Macrocyclic Spermidine Alkaloids from Androya decaryi L. Perrier

Three new spermidine alkaloids and two known compounds were isolated from the leaves of Androya decaryi. Their structures were elucidated on the basis of their spectroscopic data (NMR and mass spectrometry), by X-Ray diffraction and by comparison with literature values. Evaluation of the in vitro antiplamosdial properties of the isolated compounds revealed they did not possess any significant activity.


Introduction
Androya decaryi L. Perrier (Scrophulariaceae) [1] is an evergreen shrub endemic to the southwest of Madagascar.Traditionally used in folk medicine, the decoction of the aerial parts is used to treat yellow fever and malaria-like symptoms (according to local ethnopharmacological investigations).Considering the need for new antiplasmodial compounds due to the emergence of resistant strains of Plasmodium falciparum [2], and the fact that natural products and phytomedicines are still promising sources of antimalarial agents [3][4][5], the aerial parts of A. decaryi were phytochemically investigated.

OPEN ACCESS
Herein, we report the isolation and structural identification of three new macrocyclic spermidine alkaloids 1-2a/b, along with a lignan 3 and (E)-cinnamamide (4) from the leaves A. decaryi, as well as the evaluation of their in vitro antiplasmodial activity.To the best of our knowledge, this is the first report on phytochemistry of the genus Androya and on spermidine alkaloids from a plant belonging to the Scrophulariaceae.

Results and Discussion
Since alkaloids have been detected by TLC in the leaves of A. decaryi, a leaf alkaloid extract was studied.Compounds 1 and 2a (Figure 1) were isolated from the crude alkaloid fraction.Minor alkaloids were also detected; a second extraction was therefore performed on a larger amount of A. decaryi, leading to the isolation of the alkaloid 2b and compounds 3 and 4 (Figure 1).  1) were identified with J-modulated 13 C-NMR spectrum as eight sp 3 methylenes, one sp 3 methine, five sp 2 methines and two sp 2 quaternary carbons, including one amide carbonyl.The 1 H-NMR and HSQC spectra displayed signals for three pairs of aliphatic methylene protons between  H 1.40 and 1.85, five pairs of deshielded methylene protons between  H 2.33 and 3.74, one deshielded methine ( H 4.01), and a monosubstituted benzene ring.These data were consistent with a 13-membered cyclic spermidine alkaloid bearing an aromatic ring.Analysis of COSY and HMBC spectra, measurement of a negative optical rotation ([α] 25 D = −13 (c 3.0, MeOH), and comparison with literature values indicated that compound 1 was (-)-(2S)-2-phenyl-1,5,9triazacyclotridecan-4-one [6].Although this alkaloid has been used as a building block for the synthesis of other natural spermidine alkaloids [6][7][8], it is reported here for the first time as a natural product.Compound 2a was obtained as colorless crystals (CH 2 Cl 2 ).In the HR-ESIMS spectrum, it exhibited a quasi-molecular ion at m/z 310.2501 [M+H] + , establishing a molecular formula of C 17 H 31 N 3 O 2 .The molecular formula and the general framework of the 1 H and 13 C-NMR spectra (Table 2) indicated that compound 2a was also a macrocyclic lactam spermidine alkaloid.The J-modulated 13 C-NMR and HSQC spectra displayed signals for an amide carbonyl carbon at  C 173.2, two methylenes adjacent to an amide function at  C 37.4 (CONH-CH 2 ) and  C 41.9 (CH 2 -CONH), three nitrogenated CH 2 ( C 45.8, 46.3 and 54.4), three methylenes ( C 22.2, 22.5 and 26.9) and one methine carbon at  C 56.5.These signals were consistent with a macrocyclic spermidine moiety and the partial structure was confirmed by analysis of the 1 H-NMR spectroscopic data (Table 2), and COSY and HMBC correlations (Figure 2).Moreover, two additional methylene carbons ( C 20.5, 28.9), one methyl at  C 10.7, and four deshielded methine, including one oxygenated ( C 73.0) and two olefinic methines at  C 123.7 and 126.6, were observed.The 1 H-NMR data and COSY correlations supported the presence of an ethyl moiety adjacent to an oxymethine and a double bond ( H 5.59, d, J = 10 Hz and 5.79-5.84,m).The connectivity between H-2/H-14, H-14/H-15, H-15/H 2 -16, H 2 -16/H-17, H-17/H-18, H-18/H 2 -19 and H 2 -19/H-20 was evident from COSY correlations suggesting that an unsaturated and hydroxylated chain was linked to the macrocyclic lactam through C-2.Further HMBC correlations revealed that C-17 was also connected through N-1 to the macrocyclic lactam (C-17/H-2 and C-17/2H-13).This was in accordance with the downfield chemical shift of C-17 ( C 63.5).The structure of 2a was finally confirmed by single-crystal X-ray diffraction (Figure 3).Moreover, owing to the presence of chloride atoms in the crystal, the absolute configurations of C-2, C-17 and C-18 were assigned as 2S, 17S and 18S using the refinement of the Flack parameter [9].On the basis of the above findings and the measurement of a positive optical rotation [α] 25 D = +12 (c 0.84, MeOH), the structure of 2a was unambiguously elucidated and the trivial name (+)-decaryine A was proposed.(+)-Decaryine A is different from palustrine previously isolated from Equisetum palustre [10,11] with respect to the orientation of the spermidine moiety in the macrocyclic lactam.2) in combination with analysis of HSQC and HMBC spectra revealed the presence of 17 carbons similar to those of compound 2.These data along with 1 H-NMR data (Table 2) and extensive analysis of COSY and HMBC correlations suggested that compound 2b is a diastereoisomer of 2a.The relative and absolute configurations of C-2, C-17 and C-18 could not have been assessed by spectroscopic analyses.Nevertheless, considering the fact that all spermidine alkaloids, including those described here, exhibit the same three-dimensional orientation at C-2 [7,[12][13][14][15][16][17][18][19], we can speculate that the latter carbon is (S)-configurated.Schultz et al. [12] suggested that the first step of the biogenesis of spermidine alkaloids involves an enzyme-catalyzed Michael addition that would lead to the same stereochemistry at C-2 (Scheme 1).They also proposed a potential biogenetic pathway for palustrine [13] that can be adapted to the alkaloids of A. decaryi (Scheme 1).Indeed, a Michael addition of the primary amine of the butyl unit of spermidine to the α,β double bond of 2,4,7-decatrienoic acid, followed by amidification of the second primary amine may give a first intermediate, an alkaloid named myricoidine.Epoxidation of the double bond in the 17,18-position would then allow a nucleophilic attack by the nitrogen atom N-1 of the secondary amine at C-17 that induce the inversion of C-17 stereochemistry, with a concomitant 6-membered ring closure and hydroxyl group formation at C-18.
In that case, we can postulate that epoxidation may occur on both faces of the double bond.Epoxide ring opening could then yield both (2S, 17S, 18S) and (2S, 17R, 18R) diastereoisomers as depicted in Scheme 1.Detection of dihydromyricoidine [19] in crude alkaloidal extract by MS analysis (see Supplementary Material) and notable difference in chemical shift of C-17 ( C 63.5 and  C 57.0 for compound 2a and 2b, respectively) are in agreement with this hypothesis.Therefore, the configuration of compound 2b, named (-)-decaryine B, was proposed as 2S, 17R, 18R.

General
IR spectra were taken on a Perkin Elmer FT-IR Paragon 1000. 1 H and 13 C-NMR spectra were recorded on a Brüker Avance 500 instrument in CDCl 3 or MeOD.HR-ESIMS spectra were recorded on Waters GTC Premier and Waters LCT spectrometers.Single-crystal-X-ray diffraction analysis was performed on a Bruker AXS SMART APEX II diffractometer at low temperature (193(2)K) and using Mo K α radiation (λ = 0.71073 Å).Optical rotations were measured with a Perkin Elmer 241 polarimeter.Column chromatographies were performed over Merck silica gel 60A (40-63 µm) and Sephadex ® LH-20.Analytical and semi-preparative TLC were performed on precoated Kieselgel 60 F 254 plates (Merck, 0.5 mm and 20 × 20, 1 or 2 mm, respectively) using Dragendroff reagent for visualization.CCDC 923477 contains the supplementary crystallographic data for this paper.These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).

Plant Material
Androya decaryi was collected twice by Fré dé ric Manjary (in 2008) and Claude Moulis (in 2009) in the Saint-Augustin region, in southwest Madagascar.Voucher specimens (ADFM2008 and ADCM2009) were deposited at the herbarium of the Institut Supé rieur de Technologie of Toliara, Madagascar.

Extraction and Isolation
The dried and powdered leaves of a first sample of A. decaryi (108 g) were macerated with 100 mL of NH 4 OH (28% aqueous) and then successively percolated with dichloromethane (2 L) and MeOH (1 L).The CH 2 Cl 2 extract was evaporated under reduced pressure at 40 °C to give 3.58 g of a crude extract which was dissolved in CH 2 Cl 2 (200 mL), and extracted with HCl (0.5 M, 3 × 100 mL).The combined aqueous layers were then made alkaline by adding NaOH to pH 12 and extracted three times with CH 2 Cl 2 (200 mL) to obtain the crude alkaloidal fraction (582 mg).This fraction was subjected to Si gel CC eluting with a CH 2 Cl 2 -MeOH gradient to yield seven fractions (F1-7).F7 (110 mg) was subjected to a second Si gel CC in the same conditions to afford compound 1 (40 mg).F5 (150 mg) was rechromatographed over Si gel CC (CH 2 Cl 2 -MeOH gradient) and Sephadex LH 20 (CH 2 Cl 2 then MeOH) to yield F521 (75 mg).Compound 2a was repeatedly precipitated in MeOH/Et 2 O (1/1) from F 521 , and further crystallization of compound 2a in CH 2 Cl 2 afforded colorless crystal (34 mg).

Conclusions
The phytochemical investigation of Androya decaryi led to the isolation of three macrocyclic spermidine alkaloids named (-)-(2S)-2-phenyl-1,5,9-triazacyclotridecan-4-one (1), (+)-decaryine A (2a) and (-)-decaryine B (2b).To the best of our knowledge, compound 1 was previously synthesized, but it is reported here for the first time as a natural product.This is also the first report of spermidine alkaloids in Scrophulariaceae.The absolute configurations of alkaloids 1 and 2a were unambiguously elucidated, supporting the hypothesis described by Hesse and co-workers of a common initial biogenetic step.A similar biogenetic pathway was then postulated for compound 2a and 2b, allowing us to propose an absolute configuration for (-)-decaryine B (2b).The structural complexity of macrocyclic spermidine alkaloids has stimulated numerous syntheses, especially to unambiguously determine their absolute configuration [12,13,18,19,27,28].Thus, enantioselective synthesis of (-)-decaryine B would confirm these hypotheses.Evaluation of their antiplasmodial properties showed that compounds 1, 2a, 3 and 4 exhibited no significant activity against Plasmodium falciparum.

Figure 3 .
Figure 3. Perspective drawing of the X-Ray structure of compound 2a hydrochloride.Thermal ellipsoids are set at the 50% probability level.The solvent molecule, the chloride counter anion and all hydrogen atoms, except those on the chiral carbons, have been omitted for clarity.
a Assignment of13C-NMR data based on J-modulated, HSQC and HMBC spectra.