Angelocunhol: New Erythroxylane Diterpene and Other Compounds from Simira sampaioana (Rubiaceae)

Abstract

The first Simira sampaioana (Rubiaceae) phytochemical study allowed the isolation and structural determination of a new erythroxylane diterpene named Angelocunhol, 11β,12α-dihydroxy-2,4(18),15-eritroxilatrien-1-one, together with 14 known compounds: simirane B, harman, maxonine, isomalindine, malindine, sitost-4-en-6-ol-3-one, estigmast-4,22-dien-6-ol-3-one, campest-4-en-6-ol-3-one, sitost-4-en-3-one, stigmast-4,22-dien-3-one, campest-4-en-3-one, β-sitosterol, stigmasterol, and stigmast-4,22-dien-3-ol from the wood of a specimen of the species. The structures of these compounds were elucidated on the spectroscopic-data analysis basis, mainly ¹H and ¹³C nuclear magnetic resonance (NMR), including 2D experiments (¹H-¹H correlation spectroscopy (COSY), nuclear Overhauser spectroscopy (NOESY), heteronuclear multiple-bond correlation-HMBC and heteronuclear single-quantum correlation-HSQC), and high-resolution electrospray mass spectrometry (HRESI-MS).

Keywords:
Simira sampaioana; Rubiaceae; diterpene; alkaloid; steroid

Introduction

Species from Simira genus of Rubiaceae family have been investigated mainly due to the biological activities. Many of these species have been used by diverse communities as coloring producers, antifebrile, tonic and purgative substances, and by the phototoxic activities presented by some of their chemical constituents.^1-4Simira sampaioana (synonyms Sickingia sampaioana in the Atlantic Rainforest) is known by its common names "arariba", "canela-samambaia", "marfim" and "maiate", and economic interest is justified by use as timber and for the afforestation of streets.^1,5 As the first phytochemical study involving S. sampaioana, this article describes the isolation and structural characterization of the new diterpene 11β,12α-dihydroxy-2,4(18),15-eritroxilatrien-1-one, named Angelocunhol (1), together with 14 known compounds: simirane B (2), harman (3), maxonine (4), isomalindine (5), malindine (6), sitost-4-en-6-ol-3-one (7), estigmast-4,22-dien-6-ol-3-one (8), campest-4-en-6-ol-3-one (9), sitost-4-en-3-one (10), stigmast-4,22-dien-3-one (11), campest-4-en-3-one (12), β-sitosterol (13), stigmasterol (14), and stigmast-4,22-dien-3-ol (15) (Figure 1) from the wood of the plant. The structures of all compounds were characterized by 1D and 2D nuclear magnetic resonance (NMR) techniques, and also the high-resolution electrospray ionization mass spectrometry (HRESI-MS), infrared spectroscopy (IR) and comparisons with known compounds available in literature data.

Figure 1
Compounds isolated from S. sampaioana.

Results and Discussion

Angelocunhol (1) was obtained as yellow oil. Its IR spectrum exhibited bands at ν_max 3362 (broad, ν_OH) and 1649 cm^-1 (ν_C=O of an α,β-unsaturated carbonyl group). The molecular formula was assigned as C₂₀H₂₈O₃, based on the quasi-molecular peak at m/z 339.1934 ([M + Na]⁺, calcd. for C₂₀H₂₈O₃Na, m/z 339.1951) revealed by the HRESI-MS (positive mode). This molecular formula suggested a diterpene skeleton (Scheme 1) with seven degrees of unsaturation. Three methyl groups were identified by single signals at δ_H 1.24 (s, 3H-19), 1.21 (s, 3H-20) and 1.09 (s, 3H-17) in the ¹H NMR spectrum; the presence of an exocyclic double bond (=CH₂-18) and a vinyl group was deduced by signals at δ_H 5.33 (s, H-18a) and δ_H 5.32 (s, H-18b), indicating an AB system, and δ_H 5.90-5.97 (m, H-15), δ_H 5.08 (dd, J 17.5, 1.1 Hz, H-16a) and δ_H 5.03 (d, J 10.8, 1.1 Hz, H-16b) compatible with an ABX system of vinyl group. Two doublet signals (J 9.8 Hz) corresponding to olefinic hydrogens at δ_H 5.98 and δ_H 6.98 were attributed to H-2 and H-3, respectively. The distortionless enhancement by polarization transfer with retention of quaternaries (DEPTQ) ¹³C NMR spectrum allowed to recognize signals corresponding to 20 carbon atoms (Table 1): three methylics, five methylenics (including two sp² at δ_C 117.6 and 111.3), seven methynics (including three sp² at δ_C 147.6, 145.8 and 127.8 and two sp³ oxygenated at δ_C 79.8 and 76.9) and five quaternary (including one of ketone carbonyl at δ_C 205.9), allowing to establish the expanded molecular formula C₂₀H₂₆O₃ and the two hydrogen atoms needed to complete the molecular formula C₂₀H₂₈O₃ justified by two hydroxyl groups. These hydroxyl groups were located at CH-11 and CH-12 by the signals at δ_H 3.23 (d, J 9.0 Hz, H-11) and 3.52 (d, J 9.0 Hz, H-12) revealing vicinal spin-spin interaction each other in the ¹H-¹H correlation spectroscopy (COSY) and heteronuclear correlations in the heteronuclear single-quantum correlation (HSQC) (¹J _CH) spectrum with the ¹³C signals at δ_C 79.8 (CH-11) and 76.9 (CH-12) and in the heteronuclear multiple-bond correlation (HMBC) these ¹³C signals showed long-range heteronuclear correlations of the CH-11 (δ_C 79.8) with H-10 (δ_H 2.60, ³J _CH) and 3H-20 (δ_H 1.21, ³J _CH) and of the CH-12 (δ_C 76.9) with H-11 (δ_H 3.25, ²J _CH), H-15 (δ_H 5.97-5.90, ³J _CH) and 3H-17 (δ_H 1.09, ³J _CH), summarized in Table 1. The interaction of CH-11 (δ_C 79.8) with the 3H-20 (δ_H 1.21, ³J _CH) suggested the presence of this methyl group at carbon C-9 (δ_C 42.86), revealing its rearrangement of the carbon C-10 (δ_C 64.45) and signaling for a skeleton. Thus, the hydrogen chemical shifts and hydrogenated carbon atoms were unambiguously assigned by analysis of ¹H-¹H-COSY and HSQC spectra data (Table 1). The HMBC spectrum showed long-range heteronuclear correlations, which were used to confirm the carbon skeleton and localization of the substituents (Table 1). The HMBC spectrum also revealed correlations of hydrogen atoms H-3 (δ_H 6.98) and H-10 (δ_H 2.60) signals with carbon atom C-1 (δ_C 205.3), H-3 (δ_H 6.98) with carbon atom CH₂-18 (δ_C 117.60) and H-10 with carbon atom CH₃-19 (δ_C 24.74), in accordance with a erythroxylane skeleton containing a dienone system involving the carbon atoms C-1 (δ_C 205.3), CH-2 (δ_C 127.84), CH-3 (δ_C 145.79) and the exocyclic double bond CH₂-18 (δ_C 117.60). Thus, these data were used to postulate the structure of a diterpene with erythroxylane skeleton,⁶ corroborated by long-range correlations of CH-11 (δ_C 79.8) with the 3H-20 (δ_H 1.21) and of the CH-12 (δ_C 76.9) with H-11 (δ_H 3.25), H-15 (δ_H 5.97-5.90) and 3H-17 (δ_H 1.09) through the location of hydroxyl groups at 11 and 12 positions (Table 1). Additional heteronuclear long-range couplings are summarized in Table 1.

Scheme 1
Proposed fragmentation mechanisms of 1 (only peaks classified as principals).

Table 1
¹H NMR (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data for 1 . Chemical shifts (δ, ppm) and coupling constants ( J , Hz, in parenthesis)^a

	HSQC			HMBC
	δC / ppm	δH / ppm		2JCH /Hz	3JCH /Hz
C
1	205.29	−		H-10	H-3
4	154.30	−		H-3; 2H-18	H-2; H-10; 3H-19
5	43.33	−		H-10; 3H-20	H-3; 2H-18
9	42.86	−		H-10
13	40.69	−		H-12; H-15	2H-16
CH
2	127.84	5.98 (d, 9.8)
3	145.79	6.90 (d, 9.8)
8	40.58	1.45			3H-20
10	64.45	2.60 (s)			H-2; H-11; 3H-19; 3H-20
11	79.83	3.23 (d, 9.0)			H-10; 3H-20
12	76.91	3.52 (d, 9.0)		H-11	H-15; 3H-17
15	147.64	5.94 (m)		2H-16	H-12; H-14a; 3H-17
CH2
6	39.15	1.59 (t, 13.2)1.09			3H-19
7	24.34	1.63 (m), 1.42 (m)
12	−	−		−	−
14	37.76	1.91 (m), 1.23 (m)			3H-17
16	111.26	5.08 (dd, 17.5, 1.1) 5.03 (dd, 10.8, 1.1)
18	117.60	5.33 (m), 5.32 (s)			H-3
CH3
17	17.25	1.09 (s)			H-12; H-15
19	24.74	1.24 (s)			H-10
20	10.65	1.21 (s)			H-10; H-1

• ^a
  Number of hydrogens bound to carbon atoms deduced by DEPTQ-¹³C NMR spectrum. Chemical shifts and coupling constants (J) obtained from 1D ¹H NMR spectrum. Superimposed ¹H signals are described without multiplicity and chemical shifts deduced by ¹H-¹³C COSY ¹J_CH (HMQC), ¹H-¹³C COSYⁿJ_CH (n = 2 and 3, HMBC) and ¹H-¹H-COSY spectra; HSQC: heteronuclear single-quantum correlation; HMBC: heteronuclear multiple-bond correlation.

The relative stereochemistry of 1 (Figure 1) was determined by the relevant hydrogens coupling constants revealed by ¹H NMR and ¹H-¹H-COSY spectra and from the dipolar-dipolar interaction observed in the ¹H-¹H-NOESY spectrum (Figure 2). The value corresponding to vicinal interaction (³J

Figure 2
Nuclear Overhauser effect spectroscopy (NOESY) (↔) correlations for compounds 1.

_H,H) between the hydrogens H-11 and H-12 (J 9.0 Hz) is consistent with axial-axial interaction⁷ as appear in Figure 1, which also reveals dipolar interaction between H-12 (δ_H 3.52)/3H-20 (δ_H 1.21), H-10 (δ_H 2.60)/H-11 (δ_H 3.23) and H-11 (δ_H 3.23)/3H-17 (δ_H 1.09) revealed by ¹H-¹H nuclear Overhauser effect spectroscopy (NOESY) spectrum through the cross-peaks assigned to corresponding dipolar interaction (special proximity) shown in Figure 2 (1a and 1b) which indicates the axial positions for these hydrogens. Furthermore, coupling values observed in the ¹H NMR spectrum for these hydrogens indicate axial-axial interaction.

Thus, the analysis of the spectral data allowed the structural characterization of this new erythroxylane diterpene 11β,12α-dihydroxy-2,4(18),15-eritroxilatrien-1-one (1), named Angelocunhol as a simple tribute to colleague and friend Angelo da Cunha Pinto, excellent researcher who died in November 7, 2015.

The results of the extensive application of 1D and 2D NMR spectral techniques were also used to confirm the structure and to establish the ¹H and ¹³C resonance assignments of 1 (Table 1). Proposed fragmentation mechanisms of this new diterpene 1 (only peaks classified as principals) was summarized in Scheme 1.

The known simirane B (2),⁶ harman (3),^6,8 maxonine (4), isomalindine (5), malindine (6),⁹ sitost-4-en-6-ol-3-one (7), estigmast-4,22-dien-6-ol-3-one (8), campest-4-en-6-ol-3-one (9), sitost-4-en-3-one (10), stigmast-4,22-dien-3-one (11), campest-4-en-3-one (12),^10,11 β-sitosterol (13), stigmasterol (14),¹² and stigmast-4,22-dien-3-ol (15) were identified by spectral data, involving mainly 1D and 2D ¹H and ¹³C NMR spectra and comparison with literature values.

Conclusions

A total of fifteen compounds were isolated from S. sampaioana (Rubiaceae), among which four alkaloids, nine steroids and two diterpenes. These compounds are in agreement with the secondary metabolites produced by plants of the Rubiaceae family and of the Simira genus. It is angelocunhol compound (1) unprecedented in the literature, and isomalindine (5), malindine (6), sitost-4-en-6-ol-3-one (7), estigmast-4,22-dien-6-ol-3-one (8), campest-4-en-6-ol-3-one (9), stigmast-4,22-dien-3-one (11), campest-4-en-3-one (12) and stigmast-4,22-dien-3-ol (15) were reported by first time in the genus. And yet the harman alkaloid (2), corroborating the proposition of this alkaloid the taxonomic marker of genus.

Experimental

General experimental procedures

Measure of IR data was on Shimadzu IRAffinity-1. NMR spectra were obtained on Bruker DRX-500 and Avance IIIH (both 500 MHz for ¹H and 125 MHz for ¹³C), with CDCl₃ (0.1% tetramethylsilane, TMS) or dimethylsulfoxide (DMSO-d₆) as solvents, used as internal references (δ_H 7.24 and δ_C 70.00). Low-resolution mass analysis was done on Shimadzu GCMS-QP5050A and high-resolution mass spectrometry (HRMS) analysis on Bruker microTOF electrospray-time-of-flight mass spectrometer (ESI-TOF-MS) equipped with an ESI source in the positive and negative modes. Column chromatography was conducted using silica gel (Merck). Precoated thin layer chromatography (TLC) sheets (Merck) of silica gel 60 GF254 (0.25 mm) were used, and visualization of plates was carried out using a lamp UV 254 and 356 nm and vanillin (1%) solution in H₂SO₄ (5%).

Plant material

The S. sampaioana specimen employed in this study was collected at the Companhia Vale do Rio Doce (CVRD) Atlantic Rainforest, in Linhares city, Espírito Santo State, Brazil, and was identified by Domingos A. Folli. A voucher specimen (CVRD 8796) is deposited at the company's herbarium.

Extraction and isolation

The dried and powdered wood (6 kg) was extracted with Hexane and MeOH at room temperature and providing 0.004 kg of Hexane crude extract and 0.5 kg of MeOH crude extract after solvent evaporation.

The Hexane extract was subjected to column chromatography (CC) (SiO₂, gradient Hex/EtOAc) furnishing eleven fractions. The fraction 9 (333.7 mg) was chromatographed over a silica gel column with a hexane/ethyl acetate (9:1, v/v) to yield pure compound 1 (6.9 mg) besides a solid identified as a mixture of the compounds 7 + 8 + 9 (6.4 mg) by gas chromatography-mass spectrometry (GC-MS) analysis. The fraction 8 (138.5 mg) was chromatographed over a silica gel column with a hexane/ethyl acetate (9:1, v/v) to yield pure compound 2 (4 mg). The fraction 7 (522.3 mg) was chromatographed on silica gel column using hexane/acetone (9:1, v/v), which furnished a solid identified as a mixture of the compounds 11 + 12 (7.6 mg) by GC-MS analysis. The mixture of the compounds 10 + 15 (55.5 mg) and the mixture of the compounds 13 + 14 (94.4 mg) identified by GC-MS analysis, were isolated from the fraction 5 (515.2 mg) by chromatography using hexane/ethyl acetate (9.5:0.5, v/v).

An aliquot of this extract (2.5 g) was dissolved in a MeOH-H₂O (8:2 (v/v), 100 mL) mixture and partitioned with CH₂Cl₂ (5 × 100 mL), EtOAc (5 × 100 mL) and n-BuOH (5 × 100 mL), to give the following fractions: CH₂Cl₂ (1.3 g), EtOAc (0.2 g), n-BuOH (0.9 g). The CH₂Cl₂ fraction was subjected to CC (SiO₂, gradient MeOH/CH₂Cl₂) to yield pure compounds 3 (6.4 mg) and 4 (5.4 mg), and the n-BuOH fraction was subjected to CC (SiO₂, gradient MeOH/CH₂Cl₂) to yield pure compounds 5 (4 mg) and 6 (7.3 mg).

Supplementary Information

Supplementary information, including ¹H NMR, ¹³C NMR, COSY, NOESY, HSQC, and HMBC spectra, as well as mass spectra and IR (Figures S1-S9), are available free of charge at http://jbcs.sbq.org.br as a PDF file.

https://minio.scielo.br/documentstore/1678-4790/hVBF6bWDVWfc4G7gKyKxKXS/e80988f41db77bcb0f148c681f0ed998edb363b7.pdf

• Dedicated to Prof Angelo da Cunha Pinto (in memoriam) for his outstanding contributions to the field of Organic Chemistry in Brazil. This article was submitted to the special issue dedicated to Professor Angelo da Cunha Pinto and name Angelocunhol is also additional homage.

Acknowledgments

The authors are grateful to FAPERJ, CAPES and CNPq for the financial support and research fellowships (CNPq). To Prof Mário Geraldo de Carvalho, Departamento de Química-Instituto de Ciências Exatas, UFRRJ, for access to the NMR and GC-MS facilities through the technicians Vitor de Almeida/Maurício Lemos and Frances Regiane dos Santos, respectively.

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Publication Dates

• Publication in this collection
  Jan 2017

History

• Received
  27 Feb 2016
• Accepted
  19 May 2016