ZHANG Yuan FENG Juan YE Wen-Cai TIAN Hai-Yan JIANG Ren-Wang

?

Crystal Structure and Anticancer Properties of Cinobufagin 3-Hemisuberate Methyl Ester①

ZHANG Yuan FENG Juan②YE Wen-Cai TIAN Hai-Yan JIANG Ren-Wang③

(510632)

The title compound cinobufagin 3-hemisuberate methyl ester (1) was isolated from the venom ofgargarizans CANTOR. The crystal structure of 1, C35H48O9, was determined by single-crystal X-ray diffraction analysis. It belongs to orthorhombic, space group212121with= 8.9338(3),= 16.2970(4),= 22.4019(6) ?,= 3261.59(16) ?3,M= 612.73,= 4,D= 1.248 g/cm3,= 0.725 mm-1,(000) = 1320,= 1.040, the final= 0.0374 and= 0.0412 for 4458 unique reflections, of which 4088 were observed (> 2()). In the solid state, short intermolecular C-H×××O interactions involving a methine and the ester carbonyl group in cinobufagin moiety and a methyl in the suberate moiety linked adjacent molecules into a three-dimensional network. Detailed analysis of the1H-NMR data showed that X-ray structure of 1 would be expected to closely resemble the solution conformation in chloroform. Compound 1 was inactive for the inhibition of PC3 and HepG2 cancer cells, but the parent compound cinobufagin showed potent inhibition with IC50values of 0.145 and 5.48mM, respectively, indicating that esterification at C(3) decreased the cytotoxic effect of 1.

crystal structure, cinobufagin 3-hemisuberate methyl ester, anticancer

1 INTRODUCTION

The venom of(ChanSu in Chinese) has been used as a traditional Chinese medicine to treat superficial infections, odontalgia and heart failure for over 1000 years[1]. It was com- monly used in a number of Chinese patent drugs, e.g. Liushen pills, Houzheng pills,.[2]The venom is a rich source of bufadienolides which were the main active components ChanSu[3-4]. This kind of com- pounds showed potent cardiac tonic effect through targeting the Na+/K+ATPase[5-6].Recently, the anticancer activities of this kind of compounds have received worldwide attention[4, 7].

During the course of search for antitumor agents from natural products, we studied the chemical con- stituents in the venom of. A variety of compounds including bufadienoides[8-9]and C23 steroids[10]were identified. Further inves- tigation of low polar fraction of the venom led to the isolation of a fatty acid ester of bufadienolide, i.e. cinobufagin 3-hemisuberate methyl ester (1, Scheme 1). This compound had been synthesized before and was found to show antiviral activity against the rhinoviruses[11]. However, the crystal structure and anticancer property were not reported. We report herein the first isolation as a natural product, crystal structure and anticancer properties of 1.

Scheme 1. Structure of the title compound (1)

2 EXPERIMENTAL

2. 1 Materials and instrumentations

Ultraviolet absorption spectra were recorded using a Jasco V-550 UV/VIS spectrophotometer. Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AV-400 spectrometer. ESI-MS spectra were carried out on a Finnigan LCQ Advantage Max ion trap mass spectrometer. Thin-layer chromato- graphy (TLC) analyses were carried out using pre- coated silica gel GF254plates (Qingdao Marine Che- mical Plant, Qingdao, People’s Republic of China). Column chromatographies were performed on silica gel (200～400 mesh, Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China). All solvents used in column chromatography and high- performance liquid chromatography (HPLC) were of analytical (Shanghai Chemical Plant, Shanghai, People’s Republic of China) and chromatographic (Fisher Scienti?c, NJ, USA) grades, respectively.

2.2 Animal material

The venom ofwas collec- ted from Baoying Toad Breeding Base in Jiangsu province of China in August, 2008 and authenticated by Professor Guang-Xiong Zhou (Jinan University). A specimen (No. 2008082001) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, P. R. China.

2. 3 Isolation of the title compound

The dried venom (1.0 kg) was extracted by 95% ethanol under ultrasonic condition for 40 mins to provide a residue (620 g), which was subsequently partitioned between methylene dichloride and water. The methylene dichloride extract (135 g) was then subjected to silica gel chromatography cyclohexane- acetone (5:1, 3:1, 2:1 and 1:1) as eluents to afford 10 fractions. Fraction 4 was subjected to silica gel chro- matography again and eluted by gradient-hexane- ethyl acetate (10:1, 1:1 and 0:10) to afford three subfractions. The first subfraction was further puri- fied by preparative HPLC to afford the title com- pound (6.3 mg).

2. 4 X-ray structure determination

The crystals suitable for X-ray structure determi- nation were obtained by slow evaporation of me- thanl solution at room temperature. A colorless single crystal of the title compound with dimensions of 0.46mm × 0.37mm× 0.30mm was selected and mounted on a thin glass fiber. X-ray intensity data were measured at 230(2) K on a Bruker AXS SMART 1000 CCD diffractometer equipped with a graphite-monochromatized Cu(= 1.54184?) radiation. A total of 6312 reflections were collected in the range of 3.35<<62.42° (index ranges:-10≤≤8,-17≤≤18 and-23≤≤15) by using anscan mode. Among the 4458 independent reflections (int= 0.0223), 4088 with> 2() were considered as observed and used in the succeeding refinements. Corrections for incident and diffracted beam absorption effects were applied using SADABS. The structure was solved by direct me- thods with SHELXS-97 and expanded by using Fourier difference techniques. The non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were added according to theoretical models. The structure was refined by full-matrix least- squares techniques on2with SHELXL-97. The final refinement gave= 0.0374,= 0.0412 (= 1/[2(F2) + (0.0665)2+ 0.0000], where= (F2+ 2F2)/3) with> 2(). (Δ/)max< 0.001,= 1.040, ()max= 0.434, and ()min= –0.216 e/?3. The selected bond lengths and bond angles are listed in Table 1.

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

2. 5 Cytotoxic assay

The cytotoxic assay was done by MTT assay as described previously[12]with taxol serving as the positive control. Briefly, PC3 and HepG2 cancer cells were plated into 96-well plates at a density of 3 × 103cells per well and allowed to attach overnight. Then the cells were treated with different con- centrations of samples and maintained in the culture condition as reported[12]. Following incubation for 48 h, 20L of the MTT solution (5 mg/mL in phosphate buffered saline (PBS)) was added to each well, and the cells were further incubated for 4 h. Then the medium was removed and replaced by 150L of DMSO in each well to dissolve the formazan crystals. The relative cell viability was determined by measuring the optical densities at 570 nm on microplate reader (SPECTRAmax 250, Molecular Devices, Minnesota, USA), and was expressed as a percentage relative to the control. The experiments were performed three times, each in triplicate.

3 RESULTS AND DISCUSSION

The title compound was isolated from the venom of the Asiatic toad,, as color- less crystals from methanol solution. The quasi- molecular ion at/611 [M-H]-in the ESIMS sug- gested the molecular formula C26H40NO3. The maxi- mum UV absorbance was observed at 295 nm, which was characteristic for the bufadienolides[8].To establish the molecular structure and stereochemistry of the title compound, single-crystal X-ray diffrac- tion analysis was undertaken. The selected bond lengths and bond angles are given in Tables 1 and 2, respectively. Fig. 1 shows the molecular structure of the title compound, and Fig. 2 depicts the packing diagram. X-ray analysis revealed that the crystal belongs to orthorhombic system with space group212121, and the asymmetric unit contains four molecules of 1.

Fig. 1. Molecular structure of 1 showing 30% probability displacement ellipsoids and the atom-numbering scheme. Dashed line indicates the weak C-H×××O hydrogen bond

Table 2. Weak Hydrogen Bond Lengths (?) and Bond Angles (°)

Symmetry codes: (a) –+0.5, –,–0.5; (b) –+0.5, –,–0.5; (c),,; (d) –,+0.5, –+0.5

The molecule (Fig. 1) was characterized by a covalent linkage from the cinobufagin moiety to the suberate moiety via an ester bond. The cinobufagin moiety is composed of three cyclohexane rings (A, B and C), one five-membered ring (D), one epoxide ring (E) and one pyrone ring (F). The stereoche- mistry of the ring juncture is A/B, B/C, C/Dand D/E.

Fig. 2. A packing diagram for compound 1. Selected hydrogen atoms highlight the scheme of hydrogen bonding

The cyclohexane rings A, B and C have normal chair conformations. The five-membered ring D adopts an envelope conformation with C(17) displa- ced by 0.422(2) ? from the mean plane of the remaining four atoms. Both rings E and F are planar, and are roughly perpendicular to each other with a dihedral angle of 90.7(3)°. Final refinement resulted in a Flack parameter –0.1(1), which together with the known absolute configuration of bufalin, an analog of 1[13], permitted the assignments of the absolute configuration, as shown in Fig. 1.

Short intermolecular C-H×××O interactions[14](Table 1) involving a methine and the ester carbonyl group in cinobufagin moiety and the terminal methyl in suberate moiety (C(15)-H×××O(8), 3.0078(3) ?, C(21)-H×××O(7), 3.2079(4) ?, C(35)-H×××O(4), 3.3929(4) ?) linked adjacent molecules into a three-dimensional network.

The1H-NMR data of 1 in chloroform are in good agreement with the crystal structure of the title compound, which showed signals attributable to the cinobufagin moiety and suberate moiety. Com- parison of the1H-NMR data of 1 withthose of cino- bufagin[10, 15]showed that they were very similar except that the chemical shift of H(3) (5.10, 1H, br. s) in 1 was shifted to downfield 1.00 ppm, con- firming the esterification site at C(3). Furthermore, the broad single peak of H(3) indicated that the coupling between H(2, 2) and H(3), H(3) and H(4, 4) was small. X-ray analysis showed that the H(3) is on the equatorial position () and the sube- rate moiety is on the axial position (). Consequently, H(3) makes- and-type small couplings with H(2, 2) and H(4, 4) resulting a broad single peak. Similarly,1H-NMR spectrum of 1 showed that the coupling constant between H(17) and H(16) is 9.3 Hz, which is consistent with the small torsion angle H(17)–C(17)–C(16)–H(16) 21.5°in the crystal state. Thus X-ray structure of 1 would be expected to closely resemble the solution conformation in chloroform, which was similar to that of macrocae- salmin[16]. In contrast, the conformation of spiro- prorocentrimine in the solid state is different from that in DMSO solution[17]. A plausible reason for this difference is that the bufadienolide moiety of compound 1 and macrocaesalmin bear a rigid ring system, whereas a soft macrolide moiety is present in spiro-prorocentrimine.

The anticancer properties of 1 and the parent compound cinobufagin (isolated in our lab)[10]were compared. The results showed that com- pound 1 was inactive for the inhibition of PC3 and HepG2 cancer cells, but the parent com- pound cinobufagin showed potent inhibition with IC50value of 0.145 and 5.48mM, respectively, indicating that esterification at C(3) decreased the cytotoxic effect of 1.

(1). ZhonghuaBencao. Shanghai Science and

Technology Press: Shanghai 1999, pp. 362–367.

(2)(2010 Edition). Chemical Industry Press: Beijing 2010, 360.

(3) Steyn, P. S.; van Heerden, F. R. Bufadienolides of plant and animal origin.1998, 15, 397-413.

(4) Gao, H. M.; Popescu, R.; Kopp, B.; Wang, Z. M. Bufadienolides and their antitumor activity2011, 28, 953-969.

(5) Roger, J. B.; Brian, J. P.; Roxanne, R. S.;Amitava, D. Effects of ChanSu, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxicity due to more than Na+, K+-?ATPase blocking.2002, 72, 699-709.

(6) Wansapura, A. N.; Lasko, V.; Xie, Z.; Fedorova, O. V.; Bagrov, A. Y.; Lingrel, J. B.; Lorenz, J. N. Marinobufagenin enhances cardiac contractility in mice with ouabain-?sensitive alpha1 Na+-?K+-?ATPase..2009, 296, H1833-1839.

(7) Yu, C. H.; Kan, S. F.; Pu, H. F.; Chien, E. J.;Wang, P. S. Apoptotic signaling in bufalin- and cinobufagin-?treated androgen-?dependent and -independent human prostate cancer cells2008,99, 2467-2476.

(8) Tian, H. Y.; Wang, L.; Zhang, X. Q.; Zhang, D. M.; Wang, Y.; Jiang, R. W.; Ye, W. C. New bufadienolides and C23 steroids from the venom of Bufo bufo gargarizans.2010, 75, 884-890.

(9) Tian, H. Y.; Wang, L.; Zhang, X. Q.; Wang, Y.; Zhang, D. M.; Jiang, R. W.; Liu, Z.; Liu, J. S.; Li, Y. L.; Ye, W. C. Bufogargarizins A and B, two novel 19-norbufadienolides with unprecedented skeletons from the venom of Bufo bufo gargarizans.2010, 16, 10989-10993.

(10) Tian, H. Y.; Luo, S. L.; Liu, J. S.; Wang, L.; Wang, Y.; Zhang, D. M.; Zhang, X. Q.; Jiang, R. W.;Ye, W. C.

C23 Steroids from the venom of Bufo bufo gargarizans.. 2013, 76, 1842-1847.

(11) Kamano, Y.; Sato, N.; Nakayoshi, H.; Pettit, G. R.; Smith, C. R. Steroids and related natural products.

CIX bufadienolides. XLI. Rhinovirus inhibition by bufadienolides.. 1988, 36, 326-332.

(12) Tian, H. Y.; Yuan, X. F.; Jin, L.; Li, J.; Luo, C.; Ye, W. C.; Jiang, R. W. A bufadienolide derived androgen receptor antagonist with inhibitory activities against prostate cancer cells..2014, 207, 16-22.

(13) Rohrer, D. C.; Fullerton, D. S.; Kitatsuji, E.; Nambara, T.; Yoshii, E. Bufalin.. 1982, B38, 1865-1868.

(14) Takahashi, O.; Kohno, Y.; Nishio, M. Relevance of weak hydrogen bonds in the conformation of organic compounds and bioconjugates: evidence from recent experimental data and high-levelMO calculations.2010, 110, 6049-6076.

(15) Li, W. X.; Sun, H.; Li, Q.; Zhang, X. Q.; Ye, W. C.; Yao, X. S. Chemical constituents of the skin ofBufo bufo gargarizan2007, 38, 275-279.

(16) Jiang, R. W.; But, P. P. H.;Ma, S. C.; Ye, W. C.; Chan, S. P.;Mak, T. C. W. Structure and antiviral properties of macrocaesalmin, a novel cassane furanoditerpenoid lactone from the seeds of Caesalpinia minax Hance.. 2002, 43, 2415-2418.

(17) Lu, C. K.; Lee, G. H.; Huang, R.; Chou, H. N. Spiro-prorocentrimine, a novel macrocyclic lactone from benthic Prorocentrum sp.. 2001, 42, 1713-1716.

28 November 2013;

26 December 2013 (CCDC 975148)

①This work was supported by the National Natural Science Foundation of China (81102518)

② Zhang Yuan and Feng Juan contributed equally to this work

. E-mail: trwjiang@jnu.edu.cn or tianhaiyan1982@163.com