MS of podophyllotoxin and related compounds

Journal of Analytical and Applied Pyrolysis, 27 (1993) 187- 197 Elsevier Science Publishers B.V., Amsterdam

Pyrolysis-GC/MS compounds

of podophyllotoxin

187

and related

Robert S. Ward a,*, Andrew Pelter a, Guido C. Galletti b and Li Qianrong ’ a Chemistry Department, University of Swansea, Singleton Park, Swansea SA2 8PP (UK) b Istituto di Microbiologia e Tecnologia Agraria e Forestale, University of Reggio Calabria, 89061 Gallina, RC (Italy) ’ Structure Research Laboratory, University of Science and Technology of China, Hefei, Anhui 230 026 (China) (Received February

16, 1993; accepted in final form March 18, 1993)

ABSTRACT The pyrolysis behaviour of podophyllotoxin and a series of related compounds including epiisopodophyllotoxin, 4’-demethylepipodophyllotoxin, deoxyisopodophyllotoxin and hypophyllanthin is reported. The compounds differ markedly in their behaviour, some such as podophyllotoxin itself giving only low intensity peaks due to pyrolysis products, others such as 4’-demethylepipodophyllotoxin giving several abundant and characteristic pyrolysis products. Derivatives; podophyllotoxin;

Py-GC/MS;

pyrolysis.

INTRODUCTION

Podophyllotoxin (1) and its stereoisomers have long been known to display anti-tumour activity and derivatives of 4’-demethylepipodophyllotoxin (3) are used in cancer chemotherapy [l-3]. There is therefore widespread interest in the synthesis and properties of these compounds [4-61. Furthermore, there is a requirement to establish reliable methods for the analysis and characterisation of such compounds. Gas chromatography can only be applied after derivatisation and even then provides no structural information. As part of our investigation of the pyrolysis-GC/MS behaviour of lignans and lignin related materials [7,8], we have studied the pyrolysis of a series of podophyllotoxin derivatives in order to examine their thermal stability and to explore the possibility of using Py-GC/MS for the direct GC

* Corresponding

author.

016%2370/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved

188

R.S.

Ward et al. / J. Anal. Appl. Pyrolysis

(1) ImdmYX=H,Y=OH,R=Me (3) 4’-demethy@@odo@lylkxoxin

27 (1993) 187-197

(2) e@sopodophylbtoxinX = OH (4) deoxyisqodqhybtoxin X = H (6) x=sRlt

X=OH,Y=H,R=H

(S)X=OH,Y=H (7) X,Y = H & SIbt @)X,Y=H&SPh

analysis of such compounds. Of the compounds examined (see Table l), podophyllotoxin (1) and hypophyllanthin (10) are natural products, 4’demethylepipodophyllotoxin (3) has been prepared from podophyllotoxin, while the other compounds (2, 4-9) are of synthetic origin. TABLE

1

Compounds

studied

Structure

Name

M.W.

Formula

1 2 3 4 5 6 7 8 9 10

Podophyllotoxin Epiisopodophyllotoxin 4’-Demethylepipodophyllotoxin Deoxyisopodophyllotoxin

414 414 400 398 414 486 486 506 476 430

C&z08 C,,H,,O, C,, Hz,Os ‘G&,0, C,,H,,Os C,&,,O,S C,&b&S CZ~H&+ CwHwOJ C,,H,,O,

Hypophyllanthin

R.S. Ward et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) 187-197

MATERIALS

189

AND METHODS

Podophyllotoxin (1) and 4’-demethylepipodophyllotoxin (3) were obtained from the Hongjiang Chemical Factory, Hunan, China, and the Shanghai Institute of Pharmaceutical Industry respectively. Hypophyllanthin (10) was isolated from Phyllunthus niruri as previously reported [9]. The preparation of the other seven compounds has already been reported in full [lo]. The pyrolyses were carried out using a CDS Pyroprobe 100 coupled directly to a Varian 3400 gas chromatograph. This, in turn, was linked to a Finnegan MAT Ion Trap Detector (ITD) model 800 operating at 70 eV. The samples were applied as a solution in CH,Cl, or MeOH to the inner surface of a small diameter quartz tube and the solvent allowed to evaporate. The tube was then inserted into the heating coil of the pyroprobe and the probe inserted into the pyrolysis chamber. After an interval of 1 min the sample was pyrolysed at 800°C for 5 s. The products were swept directly onto a Supelco SPB-5 fused silica capillary column (30 m x 0.32 mm i.d., film thickness 0.225 pm) using a split injection and a temperature programme of 70 to 300°C increasing at 10°C min-‘. The Py-GC interface was maintained at 100°C and the injector at 250°C. The pyrolysis products were identified on the basis of their mass spectra and, where possible, by comparison with spectra in the NBS library of mass spectra stored on a personal computer (Olivetti M240). RESULTS

AND DISCUSSION

The pyrograms of compounds (1) -( 10) are shown in Figs. 1 - 10, details of the pyrolysis products (including mass spectra) are listed in Table 2, and the structures assigned to the pyrolysis products (i) to (xvii) are shown in Scheme 1. In view of the close similarity in structures of the substrates, and especially compounds (l), (2) and (5) which are isomers, it might have been

TOT-

400 6:41

800 13:21

1200 2O:Ol

Fig. 1. Pyrogram of podophyllotoxin (1).

1800 26:41

SCAN TIME

190

R.S. Ward et al. / J. Anal. Appl. Pyrolysis 27 (1993) 187-197

TOT-

460 8:41

860 13:21

1

12bo 2O:Ol

TOT

13:21

26:41

TIME

(2).

viii

6141

SCAN TIME

i

Fig. 2. Pyrogram of epiisopodophyilotoxin 100%

lsb0 28:41

2O:Ol

Fig. 3. Pyrogram of 4’-demethylepipodophyllotoxin

(3).

100%

TOT

I

I

.

, 400 6~41

.

I

- 8bO 13:21

I

xi

xii

1 ,

.

,

Fig. 4. Pyrogram of deoxyisopodophyllotoxin

.

, 1600 26:41

1200 2O:Ol

.

I

ISC AN TIME

(4).

expected that their behaviour on pyrolysis would be very similar. The quite significant differences which are observed therefore demonstrate that PyGC/MS is much more sensitive to small structural changes than might be anticipated. It is therefore an ideal analytical technique to apply to a series of closely related compounds of this type. Thus, while podophyllotoxin (1) and deoxyisopodophyllotoxin (4) give only a small number of low intensity peaks, epiisopodophyllotoxin (2) and 4’-demethylepipodophyllotoxin (3)

R.S.

Ward et al. 1 J. Anal. Appl. Pyrolysh

27 (1993) 187-197

191

TOT-

400

1200 2O:Ol

860 13:21

8:41

Fig. 5. Pyrogram of compound

(5).

Fig. 6. Pyrogram of compound

(6).

SCAN TIME

1600 26:41

TOT

I

’

,

400 6:41

I

I

.I

-,

Fig. 7. Pyrogram of compound

-,

.

1200 2O:Ol

800 13:21

,

I

,

1600 26:41

I,

I

SCAN TIME

(7).

give several peaks which are relatively abundant. To some extent these differences can be rationalised in terms of the different volatility and thermal stability of the compounds themselves. For example, 4’-demethylepipodophyllotoxin (3), which is the only compound having a phenolic OH group, left a black residue after pyrolysis along with a small amount of yellow distillate at the end of the quartz pyrolysis tube. Podophyllotoxin (1) in

192

R.S. Ward et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) 187-197

100%

i

li_ ./,;

TOT

I

400 6:41

860 13:21

Fig. 8. Pyrogram of compound 100%)

.

1200 2O:Ol

’

I

’

’

1600 26:41

’

SCAN TIME

(8).

1

ii

/

TOT

I

-XL

I,

*

I

800

6141

,

I,

1200 2O:Ol

13:21

Fig. 9. Pyrogram of compound

.

..

816bO 26:41

I SCAN TIME

(9).

100%

TOT

m,400 6:41

800 13:21

Fig. 10. Pyrogram of hypophyllanthin

1200 20:01

1600 26:41

SCAN TIME

(10).

contrast gave no black residue but left a white sublimate at the end of the quartz tube. Thus it would seem that the greater volatility of the methyl ether (1) allows most of it to escape from the region of the heating coil without undergoing pyrolysis, under conditions in which (3) is largely decomposed. The differences in behaviour between compounds (1) and (2) are also interesting. While (xi) and (xiii) are major products in both cases, the pyrogram of (2) contains significant peaks due to products resulting from

R.S. Ward et al. /J. Anal. Appl. Pyrolysis 27 (1993) 187-197

Me0

Q I

OMe

OMe

WI IvS’m/z 168

bMe

cw Mzfnh200 Scheme 1.

the pendant aryl ring. Thus 1,2,3-trimethoxybenzene (vii) is the main pyrolysis product from compound (2). Similar behaviour is exhibited by compound (3) except that the 1,2,34rimethoxybenzene is now replaced by 2,6-dimethoxyphenol (viii). In both (2) and (3), the main point of cleavage would seem to be at the bond linking the pendant aryl group to the tetrahydronaphthalene nucleus. The two major products from (3) are 2,6-dimethoxyphenol (viii) and 2,3_methylenedioxynaphthalene (xi), Some of the minor products are also identified as derivatives of 2,3-methylenedioxynaphthalene, although conclusive identification is not possible without the availability of mass spectra and gas chromatographic retention times of authentic samples. Nevertheless, they offer some clues as to the order of events which may be occurring. Scheme 2 presents a possible breakdown pathway which would account for the pyrolysis products formed from compounds (2) and (3). Of the other compounds studied, the three retro-lactones (S), (7) and (8) also show characteristic behaviour. Compound (5) which is isomeric with compounds (1) and (2) gives only three products, all arising from the

8.85 15:85 34.88 40.41

27.93 1.82 1.45 36.16 11.64 4.29 13.75 2.97

5.93 5.20 37.75 41.66 8.87 0.60

29.24 14.71 3.14 2.62 6.59 3.47 15:lO 10.02

16:Ol 17:29 1957 21:26

259 3:12 3:25 15:41 1954 20:16 21:24 23:17

3:oo 3:ll 16:22 19:44 21:18 26:06

3:oo 3:12 3~24 15:57 17:28 l&57 19:51 21:18

(1)

(2)

(3)

(4) 168(40) 160( 50) 174(36) 172( 100) 186( 100)

51(8) 67( 100) 56( 47) 153(41) 102( 100) 116(55) 171(68) 185(51)

45( 100) 59(9) 139(54) 171(78) 185( 51) 199(80)

154( 100) 172( 100) 186(100) 230( 52) 44( 100) 68( 54)

45( 100) 67( 100) 57( 53) 153(84) 171(76) 172(75) 185(59) 109( 57)

253( 42) 253( 37) 171(88) 185(53)

m/z(%)

_ 172(99) 186( 100) _ _ 168( 100) 172( 100) 196(67) 186( 100) _

M+(%)

45( 39) 53( 65) 55( 36) 93( 39) 51(43) 115(45) 115(36) 128(65)

44(33) 5W) lll(40) 115(37) 128(63) 55( 60)

44(32) 55( 74) 56( 20) 125(61) 115(36) 171(55) 128( 63) 65( 38)

153(45) 102( 50) 115(43) 128(69)

and mass spectra of pyrolysis products

Rel. int. (%)

Retn. time

Substrate

Retention times, relative proportions,

TABLE 2

42( 5) 42( 25) 43( 22) 44(45) 44(52) 44(44) 114(55) 102( 36)

43(8) 43( 100) 96(41) 114(49) 127(28) 43( 100)

43(2) 53(73) 43( 18) 1lO(92) 114(57) 114(54) 127(31) 44(40)

93( 48) 44( 54) 114(66) 102(29)

41(11) 41(47) 42( 39) 43(42) 43(41) 43( 32) 63( 27) 44(35)

42( 14) 42( 11) 93( 53) 113(29) 102(24) 41(96)

42(2) 43(51) 42( 17) 95( 60) 63( 28) 44(51) 63( 30) 41(32)

44(48) 43(42) 63(40) 44(32)

40(4) 40(31) 41( 100) 40( 100) 40( 100) 40( 100) 40( 45) 40( 77)

41( 18) 41(5) 65( 55) 63( 23) 40( 28) 40( 87)

40(2) 41(67) 41( 100) 93(60) 40( 32) 40( 100) 40( 51) 40( 100)

40( 100) 40( 100) 40( 100) 40( 73)

[tii) a (ix) (x) (xi) (xiii)

?

co*

(xiii) (xvii)

(xi)

;&a

-

(xii)a (xiii)

(xi)

[ii)&

(xi) (xiii)

Proposed Structure

s

2 ;”

c:

$.

Q

a

2

$.

b

F

22

5

3. z F

3

P ?

-

7.22 1.70 75.64 5.61 6.30 3.53

3:Ol 3:47 8:39 9.33 11.27 23:23

3:12 3:24 23:06

42.84 13:33 5.86 37.97

~(1~) 1lO( 100) 122(64) 124( 100) 136( 86) 218(66)

4.57 67.56 15.04 8.57 1.59 2.67

2:50 8:22 9:19 11:15 1151 23:16

i:oi

45( 100) 77( 27) ill(9) 121( 100) 109( 69) 109( 100)

90( 7) 122(66) 136(74)

45.97 37.14 11.55 5.34

3:06 3.27 9:41 12:42

68( 65) iO2( 100)

-

78( 100) 110(100) 122(64) 124( 100) 218(69)

45(22) 109(23) 121( 100) 109(62) 135( 100) 154(29)

90(7)

70.95 29.05

3:04 3:25

44(20) 67( 100) 56(46) 157(37)

57( 13) 75( 12) 121( 100) 135( 100)

.57(36) 75(11)

45( 53) 121( 100) 135( 100) 135( 100)

44( 100) 122(66) 136(80) 150(27)

16.98 55.23 18.39 9.41

185( 100) 73( 34) 355(26)

3:OO 9:33 12:oo 14:04

a Identified by comparison with library of mass spectra. b Probably due to impu~ties.

(10)

(9)

(8)

(7)

(6)

(5)

200( 40) 356(68) 370( 100)

3.88 4.62 6.61

22:34 2519 26:21

43(49) 55(45) 55( 33) 101(S)

44(32) 63(6) 109( 16) 66( 22) 91(71) 69( 50)

4313) 84( 16) 66( 14) 91(63) 78( 74) 109(98)

56( 48) 57( 100) 65( 18) 77( 59)

56( 48) 57( 100)

43(5) 6.q 18) 78( 46) 79( 32)

115(32) 55( 29) 202(31)

42( 76) 53(8) 43(31) 75( 38)

42( 20) 52(21) 69(13) 65( 28) 78(77) 65( 66)

42(6) 69( 15) 65( 53) 78( 59) 77( 67) 65( 60)

55(g) 42(g) 64(20) 73( 55)

470)

55(42)

42( 12) 64(27) 77(42) 77( 50)

44(35) 44(47) 55( 26)

41f 100) 41(69) 42( 33) 44(34)

44(43)

64(24) 45( 65)

41(7) 51(26) 66( 19)

44(45)

41fll) 66( 25) 64(20) 51(37) 51(U)

41( 100) 41(82) 63(51) 50( 49)

43(29) 41(74)

40(41) 40f41) 41( 100) 40( 86)

40(7) 50(28) 45( 10) 63( 57) 41(55) 40( 99)

40( 5) 51( 17) 63( 53) 45( 57) 50( 63) 40( 100)

40(9) 62( 18) 40( 81)

40( 14)

40(7)

41( 100)

40(44)

40(4) 62( 18) 50( 45)

40(64)

44(38) 41(4) 63( 58) 51(45) 51(33)

40( 93) 40( 100)

43( 36) 43( 38)

a

(xv)

?

(xvi)”

;z;

Benzene

-

-

ti)”

b

(xiv)

fi

b

in

P

196

R.S. Ward et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) 187-197

(w(3) Cleavage L

-%O

OR (v@R=Meor

(viiiiR=H

(xvii)

w m/z 168

wnc’z154

M???dZ230

(xi) Mi+mlz 172 Scheme

0

(xiii) w ml2 186

2.

pendant aryl ring. Thus 1,3-benzodioxole (iii) and 5methyl-1,3-benzodioxole (v) replace 1,2,3_trimethoxybenzene as the major products, and structural information is therefore provided directly about the nature of this aromatic ring. The pyrograms of the thioether derivatives (6)-(9) are dominated by the appearance of fragments due to the corresponding thiols (i) and (ii). However, the appearance of 1,3-benzodioxole (iii) and Smethyl- 1,3-benzodioxole (v) among the minor products from (7) -(9) contrasts with (6) which gives no products resulting from the pendant aryl group. Finally, hypophyllanthin (lo), which lacks the lactone ring, resembles podophyllotoxin (1) and deoxyisopodophyllotoxin (4) in that it gives very little information owing to an absence of abundant pyrolysis products, suggesting that due to either its volatility or its thermal stability it withstands the pyrolysis conditions used. In conclusion, a wide diversity of pyrolysis behaviour is observed. For the compounds which give several abundant pyrolysis products, a clear pattern

R.S. Ward et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) 187-197

191

emerges involving cleavage of the bond linking the pendant aryl ring to the tetrahydronaphthalene nucleus, followed by further degradation and proton rearrangement (Scheme 2). For the compounds with thioether substituents the pyrograms are dominated by fragments due to the thioether group, and very little information is provided about the rest of the molecule. It is noteworthy how sensitive the pyrolysis behaviour is to the detailed structural features of the substrate examined. ACKNOWLEDGEMENTS

The authors thank the Royal Society (UK) and the CNR (Italy) for funding exchange visits, and the Centro Conservazione Foraggi, CNR, Bologna (Italy) for the use of the Py-GC/MS facility. REFERENCES 1 I. Jardine, in J.M. Cassady and J.D. Douros (Eds.), Anticancer Agents based on Natural Product Models, Academic Press, New York, 1980, Chapter 9. 2 S.G. Weiss, M. Tin-Wa, R.E. Perdue and N.R. Farnsworth, J. Pharm. Sci., 64 (1975) 95. 3 C. Kellar-Juslan, M. Kuha, A. Von Wartburg and H. Stahelin, J. Med. Chem., 14 (1971) 936. 4 R.S. Ward, Synthesis, (1992) 719. 5 B.F. Issell, A.R. Rudolph, A.G. Louie and T.W. Doyle, in B.F. Issell, F.M. Muggia and SK. Carter (Eds.), Etoposide (VP-16): Current Status and New Developments, Academic Press, New York, 1984, Chapters 1 and 2. 6 D.C. Ayres and J.D. Loike, Lignans, Cambridge University Press, 1990, Chapters 3 and 4. I G.C. Galletti, R.S. Ward and A. Pelter, J. Anal. Appl. Pyrolysis, 21 (1991) 281; 23 (1992) 135. 8 G.C. Galletti, R.S. Ward and A. Pelter, Ann. Chim. (Rome), 82 (1992) 389. 9 L.R. Row, P. Satyanarayana and C. Srinivasulu, Tetrahedron, 26 (1970) 3051. 10 A. Pelter, R. S. Ward, M.C. Pritchard and I.T. Kay, J. Chem. Sot., Perkin Trans. 1, (1988) 1603, 1615.