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DOI: 10.1055/s-0043-120070
Mayombensin, a New Azadirachtin I Derivative with Unusual Structure from Guarea mayombensis
Correspondence
Publikationsverlauf
received 18. Februar 2017
revised 07. Juni 2017
accepted 08. September 2017
Publikationsdatum:
22. November 2017 (online)
Abstract
A new highly oxidized tetranortriterpenoid, named mayombensin (1), was isolated from the twigs of Guarea mayombensis together with five known compounds and, further, two ceramides, 3,4-dimethyl-secotirucalla-4(28),7,24-trien-3,21-dioic acid (3), the glucosides of stigmasterol and β-sitosterol, and the respective aglyca. The structure of 1 was elucidated by detailed NMR analysis and confirmed as a novel azadirachtin homologue.
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Key words
Guarea mayombensis - Meliaceae - tetranortriterpenoid - mayombensin - azadirachtin homologueIntroduction
The genus Guarea belongs to the family Meliaceae and has 150 species of trees and shrubs in tropical America (8 of them occur in Panama [1] and 20 in Africa [2]). Some species are used in folk medicine for the treatment of rheumatism and as an emetic and hemostatic remedy [3]. A few pharmacological studies demonstrated anti-inflammatory, antiviral, and antiprotozoal activities of some Guarea extracts [3] [4] [5] [6]. Previous phytochemical investigations on this genus led to the isolation and identification of a wide variety of constituents including sesqui-, di-, and triterpenes, limonoids, steroids, flavonoids, and coumarins [3] [7] [8] [9] [10]. Although a high number of chemical studies on the genus Guarea have been reported, none is known on Guarea mayombensis Pellegr., which is a big tree up to 10–15 m high, commonly distributed in tropical Africa. This paper deals with the structure elucidation of a new tetranortriterpenoid 1 isolated together with five known compounds and, further, two ceramides from the methanol extract of the finely powdered twigs of G. mayombensis on chromatographic separation.
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Results and Discussion
ESI HRMS of compound 1 displayed a pseudo-molecular ion peak at m/z 593.2583 ([M - H]–), consistent with the molecular formula C30H42O12. Its IR spectrum exhibited typical absorptions for hydroxy (3423 cm−1), ester carbonyl (1697 cm−1), ether (1268, 1142, 1075 cm−1), and double bond (1652 cm−1) functionalities. The UV spectrum showed a peak at 220 nm due to the π–π* transition of an α,β-unsaturated carbonyl group [8] [11]. The 13C NMR spectrum of 1 provided evidence for a conjugated ester carbonyl group by signals at δ C=167.6 (C-1') and of two olefinic carbon atoms at δ C=137.8 (C-3') and 130.1 (C-2'). The 13C NMR spectrum displayed a further 27 signals of aliphatic carbons, of which 14 between δ C=60-101 were oxygenated. This agreed well with the 1H NMR spectrum ([Table 1]), which exhibited a set of signals attributable to oxymethine and oxymethylene groups between δ H=3.50-6.50. Besides, the 1H NMR and HSQC spectra also showed three aliphatic methyl singlets of H3-18, H3-29, and H3-30, and two broadened signals of olefinic methyls, which coupled with an olefinic proton at δ H=7.34 (H-3'). In the HMBC experiment, cross-peaks from both H-3' and H3-5' to C-1' and C-2', and from H3-4' to C-2' were observed, indicating the presence of a tigloyl unit. The above data suggested 1 to be a highly oxygenated nortriterpene, structurally related to azadirachtin-type limonoids [12] [13] [14].
Position |
δ H (m, J in Hz) |
δ C |
Complete list of HMBC (H → C) correlations |
---|---|---|---|
1 |
6.01 (t, 2.8) |
75.4 |
C-3, 5, 10, 19 (w), 1' (w) |
2α |
2.70 (td, 2.3, 15.0) |
32.6 |
C-3, 4, 10 |
2β |
2.27 (td, 2.9, 15.8) |
||
3 |
4.06 (brs) |
70.8 |
C-5 |
4 |
- |
44.8 |
- |
5 |
4.16 (d, 12.6) |
35.2 |
C-3, 4, 6, 7, 10, 19, 28, 29 |
6 |
3.97 (m) |
75.6 |
C-7 (vw), 8 (vw) |
7 |
4.96 (brs) |
74.5 |
C-5, 6, 8, 9, 14, 30 |
8 |
- |
46.0 |
- |
9 |
3.47 (brs) |
50.6 |
C-1, 8, 10, 13 (w), 14, 19, 30 |
10 |
- |
49.9 |
- |
11 |
6.11 (brs) |
101.9 |
C-8, 10, 19 |
13 |
- |
66.8 |
- |
14 |
- |
70.6 |
- |
15 |
4.96 (brs) |
79.6 |
C-13, 14, 17, 21 |
16a |
2.22 (brd, 11.1) |
28.1 |
C-13, 14, 15, 17, 20 |
16b |
1.81 (ddd, 11.6, 5.5, 2.9) |
C-20 (w) |
|
17 |
2.66 (d, 5.3) |
53.9 |
C-13, 14, 15, 16 (w), 18 (w), 20, 21 |
18 |
2.38 (s) |
18.7 |
C-13, 14, 17 |
19 |
4.22 (d, 2.9) |
72.3 |
C-1, 5, 10, 11 |
20 |
- |
93.1 |
- |
21 |
6.54 (s) |
95.0 |
C-20, 22 |
22α |
4.34 (m) |
59.5 |
C-23 (vw) |
22β |
3.48 (dd, 11.4, 2.0) |
C-21 |
|
23α |
3.57 (dd, 11.3, 2.3) |
60.0 |
C-20 |
23β |
4.56 (td, 11.7, 2.7) |
C-22 |
|
28α |
4.67 (d, 7.2) |
77.9 |
C-3, 4, 6, 29 |
28β |
3.75 (d, 7.2) |
C-4 (w), 5, 6, 29 |
|
29 |
1.02 (s) |
20.5 |
C-3, 4, 5, 28 |
30 |
1.55 (s) |
22.7 |
C-7, 8, 9, 14 |
1’ |
- |
167.6 |
- |
2’ |
- |
130.1 |
- |
3’ |
7.33 (dq, 1.4, 7.1) |
137.8 |
C-1', 2', 4', 5' |
4’ |
1.48 (dd, 7.1, 1.3) |
14.7 |
C-2', 3' |
5’ |
1.83 (brs) |
12.8 |
C-1', 2', 3' |
OH |
6.55, 5.78, 5.69 (3 s br) |
- |
w=weak, vw=very weak
In particular, the NMR data of C-5–C-10 and C-13–C-18 for 1 were very similar to those of azadirachtin I (2) [12], suggesting that these two compounds were structurally related. This was supported by detailed analysis of the 2D NMR data (HSQC, HMBC). The COSY and TOCSY spectra clearly defined the spin system of H-1/H2-2/H-3, H-5/H-6/H-7, and H-15/H2-16/H-17. Furthermore, the HMBC spectrum showed correlations from H-1 to C-3, C-5, C10, C-19 and C-1', from H-3 to C-5, from H-5 to C-3, C-4, C-6, C-7, C-10, C-19, C-28, and C-29, from H-7 to C-5, C-6, C-8, C-9, C-14, and C-30, from H2-28 to C-3, C-4, C-6, and C-29, and from H3-29 to C-3, C-4, C-5, and C-28, confirming the carbon skeleton and the substitution in rings A and B.
Further comparison of the NMR data has shown that compound 1 differed from azadirachtin I (2) by the replacement of the acetyl group in the C-3 position by a hydroxy group, and of the tetrahydrofuran ring at C-20,21 by a 1,4-dioxan moiety. The latter was derived from the 1H and 13C NMR spectra of 1 by the presence of two oxymethylene groups at δ H=4.38/3.52 (δ C=59.5, C-22) and δ H=4.60/3.61 (δ C 60.0, C-23), which formed an isolated spin system in the 1H-1H TOCSY experiment. Moreover, the HMBC spectrum of 1 ([Fig. 1]) gave important correlations between H β -22 and an acetal-methine at δ C=95.0 (C-21), between H α -23 and a quaternary acetal carbon at δ C=93.1 (C-20), and between H-21 with both C-20 and C-22. Therefore, the planar structure of compound 1 was characterized as 3-deacetyl-20,21-defuranyl-20,21-dioxanyl azadirachtin I and named mayombensin.
The relative configuration of mayombensin (1) was established on the basis of NOESY analysis and comparison with data reported in the literature for related compounds [11] [14]. Accordingly, C-11, C-19, and C-30 were assigned as β, and H-5 was α-oriented based on the biosynthesis of azadirachtin-type limonoids [15]. The NOESY spectrum of 1 showed correlations from both H-5 and H-1 to H-9. Further NOESY correlations from H-9 to H3-18, from H3-18 to H-17 and H-21, and from H-21 to H-15 were observed. The same experiment also exhibited correlations between H3-30/H-7, H3-30/H-6, H-6/H3-29, and H3-29/H-3. The relative configuration of mayombensin (1) was thus defined as shown in [Fig. 2], which agrees – with exception for C-11,20 – with all other azadirachtins.
The known compounds were identified as 3,4-dimethyl-secotirucalla-4(28),7,24-trien-3,21-dioic acid (3), stigmasterol, β-sitosterol, and the glucosides of the latter two. The structures were determined by comparison of their NMR and mass spectral data with those reported in the literature [8] [16]. Additionally, two ceramides have been isolated (for data, see Supporting Information).
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Materials and Methods
General procedures
The optical rotation was measured on a Perkin-Elmer polarimeter 241 at the sodium D line. The IR spectrum was recorded on an FT/IR-4100 Jasco spectrophotometer. NMR spectra were recorded on Varian Unity 300 (300.145 MHz) and Varian Inova 500 (499.876 MHz) spectrometers. The NMR data of 1 were referenced on pyridine-d 5 with δ H 7.22 and δ C 150.35; measurements in CDCl3 were referenced to δ H 7.24 and δ C 77.00. ESI HRMS was measured on a microTOF (Bruker mass spectrometer). Melting points were determined on a Mettler FP61 melting point apparatus. Flash chromatography was performed using silica gel (Macherey Nagel & Co; 230-400 mesh). Thin-layer chromatography was performed using Merck pre-coated silica gel 60 F254 aluminum foil.
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Plant material
The twigs of G. mayombensis were collected in December 2014 from Kala Mount, Yaounde Cameroon. Authentication was carried out by M. Victor NANA at the National Herbarium Yaoundé, where a voucher specimen has been deposited (accession number: 46220HNC).
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Extraction and isolation
The air-dried powdered twigs of G. mayombensis (4 kg) were extracted with 10 L of MeOH at room temperature (twice, each 2 days). After evaporation, 75 g of extract were obtained. The crude extract was subjected to column chromatography (100×4.5 cm) over silica gel (70–230 mesh), with a gradient system of n-hexane–EtOAc. A total of 370 fractions of ca. 200 mL each were collected. The pure compounds were obtained by direct crystallization. Fractions 50-67 (2.29 g), eluted with n-hexane–EtOAc (19:1), gave a mixture of stigmasterol and β-sitosterol (36 mg). The combined fractions 117–147 (3.9 g), eluted with n-hexane–EtOAc (17:3), gave 3,4-dimethyl-secotirucalla-4(28),7,24-trien-3,21-dioic acid (3; 32 mg). Fractions 228-245 (2.21 g), eluted with n-hexane-EtOAc (3: 2), afforded ceramide A (8) (18 mg). Fractions 271-285 (1.82 g), eluted with n-hexane-EtOAc (1:1) precipitated at room temperature, gave glucosides of stigmasterol and β-sitosterol (28 mg). The combined fractions 318-326 (1.04 g), eluted with n-hexane–EtOAc (2:3), afforded mayombensin (1) (18 mg). Combined fractions 335-358 (2.99 g), eluted with n-hexane–EtOAc (1:3), yielded ceramide B (9) (8 mg).
Mayombensin (1): White powder; m.p. 209-210°C; + 10.4 (c 0.5, MeOH); IR (film): ν max 3423, 2930, 1697, 1652, 1268, 1142, 1075, 993 cm−1; UV (MeOH): λ max 220 nm (log ε=3.81); 1H and 13C NMR data, see [Table 1]; (–)-ESI HRMS: m/z 593.2583 [M - H]– (calcd. for C30H42O12 593.2598).
3,4-Dimethyl-secotirucalla-4(28),7,24-trien-3,21-dioic acid (3): For 1H, 13C NMR, and MS data, see Supporting Information.
Ceramide A (8): For 1H, 13C NMR, and MS data, see Supporting Information. (+)-ESI HRMS: m/z=704.6138 ([M + Na]+ (calcd. for C42H83NNaO5 704.6169). For the formula, see Fig. S1 in the Supporting Information.
Ceramide B (9): For 1H, 13C NMR and MS data, see Supporting Information. (+)-ESI HRMS: m/z=720.6092 ([M + Na]+ (calcd. for C42H83NNaO6 720.6118); (–)-ESI HRMS: m/z=696.6103 ([M - H]– (calcd. for C42H82NO6 696.6142). For the formula, see Fig. S1 in the Supporting Information.
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Supporting information
Spectral data of compounds 1, 3, 8 and 9 are available as Supporting Information.
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Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgments
We thank Dr. H. Frauendorf and Dr. M. John for MS and NMR measurements, respectively.
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References
- 1 D'Arcy WC. Flora de Panama. Check list and index, Part 1. Saint Louis, MO: Missouri Botanical Garden; 1987: 672
- 2 Schultes RE, Raffauf RF. The healing forest: medicinal and toxic plants of the North West Amazonia. Portland, OR: Dioscorides Press; 1990
- 3 Pereira C, Barreto Júnior CB, Kuster RM, Simas NK, Sakuragui CM, Porzel A, Wessjohann L. Flavonoids and neolignan glucoside from Guarea macrophylla . Quim Nova 2012; 35: 1123-1126
- 4 Oga S, Sertie JA, Brasile AC, Hanada S. Anti-inflammatory effect of a crude extract from Guarea guidonia . Planta Med 1981; 42: 310-312
- 5 Weniger B, Robledo S, Arango GJ, Deharo E, Aragón R, Muñoz V, Callapa J, Lobstein A, Anton R. Antiprotozoal activities of Colombian plants. J Ethnopharmacol 2001; 78: 193-200
- 6 Simoni IC, Munford V, Felicio JD, Lins AP. Antiviral activity of crude extracts of Guarea guidonia . Braz J Med Biol Res 1996; 29: 647-650
- 7 Garcez FR, Nuñez CV, Garcez WS, Almeida RM, Roque NF. Sesquiterpenes, limonoids and coumarins from the wood bark of Guarea guidonia . Planta Med 1998; 64: 79-80
- 8 Akinniyi JA, Connolly JD, Rycroft DS, Sondengam BL, Ifeadike NP. Tetranortriterpenoids and related compounds. Part 25. Two 3,4-secotirucallane derivates and 2’-hydroxyrohitukin from the bark of Guarea cedrata . Canadian J Chem 1980; 58: 1865-1868
- 9 Furlan M, Lopes MN, Fernandes JB, Pirani JR. Diterpenes from Guarea trichilioides . Phytochemistry 1996; 41: 1159-1161
- 10 Moutoo BS, Jativa C, Tinto WF, Reynolds WF, McLean S. Ecuadorin, a novel tetranortriterpenoid of Guarea kunthiana: structure elucidation by 2D-NMR spectroscopy. Canadian J Chem 1992; 70: 1260-126
- 11 Kubo I, Mataumoto A, Mataumoto T. New insect ecdysis inhibitory limonoid diacetylazadirachtinol isolated from Azadirachta indica (Meliaceae) oil. Tetrahedron 1985; 42: 485-496
- 12 Govindachari TR, Sandhya G, Ganesh Raj SP. Azadirachtins H and I: two new tetranortriterpenoids from Azadirachta indica. J Nat Prod 1992; 55: 596-601
- 13 Taylor DAH. Azadirachtin: A study in the methodology of structure determination. Tetrahedron 1987; 43: 2779-2787
- 14 Bilton JN, Broughton HB, Jones P, Ley SV, Lidert Z, Horgan ED, Rzepa HS, Sheppard RN, Slawin AMZ, Williams DJ. An X-ray crystallographic, mass spectroscopic and NMR study of the limonoid insect antifeedant azadirachtin and related derivatives. Tetrahedron 1987; 43: 2805-2815
- 15 Heasley B. Synthesis of limonoid natural products. Eur J Org Chem 2011; 1: 19-46
- 16 Khatun M, Billah M, Quader MA. Sterols and sterol glucosides from Phyllanthus species . Dhaka Univ J Sci 2012; 60: 5-10
Correspondence
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References
- 1 D'Arcy WC. Flora de Panama. Check list and index, Part 1. Saint Louis, MO: Missouri Botanical Garden; 1987: 672
- 2 Schultes RE, Raffauf RF. The healing forest: medicinal and toxic plants of the North West Amazonia. Portland, OR: Dioscorides Press; 1990
- 3 Pereira C, Barreto Júnior CB, Kuster RM, Simas NK, Sakuragui CM, Porzel A, Wessjohann L. Flavonoids and neolignan glucoside from Guarea macrophylla . Quim Nova 2012; 35: 1123-1126
- 4 Oga S, Sertie JA, Brasile AC, Hanada S. Anti-inflammatory effect of a crude extract from Guarea guidonia . Planta Med 1981; 42: 310-312
- 5 Weniger B, Robledo S, Arango GJ, Deharo E, Aragón R, Muñoz V, Callapa J, Lobstein A, Anton R. Antiprotozoal activities of Colombian plants. J Ethnopharmacol 2001; 78: 193-200
- 6 Simoni IC, Munford V, Felicio JD, Lins AP. Antiviral activity of crude extracts of Guarea guidonia . Braz J Med Biol Res 1996; 29: 647-650
- 7 Garcez FR, Nuñez CV, Garcez WS, Almeida RM, Roque NF. Sesquiterpenes, limonoids and coumarins from the wood bark of Guarea guidonia . Planta Med 1998; 64: 79-80
- 8 Akinniyi JA, Connolly JD, Rycroft DS, Sondengam BL, Ifeadike NP. Tetranortriterpenoids and related compounds. Part 25. Two 3,4-secotirucallane derivates and 2’-hydroxyrohitukin from the bark of Guarea cedrata . Canadian J Chem 1980; 58: 1865-1868
- 9 Furlan M, Lopes MN, Fernandes JB, Pirani JR. Diterpenes from Guarea trichilioides . Phytochemistry 1996; 41: 1159-1161
- 10 Moutoo BS, Jativa C, Tinto WF, Reynolds WF, McLean S. Ecuadorin, a novel tetranortriterpenoid of Guarea kunthiana: structure elucidation by 2D-NMR spectroscopy. Canadian J Chem 1992; 70: 1260-126
- 11 Kubo I, Mataumoto A, Mataumoto T. New insect ecdysis inhibitory limonoid diacetylazadirachtinol isolated from Azadirachta indica (Meliaceae) oil. Tetrahedron 1985; 42: 485-496
- 12 Govindachari TR, Sandhya G, Ganesh Raj SP. Azadirachtins H and I: two new tetranortriterpenoids from Azadirachta indica. J Nat Prod 1992; 55: 596-601
- 13 Taylor DAH. Azadirachtin: A study in the methodology of structure determination. Tetrahedron 1987; 43: 2779-2787
- 14 Bilton JN, Broughton HB, Jones P, Ley SV, Lidert Z, Horgan ED, Rzepa HS, Sheppard RN, Slawin AMZ, Williams DJ. An X-ray crystallographic, mass spectroscopic and NMR study of the limonoid insect antifeedant azadirachtin and related derivatives. Tetrahedron 1987; 43: 2805-2815
- 15 Heasley B. Synthesis of limonoid natural products. Eur J Org Chem 2011; 1: 19-46
- 16 Khatun M, Billah M, Quader MA. Sterols and sterol glucosides from Phyllanthus species . Dhaka Univ J Sci 2012; 60: 5-10