Journal of the Chinese Chemical Society, 2008, 55, 401-405 401

Acyclic Diterpene-g-lactones and Flavonoid from Salix cheilophila Omitted

Tong Shena* ( ), Yong-Qiang Tiana (), Wu-Xia Liua ( ) and Shang-Zhen Zhengb () aSchool of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou 730070, P. R. bCollege of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, P. R. China

Three new phytane-type diterpene-g-lactone shaliuin I (2), shaliuin II (3) and shaliuin III (4)were isolated along with thirteen known compounds from twigs of Salix cheilophila omitted (Chinese name “Shaliu”, ). The structures of three new compounds have been established on the basis of spec- troscopic methods and 2D NMR techniques.

Keywords: Salix cheilophila; Salicaceae; Twigs; Acyclic diterpene-g-lactone.

INTRODUCTION pyranoside (11),8 luteolin-7-O-b-D-glucopyranoside (12),8 Salix cheilophila omitted is mostly distributed in luteolin-5-O-b-D-glucopyranoside (13),8 stigmast-4-en- , , and Provinces and the north- 3b-ol-6-one (14),9 stigmast-5-en-3b-ol-7-one (15),9 b-sito- west of China. It shows a strong effect as an anti-influenza, sterol (16).10 antipruritic, antirheumatic, and antineoplastic agent among Compound 2 was obtained as a pale yellow gum with 1 a 20 ° others. However, its chemical constituents have not been [ ] D -131.7 (c, 0.26, CHCl3). Its HR-EIMS gave a peak at reported so far. We have carried out a detailed chemical in- m/z 430.2965 corresponding to the molecular formula vestigation and have isolated three new acyclic diterpene- C26H38O5 (calc 430.2972), indicating eight degrees of g-lactones (2, 3 and 4) along with thirteen known com- unsaturation. The IR absorption at 1746, 1682 cm-1 indi- pounds (1, 5-16) from the ethanol extract of the . In cated the presence of an a,b-unsaturated-g-lactone; its IR the present article, we report the isolation and structure elu- spectrum also showed bands assigned to ester carbonyl cidation of the three new compounds shaliuin I (2), shaliuin (1718 cm-1), double bond (1652 cm-1) and a hydroxyl group II (3), and shaliuin III (4). (3362 cm-1). 1Hand13C NMR and DEPT spectra (see Ta- bles 1 and 2) of 2 revealed signals due to six methyls, six RESULTS AND DISCUSSION methylenes, seven methines and seven quaternary carbons Sixteen compounds were isolated by repeated col- (five olefinic carbons, two carbonyl), which suggest the umn chromatography on silica gel and preparative TLC presence of 26 carbons. The 1H-1H COSY and HMBC from the ethanol extract of Salix cheilophila. Three new spectra indicated that 2 is an acyclic diterpene composed of compounds, structures were identified as shaliuin I, shaliuin the structural fragments i, ii and iii. In fragment i, the 1H-1H II, and shaliuin III. The known compounds (1, 5-16)were COSY and HMQC spectra strongly indicated the presence identified by comparing their spectral data with those of of an a,b-unsaturated-g-lactone skeleton;11,12 two methyl- authentic samples or with those reported in the literature as enes group, two tri-substituted double bonds, a hydroxyl hanliuin IV (1),2 8b,19-dihydroxy-3-oxopimar-15-ene group and two vinylic methyls group were present, the sig- 3 4 5 (5), feruloyl glucose (6), porusterone I (7), 6,7-dihy- nals at d 3.88 due to C12-OH, and d 73.0 due to C-12, d 4.64 6 droxy-3¢-methoxy-4¢,5¢-methylenedioxyisoflavone (8), (1H, brs, H-12) coupled with the H-9a (d 2.23). The signal 6,7-dihydroxy-3¢-methoxy-4¢,5¢-methylenedioxyisofla- at d 6.90 was ascribed to H-10, which was correlated with 6 7 vone-6-O-b-D-glucopyranoside (9), caffeic acid (10), H-8b (d 2.04), H-9a (d 2.23) and H-9b (d 2.09). The chemi- 5,3¢-dihydroxy-4¢-O-angeloxyflavone-7-O-b-D-gluco- cal shift is in accordance with the a,b-proton of an a,b-un-

* Corresponding author. E-mail: [email protected] 402 J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 Shen et al.

ment iii showed the vinyl proton at d 5.48 (J = 7.0, 2.0 Hz; H-2; d 124.1, C-2) coupled with H-1 (d 6.36, 2H, J =7.0 Hz; H-1; d 68.0, C-1) and allylically with a methyl group (d 1.59, 3H, H-20; d 23.6, C-20). In addition, its 1HNMRatd 5.66 (tq, H-2¢), 2.17 (dq, H-4¢), 1.08 (t, H-5¢), 2.18 (d, H-6¢) showed the presence of a methylsenecioyloxy group,13

which was also confirmed by the C6H10O2 unit due to the + + fragmentations [M-114 (C6H10O2)] and [C6H10O2-OH] peak at m/z 316and97intheEIMSspectrum.14 Except for the 13C NMR spectrum at d 166.7 (carbonyl, C-1¢), d 115.0 (C-2¢), 163.4 (quaternary, C-3¢), 33.7 (C-4¢), 11.8 (C-5¢), 18.8 (C-6¢) showed the presence of a methylsenecioyloxy group. The HMBC spectrum showed that there were cross peaks of C-1¢ (d 166.7) with H-1 (d 6.36). Thus, the methyl- senecioyloxy group must be attached to C-1. Besides, in the structural fragment ii, the broad triplet at d 5.32 (H-6) ex- hibited a cross peak with partially overlapped multiplets of two methylenes at d 2.10 (H-4) and d 1.90 (H-5), also with the broad singlet of the vinylic methyl group at d 1.45 (H- 19), respectively. The relative stereochemistry of 2 was supported by NOESY correlations, which showed cross peaks of H-1/H-4 and H-2/H-20 establishing the configura- tion of the D2-double bond as 2, and the cross peaks of H-9/H-12 revealed the E-configuration of the D10-double bond, also the E-configuration of the D6-double bond was

supported by the NOESY correlation for H-6/H-8b and (d 2.04, brt, J = 11.2) by a chemical shift of C-19 (d 11.3).15,16 Comparison of its NOESY spectrum of 2 with those of structurally related natural products,12 implied that their relative chemistry was the same. But absolute configura- tion at C-8, C-12 and C-13 were not determined, and cou-

pling constant J12,13 = 2.0 Hz was in agreement with a di- hedral angle of 70 or 115° which requires a trans-relation- ship of H-12 and H-13,12 thus we concluded that 2 is an acyclic deterpene-g-lactone named shaliuin I. Compound 3 was a pale yellow gum and its HR- EIMS displayed a [M+], at m/z 472.3119 (calc 472.3124) in saturated-g-lactone. This conclusion was supported by the agreement with a molecular formula of C28H40O6, indicat- 13CNMRdata(d 142.2, C-10; d 132.0, C-11; d 170.2, ing nine degrees of unsaturation. In the IR spectrum absorb- C-18). The proton at d 6.90 (H-10) correlated also with a ances for hydroxyl (3359 cm-1), a,b-unsaturated-g-lactone broad singlet at d 4.64 (H-12, d 73.0, C-12). The signal at d (1743, 1684 cm-1), ester carbonyl (1720 cm-1) and double 4.64 was coupled with a double double signal assigned to bond (1654 cm-1) were observed as in 3. the proton (d 5.40, H-13 and d 82.7, C-13), which also cor- The 1Hand13C NMR data (Tables 1 and 2) of 3 were related with a double multiplet of an olefinic proton (d found to be similar to those of 2. This suggested that 3 and 2 4.98, H-14; d 120.4, C-14). This, in turn, was coupled with have a similar skeleton. But 3 featured an additional ace- the broad singlet signals of two vinylic methyls at d 1.74, toxy group (dH 2.08, dC 170.9) at C-8; chemical shifts were (H-16) and d 1.72, (H-17). Furthermore, the structural frag- established by DEPT analysis and HMQC, HMBC. The Studies on Chemical Constituents of Salix cheilophila Schneid J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 403

1 Table 1. H NMR spectral data of compounds 2, 3 and 4 (400 MHz, d in ppm, CDCl3,TMS)* 234 Proton dH J (Hz) dH J (Hz) dH J (Hz) 1(1¢) 6.36 (d, 7.0) 6.32 (d, 7.0) 6.32 (d, 7.0) 2 5.48 (tq, 7.0, 2.0) 5.44 (tq, 7.0, 2.0) 5.46 (tq, 7.0, 2.0) 4 2.10 (tq, 7.0, 1.8) 2.10 (tq, 7.0, 1.8) 2.12 (tq, 7.0, 1.8) 5 1.90 (m) 1.89 (m) 1.90 (m) 6 5.32 (brt, 6.8) 5.29 (brt, 6.8) 5.30 (brt, 6.8) 8a 2.16 (ddd, 11.0, 6.2, 1.8) 4.20 (dd, 11.0, 6.0) 3.98 (dd, 11.0, 6.2) 8b 2.04 (brt, 11.2) 9a 2.23 (m) 2.30 (m) 2.28 (m) 9b 2.08 (m) 2.23 (m) 2.23 (m) 10 6.90 (ddd, 10.2, 6.8, 1.8) 6.89 (ddd, 10.2, 6.8, 1.9) 6.89 (ddd, 10.2, 6.8, 1.9) 12 4.64 (d, 2.0) 4.67 (brs) 4.69 (brs) 13 5.40 (brs) 5.40 (brs) 5.46 (brs) 14 4.98 (brs) 4.98 (brs) 4.94 (brs) 16 1.74 (s) 1.75 (s) 1.73 (s) 17 1.72 (s) 1.73 (s) 1.71 (s) 19 1.45 (s) 1.48 (s) 1.49 (s) 20 1.59 (s) 1.58 (brs) 1.59 (s) OH 3.88 (brs) 3.91 (brs) 3.90 (brs) Oac 2.08 (s) 2.06 (s) MeSen MeSen ang 2¢ 5.66 (tq, 1.2) 5.65 (tq, 1.2) 3¢ 6.06 (qq, 2.0, 9.6) 4¢ 2.17 (dq, 1.2, 7.4) 2.18 (dq, 1.2, 7.4) 1.94 (dq, 2.0, 10.0) 5¢ 1.08 (t, 7.4) 1.07 (t, 7.4) 1.79 (dq, 2.0, 2.0) 6¢ 2.18 (d, 1.2) 2.16 (d, 1.2) * Assignments from 1H-1H COSY, HMBC (from proton to carbon) and HMQC.

structure and relative stereochemistry of 3 was deduced groups at C-1 by the cross peaks dC-1¢ 167.1/dH-1 6.32 in the from COSY, HMBC and NOE difference experiments. The HMBC spectrum. Further, the coupling pattern of H-1 at dH 1 13 H, C NMR spectrum of 3 showed acetoxy signals (dH 6.06 (tq, J = 10 Hz) implied that the angeloyloxy group

2.08, dC 170.9), the NOE interactions for H-8/H-9, H-8/H- linked at C-1. All these observations led to the assignment 10 and H-8/H-6 suggested the acetoxy orientating at C-8. of structure 4 for shaliuin III. From the above information, shaliuin II was determined to have the structure as shown in 3. EXPERIMENTAL SECTION Compound 4 was obtained as pale yellow gum and General Methods had the molecular formula of C27H38O6 which was deduced Melting points were determined using a Kofler melt- from the HR-EIMS spectrum (M+, 458.2970), indicating ing point apparatus and optical rotations were made on a nine degrees of unsaturation. IR absorption at 3357 cm-1 in- DIP-181 instrument. IR and UV spectra were taken on dicated the presence of a hydroxy group, and at 1745, 1682 Perkin-Elmer 599B and Shimadzu UV-300 spectrometers. cm-1 for a,b-unsaturated-g-lactone unit. The 1H, and 13C 1HNMR,13C NMR and 2D NMR spectra were recorded on NMR spectral data (Tables 1 and 2) of 4 were extremely a Bruker AM-400 FT NMR with TMS as internal standard similar to those of 3 except the proton of C-1 was substi- and HR-EIMS and EIMS data were obtained on a MAT-12 tuted by a pair of angloyloxy groups. That could be con- (70 ev). Silica gel (100-200, 200-300 mesh) was used for firmed by the data of the missing methylsenecioyloxy column chromatography and silica gel GF254 for TLC. group of C-1 in 3 at dH 5.65, 2.18, 1.07, 2.16 and the present Plant Material carbon at dC 167.1, 128.1, 138.4, 20.0, 15.2 instead of dC Twigs of S. cheilophila were collected from the west 166.4, 113.9, 163.3, 33.5, 11.8, 18.9 in 3. The angeloylox of Gansu Province of P. R. China, in July 2005, and identi- 404 J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 Shen et al.

13 Table 2. C NMR spectral data of compounds 2, 3 and 4 (100 MHz, d ppm CDCl3,TMS)* 23 4 Carbon dcDEPTHMBC dcDEPTHMBC dcDEPTHMB

1 68.0 CH2 2 68.4 CH2 2 68.1 CH2 2 2 124.1 CH 1a, 1b, 20 124.8 CH 1a, 1b, 20 124.3 CH2 1a, 1b, 20 3 138.0 C 2, 4, 20 138.1 C 2, 4, 20 138.2 CH 2, 4, 20 4 31.2 CH2 2, 6, 20 30.9 CH2 2, 6, 20 30.9 CH 2, 6, 20 5 24.9 CH2 4, 6 24.8 CH2 4, 6 24.9 CH 4, 6 6 125.7 CH 4, 5, 8, 19 124.9 CH 4, 5, 8, 19 125.1 CH 4, 5, 8, 19 7 126.9 C 5, 8, 19 126.6 C 5, 8, 19 127.2 CH 5, 8, 19 8 26.5 CH2 6, 9a, 9b, 19 76.0 CH 6, 9a, 9b, 19 73.9 C 6, 9a, 9b, 19 9 32.0 CH2 8a, 8b, 10 31.9 CH2 8a, 10 31.9 C 8a, 10 10 142.2 CH 8a, 9a, 9b, 12 142.0 CH 8a, 9a, 9b, 12 141.8 C 8a, 9a, 9b, 12 11 132.0 C 9a, 9b, 10, 13 132.6 C 9a, 9b, 10, 13 132.4 C 9a, 9b, 10, 13 12 73.0 CH 10, 13, 14 72.8 CH 10, 13, 14 76.0 CH 10, 13, 14 13 82.7 CH 12, 17 82.5 CH 12, 17 82.5 CH 12, 17 14 120.4 CH 12, 16, 17 120.1 CH 12, 16, 17 120.4 C 12, 16, 17 15 140.1 C 13, 16, 17 140.0 C 13, 16, 17 140.2 CH 13, 16, 17 16 25.5 CH3 14, 17 25.3 CH3 14, 17 25.2 CH 14, 17 17 18.7 CH3 14, 16 18.5 CH3 14, 16 18.7 CH 14, 16 18 170.2 CO 10, 12, 13 170.0 CO 10, 12, 13 170.0 CH3 10, 12, 13 19 11.3 CH3 6, 8a, 8b 11.2 CH3 6, 8a 11.3 CH3 6, 8a 20 23.6 CH3 2, 4 23.7 CH3 2, 4 23.5 C 2, 4 OAc 170.9 CO 171.0 C Meseno Ango 1¢ 166.7 CO 1a, 1b, 2¢ 166.4 CO 1a, 1b, 2¢ 167.1 CO 1a, 1b, 2¢ 2¢ 115.0 CH 4¢,6¢ 113.9 CH 4¢,6¢ 128.1 C 3¢,5¢ 3¢ 163.4 C 4¢,5¢,6¢ 163.3 C 4¢,5¢,6¢ 138.4 CH 4¢,5¢ 4¢ 33.7 CH2 5¢,6¢ 33.5 CH2 5¢,6¢ 20.0 CH3 3¢ 5¢ 11.8 CH3 4¢ 11.8 CH3 4¢ 15.2 CH3 3¢ 6¢ 18.8 CH3 2¢,4¢ 18.9 CH3 2¢,4¢ *Assignments from 1H-1H COSY, HMBC (from proton to carbon) and HMQC fied by Prof Yong-shan Lian of Northwest Normal Univer- tained. From fraction A-B by rechromatography on silica sity. A voucher specimen (No. 35108) was deposited at the gel (200-300 mesh), with n-hexane-CHCl3 (20:1-0:1 v/v) herbarium of the College of Life Science, Northwest Nor- as eluent and preparative TLC, yielded 6 (14 mg), 7 (22 mal University, Lanzhou, 730070, P. R. China. mg), 8 (17 mg) and 9 (20 mg). The fraction C-D, which was Extraction and Isolation chromatographed on a silica gel column using a mixture of The dried twigs of S. cheilophila (6.0 Kg) were ex- n-hexane-EtOAc (8:1, v/v) by means of rechromatography tracted with 95% EtOH three times at room temperature on silica gel (350 g, 200-300 mesh) with n-hexane-CHCl3 (each process lasting 7 days). The extract was concentrated (4:2.5, v/v) as eluent and purified by preparative TLC, under reduced pressure; the residue was extracted with pe- yielded 1 (21 mg), 2 (16 mg), 3 (14 mg) and 4 (19 mg). The troleum ether (60-90 °C), CHCl3,Me2CO and MeOH three Me2O extract was concentrated to a syrup (60 g) and then times, respectively. The petroleum ether extract (68 g) was subjected to chromatographic separation on a silica gel col- chromatographed on silica gel (500 g, 100-200 mesh), us- umn (100-200 mesh, 850 g); the compounds of the mixture ing petrol-Et2O (40:1-0:1, v/v) gradient and purified by were eluted with CHCl3-Me2O (20:1-0:1, v/v) as eluent, preparative TLC, yielding 14 (11 mg), 15 (19 mg) and 16 and gave three fractions (E 9.8 g, F 6.3 g, G 8.7 g). Frac-

(23 mg). The CHCl3 extract was concentrated to a syrup tions D and E were repeatedly rechromatographed over sil- (66 g) which was chromatographed on silica gel (500 g, ica gel and purified by recrystallization or preparative TLC 100-200 mesh), using hexane-EtOAc (70:1-0:1) gradient, to afford 5 (18.4 mg), 10 (24.1 mg), respectively. Fraction four major fractions (A 8 g, B 11 g, C 6.4 g, 7.5 g) being ob- G was fractionated over a silica gel column with CHCl3- Studies on Chemical Constituents of Salix cheilophila Schneid J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 405

EtOAc (4:5.5, v/v) gradient to give 11 (9.1 mg), 12 (15 Received April 24, 2007. mg), and 13 (12.4 mg), respectively. Shaliuin I a 20 ° REFERENCES :C26H38O5,paleyellowgum,[ ] D -131.7 (c, 0.26, CHCl3). HR-EIMS m/z: found 430.2959, required 1. Jiangsu New Medicine College Dictionary of Traditional lMeOH Chinese Drugs; Shanghai Science and Technology Press: 430.2972. UV max = 225 nm; IR (KBr): 3362, 1746, 1718, 1682, 1652, 1460, 1382, 1121, 1030, 975 cm-1.EIMSm/z Shanghai, 1977; p 1162. 2. Wang, J.-X.; Zheng, S.-Z.; Sun, L.-P.; Shen, X.-W.; Li, Y. J. 430 (M+), 412 (M+-H O), 316 (M+-C H O ), 298 (M+- 2 6 10 2 Chin. Chem. Soc. 2002, 49, 437-441. H O-MeSen), 235, 195, 180, 164, 139, 135, 121, 107, 97, 2 3. Wang, H.; Tian, X.; Chen, Y.-Z. J. Chin. Chem. Soc. 2002, 1 13 93, 69, 55, 41. The HNMRand C NMR: see Tables 1 and 49, 434-436. 2. 4. Shen, T.; Jia, Z.-J.; Zheng, S.-Z.; Shen, X.-W. J. Chin. Shaliuin II a 20 2003 :C28H40O6,paleyellowgum,[] D Chem. Soc. , 50, 407-411. -130.9° (c, 0.26, CHCl3). HR-EIMS m/z: found 472.3119, 5. Zheng, S.-Z.; Yang, H.-P.; Ma, X.-M.; Shen, X.-W. Nat. lMeOH Prod. Res. 2004, 18, 403-405. required 472.3124. UV max = 222 nm; IR (KBr): 3359, 1743, 1720, 1684, 1654, 1459, 1380, 1120, 1031, 975 cm-1. 6. Wang, D.-Y.; Zheng, Z.-Z.; Xu, S.-Y.; Zheng, S.-Z. J. Asian + + + Nat. Prod. Res. 2002, 4, 303-305. EIMS m/z 473, 472 (M ), 454 (M -H2O), 358 (M - + 7. Liu, M.-X.; Zheng, Y.-H.; Liu, R.-Y.; Li, X.-G. J. Jilin Agr. C6H10O2), 340 (M -H2O-MeSen), 315, 294, 230, 195, 180, 1998 1 13 Univ. , 20,6-9. 139, 135, 121, 107, 97, 93, 69. The HNMRand CNMR: 8. Sun, L.-P.; Zheng, S.-Z.; Wang, J.-X.; Shen, X.-W. Indian J. see Tables 1 and 2. Chem. Sect. B. 2002, 41B, 1319-1323. Shaliuin III a 20 :C27H38O6,paleyellowgum,[ ] D 9. Gao, K.; Jia, Z.-J. J. Lanzhou Univ. 1997, 33, 77-80. -130.2° (c, 0.26, CHCl3). HR-EIMS m/z: found 458.2970, 10. Cui, H.-D.; Lin, Z.-W. Zhiwuxuebao. 1978, 30, 244-246. lMeOH 11. Maldowado, E.; Bello, M.; Villasenor, J.-L.; Ortega, A. required 458.2966. UV max = 230 nm; IR (KBr): 3357, 1745, 1720, 1683, 1650, 1460, 1381, 1122, 1032, 976 cm-1. Phytochemistry 1998, 49, 1115-1117. + + 12. Zheng, S.-Z.; Wang, J.-X.; Lu, J.-S.; Shen, T.; Sun, L.-P.; EIMS m/z 458 (M ), 440 (M-H2O), 359 (M -C5H9O2), 341 + Shen, X.-W. Planta Med. 2000, 66, 487-490. (M -ang-H2O), 313, 295, 230, 195, 180, 139, 121, 107, 93, 13. Skehan, P.; Storeng, R.; Scudiero, D.-J. J. Natl. Cancer Inst. 69. The 1HNMRand13C NMR: see Tables 1 and 2. 1990, 82, 1107-1110. 14. Ying, B.-P.; Yang, P.-M.; Zhu, H.-R. Acta. Chim. Sinica. ACKNOWLEDGEMENTS 1987, 45, 450-453. This work was supported by the ‘Qing Lan’ Talent 15. Ravi, B.-N.; Faulker, D.-J. J. Org. Chem. 1979, 44, 968-970. Engineering Funds by Lanzhou Jiaotong University and 16. Goren, N.; Kirmizigul, S.; Zdero, C. Phytochemistry 1997, the Science and Technology Foundation of Lanzhou City 44, 311-313. No. 2006-2-18.