The Alkaloids of Tribulus terrestris: A Revised Structure for the Alkaloid Tribulusterine

The fruit of the plant Tribulus terrestris is well known as a traditional Chinese medicine with a myriad of reported uses. Herbal medicine preparations are becoming available for many uses including body building and for stimulating spermatogenesis and libido. In addition, the whole live plant causes significant sheep losses in certain regions of Australia when conditions for growth are favoured. Chronic poisoning of the sheep occurs, characterised by a distressing irreversible asymmetric locomotor disorder. The symptoms, which have some similarities to those of Parkinson’s disease in humans, are indicative of a localised interference with the central nervous system, possibly with serotonin-associated neurones. On the basis of other studies, it was thought that the neurotoxic agent may be the βcarboline alkaloid, tribulusterine, which had been isolated in very low yield from the fruit of T. terrestris. This investigation developed an efficient synthesis of tribulusterine in order to confirm its structure and provide sufficient material for animal toxicity studies. A successful synthesis of the target alkaloid with structural confirmation by NMR and X-ray crystallography indicated that a structural revision for the reported natural alkaloid was necessary. The same synthetic strategy when applied to the synthesis of perlolyrine confirmed that this closely related β-carboline was the natural Tribulus constituent. INTRODUCTION Tribulus terrestris L. (Zygophyllaceae), commonly known as puncture vine or cathead because of the sharp spurs protruding from the fruit capsule, is a small prostrate annual herb bearing yellow flowers on branches to 80 cms (Harden, 1992) (Fig. 1). This drought-tolerant, summer growing medicinal plant now has a worldwide distribution but is chiefly considered a weed because of toxicity to livestock (Bourke et al., 1992). The fruits of T. terrestris are traditionally used in decoctions and infusions for the treatment of spermatorrhea, phosphaturia and disease of the genitourinary system as well as kidney, liver and eye diseases in Ayurvedic and Chinese traditional medicine (Bedir and Khan, 2000). Recent work has established aphrodisiac properties with rats (Gauthaman et al., 2002) and hepatoprotective activity with cultured mouse cells (Li et al., 1998). Preparations containing T. terrestris extract have alleviated symptoms of prostate patients (Lokesh et al., 2001) and enhanced testosterone concentrations in older men (Brown et al., 2001a, 2001b). The ability of these products to increase hormone levels is being used for performance enhancement in athletes (Antonio et al., 2000; Krcik, 2001). In addition, insect juvenile hormone activity has been ascribed to T. terrestris Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS 2005 12 extracts (e.g. Rajashekhargouda et al., 1997) which are then recommended for increasing silk production by extending the fifth-instar larval stage of Bombyx mori. T. terrestris is well-known for its toxicity (Bath et al., 1978) although only in Australia has its ingestion by sheep resulted in outbreaks of a locomotor disorder (Bourke et al., 1990, 1992). T. terrestris staggers is an asymmetric locomotor disorder in sheep which develops as a result of a functional abnormality in the central nervous system (Bourke, 1987). The course of the disease was characterised by a slowly developing, irreversible, asymmetrical weakness of the hind limbs (Bourke, 1984) (Fig. 2). Although many potentially bioactive constituents have been isolated from T. terrestris, in most cases, structure-activity relationships are yet to be established. A number of steroids (Yan et al., 1996, 1997; Wu et al., 1996), flavonoids (Bhutani et al., 1969; Saleh et al., 1982), lignanamides (Li et al., 1998; Ren et al., 1994; Wu et al., 1999) and alkaloids (Borkowski and Lutomski, 1960; Bourke et al., 1992; Wu et al., 1999) have been identified. Animal toxicity studies (Bourke et al., 1992) of alkaloid fractions have failed to identify toxins present in T. terrestris but absent in ryegrass, Lolium perenne (Bush and Jeffreys, 1975) that could be responsible for the irreversible staggers of the former. The report of the novel alkaloid tribulusterine (1) (Wu et al., 1999) provided another alkaloid for toxicity testing. With the plant material providing such low concentrations of the natural material, synthetic strategies were examined. This communication outlines the synthesis of tribulusterine (1) and related β-carboline perlolyrine (2) which confirms that the natural T. terrestris minor constituent is the latter and not the former. MATERIALS AND METHODS Melting points were determined using a Reichert hot-stage melting point apparatus and are uncorrected. NMR spectra were recorded on a Varian Unity-300 Fourier transform NMR spectrometer. Unless otherwise stated, the spectra were obtained from solutions in CDCl3 and referenced to TMS (proton) and chloroform mid-line (77 ppm) (carbon). Chemical shifts were reported in parts per million (δ). MS (CI) were obtained using a Shimadzu QP-5000 spectrometer. High resolution (CI) MS were run using a Fisons/VG Autospec-TOF spectrometer. UV spectra were recorded on a Shimadzu UV1601-PC uv-vis spectrophotometer. TLC and preparative layer chromatography was performed using Merck silica gel 60 F254. All chromatographic solvent proportions are volume for volume. Column chromatography was performed using Merck Kieselgel 60 (0.063-0.200 nm particle size) silica gel. Solvents were removed under reduced pressure by rotary evaporator, and organic solvent extracts were dried with anhydrous Na2SO4. Synthesis of 3-Acetoxymethyl-2-furaldehyde (4) To a solution of 3-furanmethanol (3) (1.0 g, 10.5 mmol) in dry THF (25 mL) at -78°C under nitrogen was added n-butyllithium in hexane (8.3 mL, 21.0 mmol). The mixture was stirred at -78°C for 2 hours and at then 0°C for 1 hour. A solution of anhydrous N,N-dimethylformamide (4.0 mL, 48.4 mmol) in anhydrous THF (10 mL) was added dropwise at -78°C. Then the solution was allowed to warm to room temperature and stirring was continued overnight. After addition of saturated ammonium chloride solution (20 mL), extraction with ethyl acetate (3 x 20 mL), drying, and solvent evaporation gave a crude yellow oil. Acetic anhydride (3 mL) and pyridine (0.1 mL) were added and the solution was stirred at room temperature overnight. Ethanol was then added and the solution mixture was evaporated to dryness. The residue was washed with water and extracted with dichloromethane (3 x 10 mL). The combined extracts were dried and evaporated to give 3-acetoxymethyl-2-furaldehyde (4) (0.7 g, 42%) as a yellow oil; H NMR (CDCl3) δ 2.12 (s, 3H, CH3), 5.34 (s, 2H, CH2), 6.64 (d, 1H, J=1.8 Hz, H-4), 7.62 (d, 1H, J=1.8 Hz, H-5), 9.86 (s, 1H, CHO); C NMR (CDCl3) δ 21.0 (CH2), 57.3 (CH2), 113.7 (C-4), 131.2 (C-3), 147.5 (C-5), 148.7 (C-2), 170.7 (CHO), 179.0 (OCOCH3); CIMS m/z 169 [MH] 100%; HRCIMS m/z 169.0497 (calcd for C8H9O4, 169.0501).