Possible anti-tumour promoters: BI- and tetraflavonoids from Lophira alata

Phytochemistry, Vol. 31, No. 8, pp. 2689-2693,1992 Printedin GreatBritain.

POSSIBLE

0031-9422/92$5.00+0.00 0 1992Pergamon PressLtd

ANTI-TUMOUR PROMOTERS: FROM LOPHIRA

BI- AND TETRAFLAVONOIDS ALATA

A. MURAKAMI, S. TANAKA, H. OHIGASHI, M. HIROTA,* R. IRIE,* N. TAKEDA,~ A. TATEMATSU~ and K. KOSHIMIZU Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan; *Department of Bioscience and Technology, Faculty of Agriculture, Shinsyu University, Minami-minowa, Kami-ina, Nagano 399-45, Japan; tFaculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan (Receioed 25 September 1991)

Key Word Index-Lophira

alata; Ochnaceae; bark; anti-tumour promoter, biflavonoid; tetrailavonoid.

Abstract-Three possible flavonoid-type anti-tumour promoters were isolated from the bark of Lophira data using an in vitro short-term assay, Epstein-Barr virus (EBV) early antigen (EA) induction test. One of the flavonoids was identified as lophirone F. The other two, named azobechalcone A and isolophirachalcone, were a new biflavonoid and a tetraflavonoid, respectively. Azobechalcone A (5 PM) and isolophirachalcone (5 PM) showed potent inhibitory activities (83% and 65%, respectively) against EBV-EA induction by a tumour promoter, teleocidin B-4 (50 nM). Lophirone F was less active (18% at the same dose) than azobechalcone A and isolophirachalcone.

INTRODUCTION

Lophira aluta (Ochnaceae) is a tall tree widely distributed

in tropical west Africa. The bark has been used medicinally as an analgesic. Because some analgesics or antiinflammatory agents are known to possess anti-tumour promoting activity, we have searched for such agents in this plant using a convenient short-term in vitro assay, Epstein-Barr virus (EBV) early antigen (EA) induction caused by a tumour promoter, teleocidin B-4 [l]. Previously, lophirone A was isolated as a major inhibitor [2] and found to exhibit inhibitory activities against two other short-term tumour promoter-induced biological responses, viz. inflammation of the mouse ear and activation of protein kinase C (PKC) [3]. The remarkable antitumour promoting activity was, then, proved by an in uiuo initiation-promotion test on mouse skin [3]. During the course of these studies, lophiraic acid, a new polyphenol structurally related to lophirone A, was also isolated, and interestingly it was found to be inactive in every shortterm biological response [2,3]. Recent phytochemical studies of L. data and a closely related species, L. lanceolata, have revealed the occurrence of various struo turally unique flavonoids [2,4-111. Therefore, L. data was thought to be a valuable source of new flavonoidrelated polyphenols, which would contribute to the study on the structure-activity relationship of the flavonoidclass of anti-tumour promoters. Herein we describe the isolation and structure elucidation of three flavonoids from the bark of L. data and an examination of their inhibitory activities toward EBV-EA induction.

EA induction test and by the characteristic red colorisation on TLC by spraying 5% sulphuric acid in ethanol followed by heating. The bark of L. alata was extracted

Ho

OH

RESULTSAND DISCUSSION

The possible flavonoid-related anti-tumour promoters were traced by both the inhibitory activities in the EBV2689

AH

OH

2690

A.

M~JRAKAMIet al.

with methanol and the extract was partitioned between ethyl acetate and water. From the active ethyl acetatesoluble part, three inhibitors l-3 were purified by a combination of several CC on silica gel and ODS gel and finally by prep. HPLC on PBondasphere C, s. Compound 1 was identified as lophirone F, recently isolated from L. lancedata by Tih et ul. [lo]. by means of its spectral data. Compound 2 named azobechalcone A, was obtained as a pale yellow amorphous solid. It showed an absorption maximum at 379 nm in the UV and absorption bands at 3380 (br, OH), 1620 (conjugated C=O) cm- ’ in the IR. The molecular formula was determined as C3rHZ60s by high-resolution-FAB mass spectrometry m/z 527.1719 OfHI +, calcd for C31H2708. 527.1706). The ‘H (500 MHz) and r3C (125 MHz) NMR data of 2 in acetone-d, are listed in Table 1. Direct C-H connectivities were solved by a ‘H detected heteronuclear multiple quantum coherence (HMQC) spectrum. Thus, the presence of three sets of 1,2,4_trisubstituted benzene rings [ring-A (rA), rB, and rD], a 1,4_disubstituted benzene ring (rC), a 1,2-disubstituted trans olefin [S, 7.39 and 7.72 (15.4 Hz)], three methines [S, 4.78 (d), 6, 3.77 (dd) and 5, 5.84 (d)] in a chain, a chelated hydroxyl (6, 13.6), a carbonyl (6, 192.4) and a methoxyl (6, 3.30). Acetylation (pyridine/Ac,O) and methylation (Me,SO,JK,CO,) of 2 gave a penta-acetyl derivative (4) [ET mass spectrum m/z 736 (CM]‘, C,,H,,O,,)] and a pentamethyl derivative (5) [EI mass spectrum m/z 586 (CM]‘, C,,H,,O,)], respectively. No more free hydroxyls were indicated in the IR spectra of both derivatives. Furthermore, an acetylation shift was not observed in the ‘HNMR of 4. Therefore, eight oxygens contained in 2 were distributed into five phenohc hydroxyls, a methoxyl, a carbonyl and an ether group. The C-H connectivities in each benzene ring were clearly analysed by the ‘H detected heteronuclear

multiple bond connectivity (HMBC) spectrum. The HMBC spectrum of 2 additionally indicated the following significant C-H connectivities through two or three bonds; between the carbonyl carbon (C-c. 6,192.4) and a proton (6u7.96) at 6-position of ring A (H-rA-6), between C-c and both H-a (6u7.72) and H-b (6, 7.39), between Hb and C-rB-1 (6, 128.2) between H-a and C-rB-6 (6, 126.3). between H-/I(&, 3.77) and both C-rB-6 and C-rC-1 (6, 134.4), between H-7, (6, 4.78) and both C-rB-5 (S, 128.9) and Cs-rD-2, 6 (S, 157.9 or 159.2 and 6, 130.2, respectively) and between H-a (Sn 5.84) and both Cs-rC2,6 (6, 127.6). Moreover, long-range coupling through an ethereal oxygen atom was observed between H-cc and CrB-4 (6, 163.9). Hence, a methoxy group was placed at C)‘. Other C-H connectivities shown by the HMBC spectrum provided evidence of the structure of 2 for azobechalcone A. The relative stereo relationship between the protons at CI-and b-position was determined as cis by the observation of an 8.4% NOE of H-B on irradiation of HY. The absolute configuration has not yet been determined. Compound 3 was suggested to be a tetraflavonoid by high-resolution-FAB mass spectrometry m/z: 1009.3099 ([MH]+, calcd for C,,H,,O,s, 1009.3070). The ‘H NMR spectrum of 3 (400 MHz, acetone-d,, Table 2) was shown to carry an azobechalcone A residue as a C,, unit. Furthermore. the ‘H and 13C srgnals showed a close analogy with those of lophirachalcone (6, which has been isolated from L. lanceolatu [S]. The structural difference between 3 and 6 was suggested to occur only in the relative configuration of the tetrahydrofuran ring part, because significant differences in chemical shifts and coupling constants in the ‘HNMR spectrum were observed in the protons around the ring part (Table 2). Relative stereo relationships between H-4 and H-5, H-2 and H-5, and H-2 and H-3 of the tetrahydrofuran ring in

Table 1. NMR data of azobechalcone A (2) (Me&O-& 125 MHz) Pos.

13C(6)

A-l 2

114.3 167.5t 103.7 165.6t 109.0

4

6 B-l 2

4 6 C-l 2 3 4 5 6

133.2 128.2 132.6 110.0 163.9 128.9 126.3 134.4 127.6 116.0 157.8 116.0 127.6

‘H(6)

m*

J(Ha

PO% D-l

-

6.35

d

2.2

6.53

dd

7.96

d

7.57

dd

6.86

d

8.3 1.8 8.3

6.89

d

1.8

-

8.8 2.2 8.8

7.11 6.77

d d

8.5 8.5

6.77 7.11

d d

8.5 8.5

*Multiplicity. t,fMay be interchanged

TMS rnt. ref.. ‘H: 500 MHz_ 13C:

‘W(S)

‘H(6)

116.8

~~

m*

J WI

d

2.4

157.9f

-

103.2

6.48

159.2f

~--

107.9

6.52

dd

6

130.2

7.14

d

8.5 2.4 8.5

: c

145.3 117.7 192.4

7.72 7.39 --

d d

15.4 15.4

;

88.7 56.9

5.84 3 77

d dd

2’

81.1

4.78

d

4.4 8.3 4.4 8.3

OMe

56.8

3.28

s

2 3 4 5

Flavonoids from Lqhira data

2691 EXPERIMENTAL

OH

lophirachalcone (6 )

r

OH

OH

‘oH mbamichalcone (7)(a-Ii at C-3) isombamichalcone (8)(&H at C-3)

lophirachalcone (6) were reported as tram, tram and cis, respectively. Recently, the biflavonoids, mbamichalcone (7) and isombamichalcone (S), epimeric to each other at C-3 of the tetrahydrofuran ring, have been isolated from L. alata and L. lanceolata, respectively [S, 81. Relative configurations of isombamichalcone were reported to be the same (trans, trans and cis) as those of the tetrahydrofuran ring part of lophirachalcone, while those of mbamichalcone were all trans. The coupling constants of the ring protons of 3 were found to coincide with those of 8 within 0.5 Hz (Table 2). Irradiation of H-2 of the tetrahydrofuran ring enhanced the signal of H-4 by 5.2%, while no enhancements of H-3 and H-5 were observed. Hence, the relative stereo relationships among the protons on the tetrahydrofuran ring in 3 were determined as trans, trans and trans, respectively. The structure of 3 was thus identified as an epimer of lophirachalcone at C-3 of the tetrahydrofuran ring part (Fig. 1), which we have named isolophirachalcone. Absolute configurations are currently being studied. The inhibitory effects of the isolated compounds against EBV-EA induction are shown in Table 3. Azobechalcone A (2) and isolophirachalcone (3) at 5 PM inhibited EA-induction caused by a potent tumour promoter, teleocidin B-4 (50 nM) by 83% and 65%, respectively. Their activities are higher than that of lophirone A, which was recently found to be a potent anti-tumour promoter [2, 31. Azobechalcone A (2) and isolophirachalcone (3) appear to be a group of the most potent inhibitors against EBV-EA induction among the flavonoid-related inhibitors so far reported [2,12-141. On the other hand, lophirone F was less active. From the above results, it could be speculated that a bitlavonoid unit (azobechalcone A unit) including rA, rB, rC and rD, might play an important role in the inhibition. The inhibitory activities of 2 and 3 on tumour promotion in uiuo will be reported elsewhere.

Inhibitory test of EBV-EA induction. This was carried out as described in ref. [2]. Raji cells (5 x lo5 cells ml-‘) were incubated in 1 ml of RPM1 1640 medium containing 50 nM teleocidin B-4, 3 mM sodium n-butyrate and the DMSO soln (5 ~1)of a known amount of test compound at 37” under a 5% CO, atm. for 48 hr. The average percentage of EA-positive cells in the control expt (only with teleocidin B-4, n-butyrate and DMSO) was usually CLI 50%. Isolation of compounds 1-3. Bark of L. alata (1.3 kg) collected in Cameroon was extd with MeOH at room temp. After the MeOH ext. was partitioned between EtOAc and HzO, the EtOAc-sol. part (63 g) was chromatographed on silica gel (0-100X EtOAc-toluene, stepwise; O-50% MezCO-CHCl,, stepwise), ODS gel (50-100% MeOH-H,O, linear gradient) and then purified by prep. HPLC on PBondasphere Cis ODS column (19 mm x 15 cm), eluted with 45% MeCN-Hz0 (flow rate: 7mlmin-‘, detection: UV& to give lophirone F (1, 23 mg) R, 13.5 min, azobechalcone A (2,70 mg) R, 22.5 min and isolophirachalcone (3, 62 mg) R, 18 min, respectively. Lophirone F (1). C,,Hz,O,, colourless amorphous solid. [a]y -97” (MeOH; c 0.37). UV #$‘:” nm (logs): 322 (3.78), 283 (3.95). IR v!$; cm-‘: 3340 (br, OH), 1620 (C=O). ‘HNMR (400 MHz, MezCO-d,)6:4.88(1H,dd,J=8.8, 8.8 Hz), 5.02(1H, dd, J=8.8, 8.8 Hz),5.15(1H,d,J=8.8 Hz),5.64(1H,d,J=8.8 Hz),6.09(1H, d, J=2.4Hz), 6.21 (lH, dd, J=8.8,2.4Hz), 6.24 (lH, d, J =2.4Hz), 6.34 (lH, d, J=8.8,2.4Hz), 6.60 (2H, d, J=8.8Hz), 6.83 (2H, d, 5=8.8 Hz), 7.21 (2H, d, 5=8.8 Hz), 7.41 (lH, d, J =8.8 Hz), 7.43 (2H, d, 5=8.8 Hz), 7.77 (lH, d, J=8.8 Hz), 12.4 (lH, s) and 12.8 (lH, s). FABMS (glycerol, probe) m/z: 511 [MH -18]+. Azobechalcone A (2). C3iHz60s, pale yellow amorphous solid. [alif’ + 143” (MeOH; c 0.54). UV 15:” nm (logs): 379 (3.90). IR v%; cm-i: 3380 (br, OH), 1630 (C=O). ‘H and ‘sCNMR; see Table 1, HR-FABMS (glycerol, probe) m/z: 527.1719 ([MH]+, calcd for C3iHa70s, 527.1706). Isolophirachalcone (3). CGOH,,O1s, pale yellow amorphous solid. [a];’ + 166” (MeOH; c 0.71). UV $!,:z” nm (logs): 380 (4,38), 283 (4.38). IR vale cm-‘: 3380 (br, OH), 1630 (C=O). ‘H NMR; see Table 2. 13CNMR (125 MHz, Me&O-d,) 6: 36.6 (secondary), 43.4, 53.4, 55.4, 60.5, 83.4, 85.9, 90.3, 103.2, 103.5, 103.7, 104.1, 107.8, 108.3, 109.3, 110.1, 115.5, 116.0 (ZC), 117.6, 126.4, 127.7, 128.3, 130.0, 131.0, 131.6, 132.3, 133.2, 133.6, 145.5 (tertiary each), 114.3, 114.7, 116.9, 121.1, 121.5, 128.0, 130.4, 132.4, 132.7, 133.8, 155.6, 156.3, 156.4, 157.0, 157.4, 157.8 (2C), 163.7, 165.7, 165.8, 166.7, 167.5 (quaternary each), 192.4, 203.6 (carbonyl each). HR-FABMS (glycerol, probe) m/z: 1009.3099 (CMHI+, calcd. for C6,,H4aO15, 1009.3070). Azobechalcone A pentaacetate (4). Usual acetylation of 2 (20 mg) with pyridine and AczO gave azobechalcone A pentaacetate (4, 18 mg). Colourless amorphous solid. [a];’ +20.7” (CHCI,; c 0.43). UV iE;c’Z nm (log E):348 (4.26), 250 (4.08), 230 (4.20) IR e;;“* cm-‘: 1760 (-oCO-k 1610 (C=O). ‘HNMR (400 MHz, MezCO-d,) 6: 2.13 (3H, s), 2.17 (3H, s), 2.20 (3H, s), 2.24(3H,s),2.31 (3H,s),3.27(3H,s),3.84(1H,dd,J=8.3,4.4Hz), 4.68 (lH, d, J=8.3 Hz), 5.95 (lH, d, J=4.4 Hz), 6.73 (lH, d, J =2.4 Hz), 6.93 (lH, d, J= 15.5 Hz), 6.96 (1H. d, 5=7.8 Hz), 7.04 (lH, d, J=2,4Hz), 7.05 (lH, d, J=2.4 Hz), 7.08 (2H, d, J=8.8 Hz). 7.16 (lH, dd, J=8.3,2.4Hz), 7.21 (IH, dd, .J =8.8,2.4 Hz), 7.27 (2H, d, J=8.8 Hz), 7.42 (lH, d, J=15.5 Hz), 7.54(1H.d,J=8.3Hz),7.57(1H,dd,5=7.8,2.4Hz),7.79(1H,d, J = 8.8 Hz). EIMS (probe) 70 eV m/z (rel. int.): 736 (6, [M] ‘), 694 (7, [M -CHzCO]+), 662 (2), 620 (2), 577 (3), 535 (2), 498 (8), 457 (12), 415 (16), 373 (lo), 263 (16), 238 (lOOk 195 (60) 179 (8), 153 (73).

2692

A. MURAKAMIet al.

Table 2. ‘HNMR

data of isolophirachalcone (3) and the differences in chemical shifts and coupling constants between 3 and lophirachalcone (6) or mbamichalcone (7) (400 MHz, Me,CO-d,, ref., TMS; 6)

Pos.

‘H

m*

J WI

A-3 5

6.34 6.58

d

6 B-2

1.92 7.51

d

3 6 c-2 3 5 6 D-3 5

6.88 6.97 7.03 6.75 6.75 7.03 6.48 6.52

d d d d d d d

6 E-3 6

7.40 6.50 7.67

d

2.6 8.8 2.6 8.8 8.4 1.8 8.4 1.8 8.4 8.4 8.4 8.4 2.6 8.8 2.6 8.8

dd

dd

dd

s s

Aat 0.01 -0.02 0 0 -0.04 0.04 0.06 0.04 0.04 0.05 -0.04 - 0.02 -0.01 0.02 0.10

AJWS

Pos.

‘H

m*

J U-M

-0.2 0.2 -0.2 0.2 - 1.6 0 -1.6 0

F-2 3 5 6 G-3 5

6.73 6.63 6.63 6.73 6.16 6.13

d d d d d dd

6 H-2 3 5 6

7.13 7.15 6.63 6.63 7.15 7.68 7.26 2.56

d d d d d d d dd

2.85

dd

8.4 8.4 8.4 8.4 2.2 8.8 2.2 8.8 8.4 8.4 8.4 8.4 15.1 15.1 13.7 9.3 13.7 4.4

2

3.40

m

3 4 5

5.39 5.17 4.03

d d dd

;

5.54 4.60

d dd

Y

4.78

d

-0.2 -0.4 -0.2 -0.4 -

t 1

A@ 0.05 -0.03 -0.04 0.05 0.03 -0.17 -0.72 0.02 0.21 0.21 0.02 0 0.02 -0.41 -0.37

AJJS

0.2 0.3 0.2 0.3

0.1 0.1 -0.1 4.3 -0.1 -0.3

- 0.20

AJ’$

0.2 0.2 0.2 0.1 -

OH 12.7 13.6

0.15 0.01

s

s

-

9.1 8.3 8.3 9.2 3.4 11.7 3.4 11.7

-0.10 - 0.03 -0.33 0.04 0.11 0.10

-3.1 - 1.8 - 1.8 -5.7 -0.3 0.1 -0.3 0.1

0 0.1 0.1 0.5 -

*Multiplicity. TDifferences m chemical shifts C(6)-(3)], ppm. fDifferences in coupling constants [(6H3], Hz. §Differences in coupling constants [mbamichalcone (7))(3)], HZ.

Table 3. Inhibitory activities of l-3 and lophirone A toward EBV-EA induction Compound

Inhibition (%)

Viability (%)*

Lophirone F (1) Azobechalcone A (2) Isolophirachalcone (3) Lophirone A

18+5 83&12 65+18 45+12

77+1op 91+ 15 82k16 85&14

*Teleocidin B-4 (50 nM) and sodium n-butyrate (3 mM) as EA-inducers and test compounds (5 PM in 5 ~1 DMSO) were used. Average percentage of EA-positive cells in control experiment (teleocidin B-4, n-butyrate and 5 ~1of DMSO) were ca 50%. tThe experiment was carrted out three times; inhibition and viability rate are expressed as mean fs.d.

Azobechalcone A pentamethyl ether (5). Azobechalcone A (2) (20 mg) in 2 ml of dry Me&O, 50 mg of K,CO, and 100 ~1 of (Me&SO, were refluxed for 2 hr Usual work up of the reaction mixt. followed by purification by prep. TLC on silica gel gave azobechalcone A pentamethyl ether (5, 15 mg). Colourless amorphous solid. [a]ii + 10.2” (CHCl,; c 0.20). UV n$c’2 nm

(logs): 352 (4.32), 284 (4.00), 233 (4.40). IR vfi; cn-‘: 1595 (C=O). ‘H NMR (400 MHz, Me&O-d,) 6: 3.25 (3H, s), 3.57 (3H, ~),3.72(3H,s),3.76(3H,s),3.86(1H,dd,J=7.8,4.0HzX3.90(3H, s), 3.94 (3H, s), 4.83 (lH, d, J=7.8 Hz), 5.82 (lH, d, J=4.0Hz), 6.24(1H,dd, J=8.3,2.0Hz),6.52(1H,d,J=2.2Hz),6.65(1H,d, J=8.8, 2.2 Hz), 6.67 (lH, d, 5~2.0 Hz), 6.76 (lH, d, J= 1.8 Hz), 6.85 (2H, d, J=8.8 Hz), 6.86 (1H. d, J=8.3 Hz), 7.15 (2H, d, J =8.8 Hz), 7.18 (IH, d, J=15.1 Hz), 7.35 (lH, d, J=8.3 Hz), 7.45 (lH, d, J=15.1 Hz), 7.47 (lH, d. J=8.3, 1.8Hz), 7.62 (lH, d, J = 8.8 Hz). EIMS (probe) 70 eV m/z (rel. int.): 596 (2, [M] ‘), 564 (88, [M-MeOH]+), 549 (5) 414 (7), 249 (6) 199 (7), 181 (lOO), 151 (40) 135 (36) 121 (42). Acknowledgements-This study was supported by a Grant-inAid from the Ministry of Education, Science and Culture of Japan. We thank Junichi Masuda and Matashige Oyabu of Shimadzu Corporation for the measurements of HMQC and HMBC spectra.

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