Ipvelutine, 7β-Acetoxy-2α-(tigloyloxy)tropane, an Unusual Tropane Alkaloid from Ipomoea velutina R. Br. (Convolvulaceae)

Convolvulaceae provide a rich source of tropane alkaloids, however, 2-substituted tropanes have been described for only few species of this taxon. In this note, 2,7-diesters such as ipvelutine [7β-acetoxy-2α-(tigloyloxy)tropane] isolated from the vegetative parts of the Australian Ipomoea velutina R. BR. are described as a new group of tropane diesters.


Introduction
During our continuous studies on secondary metabolites of the Convolvulaceae, this plant family has been shown to produce a plethora of tropane alkaloids, especially 3-tropanols and their esters (e. g. [1,2]), as well as some 3,6-disubstituted tropanes [3] or the polyhydroxylated calystegines [4]. This underlines the chemotaxonomic relationship with their sister family Solanaceae where the biosynthetic pathway of tropane alkaloids is well investigated. The main route leads to two stereoisomeric 3-hydroxytropanes, namely 3α-tropanol (basic component of the well-known atropine and other esters), and 3β-tropanol which is also precursor of the calystegines. 2-Substituted tropane alkaloids could only be found as a by-product in the Solanaceae [5]. Accordingly, amongst the tropane alkaloids of the Convolvulaceae 2-substituted ones are extremely rare, too, and could only be detected in some Calystegia, Erycibe, and Ipomoea species [6].

Results and Discussion
In the alkaloidal screening of Convolvulaceae via GC-MS analysis the basic extracts of the Australian Ipomoea velutina R. BR. revealed the presence of several unknown substances. In the basic extract of the vegetative parts seven unknown nitrogen-containing compounds were detected: one main alkaloid and six minor ones (0.7-18.7% of the main alkaloid by integration of the corresponding GC-MS peaks). The molecular formula of the main compound (1)  The substitution pattern of the tropane diester was derived from the mass spectrometric data on the basis of the specific mass fragmentation in bridge-substituted tropanes. The most important fragment is [M − X−COO−CH=CH 2 ] + after expulsion of the ethylene bridge C-6−C-7 including its substituent; this allows a prediction of the substituents' positions in 3,6/7-disubstituted tropanes [7,8]. Regarding 1, there are two possible key ions: in case of acetylation in position 7 m/z 195 or in case of acetylation in position 2 m/z 155. As there is only a veritable peak at m/z 195, 1 has to be acetylated in position 7 of the tropane.
The relative stereochemistry of 1 was deduced from characteristic coupling constants: H-7 showed a doublet-doublet with coupling constants of 3.4 Hz and 7.9 Hz that can also be observed in the 7β-substituted schizanthines C-E [9]. This corresponds with the experience that, for steric reasons, bridge substituents usually are exo-orientated. H-2 showed a trans-diaxial coupling constant J = 10 Hz which is -according to [10] and [11] specific for α-orientated substituents at C-2. These conclusions were also confirmed by Thus, 1 (ipvelutine) was identified as 7β-acetoxy-2α-(tigloyloxy)tropane. sagitattus, and Ipomoea abrupta. Both Convolvulus species afforded similar structures, as well, and, additionally, the corresponding nortropanes in the roots. Ipvelutine-related substances were also found in Ipomoea asarifolia and I. plebeia. The mass fragmentation patterns obtained by GC-MS analysis show that these variations include differences in the stereostructure at C-2 or/and C-7, alternation of the position of the substituents, methylbutyric and hydroxymethylbutyric acid as diverging acyl components, change of the bridge substituents' position from C-7 to C-6 and a hydroxy group as additional substituent (for details see [12]).
2,7-Dihydroxynortropane showing the same substitution pattern as ipvelutine is also synthesized by root cultures of Calystegia sepium (Solanaceae). Incorporation experiments with 15 N-labelled 3-tropanone revealed that, unless 2,7-dihydroxynortropane derives the regular tropane alkaloid pathway, it is not an intermediate in calystegine biosynthesis, but can be seen as a by-product [5].
From the pharmacological point of view, the finding of ipvelutine and derivatives is of interest since they show structural similarity to bao gong teng A [13] obtained from the vegetative parts of Erycibe obtusifolia (Convolvulaceae). Bao gong teng A is characterized by strong miotic properties and therefore used as an antiglaucoma agent in medicinal products. This pharmacological effect is contradictory to that of atropine/hyoscyamine having significance as a mydriatic in ophthalmology and being one of the most commonly used tropanes of natural origin.

Experimental
General procedures 1 H-NMR and 1 H-1 H-COSY spectra were obtained on a Bruker AMX 400 MHz, HSQC and HMBC spectra on a Bruker DRX 500 MHz (TMS as internal standard). EIMS and HR-EIMS were recorded on a Varian MAT 711 (80 eV), FABMS on a Varian MAT CH 5 DF. The GC-MS system consisted of a Fisons GC 8060 coupled to a quadrupole mass spectrometer Fisons MD 800c.

Plant material
Roots and vegetative parts of Ipomoea velutina R. BR. grown from seeds collected in the wild at Florence Falls, Litchfield National Park, Northern Territory/Australia, were harvested in the greenhouse of the Institut für Pharmazie, Freie Universität Berlin. A voucher specimen is deposited at the herbarium of the Berlin-Dahlem Botanical Garden -Botanical Museum (BGBM), Freie Universität Berlin, Germany.

Extraction and isolation of ipvelutine
235 g dried and ground vegetative parts of Ipomoea velutina were extracted 4 h with 3 L MeOH three times and once with a mixture of 2.4 L MeOH and 600 mL 2% aqueous tartaric acid. After evaporation of the MeOH (50°C i. V.), the residue was redissolved in 600 mL 2% aqueous tartaric acid and extracted with petrol ether, CH 2 Cl 2 , and EtOAc, respectively (3 x 500 mL each). Then, the aqueous layer was alkalinized (pH 10) with aqueous NH 3 (25%) and extracted with 4 x 500 mL CH 2 Cl 2 . The united alkaline CH 2 Cl 2 fractions gave 172 mg crude alkaloid fraction which was dissolved in 50 mL 2% aqueous tartaric acid again and extracted with petrol ether, CH 2 Cl 2 , and EtOAc (3 x 50 mL each). After addition of aqueous NH 3 (pH 10), the aqueous layer was extracted with 4 x 50 mL CH 2 Cl 2 . After drying over Na 2 SO 4 and evaporation of CH 2 Cl 2 (40°C i. V.), the alkaline fractions were united and 10 mg ipvelutine were gained (81% purity according to NMR spectra).

GC-MS analysis
Ground plant parts (50 g) were extracted three times with 500 mL MeOH (80%). After evaporation the residue was dissolved in 2% aqueous tartaric acid and extracted with petrol ether, CH 2 Cl 2 , and EtOAc. The aqueous layer was alkalinized and extracted with CH 2 Cl 2 . To purify the extracts obtained, this procedure was repeated with corresponding smaller amounts of the solvents. The resulting extracts were subjected to GC-MS analysis. Samples were injected at 240°C (split 1:20) and separated on a DB-1 column (0.32 mm x 30 m, J&W Scientific, California) by raising temperature from 70°C to 300°C at 6°C/min. Helium was used as carrier gas. Retention indices (RI): Kovats indices [14] were calculated in relation to a set of co-injected hydrocarbons.