Phytochmisrry. Vol. 35. No. 2. pp. 54-549 IW Ekvtcr S.Sena Ltd

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CINNAMRUTINOSES A AND B, GLYCOSIDES OF POPULUS TREMULA

AKINO JOSSANG, PER JOSSANG and BERNARD BODO

Laberatoire de Chimie, URA 401 CNRS, Mu&m National d’Histoire Naturelle, 63 rue Buffon, 75005 Paris, France

(Receioed 26 July 1993)

Key Word ID&X-Populus tremula; Salicaceae; stem bark; cinnamylglucosides; cinnamrutinoses A and B.

Abstract-The cinnamylrutinosides, cinnamrutinoses A and B, were isolated along with known phenolic ghrcosides, from the stem bark of Populus tremula infected with the pathogenic fungus Hypoxylon mammatum. The structures were determined from spectroscopic and chemical evidence.

INTRODUCTION

From a culture of Hypoxylon mammatum, a fungus re- sponsible for canker in Populus tremula and P. tremul- oides, five phytotoxins, hymatoxins A-E were previously isolated by using specific bioassays [ 1,2]. To ascertain the phytotoxic activity in uiuo of these compounds, we attempted to isolate them from the bark of P. tremula infected with H. mammaturn. Together with known flav- anoids and phenolic glucosides typical of the genus Populus [3,4], we isolated two new glycosides, cinnamru- tinoses A (1) and B(Z), the structural elucidations of which are reported here.

RESULTS AND DISCUS!%ON

The methanolic extract of the stem bark of P. tremula was separated into CHzCI, soluble and insoluble parts. The insoluble part was phytotoxic and is under investiga- tion. The CHzCl, soluble part was not toxic in the aspen leaf bioassay, but displayed antifungal activity against Penicilliwn crustosum. Repeated chromatography af- forded four known antifungal compounds, viz., scopole- tin, (+)liovil [S, 63, pyrocatechol and (-)7-0- methylaromadendrin [7,8]. The antifungal activity of scopoletin [9] and 7-0-methylaromadendrin [lo] is pre- viously reported. Further substances isolated were fl- sitosterol, the flavonoids, rhamnocitrin, naringenin and eriodictyol, and the phenolic glucosides, tremulacin [ 111, tremuloidin [ 123, salicyloyltremuloidin [ 133, salicortin [ll, 143, salireposide [14] and salicin. Finally, two novel compounds, named cinnamrutinoses A (1) and B (2), were isolated.

Compound 1 was obtained as an amorphous pow- der. By positive ion HR-FAB-mass spectrometry, the molecular formula was established as Cz,H30010 dedu- ced from the pseudo-molecular ion [M + Na]’ at m/z 465.1764 (calcd: 465.1737). The CI mass spectrum dis- played the quasi-molecular ion [M + NH,] + at m/z 460.

The ‘H and “C NMR chemical shifts were assigned from 2D experiments (‘H-‘HCOSY, ‘H-“CCOSY and ‘H-l% long range COSY). Signals for five protons of an aromatic AzBzM spin system and for two ethylenic E-protons (J = 16 Hz) at 66.35 and 6.68 connected with a CH, revealed the presence of a cinnamyl group (Table I). This was in agreement with an abundant ion fragment at m/t 117 in the mass spectrum. A doublet signal at 6 1.27 was assigned to the methyl at the 6-position of a rhamno- pyranose and the second sugar moiety was identified as a glucopyranose by measurement of proton vi&al coup ling constants. The 2 Hz coupling constant value of the rhamnosyl anomeric proton 1” at 64.76 indicated a /?-position, and that of 8 Hz of the, glucosyl anomeric proton (1’) at 64.37 was indicative of an x-trans disposi- tion. Finally, the ‘H- “C long range COSY optimized for J =4 Hz allowed the assignment of the cinnamyl CH, at C-9 to the I’-/I-O-glucose oxygen atom because cross- peaks were detected between 6c70.7 and 6u4.37, and between 6c103.3 and b4.28, and 4.48. The glucose 6’- CH, was linked to the 1”-a-0-rhamnose oxygen atom which showed a cross-peak between 6c68.1 and 6u4.76. Thus, the structure of cinnamrutinose A was l’-/I-O- cinnamylrutinoside, 1.

Cinnamrutinose B, 2 was obtained as an amorphous powder. By HR-FAB-mass spectrometry the molecular formula was established as CzeHJbOl I obtained from a quasi-molecular ion [M +Na]’ at m/z 569.2031 (calcd

547

548 Short Reports

Table I. “C and ‘H NMR data for cinnamrutinose A, 1 and cinnamrutinose B, 2 in CD,OD

Cinnamrutinose A, 1 Cinnamrutinose B, 2

C 6C SH In J (Hz) 6C l5H m J (Hz)

1 138.1 2 127.5

3 129.5 4 128.7

5 129.5

6 127.5

7 133.9

8 126.5

9 70.1

1’ 103.3

2 75.1 3 71.7

4 76.9

5 78.0

6 68.1

1”

2”

3”

4”

5”

6”

1 I,,

2”

3”‘. 7” 4”‘. 6” 5”’

101.3 72.2

72.3

74.0

69.8

18.0 _

-

- 7.40

7.29 7.21

7.29

7.40

6.68

6.35 4.28

4.48

4.37

3.22

3.36

3.42

3.39

3.98

3.64

4.76

3.86

3.71 3.40

3.68

1.27

- m

m In

m

zhf ddd ddd

ddd d t t

fidd dd dd d dd dd t m

d

- 138.1

- 127.5

- 129.6

- 128.7 - 129.6

127.5

16.5 2 133.9

16. 7, 5 126.4

13. 7, 2 70.9

15, 5, 2

8 103.4

8, 8 75.2 8, 8 75.7

8, 8 72.8 8, 6, 2 74.6

II, 2 67.6

11, 6

2 102.2 3, 2 71.9

9, 3 72.2

9, 9 73.8

69.8

6 17.9

167.1

131.3

130.7

129.6

134.5

- 7.42

7.30 7.25

7.30 7.42

6.70

6.38

4.33

4.53

4.49

3.40

3.73

5.11

3.78 3.76

3.52

4.62 3.85

3.67

3.34

3.58

1.14

- m m m

m

z ddd ddd ddd

d dd dd d

aw dd dd d dd dd dd dd d

8.05 m 7.47 m 7.61 m

-

- -

- 16, 2

16, 6, 5

13, 6, 2

13, 5, 2 8

8, 9 9. 9 9

9, 6, 3

17 3 12,6 2

4, 2 10, 4

10, 9

9, 6 6

569.1999). The CI mass spectrum displayed a quasi- molecular ion [M + NH,] + at m/z 564. The ion at m/z

460 [M + NH, - 1051’ was due to the loss of a benzoyl group. 2DNMR experiments (‘H-‘HCOSY, *H-13CCOSY and ‘H-“C long range COSY), revealed the signals of 10 protons in two sets of aromatic A,B,M spin systems, two ethylenic E-protons connected with a CHI and one carbonyl group at 6 167.1, the presence of a cinnamyl and a benzoyl group. Fragments for the latter groups were observed at m/z 117 and 105 in the mass spectrum. The structure of the glycoside part was deter- mined as for 1 by measurement of the proton vicinal coupling constants (Table 1). The H-4’ signal at 63.42 in 1 moves downfield to 6 5.11 on esterification of the 4’-OH in 2. The J values of 8 and 2 Hz indicate a and #I configura- tions, respectively, for the glucose and rhamnose anom- eric protons. Confirmation that the cinnamyl 9-methyl- ene was linked to l’-/3-O-glucose., and the glucose 6’-CH, to 1”-a-0-rhamaosyl was shown by a ‘H-“C long range COSY experiment optimized for J =4 Hz. The structure of cinnamrutinose B was thus 4’-0-benzoyl-I’-/?- cinnamylrutinoside, 2.

EXPERIMENTAL

Extraction and isolation. The MeOH extract of infected dried stem bark of P. tremulu (2lOOg) was evapd to

dryness (360 g) and further fractionated with CH,CI, into a soluble (65.6 g) and insoluble part (261.9 g). By chro- matography on silica gel (CH,C12-MeOH gradient), the CH,Cl, soluble part was sepd into 12 frs and each fr. further chromatographed on a Sephadex LH 20 column (MeOH) to yield /I-sitosterol (1.17 g), (-)O-methylaro- madendrin (1.05 g), scopoletin (0.14 g), (+)liovil (0.10 g), pyrocatechol (0.2 g), rhamnocitrin (0.02 g), naringenin (0.25 g), eriodictyol(O.02 g), tremulacin (0.50 g), tremuloi- din (0.13 g), salicortin (0.40 g), salireposide (0.15 g), salicin (0.05 g), 1 (0.10 g) and 2 (0.17 g).

(+)Liouil. Amorphous solid. C,,H,,O,: 376. [alo +50.8” (MeOH; c 0.65). UV INz”: 207,231,281,288sh. EIMSm/z(%): 376([M]+, 30). 358 (40), 341(35), 252 (lo), 235 (lOO), 205 (20). 151 (15), 137 (18). ‘HNMR, CDCI,: a3.06(m, H-8),3.84(s,OMe), 3.82(&,5=4and9 Hz)and 4.19(&5=7 and 9 Hz; H,-9A4.68 (d, J=5 Hz, H-7), 6.76 (dd, J = 2 and 8 Hz, H-6). 6.82 (d, J = 8 Hz, H-5), 6.85 (d, J

=2 Hz, H-2). 13CNMR, CDCI,: 655.9 (C-8), 71.6 (C-7), 85.8 (C-S), 108.6 (C-5), 114.2 (C-2), I 18.9 (C-6), 132.8 (C-l), 145.2 (C-I), 146.8 (C-3).

Cinnamrutinose A, 1. CIMS, NH,, m/z (%): 460 (CM +NH J+, lOO), 180 (60), 164 (67). 117 (92). HRMS m/z

465.1764 [M+Na]+ (calcd: 465.1737 for C2,H3,0,,Na). Cinnamrurinose B, 2. CIMS, NH,, m/z (%): 564 (CM

+NH,]+,85),547([M+H-j+,5),460(10),413(20),309 (25), 267(35), 180(37), 163(40), 117(100), lOS(50). HRMS,

Short Reports 549

m/z 569.2031 [M+Na]+ (calcd: 569.1999 for 5.

C,sHs,G, ,Na).

Acknowkvigemenzs-We are greatly indebted to Dr Jean Pinon for harvesting and identification of plant material, 6. and to Dr J. P. Brouard for mass spectra.

7. 8.

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