NEW IMIDAZOLE ALKALOIDS FROM A GLOCHIDION SPECIES (FAMILY EUPHORBIACEAE)*

By S. R. JOHNS? and J. A. LAMBERTON~

[Manuscript received October 13, 19661

The leaves of a Glochidion species, probably C-lochidion philippicum (Cav.) C . B. Rob., of the family Euphorbiaoeae, contain three new interrelated imidazole alkaloids, glochidine (I), glochidicine (11), and Na-4'-oxodecanoylhistamine (111), together with the known alkaloid Na-cinnamoylhistamine. The bark of this species contains only glochidicine and Na-oinnamoylhistamine.

The genus Glochidio?~, of the tribe Phyllantheae in the family Euphorbiaceae, has not previously been the subject of alkaloid studies, although numerous species occur in tropical Asia and Africa. An examination has now been made of the alkaloids obtained in 0.15% yield from the leaves and 0.04% yield from the bark of a New Guinea species collected near the mouth of the Busu River, where i t grows as a small tree in swampy scrub at an elevation of only 10 ft. This species is con- sidered to be Glochidion philippicum (Cav.) C. B. Rob., but there is a possibility that it may be Glochidion novoguineensis K. Sch. Leaf samples from a Glochidion spe- cies, also probably G. philippicum, collected near Fulleborn Harbour in New Britain, gave a higher yield (0.576) of the same leaf alkaloids as the New Guinea tree.

The leaves from both sources yielded three new interrelated imidazole alkaloids, together with the known dkaloid hTa-cinnamoy1histamine.l The alkaloids from a bark sample were a simpler mixture consisting of only one of the new bases and a small proportion of Na-cinnamoylhistamine. The Queensland tree Glochidion ferdinandii Muell. Arg. gave negative field tests for alkaloids.

Glochidine, m.p. 65-67", [a], &0", has been assigned the molecular formula Cl,H,,N30 from the elementary composition of the base and its picrate, and from the molecular ion at mle 261 in the mass spectrum. When hydrolysed in refluxing concentrated hydrochloric acid glochidine is converted quantitatively into histamine hydrochloride and an acid, CloHl,03, that was shown to be 4-oxodecanoic acid from

* These alkaloids have been the subject of a preliminary communication (Johns, S. R., and Lamberton, J. A., Chern. Commun., 1966, 312).

t Division of Applied Chemistry, CSIRO Chemical Research Laboratories, Melbourne.

1 Fitzgerald, J. S., Aust. J. Chem., 1964, 17, 375.

556 S. R. JOHNS AND J. A. LAMBERTON

spectroscopic data and by comparison with an authentic specimen of 4-oxodecanoic acid synthesized by a known methods2 The infrared spectrum of glochidine shows a single strong carbonyl band at 1690 cm-l that can be assigned to a lactam carbonyl group, but there are no bands in the 3500 om-l region due to the imidazole NH or to lactam NHCO. The 60-Mc/s n.m.r. spectrum (CDC1, solution) shows a signal from the C2 imidazole ring proton as a sharp singlet a t 6 7.40, and the signal from the C4 ring proton as a sharp singlet at 6 6.70, but, in agreement with the infrared evidence, no signal that could be attributed to imidazole NH. A broadened triplet at 6 0.87 (three protons) and a broad rnultiplet at 65-88 cis (eight protons) indicate the presence of a chain containing four saturated methylene groups and a terminal methyl group. This spectroscopic evidence, in conjunction with the identification of the hydrolysis products, indicated that glochidine is (I), and this structure is in accord with the remainder of the n.m.r. spectrum and with the mass spectral breakdown pattern. The C 7 methylene protons resonate as two complex one-proton multiplets at 180-205 c/s and 249-270 CIS, and the 1.10 p.p.m. difference in chemical shift between the two methylene protons is consistent with differences found between the chemical shifts of the axial and equatorial protons at C9 of 5,5a,6,7,8,9-hexa- hydropyrido[2,1-blquinazolin-11-one, at C15 of 17-oxosparteine, and a t C10 of ~-1upanine.~ The C6 methylene protons resonate as a two-proton multiplet at 160-185 c/s, while the C 10 and C 11 methylene protons almost coincide in chemical shift and resonate as a fairly narrow multiplet at 6 2.5. Of the methylene groups of the n-hexyl chain, the protons of the group adjacent to C 12 resonate at 105-129 c/s, slightly downfield with respect to the signals from the other saturated methylene groups.

The mass spectrum of glochidine has a molecular ion peak at mle 261 and a base peak at mle 176 (M-85) which corresponds to the loss of the n-hexyl chain from C 12 (ion (IVa), (IVb)). Peaks a t both m/e 81 and mle 180 can be explained in terms of the two ions (V) and (VI) produced by cleavage of both the C6,C7 and the N l,C 12 bonds, with the charge being retained on either fragment. Similarly peaks at m/e 94 and m/e 95 probably correspond respectively to the ion (VII) and the radical ion (VIII) that would be produced by cleavage of the N l,C 12 bond and the N 8,C 7 bond. The mle 168 peak corresponds to the ion (IX) formed by the same cleavage but with retention of charge on the amide nitrogen.

Glochidicine was shown to be isomeric with glochidine by the elementary composition of the crystalline hemihydrate, m.p. 102-103", [a], &OO, and by the molecular ion at m/e 261 in the mass spectrum. The close relationship of the two alkaloids was established by the formation of glochidicine from glochidine when the latter was heated in dilute aqueous acetic acid solution. Glochidicine, unlike glochidine, is stable to refluxing concentrated hydrochloric acid from which it was recovered unchanged. Together with the spectroscopic data discussed below these results indicate that glochidicine must be (11), and that the relative instability of glochidine (I) to acid hydrolysis depends upon the N-C-N system that is not found

'Lukes, R., Ohemicke' G s t y , 1930, 24, 197. a Fitzgerald, J. S., Johnw, S. R., Lamberton, J. A., and Redcliffe, A. H., dust. J . Chem.,

1966, 19, 151.

NEW IMIDAZOLE ALKALOIDS 557

in glochidicine. The substitution pattern of the irnidazole ring was confirmed by the n.m.r. spectrum which showed signals that could be attributed to an imidazole NH group and to a single imidazole CH group.

The infrared spectrum of glochidicine shows a strong amide carbonyl absorption at 1670 cm-I and a band at 3450 cm-l which can be assigned to an imidazole NH group. A broad signal in the n.m.r. spectrum at 550-575 c/s, due to a single proton and readily exchanged by deuterium on treatment with deuterium oxide, can also be assigned to this NH proton. Only one signal from an imidazole ring CH proton appears in the n.m.r. spectrum, and this signal at 6 7.38 can be assigned to the C2 proton. A broadened triplet a t 6 0.85 (three protons), a broad multiplet at 60-92 c/s (eight protons), and a two-proton multiplet at 98-120 c/s were similar to signals in the spectrum of glochidine, and indicate that glochidicine also has an n-hexyl chain. The C6 methylene protons resonate as two multiplets a t 246-276 c/s and 165-200 c/s, similar to the multiplets observed for the C7 protons in the spectrum of glochidine. The chemical shifts of the C5, C9, and C 10 methylene protons all fall in the range 120-180 c/s, and result in a complex six-proton multiplet signal in this region.

The only major peak in the mass spectrum of glochidicine is a t m/e 176, and is presumably due to the ion formed by loss of the n-hexyl chain. As there is little further breakdown it is suggested that the resulting tricyclic ring system must be more stable that it is in glochidine.

The third new base isolated from Glochidion leaves had a molecular formula C,,H,,N,O,, shown by elementary analysis and by a molecular ion at m/e 279 in the mass spectrum. Hydrolysis with concentrated hydrochloric acid at reflux temperature afforded histamine hydrochloride and 4-oxodecanoic acid. In the infrared spectrum there were absorption bands at 3440, 1710, and 1665 cm-1 that can be assigned to imidazole NH, saturated ketonic carbonyl, and amide carbonyl groups respectively. From this evidence the third base was considered to be Nff-4'-oxodecanoylhistamine (111), and this structure has been confirmed by synthesis. The reaction of histamine with 4-oxodecanoyl chloride afforded a mixture consisting of all three bases (I), (11), and (111), which were separated and shown to be identical with the naturally occurring alkaloids. When either base (I) or (111) was dissolved in aqueous acetic acid and heated at 100°, a mixture of the three bases (I), (11), and (111) was obtained, and the proportion of base (11) in the mixture was increased by more prolonged heating. Bases (I) and (11) may therefore be regarded as alternate cyclization products of (111), and they are probably formed biosynthetically in this way as condensation of the carbony1 group of (111) can take place at either the NH or CH position of the imidazole ring. The N-C-N function in (I) is unstable to hot acetic acid so that ring opening and recyclization occurs under relatively mild conditions. The ease with which equilibration occurs by ring opening and recyclization presumably accounts for the lack of optical activity of both bases (I) and (11).

The fourth base C,,H,,N,O afforded cinnamic acid and histamine hydrochloride on hydrolysis with concentrated hydrochloric acid, and it was shown to be identical

558 S. R. JOHNS AND J. A. LAMBERTON

with a sample of Ma-cinnamoylhistamine, which had previously been isolated from Acacia po1ystacha.l

The bark alkaloids of G. philippicum are less complex, and were shown to be glochidicine with a small proportion of Na-cinnamoylhistamine.

The Glochidion alkaloids (I) and (11) provide an interesting extension to the previously known types of imidazole alkaloids. Part of the ring system of (I) ie incorporated in the structure of the alkaloid zapotidine4 (X) from Casimiroa edulis

( I V ~ ) ; mje 176

(1'1); mje 180 (VII); m/e 94 (VIII); m/e 95

(family Rutaceae), and of spinacineb from the liver of the shark Ascanthias vulgaris, both of which are presumably also derived from cyclization of histamine derivatives. 4-Oxodecanoic aoid does not appear to have been isolated from plants previously, but there is a known example of the occurrence of a 4-0x0 aoid in 4-oxooctadeca- 9 , l l , 13-trienoic aoid (licanic acid) .6

Mechoulam, R., Sondheimer, F., Nelera, A., and Kincl, F. A., J. Am. chern. Soo., 1961, 83, 2022.

Ackermann, D., and Mohr, M., 2. Biol., 1937, 98, 37. Hilditch, T. P., and Williams, P. W., "The Chemical Constitution of Natural Fats."

4th Edn, p. 635. (John Wiley: New York 1964.)

NEW IMIDAZOLE ALKALOIDS

EXPERIMENTAL (a) General

Leaves and bark of the Glochidion species from the Busu River were collected by Dr T. G. Hartley and identified by Dr R. Hoogland as probably G. philippicum (Cav.) C. B. Rob.,* but with some possibility that they may be G. novoguineensis K. Soh. Leaves from New Britain were collected by ?/Ir C. D. Sayers.

Microanalyses were made by the Australian DIicroanalytical Service, Melbourne.

N.m.r. spectra were measured in deuterochloroform solutions on a Varian A60 spectro- meter, and chemical shifts are relative to tetramethylsilane (6 0.00).

(b) Extraction of the Alkaloids

Milled dried plant material was extracted with ethanol at 40' and the alkaloids isolated according to the method previously de~cribed.~

(c) Glochidine (I) The alkaloids were separated by chromatography on columns of alumina which had been

neutralized by treatment with ethyl acetate and dried a t 100' under reduced pressure. The alkaloids were added to the column in benzene solution and the fractions eluted by benzene consisted of glochidine (I) (35% of the total alkaloids). Although apparently pure (from infrared and n.m.r, spectra and the appearance of only a single spot in thin-layer chromatograms run on Kieselgel G and stained with iodine vapour), glochidine did not crystallize until it had been further purified by conversion into a crystalline picrate and the free base subsequently regenerated. The picEate crystallized readily from ethanol in yellow prisms, m.p. 143-144" (Found: C, 51.8; H, 5.4; N, 16.6. Calc. for Cl,H,,N,O,C,H3N,O,: C, 51.4; H, 5.3; N, 17.1%).

The crystalline picrate was suspended in water, dilute ammonia was added, and the free base extracted with chloroform. Glochidine recovered from the picrate crystallized readily, and afforded colourless needles, m.p. 65-67", [aID &0" in CHCl,, on recrystallization from light petroleum(l?ound: C,68.8; H,9 .1 ; N, 16.3. Calc.forC1,H,,N3O: C,68.9; H,8 .9 ; N, 16.1%). The ultraviolet absorption spectrum (ethanol solution) showed end absorption but no distinctive maximum, and in the infrared spectrum (chloroform solution) there was strong absorption at 1690 om-l (five-membered ring lactam). The major peaks in the mass spectrum appeared at m/e 261 (12%), 180 (35), 176 (loo), 168 (44), 95 (65), 94 (27).

(d) Acid Hydrolysis of Glochidine

Glochidine (100 mg) was dissolved in concentrated hydrochloric acid (5 ml) and the solution heated at reflux temperature. After 3 hr the solution was cooled and the crystalline product which had separated was extracted with diethyl ether. Purification by sublimation at reduced pressure gave 4-oxodecanoic acid, m.p. 68-69' (Found: C, 64.3; H, 9.4. Calc, for Cl,Hl,O,: C, 64.5; H, 9.7'3,). The identification of the hydrolysis product as 4-oxodecanoic acid was confirmed by comparison (mixed m.p., infrared spectrum) with an authentic specimen (m.p. 88-69") prepared from reaction of n-hexylmagnesium bromide with AT-methy1succinimide.Z

After extraction of 4-oxodecanoic acid from the acid hydrolysate the remaining aqueous hydrochloric acid solution was evaporated to dryness. The crystalline residue consisted of histamine hydrochloride, which was identified by comparison with an authentic specimen of histamine hydrochloride (mixed m.p. determination and comparison of infrared spectra and of n.m.r. spectra measured in DzO solution).

(e) G lochidicine (11) Glochidicine (11) was eluted next in sequence after glochidine by benzene/lO% chloroform

mixture, but the later fractions contained the amide base (111) and Na-cinnamoylhistamine.

* I t has been drawn to the attention of the authors that the name G. philippicum (Cav.) C. B. Rob. has been omitted from the Index Kewensis. The pertinent reference is to Robinson, C. B., Philipp. J. Sci. (Botany), 1909, 4, 103.

7 Johns, S. R., Lamberton, J. A., and Sioumis, A. A,, Aust. J. Chem., 1966, 19, 2331.

560 S. R. JOHNS AND J. A. LAMBERTON

Glochidicine crystallized readily from acetone, but was always obtained as a hemihydrate, m.p. 102-103", [aID +0° in chloroform solution, and the water of crystallization could not be completely removed even on prolonged drying (Found: C, 66.6; H, 8.9; K, 15.5. Calc. for Cl,H,,N,O,~HzO: C, 66.6; H, 8.9; N, 15.5%). The ultraviolet spectrum of (11) in ethanol showed end absorption but no distinctive maximum, while the infrared spectrum (chloroform solution) had bands at 1670 om-' (lactam carbonyl) and 3450 cm-I (imidazole NH). The only major peak in the mass spectrum occurred at m/e 176 with the molecular ion ?n/e 261 and the (M-1)+ ion less than 1% of the base peak.

Glochidicine (11) was recovered unchanged after heating in concentrated hydrochloric acid at reflux temperature under the conditions used for the hydrolysis of glochidine (I). This resistance to acid hydrolysis provided a convenient method of purifying samples of glochidicine containing (I), (111), or Na-cinnamoylhistamine, as after refluxing with hydrochloric acid and basifying the solution with ammonia glochidicine could be recovered, while the other bases were converted into non-extractable products.

( f ) Xn-4'-Oxodecanoyllzistamine (111)

Na-4'-Oxodecanoylhistamine (111) was eluted by benzene/20% chloroform mixture, but it was difficult to isolate the pure alkaloid in good yield as it was obtained as mixtures with Na-cinnamoylhistamine. Repeated crystallization from chloroform or chloroform/ethanol mixtures of fractions containing a high proportion of (111) gave pure (111) as colourless crystals, m.p. 115-117", v,,, (CHC1,) 1665 cm-l (amide carbonyl), 1710 om-I (ketonic carbonyl), 3440 cm-1 (imidazole NH) (Found: C, 64.8; H, 9 . 2 ; X, 14.9. Calc. for C,,H,,N,O,: C, 64.5; H, 9.0; N, 15.0%). The composition of the mixtures containing (111) and cinnamoylhistamine was shown by acid hydrolysis which yielded 4-oxodecanoic acid, cinnamic acid, and histamine hydrochloride.

(g) Na-Cimnamoylhistamke

A-a-Cinnamoylhistamine mixed with base (111) was eluted by benzene/20% chloroform mixture. The pure alkaloid was obtained by repeated crystallizations of fractions rich in Na-cinnamoylhistamine from acetone or ethanol, and it was identified by comparison (m.p., mixed m.p., i.r., and n.m.r. spectra) with Na-cinnamoylhistamine isolated from Acacia polystacha.1 Hydrolysis with concentrated hydrochloric acid at reflux temperature afforded cinnamic acid and histamine hydrochloride.

(h) Synthesis of Glochidine ( I ) , Glochidicine (11), and Nn-4'-Oxodecanoylhistamirte (111)

4-Oxodecanoylchloride, prepared from 4-oxodecanoic acid (1 g) by heating with oxalyl chloride in benzene solution at reflux temperature, was dissolved in dry benzene (100 rnl). To this solution was added with stirring a solution of histamine (1 g) in anhydrous pyridine (30 ml). The solvents were removed under reduced pressure, the residue dissolved in a small volume of water, and the solution basified (NH,). Extraction with chloroform gave a colourless gum (500 mg) which in thin-layer chromatograms showed three spots corresponding in R, with alkaloids (I) , (11), and (111). Chromatography on neutral alumina afforded fractions consisting of (I) (10-20%), (11) (30-40%), and (111) (30-40%) in sequence, and each compound was purified, characterized spectroscopically, and shown to be identical with the corresponding alkaloid isolated from the Glochidion extract.

(i) Reaction of Alkaloids (I) and (111) with Acetic Acid

Glochidine (I) was recovered unchanged from acid solution at room temperature, but in dilute acetic acid (10% solution) at 100' glochidine was rapidly converted into a mixture of (I), (11), and (111). The products were separated and identified as described in section (h). The separation of the three products was not quantitative, but it was observed that the proportion of the alkaloid (11), which is stable to acid hydrolysis, increased with more prolonged heating. Heating alkaloid (111) under the same conditions gave a similar mixture of (I), (11), and (111).