Two Novel Cycloartane-Type Triterpenes from Trichilia casaretti C. DC. (Meliaceae)

Abstract

Two new triterpenes cycloartane-type, named 24-methylencycloartan-12-oxo-3β,22α-diol and trichiliol, were isolated from the leaves of Trichilia casaretti C. DC. together with three known triterpenes—24-methylencycloart-3β,22-diol, 22,25-dihydrocycloart-23(E)-en-3β-ol, and 22(R)-hydroxycycloart-24-en-3-ol. These compounds were characterized on the basis of their spectral data, mainly 1D (¹H and ¹³C) and 2D NMR (¹H-¹H-COSY, ¹H-¹H-NOESY, HMQC, HSQC, and HMBC), and mass spectra (EI-MS and HR-ESI-MS), also involving comparison with data from the literature.

1. Introduction

The Meliaceae family has attracted such a great interest among phytochemists because of its relatively complex and diverse chemical structures and its biological activity, mainly against insects [1,2,3,4]. The Trichilia genus (Meliaceae) comprises about 230 species distributed throughout tropical America, which are recognized for their significant economic importance and high commercial value. Phytochemical studies revealed that this genus is a potential source of terpenoids, including triterpenes, limonoids, steroids and other terpenes 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 two novel sesquiterpenes from the stems of T. casaretti collected in Espírito Santo State, Brazil, were reported by Vieira et al. in 2010 [4].

In the present article, we describe an investigation of methanol extract from leaves of a T. casaretti specimen, which allowed the characterization of five triterpenes, including two novel cycloartane-type triterpenes named 24-methylencycloartane-12-oxo-3β,22α-diol (1) and trichiliol (2). The known triterpenes were identified as 24-methylencycloartane-3β,22-diol (3), cycloartane-22(E)-en-3β,22,25-triol (4), and 22(R)-hydroxycycloart-24-en-3-ol (5). Their structures (Figure 1) were established by spectrometric techniques, mainly 1D and 2D NMR, and HR-ESI-MS, and comparison with literature data.

2. Results and Discussion

Fractionation of the MeOH extract of the leaves from T. casaretti by classical chromatographic methods resulted in the isolation of five cycloartane-type triterpenes 1–5 whose structures are shown in Figure 1. The two novel cycloartane-type triterpenes named 24-methylencycloartan-12-oxo-3β,22α-diol (1) and trichiliol (2), along with three known triterpenes, cycloartane-type 24-methylencycloart-3β,22-diol (3) [9,10], 22,25-dihydrocycloart-23(E)-en-3β-ol (4) [9,11] and 22(R)-hydroxycycloart-24-en-3-ol (5) [12] were characterized on the basis of ¹H and ¹³C-NMR spectral data, especially 2D NMR and mass spectral data, besides comparison with values described in the cited literature.

The new triterpene named 24-methylencycloartan-12-oxo-3β,22α-diol(1) was isolated as yellow oil, ${[\mathsf{\alpha }]}_{\mathrm{D}}^{23}$ = +27.3 (CHCl₃, c 0.02). The analysis of the {¹H}- and ¹³C-APT NMR spectra (

Table 1. ¹³C and ¹H-NMR data of triterpene 1 and comparison with triterpene 3 (CDCl₃), δ in ppm, coupling constants (J in Hz, in parenthesis) *.

C	1				3
		HMQC (H→C)	HMBC (C→H)
	δ (C)	δ (H)	2J	3J	δ (C)
1	32.8	1.65–1.55 (m, 2H)		2H-19	32.0
2	32.0	1.78–1.80 (m, 1H) 1.62–1.55 (m, 1H)			30.4
3	78.8	3.28 (dd, 11.1; 4.1, 1H)		3H-28, 3H-29	78.8
4	40.7	-	3H-28, 3H-29		40.8
5	48.0	1.30–1.25 (m, 2H)		3H-28, 3H-29	47.1
6	21.6	1.58–1.62 (m, 1H) 0.78–0.80 (m, 1H)			21.1
7	25.4	1.28–1.30 (m, 1H) 1.10–1.16 (m, 1H)			27.2
8	47.1	1.30 (br s, 1H)		3H-30	48.0
9	19.5	-			20.0
10	29.9	-			26.1
11	27.2	n.d.		2H-19	26.1
12	218.0	-		3H-18	35.7
13	49.0	-	3H-18	3H-30	45.8
14	48.0	-	3H-30	3H-18	48.4
15	45.7	1.30–1.34 (m, 2H)		3H-30	32.9
16	25.4	2.01–2.05 (m, 1H) 1.30–1.34 (m, 1H)			26.4
17	49.0	1.49–1.53 (m, 1H)		3H-18, 3H-21	49.0
18	12.1	0.91 (s, 3H)		H-12, H-17	18.0
19	30.3	0.56 (d, 4.1, 1H) 0.35 (d, 4.1, 1H)		2H-1	29.9
20	40.9	1.88–1.80 (m, 1H)	3H-21		40.5
21	19.5	0.90 (d, 5.8, 3H)	H-20	H-17, H-22	12.0
22	70.3	3.78 (br d, 10.6, 1H)			70.4
23	36.1	2.24 (d, 13.5, 1H) 1.95 (dd, 13.5; 11.2, 1H)	H-22	2H-31	36.1
24	153.5	-	2H-23, 2H-31	3H-26, 3H-27	153.6
25	33.1	2.20–2.24 (m, 1H)			33.2
26	22.3	1.05 (d, 7.0, 3H)			22.3
27	21.6	1.08 (d, 7.0, 3H)			21.6
28	25.4	0.97 (s, 3H)		H-3, H-5, 3H-29	19.5 a
29	14.2	0.81 (s, 3H)		H-3, H-5, 3H-28	14.0
30	18.0	1.01 (s, 3H)		H-8, 2H-15	25.4 a
31	109.8	4.93 (s, 1H)/4.84 (s, 1H)		2H-23	109.7

* Number of hydrogens bound to carbon atoms deduced by comparative analysis of {¹H}- and APT-¹³C-NMR spectra. Chemical shifts and coupling constants (J) obtained of 1D ¹H-NMR spectrum. Superimposed ¹H signals are described without multiplicity and approximated chemical shifts deduced by HMQC, HMBC and ¹H-¹H-COSY spectra. Assignments determined by a combination of 1D and 2D (¹H-¹H-COSY, HSQC and HMBC) NMR experiments. ^a Chemical shifts with same letter cam be exchanged. n.d. = not detected.

) revealed signals corresponding to 31 C-atoms, including seven quaternary C (five sp³ and two sp², one attributed to methylidene at δ_C 153.5 (C-24) and one C=O group at δ_C 216.0 (C-12)), seven CH (all sp³, including two linked to O atom at δ_C 78.8 (CH-3) and 70.3 (CH-22), nine methylene CH₂ (including one sp² at δ_C 109.8 (CH₂-31)) and seven methyl groups CH₃, allowing to deduce the expanded partial formula C₆(C=O)CH₅(OCH)₂(CH₂)₁₀(CH₃)₇ = C₃₁H₄₈O₃ or C₆(C=O)CH₅(HOCH)₂(CH₂)₁₀(CH₃)₇ = C₃₁H₅₀O₃ based in the presence of two hydroxyl groups.

The HR-ESI-MS spectrum of 1 showed peak corresponding to the quasi molecular ion ([M + H]⁺) at m/z 471.3475, that in combination with ¹H-NMR (1D and 2D) and comparative analysis of the {¹H}- and ¹³C-APT NMR spectra (Table 1) enabled us to propose the molecular formula C₃₁H₅₀O₃ (seven degrees of unsaturation) compatible with a cycloartane-type 24-methylencycloart-3β,22-diol (3) skeleton [9]. The only difference regarding compounds 1 and 3 consists of a carbonyl group at C-12.

The signal at δ_C 216.0 in the ¹³C-APT NMR spectrum suggested the presence of a carbonyl group in the triterpene cycloartane 1. The location of the carbonyl group at C-12 was supported in the HMBC spectrum by the correlation (³J_HC) between C-12 (δ_C 216.0) and the hydrogens of the methyl group 3H-18 at δ_H 0.90. Thus, the new triterpene cycloartane-type was characterized as 24-methylidencycloartan-12-oxo-3β,22-diol (1).

Compound 2 was isolated as a yellow oil, ${[\mathsf{\alpha }]}_{\mathrm{D}}^{23}$= +48.0 (CHCl₃, c 0.001). The analysis of the {¹H}- and ¹³C-NMR spectra (

Table 2. ¹³C- and ¹H-NMR data of triterpene 2 (pyridine-d₅), δ in ppm, coupling constants (J in Hz, in parenthesis) *.

C	2
		HSQC (H→C)	HMBC (C→H)
	δ (C)	δ (H)	2J	3J
1	32.8	1.50–1.53 (m, 1H) 1.25–1.26 (m, 1H)		2H (19)
2	31.6	2.00–2.01 (m, 1H) 1.94–1.98 (m, 1H)
3	78.3	3.57 (br d, 9.0, 1H)		3H-28, 3H-29
4	41.5	-	3H-28, 3H-29
5	47.9	1.33 (m, 1H)	2H-6	3H-28, 3H-29
6	21.8	1.60–1.65 (m, 1H) 0.77–0.82 (m, 1H)	H-5
7	26.8	1.12–1.05 (m, 2H)	H-8
8	48.6	1.46–1.50 (m, 1H)	2H-7	3H-30
9	20.3	-	2H-19
10	26.9	-	H-5, 2H-19
11	27.0	2.00–2.10 (m, 1H) 1.25–1.30 (m, 1H)		2H-19
12	33.4	1.60–1.72 (m, 2H)		3H-18
13	46.2	-	3H-18-	3H-30
14	48.9	-	3H-30	3H-18
15	36.3	2.10–2.20 (m, 1H) 1.20–1.30 (m, 1H)		3H-30
16	28.0	2.22–2.38 (m, 1H) 1.90–2.00 (m, 1H)
17	49.9	1.80 (br q, 9.0, 1H)		3H-18, 3H-21
18	18.7	1.03 (s, 3H)		H-17
19	30.5	0.54 (br s, 1H) 0.33 (br s, 1H)		2H-1, 2H-11
20	43,6	2.05–2.25 (m)	3H-21-
21	12.8	1.22 (d, 6.3, 3H)	H-20	H-17, H-22
22	69.7	4.55 (br d, 10.0, 1H)		3H-21
23	34.5	2.20–2.30 (m, 1H) 1.85–1.90 (m, 1H)
24	86.6	5.32 (br s, 1H)		3H-27
25	147.8	-	3H-27
26	112.5	5.32 (br s, 1H) 5.10 (br s, 1H)		3H-27
27	18.5	2.01 (br s, 3H)	H-25	H-24, 2H-26
28	26.6	1.25 (s, 3H)		H-3, H-5, 3H-29
29	15.2	1.22 (s, 3H)		H-3, H-5, 3H-28
30	20.1	0.89 (s, 3H)		H-8, 2H-15

* Number of hydrogens bounded to carbon atoms deduced by comparative analysis of {¹H}- and APT-¹³C-NMR spectra. Chemical shifts and coupling constants (J) obtained of 1D ¹H-NMR spectrum. Superimposed ¹H signals are described without multiplicity and approximated chemical shifts deduced by HSQC, HMBC and ¹H-¹H-COSY spectra. Assignments deduced by a combination of 1D and 2D (¹H-¹H-COSY, HSQC and HMBC) NMR experiments.

) revealed signals corresponding to 30 carbon atoms, showing similarity with the triterpene cycloartane 1. Comparison of the ¹³C-NMR data of compounds 1 and 2 revealed the absence of carbonyl signal in 2, and additional differences can be justified through modifications involving only the side chain (Table 1 and Table 2).

The ¹³C-NMR spectrum of 2 with the presence of 30 carbon signals revealed additional carbon signals corresponding to remaining carbon atoms (C)(CH)(OCH)₂(CH₂)₂(CH₃)₂ = C₈H₁₃O₂ of the side chain, different of the cycloartane triterpene 1 with signals representing 31 carbon atoms including (C)(CH)₂(HOCH)(CH₂)₂(CH₃)₃ = C₉H₁₇O of the side chain.

The ¹³C-NMR spectrum involving the side chain of 2 revealed signals representing only two methyl groups when compared with those of 1 with three, one corresponding to CH₃-21 at δ_C 19.5 correlated in the HSQC with the doublet (J = 6.3 Hz) signal at δ_H 1.22 (3H-21) and one attached to sp² carbon (CH₃-27, δ_H 2.01\δ_C 18.5), two sp² carbon atoms at δ_C 147.8 (C-25) and 112.5 (CH₂-26), featuring a methylidene group =CH₂ confirmed by correlations (¹J_HC) between δ_C 112.48 (CH₂-26) with two broad singlets at δ_H 5.32 (Ha-26) and 5.10 (Hb-26) observed in the HSQC spectrum.

The location of the methylidene group at the C-25 was confirmed through the ²J_HC correlations between C-25 (δ_C 147.8) and 3H-27 (δ_H 2.01) and C-26 (δ_C 112.5) with 3H-27 (δ_H 2.01). The presence of a isopropenyl group (isopropyl in 1) attached to CH-24 (δ_C 86.6\δ_H 5.32, br s) was deduced by HSQC and HMBC correlations between CH-24 and 3H-27 [(δ_H 2.01, br s\(δ_C 18.5, ³J_HC)]. It was also possible to observe the interaction between CH-22 (δ_C 69.87.6\(δ_H 4.55, br d, J = 10.0 Hz) and 3H-21 (δ_H 1.22, d, J = 6.3 Hz\(δ_C 12.8, ³J_HC). These data allowed to postulate the presence of oxygen atoms linked to carbon 22 (HC-O-22: δ_H 4.55\69.7) and 24 (HC-O-24 δ_H 5.32\86.6), in agreement with the ¹H-¹H-COSY spectrum which showed correlations between the methylene hydrogens 2H-23 (δ_H 2.10 and 1.90\δ_C 34.5) and both hydrogen atoms H-22 (δ_H 4.55) and H-24 at δ_H 5.32 (chemical shift justified by location in position allylic and carbinolic). Other heteronuclear correlations observed in the HMBC spectrum of 2 were shown in Table 2.

The endoperoxide function in the side chain was also supported by mass spectra of low and high resolution demanding an additional ring to suit the seven degrees of unsaturation. The HRESI-MS spectrum of 2 (Scheme S1, Supplementary Materials) showed peak corresponding to the protonated molecular ion [M + H]⁺ at m/z 457.3502 which together with the NMR spectrum of ¹³C-DEPT 135° NMR allowed to propose the molecular formula C₃₀H₄₈O₃ compatible with a cycloartane-type skeleton and one heterocyclic involving the peroxide function.

The relative stereochemistry indicated in 2 was deduced by the spatial dipolar interaction observed in the ¹H-¹H-NOESY spectrum between 3H-30 and H-17 indicating H-17α, interaction of 3H-18 with H-20 and 3H-21with H-12 equatorial suggesting H-20α and CH₃-21α, respectively. Other spatial dipolar interaction was shown in Figure 2. These data allowed to identify compound 2 as the new triterpene 22,24-peroxidecycloart-25-en-3β-ol (2), named trichiliol.

3. Material and Methods

3.1. General Methods

Measures of optic rotation were obtained on a Perkin Elmer 343 digital polarimeter. Melting point was obtained on a Microquímica MQRPF. EI-MS (low resolution) mass spectra were obtained on Shimadzu QP5050A mass spectrometer. HRESI-MS (high resolution) mass spectra were obtained by using a ESI-IT-OF-MS SHIMADZU mass spectrometer, using the positive ion mode of analysis. Chromatographic purifications were carried out over silica gel 60 (70–230 mesh). Silica gel 60 F₂₅₄ was used in thin layer chromatography analysis. ¹H and ¹³C-NMR spectra were measured on a Bruker model DRX-500 and Jeol model Eclipse-400 spectrometers, equipped with inverse probes and field gradient, operating at 500 (¹H) and 125 (¹³C) and 400 (¹H) and 100 (¹³C) MHz. CDCl₃ and pyridine-d₅ were used as solvents and 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

The leaves of Trichilia casaretti C. DC. were collected at Vale Cia, Linhares City, Espirito Santo State, Brazil, and identified by Domingos Folly. A voucher specimen (CVRD-449) was deposited at the Vale Cia of Vale herbarium, Vale Cia, Linhares City, Espirito Santo State, Brazil.

3.3. Extract and Isolation

Air dried and powdered leaves (920 g) from Trichilia casaretti C. DC. were extracted with methanol at room temperature, yielding 15.0 g of crude methanol extract. The methanol extract was submitted to liquid-liquid partition (CH₂Cl₂:H₂O, 3:1, v/v). The CH₂Cl₂ fraction (10.1 g) was submitted to liquid-liquid partition (MeOH:Hexane, 1:1, v/v). The methanol fraction (8.0 g) was chromatographed over a silica gel column with a gradient of ethyl acetate/hexane, affording eight fractions. Fraction 1 (19.3 mg) was similarly rechromatographed, yielding triterpene 5 (8.0 mg). Fraction 5 (518 mg) was analogously rechromatographed, affording eleven fractions. Fraction 5.7 (66.8 mg) was similarly chromatographed, affording triterpene 4 (4.0 mg). Fraction 7 (6.736 mg) was chromatographed affording fifteen fractions. The fraction 7.5 (1.300 mg) was rechromatographed affording fourteen fractions. The triterpene 2 (10 mg) was obtained from the fractions 7.5.3 (45 mg) and 7.5.4 (100 mg). The triterpene 1 (25 mg) was obtained from the fraction 7.6 (500 mg). The fraction 7.9 (163 mg) was chromatographed affording fifteen fractions. The fraction 7.9.2 (50.3 mg) yielded triterpene 3 (11 mg).