Quorumolides AâC, Three Cembranoids from Euphorbia antiquorum

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Quorumolides A-C, Three Cembranoids from Euphorbia antiquorum Wei-Yan Qi, Jin-Xin Zhao, Wen-Jun Wei, Kun Gao, and Jian-Min Yue J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02684 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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The Journal of Organic Chemistry

Quorumolides A-C, Three Cembranoids from Euphorbia antiquorum Wei-Yan Qi,† Jin-Xin Zhao,‡ Wen-Jun Wei, † Kun Gao,*,† Jian-Min Yue*,†,‡ †

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and

Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China

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The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT:

Three highly modified cembranoids, quorumolides A−C (1−3), were isolated from Euphorbia antiquorum. Compound 1 is the first example of a cembranoid embedding an α,β-unsaturated-γ-lactone and a tetrahydro-2H-pyran motif within the 14-membered ring. Biosynthetically, it is particularly noteworthy that the stereochemistries of C-2 and C-12 in the pyran ring of 1−3 were opposite to those of marine-derived rare cembranoids.


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Cembranoids represent a big class of 14-membered carbocyclic diterpenoids, which are mainly found in the marine source of coelenterates (the genera Lobophytum, Sinularia, and Sarcophyton),1 and only a few have been isolated hitherto from a very limited number of high plants.1,2 Cembranoids have shown a broad spectrum of biological and pharmacological activities such as cytotoxic, antimicrobial, and anti-inﬂammatory activites.3 The plant genus Euphorbia (Euphorbiaceae) has more than 2000 species globally, and is distributed over both tropical and temperate zones.4 The isolation of cembranoids from this genus has so far been documented only once from E. pekinensis.2c Previous chemical studies on Euphorbia antiquorum have afforded a few diterpenes and triterpenes.5 In continuing our chemical studies on Euphorbia species,6 three highly modified cembranoids, quorumolides A-C (Figure 1) were isolated from the aerial parts of E. antiquorum. We report herein the isolation, structural elucidation, and biological evaluation of these compounds.

Figure 1. Structures of quorumolides A−C (1−3)

Quorumolide A (1) was obtained as colorless crystals. The HRMS (ESI-TOF) displayed a molecular ion at m/z 691.4197 [2M + Na]+ (calcd for C20H30O4Na 691.4186) corresponding to a molecular formula of C20H30O4 with 6 double bond



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equivalents (DBEs). The IR spectrum showed absorption bands (3434 cm−1) and (1739 cm−1) indicative of hydroxy and carbonyl groups, respectively. The 13C NMR Table 1 1H and 13C NMR Data of Compounds 1-3a

1

2

3

δH (mult, J, Hz)

δC

δH (mult, J, Hz)

δC

δH (mult, J, Hz)

δC

1

1.27 m

46.8

1.35 m

46.3

1.25 m

45.7

2

4.48 d (8.0)

70.8

4.58 dd (10.4, 4.5)

70.9

4.48 dd (7.8, 4.0)

70.4

3

5.42 d (8.0)

128.1

5.28 d (10.3)

125.5

5.18 d (8.1)

124.9

4

135.4

5α

2.92 d (14.8)

5β

2.23 dd (14.8, 5.0)

6a

5.25 d (3.4)

39.7

2.26 m

39.0

2.33 m 80.3

6b 7

138.6

2.93 m

8

146.4

6.16 t (8.3)

136.7

9a

2.56 td (13.4, 3.3)

9b

2.42 dt (13.4, 3.6)

10α

1.50 td (13.3, 4.0)

10β

1.66 m

11

3.64 d (10.5)

12

20.3

25.8

147.2

27.5

1.60 m

3.83 d (10.4)

75.1

6.63 dd (11.6, 5.2)

143.0 2.62 td (13.6, 5.6)

34.0

1.60 m

18.2

28.5

1.72 td (14.8, 5.6) 70.1

3.34 d (11.0)

75.0

74.4

13β

1.22 m

14a

1.60 m

14b

1.27 m

15

1.28 m

28.4

1.18 m

28.9

1.24 m

28.6

16

0.89 d (6.0)

20.8

0.87 d (6.5)

20.7

0.88 d (6.1)

20.8

17

0.70 d (6.0)

20.1

0.72 d (6.4)

20.3

0.72 d (6.0)

20.2

18

1.74 d (1.4)

20.4

1.71 s

15.5

1.83 d (1.4)

17.6

175.3

9.48 s

198.2

23.8

1.06 s

23.6

1.28 m 18.4

1.64 m

23.6

1.06 s

2.42 m

33.8

1.20 m 18.6

1.37 m

176.7 1.01 s

33.7

68.5

2.45 d (12.5)

20

2.41 m

158.9

13α

19

33.9

27.8

2.23 m

1.80 td (13.2, 3.4) 68.0

2.55 m

131.7 2.45 m

36.6

2.46 m

2.33 m 34.3

2.18 m 2.27 m

2.64 m 7.02 br s

140.6

1.60 m

18.8

1.25 m

a

Data were collected in CDCl3 at 400 MHz (1H) and 125 MHz (13C). Chemical shifts (δ) are in ppm being

relative to TMS


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(Table 1) of 1 with the aid of DEPT experiments resolved 20 carbon signals comprising four methyls, five methylenes, seven methines (two olefinic), one carbonyl, and three quaternary carbons (two olefinic). Two double bonds and the carbonyl group accounted for three DBEs. Thus, the remaining three degrees of unsaturation required defined 1 as a tricyclic molecule. Its 1H NMR spectrum further revealed that four methyls included one tertiary (δH 1.01, s), one olefinic (δH 1.74, s) and two secondary (δH 0.70, d, J = 6.0 Hz; δH 0.89, d, J = 6.0 Hz) methyls. The aforementioned data, in particular the NMR spectral data and the fact that isolation of cembrane-type diterpenoids from the Euphorbia genus bas been observed suggested that 1 is likely a cembranoid. The planar framework of 1 was established by 2D NMR analysis, especially the 1

H-1H COSY and HMBC spectra (Figure 2). Three spin coupling fragments a (C-1 to

C-3, and C-13 to C-17), b (C-5 to C-7), and c (C-9 to C-11) were lined out as drawn with bold bonds by 1H-1H correlations (Figure 2). The carbon resonances at δC 128.1 (C-3) and 135.4 (C-4) were assignable to a trisubstituted ∆3 double bond, which linked the a and b fragments as indicated by the key HMBC correlations from H3-18 to C-3, C-4 and C-5, and this in turn also allowed the attachment of H3-18 to C-4. The key HMBC correlations from H2-9 to C-7 (δC 146.4), C-8 (δC 136.7) and C-19 (δC 176.7) linked the fragments b and c via the ∆7 double bond, and fixed the C-19 carbonyl at C-8. In addition, the H-6 of an oxygenated methine resonated at δH 5.25 (1H, d, J = 3.4 Hz), indicating that an α,β-unsaturated-γ-lactone was formed between C-6 and C-19. This was confirmed by the key HMBC correlations from H-7 to C-19



and H-6 to C-8. The fragments a and c, and C-20 methyl were attached to C-12 (δC 75.1) of an oxygenated quaternary carbon by the multiple HMBC cross peaks from H3-20 to C-11, C-12, and C-13, and from H-11 to C-12. A key HMBC correlation from H-2 to C-12 revealed the presence of an ether bridge between C-2 and C-12 to form a tetrahydro-2H-pyran motif. Finally, a hydroxy group was assigned as 11-OH by chemical shifts of both H-11 (δH 3.64, d, J = 10.5Hz) and C-11 (δC 68.0). The planar structure of 1 was therefore established.

Figure 2. Key (a) 1H-1H COSY (

), HMBC (H

C), and (b) ROESY correlations of 1

The relative stereochemistry of 1 was deduced partially by a ROESY experiment (Figure 2). The ∆3 double bond was assigned as E by the ROESY cross peak between H-3 and H-5 (δH 2.92), and was supported by the upﬁeld shifted C-18 signal at δC 20.4.7 The ROESY cross-peak of H-1/H-13a and the large coupling constant of H-13a (J = 12.5 Hz) indicated that H-1 and H-13a took the 1,3-axial positions of the chair-conformed pyran ring, and were arbitrarily assigned an α-orientation. Subsequently, the coupling constant between H-1 and H-2 (J1,2 ≈ 0 Hz) and the key ROESY correlation between H-3 and H-11 indicated that the C-2-C-3 bond and the C-12-C-11 bond also adopted 1,3-axial positions in the chair-conformed pyran ring,


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and was definitely in the β-direction. And H-11 was α-configured towards the inside of the macrocyclic, which was supported by the ROESY correlations of H-13α/H3-20 and H-13α/H-11. The ROESY correlation networks, especially the correlations of H-5α/H-6 and H-7/H-11 showed that the five-membered lactone ring was vertically oriented towards the molecular plane of 14-membered carbocyclic diterpenoid, suggesting an α-orientation for H-6. The relative configuration of 1 was thus tentatively assigned, and finally confirmed by single crystal X-ray crystallographic analysis (Figure 3), which allowed the unambiguous assignment of its absolute stereochemistry as 1R,2R,6S,11R, and 12S.

Figure 3. Single-crystal X-ray structure of 1 (ellipsoids shown at the 50% probability level).

Quorumolide B (2), colorless crystals (in MeOH), had the molecular formula of C20H32O4 based on the HRMS (ESI-TOF) ion at m/z 335.2224 [M − H]− (calcd for C20H31O4 335.2222) indicative of five DBEs. The 13C NMR with DEPT experiments revealed the presence of two double bonds and one carbonyl group, which accounted for three of the five DBEs, indicating that 2 is bicyclic compound. Comparison of its NMR data with those of 1, showed that the major differences are the presence of an additional deshielded methylene carbon signal at δC 25.8 in 2 replacing the



oxygenated methine carbon signal at δC 80.3 (C-6) in 1, together with the deshielded H-7 at δH 6.16, suggesting that the α,β-unsaturated-γ-lactone of 1 was converted to a free carboxylic acid in 2, and this was supported by the typical broad IR absorption bands at 3421−2500 and 1673 cm-1.8 The structure of 2 was further confirmed by a combination of 2D NMR analysis, especially the 1H-1H COSY and HMBC spectra. The relative stereochemistry of 2 was fixed by its ROESY spectrum (Supporting Information, Figure S1). The chemical shift of H-7 at δH 6.16 was consistent with a Z-configuration for the ∆7 double bond,8a which was corroborated by a ROESY correlation between H-7 and H2-9. The absolute stereochemistry of 2 was finally determined as 1R,2R,11R, and 12S by an X-ray crystallographic study (Figure 4).

Figure 4. Single-crystal X-ray structure of 2 (ellipsoids shown at the 50% probability level).

Quorumolide C (3), colorless crystals, gave a molecular formula C20H32O3 as determined by HRMS (ESI-TOF) at m/z 365.2334 [M + COOH]− (calcd for C21H33O5 365.2328). The highly similar NMR spectroscopic data (Table 1) of 3 to those of 2 indicated that their structures are closely related. Comprehensive analysis of the NMR data revealed that the only difference was the presence of an aldehyde moiety (δH 9.48, H-19; δC 198.2, C-19) in 3 instead of the carboxylic acid (δC 175.3, C-19) in 2. The


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key HMBC correlation from H-7 to C-19 further proved the location of aldehyde group at C-8. The relative configuration of 3 was assigned to be identical to that of 2 based on their very similar NMR data and ROESY spectrum (Supporting Information, Figure S2). The similar specific rotations of 3 ([α]24 D = −103) and 2 ([α]24 D = −90), the consistent CD curves of 2 and 3 in the range of 200−230 nm (Supporting Information, Figure S3), and in particular biogenetic considerations, indicated that the absolute stereochemistry of 3 was identical to that of 2 as depicted. To evaluate their biological properties, quorumolides A-C were tested in a number of bioassays by following well-recognized protocols, which included inhibition of protein tyrosine phosphatase 1B (PTP1B) and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), as well as tests of cytotoxic activity against A549 (human lung adenocarcinoma) and HL-60 (human premyelocytic leukemia) cell lines, but none of them were active on these assays. In conclusion, compound 1 represents the first cembranoid possessing both an α,β-unsaturated-γ-lactone and a tetrahydro-2H-pyran motif within the 14-membered macro ring. It is particularly interesting that the stereochemistries of C-2 and C-12 forming the ether bridge of the pyran ring in compounds 1−3 are opposite to those of the rare marine-derived cembranoids, isolated from Eunicea sp. and Sinularia rigida.9 The cembranoids, a large group of diterpenoids, are produced mainly by marine organisms as defense tools to protect themselves against the natural predators. While a few of cembranoids have been isolated from a very limited number of higher plants until now.1,2 However, the function of the higher plant originated cembranoids



remains totally unknown, and a deeper research work is therefore warranted.

EXPERIMENTAL SECTION General Experimental Procedures: Melting points were measured on a SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd.). Optical rotations were measured with a Perkin-Elmer 341 polarimeter at room temperature. UV spectra were recorded on a Shimadzu UV-2550 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 IR spectrometer with KBr disks. NMR spectra were obtained on a Bruker AM-400 NMR spectrometer using TMS as internal standard. LRESIMS and HRESIMS were carried out on an Esquire 3000 plus LC-MS instrument and a Bruker Daltonics microTOF-QII mass spectrometer, respectively. Semi-preparative HPLC was carried out on a Waters (515 pump and 2487 photodiode array detector) and a YMC-Pack ODS-A column (250 × 10 mm, S-5 µm, 12 nm). All solvents were of analytical grade (Shanghai Chemical Reagents Company, Ltd.). Silica gel (300-400 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China), C18 reversed-phase silica gel (250 mesh, Merck), and MCI gel (CHP20P, 75-150 µM, Mitsubishi Chemical Industries, Ltd.) were used for column chromatography, and TLC was carried out with GF254 plates (Qingdao Marine Chemical Factory). Plant Materials. The aerial parts of Euphorbia antiquorum were collected in July 2011 from Dongnan County of Guangxi Province, People’s of Republic of China, and the plant was authenticated by Prof. S. Q. Tang, School of Life Sciences, Guangxi Normal University of P. R. China. A voucher specimen has been deposited in


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Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number Euanti-2011-GX-1Y). Extraction, Isolation, and Purification Process. The air-dried powder of E. antiquorum plant material (6.0 kg) was extracted three times with 95% EtOH at room temperature give an EtOH extract (600 g), and the crude extract was partitioned between H2O and EtOAc. The EtOAc-soluble fraction (270 g) was separated on a MCI gel column eluted with MeOH/H2O (3:7 to 9:1, v/v) to produce three fractions A-C. Fraction C (23 g) was separated on a silica gel column and gradient elution with petroleum ether/acetone (from 20:1 to 1:1) to afford three fractions (C1-C3). Fraction C1 was separated on a reversed-phase column (MeOH/H2O, 60:40 to 80:20) to yield two major parts, and each of them was purified on semipreparative HPLC by using (CH3CN/H2O 70:30, 3ml/min) as the mobile phase to give 1 (2.6 mg), 2 (16.1mg), and 3 (9.6 mg). Quorumolide A (1): colorless crystals (MeOH); mp 191－193℃; [α]24

D

+4.3 (c

0.09, MeOH); UV (MeOH) λmax (log ε) 232 (3.33) nm; IR (KBr) νmax 3434, 2919, 1643, 1444 cm−1; for 1H NMR and 13C NMR data, see Table 1; HRMS (ESI-TOF) m/z [2M + Na]+ calcd for C40H60O8Na 691.4186, found 691.4197. Quorumolide B (2): colorless crystals (MeOH); mp 184－185 ℃; [α]24 D −90 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 225 (3.48) nm; IR (KBr) νmax 3421, 2931, 1673, 1635, 1436 cm−1; for 1H NMR and

13

C NMR data, see Table 1; HRMS

(ESI-TOF) m/z [M − H]− calcd for C20H31O4 335.2222, found 335.2224. Quorumolide C (3): colorless crystals (MeOH); mp 142－144 ℃; [α]24


D

−103


(c 0.04, MeOH); UV (MeOH) λmax (log ε) 232 (3.33) nm; IR (KBr) νmax 3502, 2925, 1673, 1670, 1454 cm−1; for 1H NMR and

13

C NMR data, see Table 1; HRMS

(ESI-TOF) m/z [M + COOH]− calcd for C21H33O5 365.2328, found 365.2334. X-ray Crystallographic Analysis of 1 and 2: Colorless crystals of 1 and 2 were obtained from MeOH. Crystal data were obtained on a Bruker APEX-ⅡCCD detector employing graphite monochromated Cu-Kα radiation (λ= 1.54178 Å) at 293(2) K and 140(2) K for 1 and 2, respectively, and operated in the φ-ω scan mode. The structures were solved by direct method using SHELXS-97 (Sheldrick 2008) and refined with full-matrix least-squares calculations of F2 using SHELXL-97 (Sheldrick 2008). All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystallographic data for 1 and 2 have been deposited at the Cambridge Crystallographic Data Centre (Deposition No. CCDC 1576367 and CCDC 1576368 for 1 and 2, respectively). Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ, UK. [Fax: (+44) 1223-336-033; or email: [email protected]]. PTP1B inhibitory assay.10 The PTP1B inhibitory activities of the two compounds were measured using pNPP as a substrate. In brief, 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT) were added into the reaction buffer containing 2 mM pNPP and PTP1B (0.05–0.1 mg) dissolved in 10 % DMSO. Then the PTP1B enzyme was placed in each of 96 wells (final volume 100


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µL) with or without tested compounds. Following incubation at 37 °C for 20 min, the reaction was terminated with the addition of 10M NaOH. The amount of produced p-nitrophenol was estimated by measuring the absorbance at 405 nm. The non-enzymatic hydrolysis of 2 mM pNPP was corrected for by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. SRB assay for A-549 cell Line.11 The growth inhibitory effect of the compounds on the A-549 cell line was measured using the SRB (sulforhodamine B) assay. Briefly, A-549 cells were seeded into 96-well plates (Falcon, CA) and allowed 24 h to adhere. The cells were treated in triplicate with graded concentrations of the tested compounds at 37 °C for 72 h in 5% CO2 in culture incubator, and then fixed with 10% trichloroacetic acid in 4 °C for 1 h. The culture plates were washed and dried before stained with SRB solution (0.4 wt %/vol in 1% acetic acid) for 15 min. After 5 washings using 1% acetum and dried in air, sulforhodamine B was dissolved in 150 µL buffer containing 10 mM Tris-base. The optical density of each well was recorded by plate reader at a wavelength of 515 nm. The results were expressed as IC50 as calculated by the Logit method. MTT assay for HL-60 cell Line.12 Cytotoxic activity of the compounds against the HL-60 cell line was evaluated using the MTT method (microculture tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Briefly, optimal amount of cells in 90 µL of culture medium, which were treated in triplicate with graded concentrations of the tested compounds at 37 °C for 72 h in 5% CO2, were plated in each well of 96-well plates (Falcon, CA). And then a 20 µL aliquot of



MTT solution (5 mg/mL in saline solution) was put directly into the wells. After 4 h incubation, 100 µL of “triplex solution” (10% SDS/ 5% isobutanol /10 mM HCl) was added, and the plates were incubated at 37 °C in 5% CO2 overnight and then measured using a plate reader at 570 nm (VERSA Max, Molecular Devices). 11β-HSD1 inhibitory activity assay.13 Inhibition against human and mouse 11β-HSD1 enzymatic activities was determined via scintillation proximity assay (SPA) using microsomes containing 11β-HSD1, and glycyrrhetinic acid was used as the positive control. The human and mouse 11β-HSD1 enzymes were expressed in HEK293 cells. Briefly, the sequences of human and murine 11β-HSD1 were obtained from the clones provided by NIH Mammalian Gene Collection. The expression plasmids were constructed by inserting the murine 11β-HSD1 sequence into the multiple clone sites of pcDNA3. HEK293 cells were transfected with the expression plasmid and selected by cultivation in presence of 700 µg/mL of G418. The microsomal fraction overexpressing 11β-HSD1 was prepared from the HEK293 cells stably transfected with either human or murine 11β-HSD1, and was used as the enzyme source for SPA. 11β-HSD1 containing microsomes was first incubated with NADPH and [3H] cortisone, then the product, [3H] cortisol, was specifically captured by a monoclonal antibody coupled to protein A-coated SPA beads. The tested compounds’ inhibitory effects on 11β-HSD1 were evaluated by detecting the SPA signal. IC50 values were calculated using Prism 5 (GraphPad Software, San Diego, CA).

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Supporting Information 1

H-1H COSY, HMBC, and ROESY correlations of 2 and 3; crystallographic data

of 1 and 2; 1D and 2D NMR, HRESIMS, and IR spectra of quorumolides A-C (1-3). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (K. Gao); * E-mail: [email protected] (J. M. Yue). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (No. 21532007 & No. 21778027).

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