Caryopristimerin , the First Example of a Sesquiterpene-Triterpene Homo Diels-Alder Adduct , and a New 29-nor-Friedelane from Roots of Salacia crassifolia

Two new compounds, caryopristimerin and 2α,3α,22β-trihydroxy-21-oxo-29-nor-friedelan24-oic acid, were isolated from the hexane/ethyl ether extract of Salacia crassifolia roots. Caryopristimerin represents the first example of a homo Diels-Alder adduct of a sesquiterpene and a triterpene, and the new 29-nor-friedelane displays a highly oxygenated A ring with a carboxylic group at the unusual C-5 position. The new compounds were elucidated by infrared (IR), highresolution-atmospheric pressure chemical ionization-mass spectrometry (HR-APCI-MS), 1D/2D nuclear magnetic resonance (NMR) and single crystal X-ray diffraction analysis. Additionally, the known compounds 3-oxo-29-hydroxyfriedelane, pristimerin, tingenone and netzahualcoyonol are herein reported for the first time as constituents of S. crassifolia. Their structures were established by spectroscopic analysis.


Introduction
The genus Salacia comprises more than 100 species and belongs to the Celastraceae family. 1 It has been used in traditional medicine around many regions of the world, from South America to Asia. 2,35][6][7] The isolated triterpenoids display a variety of skeletons like friedelane, [6][7][8] ursane, oleanane and quinonemethide. 6,8,9The latter is only found in the roots and serves as a biomarker for the Celastraceae family. 10,11alacia crassifolia (Mart.ex Schult.)G. Don is native from the Brazilian biome "Cerrado" and popularly known as "bacupari", "bacupari de caapuêra" and "saputá".Its leaves, stems, seeds and fruits are used in herbal medicine for the treatments of gastric ulcers, pediculosis capitis, common kidney disorders, chronic cough, headaches, malaria and skin cancer. 12The leaves were studied by Rodrigues et al., 13 who isolated friedelanes, ursanes and oleananes, among other compounds.However, there are no further phytochemical studies of different parts of this plant.Therefore, this work aims to isolate and characterize compounds from roots of Salacia crassifolia.
The phytochemical study led to two novel and unusual compounds: a dimer named as caryopristimerin (1) and the 2α,3α,22β-trihydroxy-21-oxo-29-nor-friedelan-24-oic acid (2).Quinonemethide dimers with aromatic moieties linked together by two ether bonds between the A rings via hetero Diels-Alder reaction are common in roots of the Celastraceae family. 14Diels-Alder adducts from quinonemethide and guaiane sesquiterpene with the same kind of linkages were also described for Cheiloclinium hippocratioides (Celastraceae). 15However, this is the first time that a dimer between a quinonemethidederivative and the sesquiterpene caryophyllene (1) linked by carbon-carbon bonds via homo Diels-Alder reaction was isolated from this family.Also, compound 2 presents the uncommon skeleton 29-nor-friedelane with a highly oxidized A ring.The structures of both substances were confirmed by high-resolution-atmospheric pressure chemical ionization-mass spectrometry (HR-APCI-MS) and single crystal X-ray diffraction.In addition to these two new compounds, four known triterpenes were also isolated and identified as 3-oxo-29-hydroxyfriedelane (3), pristimerin (4), tingenone (5) and netzahualcoyonol (6) (Figure 1).All substances were characterized by Fourier transform infrared (FTIR) and 1D/2D nuclear magnetic resonance (NMR).

General experimental procedures
Optical rotations were measured with an ADP220 Bellinghan + Stanley Ltd. polarimeter.FTIR spectra (ca.1% KBr solution) were obtained on a Shimadzu FTIR 408 spectrometer.The 1 H (400 MHz, J in Hz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker Avance DRX-400 spectrometer.Tetramethylsilane (TMS) or solvent signals of CDCl 3 (d H 7.26; d C 77.00 ppm) and dimethyl sulfoxide (DMSO-d 6 , d H 2.50; d C 39.51 ppm) were used as internal standards.HR-APCI-MS analyses were carried out on a Shimadzu LCMS-IT-TOF (liquid chromatography mass spectrometry-ion trap and time-of-flight) instrument in both positive and negative modes.Column chromatography (CC) and thin layer chromatographic (TLC) plates were carried out on silica gel 60 (70-230 Mesh, Merck) and silica gel 60 G, respectively.Spots were visualized by heating after spraying with a solution of 1% ethanol-vanillin/3% aqueous perchloric acid 1:1 v/v.Single crystal X-ray diffraction data were collected with an Oxford-Diffraction GEMINI-Ultra (293 K) using Mo Kα radiation (0.71073 Å).Reduction data/analytical absorption corrections and space group identification were performed using CRYSALIS suite 16 and XPREP, 17 respectively.Structures were solved by direct methods with SIR-92 18 and refined by full-matrix least-squares against F 2 with SHELX. 17,18Hydrogen atoms were assigned riding isotropic displacement parameters and constrained to idealized geometries. 19The theoretical crystal morphology and 3D chemical structures were established with Mercury ® . 19ant material

Results and Discussion
Compound 1 was obtained as yellowish crystals.Its molecular formula C 45 H 66 O 4 was determined by HR-APCI-MS and presented 13 degrees of unsaturation.In its IR spectrum, absorption bands for carbonyl groups and terminal methylidene group at 1728 and 898 cm -1 were observed, respectively.The 1 H NMR spectrum of 1 revealed the presence of ten methyl singlets, one of which was due to an OCH 3 group (d H 3.69 ppm), and two singlets for a methylidene at d H 4.88 and 5.01 ppm.Analysis of the 13 C NMR data with the aid of distortionless enhancement by polarization transfer (DEPT)-135 experiments (Table 1) showed 45 carbon resonances (among them three C=O and four olefinic carbon), suggesting a dimer composed by a triterpene and a sesquiterpene.This hypothesis was reinforced by the mass peaks at m/z 467.3448 [C 30 H 43 O 4 ] + (consistent with the molecular formula of a methoxylated quinonemethide) and at m/z 205.0891 [C 15 H 24 ] + (consistent with a molecular formula of a sesquiterpene).In fact, the 13 C NMR data of caryophyllene oxide and 2α-hydroxypopulnonic acid methyl ester were closely related to the sesquiterpene moiety and to the C, D and E rings of compound 1, respectively. 20,21 detailed analysis was performed using 2D NMR (heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY) and nuclear Overhauser effect (NOESY) spectra).In the HMBC spectrum, the olefinic protons H-15' (d H 4.87 and d H 5.01 ppm) correlated with C-9' (d C 42.52 ppm) and C-7' (d C 36.12 ppm).This last carbon correlated with H-5' (d H 1.54/d C 44.39 ppm).C-5' is one of the links of the dimer, due to its correlations with the triterpene (correlation with H-1 (d H 3.23 ppm) The crystal structure of 1 was unambiguously obtained from single crystal X-ray diffraction experiments.For data collection, a single crystal obtained from ethanol slow evaporation was submitted to a nitrogen flow at 150 K and irradiated with Cu Kα radiation (1.5418 Å).The compound crystallized in the non-centrosymmetric orthorhombic space group P2 1 2 1 2 1 , which indicated the obtained crystals were enantiomerically pure.Figure 2 shows the representation of structure 1 demonstrating that the dimer is indeed composed by a triterpene and a sesquiterpene.Anomalous dispersion on single crystal data was insufficient to determine the absolute structure of 1.The triterpene conformations were determined for the rings as chair (C and E), semi-chair (B) and boat (A) (Figure 2, rings identification follows Figure 1).The torsion angles observed for C and E rings were greater than 50° (standard for chair conformations) and close to zero for B ring (5.5(1)°, standard for semi-chair); however, D ring presented a torsion angle of 27.Mesa-Siverio et al. 15 also reported the isolation of sesquiterpene-triterpene dimers; however, they are connected by two ether linkages.Therefore, caryopristimerin (1) represents the first example of a sesquiterpene-triterpene dimer linked by a carbon-carbon bond.Also, this is the first report of a caryophyllene-triterpene dimer.The dimerization process may occur by a homo Diels-Alder reaction, different from the hetero Diels-Alder reported for Celastraceae triterpene dimers. 23Scheme 1 shows a plausible biosynthetic pathway for caryopristimerin (1) in which a pristimerin derivative under keto-enol equilibrium reacts with caryophyllene.
In order to prove that compound 1 is in fact biosynthesized by the plant, and not an artifact formed during the extraction, two experiments were performed.The first experiment consisted in a chemical reaction between caryophyllene (commercial) and pristimerin (quinonemethide isolated in higher quantities and similar to the precursor of compound 1).Both compounds were submitted to similar conditions employed in the extraction methodology.The analysis of the reaction mixture by high-performance liquid chromatography-ultraviolet (HPLC-UV) and TLC plates after 72 h showed no changes in the starting materials, indicating that no artifact was formed during the extract preparation.
The second experiment consisted in preparing a new extract through maceration (room temperature, 72 h) and isolating compound 1.However, due to the limited amounts of roots, only a small quantity of extract was obtained and analyzed by liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC/ESI-QTOF/MS) in positive mode.The mass peak at m/z 693.4860 (calcd.693.4853) was observed and attributed to C 45 H 66 O 4 Na + [M + Na] + , coherent with compound 1 molecular formula, reinforcing the evidence that this dimer is biosynthesized by the plant.Compound 2 was obtained as white crystals.Its molecular formula C 29 H 46 O 6 was determined by HR-APCI-MS and presented seven unsaturation degrees.The IR spectrum showed absorption bands for carbonyl (1702 cm -1 ) and hydroxyls groups (3556, 3512 and 3408 cm -1 ).The 1 H NMR spectrum of 2 displayed six methyl groups, two of them as doublets (d H 0.79 and 0.90 ppm), and three oxymethine protons (d H 3.51, 3.82 and 4.41 ppm), suggesting three hydroxyl groups.The 13 C NMR spectrum presented 29 signals, which, based on DEPT-135 experiment, were associated to 6 methyl groups, 8 methylenes, 8 methines, and 7 non-hydrogenated carbons (with one keto carbonyl carbon at d C 213.22 ppm, and one carboxylic acid carbon at d C 176.48 ppm).These data suggested that compound 2 is a nor-friedelane acid triterpene.
In the HMBC spectrum, the most shielded methyl doublet at d H 0.79 ppm (H-23) correlated with the signal d C 71.21 ppm (C-3), confirming that C-3 has a hydroxyl group.C-3 correlated with the signal at d H 1.46 ppm (H-4), which correlated with the signal at d C 176.48 ppm (C-24), attributing the carboxylic group in an unusual oxidation position at C-5. 24 In the COSY spectrum, the carbinolic signal at d H 3.82 ppm correlated with H-3 (d H 3.51 ppm) and H-1 (d H 1.59 ppm), which correlated with H-10 (d H 1.72 ppm), locating the second hydroxyl group at C-2.The second methyl doublet at d H 0.90 ppm correlated with a CH 2 (d C 30.91 ppm), C=O (d C 213.22 ppm) and a CH (d C 40.81 ppm).These correlations confirmed compound 2 as a 29-nor-friedelane, similar to triptocalline A. 25 The complete chemical shift assignments of compound 2 (Table 2) were established by further detailed analysis of HSQC, HMBC and COSY spectra.Similar to compound 1, a single crystal of compound 2 was obtained from ethanol and it crystallized in the noncentrosymmetric orthorhombic space group P2 1 2 1 2 1 .The crystal data was collected under nitrogen flow at 150 K using Cu Kα radiation.The asymmetric unit of 2 shows two independent molecules with small differences in the conformation and torsions due to the hydrogen bond interactions.Anomalous dispersion effect on the single crystal data was insufficient to determine the absolute structure of 2. Analyzing one of the molecules of the asymmetric unit, compound 2 was identified as 2α,3α,22β-trihydroxy-21-oxo-29-nor-friedelan-24-oic acid with the uncommon 29-nor-friedelane skeleton with a highly oxygenated A ring.All the rings assumed a chair conformation with the exception of D ring, which presented a distorted chair with torsion angle (29.7(2)°) indicating an intermediate conformation between chair and semi-chair (Figure 3).

Conclusions
Four triterpenes, one nor-triterpene and one sesquiterpene-triterpene dimer were isolated from Salacia crassifolia roots.The 1D/2D NMR and single crystal X-ray diffraction data of caryopristimerin (1) and 2α,3α,22β-trihydroxy-21-oxo-29-nor-friedelan-24-oic acid (2) are herein described for the first time.A hypothetical pathway for caryopristimerin biosynthesis was proposed.This dimer represents the first example of a homo Diels-Alder adduct of sesquiterpene and triterpene moieties.Compound 2 presents a 29-nor-friedelane skeleton bearing an unusual C-5 oxidized to a carboxyl, a position commonly substituted by a methyl group.

Figure 2 .
Figure 2. X-ray crystal structure of 1.Some hydrogen atoms were omitted for better 3D visualization.Dotted lines represent relevant interactions observed in the NOESY spectrum.

Figure 3 .
Figure 3. X-ray crystal structure of 2. Some hydrogen atoms were omitted for better 3D visualization.

Table 1 .
15,22d13C NMR (400 and 100 MHz, respectively, CDCl 3 ) data assignments for compound 1 Alder reaction.The literature15,22reports this kind of dimer, together with quinonemethide-aromatic triterpenes, only via a hetero Diels-Alder reaction.In the NOESY spectrum, the protons of methylene H-15' were defined by the correlations H-15'a (d H 4.88 ppm)/H-7' (d H 2.32 ppm) and H-15'b (d H 5.01 ppm)/H-10' (d H 1.74 ppm). Th NOE of the methyls H-31 (d H 3.69 ppm), belonging to the carboxymethyl group, and H-27 (d H 0.82 ppm) proofs these groups are oriented on the same side.Surprisingly, both H-15' (a and b) and H-13' correlated with H-31 and H-27, suggesting a folding of the molecule.