Bioorganic & Medicinal Chemistry 22 (2014) 6515–6522

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Thiophenes, polyacetylenes and terpenes from the aerial parts of Eclipata prostrata Feng-Min Xi a,b, , Chun-Tong Li a, , Jun Han a, Shi-Shan Yu b, Zhi-Jun Wu a,⇑, Wan-Sheng Chen a,⇑ a

Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, People’s Republic of China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 4 April 2014 Revised 5 June 2014 Accepted 21 June 2014 Available online 28 June 2014 Keywords: Eclipata prostrata Thiophene Polyacetylene Terpenoid glycosides Dipeptidyl peptidase IV Anti-inflammatory

a b s t r a c t One new bithiophenes, 5-(but-3-yne-1,2-diol)-50 -hydroxy-methyl-2,20 -bithiophene (2), two new polyacetylenic glucosides, 3-O-b-D-glucopyranosyloxy-1-hydroxy-4E,6E-tetradecene-8,10,12-triyne (8), (5E)-trideca-1,5-dien-7,9,11-triyne-3,4-diol-4-O-b-D-glucopyranoside (9), six new terpenoid glycosides, rel-(1S,2S,3S,4R,6R)-1,6-epoxy-menthane-2,3-diol-3-O-b- D -glucopyranoside (10), rel-(1S,2S,3S,4R,6R)-3-O-(6-O-caffeoyl-b-D-glucopyranosyl)-1,6-epoxy menthane-2,3-diol (11), (2E,6E)2,6,10-trimethyl-2,6,11-dodecatriene-1,10-diol-1-O-b-D-glucopyranoside (12), 3b,16b,29-trihydroxy oleanane-12-ene-3-O-b- D-glucopyranoside (13), 3,28-di-O-b- D-glucopyranosyl-3b,16b-dihydroxy oleanane-12-ene-28-oleanlic acid (14), 3-O-b-D-glucopyranosyl-(1?2)-b-D-glucopyranosyl oleanlic-18ene acid-28-O-b-D-glucopyranoside (15), along with fifteen known compounds (1, 3–7, and 16–24), were isolated from the aerial parts of Eclipta prostrata. Their structures were established by analysis of the spectroscopic data. The isolated compounds 1–9 were tested for activities against dipeptidyl peptidase IV (DPP-IV), compound 7 showed significant antihyperglycemic activities by inhibitory effects on DPP-IV in human plasma in vitro, with IC50 value of 0.51 lM. Compounds 10–24 were tested in vitro against NF-jB-luc 293 cell line induced by LPS. Compounds 12, 15, 16, 19, 21, and 23 exhibited moderate anti-inflammatory activities. Ó 2014 Published by Elsevier Ltd.

1. Introduction Eclipta prostrata, family Asteraceae, is growing widely in the tropical and subtropical areas of the world. It is distributed throughout China and known as ‘Mohanlian’. The aerial parts of E. prostrata is one significant traditional folk medicine and mainly used as a tonic for enriching the blood.1 Previous phytochemical investigations of the plant have uncovered a number of bioactive compounds, such as triterpenes,2–4 coumestans,5 steroids,6 flavonoids6, thiopenes,7 polyacetylenes,8 alkaloids.9 It has attracted a great deal of attention for their wide range of biological activities, such as anti-inflammatory,10,11 antimicrobial,12 antioxidant,13 antihyperglycemic,14 immunomodulatory,15 hepatoprotective,16 analgesic17 and hair growth promoting properties.18 As part of an ongoing search for bioactive natural products from traditional folk medicine, the aerial parts of E. prostrata were investigated. Twentyfour compounds, including eight mono-, di-, and trithiophene ⇑ Corresponding authors. Tel.: +86 21 81886181; fax: +86 21 33100038 (Z.-J.W.). E-mail addresses: [email protected] (Z.-J. Wu), chenwanshengsmmu@ aliyun.com (W.-S. Chen).   These two authors had contributed equally to this paper. http://dx.doi.org/10.1016/j.bmc.2014.06.051 0968-0896/Ó 2014 Published by Elsevier Ltd.

derivatives (1–7), two polyacetylenic glucosides (8 and 9), and fifteen mono-, sesqui-, and triterpenoid glycosides (10–24), were isolated from E. prostrata. Herein, we report the isolation and structural elucidation of one new bithiophenes (2), two new polyacetylenic glucosides (8 and 9), and six new terpenoid glycosides (10–15) from E. prostrata. Compounds 1–9 were evaluated for antihyperglycemic activities, and compounds 10–24 were tested for anti-inflammatory activities. 2. Results and discussion 2.1. Structure elucidation of new compounds Compound 2 was obtained as brown-yellow oil. The positive HRESIMS showed a pseudo-molecular ion at m/z 303.0124 [M+Na]+ (calcd for C13H12O3S2Na+, 303.0120), consistent with the formula C13H12O3S2, indicating 8 degrees of unsaturation. The 1H and 13C NMR spectra of 2 were similar to those of 5-(3-butene-1ynyl)-50 -hydroxymethyl-2,20 -bithiophene. By comparison of the 1 H NMR spectroscopic data of 2 with that of 5-(3-butene-1ynyl)-50 -hydroxymethyl-2,20 -bithiophene, the obvious differences in 2 were the absence of the terminate double bond protons, and

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the presence of an oxygen-substituted methine proton (dH 4.59, 1H, dd, J = 6.8, 5.0 Hz) and one oxygen-substituted methylene protons (dH 3.70, 2H, ddd, J = 18.0, 11.2, 5.9 Hz), while the chemical shifts and coupling constants of the other protons were quite similar. This was also supported by the HMBC correlations from H-9 (dH 3.70) to C-7 (dC 94.3), from H-8 (dH 4.59) to C-6 (dC 78.8), C-7 (dC 94.3), and from H-4 (dH 7.16) to C-6 (dC 78.8), confirmed the linkage of C-9–C-8–C-7–C-6–C-5. Consequently, the structure of 3 is assigned as 5-(but-3-yne-1,2-diol)-50 -hydroxymethyl-2,20 bithiophene. Compound 8 was isolated as a brownish amorphous powder. The positive HRESIMS showed a pseudo-molecular ion at m/z 399.1418 [M+Na]+ (calcd for C20H24O7Na+, 399.1414), consistent with the formula C20H24O7, indicating the presence of 9 degrees of unsaturation. The IR spectrum showed absorptions due to hydroxy (3371 cm1) and acetylene bond (2218 cm1). The 13C NMR spectrum exhibited 20 carbon resonances, classified into six quaternary carbons, ten methines including six oxygen-substituted carbons, three methylenes including two oxygen-substituted methylenes, and one methyl group. The signals at dC 147.5, 141.6, 128.5, 107.7 were ascribed to two double bonds. By analysis of degrees of unsaturation, six quaternary carbon signals at dC 81.3, 76.3, 76.0, 68.0, 64.1, 58.7 were ascribed to three acetylene bonds. And the signals at dC 102.3, 76.82, 73.7, 70.1, 61.1 were assigned to a glucopyranosyl unit. In the 1H NMR spectrum, the signals at dH 6.93 (1H, dd, J = 15.3, 11.0 Hz), 6.38 (1H, dd, J = 15.4, 11.0 Hz), 6.07 (1H, dd, J = 15.4, 6.1 Hz) and 5.85 (1H, d, J = 15.3 Hz) implied the presence of a conjugated double bond with E–E geometry deduced by coupling constants. The signal at dH 2.06 (3H, s) suggested one methyl was attached to acetylene bond. The 1 H and 13C NMR spectra of 8 were quite similar to that of 3-O-b-Dglucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne.19 A comparison of its spectrum with that of 3-O-b-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne showed that there were one more double bond in 8 instead of two methylenes in 3-Ob-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne. The 1H–1H COSY correlations from H-1 (dH 3.55) to H-2 (dH 1.70), H-2 to H-3 (dH 4.31), H-3 to H-4 (dH 6.07), H-4 to H-5 (dH 6.38), H-5 to H-6 (dH 6.93), H-6 to H-7 (dH 5.85) were observed. HMBC correlations from H-2 (dH 1.70) to C-1 (dC 56.9), C-3 (dC 76.1), from H-3 to C-4 (dC 141.6), C-5 (dC 128.5), from H-7 to C-5, C-6 (dC 147.5), C-8 (dC 58.7), from H-14 (dH 2.06) to C-13 (dC 81.3), C-12 (dC 68.0), C-11 (dC 64.1), C-10 (dC 76.3), C-9 (dC 76.0), C-8 (dC 58.7) established the connectivity of C-1–C-2–C-3–C-4–C5–C-6–C-7 and demonstrated that C-7 and C-14 were connected to triacetylene chain of C-8–C-9–C-10–C-11–C-12–C-13. HMBC correlation from H-10 (dH 4.19, 1H, d, J = 7.9 Hz) to C-3 suggested the glucopyranosyl unit was linked at C-3. Accordingly, compound 8 was elucidated as 3-O-b-D-glucopyranosyloxy-1-hydroxy-4E,6Etetradecene-8,10,12-triyne. Compound 9 was isolated as a brownish amorphous powder. The positive HRESIMS showed a pseudo-molecular ion at m/z 385.1264 [M+Na]+ (calcd for C19H22O7Na+, 385.1258), consistent with the formula C19H22O7, indicating 9 degrees of unsaturation. Detailed comparison of the 1H and 13C NMR data of 9 with those of 8 (see Table 2) indicated they were analogues. The differences between 8 and 9 were that one methylene [dC 37.4 (CH2), C-2] in 8 was absent in 9, meanwhile the oxygen-substituted methylene [dC 56.9 (CH2), C-1] and the double bond [dC 141.6 (CH), C-4; 128.5 (CH), C-5] in 8 were replaced by one methine [dC 74.9 (CH), C-3] and one terminal double bond [dC 117.2 (CH2), C-1; 137.5 (CH), C-2] in 9. In the 1H–1H COSY spectrum of 9, correlations between H-1 and H-2, H-2 and H-3, H-3 and H-4, H-4 and H-5, and H-5 and H-6 were obviously observed. HMBC correlations from H-1 to C-2, C-3, from H-4 to C-5, C-6, from H-6 to C-7, and from H-10 to C-4 were observed, which revealed that the connectivity was as shown in Figure 1. Thus, the structure of 9

Table 1 H (600 MHz) and

1

a

13

C (150 MHz) NMR spectroscopic data of compound 2a

No.

dC

2 3 4 5 6 7 8 9 20 30 40 50 60 70 80

140.3, C 124.3, CH 134.3, CH 122.4, C 78.8, C 94.3, C 64.8, CH 67.1, CH2 137.2, C 125.0, CH 126.9, CH 146.5, C 60.0, CH2

dH (J in Hz) 7.10 (1H, d, 3.8) 7.16 (1H, d, 3.8)

4.59 (1H, dd, 6.8, 5.0) 3.70 (2H, ddd, 18.0, 11.2, 5.9) 7.12 (1H, d, 3.6) 6.95 (1H, d, 3.6) 4.75 (2H, d, 1.0)

Compound 2 was measured in CD3OD.

was characterized as (5E)-trideca-1,5-dien-7,9,11-triyne-3,4-diol4-O-b-D-glucopyranoside. Compound 10 was isolated as a white amorphous powder. The positive HRESIMS showed a pseudo-molecular ion at m/z 371.1690 [M+Na]+ (calcd for C16H28O8Na+, 371.1676), consistent with the formula C16H28O8, indicating 3 degrees of unsaturation. The 13C NMR spectrum exhibited 16 carbon resonances, ascribed to one oxygen-substituted quaternary carbon (dC 57.2), ten methines (dC 104.4, 79.3, 76.82, 73.8, 70.1, 69.4, 60.5, 35.2 and 26.8) including eight oxygen-substituted carbons, two methylenes (dC 61.3 and 24.3) including one oxygen-substituted carbon, and three methyls (dC 22.5, 21.4 and 20.8). The 1H NMR data (Table 3) displayed two methyl doublets (dH 0.83, J = 6.6 Hz and dH 0.85, J = 6.6 Hz), one methyl singlet (dH 1.27) and one anomeric proton (dH 4.13, d, J = 7.8 Hz). These findings suggested that 10 was a menthane-type monoterpenoid glycoside. A comparison of the 1H and 13C NMR data of 10 with the corresponding spectra of rel-(1R,2S,3R,4R)-pmenthane-1,2,3-triol-3-O-b-D-glucopyranoside20 shows a close relation. The only difference consists in the lack of one methylene and the presence of an oxygen-substituted methine signals in the spectra of compound 10. 1H–1H COSY correlations from dH 1.74 (H-8) to dH 0.83 (H-10), 0.85 (H-9), 1.21 (H-4), as well as HMBC correlations from dH 0.85 (H-9) to dC 35.2 (C-4), 26.8 (C-8), 20.8 (C-10), indicated that C-4, C-9 and C-10 were attached to C-8. The connectivity of C-2–C-3–C-4–C-5–C-6 was established by the observation of 1H–1H COSY correlations from dH 3.43 (H-3) to dH 3.99 (H-2), 1.21 (H-4) and from dH 1.85 (H-5) to dH 1.21 (H-4), 3.07 (H-6), as well as HMBC correlations from dH 3.99 (H-2) to dC 79.3 (C-3), 35.2 (C-4) and from dH 3.07 (H-6) to dC 35.2 (C-4), 24.3 (C-5). HMBC correlations from dH 1.27 (H-7) to dC 57.2 (C-1), 69.4 (C-2), 60.5 (C6), indicated that C-2, C-6 and C-7 were tethered to C-1. The hydroxyl proton at dH 4.34 was located at C-2, which was demonstrated by the 1H–1H COSY correlation between dH 4.34 and dH 3.99 (H-2). C-1, C-6 was oxygen-substituted due to the existence of an epoxide triatomic heterocycle, deduced by the chemical shift and degrees of unsaturation. A characteristic doublet signal of an anomeric proton with a large coupling constant (J = 7.8 Hz) at dH 4.13 and six aliphatic carbon signals at dC 104.4 (C-10 ), 76.82 (C-30 , 50 ), 73.8 (C-20 ), 70.1 (C-40 ) and 61.3 (C-60 ) suggested the presence of a b-D-glucopyranosyl unit. HMBC correlation of dH 4.13 (H-10 ) with dC 79.3 (C-3) indicated that glycosidation position was located at C-3. The relative configuration of aglycone was established on the basis of NOESY experiment. NOESY correlations from H-7 to H-2, H-6, from H-3 to H-2, H-4, and from H-4 to H-3, H-6 allowed assignment of 1S,2S,3S,4R,6R relative configuration by comparison with the literature.20 Consequently, the structure of 10 was determined to be rel-(1S,2S,3S,4R,6R)-1,6-epoxy-menthane-2,3-diol-3O-b-D-glucopyranoside.

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F.-M. Xi et al. / Bioorg. Med. Chem. 22 (2014) 6515–6522 Table 2 H (600 MHz) and

1

13

C (150 MHz) NMR spectroscopic data of compounds 8 and 9a

No.

a

8

9

dC

dH (J in Hz)

1

56.9, CH2

2 3 4 5 6 7 8 9 10 11 12 13 14 10 20 30 40 50 60

37.4, CH2 76.1, CH 141.6, CH 128.5, CH 147.5, CH 107.7, CH 58.7, C 64.1, C 68.0, C 76.3, C 76.0, C 81.3, C 4.2, CH3 102.3, CH 73.7, CH 76.8, CH 70.1, CH 76.8, CH 61.1, CH2

3.55 3.44 1.70 4.31 6.07 6.38 6.93 5.85

(1H, (1H, (2H, (1H, (1H, (1H, (1H, (1H,

dd, 10.7, dd, 11.1, m) m) dd, 15.4, dd, 15.4, dd, 15.3, d, 15.3)

2.06 4.19 2.95 3.12 3.04 3.03 3.61 3.41

(3H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

s) d, 7.9) m) d, 4.8) m) m) dd, 11.2, 5.5) m)

5.1) 5.3)

6.1) 11.0) 11.0)

dC

dH (J in Hz)

117.2, CH2

5.36 5.25 5.95 4.32 4.37 6.43 5.94

137.5, CH 74.9, CH 83.6, CH 146.6, CH 111.3, CH 59.4, C 65.1, C 67.8, C 75.4, C 74.9, C 79.5, C 3.9, CH3 104.0, CH 75.3, CH 78.0, CH 71.4, CH 77.9, CH 62.6, CH2

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

dt, 17.3, 1.6) dt, 10.7, 1.6) ddd, 17.3, 10.7, 5.5) ddd, 5.5, 3.5, 1.6) ddd, 5.6, 3.5, 1.6) dd, 16.0, 5.6) d, 16.0)

2.02 (3H, s) 4.46 3.29 3.27 3.36 3.41 3.87 3.71

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 7.8) m) m) m) m) dd, 11.9, 2.2) dd, 11.9, 5.5)

Compound 8 was measured in DMSO-d6, and 9 in CD3OD.

Compound 11 was obtained as a brown amorphous powder. The positive HRESIMS showed a pseudo-molecular ion at m/z 533.1999 [M+Na]+ (calcd for C25H34O11Na+, 533.1993), consistent with the formula C25H34O11, requiring for 9 degrees of unsaturation. The IR spectrum showed absorptions due to hydroxy (3431 cm1), carbonyl (1680 cm1) and benzene ring (1608, 1514 cm1). Detailed analysis the 13C NMR data of 11 (Table 3) with those of 10 indicated that they are closely resembled except for there were more 9 carbon signals appeared in low field region in 11. These 9 carbon signals were sorted into four quaternary carbons (dC 166.5, 148.5, 145.6, 125.5), five methines (dC 145.3, 121.5, 115.8, 114.7, 113.8). These additional signals suggested the existence of one caffeoyl group. And it was verified by a pair of olefinic protons [dH 7.49 (1H, d, J = 15.9 Hz) and 6.25 (1H, d, J = 15.9 Hz)] and one ABX spin system of aromatic ring [dH 6.76 (1H, d, J = 8.2 Hz), 7.01 (1H, dd, J = 8.2, 2.0 Hz) and 7.06 (1H, d, J = 2.0 Hz)] in the 1H NMR spectrum. HMBC correlations from H60 to C-900 (dC 166.5) demonstrated that the caffeoyl group was located at C-60 of glucopyranosyl moiety. Thus, the structure of 11 was established as rel-(1S,2S,3S,4R,6R)-3-O-(6-O-caffeoyl-b-Dglucopyranosyl)-1,6-epoxymenthane-2,3-diol. Compound 12 was isolated as a white amorphous powder. The positive HRESIMS showed a pseudo-molecular ion at m/z 423.2360 [M+Na]+ (calcd for C21H36O7Na+, 423.2353), consistent with the formula C21H36O7, indicating 4 degrees of unsaturation. In the 13C NMR spectrum of 12, 21 carbon resonances were classified into three quaternary carbons, eight methines, seven methylenes and three methyls, including one hexose unit (dC 102.5, 78.1, 77.8, 75.0, 71.7, 62.7). The 1H NMR spectrum exhibited three methyl singlets (dH 1.28, 1.64, 1.72), one anomeric proton (dH 4.28, d, J = 7.9 Hz), three terminal vinyl protons [dH 5.06 (1H, dd, J = 10.8, 1.5 Hz), 5.22 (1H, dd, J = 17.4, 1.5 Hz) and 5.95 (1H, dd, J = 17.4, 10.8 Hz)], and two olefinic protons [dH 5.17 (1H, t, J = 6.8 Hz), 5.49 (1H, t, J = 7.0 Hz)]. By comparison of the 1H and 13C NMR data of 12 (Table 3) with the reported farnesane-type sesquiterpenoid glycoside gaillardoside,21 we found that their data was quite similar and both of them possessed 21 carbons. The obvious differences were the presence of one methylene (dC 27.3) and one oxygensubstituted methylene (dC 75.9), and the absence of one methyl and one oxygen-substituted methine in 12, while chemical shifts

with coupling constants of other protons were quite similar. The HMBC correlation from H-10 (dH 4.28) to C-1 (dC 75.9) confirmed the location of b-D-glucose at C-1 position. The full assignments of the aglycon and sugar signals were carried out by HSQC, 1 H–1H COSY and HMBC experiments. Therefore, 12 was characterized as (2E,6E)-2,6,10-trimethyl-2,6,11-dodecatriene-1,10-diol-1O-b-D-glucopyranoside. Compound 13 was obtained as a white amorphous powder, with the molecular formula C36H60O8, deduced by the HRESIMS pseudo-molecular ion at m/z 638.4629 [M+NH4]+ (calcd for C36H60O8NH+4, 638.4626). The IR spectrum exhibited the presence of hydroxyl (3402 cm1) groups and double bond (1668 cm1). The 1 H and 13C NMR data of 13 (Table 4) indicated an olean-12-enetype triterpenoid glycoside, due to the signals assignable to an aglycon [seven tertiary methyls at dH 0.84 (H-25), 0.992 (H-24, 26), 1.12 (H-28), 1.16 (H-30), 1.29 (H-23), 1.38 (H-27), as well as one olefinic proton at dH 5.33 (t, J = 3.6 Hz, H-12) with two typical olefinic carbon signals at dC 122.7 (C-12) and 144.5 (C-13)] and one b-D-glycopyranosyl moiety [one anomeric proton dH 4.90 (d, J = 7.8 Hz, H-10 ), along with six carbon signals at dC 106.5 (C-10 ), 78.8 (C-30 ), 78.3 (C-50 ), 75.9 (C-20 ), 71.9 (C-4’) and 63.1 (C-60 )]. The 1H and 13C NMR data of the aglycon of 13 were in good accordance with those of maniladiol,22 except for the chemical shift of C29. The only difference was that a hydroxymethyl group (dC 66.1, CH2OH) in 13 substituted for a methyl group (dC 33.1) in maniladiol at position C-29. This was supported by HMBC correlations from to H-29 to C-19, C-20, and C-30. The position of glucopyranosyl moiety in 13 at C-3 was confirmed by HMBC correlation of anomeric proton (dH 4.90, H-10 ) with carbon at dC 89.0. Thus, the structure of 13 was assigned as 3b,16b,29-trihydroxy oleanane-12-ene-3-O-b-D-glucopyranoside. Compound 14 was isolated as a white amorphous powder, with the molecular formula C42H68O14, as referred by the HRESIMS pseudo-molecular ion at m/z 819.4508 [M+Na]+ (calcd for C42H68O14Na+, 819.4501). The IR spectrum showed the presence of hydroxyl group (3394 cm1) and an ester carbonyl group (1736 cm1). The 1H NMR spectrum showed signals for seven tertiary methyl groups [dH 0.86, 0.912, 1.00, 1.10, 1.31 and 1.36], an olefinic proton at dH 5.46 (t, J = 3.2 Hz), and two anomeric protons at dH 4.94 (d, J = 7.8 Hz) and 6.25 (d, J = 8.2 Hz). Its 13C NMR

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F.-M. Xi et al. / Bioorg. Med. Chem. 22 (2014) 6515–6522

HO

HO 4'' HO

1'' 5

3''

2

S

1'

3'

9 5

7

HO

5'

S

2 2'

5'

S

2

1

OH 6'

O

3b 1b

O

O

6' 5'

2' 2

S

5

S

7

O

9

3

1a

5''

3a

S

2'' 5'

HO 5'

HO HO

3

O O

7

9

11

13 14

HO HO

5

1'

3'

OH

OH

5'

O 3

5

3''

HO

1'

3'

HO

1

O O OH

9

5

R

3

5'

O O

5 7

OH1'

3'

9

11

13

9

7

7

HO HO

OH

8

HO

S

OH

1

1

OH

2' 2

S

4 R = CH2OH 5 R = CH2OCH3 6 R = CH2OCH2CH3 7 R = CHO

8

O

7''

1''

9''

HO HO

5''

10

O

HO O 5'

3

O O 1'

3'

OH

13

1

9

OH

5

8

HO HO

5'

O O

14 5

1

9 7

3

11

12

1'

3'

OH

10

11

10

15

HO

12 29

29

30

1 3

11 25 9 5

R1O

26 8

10 6

CH2R5

20 18 22

13

28

14 16

R2

27

R4 R3

24 23

30 29

1

OH HO HO

5'

3'

3

11 25 9 5

O O 1'

OH

26 8

10 6

13

R2 OH OH H H H H H H

R3 H H OH OH OH OH H H

R4 CH3 COO-Glc COO-Glc COOH COO-Glc COO-Glc COO-Glc COO-Glc

R5 OH H H H H H H H

19

1

OH HO OH HO HO HO

5'' 3''

O

5'

3'

3

11 25 9 5

6

O O 1'

O Glc-I

13

20 22

18 17

14

O C O O

28

27

24 23

HO

1''

OH Glc-II

15

5'''

1'''

OH 3'''

OH OH Glc-III

30 29

28

R2 R1

26 8

10

OH

20 18 22

14 16 27

R1 Glc Glc Glc Glc Glc-(1 2)-Glc Glc-2-OSO3H Glc Glc-(1 2)-Glc

13 14 16 17 18 19 22 23

30

20 21

R1 OH H

R2 CH3 CH2OH

1

OH HO HO

24 23

5'

3'

3

11 25 9 5

1'

OH

24

8

10

O O

26

6

20 19 13

18

14 27

21 22

O C 28 O O HO

23

24

5''

1''

OH 3''

OH OH

Figure 1. Structures of compounds 1–24.

spectrum was very similar to that of compound 13, except for there were the presence of one carbonyl and one b-D-glycopyranosyl, and the absence of one hydroxylmethyl in 14. The linkage positions of the carbonyl and the b-D-glycopyranosyl were confirmed by HMBC spectrum. In the HMBC experiment, the correlation between carbonyl carbon (dC 175.8) and H-18 (dH 3.30), H-22 (dH 1.74), carbonyl carbon (dC 175.8) and H-100 (dH 6.25) of the b-D-glycopyranosyl were observed, indicating that the carbonyl carbon (C-28) was linked at the C-17, and the b-D-glycopyranosyl was attached at the carbon C-28. Therefore, the structure of 14 was elucidated as 3,28-di-O-b-D-glucopyranosyl-3b,16b-dihydroxy oleanane-12ene-28-oleanlic acid. Compound 15 was obtained as a white amorphous powder, with the molecular formula C48H78O18, as implied from the HRESIMS pseudo-molecular ion at m/z 965.5067 [M+Na]+ (calcd for C48H78O18Na+, 965.5080). The 1H NMR data of 15 (Table 4) disclosed the presence of seven tertiary methyl groups at dH 0.75 (H-25), 0.92 (H-27), 1.01 (H-30), 1.05 (H-29), 1.07 (H-24), 1.11 (H-26) and 1.26 (H-23), one olefinic proton at dH 5.29 (1H, s, H-19), along with three anomeric protons at dH 4.91 (1H, d, J = 7.6 Hz, H-10 ), 5.37 (1H, d, J = 7.8 Hz, H-100 ) and 6.43 (1H, d, J = 8.2 Hz, H-1000 ). In the 13C NMR spectrum, 30 signals were assigned to the aglycone and the

remaining 18 signals were attributed to three hexose units. And a pair of olefinic carbons appeared at dC 138.3 (C) and 133.1 (CH), different from those in 15, which revealed the double bond was not located at position C-12. HMBC correlations from dC 133.1 to dH 1.33 (H-21), 1.05 (H-29), 1.01 (H-30), from dC 138.3 to dH 2.65 (H-13), 2.30 (H-22), and from dH 5.29 (H-19) to dC 49.0 (C-17), 41.6 (C-13), 33.7 (C-21), 30.9 (C-29) suggested that the double bond was assigned to the position C-18. By comparison the 13C NMR data of aglycone of 15 with those of methylmorolate,23 showed that they had the same olean-18-ene skeleton. The signals of C-3 and C-28 were observed at dC 89.3 and 175.8, respectively, which implied that compound 15 must be a bisdesmosidic triterpene saponin. This was also supported by the HMBC correlations from dH 4.91 (H-10 ) to dC 89.3 (C-3), from dH 6.43 (H-1000 ) to dC 175.8 (C-28). HMBC correlation between dH 5.37 (H-100 ) and dC 83.7 (C-20 ) indicated Glc II was connected to C-20 of Glc I. Hence, the structure of 15 was identified as 3-O-b-D-glucopyranosyl(1?2)-b-D-glucopyranosyl oleanlic-18-ene acid-28-O-b-Dglucopyranoside. Fifteen known compounds, including six thiophene derivatives (1 and 3–7) and nine terpenoid glycosides (16–24), were isolated and identified as 2-(penta-1,3-diynyl)-5-(3,4-dihydroxy-but-1-

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F.-M. Xi et al. / Bioorg. Med. Chem. 22 (2014) 6515–6522 Table 3 H (600 MHz) and

1

13

C (150 MHz) NMR spectroscopic data of compounds 10–12a

No.

10 dH (J in Hz)

dC

a

11

1

57.2, C

2

69.4, CH

3 4 5

79.3, CH 35.2, CH 24.3, CH2

6 7 8 9 10 10 20

60.5, CH 22.5, CH3 26.8, CH 21.4, CH3 20.8, CH3 104.4, CH 73.8, CH

30 40 50 60

76.8, 70.1, 76.8, 61.3,

CH CH CH CH2

No.

dH (J in Hz)

dC 57.0, C

3.99 4.34 3.43 1.21 1.85 1.68 3.07 1.27 1.74 0.85 0.83 4.13 2.91

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (3H, (3H, (1H, (1H,

dd, 7.6, 2.5) d, 7.6, OH) m) m) ddd, 14.2, 4.6, 1.7) ddd, 14.2, 12.2, 1.7) t, 1.7) s) ddd, 13.4, 9.3, 6.5) d, 6.6) d, 6.6) d, 7.8) m)

3.12 3.05 3.10 3.68 3.45

(1H, (1H, (1H, (1H, (1H,

m) dd, 9.2, 4.9) m) dd, 11.4, 1.6) m)

12 dC

dH (J in Hz)

1

75.9, CH2

4.23 (1H, d, 11.4) 4.07 (1H, d, 11.4)

69.0, CH

4.08 (1H, d, 2.0)

2

132.9, C

80.0, CH 34.8, CH 24.5, CH2

3.44 1.21 1.85 1.63 3.07 1.21 1.74 0.85 0.82 4.24 2.96

(1H, (1H, (1H, (1H, (1H, (3H, (1H, (3H, (3H, (1H, (1H,

m) m) ddd, 12.4, 4.8, 1.0) m) t, 1.6) s) ddd, 13.4, 6.6, 2.5) d, 6.7) d, 6.7) d, 7.8) m)

3 4 5

129.9, CH 27.3, CH2 40.3, CH2

6 7 8 9 10 11 12

135.6, C 125.9, CH 23.7, CH2 43.4, CH2 73.8, C 146.3, CH 112.0, CH2

3.18 3.09 3.44 4.47 4.05

(1H, (1H, (1H, (1H, (1H,

m) m) m) dd, 11.8, 1.9) dd, 11.8, 7.5)

13 14 15 10

14.1, CH3 16.0, CH3 27.6, CH3 102.5, CH

20 30 40 50 60

75.0, 78.1, 71.7, 77.8, 62.7,

60.5, CH 22.4, CH3 26.9, CH 21.3, CH3 20.8, CH3 104.3, CH 73.7, CH 76.6, 70.3, 73.7, 63.9,

CH CH CH CH2

100 200 300 400 500

125.5, 114.7, 145.6, 148.5, 115.8,

C CH C C CH

600 700 800 900

121.5, 145.3, 113.8, 166.5,

CH CH CH C

7.06 (1H, d, 2.0)

6.76 (1H, d, 8.2)

CH CH CH CH CH2

5.49 (1H, t, 7.0) 2.17 (2H, m) 2.06 (2H, t, 7.4)

5.17 (1H, t, 6.8) 2.06 (2H, m) 1.55 (2H, t, 6.4) 5.95 5.22 5.06 1.72 1.64 1.28 4.28

(1H, (1H, (1H, (3H, (3H, (3H, (1H,

dd, 17.4, 10.8) dd, 17.4, 1.5) dd, 10.8, 1.5) s) s) s) d, 7.9)

3.24 3.38 3.34 3.26 3.89 3.71

(1H, (1H, (1H, (1H, (1H, (1H,

m) m) m) m) dd, 11.8, 2.1) dd, 11.8, 5.7)

7.01 (1H, dd, 8.2, 2.0) 7.49 (1H, d, 15.9) 6.25 (1H, d, 15.9)

Compounds 10 and 11 were measured in DMSO-d6, and 12 in CD3OD.

ynyl)-thiophene (1),24 50 -isovaleryloxymethyl-5-(4-isovaleryloxybut-1-ynyl)-2,20 -bithiophene (3),25 a-terthienylmethanol (4),26 5-methoxymethyl-2,20 :50 ,200 -terthiophene (5),27 5-ethoxymethyl2,20 :50 ,200 -terthiophene (6),26 a-formylterthienyl (7),28 eclalbasaponin I (16),3 eclalbasaponin II (17),3 eclalbasaponin III (18),3 eclalbasaponin VI (19),3 eclalbasaponin VII (20),4 eclalbasaponin VIII (22),4 silphioside B (22),29 silphioside E (23),30 28-O-b-D-glucopyranosyl betulinic acid 3b-O-b-D-glucopyranoside (24),31 by comparison with literature data. 2.2. Evaluation of biological activity The antihyperglycemic activities of compounds 1–9 were evaluated in vitro by inhibitory effects on DPP-IV in human plasma, with diprotin A (DA) as positive control. As shown in Table 5, all the thiophene derivatives (1–7) were potent DPP-IV inhibitors with IC50 values ranging from 0.51 to 2.81 lM. Compound 6 exhibited the strongest inhibitory activity against DPP-IV with the IC50 value of 0.51 lM. The anti-inflammatory activities of compounds 10–24 were tested in vitro against NF-jB-luc 293 cell line induced by LPS,32 with hydrocortisone as a positive control. As shown in Table 6, the testing compounds 12, 15, 16, 19, 21, and 23 show moderate inhibitory activity. 3. Experimental 3.1. General Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR analyses were performed with a NEXUS 470 FT-IR spectrometer. UV spectra were recorded on Shimadzu UV/VIS-240 recording spectrophotometer. 1D and 2D NMR spectra were

obtained on a Bruker Avance 600 NMR spectrometer. HRESIMS spectra were acquired on an Agilent 6220 ToF LC-MS instrument. GC–MS was conducted on a Thermo Finnigan Trace GC apparatus using an L-Chirasil-Val column (25 m  0.32 mm). Column chromatography (CC) was performed by using silica gel (100–200 mesh), Sephadex LH-20 (40–70 lm), ODS (50 lm). Semi-preparative HPLC isolation was achieved with an Agilent 1200 instrument with a refractive index detector (RID), using a C18 column (250 mm  10 mm  5 lm) and eluting with MeCN–H2O at 2.0 mL/min. Precoated silica gel GF254 and HF254 plates were used for TLC, and Zones were visualized under UV light (254 nm and 365 nm) or by spraying with 10% H2SO4–EtOH followed by heating. 3.2. Plant material The whole plant of E. prostrata was collected from Xianning city, Hubei province (GPS coordinates: N 29°13.4840 , E 113°50.7610 ), during July 2011, and identified by Prof. Lian-Na Sun (School of Pharmacy, Second Military Medical University, Shanghai, China). A voucher specimen (No. 20110828) was deposited at the Department of Pharmacognosy of the Second Military Medical University. 3.3. Extraction and isolation The air-dried aerial parts of E. prostrata (15.0 kg) were extracted three times with 80% EtOH under reflux. After removal of the solvent by evaporation in vacuum, the residue was suspended in water (20 L) and then successively partitioned with petroleum ether, EtOAc, and n-BuOH (3  15 L), respectively. The petroleum ether-soluble part (235.0 g) was fractionated by silica gel CC and eluted with petroleum ether–acetone in increasing polarity (200:1 ? 100:1 ? 50:1 ? 20:1 ? 10:1 ? 5:1 ? 2:1 ? 1:1) to yield four fractions (Fr.1–Fr.4). Fr.1 (70.0 g)

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F.-M. Xi et al. / Bioorg. Med. Chem. 22 (2014) 6515–6522

Table 4 H (600 MHz) and

1

13

C (150 MHz) NMR spectroscopic data of compounds 13–15 in pyridine-d5

No.

13

14

15

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1

39.0, CH2

3 4 5 6

89.0, 39.6, 55.9, 18.6,

m) m) m) m) dd, 11.7, 4.5)

1.44 0.91 2.24 1.81 3.39

(1H, (1H, (1H, (1H, (1H,

m) m) m) m) dd, 11.6, 4.5)

39.4, CH2

26.7, CH2

(1H, (1H, (1H, (1H, (1H,

39.1, CH2

2

1.44 0.90 2.23 1.82 3.37

1.59 0.81 2.22 1.83 3.28

(1H, (1H, (1H, (1H, (1H,

m) m) m) m) dd, 11.9, 4.5)

7

33.1, CH2

0.76 1.46 1.29 1.51 1.32

(1H, (1H, (1H, (1H, (1H,

d, 11.6) m) m m) m)

0.78 1.46 1.29 1.60 1.19

(1H, (1H, (1H, (1H, (1H,

d, 11.5) dd, 11.5, 5.2) m) m) m)

0.65 1.36 1.26 1.61 0.92

(1H, (1H, (1H, (1H, (1H,

d, 10.6) m) m) m) m)

8 9 10 11 12

40.3, C 47.3, CH 23.9, C 36.9, CH2 122.7, CH

13 14 15

144.5, C 44.1, C 36.5, CH2

16

65.2, CH

17 18 19

38.1, C 49.3, CH 42.6, CH2

20 21

36.1, C 30.2, CH2

22

31.2, CH2

23 24 25 26 27 28 29

28.4, 17.2, 15.8, 17.2, 27.5, 22.5, 66.1,

30

28.7, CH3

Glc-I 10 20 30 40 50 60

106.5, CH 75.9, CH 78.8, CH 71.9, CH 78.3, CH 63.1, CH2

Glc-II 100 200 300 400 500 600

CH C CH CH2

CH3 CH3 CH3 CH3 CH3 CH3 CH2

1.56 (1H, dd, 10.0, 7.5) 1.82 (2H, m) 5.33 (1H, t, 3.6)

2.05 (1H, m) 1.63 (1H, m) 4.62 (1H, dd, 10.9, 4.6)

2.42 (1H, m) 1.88 (1H, t, 13.9) 1.65 (1H, m) 1.74 1.65 2.42 1.45 1.29 0.99 0.84 0.99 1.38 1.12 3.91 3.83 1.16

(1H, (1H, (1H, (1H, (3H, (3H, (3H, (3H, (3H, (3H, (1H, (1H, (3H,

m) m) m) m) s) s) s) s) s) s) d, 10.6) d, 10.6) s)

4.90 4.03 4.25 4.18 3.98 4.55 4.36

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 7.8) m) m) m) m) dd, 11.7, 2.5) dd, 11.7, 5.5)

26.7, CH2 89.2, 39.8, 56.2, 18.8,

CH C CH CH2

33.5, CH2 40.2, C 47.5, CH 37.2, C 24.2, CH2 123.2, CH 143.5, C 44.8, C 38.5, CH2 65.1, CH 51.7, C 44.4, CH 46.8, CH2 30.9, C 34.1, CH2 26.9, CH2 28.6, CH3 17.4, CH3 16.0, CH3 17.7, CH3 27.2, CH3 175.8, C 33.4, CH3

1.58 (1H, dd, 10.8, 6.8) 1.92 (2H, m) 5.46 (1H, t, 3.2)

2.31 (1H, t, 12.8) 1.77 (1H, m) 4.58 (1H, br s)

3.30 (1H, dd, 13.9, 4.3) 1.84 (1H, m) 1.29 (1H, m) 1.63 1.19 2.76 1.74 1.31 1.00 0.86 1.10 1.36

(1H, (1H, (1H, (1H, (3H, (3H, (3H, (3H, (3H,

dd, 13.9, 3.3) m) m) dd, 14.1, 4.2) s) s) s) s) s)

27.0, CH2 89.3, 39.9, 56.3, 18.6,

CH C CH CH2

35.2, CH2 43.2, 51.6, 37.3, 21.5, 26.6,

C CH C CH2 CH

41.6, CH 41.3, C 30.1, CH2 34.2, CH2 49.0, C 138.3, C 133.1, CH 32.5, C 33.7, CH2 34.0, CH2

1.23 (1H, m) 1.42 1.62 1.25 2.65

(2H, (1H, (1H, (1H,

m) m) m) m)

2.18 1.22 2.55 1.55

(1H, (1H, (1H, (1H,

m) m) dd, 10.3, 3.2) m)

5.29 (1H, s)

1.70 1.33 2.30 1.72 1.26 1.07 0.75 1.11 0.92

(1H, (1H, (1H, (1H, (3H, (3H, (3H, (3H, (3H,

m) m) m) m) s) s) s) s) s)

0.91 (3H, s)

28.3, CH3 16.9, CH3 17.0, CH3 16.5, CH3 15.5, CH3 175.8, C 30.9, CH3

24.2, CH3

0.91 (3H, s)

29.5, CH3

1.01 (3H, s)

107.2, CH 76.1, CH 79.1, CH 72.2, CH 78.6, CH 63.4, CH2

4.94 4.05 4.25 4.24 4.01 4.59 4.41

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 7.8) m) m) m) m) dd, 11.6, 2.3) dd, 11.6, 5.2)

105.3, CH 83.5, CH 78.6, CH 71.9, CH 78.3, CH 63.1, CH2

4.91 4.24 4.30 4.15 3.92 4.47 3.92

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 7.6) d, 9.1) m) m) m) dd, 10.4, 4.2) dd, 10.4, 5.6)

96.5, 74.4, 78.8, 71.5, 79.9, 62.6,

6.25 4.20 4.31 4.32 4.09 4.49 4.39

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 8.2) m) m) m) m) dd, 11.9, 2.2) dd, 11.9, 4.9)

106.1, CH 77.3, CH 78.5, CH 71.9, CH 78.2, CH 63.0, CH2

5.37 4.12 4.26 4.33 3.92 4.55 4.36

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 7.8) m) m) m) m) m) m)

96.2, 74.5, 79.6, 71.4, 79.2, 62.6,

6.43 4.16 4.02 4.33 4.03 4.43 4.37

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 8.2) m) m) m) m) m) m)

CH CH CH CH CH CH2

Glc-III 1000 2000 3000 4000 5000 6000

was chromatographed on silica gel with petroleum ether–acetone gradient elution (500:1 ? 400:1 ? 300:1) to give compounds 6 (27.8 mg) and 7 (6.0 mg). Fr.3 was subjected to repeated silica gel CC using a step gradient of petroleum ether–acetone

CH CH CH CH CH CH2

1.05 (3H, s)

(100:1 ? 50:1 ? 10:1 ? 3:1) and further purified by Sephadex LH-20 column eluted with CH2Cl2–MeOH (1:1) to afford compounds 3 (4.8 mg), 4 (62.0 mg) and 5 (7.0 mg). Fr.4 (60.0 g) was chromatographed on silica gel column and eluted with increasing

F.-M. Xi et al. / Bioorg. Med. Chem. 22 (2014) 6515–6522 Table 5 Antihyperglycemic activities of compounds 1–9a

a b

Compounda

IC50 (lM)

1 2 3 4 5 6 7 DAb

2.74 2.81 0.75 3.35 2.39 0.51 0.89 2.53

Compounds 8 and 9 were inactive against DPP-IV (IC50 >100 lM). Diprotin A was tested as positive control.

Table 6 Luciferase activity assay of compounds 10–24 Concentration (lg/mL)a

Compound 1

10

The EtOH extract 0.04 ± 0.06 0.03 ± 0.08 n-BuOH-soluble part 0.01 ± 0.07 0.01 ± 0.08 12 0.04 ± 0.10 0.21 ± 0.07* 15 0.01 ± 0.10 0.06 ± 0.11 16 0.08 ± 0.08 0.22 ± 0.08* 19 0.01 ± 0.06 0.16 ± 0.02 21 0.18 ± 0.11 0.24 ± 0.09* 23 0.09 ± 0.08 0.23 ± 0.07* HCb 0.15 ± 0.10 0.32 ± 0.06* a b * **

100

500

0.22 ± 0.07* 0.25 ± 0.09* 0.26 ± 0.11* 0.49 ± 0.08** 0.33 ± 0.05** 0.23 ± 0.05* 0.47 ± 0.07** 0.47 ± 0.06** 0.45 ± 0.05**

0.30 ± 0.11* 0.31 ± 0.07* 0.47 ± 0.07** 0.72 ± 0.07** 0.50 ± 0.05** 0.07 ± 0.07 0.50 ± 0.10** 0.59 ± 0.10** 0.64 ± 0.04**

Compounds 10, 11, 13, 14, 17–18, 20, 22, and 24 were inactive. HC (hydrocortisone) was tested as positive control. p

Thiophenes, polyacetylenes and terpenes from the aerial parts of Eclipata prostrate.

One new bithiophenes, 5-(but-3-yne-1,2-diol)-50-hydroxy-methyl-2,20-bithiophene (2), two new polyacetylenic glucosides, 3-O-b-D-glucopyranosyloxy-1-hy...
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