Stemonae Radix (‘Baibu’ in Chinese),the dried root ofStemona tuberosaLour. and relatedStemonaspecies (Stemonaceae family), has been used as a traditional Chinese medicine for antitussive and insecticide remedies for thousands of years ^{[1, 2]}. Plants of genus Stemonawere known to produce a variety of alkaloids with unique structures characterized by the presence of a pyrrolo^{[1, 2, a]}azepine nucleus,which were believed to be responsible for the antitussive activity of the herb ^{[3, 4, 5]}. So far,more than 140 alkaloids had been isolated from genus Stemona^[6].

During the course of studying the antitussive Stemona alkaloids in Stemonae Radix,we found that the chemical diversity in genus Stemonawas very significant ^{[7, 8, 9]}. Though alkaloids with pyrrolo^{[1, 2, a]}azepine nucleus could be observed in all Stemonaspecies,the structural-types varied in different species. Furthermore,in the sameS. tuberosaspecies,four chemical-types were observed in different places of production ^[9].

To further explore the chemical diversity ofS. tuberosa,we performed a phytochemical investigation on the ethanol extract of S. tuberosacollected in Yulin (Guangxi Province),leading to the isolation of one new alkaloid,tuberostemonine D (1) as well as seven known compounds (2-8),i.e. tuberostemonine (2) ^[9], tuberostemonine K (3) ^[9],tuberostemonine H (4) ^[10],didehydrotuberostemonin (5) ^[11],tuberostemoline (6) ^[12],bisdehydrostemonine (7) ^[13],and stemoninine (8) ^[14]. Compound 1 was a new stereoisomer of tuberostemonine. Herein,we reported the isolation and structure determination of 1. 2. Experimental

The dried roots ofStemona tuberosaLour. (3 kg) were powdered and extracted with 95% (v/v) EtOH at 90°C. The crude extract was acidified with dilute HCl (4%) to pH 1-2 and then partitioned between Et₂O and H₂O. The aqueous layer was separated and basified with CH₃·H₂O to pH 9-10 and extracted with Et₂O to afford the crude alkaloids (26.8 g). The crude alkaloids (26.8 g) was subjected to silica gel column chromatography eluted with CHCL₃-CH₃OH gradient system (100:1-1:1) to afford 10 fractions (A-J). Fr. E (3.2 g) was separated by silica gel column using CHCL₃-(CH₃)₂CO (30:1-0:30) as eluent to yield seven subfractions (Fr. E1-E7). Fr. E3 (183 mg) was subjected to preparative HPLC eluted with CH₃CN-H₂O (1:1) to give compounds 1 (34 mg) and 6 (13 mg). Fr. E6 (112 mg) was separated by Sephadex LH-20 column and preparative HPLC eluted by CH₃CN- H₂O (45: 65) to yield compounds 3 (24 mg),4 (23 mg) and 7 (18 mg). Fraction H (2.5 g) was separated by silica gel column using CHCL₃-methanol (30:1-0:30) as the eluent to yield five subfractions (Fr. H1-H5). Fractions H4 (83 mg) was purified by preparative HPLC eluted by CH₃CN-H₂O (38: 62) to afford2 (22 mg) and 8 (14 mg). Similarly,fraction H5 (123 mg) was separated using the same method as H4 to afford5(24 mg).

Fig. 1

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Fig. 1. The chemical structures of compounds 1-8.

Tuberostemonine D (1): colorless prism-like crystals; [α]_D ²⁶-11:8(c= 0.5,CH₃OH); UV (CH₃OH)lmax(loge) 208 (3.55) nm; IR (KBr,cm^-1 )ν_max 1761,1456,1199,1007; ¹H NMR and ¹³C NMR data,see Table 1; HRESIMSm/z 376.2486 [M+H]⁺(calcd. for C₂₂H₃₃NO₄,376.2482).

Table 1

Table 1 1H and 13C NMR spectral data of compound1(in pyridine-d5, 400 MHz for 1H).a

Table 1 1H and 13C NMR spectral data of compound1(in pyridine-d5, 400 MHz for 1H).a

X-Ray crystallographic analysis of 1.C₂₂H₃₃NO₄,colorless blocks,Mr = 376.25,monoclinic,P2₁,a= 10.5516(4), b= 9.4061(3),c= 11.3265(4) Å ,β= 114.205(5),V= 1025.32(6) ų, Z=2,d_x= 1.216 mg/cm³,F(0 0 0) = 408,m(Cu-Ka) = 0.661 mm^-1. Data collection was performed on a Gemini S Ultra using graphitemonochromated radiation (l= 1.54184 Å ); 2294 unique reflections were collected to u_max= 62.71°,in which 2207 reflections were observed [F²>4s(F²)]. The crystal structure was resolved by direct methods using SHELXS-97 and refined by full-matrix leastsquares method on F² using SHELXS-97. Non-hydrogen atoms were refined with anisotropic temperature factors. Hydrogen atoms bonded to carbons were placed at their geometrically ideal positions. FinalR= 0.0392,Rw= 0.1054 and S= 1.061. Crystallographic data for structure1have been deposited at the Cambridge Crystallographic Data Center (CCDC 980755). Copies of the data can be obtained free of charge on application to CCDC,12 Union Road,Cambridge CB2 1EZ,UK (Fax: +44 1223 336033; Email: deposit@ccdc.cam.ac.uk). 3. Results and discussion

Compound1was isolated as colorless prism-like crystals. Its molecular formula was established as C₂₂H₃₃NO₄ by the positive quasi-molecular ion peak atm/z376.2486 [M+H]⁺in its HR-ESIMS with seven degrees of unsaturation. The IR band at 1761 cm^-1 and ¹³C NMR datadC179.7 and 180.6 suggested the presence of twoglactone rings. The ¹H NMR spectrum showed the presence of characteristic signals for one primary methyl atδ_H0.90 (t,3H, J= 7.4 Hz,H-17),two secondary methyls at δ_H 1.26 (d,3H, J= 7.1 Hz,H-22) and 1.25 (d,3H,J= 7.4 Hz,H-15),one methylene and two methines bearing a nitrogen atom at δ_H3.08 (m,1H,H-5b), 2.53 (m,1H,H-5a),2.40 (m,1H,H-9a) and 2.87(m,1H,H-3),and two oxymethines at δ_H4.43 (m,1H,H-11) and 4.48 (m,1H,H-18). The ¹³C NMR and DEPT spectra of1displayed 22 carbon signals, including two carbonyl carbons,10 methine carbons,seven methylene carbons,and three methyl carbons. A comparison of the ¹H NMR and ¹³C NMR data of 1 with those of tuberostemonine (2) implied they have the same planar structure ^[9],suggesting that compound1might be an isomer of 2.

The full assignments and connectivities of all the H- and C-atom signals (Table 1) of 1 were further confirmed by a combination of 1^H- 1^H COSY,HSQC and HMBC spectral analyses. The 1^H- 1^HCOSY spectrum showed the spin systems involving H-9a↔H-9↔H-10↔H-11↔H-12↔H-13↔H-15; H-10↔H-16↔H-17 and H-18↔H-19↔H-20↔H-22,which were shown in bold face in Fig. 2.In the HMBC spectrum,the correlations between H-9a and C-3, between H-9a and C-5,as well as between H-3 and C-5 were observed,indicating C-3,C-9a and C-5 were connected via a nitrogen atom (N-4). The HMBC correlations between H-15 and C-14 and between H-11 and C-14 revealed the existence of ag-lactone ring (ring A). Similarly,the HMBC correlations between H-18 and C-11 and between H-22 and C-11 suggested the formation of anotherglactone ring (ring E),and the HMBC correlation between H-18 and C-3 indicated that this lactone ring was located at C-3. The relative configuration of1was determined by the ROESY spectrum,which showed NOE correlations H-9a↔H-3,H-9a↔H-9,H-9a↔H-12 and H-11↔H-12 (Fig. 3). These observations suggested H-3,H-9,H-9a,H-11 and H-12 were all b-oriented.

Fig. 2

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Fig. 2. 1H- 1H COSY and key HMBC correlations of compound 1.

Fig. 3

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Fig. 3. Key NOE correlations of compound 1.

Furthermore,NOE correlations between H-1↔H-10,and H-10↔H-13 indicated that those protons were alla-oriented.

Moreover,NOE correlation between H-18 and H3-22 suggested they were on the same side of theg-lactone (ring E). However,the relative configuration of C-18 to C-3 still could not be determined as the free rotation of the single bond between C-3 and C-18. Fortunately,crystals of 1 suitable for single crystal X-ray diffraction analysis were obtained from the methanol solution. The relative configuration of1was unambiguously established by its X-ray crystal structure (Fig. 4). Besides,a final refinement of the CuKadiffraction data resulted in a small Flack parameter 0.2(3) permitting the assignment of the absolute configuration of1as 1R, 3R,9R,9aR,10R,11S,12S,13S,18R,and 20S.

Fig. 4

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Fig. 4. X-ray crystal structure of1with atom-labeling scheme.

Up to now,there were eleven isomers of tuberostemonine reported (Supporting information). These isomers differ from each other by the configurations at C-1,C-3,C-9,C-9A,C-10,C-11,C-12, C-13,C-18 and C-20 ^{[6, 15, 16]}. Among these known isomers,3R configuration is rare except for tuberostemonine A. Compound1 represents the second isomer with the same 3Rconfiguration as tuberostemonine A,but the configuration at C-18 is S,which is different from tuberostemonine A. Thus compound1was a new stereoisomer of tuberostemonine,and was named as tuberostemonine D (Fig. 1).

Chemical diversity amongStemonagenus is very large ^[17]. Even in the same species ofS. tuberosa,chemical diversity is still significant. Previously,we studied the chemical diversity of S. tuberosa ^[9],and found that this herb from different places of production could be divided into four chemical types with tuberostemonine,neotuberostemonine,stemoninine and croomine as the major components,respectively. In the present study, compounds1-6belonged to the stenine-type (also belonged to the stichoneurine-type ^[15]) alkaloids and 7 and 8 belonged to the stemoninine-type alkaloids were identified. Thus first simultaneous isolation of stenine- and stemoninine types alkaloids added a new chemical type and increased the chemical diversity ofS. tuberosa. In addition,genusStemonais a rich source of stenine-type alkaloid ^[9],and genusStichoneuronis a rich source of stemoninine-type alkaloids ^[18]. Our previous study revealed the close taxonomic relationship between these two genera through parsimonious analysis of trnL DNA sequences ^[7]; however, chemotaxonomic evidence is weak. Our current results showed that stenine- and stemoninine types alkaloids could be simultaneously identified in Stemona plant,providing a significant chemotaxonomic clue confirming the close relationship between generaStemonaandStichoneuronas revealed by DNA analysis,and favored retaining them in the same Stemonaceae family. 4. Conclusion

From the roots of Stemona tuberosa,one new alkaloid tuberostemonine D (1) together with seven known compounds were isolated and identified by extensive spectroscopic methods. The molecular structure and absolute configuration of compound1 were further confirmed by single crystal X-ray diffraction analysis. First simultaneous isolation of stenine- and stemoninine types alkaloid not only added a new chemical type and increased the chemical diversity ofS. tuberosa,but also provided chemotaxonomic clues for the close relationship between generaStemonaand

Stichoneuronas revealed by DNA analysis,and favored retaining them in the same Stemonaceae family. Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (No. 11612603) and the Science and Technology Planning Project of Zhongshan City (No. 2013A3FC0325). Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.03.051.