Pyrrolizidine Alkaloids from Onosma erecta - Journal of Natural

Article pubs.acs.org/jnp

Pyrrolizidine Alkaloids from Onosma erecta Harilaos 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 erecta afforded four new pyrrolizidine alkaloids, 7-O-acetylechinatine N-oxide (1), a viridinatine N-oxide stereoisomer (2), 7-epi-echimiplatine Noxide (3), and onosmerectine N-oxide (4), and two additional new natural products, the acid 2,3-dimethyl-2,3,4-trihydroxypentanoic acid (5) and the acyloin 4-methyl-2-hydroxypentanone (6).

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the PAs isolated in this work are 1,2-unsaturated in their pyrrolizidine ring, it is anticipated that they exhibit this kind of toxicity and ecological function.2−7 Onosma is a genus of hairy plants distributed around the East Mediterranean Sea and in Central Asia.9 In continuation of our study of greek Onosma species (Boraginaceae) for their alkaloidal content,9 we report herein the results of the chromatographic separation of the MeOH extract components of the aerial parts of Onosma erecta Sibth. & Sm. (Boraginaceae), collected from southern Greece. O. erecta is a perennial herb with a lignified base and a cushion-like growth habit, lanceolate leaves, and yellow flowers, growing in dry rocky locations, either within low vegetation or in sparse conifer forests.

yrrolizidine alkaloids (PAs) have been identified in 300 plant species of up to 13 families. PAs have received considerable interest as a class of potentially toxic natural products, while it has been estimated that the percentage of plants containing PAs is as high as 3% of the world’s flowering plants.1 In particular, Asteraceae, Boraginaceae, Leguminosae, Orchidaceae, and Compositae families include the majority of the species that contain PAs.1−4 PAs are esters of hydroxylated 1-methylpyrrolizidines (necines), while the hepatotoxic members are esters of unsaturated necines having a Δ1,2 double bond in the pyrrolizidine moiety. The potential of PAs as hepatotoxins is determined by certain minimum structural features: (i) an unsaturated 3-pyrroline ring, (ii) one or two hydroxymethyl groups, each attached to the pyrroline ring, (iii) one or preferably two acid moieties (necic acids), esterified on the latter groups, and (iv) the presence of a branched chain on the necic acid moiety.4 Typical toxicological effects on vertebrates are hepatotoxicity, pneumotoxicity, mutagenicity, carcinogenicity, and embryotoxicity along with weak virustatic and antileukemic activity.2−7 PAs’ well-known expressed hepatotoxicity is due to the liver transformation of 1,2-unsaturated PAs into reactive alkylating agents. It is also known that PAs in low concentrations can contaminate milk or honey, although the public health implications of such exposure are still unknown. Chronic exposure to PAs can also cause a veno-occlusive disease with similarities to Budd-Chiari syndrome.4 Therefore, certain European countries have introduced restrictions to the potential daily exposure to PAs (all PAs with a 1,2-unsaturated necine moiety and the corresponding N-oxides) through medicinal products and/or foods.2−7 In addition, PAs play an important role in insect−plant relationships. Thus, while PAcontaining plants are usually avoided by herbivores, certain insect species not only have evolved resistance to their toxicity but also utilize them either for their protection from predators or transform them into insect alkaloids or sex pheromones.8 As © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION PAs are abundant in Boraginaceae plants, and usually their necine bases are esterified at C-7 and C-9 with various necic acids, giving rise mainly to acyclic retronecine/heliotridine mono- or diesters,10 while the bridgehead nitrogen atom is present as N-oxide for the majority of the naturally occurring PAs.11 Because of the high polarity of the N-oxide functionality and the hydroxy groups attached to the necyl moieties, PA Noxides have high water solubility, which impedes their partition to organic solvents during extraction2 and also causes their significant loss on silica stationary phases due to irreversible adsorption. Therefore, reduction to the corresponding amines with zinc dust at low pH is usually carried out, followed by their extraction with CHCl3 or CH2Cl2 from the alkalinized aqueous layer.12−22 Because of the known susceptibility of esters to undergo hydrolysis at extreme pH and the long reduction times needed when zinc is the reducing agent, we attempted to directly separate PA N-oxides from their original mixture, Received: November 9, 2012

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dx.doi.org/10.1021/np300785g | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

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

avoiding the need for the reduction/extraction step. For this purpose microcrystalline cellulose was used as the packing material of choice, giving efficient CC separations, with better recovery of PAs compared to silica, and without high backpressure problems compared to usual finer cellulose packings. In addition, nonchlorinated solvents were used in the partitioning process of the methanolic extract, as possible artifact formation is sometimes reported.2,23,24 Furthermore, the improved version of the Mattocks reagent as described by Molyneux et al. for PA/PA N-oxide TLC detection25 turned out to be sensitive and reliable for this purpose, in contrast to Wagner and Dragendorff reagents. For the separation of sugars from the methanolic extract, fractionation through a Diaion HP-5 column was used alternatively with the simpler solvent precipitation. Compound 1 was obtained as an optically active brown semisolid, whose presence on TLC plates was detected by the Mattocks-Molyneux reagent as a purple spot, revealing the presence of a 1,2-unsaturated necine N-oxide moiety of the retronecine/heliotridine type.25 1H NMR chemical shifts of the ring protons were in close agreement with the values reported for other acyclic diester retronecine/heliotridine N-oxides.10 Indeed, the chemical shifts of the deshielded H-3a/H-3b (δH 4.70/4.38), 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 NOESY cross-peak was observed between H-7 (δH 5.69) and H-8 (δH 4.88), indicating a heliotridine-type structure, where these protons are trans-oriented. Within the Onosma genus heliotridine PAs have been reported only for Onosma heterophylla.26 The signal at δH 2.06 (3H, s) is due to an acetyl group with the corresponding methyl carbon atom (C-9′) resonating at δC 21.1. The carbonyl carbon at δC 171.5 is assigned to the same acetyl group (C-8′), due to the HMBC correlations of C-8′ to H-7 and H-9′, revealing that O-7 is acetylated. The presence of the Δ1,2 double bond is confirmed by the downfield signals of H-2 at δH 6.00, and C-1/C-2 at δC 133.0/124.3, as well as from the COSY correlations H-3a, H-3b/H-2 and H-8/H-2. The protons at δH 4.78 (2H, s) show HMQC correlation with the carbon at δC 62.1, confirming the CH2-9 group. Corroborative evidence for the aforementioned assignment comes from the COSY H2-9/H-2 allylic correlation, along with the HMBC interactions of C-9 to H-2; C-1 to H2-9; and C-8 to H2-9. Key HMBC correlation between the carbonyl carbon at δC 175.0 (C-1′) and H2-9 indicated the presence of a necic acid unit esterified at O-9. Indeed, the signals at δH 3.96 (1H, q, J = 6.8 Hz), 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 the peaks at δC 175.0, 85.2, 72.4, 34.0, 16.7, 18.2, and 18.5 were in close agreement with those reported for the (−)-viridifloryl moiety in viridinatine.27 This assumption was further supported by COSY correlations H-3′/H3-4′and H3-6′/H-5′/H3-7′, as well as by HMBC correlations of C-1′ to H-3′, H-5′; C-2′ to H3′, H3-4′, H-5′, H3-7′; C-3′ to H3-4′; C-4′ to H-3′; C-5′ to H36′, H3-7′; and C-6′, C-7′ to H-5′. The structural formula corresponds to the molecular formula C17H27NO7 with a molecular mass of 357.4, as confirmed by the m/z 358.4072 of the pseudomolecular ion [M + H]+, obtained by HRESIMS. The absolute configuration was deduced by determining the retention index (RI) of reduced 1 (free amine), by the reduction/GC-MS method described by Witte et al.8 The mass spectrum recorded and the obtained RI value (2238) were in good agreement with those reported (RI 2235) for 7-Oacetylechinatine.20 Thus, 1 was identified as 7-O-acetylechina-

Table 1. NMR Data (600/400 MHz, methanol-d4) for Compound 1 position

δC, type

δH (J in Hz)

1 2 3a 3b 5a 5b 6a 6b 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

133.0, C 124.3, CH 79.0, CH2 70.1, CH2 33.4, CH2 73.8, 95.0, 62.1, 175.0, 85.2, 72.4, 18.2, 34.0, 18.5, 16.7, 171.5, 21.1,

CH CH CH2 C C CH CH3 CH CH3 CH3 C CH3

COSY

6.00 4.70 4.38 3.83 3.79 2.74 2.23 5.69 4.88 4.78

brs d (16.4) d (16.4) m m m m brm brs s

3a,3b,8,9 2,3b 2,3a 5b,6a,6b 5a,6a,6b 5a,5b,6b,7 5a,5b,6a,7 6a,6b,8 2,7 2

3.96 1.24 2.17 0.90 0.93

q (6.8) d (6.8) sept (6.8) d (6.8) d (6.8)

4′ 3′ 6′,7′ 5′ 5′

HMBCa 2,3a,3b,9 3a,3b,8,9 2,8 3a,6a 5a 5a,5b,6b 2,6a,6b,7,9 2 9,3′,5′ 3′,4′,5′,7′ 4′ 3′ 3′,6′,7′ 5′ 5′ 7,9′

2.06 s

a

HMBC correlations are from proton(s) stated to the indicated carbon.

Compound 2 was obtained as an optically active, brown semisolid that was detected on TLC plates by the MattocksMolyneux reagent and was recognized as a retronecine/ heliotridine N-oxide.25 The necine unit was identified as heliotridine as in 1. The remaining signals in the 1H and 13C NMR spectra were reminiscent of two viridifloryl moieties, esterified at O-7 and O-9 as in the PA viridinatine.27 Two isopropyl 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 H10′/H3-11′ fragments) were indeed recognized via COSY. The carbonyl carbons at δC 176.1 and 176.6 were attributed to C-8′ and C-1′, respectively, based on HMBC correlations of C-8′ to H-7, H-10′, and H-12′ and of C-1′ to H-9 and H-5′. In addition, peaks at δC 85.9 and 86.2 (missing from the DEPT spectrum) suggested the presence of two oxygenated quaternary carbon atoms. HMQC 13C/1H matching along with 13C/1H HMBC interactions observed among the aforementioned necyl signals (see Table 2) established their connectivity. The structural formula inferred for 2 corresponds to the molecular formula C22H37NO9 with a molecular mass of 459.5, as confirmed by the m/z 460.5388 of the [M + H]+ ion, obtained by HRESIMS. For the sake of comparison, part of 2 was reduced in an acidic zinc suspension.19 The 1H NMR spectrum of the free PA was recorded in the same specified solvent (CDCl3 saturated in D2O) and was subsequently compared with that of viridinatine.27 Viridinatine was reported to have two (−)-viridifloryl moieties esterified at O-7 and O-9, and consequently the isopropyl and 1-hydroxyethyl methine pairs appeared in the 1H NMR spectrum as close overlapping signals. Nevertheless, reduced 2 exhibited necyl protons at B



Article

δH 4.18 (1H, q, J = 6.4 Hz) and 1.14 (3H, d, J = 6.4 Hz) was recognized from 1H-1D and COSY spectra. From the 13C-1D, DEPT, and HMQC spectra, the presence of three oxygenated carbon atoms was evident, two quaternary (δC 86.9, 75.6) and one tertiary (δC 71.1), along with the ester carbonyl carbon at δC 176.1. HMQC 13C/1H matching along with 13C/1H HMBC interactions among the sites 1′−7′ established an echimidinyllike 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 the molecular formula C15H25NO7 with a molecular mass of 331.4, as confirmed by HRESIMS ([M + H]+ at m/z 332.3697). Two known PAs share the structural formula of 3, leptanthine Noxide isolated from O. leptantha (Boraginaceae) 9 and echimiplatine N-oxide, speculated from LC-MS data from extract analysis of Echium plantagineum (Boraginaceae).28 The 1 H-1D spectrum of 3 in methanol-d4 did not match that of leptanthine N-oxide.9 Part of 3 was reduced as described elsewhere,19 and the 1H-1D spectrum of the free PA was recorded in CDCl3 and subsequently was compared for its necyl signals (Table 3) with spectra of known PAs with necic acid of similar structure esterified at O-9, in the same solvent. The best matching of δH values occurred for the free PA hydroxymyoscorpine: δH 1.24/1.30 (H3-6′/H3-7′), 1.25 (H34′), and 4.19 (H-3′).10 Hence, it was inferred that the necic acid of 3 is echimidinic acid, and therefore 3 is the 7-epimer of echimiplatine N-oxide, a new PA for which the name 7-epiechimiplatine N-oxide is proposed. A portion of 3 was reduced to the corresponding PA as for 1, and its RI value (2069) was determined by GC using an HP-5MS column. Compound 4 was obtained as an optically active, brown semisolid and was recognized as a heliotridine N-oxide derivative 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′) and one as a doublet at δH 1.26 (d, J = 6.2 Hz, H3-5′) coupled to a methine group at δH 4.22 (q, J = 6.2 Hz, H-4′). The latter two signals suggest the presence of a 1-hydroxyethyl group. In the DEPT spectrum, except for the three methyl groups [δC 18.7, (C-5′); 26.6 (C-6′); 25.9 (C-7′)], two oxygenated methine groups were recognized at δC 70.7 and 70.8, ascribed interchangeably to C-4′ and C-7. The 13C NMR spectrum exhibited three additional peaks: the downfield signal at δC 175.3 due to the ester carbonyl group (C-1′) and two oxygenated quaternary carbon atoms at δC 74.6 (C-3′) and 85.7 (C-2′), the only possible locations of the two methyl groups (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 4.92 and 4.88, respectively, reveals O-9 as the necylated site of heliotridine N-oxide. The structure assigned for 4 corresponds to the molecular formula C15H25NO7 with a molecular mass of 331.4, as confirmed by HRESIMS ([M + H]+ at m/z 332.3696). Compound 4 is a novel PA, and the name onosmerectine N-oxide is proposed for it. Owing to the paucity of the compound, its hydrolysis was not attempted. The RI value of the reduced sample could not be obtained by GC, because of its thermal decomposition (one broad peak recognized as various nitrogenous compounds by the MS library, but not as a PA). Compound 5 was obtained as an optically active, amorphous, brownish solid, whose aqueous solution was tested with litmus

different chemical shifts from those of viridinatine, whereas the signals 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)] appeared as clearly separated peaks (see Table 2). Thus, the two structurally equivalent necic acids of 2 must be stereoisomers, and 2 a novel viridinatine N-oxide stereoisomer. Owing to paucity of the sample, its hydrolysis was not attempted. Furthermore, the RI value of the reduced sample could not be obtained by GC, due to its thermal instability (two overlapping peaks 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-Molyneux reagent and recognized as a retronecine/heliotridine N-oxide.25 The necine part was identified as heliotridine as in 1. The remaining signals in the 1H and 13C NMR spectra were reminiscent of an echimidinyl moiety. Two methyl groups at δH 1.16 (3H, s) and 1.21 (3H, s) bonded to a quaternary carbon atom, and a 1-hydroxyethyl group accounting for the signals at C



Article

Table 2. NMR Data (400 MHz) for Compound 2 and Its Reduced Analogue reduced 2 (tert. amine) (CDCl3 sat. in D2O)

2 (N-oxide) (D2O) δC, type

position 1 2 3a 3b 5a 5b 6a 6b 7 8 9

132.5, C 125.4, CH 79.5, CH2

1′ 2′ 3′

176.6, C 85.9, C 70.9, CH

δH (J in Hz) 5.86 4.51 4.22 3.73 3.61 2.45 2.00 4.67 4.57 4.75

70.1, CH2 35.8, CH2 71.3, CH 96.6, CH 63.3, CH2

brs d (16.6) d (16.6) m m m m brm brs brs

3a,3b,8,9 2,3b,9 2,3a,9 5b,6a,6b 5a,6a,6b 5a,5b,6b,7 5a,5b,6a,7 6a,6b,8 2,7 2,3a,3b

2,3a,3b, 8,9 3a,3b,8,9 2,5b 3a,3b,6b, 7,8 5a,5b,7,8 3a,3b,5a,5b, 6b,8 2,5a,5b,6a, 6b,7,9 2,8

4.04 q (6.4)

4′

9,5′ 3′,4′,5′ 6′,7′ 4′,5′

4′

17.6, CH3

1.04 d (6.4)

3′

3′

5′

34.6, CH

1.89 sept (6.8)

6′,7′

3′,6′,7′

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′

3.93 q (6.4)

11′

7,10′,12′ 10′,11′, 12′,13′,14′ 11′,12′

8′ 9′ 10′

176.1, C 86.2, C 72.3, CH

11′

18.6, CH3

1.10 d (6.4)

10′

10′

12′

34.2, CH

2.05 sept (6.8)

13′,14′

10′,13′,14′

a

δH (J in Hz)

HMBCa

COSY

5.93 4.41 3.65 3.86

brs d (15.4) d (15.4) m

3.12 2.16 4.80 4.56 4.94 4.89

m m m brm d (13.2) d (13.2)

4.18 4.01 1.23 1.12 2.28 1.97 0.99 0.95 0.92 0.84

q (6.8) or q (6.8) d (6.8) or d (6.8) sept (6.8) or sept (6.8) d (6.8) d (6.8) d (6.8) d (6.8)

4.18 4.01 1.23 1.12 2.28 1.97

q (6.8) or q (6.8) d (6.8) or d (6.8) sept (6.8) or sept (6.8)


Table 3. NMR Data (400 MHz) for Compound 3 and Its Reduced Analogue reduced 3 (tert. amine)a (CDCl3)

3 (N-oxide) (D2O) position 1 2 3a 3b 5a 5b 6a 6b 7 8 9 1′ 2′ 3′ 4′ 5′ 6′,7′ a

δC, type

δH (J in Hz)

132.4, C 125.9, CH 79.7, CH2 70.2, CH2 35.8, CH2 71.4, 96.7, 63.6, 176.1, 86.9, 71.1, 19.0, 75.6, 26.3, CH3 (×2)

CH CH CH2 C C CH CH3 C 27.0,

5.89 4.52 4.21 3.74 3.59 2.47 2.01 4.67 4.55 4.78

brs d (17.0) d (17.0) m m m m brm brs brs

4.18 q (6.4) 1.14 d (6.4)

COSY 3a,3b,9 2,3b,9 2,3a,9 5b,6a,6b 5a,6a,6b 5a,5b,6b,7 5a,5b,6a,7 6a,6b,8 7 2

4′ 3′

1.16 s 1.21 s

HMBCb

δH (J in Hz)

2,3a,3b, 8,9 3a,3b,8,9 2,8,5a 3a,3b,6a,7 5a,5b,7,8 5a,5b,6b,8 2,9 2,8 9,3′ 3′,4′,6′,7′ 4′ 3′ 3′,6′,7′ 6′,7′

4.19 q (6.4) 1.24 d (6.4) 1.30 s 1.22 s

Only necic acid (necyl) protons presented. bHMBC correlations are from proton(s) stated to the indicated carbon.

D



Article

Table 4. NMR Data (400 MHz, methanol-d4) for Compound 4 position 1

133.5, C

2

123.8, CH

3a 3b 5 6a 6b 7 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′ a

δC, type

78.6, CH2 69.7, CH2 35.7, CH2 70.7 or 70.8, CH 96.7, CH 62.5, CH2 175.3, C 85.7, C 74.6, C 70.7 or 70.8, CH 18.7, CH3 26.6, CH3 25.9, CH3

δH (J in Hz)

COSY

5.96 brs

3a,3b, 9a,9b

4.66 4.45 3.87 2.58 2.12 4.75 4.85 4.92 4.88

2,3b,9a,9b 2,3a,9a,9b 6a,6b 5,6b,7 5,6a,7 6a,6b,8 7 2,3a,3b,9b 2,3a,3b,9a

4.22 1.26 1.31 1.27

d (16.4) d (16.4) m m m brm brs d (16.4) d (16.4)

q (6.2) d (6.2) s s

5′ 4′

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

HMBCa 3a,3b,8, 9a,9b 3a,3b,8, 9a,9b 2,8

δH (J in Hz)

δC, type

position

COSY

HMBCa

5 1 2

177.3, C 84.0, C

3

74.9, C

4

70.8, CH

5

19.0, CH3

2-Me 3-Me

25.8, CH3 26.6, CH3

1

6 17.1 or 17.6 or 17.7, CH3

2

70.1, CH

3 4

C 33.5, CH

5

17.1 or 17.6 or 17.7, CH3 (×2)

4.17 q (6.4) 1.27 d (6.4) 1.32 s 1.26 s

5

3-Me, 4 2-Me, 3Me 2-Me, 3-Me, 5 3-Me, 5

4

4

1.18 d (6.4) 4.00 q (6.4)

2

2

1

1

2.06 sept (6.4) 0.95 d (6.4) 0.96 d (6.4)

5, 6

5, 6

4

4, 5, 6

3a,6a,6b

5,6a,6b 2,9a,9b 2 9a,9b 6′,7′ 7′ 5′ 4′

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

6

paper and was found acidic (pH = 2−3). In its 1H NMR spectrum, one 1-hydroxyethyl group [δH 4.17 (q, J = 6.4 Hz, H4); 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 atoms were recognized, as in 4. In the 13C NMR spectrum, one downfield signal (δC 177.3) was due to a carboxyl group, while small peaks at δC 84.0 and 74.9 missing from the DEPT spectrum were ascribed to two hydroxylated quaternary carbons (C-2 and C-3), bearing CH3-2 and CH3-3 groups. 13 C/1H HMBC correlations (see Table 5) established the connection among the recognized parts. The structure of 5 was identified as 2,3-dimethyl-2,3,4-trihydroxypentanoic acid, a new natural product. The molecular formula of 5 is C7H14O5 with a molecular mass of 178.2, as confirmed by HRESIMS ([M + H]+ at m/z 179.1906). It is noteworthy that 5 is structurally similar to the necic acid moiety of 4. The following considerations allowed us to infer that 5 is a natural metabolite and not a hydrolysate: (i) the known separate biosynthesis of monocarboxylic necic acids from amino acids,5 (ii) the mild conditions used during the isolation procedure, and (iii) the absence of the corresponding necic acids from the other PA Noxides. Besides, isolation of PAs and PA N-oxides along with their corresponding necic acid has been reported by Braca et al.29 In addition, it has been shown for some Boraginaceae species that PA biosynthesis takes place at several plant organs and not exclusively in the roots, as it has been proved for PAcontaining Asteraceae species.30 In this context, isolation of 5 as a PA biosynthesis intermediate from the aerial parts of a boraginaceous 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 = 6.4 Hz) (H3-5, H3-6), and 2.06 (sept, J = 6.4 Hz, H-4) indicated the 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 to a carbonyl group could explain the increased chemical shifts of

a


the two methine protons (the one at δH 2.06 being typical for α-H of carbonyl compounds), no carbonyl signal was observed downfield in the 13C NMR spectrum, possibly due to its elongated relaxation time in such a small structure. 13C/1H HMBC correlations (see Table 5) support structure 6, whereas no 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 hydrogen bonding. Two strong absorptions at 2973 and 2935 cm−1 are due to methyl C−H stretching vibrations, while a strong band at 1714 cm−1 is typical of aliphatic ketones (CO stretching). The latter is partially overlapped with a stronger band at 1758 cm−1, which is due to the same kind of vibration but shifted to a higher frequency due to α-OH rotational isomerism. Mediumintensity 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 a molecular mass of 116.2, as confirmed by HRESIMS ([M + H]+ at m/z 117.1659). 4-Methyl-2-hydroxypentanone (6) is reported 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 along with ESIMS ([M + K]+ at m/z 197.1, 13%, [M + Na]+ at m/z 180.9, 47%). Allantoin has been detected or isolated from other Boraginaceae species as well: Symphytum of f icinale L., S. uplandicum Nym.,33 Anchusa of f icinalis L., 34 Auxemma oncocalyx,35 Cordia trichotoma,36 Anchusa strigosa,29 and Ehretia thyrsif lora.37 Glycerol, D-fructose, and D-glucose were also obtained and identified on the basis of their 1H and 13C NMR spectra38 and TLC comparison with authentic samples. E



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volume of H2O, and after addition of iPrOH, the dark brown solution became turbid and finally separated into a lower brown layer and an upper orange layer, which was evaporated in vacuo and redissolved in the minimum amount of H2O. After addition of iPrOH and some cyclohexane, cooling of the solution overnight in a refrigerator allowed its separation into an upper yellow layer and a lower viscous brown one, which was combined with the previous lower brown layer obtained likewise, guided by TLC. The yellow layer after evaporation to dryness yielded 0.702 g of a yellow foam, which was subjected to CC (⦶ 1.5 × 15.7 cm, Diaion HP-20, water) to give fractions UL1A−G. Fractions UL1A,B were combined (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 contained pure 5 (11.2 mg). Fractions UL1C−E were combined (126.3 mg) and further 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), yielding pure 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 to CC (⦶ 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 was subjected to preparative TLC (silica RP-18, H2O/MeOH, 9:1), and after extraction of the zones with MeOH, glycerol (6.1 mg) was obtained. Subfraction UL2C-H was subjected to CC (Sephadex LH20, MeOH), and allantoin (27.9 mg) was obtained. Subfraction UL2IN (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 four fractions. The second and third fractions were further separated by preparative 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 brown layer was subjected to three successive CCs (cellulose, first column eluted with EtOAc, EtOAc/iPrOH mixtures, second and third columns eluted with cyclohexane/EtOAc and EtOAc/MeOH mixtures), and finally the mixture was separated using preparative TLC (silica plates, first development with EtOAc/MeOH/HOAc, 50:49.5:0.5, second development with EtOAc/MeOH/HOAc, 60:39:1). After extraction of the zones with MeOH, 4 (44.6 mg, also containing silica) was obtained. 7-O-Acetylechinatine N-oxide (1): brown semisolid; [α]20D −5.7 (c 0.105, 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 for C15H26NO7, 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

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

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EXPERIMENTAL SECTION

General 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 and 366 nm) and after spraying with a 2.5% H2SO4 and 2.5% vanillin MeOH solution, followed by heating at 105 °C for 5 min. For the special spot detection of PAs (free bases or N-oxides) on TLC plates, the visualizing reagents proposed by Molyneux and Roitman were applied successfully.25 For preparative TLC, Merck 20 × 20 cm silica gel 60 F254, silica gel 60 RP-18 F254S, and cellulose plates were used. Optical rotations were measured using a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Perkin-Elmer FT-IR Paragon 500 spectrophotometer. 1H-1D and HMQC, HMBC, and COSY 2D NMR spectra were recorded on 600 or 400 MHz while 13C-1D and DEPT spectra were recorded on 150 or 50 MHz FT NMR Bruker AVANCE spectrometers. ESIMS spectra (positive mode) were obtained by injecting samples (as MeOH solutions, except for 5, which was dissolved in H2O) directly into the spectrometer, using a 3200 QTRAP LC/MS/MS Applied Biosystems mass spectrometer and ion spray voltage of +4500 V, declustering potential of 50 V, and entrance potential of 10 V. High-resolution mass spectra (ESI+) were recorded on a Thermo Scientific LTQ Orbitrap Discovery mass spectrometer with the infusion method. The solvents or solvent combinations used in column chromatography with cellulose as stationary phase had been formerly saturated in H2O, after shaking with H2O in a separatory funnel (except for H2O-miscible solvents). The stationary phases used for column chromatography were silica gel 60H and 230−400 mesh, microcrystalline cellulose (Merck), as well as Sephadex LH-20 (Pharmacia), and styrene-divinylbenzene copolymer resin Diaion HP-20 (Supelco). GC-MS analyses were performed on a Hewlett-Packard 6890 chromatograph, connected to an electronimpact Hewlett-Packard 5973 mass spectrometer. For GC-MS PA identification, the analytical method described by Witte and coworkers8 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) (or alternatively capillary column HP-5MS 30 m × 0.25 mm i.d., film thickness 0.25 μm), injector temperature 220 °C, temperature program 150−300 °C, 6 °C/min, split ratio 1:20, carrier gas He 0.75 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 May 2004, from “Lefka Ori” mountains in western Crete, Greece. The plant was botanically identified by Dr. E. Kalpoutzakis, dried at room temperature, and subsequently deposited at the Herbarium of the Pharmacognosy Department of the Faculty of Pharmacy of the University 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 g extract), 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 room temperature. Seventeen grams of the MeOH extract was suspended in 100 mL of H2O and successively extracted with 2 × 100 mL of cyclohexane, cyclohexane/n-butanol (66:33), cyclohexane/n-butanol (33:66), and n-butanol. The latter three extracts were pooled after TLC examination, evaporated in vacuo, and partitioned between EtOAc and H2O. The mixture (0.272 g) obtained from the EtOAc layer was subjected to column chromatography (⦶ 2 × 41 cm, Sephadex LH-20, MeOH) and resulted in multicomponent fractions of only minor amounts (0.1−30 mg). The mixture (3.792 g) obtained from the H2O layer was combined with the H2O-soluble part (9.4 g) of the MeOH extract after TLC examination, and the solution was evaporated in vacuo. The residue was dissolved in the minimum F



Article

(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 for C7H15O5, 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).

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(18) Roeder, E.; Breitmaier, E.; Birecka, H.; Frohlich, M.; BadziesCrombach, A. Phytochemistry 1991, 30, 1703−1706. (19) Witte, L.; Ernst, L.; Adam, H.; Hartmann, T. Phytochemistry 1992, 31, 559−565. (20) El-Shazly, A.; Sarg, T.; Ateya, A.; Abdel Aziz, E.; Witte, L.; Wink, M. Biochem. Syst. Ecol. 1996, 24, 415−421. (21) Suau, R.; Cabezudo, B.; Rico, R.; Nájera, F.; López-Romero, J. M.; García, A. I. Biochem. Syst. Ecol. 2002, 30, 981−984. (22) Benn, M.; Gul, W. Biochem. Syst. Ecol. 2007, 35, 676−681. (23) Baerheim Svendsen, A.; Verpoorte, R. J. Chromatogr. Libr. 1983, 23A, 52−57. (24) El-Shazly, A.; Sarg, T.; Witte, L.; Wink, M. Phytochemistry 1996, 42, 1217−1221. (25) Molyneux, R. J.; Roitman, J. N. J. Chromatogr. 1980, 195, 412− 415. (26) Mellidis, A. S.; Papageorgiou, V. P. Chem. Chron. 1988, 17, 67− 73. (27) Ravi, S.; Ravikumar, R.; Lakshmanan, A. J. J. Asian Nat. Prod. Res. 2008, 10, 349−354. (28) Colegate, S. M.; Edgar, J. A.; Knill, A. M.; Lee, S. T. Phytochem. Anal. 2005, 16, 108−119. (29) Braca, A.; Bader, A.; Siciliano, T.; Morelli, I.; De Tommasi, N. Planta Med. 2003, 69, 835−841. (30) Frölich, C.; Ober, D.; Hartmann, T. Phytochemistry 2007, 68, 1026−1037. (31) Poje, M.; Sokolić-Maravić, L. Tetrahedron 1986, 42, 747−751. (32) Ferreira, D. T.; Alvares, P. S. M.; Houghton, P. J.; Braz-Filho, R. Quim. Nova 2000, 23, 42−46. (33) Fell, K. R.; Peck, J. M. Planta Med. 1968, 16, 411−420. (34) Romussi, G.; Cafaggi, S.; Mosti, L. Pharmazie 1979, 34, 751− 752. (35) Pessoa, O. D. L.; Lemos, T. L. G. Rev. Brasil. Farm. 1997, 78, 9− 10. (36) Menezes, J. E. S. A.; Lemos, T. L. G.; Silveira, E. R.; Braz-Filho, R.; Pessoa, O. D. L. J. Brazil. Chem. Soc. 2001, 12, 787−790. (37) Li, L.; Shi, R.; Wu-Lan, T.; Xu, L.; Peng, Y.; Xiao, P. Zhongguo Zhongyao Zazhi 2010, 35, 331−332. (38) Pouchert, C. J.; Behnke, J. The Aldrich Library of 13C and 1H FT NMR Spectra; The Aldrich Chemical Company: Milwaukee WI, 1993; Vol. 1; pp 283−300.

ASSOCIATED CONTENT

S Supporting Information *

IR, COSY, and 1H and 13C NMR spectroscopic data of 1−6 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (+30) 2107274595. Fax: (+30) 2107274115. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to Dr. E. Kalpoutzakis for collecting and identifying the botanical sample and Dr. M. Popova (Bulgarian Academy of Sciences, Sofia) and Dr. D. Benaki (“EKEFE Demokritos” Research Institute, Athens) for recording certain NMR spectra.

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REFERENCES

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Pyrrolizidine Alkaloids from Onosma erecta - Journal of Natural

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