Hirtinone, a Novel cycloartane-type triterpene and other compounds from Trichilia hirta L. (Meliaceae).

One novel triterpene cycloartane-type, named hirtinone (1), six protolimonoids - nilocitin (2), dihydronilocitin B (3), melianone epimers (4) and (5), piscidinol A (6) and melianone lactone (7), one tertranortriterpenoid, hirtin (8), and one sesquiterpene, spathulenol (9), were identified in the fruits of Trichilia hirta. The structures were established by 1D and 2D NMR (1H and 13C-NMR, DEPTQ, 1H-1H-COSY, 1H-1H-NOESY, HSQC and HMBC), high resolution mass spectroscopy (HR-ESI-MS) and infrared (IR) spectral data.

1. Introduction

The Meliaceae family has attracted such a great interest among phytochemists interested in bioproduction because of its very complex and diverse chemical structures and its biological activity, mainly against insects [1,2,3,4]. The Trichilia genus (Meliaceae) consists of about 230 species distributed throughout tropical America, which are recognized for their significant economic importance and high commercial value. Phytochemical studies have revealed that this genus is rich in terpenoids, including triterpenes, limonoids, steroids and other terpene derivatives [3,4,5,6]. Species of this genus have been also studied for their insecticidal activities and their isolated compounds revealed complex and interesting structures, including various limonoids [5,7,8]. The isolation and structural elucidation of the two novel limonoids from the fruits of T. hirta collected in Espírito Santo State, Brazil, was reported by Cortez et al. in 1992 [9].

In the present paper, we report an investigation of a hexane extract of fruits of a T. hirta specimen, which allowed us to characterize nine terpenoids, including a novel cycloartane-type triterpene named hirtinone (1), five protolimonoids: nilocitin (2) [10,11], dihydronilocitin B [10,11] (3), melianone epimers (4) and (5) [11,12], piscidinol A (6) [10,11,13] and melianone lactone (7) [11,12], the tertranortriterpenoid hirtin (8) [14] and the sequiterpene spathulenol (9) [15]. The structures were established by spectrometric techniques, mainly HRESIMS and 1D and 2D NMR, and comparative analysis with literature values. The structures of all the isolated compounds are shown in Figure 1.

Figure 1

Chemical structure of the compounds isolated from the fruits of T. hirta.

2. Results and Discussion

The crude hexane extract of T. hirta fruits was submitted to chromatography and furnished nine terpenoids 1–9. The known terpenoids, i.e., the six protolimonoids nilocitin (2), dihydronilocitin B (3), melianone epimers (4) and (5), piscidinol A (6) and melianone lactone (7), the tertranortriterpenoid, hirtin (8) and the sequiterpene spathulenol (9) were identified on the basis of 1H-and 13C-NMR spectral data, including 1H-1H-COSY, 1H-1H-NOESY, HSQC and HMBC NMR experiments, which were also used to complete and unambiguous 1H and 13C chemical shift assignments [16,17].

Hirtinone (1), m.p. 160–162 °C, was isolated in an amorphous form. The IR spectrum showed bands at νmax 1,713, characteristic of C=O stretching of a ketone carbonyl group, as well as absorptions at νmax 3,354, 2,925-2,852 and 1,089 cm−1, characteristics of OH , C-H and C-O stretching, respectively.

The HR-ESI-MS spectrum of 1 utilizing the ESI+ ionization mode showed a cationic base peak at m/z 479.3485 [M+Na]+, compatible with the molecular formula C30H48O3Na (calc. m/z 479.3501, Δ = 0.0016). These data and the comparative analysis of the {1H}- and DEPTQ-13C NMR spectra, that allowed us to identify signals (Table 1) corresponding to seven non-hydrogenated [including one sp2 of carbonyl group at δC 216.6 (C-3) and one sp3 oxygenated at δC 71.6 (C-25)], five methine [all sp3 including one oxygenated at δC 84.1 (CH-24)], twelve methylene [all sp3 including one oxygenated at δC 72.8 (CH2-21)] and six methyl carbon atoms, were used to propose the molecular formula C30H48O3 = (C=O)(C-O)(C)5(O-CH)(CH)4(O-CH2)(CH2)11(CH3)6, seven unsaturation degrees (one carbonyl group in a triterpenoid with a cycloartane skeleton). The fragments postulated (Scheme 1) to justify the principal peaks observed in the HR-ESI-MS are compatible with a carbon skeleton of the cycloartane-triterpene type, including the presence of the cationized adduct as a complex involving two molecules of 1.

Table 1

1H- (500 MHz) and 13C- (125 MHz) NMR of hirtinone (1), including results obtained by heteronuclear 2D shift-correlated HSQC and HMBC, in CDCl3 as solvent and TMS used as internal reference. Chemical shifts (δ, ppm) and coupling constants (J, Hz, in parenthesis) *.

		HSQC		HMBC
Atom	Type	δC	δH	2JH→C	3JH→C
1	CH2	33.4	1.87 (m), 1.58 (m)		2H-19
2	CH2	37.5	2.72 (dt, 6.4; 13.9)
			2.34 (ddd; 1.7, 6.4, 13.9)
3	C	216.6	-	H-2a	3H-28; 3H-29; H-5
4	C	50.7	-	3H-28; 3H-29
5	CH	48.6	1.73 (m)		2H-19; 3H-28; 3H-29
6	CH2	26.4	2.06 (m), 1.15 (m)
7	CH2	21.5	1.60 (m), 1.05 (m)
8	CH	49.4	1.54 (m)		3H-19; 3H-30
9	C	21.5	-	2H-19
10	C	26.1	-	2H-19
11	CH2	26.1	1.80 (m), 1.35 (m)		2H-19
12	CH2	35.5	1.45 (m), 1.30 (m)		3H-18
13	C	45.2	-	3H-18	3H-30
14	C	48.6	-	3H-30	3H-18
15	CH2	32.2	1.60 (m)		3H-30
16	CH2	26.6	1.40 (m), 1.15 (m)
17	CH	47.9	1.62 (m)		3H-18
18	CH3	18.5	0.98 (s)
19	CH2	29.6	0.82 (dl, 4.1), 0.62 (d, 4.1)
20	CH	39.2	1.55 (m)
21	CH2	72.8	4.21 (dl, 11.6), 3.06 (t, 11.6)
22	CH2	30.1	1.95 (m), 1.10 (m)
23	CH2	25.9	1.55 (m), 1.38 (m)
24	CH	84.1	3.03 (dd, 11.4, 2.0)		3H-26; 3H-27
25	C	71.6	-	3H-26; 3H-27
26	CH3	26.1	1.19 (s)		3H-22
27	CH3	24.0	1.16 (s)		3H-26
28	CH3	20.8	1.12 (s)		3H-24
29	CH3	19.2	1.07 (s)		3H-28
30	CH3	22.2	1.08 (s)

* Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPTQ-13C NMR spectra. Chemical shifts and coupling constants (J) obtained from 1D 1H-NMR spectrum. Superimposed 1H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC and 1H-1H-COSY spectra.

Scheme 1

Proposed fragmentation mechanisms of 1 (only peaks classified as principal ones, with intensity of the peaks (%) in parentheses).

The 1H-NMR spectra (1D and 1H-1H-COSY, Table 1) of 1, exhibiting a cycloartane triterpene profile, showed two doublets at δH 0.82 (J = 4.1 Hz) and δH 0.62 (J = 4.1 Hz), which were characteristic for a C-9/C-10 cyclopropyl methylene and singlet signals at δH 0.98, 1.19, 1.16, 1.12, 1.08, 1.07 corresponding to six methyl groups. The presence of the signals at δH (2.72 dt, 6.4 and J = 13.9 Hz) and 2.34 (ddd, 1.7, 6.4 and J = 13.9 Hz), correlated with the 13C signal of a methylene carbon at δC 37.5 (CH2-2) in the HSQC spectrum indicated the presence of carbonyl group at carbon C-3, characteristic of a cycloartan-3-one triterpenoid [18,19], which was confirmed by the HMBC spectrum (Table 1) with interactions between C-3 and the 3H-28, 3H-29 and H-5 (3JCH) as well as H-2 (2JH→C).

The signals at δH 4.21 (dl, 11.6 Hz, H-21eq), 3.06 (t, 11.6 Hz, H-21ax) and 3.03 (dd, 11.4 and 2.0 Hz, H-24) were attributed to hydrogen atoms of oxymethylene (CH2-21) and oxymethine (CH-24), respectively, characteristic of the tetrahydropyran ring present in protolimonoid triterpenes isolated from Trichilia species [8,20]. The absorptions at 1713 cm−1 (carbonyl group), 1089 cm−1 (carbon-oxygen bond) and 3354 cm−1 (OH) observed in the IR spectrum corroborate the NMR data. The coupling constant values J = 11.6 Hz and J = 11.4 Hz observed in the signals of hydrogens H-21ax (triplet at δH 3.06) and H-24 (dd at δH 3.03) indicated an axial-axial interaction and, consequently, were used to define the axial positions of these H-20 and H-24 [20]. The analysis of the HSQC (1JH→C) experiment support the 1H- and 13C-NMR data and led to assignment of six methyl signals at δC/δH 18.5/1.07(s), 26.1/1.19(s), 24.0/1.16(s), 20.8/1.12(s), 19.2/0.98(s) and 22.2/1.08(s)] as well as signals of methylene OCH2-21 [δC/δH 72.8/4.21 and 3.06), methine OCH-24 (δC/δH 84.1/3.03) and methylene CH2-19 (δC/δH 29.6/0.82 and 0.62), which are compatible with the presence of tetrahydropyran ring [20] and cyclopropane ring in the compound 1 [18,19]. The presence of the cyclopropane ring was confirmed by HMBC spectrum analyses which showed cross-peaks 2H-19/C-9, 2H-19/C-10. The cross-peak at 2JH→C 3H-26/C-25 and 3H-27/C-25 supporting the proposal of hydroxyl group observed in the IR spectrum (Table 1).

The relative stereochemistry of 1 was determined from the coupling constants of relevant hydrogens, from the observed 1H-1H-NOESY and from the comparison with data of analogous compounds with configuration described in the literature [20]. The relative stereochemistry of carbon atoms CH-20 and CH-24 (vide supra) was defined by coupling constants J = 11.6 Hz and J = 11.4 Hz observed in the signals of H-21ax (a triplet by coupling geminal with H-21eq–2JHH and vicinal with H-20–3JHH) by and H-24 2JHH with H-23ax) and comparison with the literature 13C-NMR values of bourjotinolone A, a triterpene isolated from T. hispida [20], with both a-orientations being consistent with the relative configuration shown in 1. Consistent with these observations, the 1H-1H-NOESY spectrum of 1 showed cross-peaks assigned to dipolar interaction (spatial proximity). Thus, the structure of the new triterpene cycloartane-type, isolated from Trichilia hirta was established as 19-methylene-25-hydroxy-20,24-oxidecycloartan-3-one named hirtinone (1).

3. Experimental

3.1. General Procedures

Melting points were obtained on a Microquímica MQRPF and were uncorrected. FTIR spectra were recorded on a FTIR-8300 Shimadzu spectrometer using KBr disk. ESI-MS (high resolution) mass spectra were obtained by using a micrOTOF-QII (Bruker) mass spectrometer, using the positive ion mode of analysis. Chromatographic purifications were carried out by using silica gel 60 (0.063–0.200 mm).

1H and 13C-NMR spectra were measured on a Bruker Utrashield 500 Plus spectrometer, operating at 500 (1H) and 125 (13C) MHz. CDCl3 was used as solvent with TMS as internal reference. Chemical shifts are given in the δ scale (ppm) and coupling constants (J) in Hz. One dimensional (1D) 1H and 13C-NMR spectra were acquired under standard conditions by using a direct detection 5 mm 1H/13C dual probe. Standard pulse sequences were used for two dimensional spectra by using a multinuclear inverse detection 5 mm probe with field gradient.

3.2. Plant Material

Fruits of Trichilia hirta were collected in May 2011, at Vale do Rio Doce Company, Linhares City, Espírito Santo State, Brazil. After botanical identification by botanist Domingos Folly. The voucher specimen of T. hirta was deposited at Vale do Rio Doce herbarium, under the code CRVD-6784.

3.3. Extraction and Isolation

Fruits of T. hirta were dried at room temperature until a constant weight was achieved (about one week). The dried and powdered fruits (438.0 g) were extracted with hexane (volume, time) at room temperature, furnishing, after solvent evaporation, 23.0 g of crude hexane extract. The hexane extract was chromatographed over a silica gel column with a gradient of hexane/ethyl acetate to afford nine fractions. Fraction 8 (1.36 g) was rechromatographed over a silica gel column with a gradient of hexane/acetone furnishing eighth fractions and 6 (52.5 mg). Fractions 8.2 (23.8 mg) and 8.3 (524.2 mg) were rechromatographed over a silica gel column with a gradient of hexane/acetone yielding compounds 1 (3.1 mg) and 9 (22.5 mg), respectively. Fraction 8.5 (49.5 mg) was rechromatographed over a silica gel column with a gradient of hexane/acetone (8:2) v/v furnishing compound 2 (4.5 mg). Fraction 8.7 (99.3 mg) was rechromatographed over a silica gel column with a gradient of hexane/acetone furnishing compound 3 (18.5 mg). Fraction 9 (4.03 g) was rechromatographed over a silica gel column with a gradient of hexane/acetone to provide compounds 7 (67.0 mg), 4 + 5 (176.0 mg) and 8 (65.7 mg).

Nilocitin (2): 13C δ (ppm): 38.56 (CH2-1); 34.93 (CH2-2); 216.96 (C-3); 47.84 (C-4); 52.34 (CH-5); 24.36 (CH2-6); 118.04 (CH-7); 145.72 (C-8); 48.47 (CH-9); 36.05 (C-10); 18.86 (CH2-11); 33.52 (CH2-12); 43.35 (C-13); 51.23 (C-14); 34.06 (CH2-15); 29.77 (CH2-16); 53.28 (CH-17); 21.78 (CH3-18); 12.80 (CH3-19); 33.69 (CH-20); 19.89 (CH3-21); 40.47 (CH2-22); 69.25 (CH-23); 68.46 (CH-24); 60.32 (C-25); 24.88 (CH3-26); 19.82 (CH3-27); 24.53 (CH3-28); 21.61 (CH3-29); 27.40 (CH3-30). 1H δ (ppm): (2.02; 1.50; 2H-1); (2.78 dt 14.6, 5.6; 1H-2); (2.26 dt 14.6, 3.8; 1H-2) (1.70; 1H-5); (2.10; 2H-6); (5.33 m; 1H-7); (2.31; 1H-9); (1.60; 2H-11); (1.65; 1.40; 2H-12); (1.85; 1.55; 2H- 15); (2.08; 1.18; 2H-16); ( 1.50; 1H-17); (0.83 s; 3H-18); (1.03 s; 3H-19); (1.30; 1H-20); (0.98 s, 6.1; 3H-21); (1.70; 1.40; 2H-22); (3.61 m; 1H-23); (2.68 d,8.3; 1H-24); (1.35 s; 3H-26); (1.34 s; 3H-27); (1.07 s; 3H-28); (1.14 s; 3H-29); (1.04 s; 3H-30).

Dihydroniloctin (3): 13C δ (ppm): 37.19 (CH2-1); 27.66 (CH2-2); 79.25 (CH-3); 38.96 (C-4); 50.61 (CH-5); 23.94 (CH2-6); 118.05 (CH-7); 145.57 (C-8); 48.91 (CH-9); 34.93 (C-10); 18.09 (CH2-11); 33.97 (CH2-12); 43.58 (C-13); 51.79 (C-14); 33.78 (CH2-15); 28.79 (CH2-16); 53.25 (CH-17); 13.11 (CH3-18); 21.71 (CH3-19); 33.59 (CH-20); 19.84 (CH3-21); 40.69 (CH2-22); 69.30 (CH-23); 68.51 (CH-24); 60.34 (C-25); 19.13 (CH3-26); 24.88 (CH3-27); 27.62 (CH3-28); 14.71 (CH3-29); 27.24 (CH3-30). 1H δ (ppm): (3.24 dd 11.1, 4.1; 1H-3); (5.26 sl; 1H-7); (0.75 s; 3H-18); (0.82 s; 3H-19); (0.96 d, 6.4; 3H-21); (3.57 m; 1H-23); (2.66 d, 8.2; 1H-24); (1.32 s; 3H-26); (1.33 s; 3H-27); (0.96 s, 3H-28); (0.86 s; 3H-29); (0.97 s; 3H-30).

Melianone (4): 13C δ (ppm): 38.49 (CH2-1); 34.91 (CH2-2); 216.96 (C-3); 47.88 (C-4); 52.35 (CH-5); 24.38 (CH2-6); 118.35 (CH-7); 145.58 (C-8); 48.37 (CH-9); 35.10 (C-10); 17.72 (CH2-11); 31.60 (CH2-12); 43.76 (C-13); 51.03 (C-14); 33.93 (CH2-15); 27.64 (CH2-16); 49.37 (CH-17); 22.62 (CH3-18); 12.75 (CH3-19); 47.04 (CH-20); 101.19 (CH-21) e 97.74 (CH-21); 34.83 (CH2-22); 77.50 (CH-23); 65.37 (CH-24); 57.30 (C-25); 24.92 (CH3-26); 19.43 (CH3-27); 24.44 (CH3-28); 21.59 (CH3-29); 27.28 (CH3-30). 1H δ (ppm): (1.98; 1.45; 2H-1); (2.77 dt 14.5, 5.4; H-2); (2.25 dt 14.5, 4.0; H-2) ; (1.73; H-5); (2.15; 2H-6); (5.34; H-7); ( 2.30 H-9); (1.62; 2H-11); (2.05; 1.75; 2H-12); (1.55; 2H-15); (1.95; 1.86; 2H-16); (1.82; H-17); (0.91 s; 3H-18); (1.02 s; 3H-19); (2.25; H-20); (5.38 dl 3.0; H-21); (2.15; 1.40; 2H-22); (3.95 m; H-23); (2.72 d 7.6; H-24); (1.34 s, 3H-26); (1.32 s; 3H-27); (1.06 s; 3H-28); (1.13 s; 3H-29); (1.05 s; 3H-30).

Piscidinol A (6): 13C δ (ppm): 38.25 (CH2-1); 34.98 (CH2-2); 47 (C-4); 52.31 (CH-5); 24.37 (CH2-6); 117.95 (CH -7); 145 (C-8); 48.46 (CH-9); 35 (C-10); 18.31 (CH2-11); 33.99 (C-12); 43 (C-13); 51 (C-14); 33.77 (CH2-15); 28.48 (CH2-16); 53.60 (CH -17); 22.06 (CH3-18); 12.80 (CH3-19); 33.69 (CH-20); 18.92 (CH3-21); 40.40 (CH2-22); 69.71 (CH-23); 74.81 (CH-24); 72 (C-25); 26.23 (CH3-26); 27.50 (CH3-27); 24.39 (CH3-28); 21.61 (CH3-29); 27.41 (CH3-30). 1H δ (ppm): (2.05; 1.52; 2H-1); (2.78 dt 14.7, 5.7; H-2); (2.27 dt 14.7, 3.8; H-2); (1.75 t 8.9; H-5); (2.15 m; 2H-6); (5.34 tl 3.1; H-7); (2.40 m; H-9); (1.60; 2H-11); (1.85; 1.52; 2H-12); (1.70; 1.45; 2H-15); (1.90; 1.25; 2H-16); (1.71; H-17); (0.85 s; 3H-18); (1.03 s; 3H-19); (1.48; H-20); (0.96 d 6.4; 3H-21); (1.90; 1.25; 2H-22); (4.15 dd 9.7, 5.0; H-23); (3.10 sl; H-24); (1.36 s; 3H-26); (1.39 s; 3H-27); (1.07 s; 3H-28); (1.19 s; 3H-29); (1.04 s; 3H-30).

Melianone lactone (7): 13C δ (ppm): 38.51 (CH2-1); 34.90 (CH2-2); 216.76 (C-3); 47.88 (C-4); 52.57 (CH-5); 24.39 (CH2-6); 118.33 (CH-7); 145.87 (C-8); 47.37 (CH-9); 35.12 (C-10); 17.64 (CH2-11); 31.02 (CH2-12); 43.91 (C-13); 50.53 (C-14); 33.89 (CH2-15); 24.22 (CH2-16); 48.49 (CH-17); 23.44 (CH3-18); 12.73 (CH3-19); 40.85 (CH-20); 178.04 (C-21); 30.21 (CH2-22); 77.96 (CH-23); 64.48 (CH-24); 57.54 (C-25); 24.82 (CH3-26); 19.47 (CH3-27); 24.52 (CH3-28); 21.55 (CH3-29); 27.59 (CH3-30) 1H δ (ppm): (2.10; 1.48; 2H-1); (2.60; 2.30; 2H-2); (1.51; H-3); (2.15; 1.95; 2H-6); (5.35 sl; H-7); (2.40; H-9); (1.60; 2H-11); (1.85; 1.75; 2H-12); (1.60; 2H-15); (1.90; 1.45; 2H-16); (2.17; H-17); (0.86 s; 2H-18); (1.05 s; 2H-19); (2.72; H-20); (2.35; 1.85; 2H-22); (4.18; H-23); (2.84; H-24); (1.40 s; 2H-26); (1.37 s; 3H-27); 1.07 s; 3H-28); (1.14 s; 3H-29); (1.09 s; 3H-30).

Hirtin (8): 13C δ (ppm): 150.52 (CH-1); 125.84 (CH-2); 195.66 (C-3); 60.43 (C-4); 129.1 (C-5); 141.90 (C-6); 196.19 (C-7); 46.15 (C-8); 42.01 (CH-9); 39.85 (C-10); 72.41 (CH-11); 77.76 (CH-12); 45.32 (C-13); 67.59 (C-14); 55.15 (CH-15); 32.15 (CH2-16); 41.37 (CH-17); 15.80 (CH3-18); 25.97 (CH3-19); 121.48 (C-20); 140.48 (CH-21); 111.22 (CH-22); 142.69 (CH-23); 22.85 (CH3-28); 170.06 (C-29); 22.52 (CH3-30); 174.49 (C-1′); 34.10 (CH-2′); 18.70 (MeO-3′); 18.68 (MeO-4′); 169.39 (C-1′′); 21.20 (MeO-2′′). 1H δ (ppm): (7.0 d 10.1; H-1); (6.19 d 10.1; H-2); (2.99 s; H-9); (5.37 s; H-11); (5.23 s; H-12); (2.93 s; H-15); (2.33 dd 13.8, 6.7; 2H-16); (2.02 dd 13.8, 11.0; 2H-16); (2.96 dd 11.0, 6.7; H-17); (0.84 s; 3H-18); (1.45 s; 3H-19); (7.15 sl; H-21); (6.2 sl; H-22); (7.32 t 1.6; H-23); (1.85 s; 3H-28); (2.47 sept 7.0; H-2′); (2.31 s; H2′′-MeO); (1.10 d 7.0; H3′-MeO); 0.98 d 7.0; H4′-MeO); (1.43 s; 3H-30); (6.47 s; H-HO).

Spathulenol (9): 13C δ (ppm): 53.42 (CH-1); 26,73 (CH2-2); 41.74 (CH2-3); 80 (CH-4); 54.33 (CH-5); 29.91 (CH-6); 27.48 (CH-7); 24.77 (CH2-8); 38.86 (CH2-9); 153,40 (C-10); 20 (C-11); 16.15 (CH3-12); 28.69 (CH3-13); 108.28 (CH2-14); 26.10 (CH3-15). 1H δ (ppm): (2.22 m; H-1); (2.08 m; 1.90 m; 2H-2); (1.80 m; 1.60 m;2H-3); (1.30 t3.5; H-5); (0.49 dd, J = 11.3, 9.5 Hz; H-6); (0.74 m; H-7); (1.70 m; 2H-8); (2.4 dd, J = 12.3, 5.3 Hz, 2H-9); (1.06 s, 3H-12); (1.08 s, 3H-13); (4.71 s; 4.69 s; 2H-14); (1.32 s; 3H-15).

4. Conclusions

The hexane extract from the fruits of T. hirta provided five protolimonoids 2–7, one tertranortriterpenoid 8 and one sesquiterpene 9, which were isolated in a previous phytochemical investigation. A cycloartane-type triterpene 1, named hirtinone, is described for the first time in the literature.