J. Nat. Prod.

2006, 69, 403-405 403

Andropanolide and Isoandrographolide, Minor Diterpenoids from Andrographis paniculata:

Structure and X-ray Crystallographic Analysis⊥

Swapan Pramanick,† Sukdeb Banerjee,† Basudeb Achari,† Binayak Das,† Ashis K. Sen, Sr.,† Sibabrata Mukhopadhyay,*,†
Alain Neuman,‡ and Thierry Prangé§
Indian Institute of Chemical Biology, Kolkata 700032, India, Laboratoire de Chimie Biomoléculaire (UMR 7013 CNRS),
93017 Bobigny Cedex, France, and Laboratoire de Cristallographie & RMN Biologiques (UMR 8015 CNRS), 75006 Paris, France

ReceiVed June 14, 2005

Phytochemical investigation of the leaves of Andrographis paniculata has led to the isolation of a new labdane type
diterpenoid, andropanolide (1), along with seven known diterpenoids including isoandrographolide (2), previously reported
as a rearrangement product of andrographolide. The structures and stereochemistry of compounds 1 and 2 were established
by X-ray crystallographic analysis.

Andrographis paniculata Nees (Acanthaceae)1 is a small herb

found throughout southeast Asia and India. Extracts of this plant
and its constituents are reported to exhibit a wide spectrum of
biological activities including antibacterial,2 antiinflammatory,3
antimalarial,4,5 antithrombotic,6 hepatoprotective,7 and immuno-
stimulant8 properties. An alcoholic extract of the plant and its major
constituents have been shown to affect the cell cycle progression
in prostrate and breast cancer cell lines9 as well as to exhibit potent
cell differentiation inducing activity in mouse myeloid leukemia
(M1) cells.10 Previous investigations on the chemical composition
of A. paniculata showed that it is a rich source of labdane
diterpenoids6,10-18 and 2′-oxygenated flavonoids.16,18-23 In the
present study, we report the isolation of andropanolide (1), a new
labdane diterpenoid, along with seven known diterpenoids,
andrographolide,6,10-18 andrograpanin,10,14,18 isoandrographolide
(2),24,25 14-deoxy-11,12-didehydroandrographolide,10,13,17,18 14-
deoxyandrographolide,10,12-14,18 isoandrographolide (2),24,25 neo- Figure 1. ORTEP drawing for compound 1.
andrographolide,10,12,14,26 and 14-deoxyandrographolide 19-gluco-
side.10,14 The structures were determined mainly through the use
of 1D and 2D NMR techniques. For 1 and 2 the deductions were singlets with fine splitting at δ 4.68 (1H) and 4.52 (1H) ascribed
confirmed by X-ray crystallographic analysis. to the exomethylene group. A set of doublets (J ) 11.0 Hz) at δ
4.24 and 3.41, each integrating for one proton, pointed to the
presence of a hydroxymethylene group, with the doublet at δ 3.41
overlapping a signal assignable to H-3. The double triplet at δ 6.52
(J ) 7.0, 1.0 Hz) and a broad singlet at δ 4.80 were attributable to
H-12 and H-14. Appropriate 13C NMR signals were observed at δ
17.0 (C-20), 25.42 (C-18), 65.9 (C-19), 70.8 (C-14), 81.6 (C-3),
109.8 (C-17), 131.5 (C-13), and 150.1 (C-12). The 13C NMR data
of 1 were comparable to those of andrographolide, except that the
signals for C-12 and C-14 appeared downfield in 1 by 3.2 and 4.9
ppm, respectively. This was accompanied by an upfield shift of
0.65 ppm for H-12, which suggests that the geometry of the double
bond may be reversed in 1, so that the carbonyl group is oriented
Andropanolide (1) displayed a quasimolecular ion peak at m/z away from H-12. The downfield shift of C-14 in 1 can then be
373 in the ESIMS (positive mode). This information, together with rationalized on the basis of the absence of a γ-effect from H-11.
data from elemental analysis and the 13C NMR DEPT spectrum, On the basis of the above data, andropanolide (1) was concluded
suggested the molecular formula to be C20H30O5. The IR spectrum to be a geometric isomer of andrographolide, although the stereo-
showed absorption bands at 3600 (hydroxy group), 1760 (R,β- chemistry at C-14 remained to be proved conclusively. Finally, the
unsaturated-γ-lactone), and 898 (exomethylene) cm-1. The 1H NMR detailed structure and stereochemistry of 1 were established
spectrum of 1 indicated the presence of two methyl groups with unambiguously from single-crystal X-ray analysis (ORTEP drawing
three-proton singlets at δ 1.29 and 0.52 ppm. It also exhibited two in Figure 1). The absolute configuration has been assumed on the
basis of that established for andrographolide from ORD studies27
⊥ Dedicated to Dr. Norman R. Farnsworth of the University of Illinois and X-ray crystallographic inferences.28
at Chicago for his pioneering work on bioactive natural products. A compound with similar structure but with unknown C-14
* To whom correspondence should be addressed. Tel: +91-33-24733491. stereochemistry has been reported earlier from A. paniculata10 and
Fax: +91-33-24735197. E-mail: sbmukh@yahoo.co.uk.
† Indian Institute of Chemical Biology. as a metabolite of andrographolide in rats.28 The spectroscopic data
‡ Laboratoire de Chimie Biomoléculaire. reported are in good agreement with those observed by us for
§ Laboratoire de Cristallographie & RMN Biologique. andropanolide (1), which settles the structure of these products.
10.1021/np050211n CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/11/2005
404 Journal of Natural Products, 2006, Vol. 69, No. 3 Notes

Table 1. X-ray Crystallographic Data Processing and

Refinement Statistics for Andropanolide (1) and
Isoandrographolide (2)
andropanolide isoandrographolide
(1) (2)
formula (asymmetric unit) C20H30O5
molecular weight 350.44
space group P212121 P212121
Z 4 4
cell params (Å, deg)
a 7.435(1) 7.004(1)
b 10.636(1) 11.484(1)
Figure 2. ORTEP drawing for compound 2. c 23.951(2) 22.831(1)
volume (Å3) 1894.1(7) 1836.3(3)
no. of reflns measd 9221 8323
Isoandrographolide (2), obtained as a colorless crystalline solid, completeness (%) 98.5 99.0
displayed 20 signals in the 13C NMR spectrum (one carbonyl, four R sym (%) overall 5.1 4.4
other nonprotonated carbons, five methines, seven methylenes, and no. of indep reflns 2796 2729
three methyls) and gave a pseudomolecular ion peak at m/z 373 in no. of obsd reflnsa 2153 2339
the ESIMS (positive mode) attributed to the [M + Na]+ ion, R factor (observed F data) 0.043 0.039
R factor (all F data) 0.062 0.052
suggesting it to be an isomer of andrographolide (C20H30O5). The R factor (observed F2 data) 0.101 0.100
1H NMR spectrum had several signals comparable to those of the
no.of params 271 232
latter, viz., a 1H downfield singlet (δ 7.29) assignable to the min./max. (e-) in last -0.16/+0.14 -0.14/+0.11
β-proton of an R,β-unsaturated carbonyl system, a signal at δ 3.44 electron density
for H-3, and a pair of AB doublets (J ) 10.8 Hz) at δ 4.26 and CCDC deposit number 259885 274843
3.36 for the hydroxymethylene group (H2-19). The major difference a
Criteria for observation: F > 2σ(F).
was in the presence of three methyl singlets (at δ 1.25, 1.10, and
0.95) coupled with the absence of signals for an exomethylene the Indian Botanical Garden, Howrah, India. A voucher specimen (No.
group, which pointed to the presence of a ring system involving 338) has been deposited at the Medicinal Chemistry Department of
C-8. this institute.
Extraction and Isolation. The air-dried powdered leaves of A.
Detailed analysis of the 2D NMR spectra provided further paniculata (1 kg) were subjected to extraction in a Soxhlet apparatus
structural information. Although correlations involving ring A and with light petroleum ether (bp 60-80 °C), CHCl3, and MeOH,
most of ring B were comparable to those of andrographolide, the respectively. The MeOH extract was partitioned between n-BuOH and
chemical shifts of C-8 and C-12 (δ 83.1 and 73.5) identified them water. The n-butanol extract (15 g), obtained after evaporation of solvent
as being oxygenated, nonolefinic carbons. Evidence for a furanoid in vacuo, was chromatographed on a column of silica gel (mesh 60-
C-ring came from the observed HMBC correlation of the C-8 signal 120). Gradient elution was carried out with chloroform followed by
with that of H-12 (δ 4.68). The H-12 triplet showed, in addition, various mixtures of CHCl3 and MeOH (19:1, 9:1, 17:3, and 4:1). A
cross-peaks with signals for the olefinic carbons (δ 143.7 and total of 50 fractions (each 250 mL) were collected, and fractions giving
138.8), pointing to branching at C-12. The 2H singlet at δ 4.81 similar spots on TLC were combined. Repeated chromatography of
the fractions followed by preparative TLC resulted in the isolation of
could then be due to the acyloxymethylene protons of the R,β-
andrographolide (2.5 g), andropanolide (14 mg) (1), andrograpanin (40
unsaturated-γ-lactone moiety constituting ring D. The location of mg), 14-deoxy-11,12-didehydroandrographolide (80 mg), 14-deoxyan-
the third methyl group at C-8 was evident from the HMBC drographolide (500 mg), isoandrographolide (25 mg) (2), neoandrogra-
correlation of C-17 with the H-9 signal. pholide (1.5 g), and 14-deoxyandrographolide 19-glucoside (40 mg).
The methyl group at C-10 (supposed to be R as in case of Andropanolide (1): colorless needles (MeOH); mp 210-211 °C;
andrographolide) showed a strong NOE interaction with H-12; the [R]25D - 31.5° (c 0.5, C5H5N); IR (KBr) νmax 3600, 1760, 898 cm-1;
latter must therefore be R-oriented. Absence of any NOE correla-
1
H NMR (C5D5N, 500 MHz) δ 6.52 (1H, dt, J ) 7.0, 1.0 Hz, H-12),
4.68 (1H, s, H-17a), 4.52 (1H, br s, H-17b), 4.33 (1H, dd, J ) 9.5, 6.0
tions further indicated that H3-17 is trans to both H3-20 and H-12.
Hz, H-15a), 4.24 (1H, d, J ) 11.0 Hz, H-19a), 4.19 (1H, dd, J ) 9.5,
The evidence suggested that the compound could be identical with 3.5 Hz, H-15b), 3.44 (1H, m, overlapped signal, H-3), 3.41 (1H, d, J
isoandrographolide, reported earlier24,25 as the acid-catalyzed rear- ) 11.0 Hz, H-19b), 2.92 (1H, brdd, J ) 16.0, 6.0 Hz, H-11a), 2.79
rangement product of andrographolide but not so far isolated from (1H, ddd, J ) 16.0, 11.0, 7.5 Hz, H-11b), 2.11 (1H, brd, J ) 12.5 Hz),
a natural source.29 However the stereochemistry, particularly at C-8 1.85-1.50 (several protons), 1.29 (3H, s, H-18), 1.2-0.9 (several
and C-12, remained to be settled. Therefore, we undertook an X-ray protons), 0.52 (3H, s, H-20), signal for H-14 apparently merged in
crystallographic study. The results prove that the stereochemistry solvent signal at 4.80 ppm; 13C NMR (C5D5N, 125 MHz) δ 171.6 (C,
at C-8 and C-12 should be as shown (8R,12S) in the ORTEP C-16), 150.1 (CH, C-12), 149.9 (C, C-8), 131.5 (C, C-13), 109.8 (CH2,
diagram (Figure 2). C-17), 81.6 (CH, C-3), 76.0 (CH2, C-15), 70.8 (CH, C-14), 65.9 (CH2,
C-19), 58.4 (CH, C-5), 57.1 (CH, C-9), 44.9 (C, C-4), 41.1 (C, C-10),
40.0 (CH2, C-7), 39.0 (CH2, C-1), 30.7 (CH2, C-2), 26.2 (CH2, C-11),
Experimental Section 25.42 (CH3, C-18), 25.36 (CH2, C-6), 17.0 (CH3, C-20); ESIMS
General Experimental Procedures. TLC was carried out on silica (positive mode) m/z 373 [M + Na]+; anal. C 68.44%, H 8.62%, calcd
gel 60 F254 (Merck) plates, and spots were visualized by spraying with for C20H30O5, C 68.54%, H 8.63%.
Liebermann-Burchard reagent followed by heating at 120 °C. Column Isoandrographolide (2): colorless prisms (ethyl acetate-hexane);
chromatography was performed on silica gel mesh 60-120 (Merck). mp 198-200 °C; [R]25D -40.4° (c 1.09, CHCl3); IR (KBr) νmax 3268,
1754, 1021 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.29 (1H, s, H-14),
Melting points were measured on a Yanagimoto micro-melting point
4.81 (2H, s, H-15), 4.68 (1H, t, J ) 4.6 Hz, H-12), 4.26 (1H, d, J )
apparatus and were uncorrected. Specific rotations were measured on
10.8 Hz, H-19a), 3.44 (1H, d, J ) 9.3 Hz, H-3), 3.36 (1H, d, J ) 10.8
a JASCO DIP-370 polarimeter. IR spectra were recorded as KBr pellets Hz, H-19b), 2.43 (1H, dd, J ) 8.3, 4.6 Hz, H-11a), 2.20 (1H, m, H-1a),
using a JASCO 7300 FTIR spectrometer. 1H and 13C NMR spectra 2.01 (1H, dd, J ) 8.3, 4.6 Hz, H-11b), 1.77 (3H, m, H-7a, H-2), 1.53
were recorded using a Bruker DRX spectrometer (500 MHz). The mass (3H, m, H-1b, H-6a, H-9), 1.44 (1H, m, H-6b), 1.25 (3H, s, H-18),
spectra were recorded on a Q-TOF-Micromass spectrometer. 1.10 (3H, s, H-17), 1.04 (1H, m, H-7b), 0.98 (1H, m, H-5), 0.95 (3H,
Plant Material. The leaves of Andrographis paniculata were s, H-20); 13C NMR (CDCl3, 125 MHz) δ 173.1 (C, C-16), 143.7 (CH,
collected from suburbs of Kolkata in January 2005 and identified at C-14), 138.8 (C, C-13), 83.1 (C, C-8), 81.2 (CH, C-3), 73.5 (CH, C-12),
Notes Journal of Natural Products, 2006, Vol. 69, No. 3 405

71.0 (CH2, C-15), 64.6 (CH2, C-19), 58.4 (CH, C-9), 53.1 (CH, C-5), (13) Balmain, A.; Connolly, J. D. J. Chem. Soc., Perkin Trans. 1 1973,
42.9 (C, C-4), 39.4 (CH2, C-7), 36.6 (CH2, C-1), 36.0 (C, C-10), 33.3 1247-1251.
(CH2, C-11), 32.0 (CH3, C-17), 27.8 (CH2, C-2), 23.2 (CH3, C-18), (14) Fujita, T.; Fujitani, R.; Takena, Y.; Takaishi, Y.; Yamada, T.; Kido,
18.6 (CH2, C-6), 16.8 (CH3, C-20); ESIMS (positive mode) m/z 373 M.; Miura, I. Chem. Pharm. Bull. 1984, 32, 2117-2125.
[M + Na]+; anal. C 68.46%, H 8.61%, calcd for C20H30O5, C 68.54%, (15) Jantan, I.; Waterman, P. G. Phytochemistry 1994, 37, 1477-1479.
H 8.63%. (16) Reddy, M. K.; Reddy, M. V. B.; Gunasekar, D.; Murthy, M. M.;
Caux, C.; Bodo, B. Phytochemistry 2003, 62, 1271-1275.
X-ray Crystallographic Analysis of Andropanolide (1) and (17) Rao, Y. K.; Vimalamma, G.; Rao, C. V.; Tzeng, Y. M. Phytochem-
Isoandrographolide (2). The two compounds were crystallized from istry 2004, 65, 2317-2321.
a methanol-anisole mixture. In both cases, a monocrystal was selected, (18) Reddy, V. L. N.; Reddy, S. M.; Ravikanth, V.; Krishnaiah, P.; Goud,
glued on a glass fiber, and mounted on a Nonius CCD Kappa T. V.; Rao, T. P.; Ram, T. S.; Gonnade, R. G.; Bhadbhade, M.;
diffractometer using Mo KR radiation (λ ) 0.7107 Å). The structures Venkateswarlu, Y. Nat. Prod. Res. 2005, 19, 223-230.
were solved using the SHELXS30 program and refined with SHELXL. (19) Govindachari, T. R.; Pai, B. R.; Srinivasan, M.; Kalyanaraman, P.
Diffraction data and refinement statistics are given in Table 1. S. Indian J. Chem. 1969, 7, 306.
(20) Jalal, M. A. F.; Overton, K. H.; Rycroft, D. S. Phytochemistry 1979,
Acknowledgment. The authors are thankful to the Director, Indian 18, 149-151.
Institute of Chemical Biology, Kolkata, India, for his keen interest in (21) Gupta, K. K.; Taneja, S. C.; Dhar, K. L.; Atal, C. K. Phytochemistry
the work. The authors also thank Sri Soumendranath Sardar and Sri 1983, 22, 314-315.
(22) Gupta, K. K.; Taneja, S. C.; Dhar, K. L. Indian J. Chem. 1996, 35B,
Rajendra Mahato of this institute for the collection of plant material
512-513.
and technical assistance. (23) Kuroyanagi, M.; Sato, M.; Ueno, A.; Nishi, K. Chem. Pharm. Bull.
1987, 35, 4429-4435.
References and Notes (24) Schwyzer, R.; Biswas, H. G.; Karrer, P. HelV. Chim. Acta 1951, 34,
(1) Gorter, K. Rec. TraV. Chim. 1911, 30, 151-160. 652-677.
(2) Gupta, S.; Choudhry, M. A.; Yadava, J. N. S.; Srivastava, V.; Tandon, (25) Cava, M. P.; Chan, W. R.; Stein, R. P.; Willis, C. R. Tetrahedron
J. S. Int. J. Crude Drug Res. 1990, 28, 273-283. 1965, 21, 2617-2632.
(3) Deng, W. L.Drugs Future 1990, 15, 809-816. (26) Chan, W. R.; Taylor, D. R.; Willis, C. R.; Fehlhaber, H. W.
(4) Misra, P.; Pal, N. L.; Guru, P. Y.; Katiyar, J. C.; Srivastava, V.; Tetrahedron Lett. 1968, 4803-4806.
Tandon, J. S. Int. J. Pharmacol. 1992, 30, 263-274. (27) Cava, M. P.; Chan, W. R.; Haynes, L. J.; Johnson, L. F. Tetrahedron
(5) Najib, N. A. R. N.; Furuta, T.; Kojima, S.; Takane, K.; Ali, M. M. 1962, 18, 397-403.
J. Ethnopharmocol. 1999, 64, 249-254. (28) He, X.; Li, J.; Gao, H.; Qiu, F.; Cui, X.; Yao, X. Chem. Pharm.
(6) Zhao, H. Y.; Fang, W. Y. Chin. Med. J. 1991, 104, 770-775. Bull. 2003, 51, 586-589.
(7) Choudhury, B. R.; Haque, S. J.; Poddar, M. K. Planta Med. 1987, (29) Although a compound of the name isoandrographolide has been
53, 135-140. reported from A. paniculata by Matsuda et al.10 and He et al.,28 the
(8) Puri, A.; Saxena, A.; Saxena, R. P.; Saxena, K. C.; Srivastava, V.; published structure is quite different from that of isoandrographolide
Tandon, J. S. J. Nat. Prod. 1993, 56, 995-999. reported earlier by Schwyzer et al.24 and Cava et al.25 We therefore
(9) Babish, J. G. WO 9617605, 1996; Chem. Abstr. 1996, 125, 132808. prefer the name isoandrographolide for the earlier reported prod-
(10) Matsuda, T.; Kuroyanagi, M.; Sugiyama, S.; Umehara, K.; Ueno, uct.24,25
A.; Nishi, K. Chem. Pharm. Bull. 1994, 42, 1216-1225. (30) Sheldrick, G. M. SHELXL97: Program for the Refinement of Crystal
(11) Kleipool, R. J. C. Nature 1952, 169, 33-34. Structures; University of Göttingen: Göttingen, Germany, 1997.
(12) Chan, W. R.; Taylor, D. R.; Willis, C. R.; Bodden, R. L. Tetrahedron
1971, 27, 5081-5091. NP050211N