Phytochemistry,Vol. 27, No. 2, pp. 439444, 1988. 0031 9422/88 $3.00+0.00 Pergamon Journals Ltd. Printed in Great Britain. ALKALOID DISTRIBUTION IN SEEDS OF ORMOSIA, PERICOPSIS AND HAPLORMOSIA* A. DOUGLAS KINGHORN, RAOUF A. HUSSAIN, EUGENE F. ROBBINS,t MANUEL F. BALANDRIN,~ CHARLES H. STIRTON§ and STEPHEN V. EVANS§,II Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, t Research Resources Center, University of Illinois at Chicago, Chicago, IL 60612, U.S.A.; :~NPI, Salt Lake City, UT 84108, U.S.A.; §Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, U.K. (Received 30 April 1987) Key Word Index--Ormosia; Pericopsis; Haplormosia; Leguminosae; chemotaxonomy; quinolizidine alkaloids; homopodopetaline. Abstract--Alkaloid profiles were determined by capillary gas chromatography/mass spectrometry in seeds of 15 Ormosia species, of both South American and Asian origin, as well as in three Pericopsis species and Haplormosia monophylla. All samples contained alkaloids, and a total of 31 compounds were identified, comprising 23 lupine-type and seven Ormosia-type quinolizidine alkaloids, and the dipiperidine alkaloid, ammodendrine. Homopodopetaline, which has not previously been characterized as a natural product, was detected in extracts prepared from O. coutinhoi, O. macrophylla and O. semicastrata seeds. Ormosia-type quinolizidine alkaloids were restricted to the genus Ormosia, but were not observed in four members of this genus. The Pericopsis species accumulated predominantly ~-pyridone quinolizidine bases, while two collections of H. monophylla contained mainly lupine-type quinolizidine alkaloids of the sparteine/lupanine class. INTRODUCTION The tribe Sophoreae is arranged taxonomically between the legume subfamily Caesalpinioideae and the remainder of the subfamily Papilionoideae. A constituent group of the Sophoreae, the Ormosia group, consists of the three genera, Ormosia (ca 100 species), Pericopsis (four species) and Haplormosia (one species). Ormosia species are found in the tropics of eastern South America and eastern Asia to northeastern Australia, but are absent in Africa [2]. Although Ormosia is generally regarded as constituting one genus, Yakovlev has recognized its segregation into six separate genera based on fruit structure and seed dispersal [3, 4]. Quinolizidine alkaloids have been reported to occur only in the 10 most primitive tribes of the Papilionoideae, and have a high chemosystematic significance [5-9]. The genus Ormosia is characterized by the occurrence of some of the most structurally complex quinolizidine alkaloids found in the Leguminosae [5-9], while only pyridone quinolizidine bases have been found to occur in Pericopsis [9-12]. There has been no prior report of alkaloids in H aplormosia. In this study, we have investigated alkaloid profiles of seeds representing 15 Ormosia species, of both South American and Asian origin, as well as of three Pericopsis and one Haplorraosia species. The objective of the investigation was to determine, in a preliminary manner, if the * Part 3 in the series 'Alkaloids of Papilionoideae'. For part 2, see ref. [1]. [IPresent address, Shell Research Ltd, Sittingbourne Research Centre, Sittingbourne, Kent, ME9 8AG, U.K. 439 distribution of different structural types of quinolizidine alkaloids would support the proposed taxonomic subdivisions of the species examined. In view of the small quantities of alkaloids present, seed alkaloidal identifications were carried out by capillary G C / M S using a combination of two stationary phases. RESULTS AND DISCUSSION The dipiperidine alkaloid, ammodendrine, and 23 lupine-type and seven Ormosia-type quinolizidine alkaloids were identified in one or more of the Ormosia, Pericopsis and Haplormosia species studied in this investigation, by G C / M S comparison with authentic samples (Table 1). G C / M S has been widely applied towards the analysis of lupine alkaloids [9], and the data in Table 1 substantiate a previous observation [12] of the diverse nature of this class of quinolizidine alkaloids in the genus Ormosia. While relatively few of the species embraced in the present study have been examined before for lupinetype quinolizidine alkaloids, the various past identifications of ~-pyridone bases in O. emarginata [13], P. laxiflora [11] and P. mooniana [12] were confirmed. The Ormosia-type quinolidizine alkaloids are pentacyclic (C2o) and hexacyclic (C2o and C21) compounds that are restricted in distribution to members of only a few genera in the Papilionoideae, including Ormosia [5-9, 14]. Such compounds are based on the same carbon skeleton and differ only in their degree of unsaturation and/or in their stereochemistry [9, 14]. While mass spectrometry is useful in assigning the molecular formulas of Ormosia alkaloids, even at low-resolution, this technique appears to be restricted in value in distinguishing between such 440 A.D. KINGHORNet al. Table 1. Quinolizidine and dipiperidine alkaloids identified in Ormosia, Pericopsis and Haplorraosia seeds in the present investigation Species Ormosia amazonica O. balansae O. cinerea O. coutinhoi O. discolor O. emarginata O. fordiana O. henryi O. macrocalyx O. macrophylla O. nobilis O. pachycarpa O. panamensis O. semicastrata O. sumatrana Pericopsis angolensis P. (Afrormosia) laxi,qora P. mooniana Haplorraosia raonophyllat H. monophylla~ Number of unidentified alkaloids Alkaloid(s) identified 5,6-Dehydrolupanine, 13-hydroxylupanine, lupanine, ormosanine, 17-oxolupanine, 17-oxosparteine, panamine, sparteine fl-Isosparteine, jamine, lupanine, 10-oxo-fl-isosparteine, 17-oxolupanine, 17oxosparteine, panamine, sparteine ct-Isolupanine, ct-isosparteine, fl-isosparteine, lupanine, sparteine, tetrahydrorhombifoline Homo-6-epipodopetaline, homopodopetaline, lupanine, podopetaline Anagyrine, 5,6-dehydrolupanine, ct-isosparteine, lupanine, 17-oxolupanine, 17-oxosparteine, sparteine Ammodendrine, cytisine, N-formylcytisine, lupanine, N-methylcytisine, tetrahydrorhombifoline Ammodendrine, anagyrine, 5,6-dehydrolupanine, ~t-isolupanine, lupanine, 17-oxolupanine Ammodendrine, cytisine, N-formylcytisine, N-methylcytisine Angustifoline, 5,6-dehydrolupanine, ct-isoangustifoline,* fl-isosparteine, lupanine, ormosanine, 17-oxolupanine, panamine, sparteine, 13cttiglyoxylupanine Homopodopetaline Angustifoline, homo-6-epipodopetaline, ct-isoangustifoline,* ct-isolupanine, lupanine, N-methylcytisine, sparteine, tetrahydrorhombifoline, 13cttigloyloxylupanine Angustifoline, ct-isoangustifoline,* lupanine, panamine, sparteine Anagyrine, baptifoline, N-formylcytisine, lupanine, N-methylcytisine, rhombifoline, thermopsine Angustifoline, 6-epipodopetaline, homopodopetaline, ct-isoangustifoline,* ct-isolupanine, jamine, lupanine, ormosanine, 1l-oxotetrahydrorhombifoline,* podopetaline, tetrahydrorhombifoline, 13ct-tigloyloxylupanine Ammodendrine, anagyrine, 5,6-dehydrolupanine, ~-isolupanine, ~isosparteine, lupanine, ormosanine, panamine, sparteine Ammodendrine, anagyrine, cytisine, N-formylcytisine, N-methylcytisine Cytisine, lupanine, N-methylcytisine, sparteine 13 5 9 6 3 3 2 1 5 3 8 2 5 7 Cytisine, N-formylcytisine, N-methylcytisine Anagyrine, 5,6-dehydrolupanine, 5,6-dehydro-~-isosparteine, ~t-isosparteine, lupanine, 10-oxo-fl-isosparteine, sparteine Anagyrine, 5,6-dehydrolupanine, 5,6-dehydro-~-isosparteine, lupanine, sparteine *Tentative identification. ?Collected in Liberia (Barker 1221). :~Collected in Nigeria (Sankey s.n.). stereoisomers on the basis of differential fragmentation patterns [9, 12, 14, 15]. However, the resolution attained on the capillary GC stationary phases used in this study was such that the available pairs of authentic Ormosia alkaloid stereoisomers were clearly separated, and, as a result, it was possible to identify seven Ormosia alkaloids among nine members of the genus (Table 1). The previous identifications of ormosanine in the seeds of O. semicastrata and O. sumatrana were confirmed, in addition to podopetaline in the former species, as established by McLean and co-workers [13, 15, 16]. To date, no C2t homo-derivative of an unsaturated pentacyclic C20H33N30rmosia alkaloid has been identified as a constituent of any species in the genus. In this study, it was possible to detect homo-6-epipodopetaline and homopodopetaline (I) in several Orraosia species (Table 1). Homo-6-epipodopetaline has previously been ! found to occur in plant parts of Acosmium panamense (Benth.) Yakovlev and A. subelegans (Mohlenbrock) Yakovlev [14]. Homopodopetaline (1) is a new natural product, although it has been produced synthetically from podopetaline isolated from Podopetalum ormondii [16], which is now known as O. ormondii. The identification of 1 in extracts of O. coutinhoi, O. raacrophylla and O. semicastrata was confirmed by G C / M S comparison with the Alkaloids of Ormosia, Pericopsisand Haplormosiaspecies aminal formed by the known reaction of podopetaline with formaldehyde [14, 16]. The Ormosia species studied embraced all four sections of the genus, as enunciated by Rudd [17], as well as representatives in each of Yakovlev's genera 1-3, 4] Ormosia, Fedorovia, Macroule, Placolobium, Ruddia and Trichocyamos. About half these species were collected in South America and half in Asia. The study also included three of the four species of the closely related genus, Pericopsis, in addition to collections of Haplormosia monophylla from both Liberia and Nigeria, a species which constitutes a monotypic genus [2]. Reference to Table 1 shows that a number of the alkaloids that occurred in the seeds of the species investigated were unidentified. Most of these compounds were Ormosiatype quinolizidine stereoisomers for which no authentic standards were available for comparison purposes, although such compounds were of an assignable molecular formula. Therefore, the alkaloid constituents of the species studied have been divided into six classes, based on structural complexity and/or postulated biogenetic advancement, namely, (i) the dipiperidine alkaloid, ammodendrine; (ii) tetracyclic alkaloids of the sparteine/lupanine type; (iii) an ester of an alkaloid in (ii); (iv) tricyclic degradation products of the alkaloids in (ii); (v) Ormosia alkaloids based on the general structures A-H shown in Fig. 1, and (vi) ~-pyridone quinolizidine bases. In ~H " A B UH H H H C L. U H o H " E F UH G H H It Fig. 1. General structural types of Ormosia-type quinolizidine alkaloids detected in this study. For elemental composition of types A-H, see text. 441 all cases, unidentified alkaloids indicated in Table 1, with uncertain stereochemistry or position of oxygenated functionalities, could be included in one of these six categories. Alkaloids in each major class, expressed as a percentage of the total alkaloids in each Ormosia, Pericopsis and the Haplormosia species studied, are shown in Table 2. In this manner, it was felt that more definitive conclusions could be made concerning variations of alkaloid profiles in relation to taxonomic subdivisions of the species represented. It was also considered that the expression of total alkaloid percentages in groups (i)-(vi) would be more valuable than determinations of the w/w yield of each alkaloid constituent in each seed investigated. As may be seen from Table 2, Ormosia-type quinolizidine alkaloids were found in 11 of the 15 Ormosia species studied, that were indigenous to both South America and Asia. These compounds Were present in species in the sections Ormosia, Macrocarpae, and Unicolores, but were absent in the two species in the section Emarginatae, namely, O. emarginata and O. henryi, which are also classified in Yakovlev's genus Fedorovia. Ormosia alkaloids were also not detected in O. fordiana and O. panamensis seeds, which are both in the section Ormosia. When Ormosia alkaloids were present, the most prevalent type was the hexacyclic C2oHaaN a variant (Fig. ID), as represented by the compound, panamine. There was evidence for the accumulation of oxygenated Ormosia alkaloids in five species, with molecular formulas C21H31N30 and C21Ha3N30 (Fig. 1, G and H, respectively). Such compounds have not hitherto been observed as natural products, and exhibited mass spectral fragmentation patterns similar to those of homoxy-6epipodopetaline (C21HaIN3 O) and homoxyormosanine (C21H33N30), respectively, that were synthesized according to previous methodology [13, 14]. None of these oxygenated Ormosia alkaloids exhibited coincident column residence times to the two standards that were prepared, and thus they could not be provided with stereochemical assignments. No trace of Ormosia-type quinolizidine alkaloids was found in any species investigated in the genera Pericopsis and Haplormosia (Table 2). As has been pointed out previously [9], among the quinolizidine alkaloid-bearing genera of the papilionates, there appears to be a mutual exclusivity in enzyme systems that elaborate Ormosia- and ~t-pyridone-base types, with the latter regarded as being more biogenetically advanced. In the present investigation, it was generally found that species that biosynthesized Ormosia alkaloids tended to produce no ct-pyridones, and vice versa. However, traces of ~-pyridones were found in seeds of O. discolor and O. nobilis that were also well represented by Ormosia alkaloids. In addition, data for O. sumatrana proved exceptional, in that anagyrine was found to constitute nearly 10~o of the total seed alkaloids, and over 4 0 ~ of the remainder were Ormosia alkaloids. The tetracyclic ~pyridone, anagyrine, however, may be regarded as being less biogenetically advanced than the tricyclic ct-pyridones [18]. Given the tendency of Ormosia and a-pyridone alkaloids not to co-occur in a given Ormosia species, the present data showing that ~-pyridones are the predominant quinolizidines in O. panamensis and O. emaroinata contradict earlier reports on Ormosia alkaloids in these species published, respectively, by Lloyd and Horning [19] and Arthur and Loo [20]. We were able to obtain a A. D. KINGHORNet al. 442 Table 2. Percentage of major classes of alkaloids occurring in Ormosia, Pericopsis and Haplormosia seeds Genus Ormosia~ Section Ormosia Series Amacrotropis O. semicastrata Series Amazonicae O. amazonica O. fordiana O. sumatrana Series Nobiles O. discolor O. macrophylla O. nobilis Series Pachycarpae O. pachycarpa Series Panamenses O. panaraensis Section Macrocarpae O. balansae O. cinerea O. coutinhoi Section Emarginatae O. eraarginata O. henryi Section Unicolores O. macrocalyx Pericopsis P. angolensis P. laxiflora P. mooniana H aplormosia H. monophylla H. monophylla Collection* location AS SA AS AS I II III IV 0 0.8 1.4 12.4 6 2 . 5 22.0 0 43.5 3.6 93.5 0.8 44.2 0 0 0 VA VB 0 0 0 0 0 1.1 14.6 0 7.3 0 0 1.5 0 0 0 0 4.5 t§ Alkaloid classt VC VD VE VF VG VH VI 0 0 0.4 0.3 0 0.2 0 0.6 0 0 38.6 0 34.5 0 0 0.2 1.2 0 2.0 0.8 0 0 0.7 0 0 0 2.9 9.9 0 0 2 6 . 9 65.0 0 91.5 0 2.1 2.5 0.1 1.5 t 0 0 0 0 0 0 0.2 0 0.1 0 SA SA SA 0 0 0 99.7 0 0 0 2.7 1.6 AS 0 68.9 0 15.8 0 6.1 0 2.7 0 6.5 0 0 SA 0 t 0 0 0 0 0 0 0 0 0 0 AS SA SA 0 0 0 94.8 25.9 1.0 0 0 0 0 t 0 0 0 88.9 0 70.4 1.6 0 0 0 0.1 0 0 0 0 7.3 4.1 3.6 1.1 0 0 0.1 1.0 t 0 0 0 0 AS AS 13.7 3.4 0.1 6.0 0 0 3.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 82.6 90.6 SA 0 46.5 0 0.4 1.0 1.1 0 47.1 0 t 0 3.9 0 AF AF AF 26.1 0 0 0.1 1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 73.8 98.5 100.0 AF AF 0 0 88.7 86.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11.3 13.2 100.0 *Key to continent in which collected: AF, Africa; AS, Asia; SA, South America or Mexico. tKey to alkaloid classification: I, dipiperidine alkaloid, II, sparteine- lupanine-type; III, an ester of an alkaloid in group 11; IV, postulated degradation products of alkaloids in group II; V, Ormosia-type, A, pentacyclic, C2oH33N3, B, pentacyclic, C20H35N3,C, hexacyclic, C2oH31Na, D, hexacyclic, C2oHa3N3, E, hexacyclic, C21H33N3, F, hexacyclic, C21H35N3, G, hexacyclic, C21H31N30, H, hexacyclic, C21H33N30; VI, ct-pyridone type. :~Classified according to Rudd [17]. §t = trace ( < 0.05 ~o w/w). sample of the seeds used by Lloyd and Horning, and it is apparent that their seeds were originally misidentified as O. panamensis and are actually O. macrocalyx Ducke. The latter species has been represented in the present study, and it is significant that among its constituents (Table 1) are the Ormosia alkaloids, ormosanine and panamine, as originally identified by Lloyd and Horning [19]. Although we were unable to check on the identity of the material used by Arthur and Loo [20], this may also be a case of taxonomic misidentification, since in a later work on O. emarginata by McLean et al. [13], the ct-pyridone base N-methylcytisine was the only quinolizidine alkaloid constituent detected, with Ormosia alkaloids being absent. Other observations concerning compound identification include the occurrence of ammodendrine in four Ormosia species, all of Asian origin (Table 1). Thus far, this dipiperidine alkaloid has not so far been detected in any papilionate genus more taxonomically primitive than Ormosia [9], having been previously identified in a South American member of the genus [12]. Esters of sparteine/lupanine alkaloids were somewhat rare among the Ormosia species, and not found in any Pericopsis species or H. monophylla (Table 2). Analysis of H. monophylla seeds from two geographical locations revealed similar alkaloid profiles, with ct-isosparteine being the most a b u n d a n t alkaloid in both cases. In conclusion, it may be pointed out that G C / M S is a very suitable approach for determining quinolizidine alkaloid profiles of papilionaceous species, in being sensitive, rapid and facile. Such methodology is particularly of value in chemosystematic studies, in that the absence of a particular class or classes of compounds may be established in a given species. Thus, since no Ormosiatype quinolizidine alkaloids were found in any of the Pericopsis species or H. monophylla in this preliminary study, support has been provided for retaining the taxonomic divisions between these genera and Ormosia. The prevalence of ct-pyridone quinolidizine bases and the exclusion of Ormosia alkaloids in the specimens investigated, substantiates the proposed taxonomic subdivi- Alkaloids of Ormosia, Pericopsis and Haplormosia species signs [17] for O. panamensis (section Ormosia, series Panamenses) and O. emarginata and O. henryi (section Emarginatae). An extended G C / M S study of all the Ormosia species could therefore help provide a further understanding of the suitability of the proposals [3, 4, 17] for the subdivision of the genus, and help establish if the South American and Asian species differ phytochemically. Such studies might also provide chemical data which could help elucidate why the genus Ormosia is absent from Africa and whether the American or Asian representatives are the more primitive group of the genus. EXPERIMENTAL GC/MS. Finnigan GC/MS 4510, equipped with INCOS data system; Varian 1440 GC/Varian MAT 112S MS, modified with a Cook interface connected to a deactivated vitreous silica capillary tube direct line, and Varian 166 data system. Plant material. Seeds of the following species (country of origin and voucher number in parenthesis) were obtained from the Herbarium, Royal Botanic Gardens, Kew [Krukoff Seed Collection (K) and the South China Institute of Botany (ISBC),1: Ormosia amazonica Ducke (Ecuador, Pennington 10787), O. balansae Drake (Guangzhou, China, Chen Pong-yu s.n., from ISBC), O. cinerea R. Ben. (Surinam, Wullschflagel 1493, K), O. coutinhoi Ducke (Surinam, Makauria and Supenaam 124a), O. discolor Spruce ex Benth. (Brazil, Krukoff 20822), O. emarginata (Hook. & Arn.) Benth. (Hong Kong, Dept. of Agric. s.n. 1975),O. fordiana Oliv. (China, Tsing Ying 1539), O. henryi Prain (Hainan, China, Cheng Pong-yu s.n., from ISBC), O. macrocalyx Ducke (Mexico, Souza 4211), O. macrophylla Benth. (Brazil, Zarucchi 1320), O. nobilis Tal. (Brazil, Murca Pires s.n.), O. pachycarpa Champ. ex Benth. (Hong Kong, Dept. of Agric, s.n. 1975), O. panamensis Benth. (Panama, Roy DB86), O. semicastrata Hance (Hong Kong, Krukoff 1974/26), O. sumatrana (Miq.) Prain (Thailand, Niyomdbam 815), Pericopsis angolensis (Bak.) Van Meeuwen (Zimbabwe, Krukoff s.n.), P. (Afrormosia) laxiflora (Benth.) Van Meeuwen (Mall, Lafemere 80), P. mooniana Thw. (Borneo, Kostermans 6122), Haplormosia monophylla Harms. (Liberia, Barker 1221; Nigeria, Sankey s.n.). Seed extractions and chromatographic methods. Each seed sample (ca 0.5 g) was ground and extracted with 75 % EtOH (2 x 10 ml) at room temp. Seed EtOH extracts were evapd to dryness in vacuo, and moistened with 28 % NH4OH. On drying and acidification with N HCI, impurities were removed with CH2C12. The aqueous portion of each extract was made alkaline with 28 % NH4OH (pH 8.5), and alkaloids were extracted into CH2C12 and subjected to GC/MS analysis. Using the Finnigan instrument, GC/MS was performed on a DB-5 column (J & W Scientific, Folsom, California) (30 m x 0.25 mm i.d. x 0.25 #m film thickness) with the column temp. held at 180° for 1 min, and then programmed 180-300° at 4 ° min. He head pressure, ca 0.70 kg/cm 2. Injector temp. 230°, interface separator temp. 270°, electron energy, 70 eV, emission current, 0.25mA, scan-to-scan ratio, 1 sec, mass range scanned, 45--475 au. With the Varian instrument, GC/MS was conducted on a DB-1 column (J & W Scientific) (30m x 0.32 mm i.d. x 0.25/zm film thickness). Other conditions were the same as those above, except that the programme was only continued to 270°, and the He head pressure was ca 0.28 kg/cm 2. In both cases, splitless injection with 1 #1 of each diluted alkaloidal extract was used. Quantitation of each alkaloid as a percentage of the total alkaloids in a given extract was performed by internal normalization. Reference alkaloids. Authentic samples of the following alkaloids, either in the form of free bases or salts, were available to 443 us, as described previously [ 1, 12, 21-24]: ammodendrine, angustifoline, anagyrine, baptifoline, cytisine, 5,6-dehydrolupanine, ll,12-dehydrosparteine (5,6-dehydro-a-isosparteine), 13-epihydroxylupanine (jamaidine), 6-epipodopetaline (sweetinine), Nformylcytisine, homo-6-epipodopetaline, 13-hydroxylupanine, ctisolupanine, ct-isosparteine, fl-isosparteine, lupanine, Nmethylcytisine, ormosanine, ormosinine, 10-oxo-fl-isosparteine, 17-oxolupanine, 17-oxosparteine, panamine, podopetaline, sparteine, templetine, tetrahydrorhombifoline and thermopsine. Jamine (homo-ormosanine), as well as a mixture of 13~tangeloyloxylupanineand 13ct-tigloyloxylupanine, and a Sophora secundiflora (Ort). Lag. ex DC. extract containing rhombifoline [251, were kindly supplied by other workers in this area. Homopodopetaline and homotempletine were synthesized from podopetaline and templetine, respectively, by reaction with formaldehyde [14, 16, 26]. 13fl-Tigloytoxylupanine was prepared by the general method of ref. [27-1, by reaction of tigloyl chloride with 13-epihydroxylupanine. Homoxy-6epipodopetaline was prepared from (+)-6-epipodopetaline (2 mg) by dissolution in benzene (3 ml), addition of triethylamine (0.3 ml), cooling, and passage of phosgene gas for 2 min; after standing overnight and flushing with N2, the homoxy derivative was obtained as a white powder [14, 28]. The homoxy derivative of ormosanine was prepared in a similar way. Identification of alkaloids. The following compounds (arranged in the classification used in Table 2) were identified by direct comparison (RRt to lupanine on DB-5 and DB-1 columns, respectively; MS) to authentic alkaloids: (i) dipiperidine alkaloid, ammodendrine, RR,: 0.57, 0.47; MS: m/z 208 [M,1+ [29]; (ii) sparteine-/lupanine-typequinolizidinealkaloids, 5,6-dehydrolupanine, RR,: 0.95, 0.94; MS: m/z 246 [MI + [30,1; 11,12dehydrosparteine, RR,: 0.46, 0.40; MS: m/z 232 [M,1+ [12]; 13hydroxylupanine, RR: 1.41, 1.69; MS: m/z 264 [M] + [30]; ~tisolupanine, RR,: 0.91, 0.90; MS: m/z 248 [Ml ÷ [30,1; ctisosparteine, RR,: 0.35, 0.34; MS: m/z 234 IM] + [9, 14]; /L isosparteine, RR,: 0.51, 0.49; MS: m/z 234 [MI ÷ [31]; lupanine, RR,: 1.00, 1.00; MS: m/z 248 [MI + [32]; 10-oxo-fl-isosparteine, RRt: 1.03, 1.12; MS: m/z 248 [M,1+ [24]; 17-oxolupanine, RRt: 1.32, 1.45; MS: m/z 262 [Ml + [32]; 17-oxosparteine, RRt: 0.86, 0.88; MS: m/z 248 [M,1+ [32]; sparteine, RR,: 0.44, 0.38; MS: m/z 234 [M] ÷ [30]; (iii)ester of alkaloid in lii), 13ct-tigloyloxylupanine, RR,: 2.05, 2.47; MS: m/z 346 [M] ÷ [27]; (iv)tricyclic degradation products of alkaloids in (ii), angustifoline, RRt: 0.88, 0.85; MS: m/z 234 [M] + missing, 193 [30]; tetrahydrorhombifoline, RR,: 0.83, 0.80; MS: m/z 248 [M] + missing, 207 [22-1; (v) Ormosia quinolizidine alkaloids, C2oH33N3 (Fig. 1A): 6-epipodopetaline, RR~:1.37, 1.41;MS: m/z 315 [M,1+ [23]; podopetaline, RR,: 1.43, 1.73; MS: m/z 315 I-M]+ [9, 14]; C2oH35N3 (Fig. 1B): ormosanine, RR,: 1.43, 1.60; MS: m/z 317 [Ml + [12]; C2oH33N3 (Fig. 1D): panamine, RR,: 1.56, 1.81; MS: m/z 315 [M] + [12,1; C21Ha3N3 (Fig. IE): homo-6-epipodopetaline, RR,: 1.55, 1.67; MS: m/z 327 [M,1+ [23]; homopodopetaline, RR,: 1.61, 1.75; MS: m/z 327 [MI ÷ (100),312 (8), 284 (8), 244 (22),243 (36), 229 (53),98 (8), 84 (3), 55 (5), 41 (12); C21H35N3 (Fig. 1F): jamine, RR,: 1.67, 1.90; MS: m/z 329 [M] ÷ [9, 14]; (vi) ct-pyridone quinolizidine bases, anagyrine, RR,: 1.42, 1.55; MS: m/z 244 [M] + [30]; baptifoline, RR,: 1.82, 1.90; MS: m/z 260 [M] ÷ [ 1,1;cytisine, RR,: 0.78, 0.71; MS: m/z 190 [M] + [33,1;N-formylcytisine RR,: 1.33, 1.24; MS: m/z 218 [M] + [34,1;N-methylcytisine, RR,: 0.78, 0.66; MS: m/z 204 [M] + [33]; rhombifoline, RR,: 1.00, 0.99; MS: m/z 244 [M] + missing, 203 [30,1; thermopsine, RR,: 1.25, 1.38; MS: m/z 244 [M,1+ [30]. Resolution of lupine-type quinolizidine alkaloid isomers. Four pairs of isomers, epimeric at C-11 or C-6, were separable by analysis of their RR, s to lupanine on both stationary phases used, namely, anagyrine and thermopsine, a-isolupanine and lupanine, ~-isosparteine and sparteine, and 10-oxoq~-isosparteine and 17- 444 A.D. KINGHORNet al. oxosparteine. The epimeric pairs, 13-epihydroxylupanine (RR, 1.47, 1.62, on DB-5 and DB-1, respectively) and 13-hydroxylupanine, and 13a-tigloyloxylupanine and 13fltigloyloxylupanine [RRt 2.43, 3.12; MS, m/z 346 [M] + (2), 281 (27), 246 (98), 207 (51), 148 (22), 134 (43), 55 (100)] were also separable with the chromatographic systems employed. In addition, 13~t-tigloyloxylupanine was resolvable from its geometrical isomer, 13ct-angeloyloxylupanine (RR, 1.99, 2.37). Ormosia-type quinolizidine alkaloid identification. In contrast to previous GC/MS work using a packed column [12], the available stereoisomers of pentacyclic and hexacyclic Ormosiatype quinolizidine alkaloids were separable on both the stationary phases employed in the present work. Hence, compounds based on the followingcarbon skeletons were resolved in this study (RR, on DB-5 and DB-1, respectively): C20Ha3N3,6epipodopetaline and podopetaline; C2oH35N3, ormosanine and templetine (RR, 1.52; 1.75; MS, m/z 317 [M] + [12]); C21HaaN 3, homo-6-epipodopetaline and homopodopetaline; C21HasN3, jamine (homo-ormosanine)and homotempletine [RRr 1.63, 1.85; MS, m/z 329 [M] + (33), 328 (100),281 (8),246 (7), 245 (2), 231 (2), 207 (6), 163 (2), 98 (1), 84 (1), 44 (21), 41 (27)]. The hexacyclic Ormosia alkaloid, panamine (C2 ~H 31Na) was resolved from the dimeric Ormosia-alkaloid, ormosinine (C4oH66N6),a compound which shows closely comparable EIMS data [9, 35]. The two homoxy-Ormosia alkaloids prepared by synthesis exhibited the following data (RRt on DB-5 and DB-1, respectively): homoxy-6-epipodopetaline (C21H31N30): RR, 2.20, 2.72; MS, m/z 341 [M] + (12),298 (1), 257 (3), 243 (8), 160 (2), 146 (3),98 (100), 67 (7), 55 (11) homoxyormosanine (C21H33N30): RR, 2.26, 2.80; MS, m/z 343 [M] + (100), 314 (5), 300 (27), 286 (4), 258 (13), 245 (10), 172 (3), 98 (64), 69 (8), 55 (14). Tentative alkaloid idemifications. One compound was tentatively identified as ~-isoangustifoline in several extracts, and exhibited RR, (DB-5, DB-1) of 0.88 and 0.80, and MS data identical to angustifoline,with which it co-eluted in all cases. This compound has previously been described as a constituent of L. polyphyllus leaflets, as a minor alkaloid [27]. Its shorter column residence time than the parent compound, angustifoline (with an 1lct-H substituent), is consistent with this isolate being epimeric at C-11 [36]. A second compound that may be classified as a degradation product of the sparteine- lupanine-type quinolizidine alkaloids, 1l-oxotetrahydrorhombifoline, was tentatively identified by comparison with literature mass spectral data, as described previously [12, 37]. This compound was first isolated from the bark of O. countinhoi [37], but was not present in the seeds of this species in the present study. Acknowledgements--We wish to thank Dr P. Naegeli, Givaudan Research Company, Zurich, Switzerland, for a sample ofjamine, Dr M. Wink, University of Munich, Munich, F.R.G. for a mixture of 13ct-angeloyloxylupanine and 13u-tigloyloxylupanine, and Professor W. J. Keller, Northeast Louisiana University, Monroe, Louisiana, U.S.A, for a legume extract containing rhombifoline. We are especially grateful to Dr H. A. 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