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Sesquiterpene lactones from Inula salsoloides
Perpmon
0031~9422(93)E0173-C
SESQUITERPENE
LACTONES
FROM
Phymchmwrry. Vol 36, No. 3. pp. 721 124, 1994 Copyright Q 1994 Elwvm Saenoe Ltd Prinlcd I” Grcac Brimin. All rights mened 0031~!I42294 s7.cQ+o.o0
ZNULA
BING-NANZHOU, NAI-SHENGBAI, LONG-ZE LIN* and
GEOFFREY
SALSOLOZDES A. CoRDELL*t
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Academia Sink, Shanghai 2CKlO31,People’s Republic of China; *Program for Collaborative Research in the Pharmaceutical Sciences. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, U.S.A. (Receioed in rekedform
11November 1993)
Key Word Index--lnula safsoloides; Inulaceae; sesquiterpene lactones; inulasalsolin; eupatolide; budlein B, NMR techniques; NMR data; cytotoxicity.
inulasalsolide;
Abstract-Two new sesquiterpene lactones, inulasalsolin and inulasalsolide, and the known compounds eupatolide and budlein B, were isolated from fnula salsoloides and their structures assigned unambiguously by COSY, ROESY, HMQC and selective INEPT experiments. The first and third compound showed cytotoxic activity against KB and P-388 cell lines.
INTRODUCnON salsoloides (Turcz.) Ostenf, is distributed in Inner Mongolia, Xin-Jian, Shan-Xi and Gan-Su Provinces of China. It has been used for the treatment of dysentry and inflammatory diseases [ 1J. Recently, Chen et al. reported on the isolation of taraxasterol, taraxasterol acetate, stigmasterol, daucosterol and a sesquiterpene, eupatolide, from this plant [23. In this paper, we report the isolation of four sesquiterpene lactones, inulasalsolin (I), inulasalsolide (2), eupatolide (3) and budlein B (4) from the ethanol extract of the whole plant. The structures and assignments of the ‘H and ’ % NMR parameters of the sesquiterpene derivatives were unambiguously assigned by means of their COSY [3], ROESY [4-61, HMQC [fl and selective INEPT [8, 93 spectra. Inula
RESULTSAND
DISCUSSION
Inulasalsolin (1) was assigned the molecular formula C,jHZOO1 (HRMS). Its IR spectrum suggested hydroxyl (3520 cn- ‘) and a&unsaturated 7-lactone (1740 cm- ‘) moieties, and the ‘H, “C and APT spectra showed the signals for two methyls, four methylenes, five methines, three quaternary carbons and one carbonyl function. The reverse detected HMQC spectrum [7-J of 1 revealed the carbon attached protons, and thus the methylene protons could be easily assigned. The structure was further deduced by the use of COSY [33, ROESY [4-61 and selective INEPT NMR [8, 9) techniques. The results of these experiments are summarized in Table 1. Thus, the structAuthor to whom correspondence should lx addressed.
ture of inulasalsolin was deduced to be 1, and the ‘H and 13C NMR signal parameters were unambiguously assigned as in Tables 3 and 4, respectively. In the ROESY experiment [4-61, the NOES were observed between H-7 and H-8, and H-8 and the methyl at C-10, thus, the C-IO methyl and H-8 might be in the aconfiguration as is H-7, thereby placing the C-8 hydroxyl function in a @-configuration. H-7 showed no NOE with H-6, therefore establishing H-6 in a /I-configuration, whereas H-8 showed a NOE with one of the C-13 methylene protons at 65.67, which is spatially closer to H8; the partner at 66.38 is closer to the carbonyl function. Thus, the stereochemistry should be assigned as shown in 1. Inulasalsolide (2) was soluble in CHCI,, (Me),CO and MeOH, etc. Like 1, the IR spectrum of 2 suggested the presence of hydroxyl (3450 and 3400 cm- ‘) and rx,fiunsaturated y-lactone (1740 cm - ‘) units. Its ’ H, ’ %Zand APT spectra showed the signals for one methyl, five methylenes, five methines, three quaternary carbons and one carbonyl function. The structure of 2 was deduced by use of a combination of COSY [3], ROESY [4-61, HMQC [7-j and selective INEPT [S, 93 experiments, and the results of these experiments are summarized in Table 2. Thus, the structure of inulasalsolide was deduced to be 2, and the ‘H, and 13C NMR signal parameters were unambiguously assigned as in Tables 3 and 4, respectively. The stereochemistry of 2 was also confirmed by a ROESY spectrum [4-61 which showed a NOE between z-H-7 and H-8, establishing H-8 to have an x-configuration, and the C-8 hydroxyl to have a fi-configuration. The NOE between z-H-8 and H-5, and the absence of a NOE between H-6 and H-7, indicated that H-S is in an a721
722
BING-NAN ZHOU et al.
Hb Ha 0
4
Table
Proton
I. Summary
NMR
of the 2D
correlations
cow
ROESY
Selective
(proton)
(proton)
INEPT
of I*
(carbon)
Table
Proton
2. Summary
of the 2D NMR
correlations
COSY
ROESY
Selective
(proton)
(proton)
INEPT
of 2*
(carbon)
lb
la. 2a. 2b
2a. 2b, 9. 15
3. 9
I
2a. 2b
9b
(2). 9, (IO). 15
2a 3
la, lb, 2b. 3 2a. 2b. (14)
lb, 2b, 3, 15 la, 2a, 5b. 14
(3). 4 1. 5, 14
2a
I. lb, 2b, 3b
2b. 3b
(I). (3). 10
3h
2a, 2b. 3a
2a. 2b 3a ’ 5
(4). 5. IS
5b
5a, 6
3.5a.6,
3. (4). (6), 7, 14
5
6
3b. 9b’
3. (4). (6). 7.
6
5a, 5b, 7
5b, 7, 14
4. (5). 8,
II
6
5, 7
9. 14
4. (7). 8, I2
7
6, 8, (13a). (13b)
5b, 8
5. (6). (8). 9, (I I), 12, 13
7
6, 8, (13a). (1%)
8, 9b
5, (6). (8). 9, 13
8
7. 9a, 9b
7, 9a, 9b, 13b, 8-OH
6. (9),
8, 9a (7). 13a
7, 8, Ya 8, 13a
1, (IO). (14) 7. (II). 12
6
3, (4). 5
8
14
II
7. 9
7, 13b. 15
6, (9). 10.
9 13b
8 (7). 13a
lb, 7 8, 13a
I. 7. (8). (IO) 7.(11), 12
14
(3)
3
3. (41. 5
lb, 2a
I. 9. (IO)
15
*Long
range couplings of protons and two-bond
between ‘H and ‘%Z
Yb 13b 14 I5
couplings
I5
(I IL 12,
10. I I
I, 9. (10)
IS-OH
10. (15)
are shown In parentheses. *Long
configuration, thus, the epoxide linkage at C-5 is in a iiconfiguration. A NOE was observed between P-H-6 and the H-15 methyl, which should be in the /I-configuration, thus, the epoxide linkage at C-4 should be in an zconfiguration. H-8 showed a NOE with one of the C-13 methylene protons at 65.78, which should be spatially closer to H-6; the partner at 66.15 being closer to the carbonyl function. This spectrum also showed NOES between the &-OH and C, ,-OH, H-3,, and H-5, H-6 and H-9b, and H-6 and H-9,. Thus, the stereochemistry should be assigned as shown in 2. Eupatolide (3) was obtained as cubic crystals and was identified by direct comparison with an authentic sample. Its ‘H and “CNMR assignments were established to be the same as those in the literature [lo--121. Budlein B (4)
range couplings of protons
between ‘H and “C
and two-bond
couplings
are shown in parentheses.
was obtained as needles and exhibited the same physical data as reported previously [ 12, 133. 1r1vitro bio-assay evaluation [14, 151 of 1-4 indicated that 1 and 3 were cytotoxic against the KB cell line at 1.2 and 1.6jcgml- I, respectively, and the P-388 cell line at 0. I and 0.5 pg ml _ I, respectively. Compounds 2 and 4 were notactiveinKB(>IOpgml. ‘)andP-388(>5pgml-‘) tests. The structure of 2 has recently been confirmed by X-ray crystallographic analysis [ I63 EXPERIMEh7AL
Mp: uncorr.; MS: MAT 711 and Hitachi instruments; HRMS: Finnigan MAT-90 instrument. ‘H, “C. COSY,
Sesquiterpene lactones from Inula salsoloides
123
Table 3. ‘H NMR data of l-3’ H la lb 2a 2b 3a 3b 5a 5b 6 1 8 9a 8b 13a 13b 14 15a 15b 8-OH 15-OH
1
2
3
2.15 (m) 1.25 (m) 2.48 (m) 2.17 (m) 5.19 (br d, 10.6) _ 2.63 (dd, 13.6, 5.0) 2.32 (dd, 13.6, 1.6) 4.59 (t. 8.2) 3.03 (m) 4.61 (m) 2.63 (dd, 13.6, 5.0) 2.32 (dd, 13.6, 1.6) 6.38 (d, 3.5) 5.67 (d, 3.5) 1.35 (s) 1.87 (s)
5.40 (dd, 12.5, 3.3) 2.46 (m) 2.25 (m) 1.99 (dd, 12.9, 5.0) 1.14 (m) 2.92 (d, 8.4) -
4.71 (dd, 11.1, 1.6)
4.30 (1, 8.4) 3.44 (m) 4.37 (1. 2.0) 2.64 (dd. 13.3, 4.8) 2.43 (dd, 13.3. 5.7) 6.15 (d, 3.2) 5.78 (d. 3.2) 1.14 (s) 3.94 (dd, 11.7, 5.3) 4.10 (dd, 11.7, 5.3) 5.57 (d, 2) 5.05 (d, 5.3)
2.28 (m) 2.06 (m) 2.06 (m) 2.22 (m) 4.77 (d. 9.5) 5.09 (dd, 7.9, 4.0) 2.82 (dd, 7.9, 4.0) 4.43 (m) 2.45 (dd, 12.6, 4.0) 2.16 (br d, 12.6) 6.10 (d, 3.0) 5.63 (d. 3.0) 1.59 (br s) 1.53 (br s) _
_
*Compound 1 was recorded in CDCI,, and 2 and 3 were recorded in DMSO-d,, chemical shift values are reported as 6 values (ppm) from TMS at 500 MHz, signal multiplicity and coupling constants (Hz) are shown in parentheses.
Table 4. “CNMR
data of l-3*
C
1
2
3
I
36.0 24.2 127.1 133.4 47.3 74.0 50.7 75.1 66.8 62.1 137.9 169.5 122.1 17.2 20.4
136.3 23.6 35.7 61.4 65.2 72.6 49.3 75.4 42.8 129.5 138.9 169.5 121.8 16.8 59.9
136.2 25.8 38.8 140.4 128.2 14.9 52.8 70.5 47.2 127.9 138.9 170.0 120.2 17.0 19.6
2 3 4 5 6 7 8 9 10 11 12 13 14 I5
*Compound 1 was recorded in CDCI,, and 2 and 3 were recorded in DMSO-d,, chemical shift values are reported as 6 values (ppm) from TMS at 126 MHz.
ROESY and HMQC NMR spectra were recorded with a General Electric GN-500 instrument, using standard programs. APT and selective INEPT spectra were recorded with a Nicolet NT 360 instrument. Plant materials. Inula salsoloides (Turn.) Ostenf. was collected from Yu-Lin County and identified by Dr Hong-Hua Yang, Yu-lin Institute for Drug Quality Con-
trol. A herbarium specimen was deposited in the herbarium of the Shanghai Institute of Materia Medica, Academia Sinica. Extraction and separation. The whole plants (17 kg) of I. salsoloides were percolated with 95% EtOH at room temp. The CHCl,-soluble part (650 g) from the EtOH extract was subjected to CC on silica gel. Compounds 1 (110 mg, 0.00065%) and 3 (3 g, 0.18%) were eluted with CHCl,-Me&O (15~1). and 2 (30mg, 0.00018%) and 4 (56 mg, 0.00033%) with CHCI,-Me&O (5: 1). Inulasalsolin (1) was obtained as needles, mp 1366138”, [a&,- 186.3” (MeOH; ~0.085). UV i.$p nm (log E): 212 (3.92); IR vi;:: 3520, 2990, 2930, 2900, 2860, 1740, 1655, 1440, 1400. 1340, 1300 and 1270cm-‘; ‘HNMR: Table 3: “CNMR: Table 4; EIMR m/z: 264 [Ml’, 246,238,213,203, 189, 188 and 177; HREIMS: observed 264.1362 for C,sHzcO,, calcd 264.1361. Inulasalsolide (2) was obtained as cubes, mp 158-l W, [z]u + 174.5”(MeOH; c 0.112). UV j.:$‘” nm (log E): 213 (3.86); IR v”,:;: 3450, 3400, 3090, 2980, 2942, 2910, 2860, 1740, 1655, 1480, 1440, 1400, 1340, 1300, 1276 and 1160 cm-‘; ‘HNMR. Table 3; 13CNMR:Table 4; EIMS m/z:262 [M - 183’. 244,216,215,204,201,194.187.177, 176 and 175; HREIMS: 262.1232 for C,sH,sO., [M -H,O]+, calcd 262.1205. Eupatolide (3) was obtained as cubes, mp 185-187”. [u]n +45.3” (MeOH; cO.l02), UV, IR and MS were the same as indicated in refs [lo-123; ‘H and 13C NMR: Tables 3 and 4. Budlein B(4) was obtained as needles. The mp 163-165’. [a&-22.7” (MeOH; c 0.088). and other physical data were the same as indicated in refs [ 12, 133.
BING-NAN ZHOU er ul.
724 Cyfotoxicity
utilizing cultured [14, IS].
Cytotoxicity was determined P-388, and KB cells as described in refs
eoaluation.
C. D. and Jeanloz,
R. W. (1984) J. Am. Chem. Sot.
106, 81 I. 5. Summers,
M. F., Marzilli,
L. G. and Bax, A. (1986)
J. Am. Chem. Sot. 108, 4285.
work was supported, in part, by a grant from the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD to the U.S.A. group, and funds from National Natural Sciences Foundation of P.R. China to the Chinese group. The authors thank Dr George Doss for the initial establishment of the selective INEPT NMR technique at the University of Illinois at Chicago [ 163, Dr H. B. Chai. MS M. You and Dr J. M. Pezzuto for the biological tests, Mr R. Dvorak for the high resolution mass spectra, and the Research Resources Center of the University of Illinois at Chicago for the provision of NMR spectroscopic facilities. Acknowledyements-This
REFEREKCFS 1.
(1977) in Dictionary o/Chinese Herbs. p. 4068. JiangSu Medical University, Shanghai Science and Technology Press. 2. Chen, J.. Huang, S.-G., Li, F.-G., Fan, S.-D. and Chen, Y. (1992) Acta Botan. Sin. 34, 62. 3. Bax, A. and Freeman, R. (1981) J. Mugn. Reson. 44.
C. and Ernst. R. R. (1987) J. Mayn. Reson.
75, 261. 7. Bax, A. and Subramanian,
S. (1986) J. Magn. Reson. 67, 565. 8. Bax, A. (1984) J. Magn. Reson. 57, 314. 9. Cordell, G. A. and Kinghorn. A. D. (1991) Tetrahedron 47, 352. 10. Lee, K. H., Huang,
H. C., Huang, E. S. and Furukawa, H. (1972) J. Pharm. Sci. 61, 629. 11. McPhail, A. T. and Onan, K. D. (1974) Tetrahedron Letters
36, 3203.
12. Deigado,
G., Alvarez, L. and Romo de Vivar, A. (1985) Phytochemistry 24, 2736. 13. Romo de Vivar, A., Guerrero, C., Diaz, E., Bratoeff, E. A. and Jimenez, L. (1976) Phyrochemistry IS, 525. 14. Kigodi, P. G. K., Blasko, G., Thebtaranonth, Y., Pezzuto, J. M. and Cordell, G. A. (1989) J. Nat. Prod. 52, 1246. 15. Lin, L.-J., Ruangrungsi,
N., Cordell, G. A., Shieh, H.-L., You, M. and Pezzuto, J. M. (1992) Phyto-
chemsitry
31.4333.
16. Abdel-Sayed,
542. 4. Bothner-By,
6. Griesinger,
A. A.. Stephens,
R. L., Lee, J., Warren,
Letters
A. N. and Bauer, L. (1986) Tetrahedron
27. 1003.
0031~9422(93)E0173-C
SESQUITERPENE
LACTONES
FROM
Phymchmwrry. Vol 36, No. 3. pp. 721 124, 1994 Copyright Q 1994 Elwvm Saenoe Ltd Prinlcd I” Grcac Brimin. All rights mened 0031~!I42294 s7.cQ+o.o0
ZNULA
BING-NANZHOU, NAI-SHENGBAI, LONG-ZE LIN* and
GEOFFREY
SALSOLOZDES A. CoRDELL*t
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Academia Sink, Shanghai 2CKlO31,People’s Republic of China; *Program for Collaborative Research in the Pharmaceutical Sciences. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, U.S.A. (Receioed in rekedform
11November 1993)
Key Word Index--lnula safsoloides; Inulaceae; sesquiterpene lactones; inulasalsolin; eupatolide; budlein B, NMR techniques; NMR data; cytotoxicity.
inulasalsolide;
Abstract-Two new sesquiterpene lactones, inulasalsolin and inulasalsolide, and the known compounds eupatolide and budlein B, were isolated from fnula salsoloides and their structures assigned unambiguously by COSY, ROESY, HMQC and selective INEPT experiments. The first and third compound showed cytotoxic activity against KB and P-388 cell lines.
INTRODUCnON salsoloides (Turcz.) Ostenf, is distributed in Inner Mongolia, Xin-Jian, Shan-Xi and Gan-Su Provinces of China. It has been used for the treatment of dysentry and inflammatory diseases [ 1J. Recently, Chen et al. reported on the isolation of taraxasterol, taraxasterol acetate, stigmasterol, daucosterol and a sesquiterpene, eupatolide, from this plant [23. In this paper, we report the isolation of four sesquiterpene lactones, inulasalsolin (I), inulasalsolide (2), eupatolide (3) and budlein B (4) from the ethanol extract of the whole plant. The structures and assignments of the ‘H and ’ % NMR parameters of the sesquiterpene derivatives were unambiguously assigned by means of their COSY [3], ROESY [4-61, HMQC [fl and selective INEPT [8, 93 spectra. Inula
RESULTSAND
DISCUSSION
Inulasalsolin (1) was assigned the molecular formula C,jHZOO1 (HRMS). Its IR spectrum suggested hydroxyl (3520 cn- ‘) and a&unsaturated 7-lactone (1740 cm- ‘) moieties, and the ‘H, “C and APT spectra showed the signals for two methyls, four methylenes, five methines, three quaternary carbons and one carbonyl function. The reverse detected HMQC spectrum [7-J of 1 revealed the carbon attached protons, and thus the methylene protons could be easily assigned. The structure was further deduced by the use of COSY [33, ROESY [4-61 and selective INEPT NMR [8, 9) techniques. The results of these experiments are summarized in Table 1. Thus, the structAuthor to whom correspondence should lx addressed.
ture of inulasalsolin was deduced to be 1, and the ‘H and 13C NMR signal parameters were unambiguously assigned as in Tables 3 and 4, respectively. In the ROESY experiment [4-61, the NOES were observed between H-7 and H-8, and H-8 and the methyl at C-10, thus, the C-IO methyl and H-8 might be in the aconfiguration as is H-7, thereby placing the C-8 hydroxyl function in a @-configuration. H-7 showed no NOE with H-6, therefore establishing H-6 in a /I-configuration, whereas H-8 showed a NOE with one of the C-13 methylene protons at 65.67, which is spatially closer to H8; the partner at 66.38 is closer to the carbonyl function. Thus, the stereochemistry should be assigned as shown in 1. Inulasalsolide (2) was soluble in CHCI,, (Me),CO and MeOH, etc. Like 1, the IR spectrum of 2 suggested the presence of hydroxyl (3450 and 3400 cm- ‘) and rx,fiunsaturated y-lactone (1740 cm - ‘) units. Its ’ H, ’ %Zand APT spectra showed the signals for one methyl, five methylenes, five methines, three quaternary carbons and one carbonyl function. The structure of 2 was deduced by use of a combination of COSY [3], ROESY [4-61, HMQC [7-j and selective INEPT [S, 93 experiments, and the results of these experiments are summarized in Table 2. Thus, the structure of inulasalsolide was deduced to be 2, and the ‘H, and 13C NMR signal parameters were unambiguously assigned as in Tables 3 and 4, respectively. The stereochemistry of 2 was also confirmed by a ROESY spectrum [4-61 which showed a NOE between z-H-7 and H-8, establishing H-8 to have an x-configuration, and the C-8 hydroxyl to have a fi-configuration. The NOE between z-H-8 and H-5, and the absence of a NOE between H-6 and H-7, indicated that H-S is in an a721
722
BING-NAN ZHOU et al.
Hb Ha 0
4
Table
Proton
I. Summary
NMR
of the 2D
correlations
cow
ROESY
Selective
(proton)
(proton)
INEPT
of I*
(carbon)
Table
Proton
2. Summary
of the 2D NMR
correlations
COSY
ROESY
Selective
(proton)
(proton)
INEPT
of 2*
(carbon)
lb
la. 2a. 2b
2a. 2b, 9. 15
3. 9
I
2a. 2b
9b
(2). 9, (IO). 15
2a 3
la, lb, 2b. 3 2a. 2b. (14)
lb, 2b, 3, 15 la, 2a, 5b. 14
(3). 4 1. 5, 14
2a
I. lb, 2b, 3b
2b. 3b
(I). (3). 10
3h
2a, 2b. 3a
2a. 2b 3a ’ 5
(4). 5. IS
5b
5a, 6
3.5a.6,
3. (4). (6), 7, 14
5
6
3b. 9b’
3. (4). (6). 7.
6
5a, 5b, 7
5b, 7, 14
4. (5). 8,
II
6
5, 7
9. 14
4. (7). 8, I2
7
6, 8, (13a). (13b)
5b, 8
5. (6). (8). 9, (I I), 12, 13
7
6, 8, (13a). (1%)
8, 9b
5, (6). (8). 9, 13
8
7. 9a, 9b
7, 9a, 9b, 13b, 8-OH
6. (9),
8, 9a (7). 13a
7, 8, Ya 8, 13a
1, (IO). (14) 7. (II). 12
6
3, (4). 5
8
14
II
7. 9
7, 13b. 15
6, (9). 10.
9 13b
8 (7). 13a
lb, 7 8, 13a
I. 7. (8). (IO) 7.(11), 12
14
(3)
3
3. (41. 5
lb, 2a
I. 9. (IO)
15
*Long
range couplings of protons and two-bond
between ‘H and ‘%Z
Yb 13b 14 I5
couplings
I5
(I IL 12,
10. I I
I, 9. (10)
IS-OH
10. (15)
are shown In parentheses. *Long
configuration, thus, the epoxide linkage at C-5 is in a iiconfiguration. A NOE was observed between P-H-6 and the H-15 methyl, which should be in the /I-configuration, thus, the epoxide linkage at C-4 should be in an zconfiguration. H-8 showed a NOE with one of the C-13 methylene protons at 65.78, which should be spatially closer to H-6; the partner at 66.15 being closer to the carbonyl function. This spectrum also showed NOES between the &-OH and C, ,-OH, H-3,, and H-5, H-6 and H-9b, and H-6 and H-9,. Thus, the stereochemistry should be assigned as shown in 2. Eupatolide (3) was obtained as cubic crystals and was identified by direct comparison with an authentic sample. Its ‘H and “CNMR assignments were established to be the same as those in the literature [lo--121. Budlein B (4)
range couplings of protons
between ‘H and “C
and two-bond
couplings
are shown in parentheses.
was obtained as needles and exhibited the same physical data as reported previously [ 12, 133. 1r1vitro bio-assay evaluation [14, 151 of 1-4 indicated that 1 and 3 were cytotoxic against the KB cell line at 1.2 and 1.6jcgml- I, respectively, and the P-388 cell line at 0. I and 0.5 pg ml _ I, respectively. Compounds 2 and 4 were notactiveinKB(>IOpgml. ‘)andP-388(>5pgml-‘) tests. The structure of 2 has recently been confirmed by X-ray crystallographic analysis [ I63 EXPERIMEh7AL
Mp: uncorr.; MS: MAT 711 and Hitachi instruments; HRMS: Finnigan MAT-90 instrument. ‘H, “C. COSY,
Sesquiterpene lactones from Inula salsoloides
123
Table 3. ‘H NMR data of l-3’ H la lb 2a 2b 3a 3b 5a 5b 6 1 8 9a 8b 13a 13b 14 15a 15b 8-OH 15-OH
1
2
3
2.15 (m) 1.25 (m) 2.48 (m) 2.17 (m) 5.19 (br d, 10.6) _ 2.63 (dd, 13.6, 5.0) 2.32 (dd, 13.6, 1.6) 4.59 (t. 8.2) 3.03 (m) 4.61 (m) 2.63 (dd, 13.6, 5.0) 2.32 (dd, 13.6, 1.6) 6.38 (d, 3.5) 5.67 (d, 3.5) 1.35 (s) 1.87 (s)
5.40 (dd, 12.5, 3.3) 2.46 (m) 2.25 (m) 1.99 (dd, 12.9, 5.0) 1.14 (m) 2.92 (d, 8.4) -
4.71 (dd, 11.1, 1.6)
4.30 (1, 8.4) 3.44 (m) 4.37 (1. 2.0) 2.64 (dd. 13.3, 4.8) 2.43 (dd, 13.3. 5.7) 6.15 (d, 3.2) 5.78 (d. 3.2) 1.14 (s) 3.94 (dd, 11.7, 5.3) 4.10 (dd, 11.7, 5.3) 5.57 (d, 2) 5.05 (d, 5.3)
2.28 (m) 2.06 (m) 2.06 (m) 2.22 (m) 4.77 (d. 9.5) 5.09 (dd, 7.9, 4.0) 2.82 (dd, 7.9, 4.0) 4.43 (m) 2.45 (dd, 12.6, 4.0) 2.16 (br d, 12.6) 6.10 (d, 3.0) 5.63 (d. 3.0) 1.59 (br s) 1.53 (br s) _
_
*Compound 1 was recorded in CDCI,, and 2 and 3 were recorded in DMSO-d,, chemical shift values are reported as 6 values (ppm) from TMS at 500 MHz, signal multiplicity and coupling constants (Hz) are shown in parentheses.
Table 4. “CNMR
data of l-3*
C
1
2
3
I
36.0 24.2 127.1 133.4 47.3 74.0 50.7 75.1 66.8 62.1 137.9 169.5 122.1 17.2 20.4
136.3 23.6 35.7 61.4 65.2 72.6 49.3 75.4 42.8 129.5 138.9 169.5 121.8 16.8 59.9
136.2 25.8 38.8 140.4 128.2 14.9 52.8 70.5 47.2 127.9 138.9 170.0 120.2 17.0 19.6
2 3 4 5 6 7 8 9 10 11 12 13 14 I5
*Compound 1 was recorded in CDCI,, and 2 and 3 were recorded in DMSO-d,, chemical shift values are reported as 6 values (ppm) from TMS at 126 MHz.
ROESY and HMQC NMR spectra were recorded with a General Electric GN-500 instrument, using standard programs. APT and selective INEPT spectra were recorded with a Nicolet NT 360 instrument. Plant materials. Inula salsoloides (Turn.) Ostenf. was collected from Yu-Lin County and identified by Dr Hong-Hua Yang, Yu-lin Institute for Drug Quality Con-
trol. A herbarium specimen was deposited in the herbarium of the Shanghai Institute of Materia Medica, Academia Sinica. Extraction and separation. The whole plants (17 kg) of I. salsoloides were percolated with 95% EtOH at room temp. The CHCl,-soluble part (650 g) from the EtOH extract was subjected to CC on silica gel. Compounds 1 (110 mg, 0.00065%) and 3 (3 g, 0.18%) were eluted with CHCl,-Me&O (15~1). and 2 (30mg, 0.00018%) and 4 (56 mg, 0.00033%) with CHCI,-Me&O (5: 1). Inulasalsolin (1) was obtained as needles, mp 1366138”, [a&,- 186.3” (MeOH; ~0.085). UV i.$p nm (log E): 212 (3.92); IR vi;:: 3520, 2990, 2930, 2900, 2860, 1740, 1655, 1440, 1400. 1340, 1300 and 1270cm-‘; ‘HNMR: Table 3: “CNMR: Table 4; EIMR m/z: 264 [Ml’, 246,238,213,203, 189, 188 and 177; HREIMS: observed 264.1362 for C,sHzcO,, calcd 264.1361. Inulasalsolide (2) was obtained as cubes, mp 158-l W, [z]u + 174.5”(MeOH; c 0.112). UV j.:$‘” nm (log E): 213 (3.86); IR v”,:;: 3450, 3400, 3090, 2980, 2942, 2910, 2860, 1740, 1655, 1480, 1440, 1400, 1340, 1300, 1276 and 1160 cm-‘; ‘HNMR. Table 3; 13CNMR:Table 4; EIMS m/z:262 [M - 183’. 244,216,215,204,201,194.187.177, 176 and 175; HREIMS: 262.1232 for C,sH,sO., [M -H,O]+, calcd 262.1205. Eupatolide (3) was obtained as cubes, mp 185-187”. [u]n +45.3” (MeOH; cO.l02), UV, IR and MS were the same as indicated in refs [lo-123; ‘H and 13C NMR: Tables 3 and 4. Budlein B(4) was obtained as needles. The mp 163-165’. [a&-22.7” (MeOH; c 0.088). and other physical data were the same as indicated in refs [ 12, 133.
BING-NAN ZHOU er ul.
724 Cyfotoxicity
utilizing cultured [14, IS].
Cytotoxicity was determined P-388, and KB cells as described in refs
eoaluation.
C. D. and Jeanloz,
R. W. (1984) J. Am. Chem. Sot.
106, 81 I. 5. Summers,
M. F., Marzilli,
L. G. and Bax, A. (1986)
J. Am. Chem. Sot. 108, 4285.
work was supported, in part, by a grant from the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD to the U.S.A. group, and funds from National Natural Sciences Foundation of P.R. China to the Chinese group. The authors thank Dr George Doss for the initial establishment of the selective INEPT NMR technique at the University of Illinois at Chicago [ 163, Dr H. B. Chai. MS M. You and Dr J. M. Pezzuto for the biological tests, Mr R. Dvorak for the high resolution mass spectra, and the Research Resources Center of the University of Illinois at Chicago for the provision of NMR spectroscopic facilities. Acknowledyements-This
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