Matairesinol as precursor of Podophyllum lignans

Phyfochemiary,Vol. 30. No. 5. pp. 1489 1492. 1991 Printed in Great Bntaln.

MATAIRESINOL A.

JANE BR~OMHEAD,

Departments

AS PRECURSOR

MAIADA

of Pharmaceutical

0031 9422/91 53.00+0.00 1991 Pergamon Press plc

Q

M. A. RAHMAN,

OF PODOPH YLLUM

PAUL M. DEWICK,

Sciences and Botany, (Recked

University 15 October

LIGNANS*

DAVID E. JACKSON

of Nottingham,

Nottingham

and

JOHN A. LUCAS

NG7 2RD, U.K.

1990)

Key Word Index--Podophyllum hexandrum; P. peltotum; Diphylleia dibenzylbutyrolactone; biosynthesis; podophyllotoxin; matairesinol.

cymosa; Berberidaceae;

lignans;

aryltetralin;

Abstract-The dibenzylbutyrolactone lignan ( -)-[14C]matairesinol was efficiently incorporated into the aryltetralin lactone lignans podophyllotoxin, 4’-demethylpodophyllotoxin, /?-peltatin, z-peltatin and 4’-demethyldesoxypodophyllotoxin in one or more of the plant systems Podophyllum hexandrum root, P. peltatum leaf or Diphylleia cymosa leaf. This demonstrates matairesinol to be a common precursor of the 3’,4’,5’-trimethoxy and 4’-hydroxy-3’,5’-dimethoxy groups of Podophyllum lignans, and it may represent the branch-point compound to the two series of compounds. No incorporation of matairesinol into the arylnaphthalene lactone lignan diphyllin in D. cymosa was detected.

INTRODUCHON

Species of Podophyllum produce a range of aryltetralin lactone lignans possessing antitumour activity Cl-33 which are exploited commercially as raw material for the semisynthesis of the anticancer drugs etoposide and teniposide [4]. In previous studies [S], we demonstrated that the aryltetralin skeleton is produced biosynthetically by oxidative cyclization of an appropriate dibenzylbutyrolactone lignan. Thus, yatein (1) is efficiently incorporated into podophyllotoxin (7) via desoxypodophyllotoxin (5) and an intermediate quinonemethide (3) was proposed in the conversion of 1 into 5 (Scheme 1). An analogous sequence from 4’-demethylyatein (2) was suggested to account for biosynthesis of the 4’-demethyl series of lignans, such as 4’-demethyldesoxypodophyllotoxin (6) and 4’-demethylpodophyllotoxin (8). The peltatins, ,!Ipeltatin (9) and I-peltatin (10) were shown to be derived from 5 and 6 respectively by aromatic hydroxylation (Scheme 1) [6]. The C,, skeleton of the Podophyllum lignans is known to be derived from two C,C, molecules, and a phenolic oxidative coupling process is generally believed to operate [7]. From the results of feeding experiments, this coupling has been shown to involve two phenylpropane precursors with the same 4-hydroxy-3-methoxy substitution pattern [8], and coniferyl alcohol (11) is almost certainly the intermediate concerned [9]. Stereospecific enzyme-catalysed coupling of free radical mesomers (12) derived from coniferyl alcohol would lead to a diquinonemethide (13) (Scheme 2), and the dibenzylbutyrolactones yatein (1) and 4’-demethylyatein (2) are presumably produced from this via reduction, lactone ring formation and appropriate modification of the aromatic substitution pattern. Matairesinol (14) is likely to be a common intermediate on the pathway to 1 and 2, and the probable

*Part

5 in the series ‘Biosynthesis

For Part 4 see ref. [S].

of Podophyhm

Lignans’.

branch-point to the two series of [S]. This has now been confirmed experiments with matairesinol in systems, Podophyllum hexandrum, phylleia

Podophyllum lignans by a series of feeding three different plant P. peltatum, and Di-

cymosa.

RESULTS

AND DISCUSSION

In order to investigate as many different aryltetralin lignan structures as possible, three plant systems were employed for feeding experiments. Podophyllum hexandrum contains principally podophyllotoxin (7), with smaller amounts of 4’-demethylpodophyllotoxin (8) in its roots, and has been routinely used in biosynthetic experiments [S, 6, 81. Podophyllum peltatum also produces podophyllotoxin in its roots, but in addition accumulates similar amounts of the two peltatins, /3- and a-peltatin (9 and 10) [I]. However, the root tissue of P. peltatum has not proved as effective a vehicle for the study of lignan biosynthesis as that of P. hexundrum, as in particular, the amounts of the peltatins are observed to be quite low during the growing season [6]. Instead, the leaf tissue was employed in these feedings, as analysis indicated the presence of satisfactory levels of the P-D-glucosides of podophyllotoxin, ,&peltatin and a-peltatin. Leaf tissue of D. cymosa was also used for the studies. This has been shown to synthesize high levels of podophyllotoxin, 4’demethylpodophyllotoxin, 4’-demethyldesoxypodophyllotoxin (6) and the arylnaphthalene lignan diphyllin (15) [3]. Glucosides of some of these lignans are also present. 14C-Labelled (-)-matairesinol was produced biosynthetically by feeding L-[U-t4C]phenylalanine to a cell suspension culture of Forsythia intermedia [9] using a cell line synthesizing high levels of matairesinol 4’-O-,&Dglucoside [lo]. Matairesinol was isolated from the extract after treatment with fi-D-glucosidase in good yield and with a specific activity suitable for further feeding experiments [9]. Samples (l-l.5 mg) were solubilized in a DMSO-NaOH-H,O mixture and were fed to appropriate plant tissues.

1489

1490

A. J. BROOMHEAD cl (11.

yalein

1

R - Me,

2

R . H, 4’.4emethyIyalein

3

R-Me

5

4

R-H

6

R-Me.

desoxypodophylbtoxm

R - H. 4’.demethy!desoxypodophylbbxin

OR

Scheme

1. Proposed

biosynthetic

7

R - Me. podophylbtoxin

a

R -

6

R - Ii.

1 0

R - H.

pathway

4’.demethylpodophylbotoxin

to Podophyllum

lignans r:ia dibenzylbutyrolactone

Me,

R-pellatin d -petlatin

intermediates.

OH

0

11

13

14

matairesinol

12

Scheme 2. Proposed

biosynthetic

pathway

IO dibenzylbutyrolactone

lignans from coniferyl alcohol.

Matairesinol as precursor of Podophyllum lignans OH lb400

\ A’ /

T

Me0

0 0

3

O.-i0

15

diphyllin

The precursor solution was fed via the roots to one P. hexandrum plant, applying the material to the soil-free root system over 6-8 hr, then growing on the plant for six days before extraction of the lignans. Single freshly cut leaves with stem from both P. peltatum and D. cymosa were dipped in precursor solutions, allowed to take up the material, and grown on for a further three days. Plant tissue in these cases was homogenized and treated with fi-D-glucosidase before extraction of lignans. In all three feedings, the major lignah aglycones were isolated by TLC and purified to constant specific activity. Wherever possible, this was achieved by dilution with inactive carrier, derivatization by acetylation, and repeated crystallizations. Resultant percentage incorporation and dilution values are given in Table 1. In P. hexnndrum, matairesinol was incorporated efficiently into both podophyllotoxin and 4’-demethylpodophyllotoxin, representatives of the trimethoxy and hydroxydimethoxy groups of lignans. In P. peltofum leaf, good incorporations into podophyllotoxin and /?-peltatin were recorded, with somewhat lower levels in qAtatin. In D. cymosa leaf, an extremely high incorporation (13.5%) into podophyllotoxin was observed, with very efficient incorporation into the 4’-demethyl compound 4’-demethyldesoxypodophyllotoxin. Unfortunately, the sample of 4’-demethylpodophyllotoxin was lost in this experiment, and no incorporation data are available. The large variations in percentage incorporation figures must be interpreted in terms of the relative amounts of the lignans present in the plant, and their rates of synthesis during the feeding experiment. Overall, it may be concluded that matairesinol is a precursor of the aryltetralin lactone lignans, and is incorporated into both 3’,4’,5’trimethoxy and 4’-hydroxy-3’,5’-dimethoxy groups of lignans. Previous evidence from the labelling patterns in methyl groups [S] had indicated that the 3’,4’,5’trimethoxy substitution did not arise via the 4’-hydroxy3’,5’-dimethoxy substitution pattern, and led to the conclusion that the branch-point compound to the two series of lignans must have either 4’-hydroxy-3’-methoxy or 4’,5’-dihydroxy-3’-methoxy substitution in the aromatic ring that ultimately becomes the pendent aryl. We propose that matairesinol (4’-hydroxy-3’-methoxy substitution) is probably this branch-point compound to the two groups of Podophyllum lignans. The extremely high incorporations recorded in D. cymosa are especially convincing, and it is puzzling that the arylnaphthalene lactone lignan diphyllin (15) isolated from the same experiment was totally inactive. This means either that no diphyllin was synthesized during the

1491

A. J. BROOMHEADPI d.

1492

feeding period, or alternatively that matairesinol is not a precursor ot’diphyllin. Arylnaphthalenes may be synthesized chemically by oxidation of aryltetralins [I I-131 and potentially a similar sequence could occur in nature, in which case, matairesinol might be anticipated to function in the biosynthesis of diphyllin. Changes in the diphyllin content of D. c~~~o.sa leaves throughout the season [3] show that levels dimimsh as the season progresses, suggesting active synthesis early in the year, followed by metabolism. The present feeding was conducted mid-season, and thus biosynthesis may have terminated by this time. The relationship of arylnaphthalene structures to other lignans thus requires further study. The results reported here support the biosynthetic pathway given in Schemes I and 2. Scheme 2 parallels that deduced for the biosynthesis of lignans in Forsythia infermedia, where matairesinol is transformed into arctigenin and glucosides of matairesinol and arctigenin [9]. Furofuran lignans such as epipinoresinol, phillygenin and their glucosides are also synthesized in Forsythia, and these are likely to be formed via stereospecific nucleophilic additions of the alcohol hydroxyls on to the quinonemethides in 13, followed by further alkylation/ glucosylation [9]. In Podophyllum, Diphylleia and Forsyrhia, therefore, phenolic oxidative coupling appears to occur with coniferyl alcohol as substrate, with further modifications. including build up of the substitution pattern, taking place after this key event.

the stem placed m a small test tube, the top of the tube being covered with Ncscofilm. as before. The leaves were supported with clamps. and placed rn a fume cupboard to assist transpiration. Drstilled H,O was added at Intervals as required over a period of 3 days. The two Ieaf,stem systems were separately macerated with H,O (IO ml) using ground glass m a mortar, then treated vvith a soln of /I-n-glucostdasc (from almonds. Sigma, S mg) dissolved rn H,O (40 ml) adJusted to pH 5.0 with drl. HCI. Hydrolysis was allowed to procccd at room temp. for 6 hr. The rcactron was halted by the addrtion of hot MeOH (80 ml), and then filtered. The plant materral was further extracted with hot MeOH (2 x 40 ml). and the combmed extracts cvapd to dryness. f.sohfion r!llignun.\. The extracts were subJcctcd to TLC using CHCI,---MeOH (25: I) and bands corresponding to authentic lignan standards [l. 3J were cluted. These were purrlied further by TLC [Mc,CO petrol (60 X0 ), I : I]. and then quanttfied by UV absorption [3]. If necessary, they were diluted wtth inactive carrier (20 mg). then acetylated by heatrng under rcffux wtth Ac,O. Acetates were purified by TLC LCHCI, -MeOH. 25: I or 50: 1; Me,CO--petrol ((i&80.), I : I or I : 21, then recrystallized IO constant specific activity from EtOH. Physical data for the acetates were as reported earlier [6. 81. Diphyllm and 4demethyldesoxypodophyllotoxm were purified entirely by repeatecf TLC (CHCI,, MeOH. IO: I; Et,O-EtOH. 30: I) Acknowledgement

search

Counctl

We thank the Science and Engtneering for financral support (to A.J B.)

Re-

REFERENCES EXPERIMENTAL

Prep. TLC was carned out using 0.5 mm layers of silica gel (Merck Kicsel gel GF254): TLC zones were located by UV light. and eluted with Mc,CO. [“Cl Motarrc~srno/. The preparation of [‘Q.I]matairesinol (3. I7 MBq mM _ ‘) using cell cultures of Fors)rhio inrermedia has been described carlrer [9]. P/ant material. /ceding techniques, and extraction oJ lignans. Soil was carefully washed from the root system of one pot-grown Podophyllum hexandrum plant. The root system was placed in a beaker, the top of the beaker covered with paraffin film (Nescofilm), and the plant placed in a well ventilated fume cupboard. The labelled precursor (I mg) was dtssolved m DMSO (0.25 ml) +0.50/u aq. KaOH (0.25 ml). then diluted to 2 ml with H,O. and the solution apphed drrcctly on to the roots by Pasteur pipette. The drarnings were re-applied over a 6 8 hr period until absorption was complete. The root system was then covered with motst vermrcuhte, the plant transferred to a cool greenhouse, and allowed to metabolize for a further 6 days. The vermiculite was removed, the plant was cut into small preces with scissors and homogenized with a little water using ground glass in a mortar. The slurry was then extracted with hot EtOH (100 ml), filtered, and the plant material m-extracted with EtOH (3 x 100 ml). The combined extracts were evapd to dryness. Labelled matairesinol (1.5 mg) dissolved as above was fed to single freshly-cut leaves with stem from garden grown Podophyllum peltarum or DiphyIIeia cxmosa plants via the cut end of Chromaroyraphy.

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