15'	32.0	1.30 (2H, m)
16'	22.4	1.31 (2H, m)
17'	14.0	0.88 (3H, t, 7.0)
OH-4		4.63 ( brs)

Table 1. ¹H- (400 MHz) and ¹³C-NMR (100 MHz) data of compound 1 (in CDCl₃; δ in ppm, J in Hz).

Position	δC	δH
2	77.1	4.00 (1H, dddd, 1.7, 5.4, 7.3,11.2)
3	35.2	2.22 (1H, ddd,2.0, 6.7, 13.5); 1.70 (1H, m)
4	62.1	4.75 (1H, dd, 7.0, 9.3)
4a	114.7
5	200.6
6	36.7	2.33 (1H, dd, 9.6, 16.8); 2.39 (1H, m)
7	20.5	1.97 (1H, dd, 6.3, 12.9); 1.96 (1H, dd, 6.1, 12.9)
8	28.4	2.40 (2H, m)
8a	173.4
1'	34.7	1.62 (1H, m); 1.73 (1H, m)
2'	25.0	1.46 (2H, m)
3'-10'	29.3–29.8	1.26–1.37 (16H, m)
11'	27.2	2.00 (2H, m)
12', 13'	129.8, 129.9	5.35 (2H, m)
14'	26.9	2.01 (2H, m)
15'	32.0	1.30 (2H, m)
16'	22.4	1.31 (2H, m)
17'	14.0	0.88 (3H, t, 7.0)
OH-4		4.63 ( brs)

Figure 2. MS fragmentation of 1.

Figure 2. MS fragmentation of 1.

Figure 3. Key ¹H-¹H COSY and HMBC correlations of 1.

Figure 3. Key ¹H-¹H COSY and HMBC correlations of 1.

Compound 2 had the molecular formula C₂₆H₄₂O₃ as deduced from the HRESI-MS peak at m/z 425.3029 [M+Na]⁺ (calcd for C₂₆H₄₂O₃Na, 425.3032). The UV maxima at 249 nm, and the IR absorptions at 1,661, 1,605 cm⁻¹ suggested the presence of a γ-pyrone ring, which was 2,3,5-trisubstituted according to the olefinic signals at δ_C 112.7 (C-3) and δ_H 6.10 (1H, s, H-3) in the NMR spectra (Table 2) [20]. Similar to compound 1, it showed evidence for one moiety, ⁵CH(O)–⁶CH₂–⁷CH₂–⁸CH₂, and a alkyl chain from the ¹H-NMR, ¹³C-NMR, and ¹H-¹H COSY and HMBC spectra (Figure 4). In the HMBC spectrum, the proton of the oxymethylene δ_H 4.91 (1H, br t, J = 3.8 Hz, H-5) showed correlation with the carbonyl carbon (δ_C 180.7), C-4a (δ_C 123.2), C-6 (δ_C 29.5), C-7 (δ_C 18.1) and C-8 (δ_C 27.6), indicating that C-5 is connected with C-4a. The protons of the methylene [δ_H 2.46 (1H, m, H-8a) and 1.98 (1H, m, H-8b)] gave cross peaks with the carbons at C-5, C-6, C-7, C-4a and C-8a, but not with C-4, suggesting that C-8 is attached to C-8a. Surprisingly, the weak four bond reciprocal H-5/C-8 and H-8/C-5 HMBC correlations signals were observed. Although these may be considered unusual, such long-range correlations have been reported, especially in constrained ring systems [21,22,23]. The HMBC spectrum also suggested the linkage of C-2 and the alkyl chain. Thus, the structure of 2 was determined to be 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8- tetrahydrochromen-4-one.

Table 2. ¹H- (400 MHz) and ¹³C-NMR (100 MHz) data of compound 2 (in CDCl₃; δ in ppm, J in Hz).

Position	δC	δH
2	169.3
3	112.7	6.10 (1H, s)
4	180.7
4a	123.2
5	63.8	4.91 (1H, br t, 3.8)
6	29.5	1.76 (1H, m); 1.96 (1H, m)
7	18.1	1.74 (1H, m); 1.97 (1H, m)
8	27.6	2.49 (1H, m); 2.57 (1H, m)
8a	165.0
1'	33.5	2.48 (2H, t, 7.6)
2'	26.8	1.61 (2H, m)
3'–10'	28.9–29.7	1.26–1.37 (16H, m)
11'	27.1	2.00 (2H, m)
12', 13'	129.8	5.35 (2H, m)
14'	26.9	2.01 (2H, m)
15'	31.9	1.30 (2H, m)
16'	22.3	1.31 (2H, m)
17'	14.0	0.89 (3H, t, 6.4)
5-OH		4.43 ( brs)

Table 2. ¹H- (400 MHz) and ¹³C-NMR (100 MHz) data of compound 2 (in CDCl₃; δ in ppm, J in Hz).

Position	δC	δH
2	169.3
3	112.7	6.10 (1H, s)
4	180.7
4a	123.2
5	63.8	4.91 (1H, br t, 3.8)
6	29.5	1.76 (1H, m); 1.96 (1H, m)
7	18.1	1.74 (1H, m); 1.97 (1H, m)
8	27.6	2.49 (1H, m); 2.57 (1H, m)
8a	165.0
1'	33.5	2.48 (2H, t, 7.6)
2'	26.8	1.61 (2H, m)
3'–10'	28.9–29.7	1.26–1.37 (16H, m)
11'	27.1	2.00 (2H, m)
12', 13'	129.8	5.35 (2H, m)
14'	26.9	2.01 (2H, m)
15'	31.9	1.30 (2H, m)
16'	22.3	1.31 (2H, m)
17'	14.0	0.89 (3H, t, 6.4)
5-OH		4.43 ( brs)

Figure 4. Key ¹H-¹H COSY and HMBC correlations of 2.

Figure 4. Key ¹H-¹H COSY and HMBC correlations of 2.

The structures of known compounds 3~13 were confirmed by detailed NMR and MS data comparison with those in the literature [10,11,12,13,14,15,16,17,18]. In addition, some similar compounds have been reported from Trichoderma [24,25].

In vitro cytotoxicity of all the isolated compounds to HUVEC was examined after 48 h incubation. It was found that compounds 2, 3, 5, 8 could dose dependently induced significant toxicity to HUVEC at different concentrations (Figure 5). The growth of HUVEC was almost completely inhibited by compound 3 at 24 μM. Compounds 5 and 8 also exhibited significant tube formation-inhibiting activity at 3, 6, 12 and 24 μM, respectively (Figure 6).

Figure 5. Effect of compounds 2 (A), 3 (B), 5 (C), 8 (D) on the viability of HUVEC after 48 h incubation. For blank control, the DMSO concentration was adjusted to below 0.1%. Values are expressed as mean ± SD, n = 4–6. * p < 0.05, ** p < 0.01 as compared with control.

Figure 5. Effect of compounds 2 (A), 3 (B), 5 (C), 8 (D) on the viability of HUVEC after 48 h incubation. For blank control, the DMSO concentration was adjusted to below 0.1%. Values are expressed as mean ± SD, n = 4–6. * p < 0.05, ** p < 0.01 as compared with control.

Figure 6. Effect of compounds 5, compound 8, and ursolic acid on HUVEC tube formation. A, B, and C: Tube length (% of control) after treatment with compound 5, compound 8, and ursolic acid (positive control), respectively. Values are expressed as mean ± SD, n = 4. * p < 0.05, ** p < 0.01 as compared with control. (D): The representative photographs of tube networks after treatment with ursolic acid (10 μM) and compounds 5 and 8 at various concentrations. Bar = 200 μm.

Figure 6. Effect of compounds 5, compound 8, and ursolic acid on HUVEC tube formation. A, B, and C: Tube length (% of control) after treatment with compound 5, compound 8, and ursolic acid (positive control), respectively. Values are expressed as mean ± SD, n = 4. * p < 0.05, ** p < 0.01 as compared with control. (D): The representative photographs of tube networks after treatment with ursolic acid (10 μM) and compounds 5 and 8 at various concentrations. Bar = 200 μm.

3. Experimental

3.1. General

Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV-2500 PC spectrophotometer. IR spectra were recorded on a Nexus 670 spectrometer. NMR spectra were measured on a Bruker DRX 400 spectrometer and Bruker AV 500 spectrometer. EI-MS (70 eV) was carried out on an Autospec Premier P708 mass spectrometer. ESI-MS was carried out on a Waters Q-Tof micro YA019 mass spectrometer. Silica gel (200–300 mesh) was used for column chromatography, and pre-coated silica gel GF254 plates (Qingdao Marin Chemical Plant, Qingdao, China) were used for TLC.

3.2. Plant Material

Peperomia dindygulensis Miq., collected in Simao Region Yunnan Province of China in June 2008, was identified by Associate Professor Guo-Hong Yang. A voucher specimen (GHY-PDM20080728) has been deposited in the Herbarium of the Laboratory of Department of Traditional Chinese Medicine, Shanghai Institute of Pharmaceutical Industry, China.

3.3. Extraction and Isolation

The air-dried whole plant (10 kg) of P. dindygulensis was extracted exhaustively with 95% EtOH at r.t. (250 L, 7 days). The EtOH extract was evaporated in vacuo to yield a semisolid (1,300 g), of which 1,290 g was suspended in H₂O (2,000 mL) and partitioned with CHCl₃ (2,000 mL × 5) to yield 756 g of extract after concentration. Part of the CHCl₃ extract (300 g) was subjected to column chromatography on Si gel (200~300 mesh, 3 kg) eluted with petroleum ether/EtOAc (100:0, 99:1, 49:1, 19:1, 9:1, 4:1, 7:3, 3:2, 1:1) and EtOAc to yield ten fractions (Frs. 1–10). Fraction 6 (13.9 g) was separated repeatedly on silica gel columns (5 × 50 cm) using n-hexane/acetone (5:1) as eluent to obtain six fractions (6A–F). Fraction 6B (227 mg) was further purified by Sephadex LH-20 and eluted with CHCl₃/MeOH (1:1) to give 1 (10 mg). Fraction 6D (139 mg) was purified repeatedly by Sephadex LH-20 using CHCl₃/MeOH (1:1) as eluent to give 3 (20 mg). Fraction 7 (21.8 g) was separated repeatedly on silica gel columns (5 × 50 cm) using n-hexane/acetone (4:1) as eluent to obtain five fractions (7A–E). Fraction 7B (1.6 g) was further purified by Sephadex LH-20 [eluted with CHCl₃/MeOH (1:1)] to give four fractions, and the second fraction (700 mg) was applied to preparative silica gel TLC using n-hexane/acetone/acetic acid (5:1:0.1) as eluent to afford 4 (40 mg), 5 (100 mg) and 6 (12 mg). Fraction 7C (7.6 g) was chromatographed over silica gel columns (3 × 50 cm) and eluted with n-hexane/EtOAc (3:1) to obtain five fractions, the second fraction (154 mg) was applied to preparative silica gel TLC using toluene/EtOAc (9:1) as eluent to give 11 (4 mg). Fraction 8 (4.5 g) was chromatographed over silica gel columns (3 × 50 cm) eluted with CHCl₃/MeOH (50:1) to obtain four fractions (8A–D). Fraction 8B (205 mg) was recrystallized from CHCl₃/MeOH (1:1) to afford 9 (51 mg). Fraction 8C (600 mg) was recrystallized from CHCl₃/MeOH (1:1) to afford 10 (246 mg). Fraction 8D (1.4 g) was chromatographed over silica gel columns (3 × 50 cm) and eluted with n-hexane/acetone/acetic acid (100:10:1) to give 2 (277 mg) and 6 (36 mg). Fraction 9 (18.9 g) was separated repeatedly on silica gel columns (5 × 50 cm) using n-hexane/acetone (4:1) as eluent to obtain eight fractions (9A–H). Fraction 9B (1.5 g) was further separated repeatedly on silica columns (3 × 50 cm) and eluted with CHCl₃/acetone (200:1) to give three fractions, and the second fraction was subjected to preparative silica gel TLC using n-hexane/acetone/acetic acid (100:10:1) as eluent to give 8 (20 mg). Fraction 9D (1.8 g) was chromatographed on silica gel columns (3 × 50 cm) using n-hexane/acetone (5:1) as eluent to obtain three fractions, and the last fraction (222 mg) was further purified by Sephadex LH-20 and eluted with CHCl₃/MeOH (1:1) to give 13 (5 mg). Fraction 9E (12.2 g) was separated repeatedly on silica gel columns (5 × 50 cm) using CHCl₃ as eluent to obtain six fractions, and the last fraction was separated repeatedly by Sephadex LH-20 and eluted with CHCl₃/MeOH (1:1) to give 12 (12 mg).

2Z-(Heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1). Yellowish oil; [α]_D¹³ 126° (c = 0.368, CHCl₃); UV λ_max (MeOH) nm (log ε): 258 (4.44); IR (NaCl) ν_max: 3466, 2925, 2854, 1613, 1427, 1370, 1249, 1188, 1083, 1054 cm⁻¹; EI-MS m/z 404 [M]⁺ (9.1), 386 (100.0), 167 (24.1), 139 (50.7), 125 (43.1), 111(41.9), 97 (5.2), 43 (21.5); ¹H- and ¹³C-NMR data: see Table 1; HRESI-MS m/z 427.3185([M+Na]⁺, calcd for C₂₆H₄₄O₃Na 427.3188).

2-(Heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen-4-one (2). Yellowish oil; [α]_D¹³ 34° (c = 0.435, CHCl₃); UV λ_max (MeOH) nm (log ε): 216 (3.66), 249 (3.68); IR(NaCl) ν_max: 3431, 3003, 2926, 2854, 1716, 1661, 1605, 1436, 1176, 1086, 950, 859, 722 cm⁻¹; ¹H- and ¹³C-NMR data: see Table 2; HRESI-MS m/z 425.3029 ([M+Na]⁺, calcd for C₂₆H₄₂O₃Na 425.3032).

3.4. Antiangiogenic Activity Assays

The effect of isolated compounds on the proliferation of HUVEC was evaluated by CCK-8. HUVEC tube formation was conducted for the assay of in vitro angiogenesis using the Chemicon in vitro angiogenesis assay kit (ECM625). Details of the assays were provided in a previous report [9].

4. Conclusions

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1), and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen-4-one (2), were isolated from Peperomia dindygulensis, together with eleven known compounds 3–13. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cells (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.