Unusual natural 9,10-dihydrophenanthrenes from roots of Toona Ciliata

The roots of Toona ciliata yielded four new 9,10-dihydrophenanthrenes which were identified on the basis of spectroscopic analysis as 9,10-dihydro-9-hydroxy-9-( tert -butoxycarbonylmethyl)- 10-oxophenanthrene, 9,10-dihydro-9-hydroxy-9-(ethoxycarbonylmethyl)-10-oxophenanthrene, 9,10-dihydro-9-hydroxy-9-( n -butoxycarbonylmethyl)-10-oxophenanthrene and 9,10-dihydro-9- hydroxy-9-(benzyloxycarbonylmethyl)-10-oxophe-nanthrene. These compounds represent a novel group of phenanthrenes lacking oxygen in the benzene rings. In addition the known limonoid cedrelone, the sterols sitosterol and stigmasterol, the coumarins isopimpinellin and siderin, the furoquinoline alkaloid skimmianine and 2-hydroxy-4-methoxycinnamaldehyde were also isolated and characterized.


Introduction
The genus Toona (Endlicher) M. J. Roemer contains approximately six poorly defined species which occur in the old world eastwards from Indian to Australia. 1 Phytochemical data are available for T. sureni (Blume) Merril.and T. ciliata M. J. Roem.3][4][5][6][7] Furthermore, it produces sesquiterpenes of the cadinene group, 8,9 α-and β-amyrins acylated with fatty acids, 5 simple coumarins 5 and proanthocyanidins. 10The constituents of T. sureni are so far four limonoids with intact carbon squeleton 11 and ring-A/ring-B dilactones. 12Toona was originally described by Endlicher 1 as a section of Cedrela.However, Roemer later recognized that they could be separated by a number of sound morphological characters, raising Toona to generic rank. 1 The known limonoids from Cedrela are mainly mexicanolides. 5Thus, the total lack of mexicanolides in all aerial parts of T. ciliata strongly supports Roemer's taxonomic conclusions.We have now examined the roots since they have never been investigated before and in order to determine if the above differences still remain also in this organ.
The major compound was identified as 1 on the basis of the following data.EI mass spectrometry gave [M] + as C 20 H 20 O 4 and the IR spectrum indicated carbonyl, ester carbonyl, and hydroxyl functional groups.The 1 H NMR spectrum run in C 6 D 6 (Table 1) was better resolved than in CDCl 3 and showed signals integrating for eight aromatic protons.The 1 H-1 H COSY and homonuclear decoupling experiments suggested the presence of two ortho disubstituted phenyl rings.One ring was established by the four mutually-coupled proton system at δ 7.88 (ddd, J = 7.4, 1.6, 0.4 Hz), 7.06 (td, J = 7.4, 1.4 Hz), 7.02 (td, J = 7.4, 1.6 Hz), 7.36 (ddd, J = 7.4, 1.4, 0.4 Hz).Some small aromatic para coupling were obscured by line broadening.
Another sequence consisted of a downfield-shifted proton signal at δ 8.00 (ddd, J = 7.5, 1.5, 0.5 Hz) which was coupled to the 1 H signals at δ 6.93 (td, J = 7.5, 1.0 Hz), 7.10 (ddd, J = 8.0, 7.5, 1.5 Hz) and 7.37 (ddd, J = 8.0, 1.0, 0.5 Hz), requiring one of the carbonyl substituents to be attached to this second ring and permitting the assignment of the signal at δ 8.00 to H-1.This was supported by the HMBC experiments (see Experimental) which showed correlations between this latter proton signal and the 13 C signal at δ 201.9 ( 3 J).The 1 H signal at δ 6.93 was ortho-coupled to the H-1 signal and showed a cross peak with the singlet resonance at δ 129.8 (J 3 ), thus these signals can be attributed to H-2 and C-1a adjacent to the ketonic carbonyl group (δ 201.9), respectively.The proton signal at δ 7.37 was para-coupled to the H-1 signal (then assigned to H-4) and showed long-range correlation with the 13 C signal at δ 129.7.The 1 H signal at δ 7.36 belonging to another phenyl ring, also showed long-range correlation with the 13 C signal at δ 129.7.If the latter signal was assigned to a carbon belonging to the second ring and shows cross peaks by 3 J with the H-4, both rings must be bound to each other.Thus, the above correlations permitted the assignments of H-5 at δ 7.36 and C-5a at δ 129.7.Furthermore, the observed correlations between the quaternary oxygen-bearing carbon at δ 78.2 and the 1 H signal at δ 7.88, which was para-coupled to the H-5, indicated a quaternary benzyl alcohol to be located at C-8a and led to their assignments as C-9 and H-8, respectively.The 1 H NMR also showed one isolated AB-type methylene at δ 2.62 and 2.72.The correlation from the latter signal to the C-9 signal and to two carbonyl signals at δ 201.9 and 167.8, resulted in the construction of an atypical 9,10-dihydro-9-hydroxy-9-(alkoylcarbonylmethyl)-10-oxophenanthrene system, lacking oxygen in the benzene rings.A tert-butoxide group must be connected at carbonyl carbon due to the observed correlation between a singlet at δ 1.28 (9H, assigned to three magnetically equivalent methyl groups) and the 13 C signal at δ 81.0.The large geminal coupling constant of the methylene protons was consistent with C-9 fully substituted.The identification of the nucleus as a 9-hydroxy-10-oxophenanthrene was also supported by the mass spectrum which gave a significant fragment at m/z 209 (Scheme 1) due to fission of the side chain between C-1' and C-9.The structure of the new natural product was thus established as 9,10-dihydro-9-hydroxy-9-(tertbutoxycarbonylmethyl)-10-oxophenanthrene (1).The structural assignment was also confirmed by comparison of the 1 H and 13 C NMR spectra (Table 1 and 2) with those of synthetic phenanthrenequinone (5). 13 1 and 2).The 1 H NMR spectrum, instead of signals for a tert-butoxide group, showed signals for a methyl triplet (δ 0.84, J = 7.2) and 1 H resonances for an AB system associated with a methylene group whose hydrogens were not equivalent and each make up an AB doublet (J = 18.4) further split by additional coupling with the methyl-hidrogens (J = 7.2).A significant fragment at m/z 89 [CH 3 COOCH 2 CH 3 + H] in the mass spectrum for 2, associated with retro-aldol cleavage of side-chain, clearly indicated the presence of a carboethoxy group at C-1' (Scheme 1).Thus, compound 2 was concluded to be 9,10-dihydro-9-hydroxy-9-(ethoxycarbonylmethyl)-10-oxophenanthrene.Compounds 1 and 2 have already been known as a reaction product obtained from phenanthrenequinone (5), [14][15][16] however, this the first time that they have been isolated as natural products.
Compound 3 was isolated in very small amounts and could not be separated from 2. This mixture gave rise to a mass spectrum which indicated a molecular formula C 20 H 20 O 4 ([M+H] + = 325) for 3, suggesting an isomer of 1. Analysis of the 1 H NMR, which in addition to signals described above for 2 showed a broad quintet at δ 1.53 and a broad sextet at δ 1.30, together with the signals at δ 64.9 (C-1''), 30.4 (C-2''), 19.1 (C-3'') and 13.7 (C-4'') in the 13 C NMR, indicated the alcohol portion to be n-butyl.Moreover, the chemical shifts of n-butyl carbons were comparable with those reported for O-alkyl group in n-butyl ethanoate (δ 63.1, 30.4,18.6 and 12.7). 17In addition, compound 3 did not give a significant fragment at m/z 57 as in 1, the ion observed being m/z 117 for [CH 3 COOCH 2 CH 2 CH 2 CH 3 + H] + .These data were consistent with the structure of 9,10-dihydro-9-hydroxy-9-(n-butoxycarbonylmethyl)-10-oxophenanthrene for 3.
This appears to be the first record of phenanthrenes from Meliaceae or from the allied families of the order Rutales (Rutaceae, Meliaceae, Simaroubaceae and Cneoraceae).In addition, these compounds represent a novel group of unsubstituted phenanthrenes.Oxidation involving phenyl rings, as hydroxyl and methoxyl substituents, has been found in Orchidaceae. 13It can of course be argued that the roots were collected with a trace of other roots which do not belong to T. ciliata.However, subsequent to this work we ourselves have found these compounds in stock of a 5-year-old tree of C. odorata grafted on T. ciliata. 19Indeed the sporadic occurrence of particular micromolecular types in unrelated taxa is a general phenomenon. 20
Plant material.T. ciliata var.australis was collected in Viçosa, M.G., Brazil, and a voucher is deposited in the Herbarium of the Departamento de Engenharia Florestal, Universidade Federal de Viçosa, Viçosa, M.G.

Table 2 .
13C NMR chemical shift for compounds 1-5 Assignments based on HMQC and HMBC for 1 and DEPT 135 for 2-4 Compound 2 exhibited similar spectra data to 1 (Table