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) is consistent with C15H23NO4 (m/z 281).

The 1H-NMR (Table 1) in combination with HSQC and HMBC experiments showed two acylic residues: a C5-acid containing a double bond, namely tiglic acid, as well as acetic acid. Both were confirmed by fragmentation ions in the EIMS as products of α-cleavage neighbouring the ester carbonyls: m/z 83 (C4H7−CO+; HRMS: [C5H7O]+ as 83.04959, calcd. 83.04969) and m/z 43 (CH3−CO+).

Tab. 1

1H- and 13C-NMR data of ipvelutine (in MeOD)

atom		1H-NMR (in MeOD)		13C-NMR* (in MeOD)
1	3.55	br d	3.2 Hz	72.9
2a	5.02	ddd	2.2 Hz; 5.9 Hz; 11.3 Hz	68.9
3e	1.98	m		22.8
3a	1.49	dtd	6.4 Hz; 12.1 Hz; 12.8 Hz
4a	1.89	m		27.5
4e	1.68	ddd	2.3 Hz; 6.7 Hz; 13.7 Hz
5	3.82	br t	5.2 Hz	64.7
6n	2.36	dd	8.0 Hz; 14.6 Hz	37.8
6x	2.27	ddd	3.5 Hz; 6.3 Hz; 14.7 Hz
7n	4.61	dd	3.4 Hz; 7.9 Hz	70.8
N−CH3	2.91	s		40.9
1’				167.8
2’				129.0
3’	6.96	dq	1.2 Hz; 6.9 Hz	139.3
CH3−4’	1.83	d	7.1 Hz	11.9
CH3−5’	1.84	d	0.9 Hz	14.1
1”				176.7
CH3−2”	1.93	s		21.9

…taken from HSQC/HMBC.

The HSQC spectrum revealed a characteristically downfield shifted N−CH3 (δC 40.9, δH 2.91) as well as three methylene signals (δC 37.8, 27.5, and 22.8) and four methine groups (δC 72.9, 70.8, 68.9, and 64.7). From the 1H-1H-COSY, the complete coupling sequence could be deduced. As a result, 1 (Fig. 1) could be identified as a 2,7-disubstituted tropane.

Fig. 1

Structure of ipvelutine [7β-acetoxy-2α-(tigloyloxy)tropane], main alkaloid from the vegetative parts of Ipomoea velutina R. Br.

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=CH2]+ 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 NOE measurements: H-2 (δH 5.02) showed correlations to H-1 (δH 3.55), to the equatorial H-3e (δH 1.98) and to the axial H-4a (δH 1.89) which is only possible if H-4a and H-2 are both axial [11]. H-7 (δH 4.61) was correlated to H-1 (δH 3.55) and – only enabled by its endo-position – to the axial H-3a (δH 1.49) and H-6n (δH 2.36).

Thus, 1 (ipvelutine) was identified as 7β-acetoxy-2α-(tigloyloxy)tropane.

In the vegetative parts and/or roots, eight minor compounds related to ipvelutine could be detected by GC-MS analysis. They were identified by their fragmentation patterns; characteristic base peaks of those 2,7-disubstituted tropanes are m/z 95 and m/z 82 or m/z 81 together with a prominent peak at m/z 156, and of their nortropane derivatives m/z 125 and m/z 81 including a half-maximal peak at m/z 108. An additional result of the systematic GC-MS screening is the detection of ipvelutine (appearing as deacetylated derivative in GC-MS analysis) in vegetative parts of Convolvulus graminetinus, C. 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 15N-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

1H-NMR and 1H-1H-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 CH5DF. 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, CH2Cl2, and EtOAc, respectively (3 × 500 mL each). Then, the aqueous layer was alkalinized (pH 10) with aqueous NH3 (25%) and extracted with 4 × 500 mL CH2Cl2. The united alkaline CH2Cl2 fractions gave 172 mg crude alkaloid fraction which was dissolved in 50 mL 2% aqueous tartaric acid again and extracted with petrol ether, CH2Cl2, and EtOAc (3 × 50 mL each). After addition of aqueous NH3 (pH 10), the aqueous layer was extracted with 4 × 50 mL CH2Cl2. After drying over Na2SO4 and evaporation of CH2Cl2 (40°C i. V.), the alkaline fractions were united and 10 mg ipvelutine were gained (81% purity according to NMR spectra).

7β-Acetoxy-2α-(tigloyloxy)tropane [(1S,2S,5R,7R)-7-(acetyloxy)-8-methyl-8-aza-bicyclo[3.2.1]oct-2-yl (2E)-2-methylbut-2-enoate, ipvelutine, 1]

Yellow oil. 1H-NMR (400 MHz, MeOD): see Table 1. 13C-NMR (100.6 MHz, MeOD): see Table 1. MS (EI, 80 eV, 110°C): m/z (%) = 281 (2) [M]+, 239 (83), 195 (7), 156 (100), 142 (60), 140 (35), 112 (11), 98 (46), 96 (84), 95 (91), 94 (50), 85 (41), 84 (31), 83 (27), 55 (22), 43 (20). (+)-FAB MS (80 eV): m/z = 282 [M+H]+. HR MS (80 eV): m/z = 281.16256 (calcd. 281.16271 for C15H23NO4), 239.15283 (calcd. 239.15214 for C13H21NO3), 156.10254 (calcd. 156.10245 for C8H14NO2+), 142.08678 (calcd. 142.08681 for C7H12NO2+), 140.10749 (calcd. 140.10754 for C8H14NO+), 98.062524 (calcd. 98.06059 for C5H8NO+), 95.072728 (calcd. 95.073499 for C6H9N).

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, CH2Cl2, and EtOAc. The aqueous layer was alkalinized and extracted with CH2Cl2. 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 ×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.