Phytochemistry 65 (2004) 2317–2321
Flavonoids and andrographolides from Andrographis paniculata
Y. Koteswara Rao a, G. Vimalamma b, C. Venkata Rao b, Yew-Min Tzeng
Department of Applied Chemistry, Chaoyang University of Technology, 168 Gifeng E. Road, Wufeng, Taichung 413, Taiwan
Natural Products Division, Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India
Received 19 December 2003; received in revised form 27 March 2004
Available online 18 August 2004
Two ﬂavonoids, identiﬁed as 5,7,20 ,30 -tetramethoxyﬂavanone and 5-hydroxy-7,20 ,30 -trimethoxyﬂavone, as well as several other
ﬂavonoids, andrographolide diterpenoids, and polyphenols, were obtained from the phytochemical investigation of the whole plant
of Andrographis paniculata, a well known medicinal plant. The structures of these compounds were established with the aid of
spectroscopic methods, including analysis by 2D NMR spectroscopy.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Andrographis paniculata; Acanthaceae; Flavonoids; Andrographolide diterpenoids; Benzenoids
Andrographis is a genus of the Acanthaceae family
comprising of about 40 species several members of
which enjoy a reputation in traditional medicine. Particularly, Andrographis paniculata Nees is used for several applications such as an antidote for snake-bite and
poisonous stings of some insects, and to treat dyspepsia,
inﬂuenza, dysentry, malaria and respiratory infections
(Kirtikar and Basu, 1975; Chopra et al., 1980). It is also
considered to be a latent-heat clearing, antipyretic, detoxicant, anti-inﬂammatory, detumescent, febrifugal,
antiphlogistic and analgesic agent for the treatment of
acute infections of the gastrointestinal tract, respiratory
organs and urinary system (Nazimudeen et al., 1978;
Choudhury and Poddar, 1985). A. paniculata is an erect
handsome herb well known in Asia. It occurs widely in
the plains of India, Sri Lanka, Mainland China and
Taiwan (Gamble, 1956). Previous investigations on the
chemical composition of this well studied herb showed
that it is a rich source of 20 -oxygenated ﬂavonoids
(Govindachari et al., 1969; Jalal et al., 1979; Gupta
et al., 1983, 1996; Kuroyanagi et al., 1987), and labdane
Corresponding author. Tel.: +886-4-23304898/23323000x3003; fax:
E-mail address: email@example.com (Y.-M. Tzeng).
0031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
diterpenoids (Kleipool, 1952; Chan et al., 1971; Balmain
and Connolly, 1973; Fujita et al., 1984; Puri et al., 1993;
Matsuda et al., 1994; Jantan and Waterman, 1994;
Munta et al., 2003). In the present study, we report the
isolation and characterization of 23 compounds (1–23),
(Fig. 1) including two new ﬂavonoids, (2S)-5,7,20 ,30 tetramethoxyﬂavanone (6) and 5-hydroxy-7,20 ,30 -trimethoxyﬂavone (12), as well as 21 known compounds
(1–5, 7–11 and 13–23) from the extracts of the whole
plant of A. paniculata.
2. Results and discussion
The MeOH extract of the whole plant of A. paniculata was divided into CHCl3 , Me2 CO and MeOH soluble fractions. Each fraction was submitted to a series of
chromatographic separations individually to yield two
new ﬂavonoids (6, 12) and twenty one known compounds (1–5, 7–11 and 13–23).
Compound 6, obtained as colourless solid, gave a
molecular ion peak at m=z 344.1331 in its HREIMS
corresponding to the molecular formula C19 H20 O6 . This
was further supported by 13 C NMR spectral analysis,
which displayed 19 signals for all carbon atoms in the
molecule including one carbonyl, seven nonprotonated,
six methine, one methylene and four methoxyl carbons.
The UV spectrum of 6 in MeOH at 263 and 336 nm
Y. Koteswara Rao et al. / Phytochemistry 65 (2004) 2317–2321
Fig. 1. Structures of compounds isolated from A. paniculata.
suggested a ﬂavanone skeleton for the molecule (Mabry
et al., 1970); its UV absorption maxima was unaﬀected
by the addition of NaOAc and AlCl3 /HCl indicating
the absence of free hydroxyls at C-7 and C-5 positions,
The 1 H NMR spectrum of 6 showed the presence of
four methoxyl groups at d 3.81, 3.86, 3.88 and 3.90. It
also exhibited three sets of double doublets of an AMX
system at d 5.76 (1H, J ¼ 13:3, 3.0 Hz), 2.98 (1H,
J ¼ 16:7, 13.3 Hz) and 2.77 (1H, J ¼ 16:7, 3.0 Hz)
which were characteristic of H-2, H-3ax and H-3eq , respectively, of the ring C of a ﬂavanone moiety (Mabry
et al., 1970). Two meta-coupled doublets (J ¼ 2:3) at d
6.09 and 6.14, each integrating for one proton, were
assigned to H-6 and H-8, respectively. The EIMS of
compound 6 displayed diagnostic peaks of retro-Diels–
Alder (RDA) cleavage of ring C at m=z 180 and 164
suggesting the presence of two methoxyl groups in ring
A, and hence the remaining two methoxyl groups should
be in ring B. Two of the four methoxyl groups at d 3.88
and 3.81 were situated on C-5 and C-7, as they showed
HMBC correlations with the carbons at 162.2 and 165.8
ppm, assignable to C-5 and C-7, respectively. These
assignments were further supported by NOE correlations of the methoxyl protons (d 3.88) with H-6 (d 6.09),
and the methoxyl protons (d 3.81) with H-6 (d 6.09) and
H-8 (d 6.14) in the NOESY spectrum. The C-2 signal in
20 -unsubstituted ﬂavanones usually appears at d 79.0.
However, in compound 6, the C-2 signal appeared at an
upﬁeld position (d 74.2), indicating the presence of C-20
oxygenation in ring B (Agrawal, 1989). Thus a methoxyl
group at d 3.90 was attached to C-20 on the basis of its
HMBC correlation with C-20 ðd 146.8), and was further
conﬁrmed by its NOE correlation with H-3eq . The ﬁnal
Y. Koteswara Rao et al. / Phytochemistry 65 (2004) 2317–2321
methoxyl group at d 3.86 was placed at C-30 , as evidenced by its HMBC correlation with C-30 at d 152.5,
and two strong NOEs with 20 -OMe and a proton at d
6.93 (H-40 ) (Fig. 2). The remaining aromatic proton
signals at d 6.93 (1H, dd, J ¼ 7:0, 2.7 Hz) and 7.15 (2H,
m) were attributed to H-40 and H-50 and H-60 , respectively, of ring B. The chemical shift values of ring B
carbons of 6 were similar to those observed for ring B
carbons of 20 ,30 -dioxygenated ﬂavanones (Kojima et al.,
1997). The absolute conﬁguration at C-2 was shown to
be S-conﬁguration (Iinuma et al., 1994), as the CD
spectrum of 6 exhibited a negative Cotton eﬀect at 292
nm (De À 1:05) and positive Cotton eﬀects at 262 nm
(De þ 0:53) and 338 nm (De þ 0:46). Thus, from the
foregoing spectral studies the structure of compound 6
was elucidated as ð2SÞ-5,7,20 ,30 -tetramethoxyﬂavanone.
Compound 12, isolated as a yellow amorphous solid,
gave a positive ferric chloride test. Its HREIMS displayed a [M]þ peak at m=z 328.1052 consistent with the
molecular formula C18 H16 O6 . This was further corroborated by the 18 carbon signals in its 13 C NMR spectrum, which include a conjugated carbonyl, eight
nonprotonated, six methine and three methoxyl carbons. The UV absorption maxima of 12 at 241 and 327
nm were typical of a ﬂavone derivative (Mabry et al.,
1970). The UV spectrum was unaﬀected by the addition
of NaOAc again suggesting the absence of a free hydroxyl at C-7. A bathochromic shift of 42 nm of the
band I absorption maximum with AlCl3 /HCl indicated
the presence of a chelated hydroxyl group at the C-5
position. The IR spectrum exhibited bands at 3158 and
1656 cmÀ1 corresponding to hydroxyl and a,b-unsaturated carbonyl functionalities, respectively.
The 1 H NMR spectrum of 12 showed a D2 O exchangeable downﬁeld signal at d 12.82 assignable to a
hydrogen-bonded hydroxyl group at C-5. A pair of
meta-coupled doublets (J ¼ 2:2 Hz) at d 6.36 and 6.44
were attributed to H-6 and H-8, respectively. It also
exhibited signals due to three methoxyl groups at d 3.92,
3.89 and 3.86. The EIMS fragmentation of the molecular ion at m=z 328 of 12 in its RDA cleavage at ring C
yielded diagnostic peaks at m=z 166 and 162 thereby
inferring that a hydroxyl and a methoxyl group were in
ring A and two methoxyl groups were in ring B of the
molecule. The methoxyl group at d 3.89 was placed at C7, on the basis of its 3 J correlation with a carbon at
d165:5 (C-7) in the HMBC spectrum and NOE crosspeaks with H-6 (d 6.36) and H-8 (d 6.44) in NOESY
spectrum. A sharp one-proton singlet at d 6.98 correlating with C-3 (110.5 ppm) in the HSQC spectrum was
characteristic of H-3 of a 20 -oxygenated ﬂavone (Tanaka
et al., 1986). A typical ABC spectrum at d 7.07 (1H, dd,
J ¼ 8:1, 1.5 Hz), 7.21 (1H, dd, J ¼ 8:1, 7.9 Hz) and 7.33
(1H, dd, J ¼ 7:9, 1.5 Hz) for three adjacent protons, 40 ,
50 and 60 protons, respectively, established a 20 ,30 -dioxygenated B ring in the molecule (Kuroyanagi et al.,
1987). Thus, the methoxyl groups at d 3.92 and 3.86
were placed at C-20 and C-30 positions, as they have
HMBC connectivities with C-20 (d 148.0) and C-30 (d
153.3), respectively. This was also inferred by the NOEs,
OCH3 -20 /OCH3 -30 , OCH3 -30 /H-40 , and H-50 /H-40 , H-60
in the NOESY experiment (Fig. 2). From these ﬁndings,
compound 12 was established as 5-hydroxy-7,20 ,30 -trimethoxyﬂavone.
The structures of known isolates (Fig. 1) from the
whole plant of A. paniculata were identiﬁed by comparison of their spectral data with literature values as
b-sitosterol (1) (Ali et al., 2002); andrographolide (2),
14-deoxy-ll, 12-dedihydroandrographolide (3) and
14-deoxyandrographolide (4) (Matsuda et al., 1994); 7O-methyldihydrowogonin (5) (Gupta et al., 1983);
dihydroskullcapﬂavone I (7) (Hari Kishore et al., 2003);
7-O-methylwogonin (8) (Kuroyanagi et al., 1987); 5hydroxy-7,8,20 ,50 -tetramethoxy-ﬂavone (9) (Mopuru et
al., 2003); 5-hydroxy-7,8,20 ,30 -tetramethoxyﬂavone (10)
(Kuroyanagi et al., 1987); 5-hydroxy-7,20 ,60 -trimethoxyﬂavone (11) (Munta et al., 2003); skullcapﬂavone
120 -methylether (13) (Jalal et al., 1979); cinnamic acid
(14), caﬀeic acid (15), ferulic acid (16) and chlorogenic
acid (17) (Satyanarayana et al., 1978); 7-O-methylwogonin 5-glucoside (18) (Kuroyanagi et al., 1987);
skullcapﬂavone I 20 -glucoside (19) (Gupta et al., 1996);
14-deoxy- 15-isopropylidene-11,12-didehydro-andrographolide (20) (Munta et al., 2003); 14-deoxy-11hydroxyandrographolide (21), neoandrographolide (22)
and andrographoside (23) (Matsuda et al., 1994).
Fig. 2. Signiﬁcant HMBC (!) and NOESY (
) correlations of 6 and 12.
Y. Koteswara Rao et al. / Phytochemistry 65 (2004) 2317–2321
Isolation of two new and nine known 20 -oxygenated
ﬂavonoids, which occur rarely in nature, in addition to
seven andrographolide diterpenoids, from A. paniculata
provided strong evidence for the statement, ‘‘Andrographis species are noted for profuse production of
20 -oxygenated ﬂavonoids and andrographolide diterpenoids’’ (Iinuma and Mizuno, 1989). Accordingly, the
above class of compounds isolated from Acanthaceae so
far were conﬁned to Andrographis species only; this
shows promise of being a useful chemotaxonomic marker for Andrographis in the Acanthaceae.
Melting points were determined on a Koﬂer hot stage
apparatus and are uncorrected. CD spectra were recorded in MeOH at 25 °C on a JASCO J 715 spectropolarimeter, where UV spectra were obtained on a
Shimadzu UV-240 spectrophotometer. Optical rotations were measured in MeOH at 25°C on a Perkin–
Elmer 241 Polarimeter, whereas IR spectra were determined KBr discs using a Perkin–Elmer 283 double
beam spectrophotometer. 1 H NMR spectra were recorded on a Bruker Avance 400 spectrometer operating
at 400.13 MHz and 13 C NMR spectra on a Bruker AC
300 spectrometer operating at 75.43 MHz in CDC13
using TMS as internal standard. 1 H–1 H COSY, HSQC,
HMBC and NOESY (with 150 ms mixing time) spectra
were recorded using the standard pulse sequences.
EIMS were obtained on a Nermag RI0-10 mass spectrometer at 70 eV by direct inlet probe, whereas
HREIMS were recorded on a Jeol JMS HX 110 mass
spectrometer. CC was performed on Acme Si gel ﬁner
than 200 mesh (0.08 mm).
3.2. Plant material
The whole plant of A. paniculata Nees was collected
in September 2002 at Tirumala Hills, Andhra Pradesh,
S. India. A voucher specimen (No. 0024/AP) has been
deposited in the Herbarium of the Department of Botany, Sri Venkateswara University, Tirupati, India.
3.3. Extraction and isolation
The air-dried and powdered whole plant (10.5 kg) of
A. paniculata was extracted with MeOH (5 Â 151, reﬂux
for 8 h) and the combined extracts were evaporated in
vacuo to yield a dark brown residue (1.2 kg). This was
fractionated by its solubility in CHCl3 , Me2 CO, and
MeOH, respectively. The residue (650 g) obtained from
evaporation in vacuo of the CHCl3 solubles was defatted with n-hexane and then separated by Si gel CC
using n-hexane–EtOAc step gradients as eluents to afford ﬁve fractions (I–V). Fraction I yielded 1 (64.5 mg)
and 3 (1.3 g) on puriﬁcation with Si gel column by
eluting with the mixtures of n-hexane and EtOAc.
Fraction II gave 2 (2.2 g), 4 (1.2 g), and 5 (25.6 mg)
after a series of chromatographic separations with
mixtures of n-hexane and EtOAc. Fraction III was
subjected to Si gel CC with n-hexane–EtOAc step gradients followed by prep. TLC, developed with benzene:acetone (9:1) to give 6 (17.1 mg) and 7 (5.2 mg).
Workup of fraction IV by Si gel column aﬀorded 7
(25.2 mg) and 8 (32.3 mg). The Me2 CO solubles were
defatted with n-hexane and the residue (150 g) obtained
was puriﬁed over a Si gel column using n-hexane and
EtOAc step gradient mixtures as eluents to give three
fractions. Workup of these three fractions, individually
by repeated Si gel CC with CHCl3 –EtOAc mixtures
followed by prep. TLC developed with benzene:acetone
(7:3) yielded 9 (18 mg) and 10 (25 mg); 11 (20 mg) and
12 (15 mg); and 13 (22 mg), respectively. The MeOH
solubles were extracted with n-hexane using a soxhlet
apparatus. The n-hexane insoluble portion was concentrated to dryness and the residue obtained (250 g)
was subjected to Si gel CC using CHCl3 –MeOH step
gradients to give four fractions A–D. Fraction A was
puriﬁed with Si gel column using CHC3 –MeOH step
gradients followed by prep.TLC with CHCl3 –MeOH
(9:1) to obtain 14 (18 mg) 15 (10 mg), 16 (5 mg), and 17
(12 mg). Fraction B on repeated CC over Si gel using
CHCl3 –MeOH step gradients aﬀorded 18 (22 mg), 19
(33 mg), and 20 (14 mg). Fraction C on puriﬁcation
with Si gel CC by eluting with CHCl3 –MeOH step
gradients gave 21 (15 mg), 22 (20 mg), and 23 (28 mg).
3.4. 5,7,20 ,30 -Tetramethoxyﬂavanone (6)
Colorless solid (CHCl3 ); m.p. 164–166 °C; [a]25
)34.8° (MeOH; c, 0.01), CD (MeOH; c, 0.01): De338
+0.46, De292 )1.05, De262 +0.53; UV (MeOH) nm
kmax ðlog eÞ: 263 (4.16), 290 (sh) (3.85), 336 (3.76); IR
max cm : 2904, 2843 (OMe), 1662 (>C@O), 1610,
1594, 1450, 1100; 1 H NMR (400 MHz, CDCl3 ): d 2.77
(1H, dd, J ¼ 16:7, 3.0 Hz, H-3eq ), 2.98 (1H, dd, J ¼ 16:7,
13.3 Hz, H-3ax ), 3.81 (3H, s, OMe-7), 3.86 (3H, s, OMe30 ), 3.88 (3H, s, OMe-5), 3.90 (3H, s, OMe-20 ), 5.76 (1H,
dd, J ¼ 13:3, 3.0 Hz, H-2), 6.09 (1H, d, J ¼ 2:3 Hz, H6), 6.14 (1H, d, J ¼ 2:3 Hz, H-8), 6.93 (1H, dd, J ¼ 7:0,
2.7 Hz, H-40 ), 7.15 (2H, m, H-50 , 60 ); 13 C NMR (75
MHz, CDCl3 ): d 45.0 (C-3), 55.4 (OMe-7), 56.2 (OMe5), 56.5 (OMe-30 ), 60.1 (OMe-20 ), 74.2 (C-2), 93.1 (C-8),
93.9 (C-6), 106.1 (C-4a), 112.0 (C-40 ), 117.3 (C-60 ), 123.2
(C-50 ), 131.6 (C-10 ) 146.8 (C-20 ), 152.5 (C-30 ), 162.2 (C5), 165.8 (C-7), 166.5 (C-8a), 189.8 (C-4); EIMS m=z (rel.
int): 344 [M]þ (100), 343 (7), 207 (16), 181 (10), 180 (65),
164 (17), 152 (6); HREIMS: found 344.1331, C19 H20 O6
Y. Koteswara Rao et al. / Phytochemistry 65 (2004) 2317–2321
3.5. 5-Hydroxy-7,20 ,30 -trimethoxyﬂavone (12)
Yellow amorphous solid (CHCl3 ); m.p. 191–192 °C;
UV (MeOH) nm kmax ðlog eÞ: 241 (3.62), 327 (3.02);
(MeOH + AlCl3 ): 241, 369; (MeOH + AlCl3 + HCl): 241,
369; IR mKBr
max cm : 3158 (OH), 2900, 2889 (OMe), 1656
(>C@O), 1607, 1508, 1450, 1135; 1 H NMR (400 MHz,
CDC13 ): d 3.86 (3H, s, OMe-30 ), 3.89 (3H, s, OMe-7),
3.92 (3H, s, OMe-20 ), 6.36 (1H, d, J ¼ 2:2 Hz, H-6), 6.44
(1H, d, J ¼ 2:2 Hz, H-8), 6.98 (1H, s, H-3), 7.07 (1H, dd,
J ¼ 8:1, 1.5 Hz, H-40 ), 7.21 (1H, dd, J ¼ 8:1, 7.9 Hz, H50 ), 7.33 (1H, dd, J ¼ 7:9, 1.5 Hz, H-60 ), 12.82 (1H, s,
OH-5); 13 C NMR (75 MHz, CDCl3 ): d 55.7 (OMe-7),
56.0 (OMe-30 ), 60.9 (OMe-20 ), 92.4 (C-8), 97.9 (C-6),
105.6 (C-4a), 110.5 (C-3), 115.2 (C-10 ), 120.6 (C-60 ),
124.2 (C-40 ), 126.0 (C-50 ), 148.0 (C-20 ), 153.3 (C-30 ),
158.0 (C-8a), 162.0 (C-5), 162.1 (C-2), 165.5 (C-7), 182.7
(C-4); EMS m=z (rel. int): 328 [M]þ (100), 300 (2), 166
(70), 165 (8), 162 (15), 161 (10), 149 (1), 138 (2), 107(1);
HREIMS: found 328.1052, C18 H16 O6 requires 328.1025.
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0 1994 E2szvicr Scicaoc Ltd
Pnnrcd in Great Entam All nghts reserved
17.00 + 0.00
DITERPENE FROM THE AERIAL PARTS OF ANDROGRAPHlS PANZCULATA
PETER G. WATERMAN*
Department of Chemistry, Forestry Research Institute of Malaysia, Kepong, 52109 Kuala Lumpur, Malaysia, lPhotochemistry
Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow Gl IXW, U.K.
Key Word Index-Andrographis
paniculata; Acanthaceae; labdane
novel diterpene has been isolated from the aerial parts of Andrographis paniculata and identified as ent14/I-hydroxy-8( 17),12-labdadien- 16,15-olide-3/I,19-oxide on the basis of 2D NMR techniques.
Andrographis paniculata Nees is a small herb found
throught southeast Asia and in India. Extracts of the
whole plant are used extensively as a tonic, an antihypertensive, a cure for snake-bite, against fever and in combination with Orthosiphon aristatus as a treatment for
diabetes [l, 23. Preparations made from A. paniculata
have also been shown to be effective in treating bacterial
infections . Previous phytochemical studies on A.
paniculata have resulted in the isolation of a number of
labdane diterpenes including andrographolide (1) . In
this paper we wish to report the presence of a further
labdane, characterized as (2), as the major constituent of a
Malaysian specimen of this species.
RFSULTS AND Dl!XXS!SlON
The whole plant was soaked in MeOH and the resulting extract concentrated and partitioned with EtOAc.
The EtOAc insoluble portion was decolourized with
charcoal and then recrystallized from MeOH to give 2 in
a yield of 1.12%. The EIMS revealed CM]’ at 332,
solving for C,,H,,O,
and the IR spectrum suggested the
presence of a hydroxyl, an x$-unsaturated lactone and an
exocyclic methylene. The ‘H NMR spectrum (Table 1)
confirmed the presence of an exomethylene and revealed
the presence of two methyls on quaternary carbons, the /Iproton of the %$-unsaturated lactone, two oxymethine
protons and an isolated oxymethylene. The ’ 3C NMR
spectrum (Table 1) indicated the presence of five quaternary carbons, five methine carbons, eight methylenes and
Correlation between proton and carbon resonances
was achieved by means of an HC-COB1 experiment
(Table I) and carbon connectivities by the HMBC tech-
nique [S] (Table 1). Important observations from the
HMBC study were as follows:
(a) Making an initial assumption that the exomethylene had arisen from the 17-methyl zJ and 3J interactions
and 13Cchemical shift data together with ‘5 and ‘J connectivities obtained from the
1.72 dr (12.9, 2.5)
ddd (12.9. 8.8, ca 2)
ddd (8.8, ca 2, 2)
2.34 dt (9.2, 1)
4.53 dd (9.9, 6.2)
4.65 dd (9.9, 2.2)
4.87, 4.90 2 x s
4.46 d (7.5)
3.66 d (7.5)
15.7 (C-20). 55.9 (C-9)
39.7 (C-10). 43.8 (C-4)
7.19 cd (6.9, 1.6)
25.5 (C-l l),
25.5 (C-l I),
15.7 (C-20). 55.9 (C-5),
39.8 (C-10). 130.7, (C-13). 148.5
56.9 (C-9). 66.5 (C-14), 171.3
37.8 (C-l). 55.9 (C-5). 56.9 (C-9)
from the exomethylene protons allowed the assignment of
C-7 (methylene), C-8 and C-9 (methine), indicating a
(b) A ‘J coupling between C-9 and the H-20 protons
identified the 20-Me which showed further couplings to
C-l, C-5 and C-10.
(c) The protons of a CH,-CH = system both showed
five long-range H -C couplings that allowed this system
to be placed at C-l 1 and C-12 and identified C13, C-14
(oxymethine) and C-16 (lactone carbonyl).
(d) The protons of an oxymethylene (64.53, 4.65) also
revealed couplings to C-14 and C-16, thus completing the
connectivities for the side chain.
(e) The remaining methyl showed four couplings, to
quaternary (C-4), methine (C-5), oxymethine (80.4 ppm)
and oxymethylene carbons.
(f) The protons of that oxymethylene (S4.46,3.66) also
correlated with C-4 and C-5 and must therefore represent
an oxidized C-4 methyl. Another ‘.J H-C coupling from
the oxymethylene protons also correlated with the oxymethine at 80.4 ppm, which must be linked to the oxymethylene by an oxygen bridge and be placed at C-3, thus
giving rise to an oxetane ring system.
With C-2 and C-6 being identified by further HMBC
correlations (Table 1) the assignment of structure 2 was
(C-7). 56.9 (C-9)
(C-5). 64.7 (C-19),
(C-18). 55.9 (C-5). 80.4 (C-3)
The COSY45 spectrum was valuable in confirming the
Hz-l l/H-12 and H14/H,-15 spin systems. A NOESY spectrum showed a
number ofspatial interactions (Fig. 1) confirming that the
20-Me and 19-oxymethylene were diaxial and on the
same face of the molecule (LXface). The diterpene would
appear to be of the ent-labdane series, as are all other
diterpenes isolated from this species  and the stereochemistry at C-14 is a-OH.
Mp: uncorr. IR: KBr disks. NMR spectra were run in
C,D,N at 400 MHz for ‘H spectra and 100.6 MHz for
‘% spectra. 2D experiments were run using the standard
Bruker microprograms .
Plant material. A. paniculata was collected in January
1992 from Masjid Tanah, Melaka. A voucher specimen
has been deposited in the Herbarium of the Department
of Botany, Universiti Kebangsaan, Malaysia.
Isolation of 2. The whole plant was air-dried in the
shade for 2 days and then ground. The resulting powder
(255 g) was macerated in MeOH for 5 days and this
process repeated twice. Evapn of the combined MeOH
extracts in uacuo gave a green solid (12.8 g) which was
repeatedly stirred with EtOAc. The insoluble residue was
redissolved in MeOH and stirred with charcoal. Concn of
the MeOH extract and storage at 0” afforded a ppt. which
on recrystallization from MeOH yielded 2 (2.8 g).
Ent-14/&hydroxy-8( 17),12-Iabdadien-16.1 S-elide-3P,l9oxide (2). Plates from MeOH, mp 221-222”. [c~]n - I2
(MeOH;c 1.00). IR v,,, cm - ‘: 3405, 1725, 1671,919; ‘H
and 13CNMR: Table 1; EIMS m/z (rel. int.): 332 CM]’
(1). 303(2), 275(2), 205(4), 173(8), 159(11),93(48), S5(59),
anonymous referee is thanked for
valuable comments. NMR spectra were obtained at the
Strathclyde University NMR Laboratory.
Perry, L. M. (1980) Medicinal P/ants ofEast and Southeast Asia: Attributed Properties and Uses. M.I.T. Press,
2. Burkill, 1. H. (1935) A Dictionary ofEconomic Products
of the Malay Peninsula, Volume 1. Crown Agents for
the Colonies, London.
3. Deng, W. L. (1978) Zhongcaiyao Tongxun. 9,459.
4. Dictionary of Natural Products (1993) Chapman and
5. Bax, A. and Summers, M. F. J. (1986) J. Am. Chem. Sot.
6. Quader, A., Gray, A. I., Waterman, P. G., Lavaud, C.,
Massiot, G. M. and Sadler, I. H. (1991) Tetrahedron 47,
Phyfochemrsrry,Vol 22, No
Pnated III Great Bntam
1, pp 314-315, 1983
FLAVONOIDS OF ANDROGRAPHIS
S C TANEJA, K L DHAR* and C K ATAL
Regional Research Laboratory (CSIR), Jammu Tawl, India
(Reorsedrecerved 20 May 1982)
Key Word Index-Andrographzs panzculata,
Acanthaceae, roots, 5-hydroxy-7&dlmethoxyflavanone, 3,7,8,2’-
separation of the petrol extract of A~rogra~h~punzcufaru
of two new flavonolds, 5-hydroxy-7,8dlmethox~avanone
methoxyflavone, as well as the known flavonold 5-hydroxy-7,8-dlmethoxyflavone
panzculuta Nees IS widely known for Its
medicinal value [l] Earher reports on Its chemical
constituents mclude flavonolds, sesqulterpene lactones
and other compounds [Z-8] In this paper we report the
isolation and charactermtlon of two new flavonolds, ( +)S-hydroxy-7,8-dimethoxyflavanone
2’-tetramethoxyflavone, from the roots In addmon, 5hydroxy-7,8-dlmethoxyflavone
also reported for the first time from this species
When subJected to column chromatography, the petrol
extract resulted m the lsolatlon of compounds l-3 (Fig 1)
Compound 1, mp 98-99” was assigned the structure ( +)5-hydroxy-?,8-dimethoxyflavanone
on the basis of the
following data It analysed for C1, H,,OS
spectrum gave bands at 288 and 342nm and UV shifts
with diagnostic reagents ascertained the presence of a 5hydroxyl group The ‘H NMR spectrum (CDCl,) gave a
multlplet centred at 63 0 assigned to C-3 methylenes,
besides ssgnats for two methoxyls A double doublet at
65 33 ( f = 5 and 10 Hz) Identified the C-2 proton The C6 proton was located at 66 13 and a broad smglet was
observed at 6746 for the aromatlc protons of rmg B
Acetylatlon gave the monoacetate, mp 13&132” In the
‘H NMR spectrum of this acetate the signal for C-6
shifted to 66 33, other signals remamed practically at then
orlgmal posttlons With dzazomethane under normal
condltlons, no methylatlon was observed However, when
methylated with DMS, a monomethyl
15&158”, was formed This confirms that the only
hydroxyl group present IS at C-5, which IS chelated
Oxidation with KMnO, m acetone gave an acid which
*To whom correspondence should be addressed
roots resulted m the lsolatron
was identified as benzolc acid, confirming the unsubstrtuted ring B Mass fragmentation fully supported the
assigned structure Therefore, 1 IS (&)-5-hydroxy-7,8dlmethoxyflavanone
Flavanones of the same structural
formula with 5,7,8- and 5,6,7_substltutlon patterns are
known synthetically [9, lo] Compound 1 agrees closely
with the physical data of the 5,7,8-substituted synthettc
compound, which ISreported to have mp 98-99”, the other
isomer havmg mp 148- 149” This 1sthe first report of 1 as
a natural substance
Compound 2, mp 209-21 l”, analysed for C19H1807
The UV spectrum m methanol showed strongabsorptlons
at 272, 362 and an mflexton at 302 nm, and a shaft with
AICi,-HCl indicated the presence of a .5-hydroxyl group
The ‘H NMR spectrum (60 MHz, CDCl,), gave, besides
the signals for four methoxyl groups, a sharp singlet at
66 46 for the C-6 proton and a multlplet centred at 67 10
for the 3’, 4’, 5’ protons C-6‘ was located separately as a
multlplet at 67 60 Acetylatton resulted m the formation
of the monoacetate, mp 15%159”, m the ‘H NMR of
which, C-6 shifted to 66 7, other signals remained practltally at then original positions Methyiatlon gave a
monomethyl ether, mp 152-154” On the basis of the
above data, 2 must be 5-hydroxy-3,7,8,~-tetramethoxyflavone The 5,7,8-substltutlon pattern of ring A
m 2 was further confirmed when the chemical shift values
of the C-6 proton of 2 were compared with dechlorochloroflavonm [ 111, a metabohte from cultures of A candzcans,
which 1s reported to have the same substitution pattern
The rmg B substitution pattern was confirmed when the
methyl ether was oxidized with KMnO, m acetone One
of the products was an acid (mp 99-loo”), which was
Identified as methylsahcyhc acid This estabhshes the
structure of 2 conclusively
Compound 3, mp 180-181”, was ldentlfi~ as 5hydroxy-7,8-dimethoxyflavone
(lit mp 173-175” )
from its spectral data and derlvatlves There appear to be
some maccuracles m the pubhshed spectral data for this
compound, so spectral details are being presented here
R’ = AC
3a R = H, R’ = AC
All the mps are uncorr Roots of Ana’rographis pawculara
(1 kg) were extracted first with petrol (bp 60-80”), followed by
EtOH The petrol extract was coned and kept at 0” A sohd mass
(700 mg) separated, which was subJected to CC over SI gel (50 g)
using C, H,, EtOAc and MeOH m different proportlons
Compound 1 was Isolated from the C,Hs fractions, crystalhzed from Me, CO-petrol as cream plates (80 mg), mp 98-99”,
analysed for C17H160s Found C, 68 07, H, 5 29 Requires C,
nm 288, 342, +NaOMe 286, 360,
+ AICI, 310,364, + AICI,-HCI 310,364, + NaOAc 288,342 IR
cm-’ 3435 (OH), 1650 (C = 0) MS m/z (rel mt) 300 (M+,
lOO),299 (16 58), 285 (27 77), 257 (6 80), 223 (32 86), 197 (21 99),
196 (l@O),181 (lOO),168 (49 44), 167 (28 47), 153 (95), 104 (23 96)
and 103 (28 20) Fragments 196 and 104 occurred due to retroDiels-Alder fragmentation of m/z 300 The monoacetate crystallized from MeOH as yellow crystals, mp 13(r132”, analysed for
C19H1806 ‘H NMR (60 MHz, CDt&) 62 4 (3H,s, -OCOMe),
3 0 (2H,m, 3-H), 3 85 and 3 95 (2 x 3H, 2s, 7,8-OMe), 5 53 (lH,dd,
J = Sand lOHz,2-H),636(1H,s,6-H),746(5H,s,2’,3’,4’,5’,6’H) The methyl ether crystalhzed from MeOH as yellow crystals,
mp 15&158”, analysed for ClsH1s05
‘H NMR (60 MHz,
CDCI,) 63 0 (2H,m,3-H), 3 75, 3 85 and 3 9 (3 x 3H, 3s, 5,7,8OMe), 5 53 (lH,dd,J = 5 and 10 Hz, 2-H), 6 15 (lH,s,6-H), 7 46
Oxadatwe degradatron of 1 The methyl ether of 1 (30 mg) was
subJected to oxldatlve degradation by KMnO, m Me,CO
Among other products, a compound crystalhzed from boihng
H, 0 (4 0 mg), mp 122”,analysed for C, H,Oz and was ldentdied
as benzolc acid by co-TLC, mmp and superimposable IR 2 was
isolated from C,H,-EtOAc
(95 5) fractions, crystalhzed from
MeOH as yellow plates, mp 209-211” (lOOmg), analysed for
C19H1807 Found C, 63 73, H, 501 Reqmres C, 63 69, H,
5 02 y0 UV I gp” nm 272, 302 mf , 362, + NaOMe 272, 360,
+ NaOMe 272, 360; + AICI, 278, 362, + AICI,-HCI 278, 362,
+ NaOAc 272, 358 IR vKBrcm-’ 3440 (OH), 1660 (C = 0),
1600,1580,1500,1370,1235,850 MS m/r (rel mt ) 358 (M+, 90),
153 (34 38), 147 (5 45) and 125 (12 14) The monoacetate crystallized from MeOH, mp 158-159”, analysed for C,, H,,08
NMR (60 MHz, CDCI,) 6 2 53 (3H,s, -OCOMe), 3 87,3 9,4 0,
403 (3H each, 4s, 3,7,8,2’-OMe), 67 (lH,s, 6-H), 7 10 (3H,s,
3’,4’,5’-H), 7 60 (lH,s,6’-H) The methyl ether was obtamed as
dark-coloured plates from Me,CO, mp 152-154”, analysed for
(15H,m,3,5,7,8,2’-OMe), 646 (lH,s,6-H), 7 10 (3H,m,3’,4’,5’-H),
7 60 (lH,s,6’-H)
Oxrdatrve degradation The methyl ether of 2 (40 mg) was
subJected to oxldatlve degradation by KMnO, in Me,CO
Among other products, a compound crystalhzed from boding
H,O (60 mg), mp 99-loo”, analysed for C,H,Os and was
identified as methylsahcylic acid by co-TLC, mmp and superimposable IR 3 was Isolated from later benzene fractions,
crystallized from MeOH, yellow needles, mp 180-181” (120mg)
and analysed for Cl7 H , 4O4 Found C, 68 46, H, 4 70 Requires
C, 68 45, H, 4 69 % UV rl$$$‘” nm 274,346, + NaOMe 274,346,
+ AICI, 280,362, + AICI,-HCI 280,362, + NaOAc 274,344 IR
,KBrcm-1 3440 (OH), 1665 (C = 0) IH NMR (60 MHz,
CDCI,) 63 97 (6H, s, 7,8-OMe), 6 43 (lH, s, 6-H), 6 66 (lH, s, 3H), 7 45-7 60 (3H, m, 3’,4’,5’-H),7 85-8 0 (2H, m, 2’,6’-H), 12 30
(1 H, s,-OH), which disappeared on D, 0 exchange MS m/z (rel
mt) 298 (M+,90),283 (lOO),255 (1003), 181, (25 12), 153 (55 85),
125 (19 71), 102 (1440) The monoacetate, crystalhzed from
MeOH as yellow crystals, mp 228-229”, and analysed for
‘H NMR (60 MHz, CDCI,) 6342 (3H, s,
OCOMe), 4 0,4 03 (3H each, 2s, 7,8-OMe), 6 66 (lH, s, 3-H), 6 70
(lH, s, 6-H), 7 45-7 60 (3H, m, 3’,4’,5’-H), 7 85-8 0 (2H, m, 2’,6’H) The methyl ether was obtained as yellow plates from MeOH,
mp 161-163”, analysed for C,,H1sOS
‘H NMR (60 MHz,
CDCI,) S4 0(9H,s(br), 5,7,8-OMe),645 (lH,s,6-H),6 70 (lH,s,
3-H), 7 45-7 60 (3H, m, 3’,4’,5’-H),7 858 0 (2H, m, 2’,6’-H)
The Wealth of Indta, Raw Marerrals, Vol I, p 77
Council of Scientific and Industrial Research, New Delhi
2 Qudrat-I-Khuda, M , Biswas, K M and Ah, A (1963) Pak J
SCI lnd Res 6, 152
3 Qudrat-l-Khuda, M , Irfan, M and Faruq, 0 (1964) SCI Res
4 Govmdachan, T R , Pal, B R , Srmlvasan, M and Kalyana
Raman, P S (1969) Indian J Chem 3, 306
5 Erfan, A M, B~swas, K M and Chowdhary, S A (1972) J
SCI Ind Res 15, 33
6 Soediro, S and Maman, H (1973) Acta Pharm 4, 36
7 Bahnam, A and Connolly, J D (1973) J Chem Sot Perkm
Trans 1, 1247
8 Satyanarayan, D, Mythuayee, C and Knshnamurthy, V
(1978) Leather Sea (Madras) 25, 250
9 Chopin, J , Chadenson, M , Gremer, G and Louise, M (1959)
Bull Sot Chum Fr 1585
10 Ramaknshnan, G, BanerJI, A and Chadha, M S (1974)
11 Marchelb, R and Vmmg, L C (1973) Can J Bzochem 51,
12 Escarna, R , Torrengera, S , Dana, R and BenJamm, A
(1977) Phytochemurry 16, 1618
Vol 27. pp. 5081 to 5091.
Pcrgamon Press 1971.
Pnntcd in Great Britain
THE STRUCTURE AND STEREOCHEMISTRY
A DITERPENE GLUCOSIDE
W. R. CHAN, D. R. TAYLOR,C. R. WILLISand R. L. BODDEN
Chemistry Department, University of the West Indies, Kingston 7, Jamaica
Bonn, West Germany
(Received in USA 9 June 1971; Received in the VKfor publication
15 June 1971)
Abstract - Neoandrographolide
(2) is shown to be the fbglucoside of ent-19-hydroxy-8( 17), 134abdadien16,15-olide. A correlation with andrographolide has been done.
Exrn~ctx of the shrub Androgruphk paniculuta Nees (Acanthaceae), common in
the West Indies and India. are extensively used as household medicines in these
areas.’ The main constituent of A. panicdata is the diterpenoid lactone, andrographolide (1).2The isolation and characterisation of a second, minor, crystalline component.
m.p. 167-168”. was described by Kleipool.3 He suggested the
molecular formula C2sH3s08 for neoandrographolide
and from solubility experiments and a positive Legal test deduced the presence of an a&unsaturated y-lactone.
The preparation of an acetate, m.p. 157”. which was considered to be an anhydrotetraacetate, was also described. We now report our further investigations on neoandrographolide which lead to the constitution 2.*
Elemental analysis of a number of derivatives combined with mass spectrometric
results (see later) indicate a molecula formula C,,H,,Os
There was a single CO band (v,, 1748 cm-‘) in the IR and this with UV data [A_
205 mn (E 10,400)] supported the presence of an a&unsaturated butenolide already
inferred by Kleipool Four OH groups were indicated by strong IR absorption near
3300 cm-’ and by the formation of a tetraacetate (3). C34H48012, m.p. 155-157”.
This acetate has a m.p. in good agreement with that reported by Kleipool for his
but a direct comparison has not been possible. The acetate (3)
has no OH absorption in the IR and can be reconverted to neoandrographolide. A
medium intensity band at 909 cm- l indicated the presence of an exocyclic methylene
group. The presence of two ethylenic bonds in neoandrographolide (2) was confirmed
by the formation of the tetrahydro derivative (4) on catalytic hydrogenation.
The NMR spectrum of the acetate (3) shows signals for four acetates and two tertiary Me groups. There is a narrow multiplet at 6 7.10 (W 3 = 4 Hz) assigned to the
B-proton of the a&unsaturated
lactone system The chemical shift is similar to
that found in analogous systems5 while the small coupling constant with the adjacent
W. R. CHAN, D. R. TAYLOR, C. R. WILLIS, R. L. RODDEN and H.-W. FEHLKUIER
protons suggests that the double bond is endocyclic rather than exocyclic.2 The
spectrum also shows a complex of signals between 6 3.1 - 5.2 integrating for 13
protons which was not amenable to analysis.
15: R = AC
Reaction of the acetate (3) with osmium tetroxide in dioxan afforded the diol (5).
C3.+Hs001+ m.p. 143-145”. which shows retention of the butenolide moiety in the
UV. The formation of the dial(5) involved the exocyclic methylene and in agreement
there are no bands associated with this group in the IR Periodate cleavage of the diol
gave, in good yield formaldehyde (isolated as the 2,4-dinitrophenylhydrazone)
m.p. 145-147”. This compound may also be
the norketone acetate (6). C,,H,,OiJ,
obtained directly from the acetate (3) by oxidation with the osmium tetroxidesodium periodate reagent.6 Hydrolysis of 6 afforded the tetro17.
Spectral data for the norketone acetate (6) are in accord with the assigned structure.
The butenolide system is associated with a maximum in the UV at 207 nm (E 8950)
while in the IR there is a broad CO band (1754-1739 cm-‘) for the acetate and lactone
functions and another at 1701 cm-’ ascribed to a cyclohexanone. In the NMR
spectrum H-14 appeared as a broadened singlet at 6 7.16 (W 4 = 4 Hz).
The location of the ketone and the stereochemistry of the bicyclic system was
established by CD data The curve for 6 shows a positive Cotton effect [As,,, + 2.74
at 293 nm. inflexions at 300 (As + 2164) and 310 nm (As + l-56)] and is virtually
superimposable on that of the andrographolide derivative (8) [As,,,, + 2.67 at 289
nm, inflexions at 297 (As + 2.41) and 305 nm (As + 1*39)]. This r&tilt places the
ketone at C(8) and indicates an identity in the relative and absolute configurations
of the decalin systems.
Oxidation of the tetraacetate (3) with the sodium periodate-potassium permanganate reagent’ gave an amorphous acid which was characterized as the crystalline
methyl ester, C31H46013, m.p. 142-144”. by treatment with diazomethane. Spectral
data (Experimental) are in agreement with the structure 9.
6: R = Glu(Ac),
7: R = Glu
The presence of a glucose moiety in neoandrographolide was established as follows.
In the iodometric estimation neoandrographolide required 158 moles of periodate
after 48 hr. With acetaldehyde and zinc chloride, an acetylidene derivative (10) was
obtained which was further characterized as the diacetate (11). Strong absorption
in the IR near 890 cm-’ indicated that the exocyclic methylene is intact in these
compounds Treatment of neoandrographolide
with hydrochloric acid in ethanol
under reflux gave the iso-aglucone (12), C,,H,,Q,,
m.p. 121-123”. together with
glucose which was identified by two dimensional paper chromatography
appropriate hexoses as markers. The @configuration of the glucoside was clearly
shown by the NMR spectrum of the norketone acetate (6) in which H-l’ appears as
a doublet (J = 7.5 Hz) at 6 4.45.
11: R = AC
W. R. CHAN, D. R. TAYLOR,C. R. WILLIS, R. L. BODDENand H.-W. FEHLHABER
Acetylation of the iso-aglucone (12) gave the acetate (13). also obtained from 2 by
treatment with acetic acid and cone sulphuric acid The structure of the acetate (13)
follows from its genesis and spectral data_ The NMR spectrum shows 3 singlet
methyls, one of which is vinylic, and one acetate. Above 6 = 3. there are a total of
five protons in distinct spin systems. An AB quartet at 6 4 10 (J = 11.5 Hz V~ 196 Hz)
can be assigned to the acetoxymethyl group. H-15 appears as a doublet (J = 1.3 Hz)
at 6 4.78 and H-14 as a narrow multiplet at 6 7.17. The chemical shift of the acetoxymethyl group is in good agreement with that found in methyl podocarpate and in
andrographolide derivatives where this group is axial.’
16:R’ = H.a-0H;R’
17: R’ = H. a-OH; R2 = CPh,
19: R’ = SCH,CH,S;
R* = H
22: R’ = 0; R2 = AC
23: R’ = NNHSO,C,H,Me:
RZ = AC
The base peak (m/e 287) in the mass spectrum of both 12 and 13 corresponds to
the ion C,,H,,O:
formed by loss of the CH,OR group. Surprisingly, there are
significant fragments which must be formed by elimination of the entire side-chain,
i.e. by a homolytic cleavage of the C(9)-C(ll) bond. Thus in 12 there is a peak at
m/e 207 (40%) and in 13, peaks at m/e 249 (25%) and 189 (249-AcOH, 54%) The
composition of all the ions mentioned was confirmed by precise mass measurements.
Since direct cleavage of a vinylic bond is not usually a favoured process, it is possible
this fragmentation is preceded by a rearrangement of the double bond to allow an
allylic cleavage or, alternatively. a Me migration, as indicated in Scheme 1, is involved.
The double bond isomerization associated with the formation of the iso-aglucone
under acidic conditions is not surprising Indeed, andrographolide (1) gave the triacetate (14) under conditions identical for the conversion of 2 to 13. In the NMR
The structure and stereochemistry of neoandrographolide
R = H. m/e 201
R = AC.m/e 249
spectrum this triacetate has two tertiary methyls, a vinylic methyl. H-14 as a diffuse
doublet at 6 5.93 and H-12 as a triplet (J = 7 Hz) at 6.92 shown to be spin-coupled
to the two protons at C (11) which appear as a diffuse doublet at 3.00. In the isomeric
triacetate (15). H-12 occurs as a triplet at 6 6.97 (J = 6.5 Hz) and is coupled to the
protons on C(11) at 2.43.
The high resolution mass spectrum of the norketone acetate (6) (Fig 1) further
delineates many of the features already established. For a sugar derivative it shows
a surprisingly strong parent ion at m/e 650 (5%) confirming the molecular formula
(&Hq6013. Three fragment ions at m/e 303, 289 and 349 are formed by loss of the
sugar moiety. These correspond to the ions C,9H270i (a), C1sHz501 (b) and
Cz0Hz90: (c) (scheme 2). The formation of (c) is initiated by cleavage of C(l’)-C(2’)
in the sugar followed by a McLafferty rearrangement as indicated in (d). An analogous
fragmentation is found in the steroidal sapogenins.”
Several metastable peaks indicate that the main fragmentation path proceeds
through the ion (e) of m/e 540. This is obtained from the parent ion through a McLafferty rearrangement involving the C(8) CO group. Subsequent loss of Me radical
yields the ion (9 at m/e 525. An alternative decomposition of(e) leads to the fragment
(8 m/e 331) representing the sugar residue. This oxonium ion. characteristic of the
glycosides of the tetraacetyl hexoses.‘* lo gives by stepwise elimination of the acetate
groups the well-known 9*l1 fragment ions at m/e 271, 169 and 109. The elemental
composition of all the fragments discussed were substantiated by high resolution
These observations lead to the structure and stereochemistry of neoandrographolide
In an attempted correlation of neoandrographolide
(2) and andrographolide (1).
(16)2 was converted to the tritylether (17) and thence to the
keto tritylether (18) by Jones oxidation. Treatment of 18 with ethane dithiol and
BF,-etherate gave the thioketal (19) together with the sulphur compounds (28 and
21). Reduction of the thioketal (19) with deactivated Raney nickel led to a complex
mixture which could not be separated.
W. R. CHAN, D. R. TAY~R,
C. R. WILLLF,R. L. E~ODDEN and H.-W.
The structure and stereochemistry of neoandrographolide
W. R. CHAN, D. R. TAYLOR,C. R. W~uts, R. L.
A successful interrelationship was achieved through the ketoacetate (22) obtained
on treatment of the keto trityl ether (18) with acetic anhydride and sulphuric acid.
A Clemmensen reduction using the conditions described by Toda, et al.” led to
no useful result. However. reduction via the p-tosylhydrazone (23)13 gave, in poor
yield, a compound identical with the iso-aglucone (12) obtained from neoandrographolide (2).
M.ps were determined on a Kofler hot stage apparatus and are uncorrected. UV data are for EtOH
solns. IR spectra for Nujol mulls and rotations in CHCl, solns unless stated otherwise. NMR spectra
were done in CDCl, with TMS as internal reference. The mass spectra were obtained with the AEI MS9
and MAT CH4 using direct insertion (ion soura 200” and 70’ respectively) at 70 eV. High resolution mass
measurements were made with the MS 9 at a resolving power of 12.000.
Isolo~ion o~neoandrographolide. After separation of andrographolide from the CHCIs extract of Andrograph paniculata Nees most of the solvent was removed and the resulting syrup was stirred repeatedly
with benzene. The residue obtained from decantation of the benzene extracts was dissolved in EtOH and
decolourized with charcoal. Concentration of the etbanolic soln and cooling afforded crude neoandrographolide, 2.20 g. from the extraction of 45 kg of crushed plant material.
recrystallized from ‘EtOH as needles. m.p. 167-168” (reported” 174-174.5”).
[a]n - 48” (c. 14 in pyridine). L,, 205 MI (E 10.400) v, 3290, 1748. 1639.900 cm-’ (Found: C. 65.3;
H. 8.6; 0. 261. CZ6H4,,0s requires: C. 65.0: H. 8.4: 0. 26.6%).
After heating under reflux with ethanolic-NNaOH
for 30 min neoandrographolide was recovered quantitatively on acidification of the alkaline soln.
tetraacetute (3). This was obtained with Ac,O:Py overnight at room temp. The
product was isolated by dilution with water and filtration. The yield of crystalline product was quantitative.
The tetraacelate (3) recrystallized from EtOH in needles. m.p. 155-157”. [aIn - 31” (c. 093). &_ 205 nm
(E 10.300). v,, 1742 1639. 909 cm-‘. 6 @68. @93 (CMe). 200. 2.02 2.07 (3H. 6H. and 3H respectively.
4 x OAc). 3.17. 3.93 (AB system J = 95 Hz 2 x H-19) 7.10 (1H. a W) 4 HZ H-14) (Found: C. 62.9;
H. 7.4. C,,H,,O,,
requires: C. 6295; H. 7.5%).
Hydrolysis of the retraacetale (3). A soln of the tctraacetate (52 mg) and KHCO, (100 mg) in MeOH
(5 ml) and water (2 ml) was kept at room tcmp for 20 hr. The ppt obtained on removal of the MeOH in
vacua and dilution with water was collected. Recrystallization from MeOH gave neoandrographolide.
40 mg (m.p.. m.m.p. and IR comparison).
Neoandrographolide (201 mg) was hydrogenated in EtOH (45 ml) with
loO/, Pd-C (192 mg). There was an uptake of 2.18 moles H,. Working up gave a gum which on treatment
with EtOH furnished the tetrahydro deriuatiw (4) as plates 140 mg. m.p. 98-100’. [a]n - w (c. @95).
v, 3226 1767 cm-’ (Found: C. 64.7; H. 92 CZ6H4,0s requires: C. 644; I% 9.15%).
The derived acetate. prepared with Ac,O-Py. crystallized from aqueous EtOH as needles m.p. 139-142”.
[a]n - 37” (c. 0.973. v,, 1761. 1748 cm-’ (Found: C. 626; H. 8.1. C3bH,20,2 requires: C. 62.6; H. 89%).
The norketone acetate (6)
(a) A soln of 0~0, (IO0 mg) and 3 (200 mg) in dry dioxan (I2 ml) was stored for 4 days. The osmate
ester was decomposed by dry H,S. the mixture filtered and the product extracted into EtOAc Chromatography on silica and elution with CHCI, gave the dial (5) 120 mg. needles from aqueous MeOH. m.p.
143-145”. [aIn - 19” (c. 0833. i,
207 nm (E SC’@@).
v,, 3333. 1745 cm-’ (Found: C. 59.2: I-L 7.2.
requires: C. 59.8; I-L 74%). A soht of 5 (474 mg) a+ndNaIO* (280 mg) in EtOH (14 ml) and
water (8 ml) was kept at 4” for 23 hr then diluted with water (20 ml) and extracted with EtOAc (2 x 30 ml).
The aqueous layer was distilled and the distillate collected in a saturated soln of 2.4-dinitrophenylhydrazine
in 2N H2S0,. The ppt was filtered off after 3 hr and recrystallized from EtOH to give ydlow needlts (51 mg.
33%). m.p. 166”. identified as formaldehyde 2.4-DNP by m.m.p. and spectral comparison.
The EtOAc extract from above was evaporated to give a gum (380 mg) which was dissolved in benzene
and filtered through a short column of alumina Evaporation of the solvent and crystallisation of the residue
from aqueous MeOH afforded the norketone acetate (6) as plate& 260 mg. m.p. 145-147” (de+ 1,207 nm
(f:895Okv, 1754-1739.1701 cm-‘. 6 07.1.05 (CMe) 1.98.2+)0.205 (3H. 6H and 3H respectively. 4 x OAc).
The structure. and stereochemistry of neoandrographolide
4.45 (1H. d J = 75 Hz H-1’). 7.15 (1H. a Wi 4 Hz H-14) [Found: C 606; H. 6.75; 0. 32.7%; M (mass
spectrum) 6502924. C,,H,,O,,
requires: C a9; H 71; 0. 32Q?/,; hf. 65029381.
(b) 0~0, (20 mg) was added to a stirred soln ~$3 (300 mg) in dioxan (10 ml) and water (2 ml) at room
temp. The 0~0, was completely dissolved after 15 min and NaIO, (2GO g) was added in portions over
30 min. After stirring overnight the mixture was colourless with a ppt of NalO,. The mixture was taken
up in etha (100 ml) and water (100 ml) and the ethereal soln was washed with water. saturated with H,S
and filtered The filtrate wss washed with loo/, NaHCO,. dried and evaporated. The residue crystal&d
from aqueous MeOH lo give 6 identical with that described above.
The Tetrol(7). A soln of 6 (1M mg) in EtOH : water (3 : 1. 12 ml) containing NaHCO, (300 mg) was kept
at room temp for 40 hr. The mixture was then passed through Dowex 5OW-X4 resin (acid form) previously
washed with 3 : 1 EtOH-water. The eluate was evaporated to a small volume in uacuo. dilute HCI (5 drops)
was added and the soln warmed on a steam bath to complete lactonisatiou After cooling the product
was collected by filtration and recrystallised from EtOAc-light petroleum to give needles. 130 mg. m.p.
137-139”. I, 206 mn (E7700). v, 3333. 1761. 1724 cm-’ (Found: C 61.7; H. 7% C,,H,sO, requires: C.
62.2; H. 7.9%).
Acetylation of 7 with AC+Py in the usual way afforded 6 in good yield.
Oxidation of neoondrographolide telraacetate with potassiwn permanganate and sodium periodate
A soln of3 (1.045 g). K,CO, (1.20 g). KMnO, (1.62 8) and NaIO, (6.38 g) in water (120 ml) and dioxan
(200 ml) was allowed to stand at room temp. After 61 hr a further quantity of KMnO, (360 mg) was added.
After 26 hr. the mixture was acidified with dil H,SO, and extracted with EtOAc (2 x 200 ml). The combined
extract was washed successively with water (2 x 100 ml) and 10% NaHCO, (2 x 100 ml). Acidification
of the bicarbonate extract with dil H$O. and extraction into EtOAc gave. after removal of the solvent
in uacuo. an acid fraction (380 mg) as a foam This was methylated in the usual way with diazomethanc to
furnish the methyl ester(91 needles from aqueous MeOH. m.p. 142-144”. v, 1745. 1710 cm-‘. d O-77.
1.01 (CMe). 201 and 2.15 (9H and 3H respectively. 4 x OAc) 366 (3H. CO,Me) (Found: C. 599; H. 7.0.
C,,H,,013 requires: C. 59.4; H. 74%).
Sodium periodate oxidation of neoandrographolide and the tetrahydro derivative
(a) A mixture of neoandrographolide (10-Omg) in EtOH (20 ml) and OOlN NalO, (50 ml) was kept at
0” in the dark. The progress of the reaction was followed by titration in the usual way. There was an uptake
of 1.58 moles of pcriodate after 48 hr.
(b) A similar estimation using 4 showed an uptake of 1.70 moles after 48 hr.
A mixture of neoandrographolidc (100 mg) freshly fused Z&l, (93 mg) and acetaldehyde (5 ml) was
stored overnight at room temp. Most of the acctaldehyde was removed by evaporation. the residue was
diluted with water and the product collected by filtration. Crystallization from aqueous EtOH and then
from EtOAc-hexane gave the acetylidene (10) as netdles m.p. 199-201”. v, 3400. 3220. 1754. 1639. 893
cm-’ (Found: C 67.1; H. 84. C2sH420s requires: C. 66.4; H 8.4%).
Tbe acetylidene diacetate (11). prepared with Ac,O-Py. crystallized as prisms from EtOAc-hexane. m.p.
166-167”. v, 1754. 1644.890 cm-‘. d 066.092 (CMe). 1.32 (3H d J = 6 Hz set-Me). P03.208 (2 OAc).
7.12 (1H. a W f = 4 Hz H-14). (Found: C. 65.4; H. 7% Cx2H,,0,0 rquires: C 65.1; H. 7.85%).
Reaction ojneoandrographolide with hydrochloric acid
The iso-a&cone (12). A soln of neoandrographolide
(1.00 g) in EtOH (m0 ml). water (50 ml) and cone
HCI (40 ml) was heated under reflux for 5 hr. After cooling and dilution with water. the product was extracted into EtOAc (3 x 100 ml). The combined extract was washed thoroughly with water. dried and
evaporated to give a gum which crystallized from aqueous MeOH as prisms. 5C0 mg m.p. 121-123”. v,
3448.1740 cm-’ [Found: C. 75.3; H. 9.5; 0.15.2x; M (mass spectrometry) 318.2195. &H,,,O,
C. 75.4; H. 9.5; 0. 15.1%; hf. 318.21951.
(a) Cone H,SO, (2 drops) was added to a soln of neoandrographolide (91 mg) in HOAc (2 ml). After 15
miu at room temp. the soln which was now a pak purple was heated on a water bath for 15 min. The
product was recovered by dilution with water and extraction into EtOAc The combined extract was
D. R. TAYLOR,C. R. WILLIS,R. L.
and H.-W. FEHJZABER
washed successively with water. 10% NaHCO, and water. Evaporation in oacuo gave a gum which was
dissolved in benzene and chromatographed on silica (4 g). Elution with benzene gave tbc title compound
which crystallized as plates from aqueous EtOH. 47 mg. m.p. 111-l 13”. [a]o - 61” (c. 1.11). v, 1754 and
1730 cm-‘. 6 097 (6H. 2 x CMe). 1.62 (vinylic Me). 205 (OAc). 4.10 (2H. q. J, = 11.5 Hz vAB= 196
Hz CH,OAc) 4.78(2H. &.I = 1.3 Hz H-15). 7.17 (If-L Wi4 HZ H-14) [Found: C. 72.8; H. 8.95; 0. 17.8%;
M (mass spectrum) 3602303. CZ2Hs204 requires: C. 73.3; H. 8.95; 0. 1775%; M. 360.2300].
(b) Acetylation of 12 with Ac,O-Py overnight at room temp gave 13 identical by m.m.p. and spectral
neatment ojandrographolide with acetic acid tmd sulphuric acid
The triacetate (14). Andrographolide (314 mg) was treated with HOAc and cone H,SO* under conditions
described in experiment (a). The product showed one major spot on TLC and was purified by PLC (3 : 1
CHCI, :EtOAc as developing solvent) to give the triacelate (14). 154 mg which crystallized as plates from
MeOH. m.p. 139-141”. v, 1760. 1720 and 1660 cm-‘. 6 1.02 1.05 (CMe). 1.59 (vinylic Me). 2Q7. 2.17
(6H and 3H respectively. 3 x OAc). 393 (2H. bd. J = 7 Hz 2 x H-11) 692 (1H. bt .I = 7 Hz H-12)
(Found: C 65.1; H. 7.55. C,,H,,Os requires: C. 65.5; H. 7.6%).
The Pity/ Ether (17). A soln of 16’ (64 g) and trityl chloride (8a g) in dry pyridine was heated under
rcflux for 42 hr then poured into water. The product. recovered with EtOAc was triturated with 1 : 1
benzene-light petroleum (6 x 50 ml) leaving a residue which chromatographed on alumina Elution with
benzene-light petroleum (1 : 1) gave the trityl ether (17) which crystallized in plates from CHCI,-light
petroleum. 664 g. m.p. 230-232”. v,, 3300. 1750 cm-’ (Found: C. 81.0; H. 7.75. Cs9H1.0. requires: C.
81.2; H. 7.7%).
The kero trityl ether (18). Jones reagent was added dropwise with stirring to a soln of the above trityl
ether (20 g) in acetone (750 ml) at 0” until a brown colour persisted The product. recovered with EtOAc.
crystallized from MeOH in prisms 1.74 g m.p. 194-196”. Y, 1750. 1700 cm-‘. 6 043. 1.38 (CMe). 3.10.
3.45 (AB system. J = 9.5 HZ 2 x H-19). 4.65 (H-17 overlapping with 2 x H-15 at 4.68). 4.85 (other H-17).
7al(lH. m w$5 HZ H-14). 7.32(15H. m w$8 Hz aromatic protons)(Found: C. 81.8; H. 7.4. (&,H,,O*
requires: C. 81.5; H. 7.4%).
The thioketal(l9). A soln of 18 (430 mg) BF,,‘Et,O (1 ml) and ethanedithiol (1 ml) in gl HOAc (18 ml)
was kept at room tentp for 12 hr. The disulphide (20) crystallised from the mixture as needks (48 mg). mp.
182-184” (from CHCl,,‘MeOH). v,, 1585 cm-‘. 6 7.25 (30H. a ArH). 2.12 (4H. s. CH,f’_& (Found: C.
8295; H. 6.0: S. 11.1. C,,H&
requires: C. 83.0: H. 5.9; S. 11.1%).
Dilution of the filtrate. after removal of the disulphidc with water until no more crystals were formed
gave a ppt (167 mg) which on PLC in light petroleum (2 developments) yielded the disulphide (45 mg) and
the thiol(21). (107 mg). needles from CHCI,/MeOH. m.p. 116-120”. v,, 2510. 1570 cm-‘. 6 7.32 (15H. m
ArH). 2.0-2.6 (4H. complex a C&CH,). 1.38 (1H. t. .I = 7.5 Hz SW (Found: C. 741; H. 5.9; S. 192.
H,O requires: C. 73.9; H. 5.9; S. 18.8%).
The filtrate was further diluted with water and extracted with EtOAc to yield a gum (333 mg) which
on PLC in CHCI, gave the thioketal(19) (150 mg) needles from MeOH/water. m.p. 158-162”. v, (CHCI,)
3500. 1751.900 cm-‘. 6 C-67. 122 (CMe). 3.27 (4H. bs. W f 3 Hz_ CH2Cu2) 22.214.171.124 (AB system J = 12
Hz 2 x H-19). 126.96.36.199 (1H each s. C==CH,). 4.78 (2H. m W 4 5 HZ 2X H-15). 7.n (1H. m W 4 5 HL
H-14) (Found: C. 64.4; H. 7.7; S. 15.8. C,,H,,O$,
requires: C 647; H. 79; S. 15.7%).
The keto acetate (22). A mixture of 18 (900 mg). HOAc (25 ml) and cone H,SO, (07 ml) was heated on
a water bath for 15 min then poured into cold loo/, NaHCO, aq. The ppt was collected and crystallized
from MeOHaq to give the ketoacetate (22) as prisms 263 mg mp. 101.5-104’. v_ 1750.1700. 125Ocm-‘.
6 1.20 (2 x CMe) 1.66 (vinylic Me). 2a (OAc). 3.98. 4.60 (AB system J = 11.5 Hz 2 x H-19). 4.80 (2H.
m. W f = 5 HZ, 2 x H-15), 7.16(1H, m W i = 5 Hz, H-14). (Found: C. 7@2: H. 8.2, C22H300s requires:
C, 7Q6: H, 8.1%).
The tosylhydrazone (23). A soln of 22 (200 mg) anrJ ptoluenesulphonylhydrazinc
(115 mg) in AcOH
(10 ml) was set aside at r.oom temp overnight. The ppt obtained on pouring the mixture into water was
collected and crystallized from MeOH in prisms. 208 mg m.p. 153-157”. v, 1760. 1735 cm-‘. 6 1.04.
1.16 (CMe). 1.62 (vinylic Me). 1.70 (OAc). 2.42 (ArMe). 3.75. 450 (AB system J = 11 Hz 2 x H-19).
4.79 (2H. m W 4 = 4 Hz 2 x H-15). 7.20 (1H. m H-14) 7.33. 7.88 (2H each. d J = 8 Hz. ArH). WI7.85
(exchangeabk with D,O. NH) (Found: C. 64.0; H. 6.9. C29H3806N2S requires: C. 642: H. 7.0%).
Reduction of the tosylhydrazone (23). A mixture of the tosylhydrazom (80 mg). NaOH (400 mg) and
NaBH, (150 g) in 65% aqueous diglyme was stirred at room temp for 17 hr. The mixture was poured into
The structure and stereochemistry of ncoandrographolide
cold dil HCI and the product recover4 with CHCI,. Purification of the product by PLC (using a sample
of 12 as marker) and crystallisation from aqueous EtOH gave plates, I5 mg, m.p. 12&122”, identical
with 12 by m.m.p_ TLC and IR spectral comparison.
Acknowledgements-We thank Dr. G. Snatzke for obtaining the CD data and for helpful comments.
Alcan Jamaica Ltd.. for a Junior Research Fellowship (to C.R.W.) and the Research and Publications Fund.
University d the West Indies. for providing the V-6058 spin decoupler. One of us (H.-W.F) also thanks the
Stiftung Volkswagenwerk for supplying the mass spectrometers.
* G. F. Asprey and P. Thornton.
W.1.med. J. 2. 233 (1953); R N. Chopra I. C. Chopra K L. Handa
and L. D. Kapur. Indigenous Drugs OfIndia p. 278. U. N. Dhur. Calcutta (1958)
2 M. P. Cava W. R. Chan. R. P. Stein and C. R. Willis. 7etrohedron 21. 2617 (1965). and refs cited
3 R. J. C. Kleipool. Nature. Land. 69. 33 (1952)
4 For a preliminary communication of some of these results sez W. R Chaa D. R. Taylor, C. R. Willis
and H.-W. Fehlhaber. Tetrahedron Letters 4803 (1968)
’ N. S Bhacca. L. F. Johnson and J. N. Shoolery. NMR Spectra Catalog Vol. I. No. 51. Varian Associates.
Palo Alto. California (1962)
6 R. Pappo. D. S. Allen. Jr., R. U. Lemieux and W. S. Johnson. J. Org. Chem. 21.478 (1956)
’ R U. Lemieux and E. von Rudloff. Conad. J. Chem. 33. 1701 (1955)
s H. Budzikiewicz D. H. Williams and C. Djerassi. Structure Elucidation dh’atural Products by Mass
Specrrometry Vol. II. p. 117. Holden-Day. San Francisco (1%4)
’ K. Heyns. H. F. Griltzmacher. H. Scharmann and D. Miiller. Forts&. them. Forsch. 5. 448 (1966)
lo I. A. Pearl and S. F. Darling. Phytochem. 7. 831 (1968)
I’ K. Heyns and D. Miiller. Tetrahedron Letters 6061 (1966)
I2 M. Toda. Y. Hirata and S. Yamamura. Chem. Comm. 919. (1969)
I’ L. Cagliotti. Tarahedron 22.487 (1966)