1H and 13C-NMR Data of the Simplest Plumeran Indole Alkaloids Isolated from Aspidosperma Species

Indole alkaloids are the chemotaxonomic markers of the Aspidosperma genera. Those that have the simplest plumeran skeleton are classified as the precursors of biosynthetic routes and the intermediates for several synthetic reactions. This work aims to review the 1H and 13C-NMR data, up to 2011, describing the skeleton of 35 different plumeran indole alkaloids, from a group of 46 of them, and highlight the main spectral differences amongst them.


Introduction
The Apocynaceae family consists of 424 genera, divided into 17 tribes that are subdivided under five subfamilies: Rauwolfioideae, Apocynoideae, Asclepioideae, Periplocoideae and Secamonioideae [1]. About 400 species of Apocynaceae have been identified and grouped into 41 genera in Brazil, 78% of which are found only in the Amazonia region [2]. Several Apocynaceae are used for landscaping. These include the arboreal Tabernaemontana and Plumeria, the climbing Alamanda and Mandevilla, and the shrubby Catharanthus and Nerium [3]. The wood of some species is of particular commercial importance for local development. Species such as the popularly named "peroba" and "guatambu" are useful for making furniture and buildings [4].
The Aspidosperma genus is included in the Rauwolfioideae subfamily, Alstoniae tribe. The genus includes about 57 species, divided according to their chemotaxonomy into eight series-Rigida,

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Nitida, Quebranchines, Polyneura, Pyricolla, Nobile, Macrocarpa and Tomentosa, all of which are restricted to the American tropical and subtropical regions [5,6]. Aspidosperma is another genus of Apocynaceae of high commercial value due the good quality of its wood.
Some species are also used in folk medicine; the infusion of its stem barks is used to treat a number of diseases [7]. For example, the extracts of the Aspidosperma stem bark, are characterized by the presence of indole alkaloids with high structural diversity. These alkaloids are responsible for the many pharmacological effects known for the plant [8]. The experimental assays [9][10][11][12][13] involving A. ramiflorum Müll. Arg., A. pyrifolium Mart., A. megalocarpon Müll. Arg., A. macrocarpon Mart. and A. quebracho-blanco Schltdl. species, attest to their popular use as an antileishmanial and an antimalarial. Alkaloids found in root bark of A. ulei Markgr. were used to evaluate and prove pro-erectile effects [14,15]. A. subincanum Mart., used in folk medicine to treat Diabetes mellitus and hypercholesterolemia [16], was proven to have a low acute toxicity in in vivo tests, resulting in its characterization as a non-toxic treatment [17]. A. ramiflorum Müll. Arg. exhibited antibacterial activity against the standard strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) [18] bacteria. The extracts of several different parts of A. polyneuron Müll. Arg. were tested against a wide range of fungi; only the ethanolic extract of the stem was able to inhibit the growth of Cladosporium herbarum [19].
The structural diversity of the indole alkaloids can be classified by examining their biosynthetic origins. The occurrence of complex alkaloids containing indole moieties is restricted to a few families; the best sources appear in the Apocynaceae, Loganiaceae and Rubiaceae families [20]. The indole alkaloids are N-methyl derivatives of tryptophan, which has a terpenic unit that originates from the mevalonic acid pathway. The metabolic origins of the remaining portion consist of a chain of ten carbon atoms, the sub-architecture of which is useful for dividing indole alkaloids into three classes: Iboga, Corynanthe and Aspidosperma. Usually, the C 9 or the C 10 chain units are shown to be of a terpenoid origin and are identified as secologanins (secoiridoids) [20,21].
Each of the three classes mentioned can be subdivided again, giving rise to nine other subclasses ( Figure 1), according to Danieli and Palmisano [22]. Vincadifformine (1), as well as tabersonine (which presents a double bond between C-14 and C-15) are of Aspidosperma type. However, the loss of the carbon atom linked to C-16 (indicated by a circle and corresponding to the carboxylate function of secologanin) by hydrolysis/decarboxylation, originates most of the plumeran indole alkaloids [21].
This work presents a review of the literature describing the 1 H and 13 C-NMR data of 35 alkaloids with a simplest plumeran skeleton. This alkaloid subclass was chosen for their structural diversity (the basic ring skeleton is representative of a large number of natural compounds) and the review of Pereira et al. (2007) [2] was adopted as a guideline. In this referred work, the criterion of classification is the same as that adopted by Manske [28]. Some structures (nomenclature and structure) were adjusted based on the literature. The numbering of the structures in this work followed the method proposed by Le Men and Taylor ( Figure 2) [20]. In many cases, it was not possible to obtain the NMR data, because the compounds' structures were elucidated by other techniques (Infrared-IR, Ultraviolet-UV, Mass Spectrometry-MS).  [20].     (2), spegazzinine (3 and 4), aspidospermidine (5 and 6) and pyrifolidine (7 to 9) skeletons [28].  and Aspidoscarpine (20 to 27) types [28].

Discussion
The structure of the alkaloid aspidospermine (8) has the basic ring skeleton typical of a large number of natural alkaloids. In addition to the characteristic signals for the aromatic methoxyl function, the 1 H-NMR spectrum shows a very common pattern amongst the indole alkaloids the N-acetyl group, the C-20 ethyl side chain, and the lone hydrogen atom attached to CH-21, which is not split by any neighboring hydrogen [56].
Thus, the 1 H and 13 C-NMR spectra of the alkaloids with the plumeran skeleton present key characteristic signals of the typical aspidospermine pattern. The hydrogen linked at CH-2 shows a signal with a double-doublet multiplicity (due the low sensibility of the 1960's NMR spectrometers, it was originally characterized as a quartet). This feature indicates that CH-2 hydrogen couples its spin with the two hydrogen atoms at C-16 [56]. The values for chemical shifts of the H-2 vary towards δ H 4.5 in the 1 H-NMR and the CH-2 appears as δ C 66 in the 13 C-NMR spectra.
For the main consulting reference molecule aspidospermine (8), the data relative to the hydrogen attached to CH-2 was missing [34] and did not appear on the spectra. Its presence was deduced by the signal of a methine carbon at δ C 64 in the 13 C spectrum. This information was then supplied by another source [35], to the best of our knowledge.
Note that for the alkaloids that present a substituent in C-16, there is a striking difference. Vincadifformine (1) is presented here as a precursor of the plumeran skeleton, by the loss of the carbon linked to C16, by a hydrolysis/decarboxylation reaction [21]. For this molecule, the presence of a double bond between C-2 and C-16 justify the absence of the signal of H-2 in the 1 H-NMR. In the 13 C-NMR, the chemical shift for C-2 appears at δ C 167.8, what may be explained by the presence of the nitrogenous atom and by its conjugation with the carbonyl group, which is also the reason for the chemical shifts of the of the carbomethoxy at δ C 169 and the carbon atom C-16 at δ C 92.8, which is very characteristic of vincadifformine [30].
Spegazzinine (3) and spegazzinidine (4) are also substituted in C-16. The signal of H-2 in the 1 H-NMR spectra appears as a doublet, due to the coupling of the hydrogen attached to CH-2 with the only hydrogen atom in CH-16. The assignments in the 1 H-NMR spectrum for spegazzinine showed signals for three aromatic hydrogen atoms, an N-acetyl function and a C-ethyl group. The consulted bibliography did not provide detailed information regarding the orientation of the aromatic hydrogen atoms [31]. However, the nature of these hydrogens is better known today due to the modern techniques available, and it is now known that there are two doublets attributed to H-9 and H-11, thus revealing ortho interactions and one triplet corresponding to H-10. The 1 H-NMR spectrum of spegazzinidine demonstrated a significant difference only in the region corresponding to aromatic hydrogens; the two aromatic hydrogens have an ortho interaction.
The relative configuration of the additional asymmetric center, C-16, in spegazzinine and spegazzinidine was defined because of the constant coupling (J = 8.0 Hz), a value typical of axial-axial hydrogens at CH-2 and CH-16 [31].
For 1,2-dehydroaspidospermidine (5), the presence of a non-substituted carbon at δ C 193.0, assigned as C-2, linked to the indolic nitrogenous atom by a double bond, characterizes it as an indolenine system. As is the case of vincadifformine (1), the presence of a double bond between C-2 and N justify the absence of the signal of H-2 in the 1 H-NMR. The aromatic hydrogens have the expected couplings for a non-substituted indolenine system: a doublet attributed to H-9 with chemical shift of δ H 7.53, two multiplets attributed to H-10 and H-11 (each with a chemical shifts of δ H 7.25), and a triplet attributed to C-12 with δ H 6.17.
Another important feature is the displacement of the chemical shift of the H-12 at non-substituted aromatic ring, as was evident for the demethoxyaspidospermidine (14) and 12-demethoxy-Nacetylcylindrocarine (43). The displacement occurs due to the attenuation of the mesomeric effect of the nitrogenous atom by the presence of an acyl group. This, in addition to the anisotropic effect of the carbonyl group at CH-12 results in the deshielding of the hydrogens at CH-10 and CH-12.
The compounds 9 and 23, isolated and characterized by Oliveira [32] as new alkaloids, were not reported in the guideline literature [2]. For these two alkaloids, the presence of the methoxy group at CH-15 is deduced by the presence of chemical shifts at δ H 3.22  and at δ C 75.5. This is a new substitution pattern observed among the Aspidosperma alkaloids.
Hitherto, all the structures have the same ethyl feature at the C-20 side chain. Due to the optical rotation [α] D , one-( 1 H-NMR, 13 C-NMR-{ 1 H}and 13 C-NMR-DEPT or APT) and two-dimensional (homonuclear 1 H-1 H-COSY, and 1 H-1 H-NOESY; heteronuclear HMQC or HSQC-1 J HC and HMBC-n J HC , n = 2 and 3) NMR experiments, the stereochemistry of this group and the absolute configuration of each of these molecules was well established.
Nevertheless, limaspermine (28) and related alkaloids 29 to 35 reveal the oxidative biotransformation of the methyl group present in the carbon atom C-20-ethyl substituent (C-20-CH 2 CH 3 ) to yield C-20-CH 2 CH 2 OH, presenting an expressive difference at the lateral chain at C-20 [23,53]. The presence of these moieties in such structures may be recognized by a comparative analysis of the 1 H-NMR spectra: -CH 2 CH 3 deduced by triplet signal at δ H 0.6 is attributed to the hydrogens of the methyl group (3H-18) and -CH 2 CH 2 OH is characterized by a triplet signal at δ H 3.5 corresponding to the carbinolic hydrogens of the methylene CH 2 OH (2H-18). The 13 C-NMR data for these compounds are not available, so the expected differences of the C-18 and the C-19 in the 13 C-NMR spectrum can only be predicted, by considering the displacement that occurs to deshielded fields.
In the alkaloids 36 to 46 ( Figure 5) the carbon atom C-20 sustains a -CH 2 COOMe containing a carbomethoxy group (COOMe), which may be explained by the additional oxidative biotransformation of CH 2 CH 2 OH to CH 2 COOH, which is then followed by a methylation reaction. The presence of the C-20-CH 2 COOMe can be recognized by 1 H and 13 C-NMR spectra as the anticipated modifications of the 1 H and 13 C chemical shifts. The singlet signal corresponding to the hydrogen of the methine carbon CH-21 demonstrates the influence induced by the proximity of the carbonyl group at C-20. This contributes to the signal displacement of the CH-21 hydrogen to δ H 2.4 (s), which is different from the observed value of δ H 2.23, found for aspidospermine [55]. The most impressive result involves the modifications observed by carbon atoms C-18 and CH 2 -19 in the 13 C-NMR spectra: δ C 171.0 and δ C 42.0, respectively. Data analysis suggests that for the alkaloids 35, 36 and 38, the values attributed to aromatics and the CH-21 hydrogens were exchanged. The chemical shifts for the aromatics remain towards δ H 6.5-7.0 and for the hydrogen attached at CH-21 remains towards δ H 2.2, in accordance with all the other compounds for the same series (36 to 46).
In regards to the aromatic hydrogens, it is important to notice that for the structures 42, 44, and 46, the aromatic signals are concerning 8 hydrogens-3 being part of the monosubstituted A ring and the other 5 of a benzyl substituent.
The expected differences associated with the N-acyl substituent also appear in the cylindrocarpidine (39) and its cynnamoyl derivative N-cynnamoyl-19-hydroxycylindrocarine (46). They exhibit the absence of the acetyl group and the appearance of lines relative to cynnamoyl system [55]. The coupling constant value (J = 16 Hz) observed in the olefinic signals of the 1 H-NMR spectra was used to confirm the trans configuration of the double bond present in the cynnamoyl moiety [50].

Conclusions
The initial purpose of this work was to emphasize a review of the 1 H and 13 C-NMR spectral data for a small group of alkaloids in a discussion that would highlight the spectral differences amongst them. However, we observed that most of the literature data were reported in the 1960's and the NMR data were incomplete or even unavailable. At that time, the structures of some alkaloids were elucidated on the basis on IR (Infrared) and/or UV (Ultraviolet) and/or MS (Mass Spectrometry) techniques, involving also chemical transformations, before the development of the NMR. Even in the beginning, the NMR spectra presented low sensitivity due to the limited equipment accuracy (frequently about 60 MHz).
Through the modern comparative analysis of 13 C-NMR-{ 1 H}-and DEPT-or APT-13 C-NMR spectra, it becomes easier to differentiate, classify, and count the signals corresponding to quaternary, methane, methylene and methyl carbon atoms. The DEPTQ (Q = quaternary), appears as a new variation of DEPT for the inclusion of signals corresponding to quaternary carbon atoms. The basic skeleton of the plumeran alkaloids (non-substituted molecules) reveals the presence of four non-hydrogenated (two sp 2 and two sp 3 ), six methine (four sp 2 aromatics and two sp 3 ), eight methylene (all sp 3 ) and one methyl carbon atoms, which may be recognized through this comparative analysis. Obviously, the modification of the numbers of CH, CH 2 and CH 3 by presence and type of substituent may be characterized with relative facility. This analysis in collaboration with high resolution mass spectrometry significantly contributes to the deduction of a molecular formula. And so, it is possible to classify an unknown alkaloidic molecule in terms of carbon patterns, which would to facilitate the elucidation of its molecular form. Also, the 2D NMR experiments ( 1 H-1 H-COSY, 1 H-1 H-NOESY, HMQC or HSQC and HMBC) are especially helpful to solve structural problems, allowing especially assignments of methylene groups that frequently appear in region revealing superimpose [34].
So, this work proposes that not only the chemical shifts for the CH-2 hydrogen (absent in the presence of a double bond between this carbon and the N-1δ C 193.0 for C-2 as reveled by alkaloid 5), but also the assignments for the CH-21 and the ethyl group (and its biosynthetic derivatives) sustained by C-20, are the main spectrometric features that characterize the presence of the plumeran alkaloidic skeleton.