LeucosceptrinesA Novel Sesterterpene with Prolylendopeptidase Inhibitory Activity from Leucosceptrum canum Muhammad Iqbal Choudhary,*,† Rosa Ranjit,† Atta-ur-Rahman,† Tirtha Maiya Shrestha,‡ Amsha Yasin,† and Masood Parvez§ H. E. J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi-75270, Pakistan, Department of Plant Resources, Ministry of Forest and Soil Conservation, Thapathali, Kathmandu, Nepal, and Department of Chemistry, University of Calgary, Alberta, Canada T2N IN4
[email protected] Received October 29, 2003
Abstract: A novel sesterterpene, leucosceptrine, was isolated from the medicinal plant Leucosceptrum canum from Nepal. The structure was determined by single-crystal X-ray diffraction and spectroscopic techniques. The biosynthesis of leucosceptrine (1) is proposed here. Leucosceptrine (1) exhibited prolylendopeptidase inhibitory activity.
Sesterterpenes (C25), a rare class of terpenes, have been obtained from widely differing sources including terrestrial fungi, plants, and insects, as well as from marine sponges and nudibranchs.1,2 Mostly sesterterpenes are derived from geranylfarnesyl pyrophosphate.3 We have isolated a novel sesterterpene, named leucosceptrine (1), from Leucosceptrum canum. Leucoscepturm canum Sm., locally known as Bhusure in Nepal, belongs to the family Laminaceae (Labiateae), a cosmopolitan family of about 200 genera and more than 3,500 species distributed all over the world. Members of this family contain essential oils, terpenoids, flavonoids, coumarins, and glycosides.4 A few sesterterpenes were also reported from this family. Leucoscepturm canum, a small tree, is distributed in the temperate Himalayans, Burma, China, and Nepal.5 The plant is used as an insecticidal agent in remote areas of Nepal. No phytochemical work is yet reported on this plant. This paper describes the isolation of the novel sesterterpene leucosceptrine along with 5-hydroxy-4,7-dimethoxyflavone, 5,6,7-trihydroxy-4′-methoxyflavone, 5-hydroxy4,6,7 trimethoxyflavone, β-sitosterol, and β-sitoglucoside. The structure of leucosceptrine (1) was elucidated with the help of X-ray crystallographic and spectroscopic * To whom correspondence should be addressed. Tel: (92-21) 9243224. Fax: (92-21) 9243190-91. † University of Karachi. ‡ Ministry of Forest and Soil Conservation. § University of Calgary. (1) Hanson, J. R. Nat. Prod. Rep. 1996, 13, 529. (2) Faulkner, D. J. Nat. Prod. Rep. 1996, 13, 75 and previous reports. (3) Wang, Y.; Dreyfuss, M.; Ponelle, M.; Oberer, L.; Riezman, H. Tetrahedron 1998, 54, 6415. (4) Hooker, J. D. Flora of British India; Bishen Singh Mahendra Pal Singh Publishers: Dehradun, 1983; Vol. IV, p 699. (5) Flora of Kathmandu Valley, Bull. Dept. Med. Plant, Nepal, No. 11; His Majesty’s Government of Nepal, Ministry of Forests and Soil Conservation, Department of Medicinal Plants: Kathmandu, Nepal, 1986; p 561.
TABLE 1.
13C
and 1H NMR Data of Leucosceptriene (1)
in CDCl3 carbon no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
13C
NMR
64.6 136.9 121.4 73.1 96.8 41.0 43.8 33.0 29.6 31.2 54.8 82.0 222.0 42.4 27.8 26.5 84.2 167.9 117.2 172.9 17.5 10.0 21.2 14.5 13.2
multiplicity CH2 C CH C C CH CH CH2 CH2 CH CH C CO CH CH2 CH2 CH C CH CO CH3 CH3 CH3 CH3 CH3
1H
NMR
3.94, 4.21 (d, 16.5) 4.92 (q, 1.6) 1.95 (m) 1.54 (m) 1.88 (m) 1.98 (m) 1.79 (m) 1.83 (m) 2.99 (m) 1.23 (m) 1.15 (m) 4.81 (bd, 6.3) 5.81 (m) 1.75 (d, 1.3) 0.99 (d, 6.8) 0.74 (d, 6.2) 1.05 (d, 6.7) 2.05 (dd, 0.8, 0.6)
techniques.6,7 It showed inhibitory activity against the enzyme prolylendopeptidase (PEP). PEP has been proposed to catalyze the degradation of proline-containing neuropeptides that are involved in the processes of learning and memory, e.g., vasopressin, substance P, and thyrotropin-releasing hormone.8 The arial parts of L. canum were collected from the Godawari, Kathmandu Valley, Nepal. Dried, powdered plant material (1.75 kg) was extracted using hexane, and the hexane extract was then chromatographed on a silica gel column. The fraction obtained by chloroform/methanol (95:5) elution was further purified by repeated silica gel column chromatography, followed by recrystallization from chloroform/hexane. The crystals were washed with diethyl ether to obtain pure compound 1. The FAB MS spectrum of compound 1 showed the (M - H)- peak at m/z 447 (C25H36O7). The HREI MS spectrum showed the highest ion at m/z 430 (C25H34O6), representing the loss of H2O from the M+. The IR spectrum of compound 1 showed a broad absorption band at 3417 cm-1, which indicated the presence of hydroxyl groups. The 13C NMR spectrum of compound 1 indicated the presence of 25 carbons including five methyl, five methylene, eight methine, and seven quaternary carbons. Table 1 presents 13C and 1H NMR correlations obtained from the 2D NMR experiments (HMBC and HMQC). The downfield signals in the 13C NMR spectrum at δ 73.1, 96.8, and 82.0 also indicated the presence of three (6) Atta-ur-Rahman; Choudhary, M. I. Solving Problems by NMR Spectroscopy; Academic Press: San Diego, 1996. (7) Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to Spectroscopy; 2nd ed., Harcourt Brace College Publishers: Fort Worth, TX, 1996. (8) Kobayashi, W.; Miyase, T.; Sano, M.; Umehara, K.; Warashina, T.; Noguchi, H. Biol. Pharm. Bull. 2002, 25 (8), 1049. 10.1021/jo030337u CCC: $27.50 © 2004 American Chemical Society
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Published on Web 03/23/2004
FIGURE 1. Proposed mass fragmentation of leucosceptrine (1).
hydroxyl groups at C-4, C-5, and C-12, respectively. The downfield shift of C-5 (δ 96.8) indicated its acetal nature. The carbon signal at δ 222.0 and IR absorption at 1732 cm-1 indicated the presence of a ketonic group. The IR and 13C NMR spectra indicated the presence an R,βunsaturated five-membered lactone ring (νmax 1732 cm-1, δC 172.9, 167.9, and 117.2). The mass fragment ion at m/z 97 (Figure 1)7,9 and a proton multiplet at δ 5.81 also indicated a lactone ring. Out of seven oxygens, three of them were hydroxyls, one was a carbonyl group, two were
involved as a lactone ring, and the remaining one might be involved in the formation of an ether ring system. One methylene carbon resonating at δC 64.6 (δH 4.21 and 3.94, d, J ) 16.5 Hz) was assigned to an oxygen-containing C-1 methylene group. The oxymethylene protons also showed clear HMBC interactions (Figure 2) with C-2 (δ 136.9) and C-5 (δ 96.8). Out of five methyl carbons, the (9) Budzkiewicz, H.; Djerassi, C.; William, D. H. Structure Elucidation of Natural Products by Mass Spectrometry; Holden-Day: San Francisco, CA, 1964; Vol. 2.
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FIGURE 3. Computer-generated final X-ray model (ORTEP) FIGURE 2. Important 1H-13C NMR long-range correlations
of compound 1.
(HMBC) in compound 1.
secondary methyl groups that resonated at δC 10.0, 21.2, and 14.5 were correlated with 1H NMR doublets at δ 0.99 (J ) 6.8 Hz), 0.74 (J ) 6.2 Hz), and 1.05 (J ) 6.7 Hz), respectively. The remaining two tertiary methyl carbons resonated at δ 17.5 and 13.2, directly correlated with signals at δ 1.75 and 2.05, respectively. The methyl carbon at δ 13.2 showed correlation with a proton resonating at δ 2.05 (dd, J ) 0.8 Hz, J ) 0.6 Hz), split as a result of the homoallylic couplings. The structure was unambiguously established by single-crystal X-ray diffraction analysis. The ORTEP diagram of the final X-ray model is presented in Figure 3. All of the fractions obtained from hexane extract exhibited potent PEP inhibitory activity (>80%) in vitro. Z-Pro-prolinal was used as a standard inhibitor in the assay (IC50 ) 1.27 nM ( 0.01).10 Compound 1 showed inhibitory activity (IC50 ) 80 µM ( 1.467) against PEP, which is a serine peptidase that catalyses the hydrolysis of peptide bond at the L-proline carboxyl terminal.11 PEP has been suggested to play an important role in the biological regulation of peptide hormones and is recognized to be involved in learning and memory. A possible biosynthetic pathway for compound 1 is proposed in Scheme 1. The linear arrangement of isoprene units as in geranylfarnesyl pyrophosphate (GFPP) was easily recognizable in 1. A series of cyclization reactions can take place in such a way as to form the novel skeleton of compound 1 through the intermediate [A]. Experimental Section Plant Material. The arial parts of L. canum Sm. were collected from the Godawari, Kathmandu, Nepal on November (10) Fan, W.; Tezuka, Y.; Ni May, K.; Kadota, S. Chem. Pharm. Bull. 2001, 49, 396. (11) Marighetto, A.; Touzani, K.; Etchamendy, N.; Cortes Torrea, C.; De Nanteuil, G.; Guez, D.; Jaffard, R.; Morain, P. Learn. Mem. 2000, 7, 159.
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26, 2000, at an altitude of 1,550 m. The plant was identified, and a voucher specimen (T037) was deposited at The National Herbarium and Research Laboratory, Department of Plant Resources, Ministry of Forests and Soil Conservation, Godawari, Kathmandu, Nepal. Extraction and Isolation. Arial parts of L. canum were cut into small pieces and dried in the shade. The air-dried and powdered plant material (1.75 kg) was soaked in hexane at room temperature for 2 days, and the resulting solution was filtered and concentrated under reduced pressure. This process was repeated three times to yield 34.03 g of hexane extract. The plant material was subsequently soaked in dichloromethane, ethyl acetate, and methanol. The resulting organic extracts were filtered and concentrated under reduced pressure. From the hexane extract, some fatty acids were removed through precipitation with acetone. The filtrate was again concentrated under reduced pressure to a semisolid extract (26.89 g). This hexane extract was repeatedly chromatographed over silica gel using solvents of various polarities starting with hexane and progressing to hexane/chloroform, chloroform/ methanol and finally methanol. The fraction LCH 207 obtained by chloroform/methanol (95:5) was recrystallized by slow evaporation of hexane/chloroform solution (9:1) containing a few drops of methanol. Thus formed colorless crystals were washed with diethyl ether, which afforded pure compound 1 (20.35 mg). The purity of crystals were checked by TLC (254 and 366 nm) by ceric sulfate spraying reagent. Compound 1. Colorless block crystals, mp 146-148 °C (dec), [R]25D +80 (c 0.04, MeOH); UV (MeOH) λmax nm (log ) 207 (4.0); IR (KBr) νmax 3417 (OH), 2954 and 2873 (CH) and 1732 (CdO) cm-1; 1H and 13C NMR data see Table 1; FAB MS m/z (M - H)447; HREI MS m/z 430.2322 (calcd for C25H36O7, m/z 430.2296); EI MS m/z (rel int %) 430 (71), 402 (2), 320 (3), 292 (2), 249 (85), 231 (69), 203 (33), 181 (41), 153 (35), 139 (48), 125 (46), 111 (84), 110 (43), 109 (84), 97 (41), 92 (25), 83 (100). X-ray Crystal Data for Compound 1. A colorless crystal of dimension 0.23 × 0.22 × 0.16 mm3 was selected for diffraction studies. Molecular formula C25H36O7; molecular mass 448.54 amu; crystal system orthorhombic; space group P212121; unit cell dimensions a ) 8.0519(10) Å, b ) 9.167(2) Å, c ) 31.314 (6) Å; V ) 2311.3(7) Å3; Dcalcd ) 1.289 mg/m3; F(000) ) 968, Z ) 4; Mo KR ) 0.71073 Å. Unit cell dimensions was determined by leastsquares fit of 5,088 reflections in the range 2.8° < θ < 27.5° which measured at 173°(2) K using Mo KR radiations on a Nonius Kappa CCD diffractometer. The total number of inde-
SCHEME 1.
Proposed Biosynthesis of Compound 1
pendent reflections measured was 2,977. The structure was solved by direct methods12 using Fourier techniques.13 The structure was refined by full matrix-least-squares calculations on F2 with the aid of the program SHELXL-97.14 The final R and wR factors were 0.038 and 0.091, respectively, and S ) 1.04. PEP Inhibitiory Activity for Compound 1. A specific inhibitor of PEP, N-benzyloxycarbony-pro-prolinal, was kindly provided by Dr. Hideaki Shimizu, Central Institute for Microbiological Research., Tokyo, Japan. The PEP inhibition activity was assayed by a modification of the method of Yoshimoto et al.15 Tris(hydroxymethyl)aminomethane HCl buffer (100 nM containing 1 mM EDTA, pH 7.0, 247 µL), PEP (0.02 unit/300 µL, 15 µL), and test sample in 8 µL of MeOH were mixed in a (12) Altomare, A.; Cascarano, M.; Giacovazzo, C.; Guagliardi, A. SIR92. J. Appl. Crystallogr. 1993, 26, 343. (13) Beurskens, P. T.; Admiraal, G.; Beurskens, G.; Bosman, W. P.; de Gelder, R.; Israel, R.; Smiths, J. M. M. The DIRDIF-94 Program System, Technical Report of the Crystallography Laboratory; University of Nijmegen: The Netherlands, 1994. (14) Sheldrick, G. M. SHELXL97; University of Go¨ttingen: Germany, 1997. (15) Yoshimoto, T.; Walter, R.; Tsuru, D. J. Biol. Chem. 1980, 255, 4786.
96-well microplate and preincubated for 10 min at 30 °C. The reaction was initiated by adding 30 µL of 2 mM N-benzyloxycarbonyl-Gly-Pro-pNA (in 40% 1,4-dioxane) as the substrate. The amount of p-nitroaniline released was determined spectrophotometrically as increase in absorption at 410 nm, on a 96-well microplate reader at 30 οC. The IC50 values were the average of at least three determinations.
Acknowledgment. Ms. Rosa Ranjit gratefully acknowledges the financial support of the Third World Organization for Women in Science (TWOWS), Trieste, Italy, which provided Ph.D. scholarship support at the H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan. Supporting Information Available: Tables of X-ray results and computer-generated diagram of final X-ray model of 1. This material is available free of charge via the Internet at http://pubs.acs.org. JO030337U
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