Five new ent-kaurane diterpenes from Annona squamosa L. pericarps

Abstract In the present study, five new ent-kaurane diterpenes including 4α-hydroxy-17,19-dinor-ent-kaurane-16-one (1), 4β-hydroxy-16β-H-18-nor-ent-kaurane-17-oic acid (2), 4β,17-dihydroxy-16α-acetoxy-18-nor-ent-kaurane (3), Annosquamosin Z (4) and 16α-H-ent-kaurane-17,18-dioic acid, 17-methy ester (5) were isolated from Annona squamosa L. pericarp. The compounds were also evaluated for their cytotoxic activities against SMMC-7721 and HepG2 cell lines, among which compound 3 exhibited potent cytotoxicity with IC50 value of less than 20 μM. Graphical Abstract


Introduction
Custard apple (A. squamosa Linn.), one of Annona species, is a famous and popular fruitage. It originates in the Americas and plants in tropics worldwide now (Gao et al. 2003). The fruit was previously used for the treatment of ''malignant sore'' (cancer) (Ma et al. 2017;Miao et al. 2016;Thang et al. 2013  . Previous studies concerning the bioactivity-directed isolation afforded important ingredients as ent-kaurane diterpenes Joy and Remani 2008), which had various viabilities, especially anti-tumor activity (Li et al. 2015;Mongelli et al. 2002;Hong et al. 2011;Wang et al. 2011). The active kaurane diterpenes were the source of potential anti-tumor agents.

Results and discussion
Compound 1 was isolated as the colorless needle (CHCl 3 ), m.p. 175-176 C. The chemical formula of 1 was determined to as C 18 (Su et al. 1994). The presence of a methyl group at C-10 was suggested by the HMBC spectrum of compound 1, in which the H-20 signal at d H 1.21 coupled to C-1 at d C 39.85, C-9 at d C 47.83 and C-10 d C 39.19. Besides, another methyl group and a hydroxyl group were suggested at C-4 by its HMBC spectrum in Figure S1. The carbonyl group was placed   Figure S1, a correlation between H-3 (d H 1.35 and d H 1.75) and H-18 (d H 1.17) confirmed the b-configuration of H-18. In addition, we the successfully obtained a single X-ray crystal structure with structure Flack value of 0.04(3), which was displayed in Figure S2. The chiral carbon atoms were 4 R, 5S, 8S, 9 R, 10S and 13 R, respectively. The. As a result, compound 1 was identified as 4a-hydroxy-17,19-dinor-ent-kaurane-16one, which was an infrequent diterpene derivative with the new skeleton compared with others in SciFinder database. The chemical data of 1 was displayed in Table S1.
Compound 2 was obtained as white amorphous powder with the melting point of 253 254 C. Comparing 1 H NMR and 13 C NMR spectrums of 2 with those of 4ahydroxy-16b-19-nor-ent-kaurane-17-oic acid (Tian et al. 2003), we found the only difference existed in the configuration of C-4 position. In NOESY spectrum, a correlation between H-3 (d H 1.24 and d H 2.83) and OH (d H 3.91), along with NOEs between H-19 (d H 0.95) and H-20 (d H 0.91) was found, which confirmed the a-configuration of OH at C-4 position. Hence, compound 2 was identified as 4b-hydroxy-16b-H-18-nor-ent-kaurane-17-oic acid according to the ESI-MS, 1 H NMR, 13 C NMR, HSQC, HMBC and NOESY data in supplementary materials. Compound 2 was a new diterpene derivative and the chemical data was displayed in Table S1.
Compound 3  ) was also found in the same spectrum. A comparison of the 13 C NMR, HSQC and HMBC data of compound 3 to those of 16b-acetoxy-17-hydroxy-19-nor-ent-kaurane-4a-ol suggested the only difference in the C-4 configuration (Yang et al. 2002). According to the NOESY spectrum of compound 3, a correlation between H-3 (d H 1.47 and d H 1.91) and OH-4 (d H 3.91) confirmed that the configuration of C-19 was a. The configuration of the acetoxy group at C-16 was determined unambiguously to be b according to the NOEs between H-14 (d H 0.93 and d H 2.02) and H-17 (d H 3.85). Therefore, compound 3 was identified as 4b,17-dihydroxy-16a-acetoxy-18-nor-ent-kaurane. Compound 3 was a new diterpene and its data were displayed in Table S1.
Compound 4 was obtained as white amorphous powder with the melting point of 284 286 C. The quasi-molecular ion peak was at m/z ¼ 317.2083[M-H] -, which had a molecular formula of C 20 H 30 O 3 , X ¼ 6. In 1 H NMR spectrum of compound 4, there were two methyl groups at d H 0.91 (3 H, s, CH 3 -20) and 1.13 (3 H, s, CH 3 -18). In 13 C NMR data of compound 4, there were a carbonyl group at d C 212.39 and a carboxyl group at d C 179.20. The presence of a methyl group at C-10 position was revealed by the HMBC spectrum of compound 4. In this spectrum, the H-20 signal (d H 0.91) coupled to C-1 (d C 41.35) and C-10 (d C 39.69). Additionally, another methyl group and a carboxyl group were illustrated at C-4 by the HMBC spectrum in Figure S5 Finally, compound 4 was identified as annonasquamosin Y according to the HSQC, HMBC and NOESY data in supplementary materials (Herz and Kulanthaivel 1983). Compound 4 was a new diterpene and its data were displayed in Table S2.
Compound 5, white powder with melting point of 174 175 C, exhibited a quasimolecular ion peak at m/z ¼ 347.2227[M-H] -, which indicated a molecular formula of C 21 H 32 O 4 . In 1 H NMR spectrum of compound 5, there were three methyl groups at d H 0.91 (3 H, s, CH 3 -20) and 1.21 (3 H,s,. A methoxyl group at d H 3.66 (3 H, s, OCOCH 3 ) was found in the same spectrum. A comparison of the 13 C NMR data of compound 5 to 16a-H-ent-kaurane-17,19-dioic acid suggested that compound 5 possessed the same ent-kaurane skeleton (Yang et al. 2002). Analysis of its HSQC spectrum revealed the direct correlation of hydrogen signals and carbon signals. The HMBC correlations between H-19 (d H 1.21) and C-3 (d C 38.01)/C-4 (d C 43.75)/C-5 (d C 56.99)/C-19 (d C 183.88) confirmed that both methyl and carboxylic groups were existed at C-4 positions. In Figure S6, the HMBC correlations between H-1' (d H 3.66) and C-17 (d C 171.14) revealed the existence of an ester group. According to the NOESY spectrum of compound 5, a correlation between H-13 and H-16 confirmed that the a-configuration of H-16 was. The configuration of the carboxyl group at C-4 was definitely determined to be b according to the NOEs between H-19 and H-20 (Yang et al. 2002;Na et al. 2006). Therefore, compound 5 was identified as a new diterpene divertive named as 16a-H-ent-kaurane-17,18-dioic acid, 17-methy ester. The chemical data of compound 5 was displayed in Table S2.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Natural Science Foundation of China [grant number 81573577, 81403082 and 81274057].