Absolute configuration of novel bioactive flavonoids from Tephrosia purpurea.

Article abstract of DOI:10.1021/ol990407c

[structure: see text] Three novel flavonoids, (+)-tephrorins A (1) and B (2) and (+)-tephrosone (3), were isolated from Tephrosia purpurea. Their structures were elucidated by NMR spectral analysis, and their absolute configurations were determined by Mosher ester methodology. Compounds 1 and 2 are flavanones containing an unusual tetrahydrofuran moiety. Compounds 1-3 were evaluated for their potential cancer chemopreventive properties using a cell-based quinone reductase induction assay.

Full text of DOI:10.1021/ol990407c

ORGANIC
LETTERS
2000
Vol. 2, No. 4
515-518
Absolute Configuration of Novel
Bioactive Flavonoids from Tephrosia
purpurea
Leng Chee Chang, Daniel Cha´vez, Lynda L. Song, Norman R. Farnsworth,
John M. Pezzuto, and A. Douglas Kinghorn*
Program for CollaboratiVe Research in the Pharmaceutical Sciences and
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
UniVersity of Illinois at Chicago, Chicago, Illinois 60612
kinghorn@uic.edu
Received December 21, 1999
ABSTRACT
Three novel flavonoids, (+)-tephrorins A (1) and B (2) and (+)-tephrosone (3), were isolated from Tephrosia purpurea. Their structures were
elucidated by NMR spectral analysis, and their absolute configurations were determined by Mosher ester methodology. Compounds 1 and 2
are flavanones containing an unusual tetrahydrofuran moiety. Compounds 1−3 were evaluated for their potential cancer chemopreventive
properties using a cell-based quinone reductase induction assay.
The genus Tephrosia (Leguminosae) comprises more than
300 members found in India and the southern part of Africa,
and extracts of some Tephrosia species have shown anti-
bacterial,1 antifungal,1 and insecticidal activities.2 In our
ongoing project directed toward the discovery of novel
naturally occurring cancer chemopreventive agents from
plants,3 the whole flowering and fruiting parts of Tephrosia
purpurea Pers. were chosen for detailed investigation, since
its petroleum ether- and ethyl acetate-soluble extracts
significantly induced quinone reductase (QR) activity with
cultured Hepa 1c1c7 (mouse hepatoma) cells.4 Induction of
Phase II drug-metabolizing enzymes such as QR is consid-
ered a major mechanism of protection against tumor initia-
tion.3,5 We earlier reported an initial phytochemical and
biological investigation on this plant, with the isolation of
several flavonoids as quinone reductase inducers.4
Many flavonoids have been reported from the genus
Tephrosia, with their structures determined by spectroscopic
analysis and chemical methods, but their absolute configura-
tions typically have not been studied, with the exception of
the recent reports on (+)-purpurin,6 (-)-semiglabrin,7 and
(+)-pseudosemiglabrin.7 Herein, we report the isolation of
three additional novel flavonoids, compounds 1-3, from
Tephrosia purpurea, in addition to their absolute configura-
tions and activity as inducers of quinone reductase.
Bioassay-guided fractionation of an EtOAc-soluble residue
of Tephrosia purpurea involving successive silica gel and
Sephadex LH-20 chromatographic steps afforded three novel
(1) Bashir, A. K.; EI-Hassan, S. S.; Amiri, M. H.; Abdalla, A. A.
Fitoterapia 1992, 63, 371.
(2) Kole, R. K.; Satpathi, C.; Chowdhury, A.; Ghosh, M. R.;
Adityachaudhury, N. J. Agric. Food Chem. 1992, 40, 1208.
(3) Pezzuto, J. M. In Recent AdVances in Phytochemistry; Vol. 29.
Phytochemistry of Medicinal Plants; Arnason, J. T., Mata, R., Romeo, J.
T., Eds.; Plenum Press: New York, 1995; p 19.
(5) Gerha¨user, C.; You, M.; Liu, J.; Moriarty, R. M.; Hawthorne, M.;
Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Cancer Res. 1997, 57, 272.
(6) Pirrung, M. C.; Lee, Y. R.; Morehead, A. T., Jr.; McPhail, A. T. J.
Nat. Prod. 1998, 61, 89.
(4) Chang, L. C.; Gerha¨user, C.; Song, L.; Farnsworth, N. R.; Pezzuto,
J. M.; Kinghorn, A. D. J. Nat. Prod. 1997, 60, 869.
(7) Pirrung, M. C.; Lee, Y. R. J. Am. Chem. Soc. 1995, 117, 4814.
10.1021/ol990407c CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/01/2000

flavonoids, namely, (+)-tephrorins A (1) and B (2) and (+)-
tephrosone (3).8
The relative stereochemistry of compound 1 in the pairs
H-2′′/H-3′′ and H-3′′/H-4′′ was established as cis by their
coupling constants and from a 1D NOE experiment. In the
1H NMR spectrum of 1, a doublet of doublets (J ) 9.4, 5.7
Hz) at δH 4.16 (H-3′′), a doublet (J ) 5.7 Hz) at δH 5.74
(H-2′′), and a doublet (J ) 9.4 Hz) at δH 5.63 (H-4′′) were
observed as an ABX system. In the 1D NOE experiment,
irradiation at OCH3-7 (δH 3.93) gave enhancements of H-2′′
and H-6, whereas irradiation at H-3′′ (δH 4.16) gave an
enhancement of H-2′′.
The absolute configuration of the stereogenic centers in 1
was determined using Mosher ester methodology.11,12 Com-
pound 1 was treated with (R)- and (S)-(-)-R-methoxy-R-
(trifluoromethyl)phenylacetyl chloride to obtain the mono-
(S)- (1s) and mono-(R)-ester (1r) C-2′′ analogues.11 The
negative values (∆δS-R) obtained for the methyls in C-5′′
and the positive difference for H-3′′ (Table 2) showed that
the absolute stereochemistry of the chiral center at C-2′′ was
S. Hence, the absolute stereochemistry for C-3′′ and C-4′′
was deduced as R and S, respectively, and C-2 is assigned
as S based on biogenetic analogy.9 Thus, (+)-tephrorin A
(1) was assigned as (2S)-8-[(2S,3R,4S)-4-(acetyloxy)tetrahy-
dro-2-hydroxy-5,5-dimethyl-3-furanyl]-2,3-dihydro-7-meth-
oxy-2-phenyl-4H-1-benzopyran-4-one.
The positive-ion HRFABMS of compound 2 showed a
molecular ion peak at m/z 485.1957 indicating a molecular
formula of C30H28O6. Hydroxyl (νmax 3422 cm-1) and gem-
dimethyl groups (νmax 1365-1261 cm-1; δC 27.1 and 26.6;
The molecular formula of compound 1 was determined
to be C24H26O7 by positive-ion HRFABMS. Comparison of
its 1H and 13C NMR data with the previously reported known
compound, (+)-purpurin,4 indicated that 1 was a flavanone
in which ring B was unsubstituted.4,9
1
δH 1.24) were observed. The H and 13C NMR spectra of
compound 2 (Table 1) were quite similar to those of 1, except
for the absence of acetate and methoxyl moieties in 2, and
additional signals appeared for a pair of methylene protons
at δH 4.87 and 4.66 (H-2′′). Moreover, additional signals at
δH 6.24 (d, J ) 16.0 Hz, 1H), δH 7.45 (d, J ) 16.0 Hz, 1H),
and δH 7.36-7.46 (m, 5H) supported the presence of a trans-
cinnamic acid group. The location of this cinnamic group at
the C-4′′ position was confirmed by HMBC NMR spectral
observations, with a cross-peak observed between H-4′′ and
166.3 (C-1′′′), and additional correlations were observed as
summarized in Table 2. The relative stereochemistry of the
pair H-3′′/H-4′′ was established as cis from the H-4′′ coupling
constant (δ 5.10, J ) 6.3 Hz, Table 1). To determine the
absolute configuration of the stereogenic centers in com-
pound 2 using the Mosher ester methodology, a hydrolysis
reaction was considered necessary. Treatment of compound
2 under mild alkaline conditions13 afforded a semisynthetic
novel chalcone (2a)13 and cinnamic acid, which is consistent
Hydroxyl (IR, νmax 3426 cm-1), acetate (δC 170.8 and 21.3;
δH 1.96), methoxyl (δH 3.93 s), and gem-dimethyl (νmax
1368-1236 cm-1; δC 27.6 and 24.3; δH 1.08 and 1.37)
1
functionalities were present. The H and 13C NMR data for
1 are shown in Table 1. The remaining portion of the
molecule of 1 was established as consisting of one furan
ring, by spectral data comparison with (+)-tepurindiol.10 This
inference was supported by a HMBC experiment in which
correlations were observed for the resonances at δH 5.63 (H-
4′′) with the signals of δC 113.5 (C-8), 170.8 (OAc-4′′), 81.8
(C-5′′), 27.6 and 24.3 (Me2-5′′), and 47.9 (C-3′′). Additional
HMBC correlations observed are summarized in Table 1.
(8) Physical and spectroscopic data. For 1: yellowish oil; [R]20 +26°
D
(c 0.5, CHCl3); UV (MeOH) λmax (log ꢀ) 239 (4.2), 281 (4.2), 314 (3.8)
nm; IR νmax (film) 3426, 2974, 2931, 1736, 1680, 1596, 1435, 1368, 1273,
1236, 1094 cm-1; FABMS m/z (rel int. %) [M + 1]+ 427 (100), 349 (95),
305 (27), 245 (99), 217 (32), 163 (21), 131 (27); HRFABMS (positive-ion
mode) m/z [M + H]+ 427.1759 (calcd for C24H27O7, 427.1749). For 2:
(11) Preparation of (S)- and (R)-MTPA ester derivatives of compounds
1, 2a (see ref 13), and 3. To a solution of 1, 2a, or 3 (1.5 mg in 0.5 mL of
CHCl3) were added sequentially pyridine (100 µL), 4-(dimethylamino)-
pyridine (0.5 mg), and (R)-(-)R-methoxy-R-(trifluoromethyl)phenylacetyl
chloride (10 mg). Each mixture was heated at 50 °C for 4 h under N2 and
then passed through a disposable pipet (0.6 × 5 cm) packed with silica gel
and eluted with 5 mL of CHCl3. The solvent was removed in vacuo, to
obtain the mono-S-Mosher ester 1s and residues from 2a and 3. These
residues were subjected to preparative TLC to give the purified mono-S-
Mosher ester 3s or the purified di-S-Mosher 2as, respectively. Treatment
of 1, 2a, or 3 (1.5 mg with (S)-(+)R-methoxy-R-(trifluoromethyl)-
phenylacetyl chloride as described above yielded the mono-R-Mosher esters
1r or 2ar and the di-R-Mosher 3r, respectively (1H NMR data, Table 2).12
(12) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092.
yellowish oil; [R]20 +28° (c 0.6, CHCl3); UV (MeOH) λmax (log ꢀ) 220
D
(4.7), 243 (4.5), 280 (4.7) nm; IR νmax (film) 3422, 2983, 1710, 1681, 1632,
1604, 1449, 1365, 1334, 1305, 1261, 1167, 1097, 1063 cm-1; FABMS m/z
(rel int. %): [M + H]+ 485 (37), 337 (10), 265 (39), 186 (67), 161 (60),
131 (100); HRFABMS (positive-ion mode) m/z [M + H]+ 485.1957 (calcd
for C30H29O6, 485.1956). For 3: yellow needles, mp 164 °C; [R]20 +26°
D
(c 0.23, CHCl3); UV (MeOH) λmax (log ꢀ) 207 (4.1), 229 (3.8), 339 (4.0)
nm; IR νmax (film) 3438, 2982, 2932, 1639, 1598, 1566, 1485, 1440, 1356,
1305, 1243, 1089 cm-1; EIMS m/z (rel int. %) [M]+ 352 (100), 334 (89),
291 (67), 230 (64), 208 (95), 131 (80), 103 (75); HREIMS m/z [M]+
352.1305 (calcd for C21H20O5, 352.1306).
(9) Venkata Rao, E.; Ranga Raju, N. Phytochemistry 1984, 23, 2339.
(10) Pelter, A.; Ward, R. S.; Venkata Rao, E.; Ranga Raju, N. J. Chem.
Soc., Perkin Trans. 1 1981, 2491.
516
Org. Lett., Vol. 2, No. 4, 2000

Table 1. 1H and 13C NMR Data and HMBC Correlations for Compounds 1 and 2 (300, 75 MHz, CDCl3, ppm)
1
2
1H no.
δH (mult)
δC
HMBCa
1′, 4, 2′/6′
δH (mult)
δC
HMBCa
1′, 4, 2′/6′
2
3
5.53 dd (4.0, 12.2)
2.83 m
80.8 d
44.7 t
5.42 dd (3.2, 12.6)
2.83 m
80.8 d
44.7 t
2, 1′, 4
2, 1′, 4
4
4a
5
6
7
191.5 s
116.1 s
128.9 d
105.9 d
164.3 s
113.5 s
161.3 s
139.2 s
126.1 d
129.1 d
129.4 d
190.7 s
115.9 s
131.1 d
105.9 d
168.9 s
114.0 s
159.5 s
138.9 s
126.0 d
128.6 d
130.7 d
7.90 d (8.9)
6.68 d (8.9)
6, 4a, 8a, 7, 4
8, 4a, 7, 3′′
7.83 d (8.6)
6.57 d (8.6)
4a, 8a, 7, 4
8, 4a, 7
8
8a
1′
2′/6′
3′/5′
4′
7.46 m
7.36 m
7.36 m
2, 3′, 4′
1′, 2′
3′, 5′, 2′/6′
7.46 m
7.36 m
7.36 m
2, 3′, 4′
1′, 2′
3′, 5′, 2′/6′
4.87 dd (9.3, 2.6)
2′′
5.74 d (5.7)
99.3 d
4′′, 3′′, 8
78.5 t
4′′, 3′′, 8
4.66 dd (9.3, 8.5)
4.07 m
5.10 d (6.3)
3′′
4′′
5′′
4.16 dd (5.7, 9.4)
5.63 d (9.4)
47.9 d
80.1 d
81.8 s
4′′, 2′′, 8, 7, 8a
Me2-5′′, 3′′, 5′′, 8
40.6 d
80.1 d
72.9 s
4′′, 5′′, 8, 7, 2′′
Me2-5′′, 3′′, 5′′, 8, 1′′′
OCH3
Me2
3.93 s
1.37 s
1.08 s
7
27.6 q
24.3 q
26.6 q
27.1 q
1.24 s
5′′, 6′′, 7′′
1′′′
2′′′
3′′′
4′′′
5′′′/9′′′
6′′′/8′′′
7′′′
166.3 s
117.4 d
146.0 d
134.2 s
129.1 d
129.3 d
131.1 d
6.24 d (16.0)
7.45 d (16.0)
C-1′′′, C-3′′′
C-1′′′, C-2′′′
7.36 m
7.46 m
7.36 m
AcO
1.96 s
170.8, 21.3
a C to H correlations.
with the structure proposed. The di-Mosher esters of the
chalcone (2as and 2ar) indicated the S configuration at C-4′′,
because of the negative difference values for H-3′′, H-2a′′,
and H-2b′′ and the positive differences for the methyls in
C-5′′ (Table 2). The absolute stereochemistry at C-3′′ was
determined as S accordingly with the cis arrangements of
the pair H-3′′/H-4′′. Therefore, (+)-tephrorin B (2) was
assigned as (3S,4S)-4-[(2S)-3,4-dihydro-7-hydroxy-4-oxo-2-
phenyl-2H-1-benzopyran-8-yl]tetrahydro-2,2-dimethyl-3-
furanyl (2E)-3-phenyl-2-propenoate.
Compound 3 was shown to possess a molecular formula
of C21H20O5 by HREIMS. Its IR spectrum showed a hydroxyl
group (3438 cm-1), a conjugated carbonyl absorption (1639
cm-1; δC 192.7), and a gem-dimethyl group (1356-1243
cm-1). The 1H and 13C NMR spectra of 3 (Table 3) exhibited
signals at δH 7.91 (d, 15.5 Hz); δC 145.2, δH 7.58 (d, 15.5
Hz); δC 120.6, and δC 192.7, consistent with the presence
of a chalcone unit in which ring B was unsubstituted.9
The remaining portion of the molecule was established as
consisting of two substituted fused furan rings, by compari-
1
Table 2. Partial H NMR Data of the (S)- and (R)-Mosher
Ester Derivatives of Compounds 1, 2a, and 3a
δH
δH
δH
(13) Alkaline hydrolysis of 2 to 2a. (+)-Tephrorin B (2) (12 mg) was
treated with 5 mL of 0.25% KOH in MeOH, and the mixture was refluxed
for 4 h. The resultant solution was adjusted to pH 8.0 and extracted with
CHCl3. The organic phase was washed with H2O, dried over Na2SO4, and
concentrated in vacuo to yield 6 mg of 2a. The aqueous phase was acidified
with 1 N HCl to pH 3.0 and extracted with CHCl3, dried over Na2SO4, and
concentrated in vacuo to give cinnamic acid (2 mg), for which the obtained
IR and 1H and 13C NMR data were consistent with literature values.15
proton 1s
1r ∆δS-R 2a s 2a r ∆δS-R 3s
3r ∆δS-R
2′′
6.60 6.62
Sb
4.84 4.90 -0.06 6.39 6.42 -0.03
4.64 4.69 -0.05
3′′
4′′
6′′
7′′
4.49 4.42 +0.07 4.13 4.16 -0.03 3.79 3.92 -0.13
5.72 5.72 ∼0
5.27 5.31 5.43 5.46
Sb Sb
Compound (2a): yellowish needles, mp 162 °C; [R]20 +70° (c 0.08,
1.17 1.20 -0.03 1.31 1.25 +0.06 1.27 1.25 +0.02
1.17 1.30 -0.13 1.21 1.10 +0.11 1.26 1.17 +0.09
D
CHCl3); UV (MeOH) λmax (log ꢀ) 215 (3.9), 345 (4.2) nm; IR νmax (film)
3400, 2962, 1640, 1566, 1480, 1440, 1356, 1310, 1242, 1095 cm-1; FABMS
positive mode m/z (rel int. %) [M + 1]+ 355 (72), 265 (31), 251 (45), 233
(36), 186 (98), 161 (100), 115 (40).
a Obtained in CDCl3 at 300 MHz. b Absolute configuration.
Org. Lett., Vol. 2, No. 4, 2000
517

Table 3. 1H and 13C NMR Data for Compounds 3 and 2a and HMBC Correlations for Compound 3 (300, 75 MHz, CDCl3, ppm)
2a
3
1H no.
δH (mult)
δC
135.0 s
HMBCa
δH (mult)
δC
1
134.9 s
129.2 d
128.8 d
131.1 d
128.8 d
129.2 d
114.8 s
133.0 d
103.5 d
169.4 s
113.9 s
161.9 s
78.8 t
42.5 d
81.5 d
72.7 s
120.3 d
145.2 d
192.5 s
26.5 q
2
3
4
5
6
1′
2′
3′
4′
5′
6′
2′′
3′′
4′′
5′′
a
7.66 m
7.44 m
7.44 m
7.44 m
7.66 m
128.9 d
129.4 d
130.9 d
129.4 d
128.9 d
114.1 s
132.8 d
102.8 d
165.9 s
115.5 s
161.8 s
113.1 d
55.5 d
7.65 m
7.44 m
7.44 m
7.44 m
7.66 m
1, 5, 4
6, 5
4, 3, 1
7.87 d (8.7)
6.47 d (8.7)
6′, 4′, 1′
2′, 4′
7.87 d (8.7)
6.51 d (8.7)
6.57 d (6.4)
4.04 d (6.4)
4.34 s
3′′, 4′′, 5′′, 4′
4′′, 5′, 5′′, 6′, 4′
5′′
4.74 m
3.85 m
3.54 br s
80.4 d
88.5 s
120.6 d
145.2 d
192.7 s
7.58 d (15.5)
7.91 d (15.5)
â′-CdO, 1
â′-CdO, 1, 6, 2
7.57 d (15.4)
7.90 d (15.4)
b
â′-CdO
Me2
OH
1.07 s, 1.40 s
13.6 s
23.4 q, 27.8 q
5′′, 4′′, 27.8, 23.4
6′, 1′, 5′
1.29 s, 1.42 s
14.7 s
a C to H correlations.
son with (+)-purpurin.4,9 This observation was supported by
an HMBC experiment, as summarized in Table 3.
this investigation. Compounds 1 and 2 bear a novel tetrahy-
drofurano ring as a side chain at the C-8 position, and in 2
an unusual cinnamic acid moiety occurs at the C-4′′ position.
It is interesting to note that the C-4′′ substituent is in the S
configuration in 1-3, as in the case also of (+)-purpurin6
and (+)-pseudosemiglabrin.7
Biological Activity. Compounds 1-3 were evaluated for
their potential as quinone reductase inducers in cultured
mouse Hepa 1c1c7 cells, according to established proto-
cols.4,5,14 Compounds with CD (concentration to double
enzyme induction) values of <10 µg/mL are considered
active.4,5 Each IC50 value (µg/mL) (half-maximal inhibitory
concentrations of cell viability) was divided by the CD value
to obtain a chemopreventive index, CI, IC50/CD.14 Compound
2 was inactive (CD value >10 µg/mL), whereas compounds
1, 2a, and 3 significantly induced QR activity, with the
observed CD values being 4.0, 5.9, and 3.1 µM, respectively.
The CI values of these compounds were 11.8, 5.5, and 6.2,
respectively. The presence of the bulky cinnamic acid group
at C-4′′ in 2 may affect its biological activity.
In addition, the location of the chelated OH proton (δH
13.6 s) at C-6′ of 3 showed correlations with the signals of
δC 161.8 (C-6′), 114.1 (C-1′), and 115.5 (C-5′). The H-2′′
and H-3′′ protons were mutually coupled (J ) 6.4 Hz) and
supported their occurrence in a cis configuration. Moreover,
the H-4′′ proton was observed as a singlet, suggesting that
it was at an opposite position relative to H-2′′ and H-3′′.4
The absolute configuration at C-2′′ of 3 was also determined
1
by analysis of the H NMR data of the (S)- and (R)-mono-
Mosher ester derivatives 3s and 3r, respectively (Table 2),
in a manner similar to the procedure described for 1. Analysis
of the ∆δH(S-R) data for compound 3 (Table 2) indicated a
negative difference in chemical shift for H-2′′ and H-3′′ and
a positive difference for the methyls at C-5′′. Thus, the
absolute configuration at C-4′′ was established as S.
Accordingly, the absolute configuration at C-3′′ and C-2′′
was deduced as S and R, respectively, because of the trans
configuration of H-4′′/H-3′′ and the cis relationship of H-3′′/
H-2′′. Venkata Rao and Ranga Raju9 reported a similar
compound obtained from alkaline hydrolysis from (+)-
purpurin without determination of its absolute configuration.
Recent work which established the absolute configuration
of (+)-purpurin as 2S,2′′R,3′′S,4S7 also supported the ster-
eochemical assignments made for this part of the molecule
of 3. Thus, the structure of 3 was determined to be (+)-
(2′′R,3′′S,4′′S)-[2′′,3′′-b]dihydrofurano-5′′,5′′-dimethyl[4′,5′-
h]-6′-hydroxy-4′′-tetrahydrofuranohydroxychalcone, to which
we have accorded the trivial name (+)-tephrosone.
Acknowledgment. We thank Dr. James P. Whitlock for
supplying the Hepa 1c1c7 cell line used herein and are
grateful to Dr. D. D. Soejarto and Mr. R. Dvorak, College
of Pharmacy, University of Illinois at Chicago, for taxonomic
assistance and for the mass spectral data, respectively. This
research was supported by program project P01 CA48112,
funded by the National Cancer Institute, NIH, Bethesda, MD.
OL990407C
(14) Prochaska, H. J.; Santamaria, A. B.; Talalay, P. Proc. Natl. Acad.
Sci. U.S.A. 1992, 89, 2394.
In conclusion, three novel compounds of the flavonoid
class (1-3) have been isolated from Tephrosia purpurea in
(15) Ikuta, A.; Itokawa, H. Phytochemistry 1988, 27, 2813.
518
Org. Lett., Vol. 2, No. 4, 2000