Cytotoxic Lathyrane-Type Diterpenes from Seeds of Euphorbia lathyris

Abstract

We isolated two new lathyrane-type diterpenes L₂₇ (1) and L₂₈ (2) along with seven known compounds (3–9) from the seeds of Euphorbia lathyris. These compounds were identified by NMR, high-resolution electrospray ionisation (HR-ESI)-MS as well as IR spectroscopy. Compounds 1 and 2 were assigned NMR spectrums with ¹H-NMR, ¹³C-NMR, distortionless enhancement by polarization (DEPT), correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond connectivity (HMBC) and nuclear Overhauser effect spectroscopy (NOESY). Stereo configuration of 1 and 2 were confirmed by comprehensive interpretation of their nuclear Overhauser effect (NOE) relationship and showed they were first natural lathyrane-type diterpenes possessing α-configuration substitutes at C-3. Cytotoxicity assay of isolated compounds were evaluated against breast cancer cell lines MCF-7 or MDA-MB-231, 786-0 and liver cancer cell lines HepG2. As a result, Euphorbia factor L₂₈ (2) showed strongly cytotoxicity to the 786-0 and HepG2 cell lines, with an IC₅₀ value of 9.43 and 13.22 µM, respectively, which preliminarily suggested that the configuration of lathyrane-type diterpene at C-3 has a significant effect on its bioactivity.

The seeds of Euphorbia lathyris (Euphorbiaceae) are a famous Chinese traditional medicine for the treatment of hydropsy, ascites, scabies and snakebites.¹⁾ Over the past few decades, phytochemical and pharmaceutical investigations on this plant have led to numerous structural diverse and bioactivity remarkable diterpenes (Euphorbia factors L₁–L₂₆),^2–17) which are mainly classified to three skeleton types: lathyrane, tigliane and ingenane.¹⁸⁾ Lathyrane-type diterpenes have been transformed to complex functionalized polycyclic structures via Lewis acid-mediated transannular cyclization.^19,20) Tigliane-type diterpenes are important starting materials for the semi-synthesis of anti-Human Immunodeficiency Virus (HIV) drug Prostratin.²¹⁾ While ingenane-type diterpene proved to have a potent of anticancer activity, anti-HIV activity; ingenol mebutate has been approved by Food and Drug Administration (FDA) for the treatment of Actinic Keratosis.^22,23) The unique skeletons of these diterpenes also aim to challenge the total synthesis field. Based on the prominent bioactivity, extraction and isolation, structural modification, total (semi) synthesis, biological evaluation and mechanism insight of these complex natural diterpenes are still a hot topic in the drug discovery fields.²⁴⁾

In our continuing investigations on the natural bioactive compounds, we are interested in the structural unique diterpenes from the seeds of E. lathyris, which led to the obtaining of nine diterpenes. Herein, we presented the extraction, isolation, structural elucidation and cytotoxic evaluation of two new lathyrane-type diterpenes, Euphorbia factors L₂₇ (1) and L₂₈ (2), as well as seven known ones: Euphorbia factors L₉ (3),³⁾ L₂ (4),⁹⁾ L₈ (5),³⁾ L₃ (6),⁹⁾ L_7a (7),⁴⁾ L₁ (8),⁸⁾ and L₂₆ (9).⁷⁾ It is notable that Euphorbia factor 1 and 2 are the first natural occurring lathyrane-type diterpenes with a α-configuration ester group at C-3 and exhibit remarkable cytotoxicity (Fig. 1).

Fig. 1. Structures of Compounds 1–9

Results and Discussion

Compound 1 was obtained as a white amorphous powder with molecular formula of C₃₇H₄₁NO₉ based on the protonated molecular ion at m/z 644.2756 [M+H]⁺ (Calcd 644.2860) in its positive-ion high-resolution electrospray ionization (HR-ESI)-MS and its ¹³C-NMR data. The typical absorption bands at 1723 and 1600 cm⁻¹, as well as 1655 cm⁻¹ in the IR spectrum suggested the existence of carbonyl and aromatic ring functionalities. The ¹H- and ¹³C-NMR spectra of 1 indicated the occurrence of a benzoyl group [δ_H 7.92 (2H, d, J=7.2 Hz, H-3′ and H-7′), 7.51 (1H, t, J=7.2 Hz, H-5′), δ_H 7.36 (2H, t, J=7.8 Hz, H-4′ and H-6′); δ_C 164.9 s, 133.3 s, 130.2 d, 129.7 d, 128.5 d], one nicotinoyl motif [δ_H 9.26 (1H, s, H-3″), 8.81 (1H, s, H-4″), 7.43 (1H, m, H-5″), 8.29 (1H, d, J=7.8 Hz, H-6″); δ_C 165.7 s, 126.3 s, 151.2 d, 153.7 d, 123.5 d, 137.2 d] and two acetyl groups [δ_H 1.28 (3H, s); δ_C 169.5 s, 21.2 q and δ_H 2.24 (3H, s); δ_C 169.9 s, 22.1 q]. The remarkable up-field shifted of acetyl methyl from 2.24 to 1.28 suggested that one acetyl groups was closed to the benzoyl group or (and) nicotinoyl motif in apace and fell into their shielding filed. The ¹H-NMR spectrum of 1 displayed two proton single peaks at δ_H 5.54 and 5.21, as well as one proton doublet at δ_H 6.52 (J=10.8 Hz), characteristic of olefin H-17 and H-12 of typical lathyrane-type diterpene. ¹³C-NMR resonances for two exocyclic terminal sp² carbons at δ_C 141.7 and 120.5, along with two endocyclic olefinic carbons at δ_C 143.4 and 135.5 further indicated the existence of two double bonds in the structure of 1. Additionally, the presence of four methyl signals at δ_H 1.79 (3H, s), 1.26 (3H, s), 1.20 (3H, s), 0.94 (3H, d, J=5.4 Hz) in ¹H-NMR spectrum also supported the lathyrane-type diterpene skeleton of 1. The heteronuclear multiple bond connectivity (HMBC) corrections observed for the benzoyl’s carbonyl signal at δ_C 164.9 to a typical proton at δ_H 5.79 in the lathyrane skeleton assigned its location at C-3. Similarly, the HMBC corrections of H-5 (δ_H 6.36, 1H, d, J=8.4 Hz) to the carbonyl carbon signal of acetyl at δ_C 169.5 and H-7 (δ_H 5.55, 1H, d, J=1.8 Hz) to the its vicinal carbonyl carbon of nicotinoyl at δ_C 165.7 suggested the presence of 5-OAc and 7-O-nicotinayl moiety, respectively (Fig. 2). The second acetoxyl group and the ketone also could be assigned at C-15 and C-14 via comprehensive analysis of the heteronuclear multiple quantum coherence (HMQC) and HMBC corrections in two dimensional (2D)-NMR spectrum of 1. Therefore, the structures of 1 could be deduced as 5,15-diacetoxyl-3-benzoyloxy-7-nicotinoyloxylathyol, which suggested that compound 1 possessed the same planar structure as that of Euphorbia factor 3.⁷⁾ A careful comparison of the ¹H- and ¹³C-NMR data of 1 with those of 3 revealed the almost identical NMR data except for the ¹³C-NMR data of C-3: δ_C at 80.6 for C-3 of 1 had a down field shift than C-3 (δ_C 79.5) of Euphorbia factor 3, which suggested the different substituted configuration at C-3 between these two compounds. Then ¹H–¹H correlation spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectrums were analyzed carefully and the α-orientation of benzoxyloxy at C-3 was confirmed based on key NOESY correction of H-2α with H-4α and the absence of nuclear Overhauser effect (NOE) cross peaks between H-4α and H-3, H-1β and H-3β (Fig. 3). The configurations of 5-OAc, 15-OAc and 7-nicotinoyloxy in 1 were also can be determined by its NOESY corrections and comparison with 3. Consequently, the structure of 1 was identified as 5α,15β-diacetoxy-3α-benzoyloxy-7β-nicotinoyloxylathyol. All the NMR data of 1 were also been assigned (Table 1) on the basis of 2D-NMR (¹H–¹H COSY, HMQC, HMBC, NOESY) observation distinctly.

Fig. 2. ¹H–¹H COSY (bold), Key HMBC of 1 and 2

Fig. 3. Key NOSEY Correlations of Compounds 1 and 2

Table 1. ¹H- and ¹³C-NMR Spectroscopic Data of Compounds 1 and 2 in CDCl₃

No.	1		2		No.	1		2
	H	C	H	C		H	C	H	C
1α	3.46 m	48.3	2.87 m	47.5	19	1.26 s	16.9	1.22 s	15.6
1β	1.75 m		1.81 m		20	1.79 s	12.8	2.15 s	14.1
2	2.38 m	37.6	2.55 m	37.3	3-Benzoyl
3	5.79 t (3.0)	80.6	5.83 t (3.0)	77.2	1′		164.9		164.8
4	2.96 dd (3.0)	53.2	2.91 t (3.6)	52.0	2′		133.3		133.4
5	6.36 d (8.4)	64.1	5.45 d (3.6)	69.5	3′,7′	7.92 d (7.2)	129.7	8.06 d (7.2)	129.8
6		141.7		143.9	4′,6′	7.36 t (7.8)	128.5	7.46 t (7.8)	128.6
7	5.55 d (1.8)	78.9	5.26 br s	73.8	5′	7.51 t (7.2)	130.2	7.58 t (7.8)	130.1
8α	2.22 br s	28.9	2.05 m	30.2	5-Acetoxy
8β	1.22 br s		1.69 t (13.8)		OCOCH3		169.5		169.6
9	1.36 m	31.7	1.42 m	32.0	OCOCH3	1.28 s	21.2	1.32 s	20.8
10		24.9		24.3	7-Nicotinoyl
11	1.51 m	27.9	1.48 t (9.6)	26.3	1″		165.7		166.4
12	6.52 d (10.8)	143.4	5.95 d (10.2)	139.9	2″		126.3		126.6
13		135.5		138.4	3″	9.26 br s	151.2	9.35 brs	151.7
14		197.4		207.3	4″	8.81 br s	153.7	8.78 brs	153.5
15		92.1		85.5	5″	7.43 m	123.5	7.40 m	123.5
16	0.94 d (5.4)	14.3	1.08 d (6.6)	14.6	6″	8.29 d (7.8)	137.2	8.42 d (7.8)	137.6
17α	5.54 s	120.5	5.18 s	113.5	15-Acetoxy
17β	5.21 s		5.25 s		OCOCH3		169.9		—
18	1.20 s	28.8	1.14 s	28.5	OCOCH3	2.24 s	22.1		—

* Means overlapped.

Compound 2, a white amorphous powder with [α]_D²⁰−25.3 (c=0.10, CHCl₃), gave the molecular formula C₃₅H₃₉NO₈ based on the HR-ESI-MS ion at m/z 602.2750 [M+H]⁺ (Calcd for C₃₅H₄₀NO₈, 602.2754) and 35 carbon signals in ¹³C-NMR spectrum. The ¹H- and ¹³C-NMR spectroscopic data (Table 1) revealed the occurrence of one benzoyloxy motif, one nicotinoyloxy group and one acetoxy substitute in 2. The presence of two carbon–carbon double bonds signals [δ_H 5.18 (1H, s), 5.25 (1H, s), 5.95 (1H, d, J=10.2 Hz); δ_C 143.9 s, 139.9 d, 138.4 s, 113.5 t] and four methyl resonances [δ_H 1.08 (3H, d, J=6.6 Hz), 1.14 (3H, s), 1.22 (3H, s), 2.15 (3H, s)] in NMR spectrum supported the same lathyrane-type diterpene skeleton of 2. The NMR data for 2 were similar to those for 1 except that an acetoxy group in 1 at C-15 was replaced by a hydroxyl in 2, which was further confirmed by the observation of upfield shift of C-15 (from δ_C 92.1 in 1 to δ_C 85.5 in 2) and HMBC corrections of H-2 and H-4 with C-15. In the same way, the locations of acetoxy, benzoyloxy and nicotinyloxy were assigned to C-5, C-3 and C-7, respectively via analysis of HMBC corrections (Fig. 2). NOE experiments showed that 2 adopt as the same configuration as 1 (Fig. 3). Hence, compound 2 was difined as 15β-hydroxy-5α-acetoxy-3α-benzoyloxy-7β-nicotinoyloxylathyol.

The known compounds were identified by comparing their spectroscopic data with that reported in the literatures as Euphorbia factors L₉ (3),³⁾ L₂ (4),⁹⁾ L₈ (5),³⁾ L₃ (6),⁹⁾ L_7a (7),⁴⁾ L₁ (8),⁸⁾ and L₂₆ (9)⁷⁾ (Fig. 1).

These compounds were evaluated for their cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against MCF-7, MDA-MB-468, 786-0 and HepG2 (Fig. 4). Among them, 2 exhibited the highest cytotoxic activities with IC₅₀ value of 9.43 µM against MCF-7 and 13.22 µM against HepG2 (Table 2), which suggested that the configurations and variety of substitutes at lathyrane-type diterpene impact its bioactivity significantly. From current bioassay results, we can draw that esterification at C-3 with α-configuration and substituting with heterocyclic ester group would improve the cytotoxicity of lathyrane-type diterpene. Of course, more and deep bioassay should be accomplished to reveal the comprehensive structure–activity relationship of these diterpenes.

Fig. 4. Antiprolifelative Activities against Human Cancer Cell Lines Treated with Compounds 1–9 at 50 µM for 24 h

Table 2. Cytotoxicity of Compounds 1, 2, 5 and 7 against MCF-7 and HepG2

Compounds	Cytotoxicity (IC50, µM)
	MCF-7	HepG2
1	>50	>50
2	9.43	13.22
5	>50	>50
7	>50	>50
DDP (cisplatin)*	16.55	2.29

* DDP as positive control.

Conclusions

In summary, two new lathyrane-type diterpenes Euphorbia factor 1 and 2, together with seven known compounds (3–9) were isolated from a famous Traditional Chinese Medicine, the seeds of Euphorbia lathyris, also known as QianJinZi in China. Euphorbia factor 1 and 2 were firstly natural occurring lathyrane diterpenes with α-orientation substitutes at C-3. Cytotoxicity assay treated test compounds showed that 2 exhibited stronger cytotoxicity against MCF-7 cell lines than classic anticancer drug cisplatin, which suggested that the configuration and variety of substituent groups in lathyrane-type diterpene are of great importance for the bioactivity. Current results also enlightened us that: 1) Deeply phytochemical investigation on Euphorbia lathyris may still lead to new diterpenes with promising bioactivities; 2) In the field of natural medicine chemistry, structural modification of lathyrane should include not only the change of function groups, but also the inversion of configuration at specific position.

Experimental

General Experimental Procedures

Optical rotations were measured in CHCl₃ using a PerkinElmer, Inc., U.S.A. polarimeter with a sodium lamp operating at 598 nm and 20°C. IR spectra were obtained using a Thermo Fisher Nicolet 6700 spectrometer. HR-ESI-MS data were measured using a Q-TOF micro mass spectrometer (Waters, U.S.A.). NMR spectra were recorded on a Bruker AV 600M spectrometer. Silica gel (Qingdao Haiyang Chemical Co., Ltd., China, 200–300 mesh), RP-18 silica gel (Merck, 40–60 mm) and Sephadex LH-20 (Pharmacia Co., U.S.A.) were used for column chromatography (CC).

Plant Material

The seeds of E. lathyris were purchased from Yuzhou, Henan Province, in November 2016, China. Voucher specimens have been deposited in the School of life Science and Engineering at Southwest Jiaotong University.

Extraction and Isolation

The air-dried and powdered seeds (10 kg) were extracted exhaustively with EtOH (5×60 L) at room temperature for 5 d. The EtOH extract was concentrated in vacuum to generate a crude residue (800 g), which was partitioned successively with petroleum ether and MeCN (each three times). The MeCN portion (360 g) was separated by a silica gel CC eluted with petroleum ether–EtOAc (from 100 : 0 to 0 : 100) to yield 8 fractions (Frs. A1–A8). Franction A2 (122 g) was purified by crystallizing from petroleum ether–acetone to obtain compounds 6 (55.8 g), 4 (20.5 g) and 8 (36.4 g). Franction A3 (14.7 g) was loaded onto a silica gel column and eluted with petroleum ether–EtOAc (20 : 1 to 7 : 1) to give compound 5 (6.5 g). Franction A5 (0.85 g) was isolated on the same condition and eluted with a gradient of petroleum ether–EtOAc (6.5 : 1 to 3 : 1), to produce compounds 3 (45 mg), 1 (2.8 mg) and 2 (4.5 mg), respectively. Franction A6 (50 mg) was subjected on a silica gel column and eluted with petroleum ether–EtOAc (3 : 1 to 1 : 1) to yield compounds 7 (42 mg) and 9 (15 mg).

Euphorbia Factor L₂₇ (1)

5α,15β-Diacetoxy-3α-benzoyloxy-7β-nicotinoyloxylathyol

White amorphous powder; [α]_D²⁰−12.5 (c=0.10, CHCl₃); IR (KBr) ν_max 3519, 3446, 3358, 2924, 2854, 1723, 1655, 1600, 1458, 1371, 1282, 1105, 1022, 799, 713, 546 and 448 cm⁻¹; ¹H- and ¹³C-NMR data see Table 1; HR-ESI-MS data m/z 644.2856 [M+H]⁺ (Calcd for C₃₇H₄₂NO₉, 644.2860)

Euphorbia Factor L₂₈ (2)

15β-Hydroxy-5α-acetoxy-3α-benzoyloxy-7β-nicotinoyloxylathyol

White amorphous powder; [α]_D²⁰−25.3 (c=0.10, CHCl₃); IR (KBr) ν_max 3610, 3523, 3443, 3332, 3203, 2925, 2854, 1723, 1671, 1629, 1595, 1456, 1374, 1284, 1110, 1069, 1026, 932, 741, 715, 539 and 455 cm⁻¹; ¹H- and ¹³C-NMR data see Tables 1; HR-ESI-MS data m/z 602.2750 [M+H]⁺ (Calcd for C₃₅H₄₀NO₈, 602.2754)

Cytotoxicity Assay

The MCF-7, MDA-MB-468, 786-0 and HepG2 were obtained from Peking Union Medical College, Beijing, China. The cells were cultured in Dulbecco’s Modified Eagle’s Medium–High (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin, and 0.03% L-glutamine and maintained at 37°C with 5% CO₂ in a humidified atmosphere. Cisplatin (Pt-amount 65%, Shanghai Aladdin Co., Ltd., China) was used as positive control.

The cytotoxicity assay of compounds 1–9 isolated from the seeds of E. Lathyris was performed via the MTT method using four kinds of breast cancer cells MCF-7 and MDA-MB-468, human kidney cells 786-0 and liver cancer cells HepG2. Cells were cultured at a density of 5×10³ cells/mL in 96-well microtiter plate and supplemented with 10% FBS, incubated for 24 h.

If the cells are still attached, the compound solution was added with concentration gradient (1.56, 3.13, 6.25, 12.50, 25, 50, 100, 200, and 400 µM) of 100 µL in every well. After 24 h of treatment, 20 µL of 5 mg/mL MTT solution was added to each well, and further incubated for 3h in the dark. The cells in each well were then solubilized with dimethyl sulfoxide (DMSO) and the optical density was recorded at 490 nm.

Acknowledgments

This work was supported by Grants from National Nature Science Foundation of China (NSFC) (31570341 and 81773605), the Educational Commission of Sichuan Province (15TD0048) and the Interdisciplinary Frontier Basic Research Project of Southwest Jiaotong University (SWJTU) (2682017QY04).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Medica CAoSaIoM, Institute of Botanica., 44, 69 (1997).
• 2) Adolf W., Hecker E., Z. Krebsforsch. Klin. Onkol. Cancer Res. Clin. Oncol., 84, 325–344 (1975).
• 3) Itokawa H., Ichihara Y., Yahagi M., Watanabe K., Takeya K., Phytochemistry, 29, 2025–2026 (1990).
• 4) Adolf W., Hecker E., Becker H., Planta Med., 50, 259–261 (1984).
• 5) Appendino G., Della Porta C., Conseil G., Sterner O., Mercalli E., Dumontet C., Di Pietro A., J. Nat. Prod., 66, 140–142 (2003).
• 6) Liao S. G., Zhan Z. J., Yang S. P., Yue J. M., Org. Lett., 7, 1379–1382 (2005).
• 7) Seip E. H., Hecker E., Phytochemistry, 22, 1791–1795 (1983).
• 8) Appendino G., Belloro E., Tron G. C., Jakupovic J., Ballero M., J. Nat. Prod., 62, 1399–1404 (1999).
• 9) Appendino G., Tron G. C., Cravotto G., Palmisano G., Jakupovic J., J. Nat. Prod., 62, 76–79 (1999).
• 10) Jiao W., Yu K.-B., Lu R.-H., Chinese Journal of Structural Chemistry, 27, 1431–1434 (2008).
• 11) Duarte N., Gyemant N., Abreu P. M., Molnar J., Ferreira M. J. U., Planta Med., 72, 162–168 (2006).
• 12) Park S.-J., Park J. H., Han A., Davaatseren M., Kim H. J., Kim M.-S., Hur H. J., Sung M.-J., Hwang J.-T., Yang H. J., Kwon D. Y., Cell Biochem. Funct., 33, 220–225 (2015).
• 13) Fortin J.-L., Bitar M.-P., Clin. Toxicol., 52, 384–385 (2014).
• 14) Escrig P. V., Iglesias D. J., Corma A., Primo J., Primo-Millo E., Cabedo N., J. Agric. Food Chem., 61, 10096–10109 (2013).
• 15) Meng X., Zhao X., Long Z., Yuan Y., Zhuang H., Bi K., Chen X., J. Pharm. Biomed. Anal., 72, 299–305 (2013).
• 16) Choi J. S., Kang N. S., Min Y. K., Kim S. H., Phytother. Res., 24, 1042–1046 (2010).
• 17) Zhu H.-Y., Meng X.-Y., Bao Y.-L., Yu C.-L., Wu Y., Li Y.-X., Chromatographia, 71, 617–622 (2010).
• 18) Vasas A., Hohmann J., Chem. Rev., 114, 8579–8612 (2014).
• 19) Appendino G., Tron G. C., Jarevang T., Sterner O., Org. Lett., 3, 1609–1612 (2001).
• 20) Jiao W., Wan Z., Chen S., Lu R., Chen X., Fang D., Wang J., Pu S., Huang X., Gao H., Shao H., J. Med. Chem., 58, 3720–3738 (2015).
• 21) Wender P. A., Kee J. M., Warrington J. M., Science, 320, 649–652 (2008).
• 22) Nothias-Scaglia L.-F., Pannecouque C., Renucci F., Delang L., Neyts J., Roussi F., Costa J., Leyssen P., Litaudon M., Paolini J., J. Nat. Prod., 78, 1277–1283 (2015).
• 23) Lebwohl M., Swanson N., Anderson L. L., Melgaard A., Xu Z., Berman B., N. Engl. J. Med., 366, 1010–1019 (2012).
• 24) Rodrigues T., Reker D., Schneider P., Schneider G., Nat. Chem., 8, 531–541 (2016).