ZHOU Lifan, CHEN Bao, ZHANG Yahui, ZHANG Xiuli, LI Xin,and WANG Changyun

New Anti-HSV-1 Guaiane Lactone from Hainan Gorgonian

ZHOU Lifan1), 2), CHEN Bao1), 2), ZHANG Yahui1), 2), ZHANG Xiuli1), 2), LI Xin1), 2),and WANG Changyun1), 2), 3), *

1),,,,266003,2),,266237,3),,266003,

A new highly oxygenated guaiane lactone 5--menverin C (1), along with four known compounds including one ses- quiterpene (2) and three steroids (3–5), were isolated from gorgonianwhich were collected from the South China Sea. Through anlyzing the NMR data and comparing with other reported compounds, their structures were determined. The absolute configuration of compound 1 was determined by comparing its experimental ECD with that obtained by calculation. In a bioassay, 5--menverin C (1) displayed antiviral activity against the HSV-1 virus with an inhibition rate of 69.2% (=25μmolL?1).

gorgonian coral;; guaiane lactone; anti-HSV-1

1 Introduction

Gorgonian corals are recognized as the main source of natural products like steroids, acetogenins, prostanoids, di- terpenoids, and sesquiterpenoids (Chung., 2018; Su., 2020; Tin., 2020; Xu., 2020). Until now, many guaiane sesquiterpenoids have been obtained from gorgonians and other marine sources. Gorgonian corals con- tain the majority (60%) of marine-derived guaiane sesqui- terpenoids (Diep., 2015; Lyakhova., 2016), while the remaining guaiane analogs have been mainly obtained from soft corals (Liang and Guo, 2013) and sponges (Hlro- ta., 1998).

To search for novel bioactive metabolites from marine fauna and flora, a batch of gorgonianwas collected from the South China Sea. In our previous study, we reported two new steroids isolated from this gorgonian (Cao., 2012). To discover more bioactive metabolites, our group recently performed further chemical studies on this species. From the organic extract of the title animal, five compounds were isolated, including one highly oxygenated guaiane-type sesquiter- pene lactone, namely, 5--menverin C (1), together with a sesquiterpene (2) and three steroids (3–5).

2 Materials and Methods

2.1 General Methods

Optical rotations were performed using a JASCO P-1020 digital polarimeter. ECD spectra were obtained with a Jas- co J-815-150S circular dichroism spectrometer. IR spec- tra were obtained using a Nicolet-Nexus-470 spectrome- ter. UV spectra were obtained using a Beckman DU 640 spectrophotometer. NMR spectra were obtained using a JEOL Eclips-600 spectrometer at 600 MHz for1H and 150 MHz for13C, with TMS as an internal standard. ESIMS was performed using a Micromass Q-TOF spectrometer. HRESIMS spectra were obtained using a Thermo Scientific LTQ Orbitrap XL spectrometer. Semi-preparative HPLCwas performed on a Waters 1525 system using a semi-pre- parative C18 (Kromasil, 5μm, 10mm×250mm) column coupled with a Waters 2996 photodiode array detector. CCsilica gel (200–300 mesh) was obtained from the QingdaoOcean Chemical Group Co. Analytical TLC was perform-ed using precoated silica gel plates (G60, F-254) from Yan- tai Zifu Chemical Group Co.

2.2 Computational Section

The OPLS_2005 force field was employed for confor- mational searches using the torsional sampling (MCMM) method. At the B3LYP/6-311G(d,p) level, conformers above 1% population were re-optimized with a PCM solvent mo-del for acetonitrile. ECD spectra were obtained by TDDFTcalculations, performed with the same basis set, solventmodel, and function as the energy optimization. The Boltz- mann-averaged ECD spectrum of (1,5,10)-1 was then obtained with SpecDis1.62.

2.3 Animal Material

The gorgonian(GXWZ-25) was col- lected from the Xisha Islands in the South China Sea in September 2018. The gorgonianwas identified by Prof. H. Huang from the South China Sea Ins- titute of Oceanology, Chinese Academy of Sciences. The gorgonian coral was deposited in the Key Laboratory ofMarine Drugs, the Ministry of Education of China, Schoolof Medicine and Pharmacy, Ocean University of China, Qingdao, P. R. China.

2.4 Extraction and Isolation

The frozen specimens (457g, dried weight)were crush- ed mechanically and extracted by EtOAc (5L×2.0L) un- til completion.The EtOAc extract (48.3g)was isolated by CCon silica gel and eluted using petroleum ether (PE)- EtOAc (10%, 20%, 30%, 50%, and 100%) to obtain fivefractions (Fr.1–Fr.5). Through CC over silica gel,Fr.3 wasisolatedwith PE-EtOAc (2:1) to give1 (1.5mg), and Fr.4 was isolated under PE-EtOAc (1:1) to provide 2 (3.2mg). Fraction 5 was elutedusing PE-EtOAc (1:2) to obtaintwo sub-fractions SFr.5-1 and SFr.5-2. SFr.5-1 was subjected to HPLC (MeOH:H2O, 4:1; 2.0mLmin?1) to give 3(13.5mg,R=13.6min), and SFr.5-2 was also purified onHPLC(MeOH:H2O, 3:1; 2.0mLmin?1) to give4(21.9mg,R=10min) and 5(24.7 mg,R=22.3min).

5--menverin C (1): colorless oil; []D25=?34.6 (0.15, MeOH); UV (MeOH):max(log) 276 (2.09)nm; IR (KBr)v: 3749, 2361, 1747, 1651cm?1;1H and13C NMR, Table1; HRESIMS: 263.1281 [M+H]+(calculated for C15H19O4, 263.1278).

2.5 Antiviral Bioassays

The anti-HSV-1 activity of compounds (1–5) was ex- amined using the MTT test (Grela., 2018) to inhibit virus-induced CPE on Vero cells. Acyclovir was used as the positive control, with an HSV-1 virus inhibition rate found to be equal to 94.7% (=25μmolL?1).

3 Results and Discussion

The frozen specimens of gorgonianwere crushed mechanically and extracted by ethyl acetate (EtOAc) until exhaustion. The EtOAc extract was repeat- edly isolated by column chromatography (CC) and high performance liquid chromatography (HPLC) to afford one new guaiane-type sesquiterpene lactone 5--menverin C (1), together with four reported compounds (2–5) (Fig.1). The four known compounds (2–5) were identified as men- verin B (2) (Zhang., 2004), cerevisterol (3) (Qin., 2009), cholesterol (4) (Gao., 2011), and 1,3,5, 6-tetrahydroxycholestane (5) (Parameswaran., 2002) through comparison with related spectroscopic information.

Fig.1 Structures of compounds 1?5 and menverin C.

5-menverin C (1)was determined as C15H18O4through high resolution electrospray ionization mass spectroscopy (HRESIMS), implying seven degrees of unsaturation. Aguaiane-type sesquiterpene lactone framework involving two OH at C-1 and C-10, and an exocyclic olefin moiety group between C-4 and C-14 through the analysis of its NMR spectroscopy (Table 1, Fig.2) was identified. The above-mentioned structural characteristics of compound 1 were strongly similar to those of menverin C (Zhang., 2004). In fact, compound 1 differed from menverin C only in the configuration of its stereogenic center C-5. Two hy- droxyl groups at C-1 (OH-1H4.74) and C-10 (OH-10H5.05) were revealed by1H-NMR spectroscopy recorded in DMSO-6. The presence of the NOESY cross-peaks of OH-1/H-5 and Me-15/H-5 indicated that H-5, OH-1, and Me-15 were-oriented, while OH-10 was-oriented (Fig.2).The absolute stereochemistry of 5--menverin C (1) was confirmed through the TDDFT-ECD calculation (Nugro- ho and Morita, 2014). The ECD spectrum of (1,5,10)-1 was found to be identical to its experimental one, imply- ing that the absolute stereochemistry of 5--menverin C (1) should be 1,5,10(Fig.3). Therefore, compound 1 was the 5-epimer of menverin C.

The anti-HSV-1 activity of compounds (1–5) was ex- amined using the MTT test to inhibit virus-induced CPE on Vero cells. 5--menverin C (1) displayed an- tiviral activity against the HSV-1 virus with an inhibition rate of 69.2% (=25μmolL?1).

Table 1 1DNMR data of 5-epi-menverin Cand menverin C in CDCl3

Fig.2 2D NMR correlations for 5-epi-menverin C.

Fig.3 ECD spectra of 5-epi-menverin C.

4 Conclusions

In the course of our research on novel bioactive natural products from the South China Sea, one new guaiane ses- quiterpenoid 5--menverin C (1) and four reported com- pounds (2–5) were obtained from the gorgonian. The absolute configurations of 5--menve- rin C (1) were successfully confirmed by the TDDFT-ECD calculation. The discovery of antiviral guaiane sesquiter- penoid has added to the complex, diverse, and rapidly ex- panding range of marine terpenoids that exhibit antiviral properties.

Acknowledgements

Our project was supported by the National Key Re- search and Development Program of China (No. 2018YF C0310900), the National Natural Science Foundation of China (No. 41830535), the Fundamental Research Funds for the Central Universities of China (No. 201962002), and the Taishan Scholars Program, China.

Cao, F., Wang, C. Y., and Shao, C. L., 2012. Studies on chemi- cal constituents of gorgonianfrom the South China Sea.. Nanjing, China, 1460-1466 (in Chinese).

Chung, H. M., Chen, N. F., Tseng, C. C., Wu, Y. C., Wen, Z. H., Wang, Y. C.,., 2018. Natural product chemistry of gor- gonian corals of genus-Part III., 16: 339, DOI: 10.3390/md16090339.

Diep, C. N., Berdyshev, D. V., Minh, C. V.,Tu, V. A., Cuong, N. X., Kalinovsky, A. I.,., 2015. Structures and absolute stereochemistry of guaiane sesquiterpenoids from the gorgo- nian., 56: 7001-7004.

Gao, C. H., Wang, P., Luo, X. M., Qi, S. H., and Zhang, S., 2011.

Sterols from the South China Sea gorgonian., 23: 1006-1010, DOI: 10.16333/j.1001-6880.2011.06.002(in Chinese with English abstract).

Grela, E., Grabowiecka, A., and Koz?owskab, J., 2018. Current methodology of MTT assay in bacteria., 120: 303-311, DOI: 10.1016/j.acthis.2018.03.007.

Hlrota, H., Fusetani, N., Yoshimura, E., and Okino, T., 1998. Fivenew antifouling sesquiterpenes from two marine sponges of the genusand the nudibranch., 54: 13971-13980.

Liang, L. F., and Guo, Y. W., 2013. Terpenes from the soft corals of the genus: Chemistry and biological activities.,10 (12): 2161-2196.

Lyakhova, E. G., Kolesnikova, S. A.,Diep, C. N.,Stonika, V. A., Berdyshev, D. N., Dmitrenok, P. S.,.,2016. Guaiane se- squiterpenoids from the gorgonian.,11 (7): 913-916.

Nugroho, A. E., and Morita, H., 2014. Circular dichroism calcu- lation for natural products., 68 (1): 1-10.

Parameswaran, P. S., Hegde, V. R., Govenkar, M., and Naik, C. G., 2002. Secondary metabolites from the gorgonian(Thomson & Simon)., 41B (5): 1093-1096.

Qin, J. C., Laatsch, H., Ma, Y. T.,Bai, M. S.,Yang, S. X., Zhang, Y. M.,., 2009. Polyhydroxylated steroids from an endo- phytic fungus,ZY-22 isolated from., 74: 786-790.

Su, T. P., Sung, P. J., Chen, J. J., Lee, G. H., Wang, Y. C., Hwang, T. L.,., 2020. 11,20-Epoxybriaranes from the gorgo- nian coral(Ellisellidae)., 18: 183, DOI: 10.3390/md18040183.

Tin, N. N., Van, M. C., Hoai, N. N., Xuan, C. N., Van, T. N., Cong, T. D.,., 2020. Briarane-type diterpenoids from the viet- namese gorgonian.,34 (3): 385-389.

Xu, C. C., Zhang, W., Tang, H., Su, L., and Li, J., 2020. Osteo- clastogenesis modulatory steroids from the South China Sea gorgonian coralsp.,17: e2000266, DOI: 10.1002/cbdv.202000266.

Zhang, W., Guo, Y. W., Mollob, E., and Ciminob, G., 2004. Men- verins A-D, new highly oxygenated guaiane lactones from Hainan gorgonian(BRUNDIN)., 87: 2919-2925.

December 13, 2020;

January 25, 2021;

June 8, 2021

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2022

. Tel: 0086-532-82031536

E-mail: changyun@ouc.edu.cn

(Edited by Qiu Yantao)