Synthesis, Crystal Structure and Na+/K+-ATPaseInhibitory Activity of Δ14,15-anhydro-24-thiocarbonylbufalin①

ZHANG Peng-Wei TANG Hong-Jin TIAN Hi-YnZHANG Rong-Rong JIANG Ren-Wng, b

?

Synthesis, Crystal Structure and Na+/K+-ATPaseInhibitory Activity of Δ14,15-anhydro-24-thiocarbonylbufalin①

ZHANG Peng-Weia②TANG Hong-Jina②TIAN Hai-YanaZHANG Rong-RongaJIANG Ren-Wanga, b③

a(510632)b(518057)

The title compoundD14,15-anhydro-24-thiocarbonylbufalin (1) was prepared by the reaction of natural product bufalin with Lawesson reagent. The crystal structure of 1, C24H30O2S·C24H30O2S, was determined by single-crystal X-ray diffraction analysis. It belongs to monoclinic, space group2, with= 30.9845(2),= 6.8036(3),= 22.5791(15) ?,= 4241.7(4) ?3,M= 384.21,= 4,D= 1.204 g/cm3,= 1.463 mm-1,(000) = 1664,= 1.064,=0.0487 and= 0.0645 for 4683 unique reflections, of which 3757 were observed (> 2()). The asymmetric unit contains two independent molecules (I and II), which are closely similar to each other except for the orientation of the lactone ring. Both conformations of I and II are in good agreement with the solution structure in methanol as indicated by1H-NMR analysis. Due to the presence of heavy atom sulfur in the molecules, the final refinement resulted in a small Flack parameter 0.02(3), permitting the assignments of the absolute configuration. In the solid state, intermolecular hydrogen bonds involving thiocarbonyl group in the lactone moiety and the hydroxyl groups in the steroid moiety ester linked adjacent molecules into a three-dimensional network. Compound 1 showed weak inhibition on Na+/K+-ATPase in contrast to the strong inhibitory activity of the parent compound bufalin, suggesting that the carbonyl group in lactone moiety and the hydroxyl group at C-14 play important roles for the inhibition of Na+/K+-ATPase.

crystal structure,D14,15-anhydro-24-thiocarbonylbufalin, Na+/K+-ATPase

1 INTRODUCTION

The venom of, known as Chansu in Chinese, is often used as a traditional Chinese medicine for the treatment of superficial infections and heart failure[1]. Bufadienolides are the main active ingredients of the toad venom and specific inhibitors of Na+/K+ATPase[2, 3]. The pro- minent clinical and pharmacological properties of bufadienolides have prompted many phytochemical studies, and over one hundred bufadienolides were isolated from the toad venom of different places of origin[2, 3]. The complexity and structural diversity of this fascinating class of compounds have attracted considerable attention, and a number of ingenious strategies toward the total syntheses of bufadieno-lides[4, 5]and structural modificationshave appeared in recent years[6, 7].

Our phytochemical studies on the venom ofresulted in a variety of compounds including bufadienoides[8, 9]and C23 steroids[10]. Subsequent structural modification of the lactone moiety of bufalin led to the synthesis of bufadieno- lactam with inhibitory activities against the prostate cancer cells but weak inhibition on Na+/K+- ATPase[11]. Similarly, the modification of hydroxyl group at C-14 led to the synthesis of two dehydrate derivatives with moderate inhibition on Na+/K+- ATPase[12]. In this context, simultaneous modifica- tions on the lactone moiety and 14-hydoxyl group were carried out, which led to the synthesis ofD14,15-anhydro-24-thiocarbonylbufalin (1, Scheme 1). We report herein the synthesis and crystal structure and Na+/K+-ATPase inhibitory activity of compound 1.

Scheme 1. Synthesis of compound 1

2 EXPERIMENTAL

2.1 Materials and instruments

Bufalin was isolated from the Chinese Traditional Medicine Chansu in our lab[8]. The purity was determined to be > 99.5% by HPLC analysis. All other chemicals were commercially available. UV spectrum was determined in CH3OH on a JascoV- 550 UV/vis spectrophotometer. NMR spectra were recorded on a Bruker AV-300 spectrometer. ESI-MS was measured on a Finnigan LCQ Advantage Max ion trap mass spectrometer. HR-ESI-MS data were obtained on an Agilent 6210 ESI/TOF mass spectro- meter. Column chromatography was performed on silica gel (200～400 mesh, Qingdao Marine Che- mical Plant, Qingdao, People’s Republic of China).

2.2 Synthesis of D14,15-anhydro-24-thiocarbonylbufalin

Bufalin (77.2 mg, 0.2 mmol)and Lawesson's Reagent (161.6 mg, 0.4 mmol) were added into anhydrous dichloromethane (10 mL), and the mix- ture was stirred for 10 h at 120 ℃. After cooling down, the reaction mixture was poured into satura- ted Na2CO3solution. The dichloromethane layer was separated and evaporated under reduced pressure. The residue was purified by silica gel column chromatography eluted by chloroform-acetone (90:5) to afford compound 1 (33.2mg, yield 43.2%), which was further recrystallized from methanol solution.

D14,15-anhydro-24-thiocarbonylbufalin (1): UV (MeOH)max239, 283 and 383 nm.1H NMR (CD3OD, 300 MHz): 4.03 (1H, br s, H-3), 2.30 (1H, m, H-5), 5.29 (1H, m, H-15), 2.87 (1H, dd,= 10.8 Hz,= 8.4 Hz, H-17), 0.75 (3H, s, H-18), 0.98 (3H, s, H-19), 7.92 (1H, br s, H-21), 7.33 (1H, dd,= 9.3, 2.1 Hz, H-22), 7.20 (1H, d,= 9.3 Hz, H-23); ESI-MS: [M+H]+= 385.4; HR-ESI-MS: [M+H]+385.2195 (calculated 385.2197); [2M+Na]+791.4154 (calculated 791.4138).

2.4 X-ray structure determination

The crystals suitable for X-ray structure determi- nation were obtained by slow evaporation of me- thanol solution at room temperature. A red prismatic single crystal of the title compound with dimensions of 0.18mm × 0.20mm× 0.41mm was selected and mounted on a thin glass fiber. X-ray diffraction intensity data were measured at 153(2) K on an Agilent Gemini S Ultra Sappharie CCD diffracto- meter equipped with a graphite-monochromatized Cu(= 1.54184 ?) radiation. A total of 9075 reflections were collected in the range of 2.95<<60.79° (index ranges:-33≤≤34,-5≤≤7 and-25≤≤25) by using anscan mode. Among the 4683 independent reflections (int= 0.0223), 3757 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 methods with SHE- LXS-97 and expanded by usingdifference 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= 0.0487,= 0.1207 (= 1/[2(F2) + (0.0679)2+ 0.3283], where= (F2+ 2F2)/3) for the observed reflections with> 2(). (Δ/)max= 0.000,= 1.064, ()max= 0.210 and ()min=－0.226 e/?3. The selected bond lengths and bond angles are listed in Table 1 and torsion angles in Table 2.

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

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

Symmetry codes: (a)+ 0.5,– 0.5,; (b) –+1,, –+1

2.5 Na+/K+-ATPase inhibitory activity

The Na+/K+-ATPase inhibitory activities were determined according to the known colormetric method[10]. Na+/K+-ATPase (from porcine cerebral cortex) was obtained from Sigma-Aldrich. The inhibitory activities of different concentrations of compound 1 on 0.03～0.06 U/ml of Na+/K+-ATPase were determined by measuring the release of inorganic phosphate from ATP over a 15 min period at 37 ℃ in an assay medium containing Na+125 mM, K+2.8 mM, Mg2+4.5 mM, EDTA 0.5 mM, ATP 5 mM, buffered to pH 7.80 using Tris (24 mM). The absorbance at 655 nm was measured by a microplate reader (SpectraMax 340PC384, Molecular Devices, USA) after being colorized by using the Taussky- Shorr Reagent. The GraphPad Prism software (Version 5) was used to fit sigmoid curves to esti- mate the slopes and IC50for the dose/inhibition relationships.

3 RESULTS AND DISCUSSION

Compound 1 was obtained by the reaction of bufalin with Lawesson's Reagent[13]in anhydrous dichloromethane, and re-crystallized as red prismatic crystals from methanol solution. High resolution ESIMS analysis of 1 showed a quasi-molecular ion peak at [2M+Na+] 791.4150 (calculated for C48H60O4S2Na, 791.4144), corresponding to a mole- cular formula C24H30O2S. Comparison of the1H- NMR data of 1 withthose of the parent compound bufalin[14]showed that they were very similar except that the chemical shift of H-21 and H-23 on the-pyrone ring in 1 were shifted to lower field (7.92 (1H,= 2.1 Hz, H-21; 7.33 (1H, dd,= 9.3, 2.1 Hz, H-22), while H-22 (7.92 (1H,= 2.1 Hz)) was shifted to a higher field. In addition, the single bond between C-14 and C15 was replaced by a double bond as indicated by the replacement of a methine and methylene signals with an olefinic proton signal5.29 (1H, m, H-15). These spectroscopic data indicated that compound 1 was a sulfur substituted dehydrate of bufalin.

The single crystal of the title compound was ob- tained by slow evaporation of its methanol solution.To establish the three-dimensional structure of the title compound, a single crystal of 1 was used for X-ray diffraction analysis. X-ray analysis revealed that the crystal belongs to monoclinic system with space group2, and the asymmetric unit contains two molecules of 1 (molecules I and II). A com- parison of the selected bond lengths and bond angles of molecules I and II are given in Table 1. Fig. 1 shows the molecular structure of the title compound, and Fig. 2 depicts the packing diagram.

Fig. 1. Molecular structure of 1 showing 30% probability displacement ellipsoids and atom numbering scheme

Fig. 2. A packing diagram for compound 1. Hydrogen-bonding network of 1 viewed roughly down the-axis. Selected hydrogen atoms highlight the scheme of hydrogen bonding

Both molecules I and II are characterized by a thiocarbonyl group at C-24 and a double bond between C-14 and C-15, indicating that the carbonyl group at C-24 was replaced by a thiocarbonyl group and the hydroxyl group at C-14 underwent dehydra- tion with the methylene at C-15. Both I and II are composed of three cyclohexane rings (A, B and C), one unsaturated five-membered ring (D) and one lactone ring (E). The stereochemistry of the ring juncture is A/Band B/C.

The cyclohexane rings A and B have normal chair conformations. Ring C exists in a twisted chair conformation due to the fusion with unsaturated ring D. The five-membered ring D adopts an envelope conformation with C-17 displaced by 0.4307(3) and 0.4734(4) ? from the mean plane of the remaining four atoms for molecules I and II, respectively. The lactone ring E is planar for both molecules. Mole- cules I and II are closely similar to each other as indicated by the bond length and bond angles shown in Table 1; however, difference is observed in the orientation of the lactone ring. The dihedral angle between the lactone ring E and the envelope plane of ring D is 77.7°for molecule I and 64.9°for molecule II. Due to the presence of heavy atom sulfur in the molecules, the final refinement resulted in a small Flack parameter 0.02(3), permitting the assignments of the absolute configuration as shown in Fig. 1.

In solid state, intermolecular hydrogen bond (Table 2) involving thiocarbonyl group in the lactone moiety and the hydroxyl groups in the steroid moiety (O(2)-H(2)×××S(1), 3.327(4) ?, O(2A)-H(2A3)×××O(2), 2.934(3) ?) linked adjacent molecu- les into a three-dimensional network (Fig. 2).

Normally, molecules exert biological functions in solution state. Thus, it is necessary to compare the solution conformation with the solid state con- formation. The broad single peak of H(3) indicated that the coupling between H(2, 2) and H(3), H(3) and H(4, 4) were small. X-ray analysis showed that H(3) is on the equatorial position () and the hydroxyl group is on the axial position (). Conse- quently, H(3) makes- and-type small couplings with H(2, 2) and H(4, 4), resulting in a broad single peak. Similarly,1H-NMR spectrum of 1 showed that the coupling constant between H(17) and H(16and 16) is 10.6 and 8.4 Hz. The large coupling constants are consistent with the small torsion angles H(17)–C(17)–C(16)–H(16) 32.3°and H(17)–C(17)–C(16)–H(16) 25.1°for molecule I and the corresponding torsion angles for molecule II 34.4°and 22.9°, respectively. Organic molecules sometimes form two or more conformations in crystal states[15, 16]. Similarly, there are two confor- mations in the crystal structure of compound 1; however, both of them are consistent with the solu- tion structure in methanol, which is similar to cino- bufagin 3-hemisuberate methyl ester[17].

The inhibition on Na+/K+-ATPase property of 1 and the parent compound bufalin (isolated in our lab)[8]were compared. Compound 1 showed weak inhibition on Na+/K+-ATPase with IC50value of 35.8mM, but the parent compound bufalin showed potent inhibition with IC50value of 0.011M[10], indicating that replacement of the carbonyl group with a thio- carbonyl group and dehydration at C-14 signifi- cantly decreased the inhibition on the Na+/K+- ATPase effect of 1.

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10 January 2014;

20 June 2014 (CCDC 980546)

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

② Zhang Peng-Wei and Tang Hong-Jin contributed equally to this work

. E-mail: trwjiang@jnu.edu.cn