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Gomphostenins: Two new antimalarial compounds from the leaves of Gomphostemma niveum
Bioorganic & Medicinal Chemistry Letters 20 (2010) 1312–1314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Gomphostenins: Two new antimalarial compounds from the leaves of Gomphostemma niveum Manisha Sathe, M. P. Kaushik * Discovery Center, Process Technology Development Division, Defence R&D Establishment, Jhansi Road, Gwalior 474 002 (MP), India
a r t i c l e
i n f o
Article history: Received 26 January 2009 Revised 24 February 2009 Accepted 27 February 2009 Available online 4 March 2009 Keywords: Antimalarials Gomphostemma niveum Labiatae Clerodane XRD
a b s t r a c t Phytochemical investigation of CHCl3 extract of the Gomphostemma niveum leaves led to the isolation of two new diterpene, compound 1 and 2. Their structures were elucidated by spectroscopic procedures and single crystal XRD. Compound 1 named as Gomphostenin 1 and structure was established as 8-ethyl (5Hfuran-2-one, 14-hydroxy, 2-oxo 3, 20 Z(17) diene clerodane, while compound 2 named as GomphosteninA; was found to be the acetyl derivative of compound 1 and revealed as 8-ethyl (5H-furan-2-one, 14-acetoxy, 2-oxo 3, 20 Z(17) diene clerodane. In vitro antimalarial activity against Plasmodium falciparum showed that compound 2 was more active than compound 1 and CHCl3 extract as well; with IC50 value of 3.4 lg/mL. Ó 2009 Published by Elsevier Ltd.
Malaria is one of the most important parasitic infections of humans due to its high morbidity and mortality with major consequent impact on economic productivity and livelihood.1 Approximately 40% of the world population live in areas with the risk of malaria. Each year 300–500 million people suffer from acute malaria and 1.5–2.5 million die from the disease.2–4 Plasmodium falciparum, the causative agent of the malignant form of malaria, has high adaptability by mutation and is resistant to various types of antimalarial drugs.5 Thus, low cost malaria treatments such as chloroquine and fansidar (sulfadoxine/pyrimethamine) become ineffective with the selection and spread of mutant drug-resistant parasites and then to multidrug resistance emergence.5–7 Artemisinin (qinghaosu) has become increasingly important as a malaria treatment but the current routes for its total chemical synthesis8–11 remain too complex for commercial production.12,13 There is no doubt that intensive use of artemisin-classes combinations therapy is an invaluable opportunity that decrease the burden of malaria and prolong drug life span of effective use. However, in the absence of substitute, the appearance of artemisinin-resistant malaria would lead to untreatable malaria and to a potential humanitarian disaster. These facts combined with the absence of a vaccine and the lack of systematic vector control strategies provides the rationale for the development of novel drugs against malaria. Therefore, new drugs with novel mechanisms of action and
* Corresponding author. Tel.: +91 751 2343972; fax: +91 751 2340042. E-mail addresses: [email protected], [email protected] (M.P. Kaushik). 0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.bmcl.2009.02.120
that are structurally unrelated to existing antimalarial agents are thus urgently required.14–16 The priority is the identification of original target(s) leading to novel antimalarial compounds which would a priori not allow cross-resistance with pre existing antimalarials. Natural products are extremely successful in providing mankind with substances to combat diseases, and today, roughly 50% of all small molecules as drugs on the market addressing infectious diseases are natural products or derivatives thereof.17 Hence, the ethnopharmacologial approach for the search of new antimalarial has proved to be more predictive with respect to malaria, quinine and its derivatives are still in use today, and new combination therapies based on artemisinin were recently introduced in the clinic.18,19 As part of our continuing efforts directed towards the discovery of the structurally interesting and biologically active compounds from the Indian medicinal plants,20 it was found that aqueous and CHCl3 extract of Gomphostemma niveum showed the antimalarial properties against P. falciparum. Gomphostemma niveum is a coarse, stellatelly pubescent or tomentose herb belonging to the Labiatae family, seen in small pockets at the foothills of the Himalayas in the northeastern part of India. Local people have used aqueous leaf extracts of this plant in acute malaria cases. Due to its highly localized presence and knowledge of its effectiveness against malaria, no systematic efforts have been reported before this. Here in we report the phytochemical analysis of chloroform extract of the Gomphostemma niveum leaves resulted in the isolation of two new clerodame diterpenes (1 and 2) compounds and there antimalarial activity against P. falciparum was studied.21
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M. Sathe, M. P. Kaushik / Bioorg. Med. Chem. Lett. 20 (2010) 1312–1314
19
20
Table 1 1 H NMR and
H
O
C NMR data of compound 1 and 2 in CDCl3
Position 17
18
15
14 7
Compound 1 dC
16
O
RO
13
13 CH3
8
H
1
10 9
6
5
O
2
3
4 CH3 12 CH3 11
Gomphostenin 1 R=H Gomphostenin-A 2 R= COCH 3
The structure of the new compounds was established using IR, MS, 1H, 13C and DEPT NMR. The final structure of compound 1 was revealed by the X-ray crystallographic analysis. Since the in vitro antimalarial efficacy of CHCl3 extract was far better than the aqueous extract, hence CHCl3 extract was used for the isolation of active compounds. The dried powdered leaves (500 g) were defatted with 3 1 L of petroleum ether followed by extraction with 3 1 L of CHCl3 extracted three times with CHCl3 in a soxhlet apparatus. The combined extracts were concentrated under vacuum. The portion of active CHCl3 extract (5 g) was subjected to column chromatography (silica gel, 60–120 mesh) using step gradient of CHCl3/MeOH to yield two major fractions (F1–F2). Fraction F1 was subjected to repeated silica gel (100–200 mesh) column chromatography (CC) by eluting with MeOH/CHCl3 (10:90) to yield compound 1 (25 mg). Similarly fraction F2 was also subjected to silica gel column chromatography eluting with MeOH/CHCl3 (4:96) to get compound 2 (1.22 g). Gomphostenin 1 was identified as 8-ethyl (5H-furan-2-one,14hydroxy, 2-oxo 3, 20 Z(17) diene clerodane. It was isolated as a white solid with the positive optical rotation ½a25 D +98.64 (c 1.64, CHCl3). The HRESI-MS of compound 1 revealed a molecular ion peak corresponding to (M+H)+ at m/z 333.2068 (calcd 333.2066) indicating the molecular formula C20H28O4. The ring and double bond factor was calculated to be 6. The UV (MeOH) absorbance at kmax 207 nm (e 9800) and 220 nm (e 9100) supported the presence of a,b-unsaturated carbonyl group. The IR spectrum displayed absorptions bands at 3434 cm1 (OH), 17331 (a,b-unsaturated clactone) and 1667 cm1 (a,b-unsaturated C@O). The 1H and 13C NMR spectra of compound 1 assigned as shown in Table 1. 1H NMR exhibited three ternary methyl groups at d 1.84, 1.07 and 0.8 (3H each, singlet), two oxygenated methylene groups at d 2.25 (2H, multiplet) and 4.75 (2H, singlet). Two olefinic protons showed typical downfield shift at d 5.77 (H-3) and d 7.08 (H-20) as singlet. The 13C NMR of compound 1 showed the presence of 20 C-atoms and also indicated the presence of a,b-unsaturated ketone (d 199.9), substituted olefin (d 125, 172), and three methyl groups (d 18.1, 18.4, 18.9). Further it also displayed signal at d 174 is due to C@O of lactone ring, d 133.4, 144.4 are corresponding to disubstituted olefin and d 70.4 is assignable to methylene carbon in the lactone ring. DEPT-135 confirmed the presence of three quartet carbon, three methyl, seven methylene and four methine carbon atoms. Suitable colourless X-ray quality crystals of compound 1 were obtained by crystallization from hexane/CHCl3 (7:3), The X-ray diffraction analysis of 1 was carried out on a single crystal,22 Figure 1. This study confirmed the structure of 1 and clearly establishes that
1 2 3 4 5 6 7 8 9 10 11 12 13 14a 14b 15 16 17 18 19 20 21 22 a b
a
35.0 199.9 125.4 172.6 21.7 18.9 45.4 38.1 39.6 45.2 18.1 18.4 18.9 63.6 34.3 35.4 133.9 174.6 70.4 144.4
dHb
multiplicity
2.25 (m) — 5.77 (m) — 1.40 (m) 1.6 (m) 1.89 (m) — — 1.25 (m) 1.84 (s) 1.07 (s) 0.8 (s) 3.24–3.32 (dd, J = 7.5, 7.5) 3.76–3.80 (dd, J = 4.0, 4.0) 1.4 (m) 2.25 (m) — — 4.75 (s) 7.08 (s)
Compound 2 dC
a
38.0 199.4 125.6 171.25 34.8 18.9 45.3 34.7 39.53 40.5 18.35 18.7 18.62 65.3 34.3 21.75 134.0 171.9 70.3 143.8 174.1 20.9
dHb multiplicity 2.24 (m) — 5.74 (s) — 1.48 (m) 1.69 (m) 1.86 (m) — — 1.25 (m) 1.80 (m) 1.40 (m) 0.92 (m) 3.80–3.86 (dd, J = 7.5, 7.5) 4.20–4.25 (dd, J = 4.0, 4.0) 1.4 (m) 1.9 (m) — — 4.7 (s) 7.1 (s) — 2.1 (s)
100 MHz. 400 MHz.
the relative configuration of the four stereogenic carbons are in fact 7R, 8S, 9S and 10S (or 7R, 8S, 9S and 10S). Gomphostenin-A 2 was isolated as a brown viscous liquid, with positive optical rotation ½a25 D +96.64 (c 1.64, CHCl3). The HRESI-MS of compound 2 revealed a molecular ion peak corresponding to (M++H) at m/z 375.2180 (calcd 375.2185). indicating the molecular formula C22H30O5 The IR spectrum showed absorption at 1714 cm1 (a,b-unsaturated c-lactone) and 1673 cm1 (a,b-unsaturated C@O). The 1H NMR and 13C NMR spectroscopic data of the compound 2 (Table 1) which is similar to that of compound l. In
Figure 1. ORTEP diagram of Gomphostenin 1.
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M. Sathe, M. P. Kaushik / Bioorg. Med. Chem. Lett. 20 (2010) 1312–1314
Table 2 IC50 values of CHCl3 extracts, compound 1, 2, and chloroquine diphosphate (CQ) against P. falciparum Compound
IC50 ± SD (lg/mL) CQ-S
IC50 ± SD (lg/mL) CQ-R
Aqueous extract CHCl3 extract 1 2 CQ
151.6 ± 2.9 9.7 ± 1.3 38.2 ± 1.2 3.4 ± 1.0 0.25
133.3 ± 5.8 9.5 ± 1.4 37.7 ± 0.466 3.2 ± 0.057
tion was calculated by the formula: (1 Nt/Nc) 100, where Nt and Nc represent the number of schizonts in the test and control. In summary, two clerodane diterpenes were isolated from the chloroform extract of Gomphostemma niveum. The structure of compound 1 and 2 was established on the basis of NMR spectral data and confirmed by X-ray crystallographic analysis. The IC50 of compound 1 and 2 has been found to be 38 lg/mL and 3.4 lg/ mL (Table 2). The reference drug chloroquine has exhibited 100% inhibition at 0.25 lg/mL concentration in the same test system.
IC50 values are expressed as the mean microgram concentrations ± standard deviations. All experiments were realized in triplicate.
Acknowledgements case of 1H NMR the sharp singlet at dH 2.1 indicated the presence of methyl attached to a carbonyl functionality. The 13C NMR showed a characteristic peak at dC 171.9 suggested the presence of lactone ring in the structure. MS–MS fragmentation of 2 showed the loss of one acetoxy group. To the best of our knowledge, this is the first report on chemical analysis of isolation of compound 1 and 2 from G. niveum. The antimalarial efficacy of the crude extracts and the pure compound 1 and 2 were evaluated by conventional in vitro parasite culture method, using the (chloroquine sensitive) MRC-02 strain of P. falciparum obtained from the National Institute of Malaria Research, New Delhi, India, maintained in a continuous culture using the standard method described by Trager and Jensen.23 Parasites were cultured in human B(+) erythrocytes in Rosewell Park Memorial Institute (RPMI)-1640 media (GIBCOBRL, Paisely, Scotland) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) buffer, 10% human AB(+) serum, and 0.2% sodium bicarbonate (Sigma), and maintained at 5% CO2. Cultures containing predominantly early ring stages were synchronized by addition of 5% D-sorbitol (Sigma) lysis, used for testing. Initial culture was maintained in small vials with 10% haematocrit, that is, 10 lL erythrocytes containing 1.0% ring-stage parasite in 100 lL complete media. The culture volume per well for the assay was 100 lL. The number of parasites for the assay was adjusted to 1–1.5% by diluting with fresh human B(+) red blood cells (RBC). Assays were carried out in 96-well microtitre flat-bottomed tissue culture plates incubated at 37 °C for 24 h in the presence of twofold serial dilutions of compounds and chloroquine diphosphate (CQ) for their effect on schizont maturation. Crude extract and pure compound GC-7 were dissolved in ethanol and further diluted with RPMI 1640 medium (the final ethanol concentration did not exceed 0.5%, which did not affect parasite growth). Chloroquine diphosphate was dissolved in aqueous medium. Tests were carried out in duplicate wells for each dose of the drugs. Solvent control cultures containing the same concentrations of the solvent present in the test wells were carried out with RPMI1640 containing 10% AB(+) serum. Parasite growth was found to be unaffected by the solvent concentrations. Growth of the parasites from duplicate wells of each concentration was monitored in Giemsa-stained blood smears by counting the number of schizonts per 100 asexual parasites. Percentage schizont maturation inhibi-
The authors would like to thank Dr. R Vijayaraghavan, Director, DRDE, Gwalior, and Professor Guru Row and Dr. Deepak Chopra of IISC, Bangalore for proving the single crystal XRD of compound 1. References and notes 1. Sachs, J.; Malaney, P. Nature 2002, 415, 680. 2. Hay, S. I.; Guerra, C. A.; Tatem, A. J.; Noor, A. M.; Snow, R. W. Lancet Infect Dis. 2004, 4, 327. 3. Carter, R.; Mendis, K. N. Clin. Microbiol. Rev. 2002, 15, 564. 4. Breman, J. G.; Egan, A.; Keusch, G. T. Am. J. Trop. Med. Hyg. 2001, 64, IV. 5. White, N. J. J. Clin. Invest. 2004, 113, 1084. 6. Olliaro, P. Pharmacol. Ther. 2001, 89, 207. 7. Biagini, G. A.; O’Neill, P. M.; Nzila, A.; Ward, S. A.; Bray, P. G. Trends Parasitol. 2003, 19, 479. 8. Schmid, G.; Hotheinz, W. J. Am. Chem. Soc. 1983, 105, 624. 9. Avery, M. A.; Jennings-White, C.; Chong, W. K. M. Tetrahedron Lett. 1987, 28, 4629. 10. Liu, H.; Yeh, W. L.; Chew, S. W. Tetrahedron Lett. 1993, 35, 4435. 11. Yadav, J. S.; Satheesh Babu, R.; Sabitha, G. Tetrahedron Lett. 2002, 44, 387. 12. Haynes, R. K. Curr. Opin. Infect. Dis. 2001, 14, 719. 13. Borstnik, K.; Paik, I. H.; Posner, G. H. Mini-Rev. Med. Chem. 2002, 2, 573. 14. Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M. Angew. Chem., Int. Ed. 2003, 42, 5274. 15. Ridley, R. G. Nature 2002, 415, 686. 16. Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S. Nat. Rev. Drug Disc. 2004, 3, 509. 17. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461. 18. The Wealth of India; C.S.I.R.: New Delhi, 1959; Vol. V, p 13. 19. Hutagalung, R.; Paiphum, L.; Ashley, E. A.; McGready, R.; Brockman, A.; Thwai, K. L.; Singhasivanon, P.; Jelinek, T.; White, N. J.; Nosten, F. H. Malar. J. 2005, 4, 46. 20. Chopra, R. N.; Nayar, S. L.; Chopra, I. C. Glossary of Indian Medicinal Plant; C.S.I.R.: New Delhi, 1956. 21. Kaushik, M. P.; Selvam, D. T.; Nivsarkar, M.; Acharya, B. N.; Subramaniam, P.; Sekhar, K. PCT/IN2006/000317, 2006. 22. Crystal data for compound 1: C20H28O4 were collected at room temperature using a Bruker AXS SMART APEX CCD diffractometer, M = 332.4, monoclinic, P212121, a = 11.051(8) Å, b = 12.553(9) Å, c = 13.039(9) Å, V = 1804.4(2) Å3, Z = 4, Dcalcd = 1.22 g/mL, X-ray source Mo-Ka (radiation), k = 0.71073 Å, l = 0.084 mm1, F(0 0 0) = 719.9, T = 290(2) K, colourless prism 0.48 0.31 0.15 mm. The structure solution was obtained by direct methods and refined with anisotropic thermal parameters using full-matrix least-squares procedures on F2 to give R = 0.085, wR = 0.106 for 14,066 independent observed reflections and 221 parameters. Crystallographic data (excluding structure factors) for the structure in this Letter have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 691114. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0) 1223 336033 or e-mail: [email protected]). 23. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Gomphostenins: Two new antimalarial compounds from the leaves of Gomphostemma niveum Manisha Sathe, M. P. Kaushik * Discovery Center, Process Technology Development Division, Defence R&D Establishment, Jhansi Road, Gwalior 474 002 (MP), India
a r t i c l e
i n f o
Article history: Received 26 January 2009 Revised 24 February 2009 Accepted 27 February 2009 Available online 4 March 2009 Keywords: Antimalarials Gomphostemma niveum Labiatae Clerodane XRD
a b s t r a c t Phytochemical investigation of CHCl3 extract of the Gomphostemma niveum leaves led to the isolation of two new diterpene, compound 1 and 2. Their structures were elucidated by spectroscopic procedures and single crystal XRD. Compound 1 named as Gomphostenin 1 and structure was established as 8-ethyl (5Hfuran-2-one, 14-hydroxy, 2-oxo 3, 20 Z(17) diene clerodane, while compound 2 named as GomphosteninA; was found to be the acetyl derivative of compound 1 and revealed as 8-ethyl (5H-furan-2-one, 14-acetoxy, 2-oxo 3, 20 Z(17) diene clerodane. In vitro antimalarial activity against Plasmodium falciparum showed that compound 2 was more active than compound 1 and CHCl3 extract as well; with IC50 value of 3.4 lg/mL. Ó 2009 Published by Elsevier Ltd.
Malaria is one of the most important parasitic infections of humans due to its high morbidity and mortality with major consequent impact on economic productivity and livelihood.1 Approximately 40% of the world population live in areas with the risk of malaria. Each year 300–500 million people suffer from acute malaria and 1.5–2.5 million die from the disease.2–4 Plasmodium falciparum, the causative agent of the malignant form of malaria, has high adaptability by mutation and is resistant to various types of antimalarial drugs.5 Thus, low cost malaria treatments such as chloroquine and fansidar (sulfadoxine/pyrimethamine) become ineffective with the selection and spread of mutant drug-resistant parasites and then to multidrug resistance emergence.5–7 Artemisinin (qinghaosu) has become increasingly important as a malaria treatment but the current routes for its total chemical synthesis8–11 remain too complex for commercial production.12,13 There is no doubt that intensive use of artemisin-classes combinations therapy is an invaluable opportunity that decrease the burden of malaria and prolong drug life span of effective use. However, in the absence of substitute, the appearance of artemisinin-resistant malaria would lead to untreatable malaria and to a potential humanitarian disaster. These facts combined with the absence of a vaccine and the lack of systematic vector control strategies provides the rationale for the development of novel drugs against malaria. Therefore, new drugs with novel mechanisms of action and
* Corresponding author. Tel.: +91 751 2343972; fax: +91 751 2340042. E-mail addresses: [email protected], [email protected] (M.P. Kaushik). 0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.bmcl.2009.02.120
that are structurally unrelated to existing antimalarial agents are thus urgently required.14–16 The priority is the identification of original target(s) leading to novel antimalarial compounds which would a priori not allow cross-resistance with pre existing antimalarials. Natural products are extremely successful in providing mankind with substances to combat diseases, and today, roughly 50% of all small molecules as drugs on the market addressing infectious diseases are natural products or derivatives thereof.17 Hence, the ethnopharmacologial approach for the search of new antimalarial has proved to be more predictive with respect to malaria, quinine and its derivatives are still in use today, and new combination therapies based on artemisinin were recently introduced in the clinic.18,19 As part of our continuing efforts directed towards the discovery of the structurally interesting and biologically active compounds from the Indian medicinal plants,20 it was found that aqueous and CHCl3 extract of Gomphostemma niveum showed the antimalarial properties against P. falciparum. Gomphostemma niveum is a coarse, stellatelly pubescent or tomentose herb belonging to the Labiatae family, seen in small pockets at the foothills of the Himalayas in the northeastern part of India. Local people have used aqueous leaf extracts of this plant in acute malaria cases. Due to its highly localized presence and knowledge of its effectiveness against malaria, no systematic efforts have been reported before this. Here in we report the phytochemical analysis of chloroform extract of the Gomphostemma niveum leaves resulted in the isolation of two new clerodame diterpenes (1 and 2) compounds and there antimalarial activity against P. falciparum was studied.21
1313
M. Sathe, M. P. Kaushik / Bioorg. Med. Chem. Lett. 20 (2010) 1312–1314
19
20
Table 1 1 H NMR and
H
O
C NMR data of compound 1 and 2 in CDCl3
Position 17
18
15
14 7
Compound 1 dC
16
O
RO
13
13 CH3
8
H
1
10 9
6
5
O
2
3
4 CH3 12 CH3 11
Gomphostenin 1 R=H Gomphostenin-A 2 R= COCH 3
The structure of the new compounds was established using IR, MS, 1H, 13C and DEPT NMR. The final structure of compound 1 was revealed by the X-ray crystallographic analysis. Since the in vitro antimalarial efficacy of CHCl3 extract was far better than the aqueous extract, hence CHCl3 extract was used for the isolation of active compounds. The dried powdered leaves (500 g) were defatted with 3 1 L of petroleum ether followed by extraction with 3 1 L of CHCl3 extracted three times with CHCl3 in a soxhlet apparatus. The combined extracts were concentrated under vacuum. The portion of active CHCl3 extract (5 g) was subjected to column chromatography (silica gel, 60–120 mesh) using step gradient of CHCl3/MeOH to yield two major fractions (F1–F2). Fraction F1 was subjected to repeated silica gel (100–200 mesh) column chromatography (CC) by eluting with MeOH/CHCl3 (10:90) to yield compound 1 (25 mg). Similarly fraction F2 was also subjected to silica gel column chromatography eluting with MeOH/CHCl3 (4:96) to get compound 2 (1.22 g). Gomphostenin 1 was identified as 8-ethyl (5H-furan-2-one,14hydroxy, 2-oxo 3, 20 Z(17) diene clerodane. It was isolated as a white solid with the positive optical rotation ½a25 D +98.64 (c 1.64, CHCl3). The HRESI-MS of compound 1 revealed a molecular ion peak corresponding to (M+H)+ at m/z 333.2068 (calcd 333.2066) indicating the molecular formula C20H28O4. The ring and double bond factor was calculated to be 6. The UV (MeOH) absorbance at kmax 207 nm (e 9800) and 220 nm (e 9100) supported the presence of a,b-unsaturated carbonyl group. The IR spectrum displayed absorptions bands at 3434 cm1 (OH), 17331 (a,b-unsaturated clactone) and 1667 cm1 (a,b-unsaturated C@O). The 1H and 13C NMR spectra of compound 1 assigned as shown in Table 1. 1H NMR exhibited three ternary methyl groups at d 1.84, 1.07 and 0.8 (3H each, singlet), two oxygenated methylene groups at d 2.25 (2H, multiplet) and 4.75 (2H, singlet). Two olefinic protons showed typical downfield shift at d 5.77 (H-3) and d 7.08 (H-20) as singlet. The 13C NMR of compound 1 showed the presence of 20 C-atoms and also indicated the presence of a,b-unsaturated ketone (d 199.9), substituted olefin (d 125, 172), and three methyl groups (d 18.1, 18.4, 18.9). Further it also displayed signal at d 174 is due to C@O of lactone ring, d 133.4, 144.4 are corresponding to disubstituted olefin and d 70.4 is assignable to methylene carbon in the lactone ring. DEPT-135 confirmed the presence of three quartet carbon, three methyl, seven methylene and four methine carbon atoms. Suitable colourless X-ray quality crystals of compound 1 were obtained by crystallization from hexane/CHCl3 (7:3), The X-ray diffraction analysis of 1 was carried out on a single crystal,22 Figure 1. This study confirmed the structure of 1 and clearly establishes that
1 2 3 4 5 6 7 8 9 10 11 12 13 14a 14b 15 16 17 18 19 20 21 22 a b
a
35.0 199.9 125.4 172.6 21.7 18.9 45.4 38.1 39.6 45.2 18.1 18.4 18.9 63.6 34.3 35.4 133.9 174.6 70.4 144.4
dHb
multiplicity
2.25 (m) — 5.77 (m) — 1.40 (m) 1.6 (m) 1.89 (m) — — 1.25 (m) 1.84 (s) 1.07 (s) 0.8 (s) 3.24–3.32 (dd, J = 7.5, 7.5) 3.76–3.80 (dd, J = 4.0, 4.0) 1.4 (m) 2.25 (m) — — 4.75 (s) 7.08 (s)
Compound 2 dC
a
38.0 199.4 125.6 171.25 34.8 18.9 45.3 34.7 39.53 40.5 18.35 18.7 18.62 65.3 34.3 21.75 134.0 171.9 70.3 143.8 174.1 20.9
dHb multiplicity 2.24 (m) — 5.74 (s) — 1.48 (m) 1.69 (m) 1.86 (m) — — 1.25 (m) 1.80 (m) 1.40 (m) 0.92 (m) 3.80–3.86 (dd, J = 7.5, 7.5) 4.20–4.25 (dd, J = 4.0, 4.0) 1.4 (m) 1.9 (m) — — 4.7 (s) 7.1 (s) — 2.1 (s)
100 MHz. 400 MHz.
the relative configuration of the four stereogenic carbons are in fact 7R, 8S, 9S and 10S (or 7R, 8S, 9S and 10S). Gomphostenin-A 2 was isolated as a brown viscous liquid, with positive optical rotation ½a25 D +96.64 (c 1.64, CHCl3). The HRESI-MS of compound 2 revealed a molecular ion peak corresponding to (M++H) at m/z 375.2180 (calcd 375.2185). indicating the molecular formula C22H30O5 The IR spectrum showed absorption at 1714 cm1 (a,b-unsaturated c-lactone) and 1673 cm1 (a,b-unsaturated C@O). The 1H NMR and 13C NMR spectroscopic data of the compound 2 (Table 1) which is similar to that of compound l. In
Figure 1. ORTEP diagram of Gomphostenin 1.
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M. Sathe, M. P. Kaushik / Bioorg. Med. Chem. Lett. 20 (2010) 1312–1314
Table 2 IC50 values of CHCl3 extracts, compound 1, 2, and chloroquine diphosphate (CQ) against P. falciparum Compound
IC50 ± SD (lg/mL) CQ-S
IC50 ± SD (lg/mL) CQ-R
Aqueous extract CHCl3 extract 1 2 CQ
151.6 ± 2.9 9.7 ± 1.3 38.2 ± 1.2 3.4 ± 1.0 0.25
133.3 ± 5.8 9.5 ± 1.4 37.7 ± 0.466 3.2 ± 0.057
tion was calculated by the formula: (1 Nt/Nc) 100, where Nt and Nc represent the number of schizonts in the test and control. In summary, two clerodane diterpenes were isolated from the chloroform extract of Gomphostemma niveum. The structure of compound 1 and 2 was established on the basis of NMR spectral data and confirmed by X-ray crystallographic analysis. The IC50 of compound 1 and 2 has been found to be 38 lg/mL and 3.4 lg/ mL (Table 2). The reference drug chloroquine has exhibited 100% inhibition at 0.25 lg/mL concentration in the same test system.
IC50 values are expressed as the mean microgram concentrations ± standard deviations. All experiments were realized in triplicate.
Acknowledgements case of 1H NMR the sharp singlet at dH 2.1 indicated the presence of methyl attached to a carbonyl functionality. The 13C NMR showed a characteristic peak at dC 171.9 suggested the presence of lactone ring in the structure. MS–MS fragmentation of 2 showed the loss of one acetoxy group. To the best of our knowledge, this is the first report on chemical analysis of isolation of compound 1 and 2 from G. niveum. The antimalarial efficacy of the crude extracts and the pure compound 1 and 2 were evaluated by conventional in vitro parasite culture method, using the (chloroquine sensitive) MRC-02 strain of P. falciparum obtained from the National Institute of Malaria Research, New Delhi, India, maintained in a continuous culture using the standard method described by Trager and Jensen.23 Parasites were cultured in human B(+) erythrocytes in Rosewell Park Memorial Institute (RPMI)-1640 media (GIBCOBRL, Paisely, Scotland) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) buffer, 10% human AB(+) serum, and 0.2% sodium bicarbonate (Sigma), and maintained at 5% CO2. Cultures containing predominantly early ring stages were synchronized by addition of 5% D-sorbitol (Sigma) lysis, used for testing. Initial culture was maintained in small vials with 10% haematocrit, that is, 10 lL erythrocytes containing 1.0% ring-stage parasite in 100 lL complete media. The culture volume per well for the assay was 100 lL. The number of parasites for the assay was adjusted to 1–1.5% by diluting with fresh human B(+) red blood cells (RBC). Assays were carried out in 96-well microtitre flat-bottomed tissue culture plates incubated at 37 °C for 24 h in the presence of twofold serial dilutions of compounds and chloroquine diphosphate (CQ) for their effect on schizont maturation. Crude extract and pure compound GC-7 were dissolved in ethanol and further diluted with RPMI 1640 medium (the final ethanol concentration did not exceed 0.5%, which did not affect parasite growth). Chloroquine diphosphate was dissolved in aqueous medium. Tests were carried out in duplicate wells for each dose of the drugs. Solvent control cultures containing the same concentrations of the solvent present in the test wells were carried out with RPMI1640 containing 10% AB(+) serum. Parasite growth was found to be unaffected by the solvent concentrations. Growth of the parasites from duplicate wells of each concentration was monitored in Giemsa-stained blood smears by counting the number of schizonts per 100 asexual parasites. Percentage schizont maturation inhibi-
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