A secoiridoid glucoside and a phenolic compound from Fraxinus ornus bark

Phytochemistry,Vol. 34, No. 5, pp. 1373 1376,1993 Printed in Great Britain.

A SECOIRIDOID

TANYA

003I -9422/93$6.00+ 0.00 0 1993Pergamon Press Ltd

GLUCOSIDE AND A PHENOLIC FROM FRAXINUS ORNUS BARK

COMPOUND

IOSSIFOVA, BOZHANA MIKHOVA and IVANKA KOSTOVA*

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria (Received in revised form 11 May 1993)

Key Word Index-Fraxinus

hydroxyphenyl)ethanol

ornus; Oleaceae; bark; derivatives; ornosol; tyrosol.

secoiridoids;

ornoside;

ligstroside;

2-(4-

Abstract-A new secoiridoid glucoside named ornoside and a new tyrosol derivative named omosol have been isolated from Fraxinus ornus bark, along with the known compounds ligstroside, 2-(4-hydroxyphenyl)ethanol and caffeic acid.

INTRODUCTION

A characteristic feature of oleaceaous plants is the high content of secoiridoid derivatives. A number of oleoside (1)-type glucosides, including ligstroside (2) and fraxiformoside (3) have been isolated from the genus Fraxiraus

Cl-4 In the course of systematic phytochemical studies on the total ethanolic extract of F. ornus bark, we isolated a new secoiridoid glucoside, ornoside (4) and a new phydroxyphenylethanol derivative, ornosol (5) together with four known substances. In this paper we describe the isolation and the structure elucidation of the new compounds. The work is connected with our interest in the chemistry and biological activity of Fraxinus species t-5, 61. RESULTS AND DISCUSSION

total ethanolic extract of F. ornus bark was worked-up as described in the Experimental to give two secoiridoid glucosides ligstroside (2) and ornoside (4) along with /I-sitosterol glucoside, three phenolic compounds, 2-(4-hydroxyphenyl)ethanol (tyrosol) and 3-[4(2-hydroxyethyl)-phenoxyl-4-hydroxyphenylethanol (ornosol) (5), and caffeic acid. The structure of 2 was established by a careful comparison of its UV, IR, ‘H and 13CNMR spectra with literature data [3] and alkaline hydrolysis. Secoiridoid glucoside (4), named ornoside, C32H36013r was obtained as a hygroscopic amorphous powder. It showed UV maxima at 229 and 274 nm in ethanol and IR bands at 3412,1734,1709,1620,1506 and 1435 cm-‘. The FAB mass spectrum of 4 exhibited a [M +H]+ at m/z 629. Its ‘HNMR spectrum revealed typical signals of an oleoside nucleus: a singlet at 67.55

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*Author to whom correspondence

should be addressed.

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characteristic for H-3 of a secoiridoid glucoside, an anomeric proton at 4.87 (d) and an olefinic proton at 66.06 (br q), coupled to a methyl signal at 1.62 (br d) and an acetal proton at 5.87 (br s). The ‘HNMR spectrum

1373

T. IOSSIFOVA et al.

1314

Table 1. “C-chemical shifts of ligstroside (2)* and ornoside (4) in CD,OD 2

C

95.3 d 155.2 d 109.5 s 31.9 d 41.3 t 173.3 s 125.0 d 130.6 s 13.6 q 168.7 s 101.0 d 74.9 d 78.0 d 71.6 d 78.5 d 62.9 t 51.9 q 66.9 t 35.2 t 130.2 s 131.1 d 116.4 d 157.2 s 116.4 d 131.1 d

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also suggested the presence of an aromatic AA’BB’ system centred at 67.21 and 6.94, a characteristic pattern of a 1,2,4_trisubstituted benzene ring at 6 6.85 (d, J = 8.0 Hz), 6.78 (dd, J=8.0, 1.8 Hz) and 6.53 (d, J- 1.8 Hz), two sets of OCH,CH,Ph moieties, which appeared as an ABMX spin system at 64.51 (m) and two multiplets in the region 2.80-3.0 as an ABX, spin system at 64.25 (dt), 4.00 (dt) and 2.76 (t). The ‘HNMR spectrum of the acetate 4a exhibited signals for only four alcoholic and one phenolic acetyl group in the trisubstituted benzene ring. A careful comparison of the ‘H and ’ 3C NMR spectra (Table 1 and Experimental) of 2 and 4 and their corresponding acetates revealed a close similarity regarding the oleoside (1) moiety, but instead of the C-l 1 carbomethoxy group the presence of an additional lJ,,Ctrisubstituted benzene ring and one PhCH,CH,O group was observed. The NMR data suggested that there were no free groups. PhCH,CH,OH Alkaline hydrolysis of 4 afforded glucose and the new phenolic compound, named ornosol, whose structure was elucidated as 5 on the basis of its spectral data and chemical behaviour. The ‘HNMR spectra of 5 and its acetate (5a) suggested the presence of two sets of PhCH ,CH ,OH units, one 1,Cdisubstituted benzene ring, one 1,2,4_trisubstituted benzene ring and only one phenolic OH group in the molecule. Further NOE experiments on 5a confirmed the exact position of all substituents. The 70eV mass spectrum of 5 exhibited a [M]’ at m/z 274 and the ions at m/z 256 [M-H20]+, 243 [M-.CHzOH]’ and 225 [M-.CHzOH-H,O]+ were in accordance with a HOCH,CH,PhOPh(OH) CH,CH,OH structure. These findings implied that in ornoside (4), the ornosol (5) moiety is linked to the oleoside (1) through the nonphenolic CH,CH,OCO ester bonds at C-7 and C-l 1. The position of attachment was unambiguously established by detailed NOE experiments on 4. The most characteristic NOES are presented in Fig. 1. On irradiation of CH,-2”, an enhancement of the H-3 singlet, the doublet for H-4” and H-8” and CH,-1” was observed. Irradiation of CH,-1”’ leads to enhancement of CH,-2”’ and H-4”‘. This confirms the assignment of CH,-l”, 2”, 1”’ and 2”’ and proves, that the disubstituted (tyrosol) moiety is linked to C-11. Recently, the isolation of several secoiridoids having two tyrosol-type molecules linked to the oleoside-COOH groups, have been reported [l, 31. Ornoside (4) is the first

3 4 5 6 7 8 9 10 11 1’ 2

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4 95.1 d 155.3 d 109.7 s 31.3 d 40.8 t 172.6 s 124.9 d 129.9 s 13.7 q 167.9 s 100.9 d 14.2 d 78.3 d 71.4 d 77.9 d 62.7 t 65.9 34.8 132.2 131.5 120.9 157.4 120.9 131.5 66.3 35.8 135.4 120.0 147.6 147.0 117.7 125.1

t t s d d s d d

t t s d s s d d

*Ref. [S]. example of a secoiridoid derivative in Fraxinus species, in which a flexible microcyclic ring (Fig. 1) is formed between one phenolic compound and the oleoside (1) aglucone. Most probably, in the preferred conformation of this ring, H,-6 and H,-6 are specifically influenced by the C-7 and C-l 1 carbonyl groups and this could explain the observed difference of their chemical shifts in 2 and 4 without a significant change of the coupling constants. This fact and the observed NOE enhancement of H-5 and H-l on irradiation of H,-6 and H,-6, respectively. (Fig. l), indicate that the configuration at C-5 and C-l in ornoside (4) is the same as that described for oleoside derivatives

lx. GC-MS analysis of the ethanol extract (see Experimental) revealed the presence of tyrosol and ornosol (!I)in trace amounts. EXPERIMENTAL General. Mps: uncorr; ‘H and i3CNMR spectra [S (ppm), J (Hz)] were obtained at 250 MHz (‘H NMR) and at 63 MHz (i3C NMR) using TMS as int. standard; NOE

Constituents of Fraxinus ornus bark

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Fig. 1. Most important enhancements of the signals in the homonuclear ‘H NOE spectra of 4. Arrows ( n) designate mutual effect between the indicated protons.

were performed using standard Bruker software. MS: 70 eV, FAB-MS: monothioglycerol as matrix. TLC: aluminium sheets, silica gel 60 F,,, (Merck), bands detected under UV light, after exposure to I, vapour or by spraying with H,SO* and heating. Prep. TLC: 20 x 20 cm plates coated with 1 mm of silica gel PF,,, (Merck), bands detected in UV light and after exposure to I, vapour. Liquid vacuum chromatography (LVC): silica gel LS 5-40 p (Chemapol); solvent systems: CHCl,-MeOH-H,O (30: 10:3), to 25 ml of the lower layer 0.5 ml of HCO,H is added (A), CHCl,-MeOH (10: 1) (B), toluene-Et,0 (1: 2), satn with 10% HOAc (C), CHCl,-MeOH-H,O (30: 12:4), to 9 ml of the lower layer 1 ml of HOAc is added (D). GC: column SPB-1 (0, 25 mm x 15 m), temp. prog. 150-300” at lo” min-‘, with a 10 min hold at 300”, He 3 ml min-‘. GC-MS was carried out at 70 eV using the same column as that described above. Plant material. A commercial sample of F. OIWJSL. bark collected in 1991 from the region of Dragoman, was investigated. A voucher specimen is deposited in the Herbarium of the Institute of Botany, BAS, Sofia. Isolation of glucosides. Dried and well-ground bark (1 kg) was extracted with hot EtOH (3 x 7 1). The insol. material was removed by filtration and the extract coned under red. pres. to a small vol. After filtration of the deposited esculin (30 g), the mother liquor was coned under red. pres. and subjected to solvent-solvent partition using PE and EtOAc to afford R-l (20 g) and R-2 (50 g), respectively. R-2 (6.2 g) was further worked-up by LVC over 70 g silica gel using DXE and DXE-MeOH with increasing polarity (10: 1, 5: 1, 3: 1). Frs eluted with DXE and DXE-MeOH (10: 1) were coned under red. pres. to give residues R-3 (0.182 g) and R-4 (1.54 g). R-4 (1.54 g) was subjected again to LVC over 70 g silica gel eluting with DXE-MeOH (1O:l). Frs showing a main spot with R,=0.6 in solvent A were combined and purified by prep. TLC in system B (3 developments) to give 4 (160 mg). Frs exhibiting a main spot with R, =0.5 were combined and purified by prep. TLC in system A (1 experiments

PHYTO 34:5-n

development) and in B (2 developments), to obtain 2 (30 mg). Ornoside (4). Powder. [!x]hO-67.70” (EtOH; c 0.011). UV ikt” nm (log E):229 (4.26), 274 (3.51). IR vi:; cm-‘: 3412, 1734, 1709, 1620, 1506, 1439. ‘HNMR (CD,OD): 67.55 (lH, s, H-3), 7.21 (2H, d, 3=8.5 Hz, H-4”, H-8”), 6.94 (2H, d, J=8.5 Hz, H-5”, H-7”), 6.85 (lH, d, .I = 8.0 Hz, H-7”‘), 6.78 (lH, dd, J = 8.0 and 1.8 Hz, H-8”‘), 6.53 (lH, d, 5=1.8 Hz, H-4”‘), 6.06 (lH, br q, J=7,0Hz, H-8), 5.87 (lH, br s, H-l), 4.87 (lH, d, J=8.0 Hz, H-l’), 4.51(2H, m, CH,-l”), 4.29 (lH, dt, J=6.0and 5.5 Hz, H,1”‘),4.0(1H,dt,J=6.0and5.5 Hz,H,-1”‘), 3.88(1H, brd, J=12.2Hz,H,-6’),3.80(1H,dd,J=10.7and3.8Hz,H-5), 3.66 (lH, dd, 5=12.2 and 5.1 Hz, H,-6’), 3.4-3.2 (4H, obscured by the signal of solvent, H-2’, H-3’, H-4’, H-S), 3.1-2.8 (2H, m, CH,-2”), 2.76 (2H, t, J=5.5 Hz, CH,-2”‘), 2.32 (lH, dd, .I= 15.0 and 3.8 Hz, H,-6), 2.18 (lH, dd, J = 15.0 and 10.7 Hz, H,-6), 1.62 (3H, br d, J = 7.0 Hz, Me10). 13C NMR see Table 1. FAB-MS m/z: 629 [M + HI+, 651 [M+Na]*. Acetylation of ornoside (4). Compound 4 (28 mg) was acetylated with pyridine-Ac,O (each 0.5 ml) at room temp. for 24 hr. The product (36.7 mg) was purified by prep. TLC (system C) to give ornoside pentaacetate (4a, 26 mg). [a]io -65.58” (EtOH: c 0.011). UV 2::” nm (log E): 230 (4.19), 274 (3.45). ‘HNMR (CDCI,): 67.53 (lH, s, H-3), 7.16 (2H, d, J= 8.5 Hz, H-4”, H-8”), 7.03 (lH, d, J=8.0 Hz, H-7”‘), 6.99 (2H, d, 5=8.5 Hz, H-5”, H-7”), 6.85 (lH, dd, J=8.0 and 1.8 Hz, H-8”‘), 6.52 (lH, d, J = 1.8 Hz, H-4”‘), 6.02(1H, br q, J=7.0 Hz,H-8), 5.65(1H, br s, H-l), 5.25 (lH, d, J=9.3 Hz, H-3’), 5.12 (2H, br t, J =9.0 Hz, H-2’, H-4’), 5.01 (lH, d, J=8.0 Hz, H-l’), 4.66 (lH,dt,J=6.0and 5.5 Hz,H,-1”), 4.4-4.2(3H, H,,-1”, H,l”‘, H-6’), 4.2-4.0(2H, H,-5, Hb-1”‘), 3.8-3.7 (2H, H-S, H6’),3.06(1H,dt,J=9.0and5.5Hz,H,-2”),2.9O(lH,dt,J =9.0and 5.5 Hz, H,,-2”), 2.80(2H, a,.T=6.0 Hz,CH,Z”‘), 2.41 (lH, dd, J= 15.8 and 3.4 Hz, H,-6), 2.31 (3H, s, OAc), 2.10 (lH, dd, J= 15.8 and 10.0 Hz, H,-6), 2.04 (6H, s, OAc), 2.02 (6H, s, OAc), 1.67 (3H, br d, J = 7.0 Hz, Me-lo). Alkaline hydrolysis of ornoside (4). Compound 4 (16 mg)

1376

T. IOSSIFOVA

was dissolved in 2 ml 2 M KOH in MeOH and refluxed for 2 hr at 60”. The reaction mixt. was diluted with H,O (8 ml), adjusted to pH 4 (cont. HCl) and extracted with Et,0 and EtOAc. TLC examination (system D) of the Hz0 layer and comparison with an authentic sample confirmed the presence of glucose. The combined Et,0 and EtOAc extracts were dried (NaSO,) and evapd to give a residue (7.8 mg), which on recrystallization from CHCl, gave pure ornosol(5, 4.8 mg), mp 114-l 16”. UV E‘max EtoHnm (log E): 208 (4.31), 221 (4.24), 277 (3.74). IR ~2: cm _ ‘: 3386, 3320, 1593, 1507, 1440; ‘HNMR (CD,OD): 67.22 (2H, d, J=8.0 Hz, H-4’, H-8’), 7.00 (lH, d, J=8.0Hz, H-7), 6.91 (lH, dd,.l=g.Oand 2.0Hz, H-8), 6.84(2H,d, J=8.0 Hz,H-S, H-7’).6.76(1H, d,J=2.0Hz, H-4), 3.87, 3.78 (each 2H, t, J = 7.0 Hz, CH,-1 and CH2l’), 2.87, 2.73 (each 2H, t, J = 7.0 Hz, CHz-2 and CH,-2’). EIMS (70 eV), m/z (rel. int.): 274 [M]’ (41), 256 [M-H,O]+ (2), 243 [M-.CH,OH]+ (lOO), 225 [M-.CH,OH-H,O]+ (29), 107 (39), 91 (18), 77 (27). Acetylation of5. Compound 5 (5 mg) was acetylated in the usual way and the acetate (7 mg) obtained purified by prep. TLC in system C to yield Sa (4.5 mg). ‘HNMR (CDCI,): 67.16 (2H, d, J= 8.0 Hz, H-4’ and H-8’), 7.07 (lH, d, 5=8.0 Hz, H-7), 6.96 (lH, dd, J=S.O and 2.0 Hz, H-8), 6.93 (2H, d, J = 8.0 Hz, H-5’ and H-7’), 6.83 (lH, d, J =2.0 Hz, H-4), 4.25, 4.22 (each 2H, t, J= 7.0 Hz, CH,-1 and CH,-l’), 2.91,2.86 (each 2H, t, 5=7.0 Hz, CH,-2 and CH,-2’), 2.17 (3H, s, OAc), 2.05 (3H, s, OAc), 1.99 (3H, s, OAc). Alkaline hydrolysis of ligstroside (2). Compound 2 (16 mg) under the same reaction conditions as described above afforded tyrosol (2 mg) and glucose.

et al.

Detection of minor compounds. R-3, already described in the Experimental, was subjected to prep. TLC (system C) and the bands corresponding to tyrosol and ornosol (5) were removed to obtain residues R-6 and R-7. GC-MS of R-6 and R-7 confirmed the presence of tyrosol and ornosol (5), respectively. Acknowledgements-We

are grateful to Mrs B. Wundrak and to Dr B. Vogler, Institute of Chemistry, University of Hohenheim, Germany for NOE measurements, to Prof. Dr H. Budzikiewicz, University of K61n, Germany for the FAB-mass spectra and to the staff of the Institute of Organic Chemistry, Bulgarian Academy of Sciences, for recording the mass, GC-mass UV and IR spectra.

REFERENCES

1. Tanahashi, T., Watanabe,

H., Itoh, A., Nagakura, N., Inoue, K., Ono, M., Fujita, T., Morita, M. and Chen, C.-C. (1992) Phytochemistry 32, 133. 2. Kuwajima, H., Morita, M., Takaishi, K., Inoue, K., Fujita, T., He, Z.-D. and Yang, C.-R. (1992) Phytochemistry 31, 1277. 3. Tanahashi, T., Watanabe, H., Itoh, A., Nagakura, N., Inoue, K., Ono, M., Fujita, T. and Chen, C.-C. (1992) Phytochemistry 31, 2143. 4. Damtoft, S., Franzyk, H. and Jensen, S. R. (1992) Phytochemistry 31, 4197. 5. Kostova, I. (1992) Planta Med. 58, 484. 6. Nykolov, N., Iossifova, T., Vassileva, E., Kostova, I. and Stoev, G. (1992) Phytochemical Analysis 4, 86.