Curcinomarcoide,a Novel Limonoid From Trichilia hirta (Meliaceae)—Complete 1 H and 13 C Chemical Shift Assignments

Abstract

A novel limonoid named curcinomarcoide (1) was isolated from Trichilia hirta and characterized on the basis of its nuclear magnetic resonance and mass spectrometry spectral data and comparison with literature data.

The family Meliaceae belongs to the order Rutales formed by 51 genera of approximately 1400 species found in the tropics and subtropics. ¹ Species of this family, for example, Trichilia hirta, are characterized chemically by the presence of limonoids and also by their insecticidal activity. ²

A phytochemical revision of the Trichilia genus ³ showed that 334 different terpenoids representing 87.7% of the compounds were isolated and identified from various species of Trichilia.

This paper describes the isolation and characterization of the novel limonoid, named curcinomarcoide (1, Figure 1), whose structure was established from ¹H and ¹³C (1D and 2D) nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) spectra involving comparison with literature data.

Figure 1

Structures of curcinomarcoide (1) isolated from T. hirta, diacetyl derivative (1a) and model compounds 2 and 3. ⁴

The methanol extract of fruit of T. hirta was subjected to classical chromatographic methods to yield curcinomarcoide (1) as an amorphous solid. The DEPTQ-¹³C NMR spectrum (Table 1) revealed signals corresponding to 33 carbon atoms. With the aid of 2D heteronuclear single quantum coherence (HSQC) ^5,6 it was possible to recognize ¹³C signals from 12 nonhydrogenated carbons [(C)₁₂: 6 sp³ and 6 sp² corresponding to 1 olefinic at δ _C 129.3, 1 oxygenated at δ _C 142.0, 2 carbonyl ketonic α,β-unsaturated at δ _C 195.9 and 196.7, and 2 carboxyl groups of esters at δ _C 170.4 and 178.0] and 21 hydrogenated (CH _n , n = 1, 2, and 3) carbons corresponding to 11 methine [(CH)₁₁: 9 sp³ including 4 oxygenated at δ _C/δ _H 56.4/3.93 (CH-15), 72.2/4.17 (CH-11), 78.2/4.16 (CH-22), and 81.6/4.99 (CH-12) and 2 di-oxygenated at δ _C 107.9/4.82 (CH-23) and 109.3/4.82 (CH-21); and 2 sp² olefinics conjugated with the carbonyl group at δ _C 151.9/6.94 (CH-1) and 126.3/6.13 (CH-2)], only 1 methylene [(CH₂)] and 9 methyl [(CH₃)₉: including 2 methoxyl groups at δ _C 54.7/3.34 and 56.4/3.48 and methyl of a carbomethoxy group at δ _C 53.0/3.78] carbon atoms; additional cross-peaks corresponding to heteronuclear direct couplings (¹ J _CH) observed in the HSQC spectrum are summarized in Table 1. These data allowed the deduction of the expanded molecular formula (C)₆(C = C O)(C = O)₄(CH)₃(HC = CH)(HC-O)₄(O-CHO)₂(CH₂)(CH₃)₆(CH₃O)₃ = C₃₃H₄₀O₁₆, which after considering the possibility of the presence of epoxy [C-O-CH: δ _C/δ _H 68.4/- (C-14) and 56.4/3.93 (CH-15)], ether [(O-CH-O)₂ = CH₂O₂ = (HC-O-CH = CH₂O) δ _C/δ _H 109.3/4.82 (CH-21) and 107.9/4.82 (CH-23)], and 4 hydroxyl groups it was possible to propose the molecular formula C₃₃H₄₄O₁₄, which was confirmed by the HRESIMS spectrum that showed a peak corresponding to a molecular formula of C₃₃H₄₄O₁₄ + Na⁺ ([M + Na]⁺) at m/z 687.2621 and calculated value [M + Na]⁺ at m/z 687.2629 (Δ _m/z 0.0008) Da (Scheme 1). Thus, all these data in combination with the NMR spectral data (DEPTQ-¹³C, ¹H-¹H correlation spectroscopy [COSY], HSQC, and heteronuclear multiple bond correlation [HMBC]) were used to propose a molecular formula of C₃₃H₄₄O₁₄ of 1, with 12 degrees of unsaturation (C₃₃H₆₈O₁₄ − C₃₃H₄₄O₁₄ = 24) corresponding to 4 carbonyl groups, 2 double bonds, and 6 rings.

Table 1

Spectral Data for 1 (¹H: 400 MHz; ¹³C: 100 MHz; in CDCl₃), Including Results of the 2D Experiments (HSQC and HMBC).

	1				Model compounds 6
	HSQC		HMBC		2	3
	δ C	δ H	2 J HC	3 J HC	δ C	δ C
C
3	195.9	-	H-2	H-1; 3H-28	195.7	-
4	59.3	-	3H-28	H-2	59.3	-
5	129.3	-		H-1; HO-6; H-9; 3H-19; 3H-28	129.6	-
6	142.0	-	HO-6		141.9	-
7	196.7	-		HO-6; H-9;3H-30	196.5	-
8	45.9	-	H-9; H-30	H-11	45.9	-
10	40.4	-	H-1 H-9; 3H-19	H-2	40.5	-
13	45.0	-	H-12; H-17; 3H-18	H-20	44.8	-
14	68.4	-		H-17; 3H-18; 3H-30	68.3	-
20	80.6	-	H-17; HO-20; H-21		121.9	-
29	170.4	-		3H-28; MeO-29	170.2	-
1′	177.8	-	H-2′	H-12; 3H-3′; 3H-4′	173.9	-
1″	-	-			-	-
CH
1	151.9	6.94(d, 10.4)			151.4	6.87(d, 10.2)
2	126.3	6.13(d, 10.4)			126.5	6.12(d, 10.2)
9	43.2	2.70 (br s)		H-1; H-12;3H-19; 3H-30	43.9	2.69 (s)
11	72.2	4.17 (m)	H-9;H-12		73.2	4.12 (br s)
12	81.6	4.82 (s)	H-11	H-9; H-17;3H-18	82.8	5.10 (br s)
15	56.4	3.93 (s)		H-17	56.5	3.98 (s)
17	50.2	2.12 (m)		H-12; H-15;3H-18	42.2	2.94 (dd, 11.0, 6.7)
21	109.3	4.99 (s)		H-17; HO-20; H-23; MeO-21	140.1	7.13 (m)
22	78.2	4.16 (m)	H-23	HO-20; H-21	111.0	6.08 (dd, 1.6, 0.7)
23	107.9	4.82 (d, 4.1)		H-21; MeO-23	142.9	7.32 (d, 1.6)
2′	34.5	2.64(sep, 7.0)	3H-3′; 3H-4′		-	2.48 (sextet, 7.0)
CH2
16	27.9	2.55(dd, 15.5, 13.5);2.08 (m)	H-15;H-17	H-20	31.6	2.34 (dd, 13.8, 6.7)2.01 (dd, 13.8, 11.1)
2′					27.8	2.27 (m, 2)
CH3
18	15.4	1.07 (s)			15.3	0.77 (s)
19	26.1	1.65 (s)			26.5	1.66 (s)
28	22.6	1.85 (s)			22.8	1.83 (s)
30	22.8	1.39 (s)		H-9	22.5	1.41 (s)
3′	19.0	1.22 (d, 7.0)	H-2′		9.0	1.08 (d, 7.0)
4′	19.1	1.22 (d, 7.0)	H-2′		-	1.03 (d, 7.0)
2″	-	-			-
MeO-21	54.7	3.34 (s)		H-23	-
MeO-23	56.4	3.48 (s)		H-21	-
MeO-29	53.0	3.78 (s)			52.9	3.76 (s)

Chemical shifts in δ (ppm) and coupling constants (J in parentheses) in hertz.

Number of hydrogens bound to carbon atoms deduced from DEPTQ-¹³C NMR spectrum. Chemical shifts and coupling constants (J, in parentheses) were obtained from 1D-¹H NMR spectrum. 2D-¹H-¹H-COSY and ¹H-¹H-NOESY spectra were also used in these assignments.

Scheme 1

Proposed fragmentations to justify principal positive mode peaks of curcinomarcoide 1).

Comparison of the NMR data with those in the literature ⁴ characterized the A, B, C, and D rings (Table 1), which was also confirmed by the spectral data of diacetyl derivative 1a (Table 2).

Table 2

Spectral Data for 1a (¹H: 500 MHz; ¹³C: 125 MHz; in CDCl₃), Including Results of the 2D Experiments (HSQC and HMBC).

	1a
	HSQC		HMBC
	δ C	δ H	2 J HC	3 J HC
C
3		-	H-2	H-1
4	59.0	-	3H-28	H-2
5	123.9	-		H-1; 3H-19
6	145.0	-
10	40.0	-	H-1	H-2
13	45.0	-	3H-18
14	68.0	-		3H-18
20	81.3	-	H-2′; 3H-3′; 3H-4′	H-12
29	169.3	-		3H-28
1′	177.6	-
CH
1	150.9	6.93		3H-19
2	127.0	6.22
9	43.0	2.74
11	72.9	5.32
12	80.7	5.03
15	57.2	3.86
17	50.5	1.90		H-12; 3H-18
21	108.7	4.90		H-17; H-23; MeO-21
22	78.3	5.38
23	105.3	4.91	H-22	H-21; MeO-23
2′	34.3	2.63	3H-3′; 3H-4′
CH2
16	27.7	2.56	H-15; H-17
CH3
18	15.1	1.06		H-12
19	23.9	1.75
28	21.0	1.78
30	21.3	1.45
3′	19.1	1.22		H-2′
4′	19.1	1.21		H-2′
MeO-21	54.8	3.34
MeO-23	56.1	3.44
MeO-29	53.2	3.73

Chemical shifts in δ (ppm) and coupling constants (J in parentheses) in hertz.

Number of hydrogens bound to carbon atoms deduced from DEPTQ-¹³C NMR spectrum. Chemical shifts and coupling constants (J, in parentheses) were obtained from 1D-¹H NMR spectrum. 2D-¹H-¹H-COSY and ¹H-¹H-NOESY spectra were also used in these assignments.

The DEPTQ-¹³C NMR spectrum displayed 2 methine (CH) signals at δ _C 109.3 (CH-21) and 107.9 (CH-23) from the C-O links of a tetrasubstituted 5-membered ring. The 2 methoxyl groups at CH-21 and CH-23 were confirmed by heteronuclear correlations ³ J _HC of CH-21 with 3 hydrogen atoms of methoxyl group 3H-MeO-21 at δ _H 3.34 (s), H-17 at δ _H 2.12 (m), H-23 at δ _H 4.82 (d, 4.1 Hz), and HO-20 at δ _H 2.63 (s), as well as CH-23 at δ _C 107.9, 3H-MeO-23 at δ _H 3.48 (s), and H-21 at δ _H 4.99 (s) observed in the HMBC spectrum (Table 1). The remaining heteronuclear correlations are summarized in Table 1. The ¹H-¹H-COSY spectrum also certifies the presence of this ring through the coupling of the H-23 hydrogen at δ _H 4.82 with the H-22 hydrogen at δ _H 4.16. The NOESY spectrum of 1 confirmed the stereochemistry of the A, B, C, and D rings, as well as suggesting the configurations of carbon atoms of the tetraoxygenated tetrahydrofuran ring (Figure 2).

Figure 2

Principal dipolar interactions revealed by ¹H-¹H-NOESY of 1.

Thus, the 2D NOESY spectrum of 1 revealed dipolar interactions of H-23 (δ _H 4.82, d, J = 4.1 Hz) with MeO-21 (δ _H 3.34, s) and H-22 (δ _H 4.16, m) with H-16b (δ _H 2.08, m) which were used to postulate the location of MeO-23 and HO-22 at positions α and β, respectively. These data together with data from the literature ^4,7,8 allowed us to propose the structure of compound 1 which was acetylated to give 1a whose 1D and 2D NMR are given in Table 2.

These results confirm the presence of hydroxyl groups on CH-11 and CH-22 through the anticipated protective effects on the following carbons atoms: CH-12 before at δ _C 81.6 and now δ _C 80.7; CH-21 before at δ _C 109.3 and now δ _C 105.7; CH-23 before at δ _C 107.9 and now δ _C 105.3, in accordance with the signals of the hydrogens H-11 and H-22 observed in the ¹H NMR spectrum of 1a at δ _H 5.32 and 5.38, respectively (Table 2).

The high-resolution ESI-MS spectrum of 1a {[M + Na]⁺ at m/z 771.2701 – m/z 687.2621 [M + Na]⁺of 1} = (Δ _m/z = 84.008) Da revealed the acetylation of 2 secondary hydroxyl groups, in accordance with the presence of the HO-11 and HO-22 (Scheme 2).

Scheme 2

Proposed fragmentations to justify principal positive mode peaks of the acetylated curcinomarcoide (1a).

Thus, curcinomarcoide (1) was characterized as a new limonoid.

Experimental

Plant Material

The fruits from T. hirta were collected in May 2011, at Vale Cia, Linhares City, Espírito Santo State, Brazil. After botanical identification by Domingos Folly a voucher specimen (registry number 12022) was deposited at the herbarium of Vale Cia.

Preparation of Organic Extracts

Fruits of T. hirta were dried at room temperature until a constant weight was achieved. The fruits (438.4 g) were exhaustively extracted at ambient temperature by maceration with methanol and evaporated under reduced pressure to obtain the methanolic extract (40.8 g).

General Experimental Procedures

ESI-MS (high resolution) mass spectra were obtained with a micrOTOF-10368 (Bruker) mass spectrometer in positive ion mode. GC/EIMS analysis was obtained using QP2010 Plus (Shimadzu), column Factor Four/VF-5ms (30 × 0.25 × 0.25) and CHCl₃ as solvent (2 µL). Chromatographic purifications were carried out by using silica gel 60 (0.063-0.200 mm). ¹H and ¹³C NMR spectra were measured on Bruker Avance III, operating at 400 (¹H) and 100 (¹³C) MHz and 500 (¹H) and 125 (¹³C) MHz in CDCl₃ with TMS as internal reference. Chemical shifts are given in the δ scale (ppm) and coupling constants (J) in hertz. One-dimensional 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 2-dimensional spectra by using a multinuclear inverse detection 5 mm probe with field gradient.

Purification and Isolation

A 27.8 g portion of the methanolic extract of the fruit was chromatographed on a silica gel column and eluted with a gradient of dichloromethane and methanol to yield 7 fractions. Fraction 4 (9.69 g) was rechromatographed on a silica gel column and eluted with a gradient of dichloromethane/methanol to yield 9 subfractions of which subfraction 4.8 (2.83 g) was rechromatographed on a silica gel column and eluted with a gradient of dichloromethane/methanol to obtain compound 1 (31.1 mg).

Acetylation of 1

Compound 1 (10 mg) was heated with a 2:1 mixture of acetic anhydride and pyridine for approximately 10 minutes and left overnight at room temperature. The reaction was terminated by addition of water, extracted with 3 × 30 mL dichloromethane, dried with anhydrous sodium sulfate, and evaporated to yield 15 mg of 1a.

Curcinomarcoide (1)

Rf: 0.4 (dichloromethane/methanol, 9:1).

¹H NMR (400 MHz, CDCl₃): Table 1

¹³C NMR (100 MHz, CDCl₃): Table 1

HRESIMS: m/z = 687.2621 [M + Na⁺].

Diacetyl Derivative of 1 (1a)

Rf: 0.4 (hexane/ethyl acetate, 1:1).

¹H NMR (500 MHz, CDCl₃): Table 2

¹³C NMR (125 MHz, CDCl₃): Table 2

HRESIMS: m/z = 771.2701 [M + Na⁺].