Structure and synthesis of the first procassinidin dimers based on epicatechin, and gallo- and epigallo-catechin

Article abstract of DOI:10.1016/S0031-9422(00)00017-0

The range of natural dimeric procassinidins is extended by identification of cassiaflavan-(4α → 8)-epicatechin, cassiaflavan-(4α → 8)-epigallocatechin, cassiaflavan-(4β → 8)-epicatechin, cassiaflavan-(4β → 8)-epigallocatechin, cassiaflavan-(4β → 8)-galloc

Full text of DOI:10.1016/S0031-9422(00)00017-0

Phytochemistry 53 (2000) 795±804
www.elsevier.com/locate/phytochem
Structure and synthesis of the ®rst procassinidin dimers based on
epicatechin, and gallo- and epigallo-catechinp
Johan Coetzeea, 1, Lulama Mcitekaa, Elfranco Malana, 1, Daneel Ferreirab,*
aDepartment of Chemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa
bNational Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi,
University, MS 38677, USA
Received 10 September 1999; received in revised form 20 December 1999
Abstract
The range of natural dimeric procassinidins is extended by identi®cation of cassia¯avan-(4a 4 8)-epicatechin, cassia¯avan-(4a
4 8)-epigallocatechin, cassia¯avan-(4b 4 8)-epicatechin, cassia¯avan-(4b 4 8)-epigallocatechin, cassia¯avan-(4b 4 8)-
gallocatechin, ent-cassia¯avan-(4b 4 8)-epicatechin and cassia¯avan-(4a 4 6)-epicatechin in the bark of Cassia petersiana. Their
structures and absolute con®guration were con®rmed by synthesis. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Cassia petersiana; Leguminosae; Flavanoids; Proanthocyanidins; Procassinidins; Synthesis
1. Introduction
gallo- and epigallo-catechin from the bark of C.
petersiana, an aqueous extract which is used in tra-
ditional African medicine to treat fevers, gonorrhoea
and skin infections (Palgrave, 1983).
The occurrence of ¯avan containing dimeric
proanthocyanidins in nature is rare (Porter, 1988,
1994). Only four sources of these compounds were
reported, viz cassia¯avan-epiafzelechin analogues from
Cassia ®stula, (Morimoto, Nonaka, Chen & Nishioka,
1988), (2S)-3',4',7-trihydroxy¯avan-catechin analogues
with lipase inhibitory activity from Cassia nomame
(Hatano et al., 1997), (2S)-4',7,8-trihydroxy¯avan-(4b
4 6)-epioritin-4a-ol from Acacia ca€ra (Malan, Siree-
parsad, Swinny & Ferreira, 1997) and butini¯avan-epi-
catechin and -epigallocatechin probutinidins from
Cassia petersiana (Coetzee, Mciteka, Malan & Fer-
reira, 1999). We now report the structure and synthesis
of the ®rst procassinidins based on epicatechin and
2. Results and discussion
The bark of C. petersiana was extracted with
acetone and yielded a complex mixture of phenolic
monomers and dimeric proanthocyanidins. Catechin,
epicatechin, gallocatechin, epigallocatechin and the
three novel butini¯avan-epicatechin and -epigallocate-
chin probutinidins (Coetzee et al., 1999) were ac-
companied by seven dimeric procassinidins 1, 3, 5, 7,
9, 11 and 13. The mixture could be resolved by gel col-
umn chromatography and the compounds puri®ed by
TLC as their permethylaryl ether acetate derivatives 2,
4, 6, 8, 10, 12 and 14, respectively.
p Part 31 in the series `Oligomeric Flavanoids'. Part 30 (Coetzee,
Mciteka, Malan and Ferreira, Phytochemistry, 1999, 52, 737±743).
* Corresponding author. Tel.: +1-91-601-232-1572; fax: +1-91-
601-232-7062.
The structures and relative con®guration of these
1
procassinidin derivatives were determined by H NMR
(Tables 1 and 2). Owing to the adverse e€ects of ro-
tational isomerism about the C-4(C) 4 C-8(D) inter¯a-
vanyl bond the spectra of compounds 6, 8, and 10
E-mail addresses: coetzeej@cem.nw.uovs.ac.za (J. Coetzee), dfer-
reir@olemiss.edu (D. Ferreira).
1 Correspondence may also be addressed to Coetzee or Malan.
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00017-0

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J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
were broadened and had to be recorded at elevated
temperatures (708C, C6D6) to obtain sharp sets of res-
onances. The spectra of compounds 2, 4, 12 and 14
showed two distinct rotamers at room temperature
(208C) for 2, 12 (in CDCl3) and 14 (C6D6), and at
408C for 4 (C6D6). The close proximity of the 4-H(C)
resonances, however, precluded de®nition of the two
rotamers since observation of NOE e€ects between
these protons and 7-OMe(D) is a prerequisite for rota-
mer di€erentiation.
The 1H NMR spectral data (Tables 1 and 2) for
compounds 2, 4, 6, 8, 10, 12 and 14 showed similarly
substituted ABC-units comprising AA'BB'- and ABX-
systems for the aromatic protons and an AMNX-
coupled pattern, reminiscent of the heterocyclic pro-
tons of a C-4 substituted ¯avan moiety (Hatano et al.,
1997; Malan et al., 1997; Coetzee et al., 1999). The
spectra of all the compounds additionally displayed a
one-proton singlet in the high ®eld aromatic region
and an AMXY-system for protons in the heterocyclic

J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
797

798
J. Coetzee et al. / Phytochemistry 53 (2000) 795±804

J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
799

800
J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
region. When taken in conjunction with the presence
of an aromatic ABX-system for derivatives 2, 6, 12
and 14, an aromatic A2-spin system for derivatives 4, 8
and 10 and the relevant number of aromatic O-methyl
and aliphatic O-acetyl resonances, the data collectively
indicated 5,7,3',4'-tetrahydroxy¯avan-3-ol DEF units
for compounds 1, 5, 11 and 13, and 5,7,3',4',5'-penta-
hydroxy¯avan-3-ol units for 3, 7 and 9. The dimeric
structures were supported by FAB-MS data which
indicated molecular ions m/z 656 (C38H40O10) and 686
(C39H42O11), for derivatives 2, 6, 12 and 14, and 4, 8
and 10, respectively.
between 2- and 4-H(C), indicating a 2,4-cis relative
con®guration of the C-ring. The conspicuous absence
of any NOE association between 2- and 4-H(C) in
compounds 6, 8 and 10 strongly suggested 2,4-trans
relative con®guration of their C-rings. These allo-
cations of relative con®guration were corroborated by
the multiplicity and coupling constants of 2- and 4-
H(C) for the 2,4-cis con®guration, e.g. 2, where both
3
2- and 4-H(C) resonate as double doublets ꢀ J2, 3 ˆ 2:0
3
, 12.0 Hz; J3, 4 ˆ 5:5, 12.0 Hz). Analogues with a 2,4-
trans relative con®guration, e.g. 6, show a double
3
doublet for both 2- ꢀ J2, 3 ˆ 3:5, ca. 7.0 Hz) and 4-
3
COSY experiments indicated coupling of 2-H(C)
with 2',6'-H(B) and 2-H(F) with 2'- and 6'-H(E) for
derivates 2, 4, 6, 8, 10, 12 and 14. The same exper-
H(C) ꢀ J3,4 ˆ 6:0, 7:0 Hz) and in some instances a tri-
3
plet for 4-H(C) ꢀ J3, 4 ˆ 6:0 Hz) (Coetzee et al., 1999).
The chemical shifts of the C-2 (C-ring) resonances in
the 13C NMR spectra of derivatives 2, 4, 6, 8, 10 and
12 fully supported these relative con®gurations. Those
compounds with a 2,4-trans con®guration (6, 8 and
10) possess shielded 2-C(C) signals (ca. 4 ppm) com-
4
iment revealed JHH coupling between 4-H(C) and 5-
H(A) which de®ned the ABC- and DEF-units for all
the compounds. Phase sensitive NOESY experiments
of derivatives 2, 4, 12 and 14 showed association
Scheme 1. Reagents and conditions: (i) NaBH4, EtOH; (ii) 17 then, TiCl4/CH2Cl2; (iii) Ac2O, pyridine.

J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
801
pared to the chemical shifts of these carbons in deriva-
tives with a 2,4-cis con®guration (Table 3) as a result
of the g-gauche e€ect (Fletcher, Porter, Haslam &
Gupta, 1977) operating in the former group of com-
pounds. The same shielding phenomena are also evi-
dent from the results of Hatano et al. (1997). The
chemical shifts of the hydrogen-bearing carbons in
compounds 2, 4, 6, 8, 10 and 12 are collated in
Table 3. Sample size precluded the collection of 13C
NMR data for the 4,6-linked analogue 14.
Bonding positions to the D-ring were established for
all the derivatives by the familiar NOE associations
exhibited by `residual' D-ring protons with methoxyl(s)
i.e. the D-ring singlet showing strong association with
the 5- and 7-methoxy groups of the D-ring in deriva-
tives 2, 4, 6, 8, 10 and 12 and selectively with 7-OMe
in 14 (Young, Brandt, Young, Ferreira & Roux, 1986).
The CD spectra of compounds 2, 4 and 14 exhibited
high amplitude negative Cotton e€ects at ca. 240 nm,
indicating a 4a-¯avanyl C-ring substituent (De Angelis
& Wildman, 1969; Van der Westhuizen, Ferreira &
Roux, 1981). Derivatives 6, 8, 10 and 12 showed posi-
tive Cotton e€ects near 240 nm which is reminiscent of
a 4b C-ring substituent. When taken in conjunction
with the relative 2,4-cis con®guration of derivatives 2,
4, 12 and 14, the negative Cotton e€ects for 2, 4 and
14 indicated their 2S,4S absolute con®guration and the
positive Cotton e€ect for 12 its 2R,4R absolute stereo-
chemistry. The positive Cotton e€ects similarly sup-
ported the 2S,4R absolute con®guration of derivatives
6, 8 and 10 with their 2,4-trans relative con®gurations.
Derivatives 2, 4, 6, 8, 12 and 14 showed 2-H(F) as a
broad singlet ‰3J2, 3ꢀF† ca 1.0 Hz] while 10 exhibited a
coupling constant of 7.5 Hz. This indicated a 2,3-cis
relative con®guration of the F-rings (Steynberg, Bur-
ger, Malan, Cronje, Young & Ferreira, 1990) of the
former group and 2,3-trans for derivative 10. Thus, we
opted to synthesize these derivatives in order to estab-
Scheme 2. Reagents and conditions: (i) NaBH4, EtOH; (ii) 20 then, TiCl4/CH2Cl2; (iii) 21 then, TiCl4/CH2Cl2; (iv) Ac2O/pyridine.

802
J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
lish the absolute con®guration (Nonaka, Miwa &
Nishioka, 1982) and constitution of the natural pro-
ducts 1, 3, 5, 7, 9, 11 and 13.
®rst compound in the class of ¯avan 4 ¯avan-3-ol
proanthocyanidins possessing a ¯avan top unit with
2R absolute con®guration, hence presumably also indi-
cating the involvement of a 2R-¯avanone in their bio-
synthesis (Stich & Forkmann, 1988).
Racemic 4',7-dimethoxy¯avanone 15 was reduced by
sodium borohydride (Hatano et al., 1997; Malan et al.,
1997) to give the diastereomeric mixture of the ¯avan-
4-ol 16 in 96% yield (Scheme 1). Treatment of this
mixture with optically pure tetra-O-methylepicatechin
17 in dichloromethane containing titanium tetrachlor-
ide (TiCl4) as Lewis acid (Kawamoto, Nakatsubo &
Murakami, 1991) a€orded a mixture of compounds,
which were puri®ed by PLC to give three procassini-
din-type dimers in fair yields. Acetylation yielded the
permethylaryl ether acetates 2, 6 and 12 which had
identical 1H NMR and CD spectral data when com-
pared to derivatives obtained from the natural com-
pounds 1, 5 and 11. We could not detect any of the
expected permethyl aryl ether of the (4a 4 6)-analogue
13, an observation which is supported by previous
reports of absence (Hatano et al., 1997; Coetzee et al.,
1999) or formation in low yields (Young, Cronje,
Botes, Ferreira & Roux, 1985) of the (4 4 6)-linked
dimers. Compounds 1, 5 and 11 were hence unequivo-
cally identi®ed as cassia¯avan-(4a 4 8)-epicatechin,
cassia¯avan-(4b 4 8)-epicatechin, and ent-cassia¯avan-
(4b 4 8)-epicatechin, respectively, and 13 tentatively as
cassia¯avan-(4a 4 6)-epicatechin.
3. Experimental
1H NMR spectra were recorded at 300 MHz for sol-
utions in CDCl3 or C6D6, with TMS as int. standard.
FAB-MS were recorded on a VG 70-70E instrument
with a VG 11-250J data system and an iontech saddle-
®eld FAB gun. CD data were obtained in MeOH.
TLC was performed on precoated Merck plastic sheets
(silica gel 60 PF254 0.25 mm) and the plates were
sprayed with H2SO4±HCHO (40:1, vol/vol) after devel-
opment. Prep. TLC plates, Kieselgel PF254 (1.0 mm)
were air dried and used without prior activation. Com-
pounds were recovered from the absorbent with
Me2CO. CC was on Sephadex LH-20 in EtOH.
Methylations were performed with an excess of CH2N2
in MeOH±Et2O over a period of 48 h at 158C, while
acetylations were in Ac2O-pyridine at ambient tem-
perature. Evaporations were done under reduced press-
ure at ambient temperatures in a rotary evaporator,
and freeze drying of aqueous solutions on a Virtis
12SL freezemobile.
In contrast to the above and our previous syntheses
of dimeric proanthocyanidins with ¯avan constituent
units (Malan et al., 1997; Coetzee et al., 1999) where
racemic ¯avanones were utilized as source of the ¯avan
molecular backbone, compounds 4 and 8 were syn-
thesized from optically pure (2S)-di-O-methylliquiriti-
genin 18 and penta-O-methylepigallocatechin 20
(Scheme 2). Initial NaBH4-reduction of 18 gave the in-
termediate 4',7-dimethoxy¯avan-4-ol 19 (diastereo-
mers, quantitative yield) which was subsequently
treated with penta-O-methylepigallocatechin 20 in
3.1. General procedure for the synthesis of procassinidin
derivatives
The reduction of ¯avanones 15 and 18 was done
according to standard procedures (Hatano et al., 1997;
Malan et al., 1997). To a dry solution of 4',7-
dimethoxy¯avan-4-ol 16 (90.0 mg) in CH2Cl2 (20 ml)
was added the permethylaryl ether 17 of epicatechin
(296 mg) and TiCl4 (0.04 ml, 1.2±1.4 equivalent). The
mixture was stirred at 08C under N2 for 60 min and
the temperature was allowed to rise to 408C for a
further 6 h. An excess of cold H2O (40 ml) was added
and the mixture was extracted with Et2O ꢀ3 Â 20 ml).
After drying (Na2SO4) the Et2O was removed under
vacuum and the mixture was resolved by prep. TLC in
hexane±C6H6±Me2CO (5:2:3) to give three bands at Rf
0.53, 0.40 and 0.33. After acetylation of the Rf 0.33
band (42 mg) it was further separated in hexane±
C6H6±Me2CO (5:4:1) to give four bands at Rf 0.57
(5.5 mg), 0.37 (5.7 mg), 0.29 (8.2 mg) and 0.22 (14.1
mg). The Rf 0.57 band yielded starting material 17.
The Rf 0.37 band yielded 4'7-di-O-methyl-ent-cassia¯a-
van-(4b 4 8)-3',4',5,7-tetra-O-methyl-3-O-acetyl-epica-
techin 12 as a light-brown amorphous solid. The Rf 0.29
1
dichloromethane containing TiCl4. The H NMR and
CD spectra of the O-acetyl derivatives of the synthetic
dimer derivatives 4 and 8 corresponded with those of
the same derivatives of the procassinidins from C.
petersiana. The structures and absolute stereochemistry
of the natural products 3 and 7 could thus be de®ned
as cassia¯avan-(4a 4 8)-epigallocatechin and cassia¯a-
van-(4b 4 8)-epigallocatechin, respectively.
A similar procedure (Scheme 2) was employed to
synthesize 10 by using (2S )-4',7-di-O-methylliquiriti-
genin 18 and penta-O-methylgallocatechin 21. Sub-
sequent acetylation a€orded the cassia¯avan-(4b 4 8)-
gallocatechin and cassia¯avan-(4a 4 8)-gallocatechin
derivatives 10 and 22. The lower yields of 4 and 22
compared to those of 8 and 10 indicated that the
stereochemical course of coupling is directed by the C-
2 eq. aryl group.
band gave 4',7-di-O-methylcassia¯avan-(4b
3',4',5,7-tetra-O-methyl-3-O-acetylepicatechin
4
6
8)-
and
The ent-cassia¯avan-(4b 4 8)-epicatechin 11 is the

J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
803
the Rf 0.22 band 4',7-di-O-methylcassia¯avan-(4a 4
8)-3',4',5,7-tetra-O-methyl-3-O-acetylepicatechin 2.
(2S)-4',7-dimethoxy¯avan-4-ol 19 (90.0 mg) was dis-
solved in dry CH2Cl2 (20 ml), the TiCl4 (0.04 ml)
added to give a dark purple solution after which the
epigallocatechin or gallocatechin permethylaryl ethers
20/21 (290 mg) was added during separate exper-
iments. The mixture was treated and worked up as
above, and the residue was separated by prep. TLC in
C6H6±Me2CO (8:2) to give two bands at Rf 0.39 (40
mg) and 0.26 (17.3 mg). The Rf 0.39 band yielded
starting material 20. Acetylation of the Rf 0.26 band
followed by prep. TLC in hexane±C6H6±Me2CO
(5:4:1, Â4) gave two bands which comprised 4',7-di-O-
methylcassia¯avan-(4b 4 8)-epigallocatechin penta-O-
methylether acetate 8 and 4',7-di-O-methylcassia¯a-
21.2 ‰CH3COO±], 26.6 [4-C(F)], 32.0 [4-C(C)], 35.0 [3-
C(C)], 55.7 [±OCH3], 55.8 [±OCH3], 56.1 [±OCH3],
56.3 [±OCH3], 56.4 [±OCH3], 56.9 [±OCH3], 68.0 [3-
C(F)], 77.3 [2-C(F)], 79.4 [2-C(C)], 88.6 [6-C(D)], 101.5
[8-C(A)], 107.3 [6-C(A)], 109.6 [2'-C(E)], 110.8 [5'-
C(E)], 114.2 (Â2) [3',5'-C(B)], 119.1, 119.4 [6'-C(E)],
119.5, 127.9 (Â2) [2',6'-C(B)], 128.2, 128.5, 128.7 [5-
C(A)], 130.6, 134.4, 148.8, 156.3 ‰Ar-OCH3], 157.7 ‰Ar-
OCH3], 158.0 ‰Ar-OCH3], 158.6 ‰Ar-OCH3], 158.9 ‰Ar-
OCH3], 159.6 ‰Ar-OCH3], 170.5 [CH3COO±]; CD ‰y
]
2704, ‰y]243.9 16710, ‰y]234.7 1016.
276.1
4',7-Di-O-methyl-ent-cassia¯avan-(4b 4 8)-3',4',5,7-
tetra-O-methyl-3-O-acetylepicatechin 12 ꢀRf 0.61, 4.5
mg) from B8.1 as a white-yellowish amorphous solid.
(Found: M+, 656.2621. C38H40O10 requires M+,
656.2620. dH (Table 2); 13C NMR (CDCl3, 208C): d
(major rotamer) 21.5 ‰CH3COO±], 26.7 [4-C(F)], 32.0
[4-C(C)], 35.8 [3-C(C)], 55.5 [±OCH3], 55.7 [±OCH3],
55.7 [±OCH3], 56.2 [±OCH3], 56.3 [±OCH3], 56.8 [±OC
H3], 67.9 [3-C(F)], 77.6 [2-C(F)], 78.8 [2-C(C)], 88.7 [6-
C(D)], 101.5 [8-C(A)], 107.9 [6-C(A)], 109.9 [2'-C(E)],
110.7 [5'-C(E)], 114.2 (Â2) [3',5'-C(B)], 119.0, 119.2
[6'-C(E)], 119.5, 127.86, 127.9 (Â2) [2',6'-C(B)], 128.1
[5-C(A)], 128.5, 130.4, 134.4, 148.7, 156.3 ‰Ar-OCH3],
157.5 ‰Ar-OCH3], 157.8 ‰Ar-OCH3], 158.6 ‰Ar-OCH3],
158.7 ‰Ar-OCH3], 159.6 ‰Ar-OCH3], 170.9 [CH3COO±];
CD ‰y]286.5 4178, ‰y]273.3 4770, ‰y]246.5 6630.
van-(4a
4
8)-epigallocatechin-penta-O-methylether
acetate 4 at Rf 0.29 (4.0 mg) and 0.39 (10 mg), respect-
ively. Derivatives 4 and 8 showed identical NMR and
CD spectral data as the same derivatives of the corre-
sponding natural products 3 and 7. The yield of com-
pound 10 and very likely also that of 22 during a
similar procedure using the gallocatechin permethylaryl
ether 21 were low and after initial prep. TLC separ-
ation in hexane±C6H6±Me2CO (5:2:3) only 6 mg of an
inseparable mixture of 10 and 22 was obtained. How-
ever, it was possible to identify compound 10 by using
1
the H NMR spectral data of the natural compound to
®ngerprint the synthetic mixture.
4',7-Di-O-methylcassia¯avan-(4a 4 6)-3',4',5,7-tetra-
O-methyl-3-O-acetylepicatechin 14 ꢀRf 0.36, 8.3 mg)
from B8.2 as a white amorphous solid. (Found: M+,
656.2620. C38H40O10 requires M+, 656.2620. dH
(Table 2); CD ‰y]286.8 4591, ‰y]244.1 15230, ‰y]230.3
3321.
3.2. Isolation of phenolic compounds
Milled bark (6.3 kg) of C. petersiana was repeatedly
extracted with Me2CO ꢀ3 Â 7:5 l) for 48 h periods at
258C. The Me2CO was removed under vacuum at
358C and the residue dissolved in H2O and freeze
dried to give a brown powder (370 g).
The extract (two batches of 25 g each) was subjected
to CC on Sephadex LH-20 in EtOH ꢀ6 Â 180 cm) col-
umn, 0.5 ml/min ¯ow rate, 32 min fractions) to give
the following fractions: B1 (tubes 225±264, 82 mg), B2
(265±279, 62 mg), B3 (280±285, 10 mg), B4 (286±319,
70 mg), B5 (320±354, 110 mg), B6 (355±364, 29 mg),
B7 (365±399, 260 mg) and B8 (400±414, 117 mg). A
portion (200 mg) of fraction B8 was methylated and
the mixture was separated by prep. TLC in C6H6±
4',7-Di-O-methylcassia¯avan-(4b 4 8)-3',4',5,7-tetra-
O-methyl-3-O-acetylepicatechin 6 ꢀRf 0.47, 2.5 mg)
from B8.3 as a white amorphous solid. (Found: M+,
656.2621. C38H40O10 requires M+, 656.2620. dH
(Table 1); 13C NMR (C6D6, 708C): d 20.5 ‰CH3COO±],
27.1 [4-C(F)], 28.9 [4-C(C)], 36.2 [3-C(C)], 55.0 [±OC
H3], 55.3 [±OCH3], 56.2 [±OCH3], 56.4 [±OCH3], 56.7
[±OCH3], 56.9 [±OCH3], 68.1 [3-C(F)], 75.8 [2-C(C)],
78.1 [2-C(F)], 90.3 [6-C(D)], 101.9 [8-C(A)], 102.6,
104.3 [2'-C(E)], 106.7 [5'-C(E)], 107.7 [6-C(A)], 111.4,
114.2 [6'-C(E)], 114.4 (Â2) [3',5'-C(B)], 118.4, 119.4,
127.2 (Â2) [2',6'-C(B)], 128.7 [5-C(A)], 133.4, 136.8,
140.2, 154.6 ‰Ar-OCH3], 156.6 ‰Ar-OCH3], 157.9 ‰Ar-
OCH3], 158.4 ‰Ar-OCH3], 159.8 ‰Ar-OCH3], 159.8 ‰Ar-
OCH3], 169.4 [CH3COO±]; CD ‰y]275.8 7354, ‰y]244.0
15780, ‰y]230.6 1358.
Me2CO (9:1, Â3) to give four bands at Rf 0.64 (B8.1
,
33 mg), 0.40 (B8.2, 24.5 mg), 0.37 (B8.3, 12.5 mg) and
0.13 (B8.4, 15.6 mg). All four fractions were separately
acetylated and puri®ed by prep. TLC in C6H6±Me2CO
(24:1, Â3) to yield the following compounds.
Fraction B8.4 ꢀRf 0.29, 8.5 mg) comprised the hepta-
methyl ether diacetate of ®setinidol-(4b 4 8)-epicate-
chin (Steynberg et al., 1990).
A portion of fraction B7 (200 mg) was methylated
and the mixture was separated by PLC in C6H6±
Me2CO (8:2), to give a band at Rf 0.67 (47 mg) which
was subsequently acetylated. Further separation in
4',7-Di-O-methylcassia¯avan-(4a 4 8)-3',4',5,7-tetra-
O-methyl-3-O-acetylepicatechin 2 ꢀRf 0.64, 15 mg)
from B8.1 as a white amorphous solid. (Found: M+,
656.2623. C38H40O10 requires M+, 656.2620; dH
(Table 1); 13C NMR (CDCl3, 208C): d (major rotamer)

804
J. Coetzee et al. / Phytochemistry 53 (2000) 795±804
hexane±Me2CO±EtOAc (12:5:3, vol/vol) a€orded two
bands at Rf 0.51 (5.2 mg) and 0.45 (6.0 mg) compris-
ing compounds 10 and 8, respectively. 4',7-Di-O-
‰Ar-OCH3], 154.3 ‰Ar-OCH3], 156.4 ‰Ar-OCH3], 157.8
‰Ar-OCH3], 158.5 ‰Ar-OCH3], 159.7 ‰Ar-OCH3], 159.8
‰Ar-OCH3], 169.5 [CH3COO±]; CD ‰y]275.5 2535, ‰y
methylcassia¯avan-(4b
4
8)-3',4',5',5,7-penta-O-
]
11860 and ‰y]222.9 2205.
243.7
methyl-3-O-acetylgallocatechin 10 was obtained as col-
orless amorphous ¯akes. (Found: M+, 686.2723.
C39H42O11 requires M, 686.2724; dH (Table 2); 13C
NMR (C6D6, 708C): d 20.5 ‰CH3COO±], 26.0 [4-C(F)],
29.2 [4-C(C)], 36.7 [3-C(C)], 54.9 [±OCH3], 55.0 [±OC
H3], 55.2 [±OCH3], 56.1 [±OCH3], 56.3 (Â2) [±OCH3],
60.5 [±OCH3], 70.0 [3-C(F)], 75.6 [2-C(C)], 79.7 [2-
C(F)], 90.3 [6-C(D)], 101.9 [8-C(A)], 103.7, 105.8 (Â2)
[2',6'-C(E)], 107.7 [6-C(A)], 110.2, 114.4 (Â2) [3',5'-
C(B)], 114.9, 119.4, 127.8 (Â2) [2',6'-C(B)], 128.9 [5-
C(A)], 133.4, 135.4, 140.1, 154.1 ‰Ar-OCH3], 154.2 ‰Ar-
OCH3], 156.5 ‰Ar-OCH3], 157.9 ‰Ar-OCH3], 158.5 ‰Ar-
OCH3], 159.7 ‰Ar-OCH3], 160.0 ‰Ar-OCH3], 169.5
[CH3COO±]; CD ‰y]275.8 4174, ‰y]238.9 14440 and ‰y
Acknowledgements
Financial support by the Foundation for Research
Development, Pretoria and the `Sentrale Navorsings-
fonds' of the UOFS, is gratefully acknowledged. This
work was supported in part by the United States
Department of Agriculture, Agricultural Research Ser-
vice Speci®c Cooperative Agreement No. 58-6408-7-
012. We thank Mrs. L Davies from Skukuza who
identi®ed Cassia petersiana after it was collected in the
vicinity of Hazyview, Mpumalanga. A voucher speci-
men is being kept in the Chemistry Department at
UOFS.
]
313. 4',7-Di-O-methylcassia¯avan-(4b 4 8)-
227.2
3',4',5',5,7-penta-O-methyl-3-O-acetylepigallocatechin
8 was isolated as colorless amorphous platelets. (Found:
M+, 686.2722. C39H42O11 requires M, 686.2724); dH
(Table 1); 13C NMR (C6D6, 708C): (20.5 ‰CH3COO±],
27.0 [4-C(F)], 29.1 [4-C(C)], 36.3 [3-C(C)], 55.0 (Â2) [±
OCH3], 55.1 [±OCH3], 56.1 [±OCH3], 56.4 (Â2) [±OC
H3], 60.5 [±OCH3], 68.1 [3-C(F)], 75.8 [2-C(C)], 78.3
[2-C(F)], 90.3 [6-C(D)], 102.0 [8-C(A)], 103.6, 105.7
(Â2) [2',6'-C(E)], 107.8 [6-C(A)], 110.2, 114.4 (Â2)
[3',5'-C(B)], 114.8, 119.3, 127.2 (Â2) [2',6'-C(B)], 128.7
[5-C(A)], 133.4, 135.3, 140.1, 154.1 ‰Ar-OCH3], 154.6
‰Ar-OCH3], 156.4 ‰Ar-OCH3], 157.9 ‰Ar-OCH3], 158.4
‰Ar-OCH3], 159.7 ‰Ar-OCH3], 159.8 ‰Ar-OCH3], 169.4
[CH3COO±]; CD ‰y]275.0 1254, ‰y]248.1 12180 and ‰y
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