Abietane Diterpenes from Hyptis crassifolia Mart . ex Benth . ( Lamiaceae )

O estudo fitoquímico do extrato etanólico das raízes de Hyptis crassifolia Mart. ex Benth. (Lamiaceae) levou ao isolamento e elucidação estrutural de nove diterpenos identificados como 11,12,15-tri-hidroxi-8,11,13-abietatrien-7-ona, 6α,11,12,15-tetra-hidroxi-8,11,13-abietatrien7-ona, 11,12,16-tri-hidroxi-17(15→16)-abeo-abieta-8,11,13-trien-7-ona, (16S)-12,16-epoxi11,14-di-hidroxi-17(15→16)-abeo-abieta-8,11,13-trien-7-ona, incanona, ferruginol, sugiol, óxido de 11-oxomanoíla e óxido de 11β-hidroximanoíla. Os compostos 11,12,16-tri-hidroxi17(15→16)-abeo-abieta-8,11,13-trien-7-ona e 6α,11,12,15-tetra-hidroxi-8,11,13-abietatrien-7-ona são inéditos na literatura, a 11,12,15-tri-hidroxi-8,11,13-abietatrien-7-ona está sendo relatada pela primeira vez como um novo diterpeno abietano natural, enquanto que para a (16S)-12,16-epoxi11,14-di-hidroxi-17(15→16)-abeo-abieta-8,11,13-trien-7-ona, isolada anteriormente de Teucrium divaricatum Subsp. villosum, propõe-se uma revisão dos dados de ressonância magnética nuclear (NMR) H e C. A determinação estrutural de todos os constituintes foi realizada através de técnicas espectroscópicas como espectrometria de massas de alta resolução (HRMS), infravermelho (IR), NMR de H e C, incluindo sequências de pulsos uni e bidimensionais, e comparação com dados descritos na literatura.


Introduction
Lamiaceae consists of about 258 genera and 6970 species distributed around the world, 1 mainly centered in the Mediterranean region despite reports of their occurrence in Australia and South America. 2 The Hyptis genus includes around 400 species distributed throughout the Americas, West Africa, Fiji Island (Oceania), and western India. 3hey are found in Northern and Northeastern Brazil, especially in the cerrado. 4Plants from this genus are used in folk medicine, especially in the treatment of fever, colds, asthma, headache, cramps, gastrointestinal infections, rheumatism, skin diseases and malaria, and also have antibacterial, antifungal and insect repellent properties. 2 Vol.26, No. 1, 2015   Previous studies on the chemical constitution of Hyptis species report the isolation of triterpenoids, 5 flavonoids, 6,7 lignans, 7,8 α-pyrone derivatives 9,10 and diterpenoids, [11][12][13][14] namely labdanes and abietanes.The abietane diterpenes have attracted particular attention, due to their biological activities, in particular as antioxidant, [15][16][17] antibacterial, [18][19][20] cytotoxic, [21][22][23][24] antiviral 25,26 and antimalarial. 27Among the diterpenes that exhibit these activities, were observed several highly oxygenated compounds that can present either the C-ring saturated or unsaturated, aromatic, and also with an ortho or para-naphtoquinone pattern.Previous works carried out on plants of the genus Hyptis from the Northeast of Brazil flora, reported the isolation of many abietane diterpenes, for example those isolated from roots of H. martiusii, 11 H. platanifolia 12 and H. carvalhoi. 28n this paper, it is reported the phytochemical analysis of the ethanol extract from roots of Hyptis crassifolia Mart.ex Benth., a small shrub that grows in abundance in the state of Bahia to which no phytochemical investigation has been reported so far.This study led to the isolation and structure elucidation of nine diterpenes: four abietanes (1, 2, 6 and 7), three rearranged abietanes (3-5) and two labdanes (8 and 9).Compounds 2 and 3 have not yet being reported in the literature, whereas 1 is being reported for the first time from a natural source.A revision of the previously published nuclear magnetic resonance (NMR) data in the literature 29 is being proposed for compound 4.

General procedures
Melting points were measured on a digital Marconi MA-381 apparatus and are uncorrected.Optical rotations [α] D 20 were determined with a Jasco P-2000 digital polarimeter, operating with tungsten lamp at a wavelength of 589 nm at 20 °C.Infrared (IR) spectra were recorded using a Perkin-Elmer FTIR 100 spectrometer using the universal attenuated total reflectance accessory (UATR).High-resolution electrospray ionization mass spectra (HR-ESI-MS) were performed with a SHIMADZU LCMS-IT-TOF (225-07100-34) equipped with a Z-spray ESI (electrospray) source operating either in the negative or positive mode. 1 H and 13 C NMR (1D and 2D) spectra were performed on Bruker Avance DPX-300 and/or DRX-500 spectrometers equipped with 5 mm direct probe or inverse detection Z-gradient probe, respectively. 1H NMR (300.13 and 500.13MHz) and 13 C NMR (75.47 and 125.75 MHz) spectra were measured at 25 °C.Chemical shifts (d), expressed in parts per million (ppm), are referenced by the signal of the residual non-deuterated hydrogens ( 1 H NMR) and the central peak of carbon-13 ( 13 C NMR) of the deuterated solvents.Flash column chromatography and column chromatography (CC) were carried out on silica gel 60 A (Whatman, 70-230 mesh) and silica gel 60 A (Acros Organic, 0.035-0.070mm), respectively.Thin layer chromatography (TLC) was performed on precoated silica gel polyester sheets (Kieselgel 60 F 254 , 0.20 mm, Merck) by detection with a spray reagent of vanillin/perchloric acid/EtOH solution followed by heating at 100 °C.Normal phase semi-preparative HPLC separations were performed with a Shimadzu LC-20AT pump, UV-PDA (SPD-M20A) detector and WATERS-1525 pump, UV-PDA (WATERS 2996) detector, a Phenomenex Silica column (10 × 150 mm, 5 µm particle size), with a flow rate of 4.72 mL min −1 and column oven temperature 40 °C, monitoring at 273.8 nm.

Plant material
The roots of Hyptis crassifolia Mart.ex Benth.were collected in July 2010, at Mucujê county (Diamantina Plateau, Bahia State).The plant material was identified by Prof Maria Lenise Silva Guedes of Instituto de Biologia, Departamento de Botânica, Universidade Federal da Bahia.A voucher specimen (No. 95.183) is deposited in the Herbário Alexandre Leal Costa of the Departamento de Botânica, Universidade Federal da Bahia.

Results and Discussion
The EtOH extract from roots of Hyptis crassifolia Mart.ex Benth.was fractionated by silica gel column chromatography after elution with pure or binary mixtures of hexane, CH 2 Cl 2 , EtOAc and MeOH.The hexane-CH 2 Cl 2 (1:1) and CH 2 Cl 2 fractions were subjected to various chromatographic procedures leading to the isolation of nine compounds (1-9, Figure 1), whose structures were elucidated by spectroscopic methods, such as IR, high resolution mass spectrometry (HRMS) and particularly, 1 H and 13 C NMR (1D and 2D).
Compound 1 was obtained as a yellowish resin.The IR spectrum exhibited absorption bands for hydroxyl group at 3385 cm −1 , Csp 3 2).Furthermore, the pattern of substitution of the pentasubstituted benzene ring Vol. 26, No. 1, 2015   was determined by correlations of the benzene proton at d H 7.49 (H-14) with the oxygenated carbon at d C 76.9 (C-15, 3 J), with the sp 2 carbons at d C 147.6 (C-12, 3 J) and 139.4 (C-9, 3 J) and with the carbonyl group at d C 199.4 (C-7, 3 J) (Figure 2).Then, compound 1 was characterized as the 11,12,15-trihydroxy-8,11,13-abietatrien-7-one, a diterpene previously synthesized during the structural determination of callicarpone by Kawazu et al., 34 but that is being reported for the first time in the literature as a new natural abietane.
Compound 2 was also obtained as a yellowish resin.The molecular formula C 20 H 28        The other expected changes were the shielding of C-13 (d C 122.9) due to the replacement of the deshielding β-hydroxy and β-methyl effect on compound 1, for the shielding γ-effect of both groups on compound 3.The release of the crowding steric hindrance of the branched side chain on compound 1 through the rearrangement for a normal chain on compound 3 also affects the chemical shift of C-14 (d C 122.4), now with a remarkable deshielding effect (∆d C = 5.9).The spectral data discussed above are consistent with a 17(15→16)-abeoabietane diterpenoid. 32,35The skeleton of 3 was confirmed through a detailed analysis of the HMBC spectrum by long-range correlations of the aromatic proton at d H 7.43 (H-14) with the carbons at d C 148.1 (C-12, 3 J), 139.2 (C-9, 3 J) and 40.2 (C-15, 3 J), and with the carbonyl carbon in d C 199.7 (C-7, 3 J) (Figure 2).Thus, compound 3 was identified as a new 17(15→16)-abeo-abietane diterpene, the 11,12,16-trihydroxy-17(15→16)-abeo-abieta-8,11,13trien-7-one.
Compound  1).Its HR-ESI-MS quasi-molecular ion at 347.1879 [M − H] − , 18 Da higher than that of compound 4, suggested that compound 4 could be a dehydrated derivative.The IR spectrum of 4 exhibited absorption bands for hydroxyl groups at 3382 cm −1 and a conjugated ketone carbonyl group at 1639 cm −1 .The 13 C CPD NMR spectrum exhibited 20 signals, that after the 1 H, 13 C-HSQC spectrum analysis allowed to determine the presence of an aryl ketone carbonyl, chelated to the hydroxyl at C-14, at d C 206.4, six sp 2 carbons at the region of d C 111.3-158.9,one very deshielded oxymethine carbon at d C 83.9, and 12 sp 3 saturated carbons (d C 18.2-51.9)(Table 1).Analysis of the 1 H NMR spectrum did not show any aromatic proton, but three singlets of methyl groups attached to quartenary carbons at d H 0.96 (Me-18), 0.99 (Me-19) and 1.39 (Me-20), and a methyl doublet at d H 1.48 (d, J 6.2 Hz, Me-17).In addition, an oxymethine proton at d H 5.08 (bq, J 6.9 Hz, H-16) and a diastereotopic methylene at d H 3.27 (H-15a); 2.75 (dd, J 15.3, 7.3 Hz, H-15b) have been observed.The main difference was the deshielding of C-16 (d C 69.9 in 5) to d C 83.9 due the formation of an α-methyldihydrofuran ring condensed with the fully substituted benzene ring at the positions C-12 and C-13.The appearance of a chelated hydroxy at d H 13.44 in the 1 H NMR spectrum (CDCl 3 ) of compound 4 (see Supplementary Information (SI) section) evidenced that the ring closure has been done through the C-12 hydroxy.The substitution pattern of the C aromatic ring was definitively established from the HMBC analysis by long-range correlations of the methylene protons at d H 3.27 (H-15a) and 2.75 (H-15b) with the carbons at d C 158.9 (C-12, 3 J), 112.2 (C-13, 2 J) and 22.3 (C-17, 3 J).In addition, the correlations of the methylene protons at d H 2.63-2.51(H-6) with the carbons at d C 34.5 (C-4, 3 J), 206.4 (C-7, 2 J), 111.3 (C-8, 3 J) and 42.2 (C-10, 3 J) (Figure 2) were also observed.To the C-16 stereogenic center was attributed the same relative stereochemistry as that in either teuvincenone A or E (αH, βMe), taking in consideration the similar chemical shifts and coupling constant values of the 2H-15, H-16 and Me-17 protons, 35 as well as the carbon-13 chemical shifts of the dihydrobenzofuran system (C-8 to C-17) (see Table 1).This, undoubtedly, indicated for compound 4 the structure of the new rearranged abietane diterpene, (16S)-12,16-epoxy-11,14-dihydroxy-17(15→16)-abeo-abieta-8,11,13-trien-7-one.Comparison of the NMR data of 4, with those published for villosin A, a compound previously isolated from Teucrium divaricatum Subsp.villosum by Ulubelen et al. 29 to which the structure suggested is the same as the one proposed for 4, did not show a good matching.For instance, the carbonyl chemical shift at d C 185.6 is not compatible with the C-7 aryl ketone carbonyl of compound 4 (d C 206.4), once other aryl ketones described in the literature have carbon ressonances of approximately d C 205.0. 32,36,37It appears that the carbonyl chemical shift at d C 185.6 previously reported, 29 is more compatible with a cross conjugated aryl ketone (ca.d C 188.0) 36 or α-hydroxy cross conjugated aryl ketones (ca.d C 183.0). 29,36In addition, several other inconsistencies can be pointed out, for example, the 13 29 Thus, the NMR data assignment previously published for villosin A should be revised, or an alternative structure should be considered.According to the analysis described above, the structure of 4 was assigned as (16S)-12,16-epoxy-11,14dihydroxy-17(15→16)-abeo-abieta-8,11,13-trien-7-one.

Conclusions
The unprecedent phytochemical analysis of Hyptis crassifolia, a herb wildly dispersed through the neighborhood of Mucujê, a small town at Chapada Diamantina, BA, Brazil, has led to the isolation of four abietane diterpenes, two of which are new, 1 and 2. This is in agreement with the chemotaxonomic profile of the genus Hyptis already reported in the literature.On the other hand, rearranged abietanes, two of which are new (3 and 4), are being reported for the first time from Hyptis.The known labdane diterpenes 8 and 9, isolated from other genera like Salvia 33 and Kyllinga, 40 have not been reported from Hyptis previously.In addition, the NMR data of the rearranged abietane 4, previously identified as villosin A, 29 was reassigned.
O 5 was determined by HR-ESI-MS, based on the quasi-molecular ion at m/z 349.2007 [M + H] + (calcd.for C 20 H 29 O 5 m/z 349.2010).The IR spectrum showed stretching bands consistent with the
H 1.70 (Me-16) and 1.68 (Me-17) correspondent to the geminal methyls of the benzene side chain of 1 are missing, while a doublet for a primary methyl C-17 [d C 23.5; d H 1.29 (d, J 6.2 Hz)] appears on the 1 H NMR. This information is in agreement with a rearrangement of the aromatic side chain of compound 1.The 13 C NMR data is totally in accordance with the suggested change, appearing the extra methylene (C-15) at d C 40.2, the oxymethine (C-16) at d C 71.3, and the methyl (C-17) at d C 23.5.

Table 1 )
-H groups at 2927 and 2865 cm −1 , skeletal bands of benzene ring at 1609 and 1564 cm −1 , conjugated C=O at 1669 cm −1 and C-O of phenol and alcohols at 1254 and 1145 cm −1 , respectively.The molecular formula C 20 H 28 O 4 was determined by HR-ESI-MS, based on the quasi-molecular ion at m/z 333.2062 [M + H] + (calcd.for C 20 H 29 O 4 m/z 333.2060).The CPD and DEPT 135° .The 1 H NMR spectrum showed five characteristic singlets of methyl groups attached to non hydrogenated carbons at d H 0.95 (Me-18), 0.98 (Me-19), 1.40 (Me-20), 1.68 (Me-17) and 1.70 (Me-16).A signal at d H 7.49 (H-14) was ascribed to one proton of a pentasubstituted benzene ring.In addition, a pair of double doublets at d H 2.53 (J 17.0, 14.4 Hz, H-6b) and 2.63 (J 17.0, 2.8 Hz, H-6a) were observed, corresponding to one methylene group coupled to a methine at d H 1.85 (dd, J 2.8, 14.4 Hz, H-5).The presence of a hydroxyl group at C-15 was accomplished by the HMBC spectrum analysis, showing long-range correlations of the methyl groups at d H 1.70 (Me-16) and 1.68 (Me-17) with the oxygenated carbon at d C 76.9 (C-15, 2 J) (Figure

Table 1 .
13 and13C NMR data (d in ppm, J in Hz) for compounds 1-5 13H groups at 2928 and 2869 cm −1 , and as for compound 1, a conjugated aryl ketone group at 1675, 1611 and 1566 cm −1 .Stretching bands at 1258 and 1080 cm −1 were consistent with the presence of C-O group of phenol and alcohols, respectively.Comparative analysis of 1 H and13C NMR data of the compounds 1 and 2 revealed several similarities.The major diferences in the13C NMR spectrum were the disappearance of a methylene at d C 35.8 for 1 and the appearance of an oxymethine at d C 73.4 for 2. The deshielding of C-5 (d C 50.5 in 1) to d C 55.6 and the shielding of C-8 (d C 124.6 in 1) to d C 121.6 are in accordance with β and γ effects, respectivelly, of the hydroxyl positioned at C-6.The stereochemistry of the hydroxyl at C-6 was assigned as α-equatorial on the basis of the coupling constants of H-5 [d H 1.81 (d, J 13.5 Hz)] and H-6 [d H 4.60 (d, J 13.5 Hz)], a typical axial-axial hydrogen coupling of cyclohexane rings.The changes in the 1 H NMR spectrum of 2, relatively to 1, are all in agreement with the above discussion.The axial oxymethine hydrogen attached to the α-carbon to the carbonyl appeared at d H 4.60 (d, J 13.5 Hz), while all methyls attached to quaternary carbons underwent a light deshielding.Accordingly with the afore mentioned spectral data, the structure of 2 was characterized as the new diterpene 6α,11,12,15-tetrahydroxy-8,11,13-abietatrien-7-one.Compound3was obtained as a yellow resin.The molecular formula C 20 H 28 O 4 was determined by HR-ESI-MS, based on the quasi-molecular ion at m/z 333.2064 [M + H] + (calcd.for C 20 H 29 O 4 m/z 333.2060).The NMR data of compound 3 showed remarkable similarities with those of compound 1 (see Table 1).The major diferences account for an oxymethine at C-16 (d C 71.3; d H 4.31 m) and an extra diastereotopic methylene at C-15 [d C 40.2; d H 2.90 (dd, J 1.9, 14.8 Hz, H-15a) and d H 2.78 (dd, J 7.4, 14.8 Hz, H-15b)].The singlets at d 4 [α] D 20 +26.79º (c 0.24, CHCl 3 ) was isolated as a yellow solid (mp 195.7-197.7 °C).The molecular formula, C 20 H 26 O 4 , was established after HR-ESI-MS analysis based on the quasi-molecular ion at m/z 331.1889 [M + H] + (calcd.C 20 H 27 O 4 m/z 331.1909).Compound 5, already known, showed very similar spectroscopic data to those of compound 4 (see Table