- Email: [email protected]
Revised structure of 20-hydroxytingenone and 13C NMR assignments of 22β-hydroxytingenone
Phytochemistry,Vol. 34, No. 3, pp. 159-763,1993
003l-9422/93 $6.00+ 0.00 0 1993Pergamon Press Ltd
Printed in Great Britain.
REVISED STRUCTURE OF 20-HYDROXYTINGENONE AND 13C NMR ASSIGNMENTS OF 22/?-HYDROXYTINGENONE KITTISAK
LIKHITwmYAwum,*tRAPEPOL BAvovADA,t LONG-ZE LIN and GEOFFREYA. CORDELLS
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Phannacognosy, College of Pharmacy, University of Illinois at Chicago, IL 60612, U.S.A.;TDepartment of Pharmaceutical Botany, Chulalongkom University, Bangkok, Thailand (Received 1 December
1992)
Key Word Index-20-Hydroxytingenone; Glyptopetalum
sclerocarpum;
20-hydroxy-20-epi-tingenone; NMR spectroscopy.
228-hydroxytingenone;
Abstract-The
structure of 20-hydroxytingenone was revised to be 20-hydroxy-20-epi-tingenone, based on the results from extensive NMR studies (COSY, ROESY, HMQC, HMBC and selective INEPT experiments). Examination of the HMQC and HMBC spectra of 22/3-hydroxytingenone led to the revision of its previous 13C NMR assignments.
INTRODUCTION
Biological studies on some quinone-methide triterpenoids, including 20-hydroxytingenone (1) and 22/?-hydroxytingenone (2), have revealed the anticancer potential of this group of natural products [l-3]. Compound 1 was first isolated as an orange pigment from Euonymus tingem, and its structure was assumed to be 1, despite the lack of conclusive evidence to establish the stereochemistry at C-20 [4]. Although subsequent reports on the isolation of this compound from other plants [3, 51 and detailed analyses of the ‘H and 13C NMR data [6] have appeared, determination of the configuration of C-20 has never been attempted, probably due to the complexity of the ‘H NMR spectrum (vi& infra). The structure of 2, on the other hand, has been well established [2,7], but there have been some discrepancies between the 13CNMR assignments of 2 [2] and those reported for some structurally related compounds [6], especially the assignments of the carbons in rings A and B. We demonstrate here that 20_hydroxytingenone, in fact, is 20-hydroxy-20-epitingenone, being represented by structure 3, not 1. In addition, the revised 13C NMR assignments of 2 are also presented.
suggested a quinone-methide structure [6, 71, and this was substantiated by the proton signals at 66.53 (br s, HI), 7.01 (br d, J = 6.9 Hz, H-6) and 6.36 (d, J =6.9 Hz, H-7) in the ‘HNMR spectrum (CDCl,) [4-61. Compound 3 was similar to tingenone (4) [6] in its ‘H NMR spectrum (CDCl,), except that all six methyl groups appeared as singlets, resonating at 62.22 (4-Me), 1.48 (9-Me), 1.36 (14and 20-Me), 1.13 (17-Me) and 0.89 (13-Me), and thereby suggesting the presence of a substituent at C-20. The molecular weight of 3, being 16 amu higher than that of 4, necessitated the inclusion of a hydroxy functionality, which was consequently placed at C-20. Determination of the stereochemistry at this carbon centre (C-20) was not directly feasible since distinction between the 14-Me and 20-Me resonances could not be made, due to their
RESULTS AND DISCUSSION
Compound 3, in the HRMS, exhibited a molecular ion at m/z 436.2606, corresponding to the molecular formula C28H3604 (calcd for 436.2614). The UV absorptions at 252 and 420 nm and IR bands at 1715 and 1591 cm-’ *Present address: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. $Author to whom correspondence should be addressed.
PHYTO 34:3-L
RI
R2
1
OH
2
H
3
CH,
4
H
R,
R*
CH,
H
H
CR,
OH
H
OH
H
H
CH,
H
H
160
K.
LIKHITWITAYAWUID~~
al.
Table 1. Summary of the ROESY, HMBC and selective INEPT correlations for 3
Proton 1 6 7 lla llfi 12a
128 15a 15s 16a 168 18 19a 198 22a 228 23 25 26 27 28 29
ROESY (proton)
HMBC* (carbon)
Selective INEPT* (carbon)
11a I, 23 6, 15/3 1l/3,27 1, lla, 128, 25 128, 19a, 198, 27 llg, 12a, 26 15g, 22a I. 15a, 26 16/L 228 16a, 26,28 19/I, 26,28 12a, 198, 27, 29 12a, 18, 19a 15a, 228, 27, 29 16a, 22a, 28 6 118, 26 128, IS/?, 16/I, 18, 25 lla, 121, 19a, 22a, 29 18, 168, 228 19a, 22a, 27
3, 5, 9 4, 8, 10 5, 9. 14
3, 5, 9 4, 8, 10 5, 9, 14
U6h 26
(17) (17), 22, 28 (13), (17), (19), 20, 27,28 13, 17, (18), (20), 21, 29 13, 17, (18), (20). 21. 29 (17), (21), 28 (17), 18, (21), 28 3, (4), 5 8, (9b 10, 11 8, 13, (14), 15 12, (13) 14, 18 16, (17), 18, 22 19, (20). 21
(t7), (2t), 28 3, (4). 5 8, (9b 10, 11 8, 13, (14), 15 12, (13), 14, 18 16, (17), 18, 22 19, (20), 21
*A parenthesis indicates two-bond coupling.
identical chemical shifts (6 1.36 in CDCI,). In this study, a number of mixed NMR solvents consisting of CDCl, and C,D, in different proportions were examined, and it was found that a sample of 3 in the solution of CDCl, : C,D, (1: 1) produced the best resolved ‘H NMR spectrum. Diamagnetic shifts with varying magnitudes were observed for almost all of the protons, as expected. Under this condition (CDCl,: C,D, = 1: l), the signals of the 14-Me and 20-Me protons were well separated from each other, with the former resonating at 6 1.06, and the latter 61.12. The definitive assignment of these two methyl signals and all other methyl resonances was achieved through analysis of the homonuclear COSY, ROESY [8, 93, HMQC [lo], HMBC [ll, 121 and selective INEPT [13, 141 spectra of 3. The results of the ROESY, HMBC and selective INEPT experiments are summarized in Table 1. From the ROESY spectrum, the most downfield methyl resonance at 62.11 showed a NOE contour with H-6 (66.66) and was therefore assigned to H,-23 (4-Me). The NOE between the methyl signal at 6 1.25 and H-11/I (6 1.80) placed this methyl group at C-9. The three-bond couplings between H,-25 (9-Me) and C-8, H,-25 and C10, and Ha-25 and C-11 observed in the HMBC and selective INEPT experiments confirmed this assignment. The H,-25 protons, in the ROESY spectrum, also exhibited a NOE with the methyl resonance at 61.06, which was consequently assigned to Ha-26 (14-Me). This assignment was corroborated by the three-bond connectivities demonstrated between H,-26 and C-8, H,-26 and C-13, and H,-26 and C-15 in the HMBC and selective INEPT
fortuitously
spectra. The H,-27 (13-Me) was assigned to the most upfield methyl resonance (60.60) according to its NOES with H-llsr and H-22a in the ROESY spectrum, and its three-bond couplings with C-12, C-14 and C-18 in the HMBC and selective INEPT experiments. The methyl signal at 6 1.00 was assigned to H,-28 (17-Me) from its NOE with H-22/I. The three-bond couplings of Ha-28 with C-16, C-18 and C-22 in the HMBC and selective INEPT spectra substantiated this assignment. The methyl signal at 6 1.12 was assigned to the 20-Me protons, based on the three-bond couplings observed between 20-Me (H,-29) and C-19, and between 20-Me (Ha-29) and C-21. The NOE observed between 13-Me and 20-Me in the ROESY spectrum (Fig. 1) suggested an a-axial orientation for the 20-Me group. The configuration of C-20 (20fi-hydroxy-2Ou-methyl) of 3 was further co&rued by the NOES displayed between H-22a and 20-Me, and
Fig 1. NOEs observed for the protons in rings D and E of 3.
2~Hydroxytingenone
and 22~-hydroxytingenone
761
Table 2. ‘H and 13CNMR assignments of 3*
C 1 2 3 4 5 6 7 8 9 10 11% B 12a B 13 14 15a B 16% B
17 18 19u
‘HS (CDCl,)
1% (CDCI,:C,D,)
(CDW
6.52 (br s)
6.53
6.66 (br d, 6.9) 6.02 (d, 6.9)
7.01 6.36
1.62 1.80 1.32 1.34
1.95 2.21 1.76 1.82
119.7 178.3 146.2 116.8 127.6 133.1 118.0 168.4 42.7 164.0 32.9
119.8 178.4 146.2 117.1 127.9 133.3 118.3 168.7 42.9 164.2 33.2
29.4
29.9
39.6 43.9 29.1
40.0 44.2 29.4
35.4
35.7
35.6 42.9 36.7
35.9 43.3 36.9
73.4 214.5 50.2
73.7 214.9 50.5
10.2 38.2 23.0 19.1 32.9 28.7
IO:3 38.5 23.3 19.4 33.2 29.0
(m) (ddd, 14.4, 3.8, 3.8) (m) (m)
1.45 1.30 1.25 1.52
(14.4, 12.0, 4.8) (m) (ddd, 14.4, 5.8, 3.8) (ddd, 14.4, 12.0, 5.3)
1.84 1.73 1.65 1.91
1.58
(dd, 9.0, 4.5)
1.93 2.28 2.20
B
1.88 (dd, 15.5, 4.5) 2.06 (dd, 15.5, 9.0)
B
2.58 (d, 14.0) 1.70 (d, 14.0)
20 21 22a 23 25 26 27 28 29
‘Ht (CDCI,:C,D,)
2.11 (3H, s) 1.25(3H, s) 1.06(3H, s) 0.60 (3H, s) 1.00 (3H, s) 1.12(3H, s)
2.99 1.95 2.22 1.48 1.36 0.89 1.13 1.36
‘%I$
*Chemical shifts are reported as ppm (6) from TMS. Wignal multiplicity and coupling constants (Hz) are in parentheses. $.Data from [6].
between H-19cr and 20-Me. On the basis of the above spectral evidence, it was, therefore, concluded that 3 has the structure of 20-hydroxy-20-epi-tingenone. Further analysis of the COSY, ROESY, HMQC and HMBC spectra of 3 led to the unequivocal ‘H and 13CNMR assignments of 3 (Table 2). The physical properties (mp, [~]g’, UV, IR, ‘H and 13CNMR in CDCI,, and MS) of 3, however, were su~~mposable with those published for 1 [4--6]. Direct comparison of 3 with an authentic sample of 1 obtained from Kokoona ochracea [33 (mmp, co-TLC, UV, IR, ‘H and 13C NMR and MS) showed that the two compounds are identical. Hence, it was clearly established that the structure of the so called ‘20-hydroxytingenone’ was incorrectly assigned and should be revised as 20-hydroxy20-epi-tingenone (3). The previous 13CNMR assignments of (2) [2), especially those of C-3, C-4, C-8 and C-10, have raised some
doubts since they are not in agreement with those reported for other structurally closely related compounds, such as pristimerin, 4 and 3 [6]. In an attempt to dissipate this confusion, we re-investigated the ‘H and i3C assign ments of 2 by using a series of NMR experiments which comprised the COSY, ROESY, HMQC and HMBC techniques. The results from analysis of the ROESY and HMBC spectra (Table 3) indicated that the earlier assignments of C-3, C-4, C-8, C-10, C-27 and C-28 [2] should be revised (Table 4). EXPERIMIZNTAL
Mps were determined on a Kofler hot-stage apparatus and are uncorr. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UV spectra were taken in MeOH on a Beckman DU-7 spectrometer. IR spectra were recorded in a KBr pellet on a MIDAC FT-IR
762
K. LIKHITWITAYAWUID et al. Table 4. ‘H and 13CNMR assignments of 2*
Table 3. Summary of the ROESY and HMBC correlations for 2 Proton
ROESY (proton)
HMBC* (carbon)
1 6 7 11% lib 12a 128 15% 15g 16a 168 18 19a 198 20 22a 23 25 26 27 28 30
11s 7, 23 6,15fi lib, 12q 27 1, lla, 128, 25 lla, 12p, 19a.,27 11/3, 12a, 26 15/J, 16a, 221 7, 15a, 168, 26 15a, 168, 28 15a. 16a, 26, 28 19a, 19/J. 26, 28 12a. 18, 198, 27, 30 18, 19a, 28, 30 27, 30 15a, 27, 6 11/3,26 128, 15/I, 168, 18,25 lla, 126 19a, 20,22a 16a, 168, 18, 19/? 19a, 198, 20
3, 5, 9 4, 8, 10 5, 9, 14
C
‘Ht
13C
‘3C$
6.53 (br s)
119.8 178.4 146.1 117.3 127.7 133.6 118.1 168.5 42.6 164.7 34.0
119.8 178.4 168.5 164.7 127.7 133.6 118.1 117.3 42.6 146.1 34.0
29.9
29.9
40.6 44.3 28.3
40.6 44.3 28.3
29.5
29.5
44.8 45.0 32.0
44.8 45.0 32.0
40.9 213.5 76.4 10.3 39.1 21.6 20.5 24.9 14.7
40.9 213.5 76.4 10.3 39.1 21.6 24.9 20.5 14.7
7.03 (br d, 6.7) 6.38 (d, 6.7)
8
(13), (17) (19), 20, 27 17
(17), 28 3, (4b 5 8, (9), 10, 11 8, 13, (14), 15 12, (13), 14, 18 16, (17), 18 19, (20), 21
*A parenthesis indicates two-bond coupling
interferometer. ‘H and 13CNMR spectra were recorded with a General Electric GEn-500 instrument at 500.12 and 125.76 MHz, respectively. APT spectra were re-
corded with a Varian XL-300 spectrometer. COSY and ROESY spectra were recorded at 500.12 MHz using standard GE programs. HMQC and HMBC spectra were obtained at 500.12/125.76 MHz using standard programs from the GE library: “J,_,=6 Hz was used for
HMBC experiments. Plant material and isolation. The plant material and the isolation of 2 were described elsewhere [2]. ‘H and ’ 3C NMR: Table 4. The mother liquor obtained from recrystallization of 2 was further purified by flash CC (silica gel and CHCl,) and prep. TLC (silica gel, CHCl,-benzene, 9: 1) to afford 3 (9 mg). Compound 3. Orange crystals, mp 202-205”, [a];’ + 102” (CHCl,; c 0.1); UV ngfr’ nm (E): 252 (3200x 420 (12690); IR vz cm-i: 1715, 1591, 1516, 1439; ‘H and “CNMR: Table 2; EIMS m/z (rel. int.): 436 [M]’ (47), 422 (14), 267 (6), 253 (14), 241 (20), 227 (14), 215 (1 l), 202 (l@O), 201 (58), 187 (12), 109 (6). HREIMS m/z found 436.2606 [Ml’, calcd C,sH,,O, 436.2614. These physical properties were in excellent agreement with those reported for 1 [4-61, and identical with the authentic 1 obtained from K. ochracea [3].
9 10 lla B 12a B 13 14 15a B 16U B 17 18 19a B 20 21 22a 23 25 26 27 28 30
2.00 (in) 2.23 (m) 1.85 (m) 1.83 (in)
1.86 (m) 1.65 (m) 1.61 (m) 2.23 (m) 1.80 (m) 2.20 (m) 1.77 (m) 2.65 (m) 4.53 (s) 2.21 (3H, 1.50 (3H, 1.35 (3H, 0.96 (3H, 0.85 (3H, 1.05 (3H,
s) s) s) s) s) d, 6.3)
*Recorded in CDCI,; chemical shifts are reported in ppm (6) from TMS. tSigna1 multiplicity and coupling constants (Hz) am in parentheses. $Data from [2].
authentic sample of 1, and Dr G. Doss for the initial establishment of the selective INEPT technique at UIC Cl51.
RFFERENCES
Delle Monache, F. D., Marini Bettolo, G. B., de Lima, 0. G., d’Albuquerque, I. L. and de Barros Co&lho (1973) J. Chem. Sot. Perkin Trans I 2725. Bavovada, R., Blaslcb, G., Shieh, H.-L., Pezzuto, J. M. and Cordell,
G. A. (1990) Planta Med. 56, 380.
Ngassapa, 0. D., Soejarto, D. D., Che, C.-T., Pezzuto, J. M. and Farnsworth, N. R. (1991) 32nd Annual Meeting of the American Society of Pharmacognosy,
thank Research Resources Center of the University of Illinois at Chicago for the provision of the NMR facilities. We are also grateful to Prof. N. R. Farnsworth and Dr 0. D. Ngassapa for an AcknowledgementsWe
Chicago, abstract no. P-24. Brown, P. M., Moir, M., Thomson, R., King, T. J., Krishnamoorthy, V. and Seshadri, T. R. (1973) J. Chem. Sot. Perkin Trans 1 2721.
20-Hydroxytingenone
and 22/Shydroxytingenone
5. Fernando, H. C., Gunatilaka, A. A. L., Tezuka, Y. and Kikuchi, T. (1989) Tetrahedron 45, 5867. 6. Gunatilaka, A. A. L., Fernando, H. C., Kikuchi, T. and Tezuka, Y. (1989) Magn. Reson. Chem. 27, 803. 7. Nakanishi, K., Gullo, V. P., Miura, I., Govindachari, T. R. and Viswanathan, N. (1973) J. Am. Chem. Sot. 95, 6473. 8. Bothner-By, A. A., Stephens, R. L., Lee, L., Warren, C. D. and Jeanloz, R. W. (1984) J. Am. Chem. Sot. 106, 811. 9. Griesinger, G. and Ernst, R. R. (1987) J. 1Wugn.Reson. 75, 261.
163
10. Bax, A. and Subramanian, S. (1986) J. Magn. Reson. 67, 565. 11. Summers, M. F., Marzilli, L. G. and Bax, A. (1986) J. Am. Chem. Sot. 108, 4285. 12. Bax, A., Aszalos, A., Dinya, Z. and Sudo, K. (1986) J. Am. Chem. Sot. 108, 8056. 13. Cordell, G. A. (1991) Phytochem. Anal. 2,49. 14. Cordell, G. A. and Kinghorn, A. D. (1991) Tetrahedron 47, 3521. 15. Abdel-Sayed, A. N. and Bauer, L. (1986) Tetrahedron Letters 27, 1003.
003l-9422/93 $6.00+ 0.00 0 1993Pergamon Press Ltd
Printed in Great Britain.
REVISED STRUCTURE OF 20-HYDROXYTINGENONE AND 13C NMR ASSIGNMENTS OF 22/?-HYDROXYTINGENONE KITTISAK
LIKHITwmYAwum,*tRAPEPOL BAvovADA,t LONG-ZE LIN and GEOFFREYA. CORDELLS
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Phannacognosy, College of Pharmacy, University of Illinois at Chicago, IL 60612, U.S.A.;TDepartment of Pharmaceutical Botany, Chulalongkom University, Bangkok, Thailand (Received 1 December
1992)
Key Word Index-20-Hydroxytingenone; Glyptopetalum
sclerocarpum;
20-hydroxy-20-epi-tingenone; NMR spectroscopy.
228-hydroxytingenone;
Abstract-The
structure of 20-hydroxytingenone was revised to be 20-hydroxy-20-epi-tingenone, based on the results from extensive NMR studies (COSY, ROESY, HMQC, HMBC and selective INEPT experiments). Examination of the HMQC and HMBC spectra of 22/3-hydroxytingenone led to the revision of its previous 13C NMR assignments.
INTRODUCTION
Biological studies on some quinone-methide triterpenoids, including 20-hydroxytingenone (1) and 22/?-hydroxytingenone (2), have revealed the anticancer potential of this group of natural products [l-3]. Compound 1 was first isolated as an orange pigment from Euonymus tingem, and its structure was assumed to be 1, despite the lack of conclusive evidence to establish the stereochemistry at C-20 [4]. Although subsequent reports on the isolation of this compound from other plants [3, 51 and detailed analyses of the ‘H and 13C NMR data [6] have appeared, determination of the configuration of C-20 has never been attempted, probably due to the complexity of the ‘H NMR spectrum (vi& infra). The structure of 2, on the other hand, has been well established [2,7], but there have been some discrepancies between the 13CNMR assignments of 2 [2] and those reported for some structurally related compounds [6], especially the assignments of the carbons in rings A and B. We demonstrate here that 20_hydroxytingenone, in fact, is 20-hydroxy-20-epitingenone, being represented by structure 3, not 1. In addition, the revised 13C NMR assignments of 2 are also presented.
suggested a quinone-methide structure [6, 71, and this was substantiated by the proton signals at 66.53 (br s, HI), 7.01 (br d, J = 6.9 Hz, H-6) and 6.36 (d, J =6.9 Hz, H-7) in the ‘HNMR spectrum (CDCl,) [4-61. Compound 3 was similar to tingenone (4) [6] in its ‘H NMR spectrum (CDCl,), except that all six methyl groups appeared as singlets, resonating at 62.22 (4-Me), 1.48 (9-Me), 1.36 (14and 20-Me), 1.13 (17-Me) and 0.89 (13-Me), and thereby suggesting the presence of a substituent at C-20. The molecular weight of 3, being 16 amu higher than that of 4, necessitated the inclusion of a hydroxy functionality, which was consequently placed at C-20. Determination of the stereochemistry at this carbon centre (C-20) was not directly feasible since distinction between the 14-Me and 20-Me resonances could not be made, due to their
RESULTS AND DISCUSSION
Compound 3, in the HRMS, exhibited a molecular ion at m/z 436.2606, corresponding to the molecular formula C28H3604 (calcd for 436.2614). The UV absorptions at 252 and 420 nm and IR bands at 1715 and 1591 cm-’ *Present address: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. $Author to whom correspondence should be addressed.
PHYTO 34:3-L
RI
R2
1
OH
2
H
3
CH,
4
H
R,
R*
CH,
H
H
CR,
OH
H
OH
H
H
CH,
H
H
160
K.
LIKHITWITAYAWUID~~
al.
Table 1. Summary of the ROESY, HMBC and selective INEPT correlations for 3
Proton 1 6 7 lla llfi 12a
128 15a 15s 16a 168 18 19a 198 22a 228 23 25 26 27 28 29
ROESY (proton)
HMBC* (carbon)
Selective INEPT* (carbon)
11a I, 23 6, 15/3 1l/3,27 1, lla, 128, 25 128, 19a, 198, 27 llg, 12a, 26 15g, 22a I. 15a, 26 16/L 228 16a, 26,28 19/I, 26,28 12a, 198, 27, 29 12a, 18, 19a 15a, 228, 27, 29 16a, 22a, 28 6 118, 26 128, IS/?, 16/I, 18, 25 lla, 121, 19a, 22a, 29 18, 168, 228 19a, 22a, 27
3, 5, 9 4, 8, 10 5, 9. 14
3, 5, 9 4, 8, 10 5, 9, 14
U6h 26
(17) (17), 22, 28 (13), (17), (19), 20, 27,28 13, 17, (18), (20), 21, 29 13, 17, (18), (20). 21. 29 (17), (21), 28 (17), 18, (21), 28 3, (4), 5 8, (9b 10, 11 8, 13, (14), 15 12, (13) 14, 18 16, (17), 18, 22 19, (20). 21
(t7), (2t), 28 3, (4). 5 8, (9b 10, 11 8, 13, (14), 15 12, (13), 14, 18 16, (17), 18, 22 19, (20), 21
*A parenthesis indicates two-bond coupling.
identical chemical shifts (6 1.36 in CDCI,). In this study, a number of mixed NMR solvents consisting of CDCl, and C,D, in different proportions were examined, and it was found that a sample of 3 in the solution of CDCl, : C,D, (1: 1) produced the best resolved ‘H NMR spectrum. Diamagnetic shifts with varying magnitudes were observed for almost all of the protons, as expected. Under this condition (CDCl,: C,D, = 1: l), the signals of the 14-Me and 20-Me protons were well separated from each other, with the former resonating at 6 1.06, and the latter 61.12. The definitive assignment of these two methyl signals and all other methyl resonances was achieved through analysis of the homonuclear COSY, ROESY [8, 93, HMQC [lo], HMBC [ll, 121 and selective INEPT [13, 141 spectra of 3. The results of the ROESY, HMBC and selective INEPT experiments are summarized in Table 1. From the ROESY spectrum, the most downfield methyl resonance at 62.11 showed a NOE contour with H-6 (66.66) and was therefore assigned to H,-23 (4-Me). The NOE between the methyl signal at 6 1.25 and H-11/I (6 1.80) placed this methyl group at C-9. The three-bond couplings between H,-25 (9-Me) and C-8, H,-25 and C10, and Ha-25 and C-11 observed in the HMBC and selective INEPT experiments confirmed this assignment. The H,-25 protons, in the ROESY spectrum, also exhibited a NOE with the methyl resonance at 61.06, which was consequently assigned to Ha-26 (14-Me). This assignment was corroborated by the three-bond connectivities demonstrated between H,-26 and C-8, H,-26 and C-13, and H,-26 and C-15 in the HMBC and selective INEPT
fortuitously
spectra. The H,-27 (13-Me) was assigned to the most upfield methyl resonance (60.60) according to its NOES with H-llsr and H-22a in the ROESY spectrum, and its three-bond couplings with C-12, C-14 and C-18 in the HMBC and selective INEPT experiments. The methyl signal at 6 1.00 was assigned to H,-28 (17-Me) from its NOE with H-22/I. The three-bond couplings of Ha-28 with C-16, C-18 and C-22 in the HMBC and selective INEPT spectra substantiated this assignment. The methyl signal at 6 1.12 was assigned to the 20-Me protons, based on the three-bond couplings observed between 20-Me (H,-29) and C-19, and between 20-Me (Ha-29) and C-21. The NOE observed between 13-Me and 20-Me in the ROESY spectrum (Fig. 1) suggested an a-axial orientation for the 20-Me group. The configuration of C-20 (20fi-hydroxy-2Ou-methyl) of 3 was further co&rued by the NOES displayed between H-22a and 20-Me, and
Fig 1. NOEs observed for the protons in rings D and E of 3.
2~Hydroxytingenone
and 22~-hydroxytingenone
761
Table 2. ‘H and 13CNMR assignments of 3*
C 1 2 3 4 5 6 7 8 9 10 11% B 12a B 13 14 15a B 16% B
17 18 19u
‘HS (CDCl,)
1% (CDCI,:C,D,)
(CDW
6.52 (br s)
6.53
6.66 (br d, 6.9) 6.02 (d, 6.9)
7.01 6.36
1.62 1.80 1.32 1.34
1.95 2.21 1.76 1.82
119.7 178.3 146.2 116.8 127.6 133.1 118.0 168.4 42.7 164.0 32.9
119.8 178.4 146.2 117.1 127.9 133.3 118.3 168.7 42.9 164.2 33.2
29.4
29.9
39.6 43.9 29.1
40.0 44.2 29.4
35.4
35.7
35.6 42.9 36.7
35.9 43.3 36.9
73.4 214.5 50.2
73.7 214.9 50.5
10.2 38.2 23.0 19.1 32.9 28.7
IO:3 38.5 23.3 19.4 33.2 29.0
(m) (ddd, 14.4, 3.8, 3.8) (m) (m)
1.45 1.30 1.25 1.52
(14.4, 12.0, 4.8) (m) (ddd, 14.4, 5.8, 3.8) (ddd, 14.4, 12.0, 5.3)
1.84 1.73 1.65 1.91
1.58
(dd, 9.0, 4.5)
1.93 2.28 2.20
B
1.88 (dd, 15.5, 4.5) 2.06 (dd, 15.5, 9.0)
B
2.58 (d, 14.0) 1.70 (d, 14.0)
20 21 22a 23 25 26 27 28 29
‘Ht (CDCI,:C,D,)
2.11 (3H, s) 1.25(3H, s) 1.06(3H, s) 0.60 (3H, s) 1.00 (3H, s) 1.12(3H, s)
2.99 1.95 2.22 1.48 1.36 0.89 1.13 1.36
‘%I$
*Chemical shifts are reported as ppm (6) from TMS. Wignal multiplicity and coupling constants (Hz) are in parentheses. $.Data from [6].
between H-19cr and 20-Me. On the basis of the above spectral evidence, it was, therefore, concluded that 3 has the structure of 20-hydroxy-20-epi-tingenone. Further analysis of the COSY, ROESY, HMQC and HMBC spectra of 3 led to the unequivocal ‘H and 13CNMR assignments of 3 (Table 2). The physical properties (mp, [~]g’, UV, IR, ‘H and 13CNMR in CDCI,, and MS) of 3, however, were su~~mposable with those published for 1 [4--6]. Direct comparison of 3 with an authentic sample of 1 obtained from Kokoona ochracea [33 (mmp, co-TLC, UV, IR, ‘H and 13C NMR and MS) showed that the two compounds are identical. Hence, it was clearly established that the structure of the so called ‘20-hydroxytingenone’ was incorrectly assigned and should be revised as 20-hydroxy20-epi-tingenone (3). The previous 13CNMR assignments of (2) [2), especially those of C-3, C-4, C-8 and C-10, have raised some
doubts since they are not in agreement with those reported for other structurally closely related compounds, such as pristimerin, 4 and 3 [6]. In an attempt to dissipate this confusion, we re-investigated the ‘H and i3C assign ments of 2 by using a series of NMR experiments which comprised the COSY, ROESY, HMQC and HMBC techniques. The results from analysis of the ROESY and HMBC spectra (Table 3) indicated that the earlier assignments of C-3, C-4, C-8, C-10, C-27 and C-28 [2] should be revised (Table 4). EXPERIMIZNTAL
Mps were determined on a Kofler hot-stage apparatus and are uncorr. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UV spectra were taken in MeOH on a Beckman DU-7 spectrometer. IR spectra were recorded in a KBr pellet on a MIDAC FT-IR
762
K. LIKHITWITAYAWUID et al. Table 4. ‘H and 13CNMR assignments of 2*
Table 3. Summary of the ROESY and HMBC correlations for 2 Proton
ROESY (proton)
HMBC* (carbon)
1 6 7 11% lib 12a 128 15% 15g 16a 168 18 19a 198 20 22a 23 25 26 27 28 30
11s 7, 23 6,15fi lib, 12q 27 1, lla, 128, 25 lla, 12p, 19a.,27 11/3, 12a, 26 15/J, 16a, 221 7, 15a, 168, 26 15a, 168, 28 15a. 16a, 26, 28 19a, 19/J. 26, 28 12a. 18, 198, 27, 30 18, 19a, 28, 30 27, 30 15a, 27, 6 11/3,26 128, 15/I, 168, 18,25 lla, 126 19a, 20,22a 16a, 168, 18, 19/? 19a, 198, 20
3, 5, 9 4, 8, 10 5, 9, 14
C
‘Ht
13C
‘3C$
6.53 (br s)
119.8 178.4 146.1 117.3 127.7 133.6 118.1 168.5 42.6 164.7 34.0
119.8 178.4 168.5 164.7 127.7 133.6 118.1 117.3 42.6 146.1 34.0
29.9
29.9
40.6 44.3 28.3
40.6 44.3 28.3
29.5
29.5
44.8 45.0 32.0
44.8 45.0 32.0
40.9 213.5 76.4 10.3 39.1 21.6 20.5 24.9 14.7
40.9 213.5 76.4 10.3 39.1 21.6 24.9 20.5 14.7
7.03 (br d, 6.7) 6.38 (d, 6.7)
8
(13), (17) (19), 20, 27 17
(17), 28 3, (4b 5 8, (9), 10, 11 8, 13, (14), 15 12, (13), 14, 18 16, (17), 18 19, (20), 21
*A parenthesis indicates two-bond coupling
interferometer. ‘H and 13CNMR spectra were recorded with a General Electric GEn-500 instrument at 500.12 and 125.76 MHz, respectively. APT spectra were re-
corded with a Varian XL-300 spectrometer. COSY and ROESY spectra were recorded at 500.12 MHz using standard GE programs. HMQC and HMBC spectra were obtained at 500.12/125.76 MHz using standard programs from the GE library: “J,_,=6 Hz was used for
HMBC experiments. Plant material and isolation. The plant material and the isolation of 2 were described elsewhere [2]. ‘H and ’ 3C NMR: Table 4. The mother liquor obtained from recrystallization of 2 was further purified by flash CC (silica gel and CHCl,) and prep. TLC (silica gel, CHCl,-benzene, 9: 1) to afford 3 (9 mg). Compound 3. Orange crystals, mp 202-205”, [a];’ + 102” (CHCl,; c 0.1); UV ngfr’ nm (E): 252 (3200x 420 (12690); IR vz cm-i: 1715, 1591, 1516, 1439; ‘H and “CNMR: Table 2; EIMS m/z (rel. int.): 436 [M]’ (47), 422 (14), 267 (6), 253 (14), 241 (20), 227 (14), 215 (1 l), 202 (l@O), 201 (58), 187 (12), 109 (6). HREIMS m/z found 436.2606 [Ml’, calcd C,sH,,O, 436.2614. These physical properties were in excellent agreement with those reported for 1 [4-61, and identical with the authentic 1 obtained from K. ochracea [3].
9 10 lla B 12a B 13 14 15a B 16U B 17 18 19a B 20 21 22a 23 25 26 27 28 30
2.00 (in) 2.23 (m) 1.85 (m) 1.83 (in)
1.86 (m) 1.65 (m) 1.61 (m) 2.23 (m) 1.80 (m) 2.20 (m) 1.77 (m) 2.65 (m) 4.53 (s) 2.21 (3H, 1.50 (3H, 1.35 (3H, 0.96 (3H, 0.85 (3H, 1.05 (3H,
s) s) s) s) s) d, 6.3)
*Recorded in CDCI,; chemical shifts are reported in ppm (6) from TMS. tSigna1 multiplicity and coupling constants (Hz) am in parentheses. $Data from [2].
authentic sample of 1, and Dr G. Doss for the initial establishment of the selective INEPT technique at UIC Cl51.
RFFERENCES
Delle Monache, F. D., Marini Bettolo, G. B., de Lima, 0. G., d’Albuquerque, I. L. and de Barros Co&lho (1973) J. Chem. Sot. Perkin Trans I 2725. Bavovada, R., Blaslcb, G., Shieh, H.-L., Pezzuto, J. M. and Cordell,
G. A. (1990) Planta Med. 56, 380.
Ngassapa, 0. D., Soejarto, D. D., Che, C.-T., Pezzuto, J. M. and Farnsworth, N. R. (1991) 32nd Annual Meeting of the American Society of Pharmacognosy,
thank Research Resources Center of the University of Illinois at Chicago for the provision of the NMR facilities. We are also grateful to Prof. N. R. Farnsworth and Dr 0. D. Ngassapa for an AcknowledgementsWe
Chicago, abstract no. P-24. Brown, P. M., Moir, M., Thomson, R., King, T. J., Krishnamoorthy, V. and Seshadri, T. R. (1973) J. Chem. Sot. Perkin Trans 1 2721.
20-Hydroxytingenone
and 22/Shydroxytingenone
5. Fernando, H. C., Gunatilaka, A. A. L., Tezuka, Y. and Kikuchi, T. (1989) Tetrahedron 45, 5867. 6. Gunatilaka, A. A. L., Fernando, H. C., Kikuchi, T. and Tezuka, Y. (1989) Magn. Reson. Chem. 27, 803. 7. Nakanishi, K., Gullo, V. P., Miura, I., Govindachari, T. R. and Viswanathan, N. (1973) J. Am. Chem. Sot. 95, 6473. 8. Bothner-By, A. A., Stephens, R. L., Lee, L., Warren, C. D. and Jeanloz, R. W. (1984) J. Am. Chem. Sot. 106, 811. 9. Griesinger, G. and Ernst, R. R. (1987) J. 1Wugn.Reson. 75, 261.
163
10. Bax, A. and Subramanian, S. (1986) J. Magn. Reson. 67, 565. 11. Summers, M. F., Marzilli, L. G. and Bax, A. (1986) J. Am. Chem. Sot. 108, 4285. 12. Bax, A., Aszalos, A., Dinya, Z. and Sudo, K. (1986) J. Am. Chem. Sot. 108, 8056. 13. Cordell, G. A. (1991) Phytochem. Anal. 2,49. 14. Cordell, G. A. and Kinghorn, A. D. (1991) Tetrahedron 47, 3521. 15. Abdel-Sayed, A. N. and Bauer, L. (1986) Tetrahedron Letters 27, 1003.