Pyrrolizidine Alkaloids from Onosma erectaHarilaos Damianakos,† Georgios Sotiroudis,‡ and Ioanna Chinou*,†

†Department of Pharmacognosy, Faculty of Pharmacy, University of Athens, University Campus of Zografou, 15771 Zografou,Athens, Greece‡Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48, Vas. Konstantinou s.,11635 Athens, Greece

*S Supporting Information

ABSTRACT: The MeOH extract of the aerial parts of Onosma erectaafforded four new pyrrolizidine alkaloids, 7-O-acetylechinatine N-oxide(1), a viridinatine N-oxide stereoisomer (2), 7-epi-echimiplatine N-oxide (3), and onosmerectine N-oxide (4), and two additional newnatural products, the acid 2,3-dimethyl-2,3,4-trihydroxypentanoic acid(5) and the acyloin 4-methyl-2-hydroxypentanone (6).

Pyrrolizidine alkaloids (PAs) have been identified in 300plant species of up to 13 families. PAs have received

considerable interest as a class of potentially toxic naturalproducts, while it has been estimated that the percentage ofplants containing PAs is as high as 3% of the world’s floweringplants.1 In particular, Asteraceae, Boraginaceae, Leguminosae,Orchidaceae, and Compositae families include the majority ofthe species that contain PAs.1−4 PAs are esters of hydroxylated1-methylpyrrolizidines (necines), while the hepatotoxic mem-bers are esters of unsaturated necines having a Δ1,2 doublebond in the pyrrolizidine moiety. The potential of PAs ashepatotoxins is determined by certain minimum structuralfeatures: (i) an unsaturated 3-pyrroline ring, (ii) one or twohydroxymethyl groups, each attached to the pyrroline ring, (iii)one or preferably two acid moieties (necic acids), esterified onthe latter groups, and (iv) the presence of a branched chain onthe necic acid moiety.4 Typical toxicological effects onvertebrates are hepatotoxicity, pneumotoxicity, mutagenicity,carcinogenicity, and embryotoxicity along with weak virustaticand antileukemic activity.2−7

PAs’ well-known expressed hepatotoxicity is due to the livertransformation of 1,2-unsaturated PAs into reactive alkylatingagents. It is also known that PAs in low concentrations cancontaminate milk or honey, although the public healthimplications of such exposure are still unknown. Chronicexposure to PAs can also cause a veno-occlusive disease withsimilarities to Budd-Chiari syndrome.4 Therefore, certainEuropean countries have introduced restrictions to thepotential daily exposure to PAs (all PAs with a 1,2-unsaturatednecine moiety and the corresponding N-oxides) throughmedicinal products and/or foods.2−7 In addition, PAs play animportant role in insect−plant relationships. Thus, while PA-containing plants are usually avoided by herbivores, certaininsect species not only have evolved resistance to their toxicitybut also utilize them either for their protection from predatorsor transform them into insect alkaloids or sex pheromones.8 As

the PAs isolated in this work are 1,2-unsaturated in theirpyrrolizidine ring, it is anticipated that they exhibit this kind oftoxicity and ecological function.2−7

Onosma is a genus of hairy plants distributed around the EastMediterranean Sea and in Central Asia.9 In continuation of ourstudy of greek Onosma species (Boraginaceae) for theiralkaloidal content,9 we report herein the results of thechromatographic separation of the MeOH extract componentsof the aerial parts of Onosma erecta Sibth. & Sm.(Boraginaceae), collected from southern Greece. O. erecta is aperennial herb with a lignified base and a cushion-like growthhabit, lanceolate leaves, and yellow flowers, growing in dryrocky locations, either within low vegetation or in sparse coniferforests.

■ RESULTS AND DISCUSSION

PAs are abundant in Boraginaceae plants, and usually theirnecine bases are esterified at C-7 and C-9 with various necicacids, giving rise mainly to acyclic retronecine/heliotridinemono- or diesters,10 while the bridgehead nitrogen atom ispresent as N-oxide for the majority of the naturally occurringPAs.11 Because of the high polarity of the N-oxide functionalityand the hydroxy groups attached to the necyl moieties, PA N-oxides have high water solubility, which impedes their partitionto organic solvents during extraction2 and also causes theirsignificant loss on silica stationary phases due to irreversibleadsorption. Therefore, reduction to the corresponding amineswith zinc dust at low pH is usually carried out, followed by theirextraction with CHCl3 or CH2Cl2 from the alkalinized aqueouslayer.12−22 Because of the known susceptibility of esters toundergo hydrolysis at extreme pH and the long reduction timesneeded when zinc is the reducing agent, we attempted todirectly separate PA N-oxides from their original mixture,

Received: November 9, 2012

Article

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avoiding the need for the reduction/extraction step. For thispurpose microcrystalline cellulose was used as the packingmaterial of choice, giving efficient CC separations, with betterrecovery of PAs compared to silica, and without highbackpressure problems compared to usual finer cellulosepackings. In addition, nonchlorinated solvents were used inthe partitioning process of the methanolic extract, as possibleartifact formation is sometimes reported.2,23,24 Furthermore,the improved version of the Mattocks reagent as described byMolyneux et al. for PA/PA N-oxide TLC detection25 turnedout to be sensitive and reliable for this purpose, in contrast toWagner and Dragendorff reagents. For the separation of sugarsfrom the methanolic extract, fractionation through a DiaionHP-5 column was used alternatively with the simpler solventprecipitation.Compound 1 was obtained as an optically active brown

semisolid, whose presence on TLC plates was detected by theMattocks-Molyneux reagent as a purple spot, revealing thepresence of a 1,2-unsaturated necine N-oxide moiety of theretronecine/heliotridine type.25 1H NMR chemical shifts of thering protons were in close agreement with the values reportedfor other acyclic diester retronecine/heliotridine N-oxides.10

Indeed, the chemical shifts of the deshielded H-3a/H-3b (δH4.70/4.38), H-5a/H-5b (δH 3.83/3.79), and H-8 (δH 4.88)suggested the presence of the N-oxide moiety.9,10 No NOESYcross-peak was observed between H-7 (δH 5.69) and H-8 (δH4.88), indicating a heliotridine-type structure, where theseprotons are trans-oriented. Within the Onosma genus helio-tridine PAs have been reported only for Onosma heterophylla.26

The signal at δH 2.06 (3H, s) is due to an acetyl group with thecorresponding methyl carbon atom (C-9′) resonating at δC21.1. The carbonyl carbon at δC 171.5 is assigned to the sameacetyl group (C-8′), due to the HMBC correlations of C-8′ toH-7 and H-9′, revealing that O-7 is acetylated. The presence ofthe Δ1,2 double bond is confirmed by the downfield signals ofH-2 at δH 6.00, and C-1/C-2 at δC 133.0/124.3, as well as fromthe COSY correlations H-3a, H-3b/H-2 and H-8/H-2. Theprotons at δH 4.78 (2H, s) show HMQC correlation with thecarbon at δC 62.1, confirming the CH2-9 group. Corroborativeevidence for the aforementioned assignment comes from theCOSY H2-9/H-2 allylic correlation, along with the HMBCinteractions of C-9 to H-2; C-1 to H2-9; and C-8 to H2-9. KeyHMBC correlation between the carbonyl carbon at δC 175.0(C-1′) and H2-9 indicated the presence of a necic acid unitesterified at O-9. Indeed, the signals at δH 3.96 (1H, q, J = 6.8Hz), 2.17 (1H, sept, J = 6.8 Hz), 1.24 (3H, d, J = 6.8 Hz), 0.93(3H, d, J = 6.8 Hz), and 0.90 (3H, d, J = 6.8 Hz) along with thepeaks at δC 175.0, 85.2, 72.4, 34.0, 16.7, 18.2, and 18.5 were inclose agreement with those reported for the (−)-viridiflorylmoiety in viridinatine.27 This assumption was further supportedby COSY correlations H-3′/H3-4′and H3-6′/H-5′/H3-7′, aswell as by HMBC correlations of C-1′ to H-3′, H-5′; C-2′ to H-3′, H3-4′, H-5′, H3-7′; C-3′ to H3-4′; C-4′ to H-3′; C-5′ to H3-6′, H3-7′; and C-6′, C-7′ to H-5′. The structural formulacorresponds to the molecular formula C17H27NO7 with amolecular mass of 357.4, as confirmed by the m/z 358.4072 ofthe pseudomolecular ion [M + H]+, obtained by HRESIMS.The absolute configuration was deduced by determining theretention index (RI) of reduced 1 (free amine), by thereduction/GC-MS method described by Witte et al.8 The massspectrum recorded and the obtained RI value (2238) were ingood agreement with those reported (RI 2235) for 7-O-acetylechinatine.20 Thus, 1 was identified as 7-O-acetylechina-

tine N-oxide, obtained as the N-oxide form for the first time.Furthermore, the RI value of reduced 1 was additionallyobtained with the more widely used HP-5MS column (2085)using the same conditions, to be used as a future reference byothers.

Compound 2 was obtained as an optically active, brownsemisolid that was detected on TLC plates by the Mattocks-Molyneux reagent and was recognized as a retronecine/heliotridine N-oxide.25 The necine unit was identified asheliotridine as in 1. The remaining signals in the 1H and 13CNMR spectra were reminiscent of two viridifloryl moieties,esterified at O-7 and O-9 as in the PA viridinatine.27 Twoisopropyl groups (H3-6′/H-5′/H3-7′ and H3-13′/H-12′/H3-14′fragments) and two 1-hydroxyethyl groups (H-3′/H3-4′ and H-10′/H3-11′ fragments) were indeed recognized via COSY. Thecarbonyl carbons at δC 176.1 and 176.6 were attributed to C-8′and C-1′, respectively, based on HMBC correlations of C-8′ toH-7, H-10′, and H-12′ and of C-1′ to H-9 and H-5′. Inaddition, peaks at δC 85.9 and 86.2 (missing from the DEPTspectrum) suggested the presence of two oxygenatedquaternary carbon atoms. HMQC 13C/1H matching alongwith 13C/1H HMBC interactions observed among theaforementioned necyl signals (see Table 2) established theirconnectivity. The structural formula inferred for 2 correspondsto the molecular formula C22H37NO9 with a molecular mass of459.5, as confirmed by the m/z 460.5388 of the [M + H]+ ion,obtained by HRESIMS. For the sake of comparison, part of 2was reduced in an acidic zinc suspension.19 The 1H NMRspectrum of the free PA was recorded in the same specifiedsolvent (CDCl3 saturated in D2O) and was subsequentlycompared with that of viridinatine.27 Viridinatine was reportedto have two (−)-viridifloryl moieties esterified at O-7 and O-9,and consequently the isopropyl and 1-hydroxyethyl methinepairs appeared in the 1H NMR spectrum as close overlappingsignals. Nevertheless, reduced 2 exhibited necyl protons at

Table 1. NMR Data (600/400 MHz, methanol-d4) forCompound 1

position δC, type δH (J in Hz) COSY HMBCa

1 133.0, C 2,3a,3b,92 124.3, CH 6.00 brs 3a,3b,8,9 3a,3b,8,93a 79.0, CH2 4.70 d (16.4) 2,3b 2,83b 4.38 d (16.4) 2,3a5a 70.1, CH2 3.83 m 5b,6a,6b 3a,6a5b 3.79 m 5a,6a,6b6a 33.4, CH2 2.74 m 5a,5b,6b,7 5a6b 2.23 m 5a,5b,6a,77 73.8, CH 5.69 brm 6a,6b,8 5a,5b,6b8 95.0, CH 4.88 brs 2,7 2,6a,6b,7,99 62.1, CH2 4.78 s 2 21′ 175.0, C 9,3′,5′2′ 85.2, C 3′,4′,5′,7′3′ 72.4, CH 3.96 q (6.8) 4′ 4′4′ 18.2, CH3 1.24 d (6.8) 3′ 3′5′ 34.0, CH 2.17 sept (6.8) 6′,7′ 3′,6′,7′6′ 18.5, CH3 0.90 d (6.8) 5′ 5′7′ 16.7, CH3 0.93 d (6.8) 5′ 5′8′ 171.5, C 7,9′9′ 21.1, CH3 2.06 s

aHMBC correlations are from proton(s) stated to the indicatedcarbon.

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different chemical shifts from those of viridinatine, whereas thesignals of the methine pairs CH-3′/CH-10′ [4.01 (1H, q, 3J =6.8 Hz)/4.18 (1H, q, 3J = 6.8 Hz)] and CH-5′/CH-12′ [1.97(1H, sept, 3J = 6.8 Hz)/2.28 (1H, sept, 3J = 6.8 Hz)] appearedas clearly separated peaks (see Table 2). Thus, the twostructurally equivalent necic acids of 2 must be stereoisomers,and 2 a novel viridinatine N-oxide stereoisomer. Owing topaucity of the sample, its hydrolysis was not attempted.Furthermore, the RI value of the reduced sample could not beobtained by GC, due to its thermal instability (two overlappingpeaks instead of one, recognized as PAs by the MS library).Compound 3 was obtained as an optically active, pale yellow

semisolid, also detected on TLC by the Mattocks-Molyneuxreagent and recognized as a retronecine/heliotridine N-oxide.25

The necine part was identified as heliotridine as in 1. Theremaining signals in the 1H and 13C NMR spectra werereminiscent of an echimidinyl moiety. Two methyl groups at δH1.16 (3H, s) and 1.21 (3H, s) bonded to a quaternary carbonatom, and a 1-hydroxyethyl group accounting for the signals at

δH 4.18 (1H, q, J = 6.4 Hz) and 1.14 (3H, d, J = 6.4 Hz) wasrecognized from 1H-1D and COSY spectra. From the 13C-1D,DEPT, and HMQC spectra, the presence of three oxygenatedcarbon atoms was evident, two quaternary (δC 86.9, 75.6) andone tertiary (δC 71.1), along with the ester carbonyl carbon atδC 176.1. HMQC 13C/1H matching along with 13C/1H HMBCinteractions among the sites 1′−7′ established an echimidinyl-like structure for the necic acid part. HMBC correlation of C-1′to H2-9 indicated O-9 as the acylated necine site (see Table 3).As a result, the structural formula of 3 corresponds to themolecular formula C15H25NO7 with a molecular mass of 331.4,as confirmed by HRESIMS ([M + H]+ at m/z 332.3697). Twoknown PAs share the structural formula of 3, leptanthine N-oxide isolated from O. leptantha (Boraginaceae)9 andechimiplatine N-oxide, speculated from LC-MS data fromextract analysis of Echium plantagineum (Boraginaceae).28 The1H-1D spectrum of 3 in methanol-d4 did not match that ofleptanthine N-oxide.9 Part of 3 was reduced as describedelsewhere,19 and the 1H-1D spectrum of the free PA wasrecorded in CDCl3 and subsequently was compared for itsnecyl signals (Table 3) with spectra of known PAs with necicacid of similar structure esterified at O-9, in the same solvent.The best matching of δH values occurred for the free PAhydroxymyoscorpine: δH 1.24/1.30 (H3-6′/H3-7′), 1.25 (H3-4′), and 4.19 (H-3′).10 Hence, it was inferred that the necic acidof 3 is echimidinic acid, and therefore 3 is the 7-epimer ofechimiplatine N-oxide, a new PA for which the name 7-epi-echimiplatine N-oxide is proposed. A portion of 3 was reducedto the corresponding PA as for 1, and its RI value (2069) wasdetermined by GC using an HP-5MS column.Compound 4 was obtained as an optically active, brown

semisolid and was recognized as a heliotridine N-oxidederivative as in 1. Apart from the necine moiety protons,three methyl groups were identified in the 1H NMR spectrum,two of them as singlets at δH 1.27 (H3-7′) and 1.31 (H3-6′) andone as a doublet at δH 1.26 (d, J = 6.2 Hz, H3-5′) coupled to amethine group at δH 4.22 (q, J = 6.2 Hz, H-4′). The latter twosignals suggest the presence of a 1-hydroxyethyl group. In theDEPT spectrum, except for the three methyl groups [δC 18.7,(C-5′); 26.6 (C-6′); 25.9 (C-7′)], two oxygenated methinegroups were recognized at δC 70.7 and 70.8, ascribedinterchangeably to C-4′ and C-7. The 13C NMR spectrumexhibited three additional peaks: the downfield signal at δC175.3 due to the ester carbonyl group (C-1′) and twooxygenated quaternary carbon atoms at δC 74.6 (C-3′) and85.7 (C-2′), the only possible locations of the two methylgroups (CH3-6′, CH3-7′) resonating as singlets.The assembly of the recognized fragments of the necyl part

was accomplished on the basis of 13C/1H HMBC correlations(see Table 4). Correlation of C-1′ to H-9a and H-9b at δH 4.92and 4.88, respectively, reveals O-9 as the necylated site ofheliotridine N-oxide. The structure assigned for 4 correspondsto the molecular formula C15H25NO7 with a molecular mass of331.4, as confirmed by HRESIMS ([M + H]+ at m/z332.3696). Compound 4 is a novel PA, and the nameonosmerectine N-oxide is proposed for it. Owing to thepaucity of the compound, its hydrolysis was not attempted. TheRI value of the reduced sample could not be obtained by GC,because of its thermal decomposition (one broad peakrecognized as various nitrogenous compounds by the MSlibrary, but not as a PA).Compound 5 was obtained as an optically active, amorphous,

brownish solid, whose aqueous solution was tested with litmus

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Table 2. NMR Data (400 MHz) for Compound 2 and Its Reduced Analogue

2 (N-oxide) (D2O)reduced 2 (tert. amine) (CDCl3 sat. in

D2O)

position δC, type δH (J in Hz) COSY HMBCa δH (J in Hz)

1 132.5, C 2,3a,3b, 8,92 125.4, CH 5.86 brs 3a,3b,8,9 3a,3b,8,9 5.93 brs3a 79.5, CH2 4.51 d (16.6) 2,3b,9 2,5b 4.41 d (15.4)3b 4.22 d (16.6) 2,3a,9 3.65 d (15.4)5a 70.1, CH2 3.73 m 5b,6a,6b 3a,3b,6b, 7,8 3.86 m5b 3.61 m 5a,6a,6b6a 35.8, CH2 2.45 m 5a,5b,6b,7 5a,5b,7,8 3.12 m6b 2.00 m 5a,5b,6a,7 2.16 m7 71.3, CH 4.67 brm 6a,6b,8 3a,3b,5a,5b, 6b,8 4.80 m8 96.6, CH 4.57 brs 2,7 2,5a,5b,6a, 6b,7,9 4.56 brm9 63.3, CH2 4.75 brs 2,3a,3b 2,8 4.94 d (13.2)

4.89 d (13.2)1′ 176.6, C 9,5′2′ 85.9, C 3′,4′,5′ 6′,7′3′ 70.9, CH 4.04 q (6.4) 4′ 4′,5′ 4.18 q (6.8) or

4.01 q (6.8)4′ 17.6, CH3 1.04 d (6.4) 3′ 3′ 1.23 d (6.8) or

1.12 d (6.8)5′ 34.6, CH 1.89 sept (6.8) 6′,7′ 3′,6′,7′ 2.28 sept (6.8) or

1.97 sept (6.8)6′,7′,13′, 14′ 18.9, 18.4, 18.0, 17.2, CH3 (×4) 0.75−0.78 m, 0.81 d (6.8) 5′,12′ 5′,12′ 0.99 d (6.8)

0.95 d (6.8)0.92 d (6.8)0.84 d (6.8)

8′ 176.1, C 7,10′,12′9′ 86.2, C 10′,11′, 12′,13′,14′10′ 72.3, CH 3.93 q (6.4) 11′ 11′,12′ 4.18 q (6.8) or

4.01 q (6.8)11′ 18.6, CH3 1.10 d (6.4) 10′ 10′ 1.23 d (6.8) or

1.12 d (6.8)12′ 34.2, CH 2.05 sept (6.8) 13′,14′ 10′,13′,14′ 2.28 sept (6.8) or

1.97 sept (6.8)aHMBC correlations are from proton(s) stated to the indicated carbon.

Table 3. NMR Data (400 MHz) for Compound 3 and Its Reduced Analogue

3 (N-oxide) (D2O) reduced 3 (tert. amine)a (CDCl3)

position δC, type δH (J in Hz) COSY HMBCb δH (J in Hz)

1 132.4, C 2,3a,3b, 8,92 125.9, CH 5.89 brs 3a,3b,9 3a,3b,8,93a 79.7, CH2 4.52 d (17.0) 2,3b,9 2,8,5a3b 4.21 d (17.0) 2,3a,95a 70.2, CH2 3.74 m 5b,6a,6b 3a,3b,6a,75b 3.59 m 5a,6a,6b6a 35.8, CH2 2.47 m 5a,5b,6b,7 5a,5b,7,86b 2.01 m 5a,5b,6a,77 71.4, CH 4.67 brm 6a,6b,8 5a,5b,6b,88 96.7, CH 4.55 brs 7 2,99 63.6, CH2 4.78 brs 2 2,81′ 176.1, C 9,3′2′ 86.9, C 3′,4′,6′,7′3′ 71.1, CH 4.18 q (6.4) 4′ 4′ 4.19 q (6.4)4′ 19.0, CH3 1.14 d (6.4) 3′ 3′ 1.24 d (6.4)5′ 75.6, C 3′,6′,7′6′,7′ 26.3, 27.0, 1.16 s 6′,7′ 1.30 s

CH3 (×2) 1.21 s 1.22 saOnly necic acid (necyl) protons presented. bHMBC correlations are from proton(s) stated to the indicated carbon.

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paper and was found acidic (pH = 2−3). In its 1H NMRspectrum, one 1-hydroxyethyl group [δH 4.17 (q, J = 6.4 Hz, H-4); 1.27 (d, J = 6.4 Hz, H3-5)] and two methyl groups [δH 1.32(s, H3-2); 1.26 (s, H3-3)] attached to quaternary carbon atomswere recognized, as in 4. In the 13C NMR spectrum, onedownfield signal (δC 177.3) was due to a carboxyl group, whilesmall peaks at δC 84.0 and 74.9 missing from the DEPTspectrum were ascribed to two hydroxylated quaternarycarbons (C-2 and C-3), bearing CH3-2 and CH3-3 groups.13C/1H HMBC correlations (see Table 5) established theconnection among the recognized parts. The structure of 5 wasidentified as 2,3-dimethyl-2,3,4-trihydroxypentanoic acid, a newnatural product. The molecular formula of 5 is C7H14O5 with amolecular mass of 178.2, as confirmed by HRESIMS ([M +H]+ at m/z 179.1906). It is noteworthy that 5 is structurallysimilar to the necic acid moiety of 4. The followingconsiderations allowed us to infer that 5 is a natural metaboliteand not a hydrolysate: (i) the known separate biosynthesis ofmonocarboxylic necic acids from amino acids,5 (ii) the mildconditions used during the isolation procedure, and (iii) theabsence of the corresponding necic acids from the other PA N-oxides. Besides, isolation of PAs and PA N-oxides along withtheir corresponding necic acid has been reported by Braca etal.29 In addition, it has been shown for some Boraginaceaespecies that PA biosynthesis takes place at several plant organsand not exclusively in the roots, as it has been proved for PA-containing Asteraceae species.30 In this context, isolation of 5 asa PA biosynthesis intermediate from the aerial parts of aboraginaceous plant is a plausible finding.Compound 6 was obtained as an optically active, colorless,

oily residue. Signals at δH 0.95 (d, J = 6.4 Hz), 0.96 (d, J = 6.4Hz) (H3-5, H3-6), and 2.06 (sept, J = 6.4 Hz, H-4) indicatedthe presence of an isopropyl group in the 1H NMR spectrum,while signals at δH 1.18 (d, J = 6.4 Hz, H3-1) and 4.00 (q, J =6.4 Hz, H-2) suggested the presence of a 1-hydroxyethyl group.Although the attachment of the two aforementioned groups toa carbonyl group could explain the increased chemical shifts of

the two methine protons (the one at δH 2.06 being typical forα-H of carbonyl compounds), no carbonyl signal was observeddownfield in the 13C NMR spectrum, possibly due to itselongated relaxation time in such a small structure. 13C/1HHMBC correlations (see Table 5) support structure 6, whereasno C-3 correlation with any hydrogen atom was evident.Nevertheless, various IR data support the proposed structure.The broad band at 3504 cm−1 is attributed to O−H stretching,shifted to a lower frequency due to intramolecular hydrogenbonding. Two strong absorptions at 2973 and 2935 cm−1 aredue to methyl C−H stretching vibrations, while a strong bandat 1714 cm−1 is typical of aliphatic ketones (CO stretching).The latter is partially overlapped with a stronger band at 1758cm−1, which is due to the same kind of vibration but shifted to ahigher frequency due to α-OH rotational isomerism. Medium-intensity bands at 1158 and 1029 cm−1 are attributed to C−CO−C stretching/bending vibrations and C−OH stretching,respectively. The molecular formula of 6 is C6H12O2 with amolecular mass of 116.2, as confirmed by HRESIMS ([M +H]+ at m/z 117.1659). 4-Methyl-2-hydroxypentanone (6) isreported for the first time as a natural product.Allantoin was obtained as an amorphous, white solid and was

recognized by means of its 1H and 13C NMR spectra,31,32 alongwith ESIMS ([M + K]+ at m/z 197.1, 13%, [M + Na]+ at m/z180.9, 47%). Allantoin has been detected or isolated from otherBoraginaceae species as well: Symphytum of f icinale L., S.uplandicum Nym.,33 Anchusa of f icinalis L.,34 Auxemmaoncocalyx,35 Cordia trichotoma,36 Anchusa strigosa,29 and Ehretiathyrsif lora.37

Glycerol, D-fructose, and D-glucose were also obtained andidentified on the basis of their 1H and 13C NMR spectra38 andTLC comparison with authentic samples.

Table 4. NMR Data (400 MHz, methanol-d4) for Compound4

position δC, type δH (J in Hz) COSY HMBCa

1 133.5, C 3a,3b,8,9a,9b

2 123.8, CH 5.96 brs 3a,3b, 9a,9b 3a,3b,8,9a,9b

3a 78.6, CH2 4.66 d (16.4) 2,3b,9a,9b 2,83b 4.45 d (16.4) 2,3a,9a,9b5 69.7, CH2 3.87 m 6a,6b 3a,6a,6b6a 35.7, CH2 2.58 m 5,6b,76b 2.12 m 5,6a,77 70.7 or 70.8, CH 4.75 brm 6a,6b,8 5,6a,6b8 96.7, CH 4.85 brs 7 2,9a,9b9a 62.5, CH2 4.92 d (16.4) 2,3a,3b,9b 29b 4.88 d (16.4) 2,3a,3b,9a1′ 175.3, C 9a,9b2′ 85.7, C 6′,7′3′ 74.6, C 7′4′ 70.7 or 70.8, CH 4.22 q (6.2) 5′ 5′5′ 18.7, CH3 1.26 d (6.2) 4′ 4′6′ 26.6, CH3 1.31 s7′ 25.9, CH3 1.27 s

aHMBC orrelations are from proton(s) stated to the indicated carbon.

Table 5. NMR Data (400 MHz, methanol-d4) forCompounds 5 and 6

position δC, typeδH (J inHz) COSY HMBCa

51 177.3, C 3-Me, 42 84.0, C 2-Me, 3-

Me3 74.9, C 2-Me,

3-Me, 54 70.8, CH 4.17 q

(6.4)5 3-Me, 5

5 19.0, CH3 1.27 d(6.4)

4 4

2-Me 25.8, CH3 1.32 s3-Me 26.6, CH3 1.26 s

61 17.1 or 17.6 or 17.7, CH3 1.18 d

(6.4)2 2

2 70.1, CH 4.00 q(6.4)

1 1

3 C4 33.5, CH 2.06

sept(6.4)

5, 6 5, 6

5 17.1 or 17.6 or 17.7, CH3 (×2) 0.95 d(6.4)

4 4, 5, 6

6 0.96 d(6.4)

aHMBC correlations are from proton(s) stated to the indicatedcarbon.

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In conclusion, the application of a modified fractionation/separation protocol for the MeOH extract of O. erecta (aerialparts) led to the isolation of six new natural products (1−6),along with allantoin, a secondary metabolite of potentialchemotaxonomic value in the Boraginaceae family.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Kieselgel 60 F254 TLC plates

with 0.2 mm layer thickness were purchased from Merck Chemical Co.Bands on TLC silica plates were detected under UV light (254 and366 nm) and after spraying with a 2.5% H2SO4 and 2.5% vanillinMeOH solution, followed by heating at 105 °C for 5 min. For thespecial spot detection of PAs (free bases or N-oxides) on TLC plates,the visualizing reagents proposed by Molyneux and Roitman wereapplied successfully.25 For preparative TLC, Merck 20 × 20 cm silicagel 60 F254, silica gel 60 RP-18 F254S, and cellulose plates were used.Optical rotations were measured using a Perkin-Elmer 341 polar-imeter. IR spectra were recorded on a Perkin-Elmer FT-IR Paragon500 spectrophotometer. 1H-1D and HMQC, HMBC, and COSY 2DNMR spectra were recorded on 600 or 400 MHz while 13C-1D andDEPT spectra were recorded on 150 or 50 MHz FT NMR BrukerAVANCE spectrometers. ESIMS spectra (positive mode) wereobtained by injecting samples (as MeOH solutions, except for 5,which was dissolved in H2O) directly into the spectrometer, using a3200 QTRAP LC/MS/MS Applied Biosystems mass spectrometerand ion spray voltage of +4500 V, declustering potential of 50 V, andentrance potential of 10 V. High-resolution mass spectra (ESI+) wererecorded on a Thermo Scientific LTQ Orbitrap Discovery massspectrometer with the infusion method. The solvents or solventcombinations used in column chromatography with cellulose asstationary phase had been formerly saturated in H2O, after shakingwith H2O in a separatory funnel (except for H2O-miscible solvents).The stationary phases used for column chromatography were silica gel60H and 230−400 mesh, microcrystalline cellulose (Merck), as well asSephadex LH-20 (Pharmacia), and styrene-divinylbenzene copolymerresin Diaion HP-20 (Supelco). GC-MS analyses were performed on aHewlett-Packard 6890 chromatograph, connected to an electron-impact Hewlett-Packard 5973 mass spectrometer. For GC-MS PAidentification, the analytical method described by Witte and co-workers8 was applied [capillary fused-silica WCOT DB-1 column,J&W Scientific CA (30 m × 0.32 mm i.d., film thickness 0.25 μm) (oralternatively capillary column HP-5MS 30 m × 0.25 mm i.d., filmthickness 0.25 μm), injector temperature 220 °C, temperatureprogram 150−300 °C, 6 °C/min, split ratio 1:20, carrier gas He0.75 bar, injection volume 1−2 μL (MeOH), ionization voltage 70 eV,MS library Wiley 275], while the same N-oxide reduction method(method A) of this team was used.8

Plant Material. O. erecta was collected at flowering stage in May2004, from “Lefka Ori” mountains in western Crete, Greece. The plantwas botanically identified by Dr. E. Kalpoutzakis, dried at roomtemperature, and subsequently deposited at the Herbarium of thePharmacognosy Department of the Faculty of Pharmacy of theUniversity of Athens (voucher number KL 025c/15-05-2004).Extraction and Isolation. A 410.6 g amount of the dry aerial parts

of the plant was successively extracted with cyclohexane (9.6 gextract), CH2Cl2 (6.4 g extract), MeOH (59.5 g extract), and H2O(29.7 g extract), by immersion in the solvent 2 × 24 h at roomtemperature. Seventeen grams of the MeOH extract was suspended in100 mL of H2O and successively extracted with 2 × 100 mL ofcyclohexane, cyclohexane/n-butanol (66:33), cyclohexane/n-butanol(33:66), and n-butanol. The latter three extracts were pooled afterTLC examination, evaporated in vacuo, and partitioned betweenEtOAc and H2O. The mixture (0.272 g) obtained from the EtOAclayer was subjected to column chromatography (⦶ 2 × 41 cm,Sephadex LH-20, MeOH) and resulted in multicomponent fractions ofonly minor amounts (0.1−30 mg). The mixture (3.792 g) obtainedfrom the H2O layer was combined with the H2O-soluble part (9.4 g)of the MeOH extract after TLC examination, and the solution wasevaporated in vacuo. The residue was dissolved in the minimum

volume of H2O, and after addition of iPrOH, the dark brown solutionbecame turbid and finally separated into a lower brown layer and anupper orange layer, which was evaporated in vacuo and redissolved inthe minimum amount of H2O. After addition of iPrOH and somecyclohexane, cooling of the solution overnight in a refrigerator allowedits separation into an upper yellow layer and a lower viscous brownone, which was combined with the previous lower brown layerobtained likewise, guided by TLC.

The yellow layer after evaporation to dryness yielded 0.702 g of ayellow foam, which was subjected to CC (⦶ 1.5 × 15.7 cm, DiaionHP-20, water) to give fractions UL1A−G. Fractions UL1A,B werecombined (328 mg) and further subjected to CC (⦶ 3 × 27 cm,cellulose, cyclohexane/EtOAc, 15:85, 10:90, 5:95, EtOAc, EtOAc/MeOH, 95:5, 85:15), leading to fractions UL2A−P. UL2A containedpure 5 (11.2 mg). Fractions UL1C−E were combined (126.3 mg) andfurther subjected to CC (⦶ 1.5 × 27 cm, cellulose, cyclohexane,cyclohexane/EtOAc, 80:20, 70:30, 60:40, 40:60, 30:70, 20:80, 10:90,EtOAc, EtOAc/MeOH, 99:1, 97:3, 95:5, 93:7, 90:10, 80:20), yieldingpure 2 (5.0 mg) and 3 (70.5 mg), both eluted with EtOAc/MeOH,90:10. Fractions UL1F,G were combined (287.2 mg) and subjected toCC (⦶ 2 × 27 cm, cellulose, cyclohexane, cyclohexane/EtOAc 95:5,90:10, 85:15, 80:20, 75:25, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80,10:90, 5:95, EtOAc, EtOAc/MeOH, 98:2, 97:3, 96:4, 95:5, 90:10,MeOH, H2O), furnishing pure 6 (6.0 mg) (eluted with cyclohexane/EtOAc, 75:25), 1 (4.4 mg, eluted with EtOAc/MeOH, 98:2), and 2(154.0 mg, eluted with EtOAc/MeOH, 97:3). Subfraction UL2B wassubjected to preparative TLC (silica RP-18, H2O/MeOH, 9:1), andafter extraction of the zones with MeOH, glycerol (6.1 mg) wasobtained. Subfraction UL2C-H was subjected to CC (Sephadex LH-20, MeOH), and allantoin (27.9 mg) was obtained. Subfraction UL2I-N (148 mg) was subjected to CC (⦶ 1.7 × 18.5 cm, cellulose,cyclohexane/EtOAc, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95,EtOAc, EtOAc/MeOH, 99:1, 98:2, 97:3, 96:4, 95:5), leading to fourfractions. The second and third fractions were further separated bypreparative TLC (cellulose plates, development with THF/H2O/HOAc, 70:20:10), yielding D-fructose (37 mg) and D-glucose (25 mg)after extraction of the zones with H2O.

One portion (1.4 g) of the condensation residue of the lower brownlayer was subjected to three successive CCs (cellulose, first columneluted with EtOAc, EtOAc/iPrOH mixtures, second and third columnseluted with cyclohexane/EtOAc and EtOAc/MeOH mixtures), andfinally the mixture was separated using preparative TLC (silica plates,first development with EtOAc/MeOH/HOAc, 50:49.5:0.5, seconddevelopment with EtOAc/MeOH/HOAc, 60:39:1). After extractionof the zones with MeOH, 4 (44.6 mg, also containing silica) wasobtained.

7-O-Acetylechinatine N-oxide (1): brown semisolid; [α]20D −5.7 (c0.105, MeOH); ESIMS m/z 396.3 [M + K]+ (12), 381.3 [M + Na +H•]+ (98), 358.5 [M + H]+ (100), 342.3 (26), 316.5 (23), 291.3 (16),214.4 (7), 185.2 (28), 136.2 (7), 120.2 (17); HRESIMS m/z 358.4072[M + H]+ (calcd for C17H28NO7, 358.4067).

Viridinatine N-oxide stereoisomer (2): brown semisolid; [α]20D+0.65 (c 1.54, H2O); ESIMS m/z 919.3 [2M + H − H•]+ (1) 339.4(26), 338.4 (89), 317.6 (100), 316.6 (75), 302.3 (26), 300.4 (17),272.3 (3), 172.2 (9), 136.1 (5); HRESIMS m/z 460.5388 [M + H]+

(calcd for C22H38NO9, 460.5384).7-Epi-echimiplatine N-oxide (3): pale yellow semisolid; [α]20D

+20.7 (c 0.14, H2O); ESIMS m/z 354.4 [M + Na]+ (36), 334.1 [M+ H + 2H•]+ (50), 333.1 [M + H + H·]+ (100), 332.1 [M + H]+ (61),316.4 (82), 314.4 (22), 300.4 (60), 270.2 (14), 256.2 (7), 172.2 (29),138.4 (6), 86.2 (6); HRESIMS m/z 332.3697 [M + H]+ (calcd forC15H26NO7, 332.3694).

Onosmerectine N-oxide (4): brown semisolid; [α]20D −3.2 (c 0.88,MeOH); ESIMS m/z 370.4 [M + K]+ (3), 354.4 [M + Na]+ (68),333.3 [M + H + H•]+ (100), 331.9 [M + H]+ (68), 316.4 (76), 270.2(9), 256.3 (4), 172.2 (47), 167.4 (3), 159.2 (13), 111.2 (3), 99.1 (3);HRESIMS m/z 332.3696 [M + H]+ (calcd for C15H26NO7, 332.3694).

2,3-Dimethyl-2,3,4-trihydroxypentanoic acid (5): amorphous,brownish solid; [α]20D +4.4 (c 0.275, MeOH), ESIMS m/z 201.4[M + Na]+ (100), 196.0 (7), 186.1 (26), 184.2 (21), 183.1 (14), 155.3

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(21), 139.5 (29), 117.0 (12), 108.0 (36), 102.1 (45), 99.2 (24), 71.1(10), 57.1 (19); HRESIMS m/z 179.1906 [M + H]+ (calcd forC7H15O5, 179.1910).4-Methyl-2-hydroxypentanone (6): colorless, oily residue; [α]20D

−1.7 (c 0.6, MeOH); IR (CH2Cl2 solution) νmax 3504, 2973, 2935,1758, 1714, 1158, 1029 cm−1; ESIMS m/z 117.3 [M + H]+ (100), 99.2(50); HRESIMS m/z 117.1659 [M + H]+ (calcd for C6H13O2,117.1662).

■ ASSOCIATED CONTENT

*S Supporting InformationIR, COSY, and 1H and 13C NMR spectroscopic data of 1−6 areavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: ichinou@pharm.uoa.gr. Tel: (+30) 2107274595. Fax:(+30) 2107274115.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThanks are due to Dr. E. Kalpoutzakis for collecting andidentifying the botanical sample and Dr. M. Popova (BulgarianAcademy of Sciences, Sofia) and Dr. D. Benaki (“EKEFEDemokritos” Research Institute, Athens) for recording certainNMR spectra.

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