Abietane Diterpenes from Hyptis platanifolia

Das raízes de Hyptis platanifolia foram isolados três novos diterpenos abietanos 19-oxoinuroyleanol, 11,14-di-hidroxi-12-metoxi-7-oxo-8,11,13-abietatrien-19,20β-olídeo and 19,20-epoxi12-metoxi-11,14,19-tri-hidroxi-7-oxo-8,11,13-abietatrieno, em adição ao inuroyleanol e a coulterona. Investigação das partes aéreas forneceu a mistura de esteróides estigmasterol e β-sitosterol, e os triterpenos ácido betulínico e ácido ursólico. A caracterização estrutural dos compostos foi estabelecida com base em métodos espectroscópicos, principalmente RMN uni e bidimensional, e comparação com dados da literatura.


Introduction
3][4][5][6][7] Despite their reported use for medicinal purposes, a literature survey revealed that the phytochemical analysis of plants belonging to the Hyptis genus have been limited to a few species.
During the course of previous work on Brazilian Hyptis species, we have reported the isolation and cytotoxic activity of abietane diterpenoids from roots of Hyptis martiusii. 8In continuation to the investigation of plants from this genus, we present now the phytochemical study of Hyptis platanifolia Mart.ex Benth, a small shrub that grows in abundance in the Northeast of Brazil.From the roots extract three new abietane diterpenoids were isolated 1-3, in addition to the known diterpenes inuroyleanol 4 and coulterone 5. 9 From the aerial parts the triterpenes betulinic 6 and ursolic acids 7 10 and a mixture of the steroids stigmasterol 8 and β-sitosterol 9, 11 were characterized.
Comparative analysis of BB and DEPT- 13 C NMR spectra (Table 1) revealed 21 lines, in agreement with the C 21 H 28 O 5 suggested molecular formula from the EIMS data (m/z [M •+ ] 360).From this analysis, one can easily deduce the presence of two carbonyls (δ 205.0 and 204.5), one of which of an aldehyde, five sp 3 methyl carbons (one methoxyl and four non functionalized methyls), four methylenes, two methine, and eight non-protonated carbons, six of which were unsaturated.
The substitution pattern of the fully substituted C aromatic ring was established from the HMBC analysis (Table 1).The suggestion of the location of one hydroxyl group at C-11 was determined by correlations of the OH hydrogen at δ 5.76 (br s) with the carbons at δ 152.7 (C-12, 3 J), 139.1 (C-11, 2 J) and 134.1(C-9, 3 J).The deshielded resonance of one hydrogen at δ 3.38 of the methylene group at C-1, allowed its assignment to the C-1β equatorial hydrogen as already observed for abietane derivatives having a C-11 phenol group on ring C. 13 Long-range correlations between the chelated hydroxyl group at δ 13.30 with the carbons at δ 159.0 (C-14, 2 J), 127.1).The equatorial orientation of the aldehyde group at C-4, was based on the chemical shift of the aldehyde hydrogen at the δ 9.26 in the 1 H NMR, whereas an axial substitution would take that hydrogen to an usual proton range, lower than δ 9.7, in agreement with a previous report by Bohlmann et al. 14 The above information and comparison of the 13 C NMR data found to compound 1 with to those published for inuroyleanol 4 and 20-oxo-inuroyleanol 10, isolated from Salvia coulteri (Labiatae), 9 allowed to establish its structure as the new abietane diterpene 19-oxo-inuroyleanol.Compound 2 was obtained as an yellow solid with mp 248-250 o C. The molecular formula C 21 H 26 O 6 was established by EIMS (m/z [M •+ ] 374).The IR spectrum showed the presence of hydroxyls (3351 cm -1 ) and two carbonyl groups relative to an ester (1735 cm -1 ) and a conjugated ketone (1618 cm -1 ).Analysis in the 13 C NMR spectrum of 2 were similar to those observed for compound 1 (Table 2), except for the absence of the signals relative to the aldehyde and a methyl carbon and the presence of an extra oxygenated methylene at δ 72.8.The 1 H NMR spectrum corroborated with these observations, through the absence of the singlets relative to both methyl and aldehyde hydrogens, besides the presence of additional signals for an AB system at δ 5.03 (d, J 12.5, H-20 pro-S) and 4.59 (dd, J 12.5, 2.0 Hz, H-20, pro-R).Significant long-range correlations in the HMBC spectrum were observed between the hydrogen of the AB system at δ 5.03 (H-20 pro-S) with the carbons at δ 175.6 (C-19, 3 J), 46.7 (C-5, 3 J) and 33.8 (C-10, 2 J), while the other at δ 4.59 (H-20 pro-R) showed correlations with the carbons at δ 175.6 (C-19, 3 J), 35.1 (C-1, 3 J) and 33.8 (C-10, 2 J) (Figure 2).The above spectral informations suggested the presence of a δ-lactone moiety for compound 2, with the ring closure through the oxygen between C-19 and C-20.The relative stereochemistry was defined by the small value of the coupling constant (J 2.0 Hz) observed for the B part of the AB system at δ 4.59, that was ascribed to the long range coupling between the H-20 pro-S with the hydrogen H-1α, only possible in structures where the ethereal oxygen C-19 and C-20 is oriented towards C-2. 15These data suggested the final structure of compound 2 as the new abietane 11,14-dihydroxy-12-methoxy-7-oxo-8,11,13-abietatrien-19,20β-olide.Compound 3 was obtained as yellow crystals, mp 175.0-181.0o C. The IR spectrum showed bands relative to hydroxyls (3411 cm -1 ) and one conjugated carbonyl group (1621 cm -1 ).The 1 H NMR spectrum displayed duplicated signals for the majority of the spin systems with a molar ratio 3:1, what indicated a close relationship with those observed for compound 2. A slight difference was found by the presence of extra singlets at δ 4.88 and 4.91 suggesting a diastereoisomeric mixture.The proposition that the δlactone moiety of compound 2 was reduced on 3 was suggested by comparison of their 13 C NMR data, since the carbonyl group of δ-lactone of the compound 2 (δ 175.6) was substituted by the hemiaketal methine carbons at δ 98.4 and 99.2 (C-19) on 3, and by the respective 3 J longrange conectivities in the HMBC spectrum between the H-19 hemiaketal hydrogens of both epimers at δ 4.91 and 4.88 with the C-5 carbons at δ 47.7 and 44.4, and C-20 carbons at δ 66.4 and 60.1, respectively.The stereochemistry at C-19 to the two isomers was proposed by the chemical shifts observed for the AB part of the ABX systems.The double doublets at δ 4.17 and 4.02 were ascribed to both H-20 pro-S signals.The more deshielded signal at δ 4.17, relative to the most abundant isomer, was related to the compound which contains the hydroxyl group at C-19 in a 1-3 diaxial relationship with the H-20 pro-S, as observed in the structure of the abietane diterpenoid 19-dihydro-deacetyl sesseine 11, also isolated as an diastereoisomeric mixture from Salvia candicans (Labiatae). 15Thus, compound 3 was determined as the diastereoisomeric mixture of the new diterpene 19,20epoxy-12-methoxy-11,14,19-trihydroxy-7-oxo-8,11,13abietatriene.
Abietane diterpenes are widespread in the Labiatae family, and well represented in the genus Hyptis through the species H. martiusii, 8 H. suaveolens 16 and H. dilatata. 17n the other hand, icetexane skeletons have been isolated only in the Labiatae genus Salvia, Coleus and Plectranthus. 9,15,18The co-occurrence of coulterone 5 and inuroyleanol 4 in Hyptis martiusii, confirmed the suggestion that icetexane diterpenes has a rearranged abietane skeleton, where the C-20 Me group was included in the ring B by migration of the C 9 -C-10 bond into the C 9 -C 20 position, as previously observed by Kelecom in Coleus barbatus. 18

General procedures
Melting point were obtained on a Mettler FP82HT and are uncorrected.IR spectra were recorded using a Perkin Elmer 1000 spectrophotometer.Optical rotations were measured on a Perkin Elmer 341 polarimeter.The mass spectra were obtained on a Hewlett-Packard 5971 mass spectrometer by electron impact ionization (70 eV). 1 H and 13 C NMR where recorded on a Bruker Avance DRX-500; chemical shifts were given in δ (ppm) relative to TMS as an internal standard.All compounds were visualized on TLC using the vanillin-sulfuric acid reagent.

Plant material
Roots and aerial parts of Hyptis platanifolia Mart ex Benth were collected in september 2002, at the flowering stage, from plant populations growing wild in Crato County, Ceará State, Northeast of Brazil.A voucher specimen (n o 31674) has been identified by Dr. Edson Paula Nunes and deposited at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Brazil.

Extraction and isolation
Roots (2470 g) and aerial parts (330 g) of H. platanifolia were separately pulverized and extracted with hexane at room temperature.The solvent was removed under reduced pressure to give the correspondent extracts.Both residues obtained after hexane extraction were extracted with EtOH.
Chromatography on a Si gel column of the hexane extract from the roots (11.6 g) by elution with hexane, CHCl 3 , EtOAc and MeOH yielded four fractions.The hexane fraction yielded small crystals, which were collected by removal of the supernatant liquid and recrystallized from MeOH yielding inuroyleanol 4 (8.0 mg).Sucessive flash chromatography of the CHCl 3 fraction using CH 2 Cl 2 :EtOAc as a binary mixture with increasing polarity, yielded compounds 2 (17.0 mg) and 3 (12.0mg).The EtOH extract (19.0 g) was submitted to partition with hexane, CH 2 Cl 2 , EtOAc and MeOH to give four fractions.The CH 2 Cl 2 fraction (4.6 g) was submitted to the flash chromatography using hexane:EtOAc(90:10) as the isocratic eluting mixture, to afford compound 1 (7.0 mg) and coulterone 5 (8.0 mg).