Electrochemistry of podophyllotoxin derivatives

J Electroanal Chen~, 184 (1985) 3 1 7 - 3 2 9

317

Elsevier S e q u o m S A , L a u s a n n e - P n n t e d m T h e N e t h e r l a n d s

ELECFROCHEMISTRY OF PODOPHYLLOTOION DERIVATIVES P A R T I. O X I D A T I O N IVIECHANISM O F E T O P O S I D E (VP 16-213)

? J M H O L T H U I S , W J V A N O O R T , F M (3 M R O M K E N S a n d J R_ENEMA

Department of Mnal)ncul Pharmac), Faculty of Pharmacy, State Unwerstty of Utrecht, Cathartjnesmgel 60, 3511 GH Utrecht (The Netherlands) P ZUMAN

Department of Chemtstry, Clark.son Untue.sto', Potsdam, N Y 13676 (U S .4 ) (Received 4 t h S e p t e m b e r 1984 m revtsed f o r m 2nd O c t o b e r 19841

ABSTRACT T h e electrochermcal oxadataon o f the a n U n e o p l a s u c agent etoposade m a q u e o u s s o l u t i o n at a glassy c a r b o n electrode s h o w s an overall t ~ o - e l e c t r o n t r a n s f e r At p H < 2 5, the o ' u d a t i o n p r o c e e d s m o n e v o l t a m m e m c oxadaUon step At p H > / 2 5, the o x a d a u o n p r o c e e d s m t w o ~ o l t a m m e t n c o x J d a u o n s t e p s T h e first v o l t a m m e t n e oxadatlon step ( p e a k ll) Is a reversable 1 e - t r a n s f e r resulting m a stable rad,eal T h e s e c o n d s t e p ( p e a k IH) c o r r e s p o n d s to the t r a n s f e r o f the s e c o n d electron T h e p r o d u c t f o r m e d after 2 e - o x a d a u o n is a n uns'.able cation w i n c h u n d e r g o e s r a p i d conversaon i n t o *..he o r t h o q u m o n e T h e o r t h o q u m o n e h a s been tsolated a.ad chaxactenzed.

INTRODUCTION

Etoposide (Ia) (VP 16-213, Vepesid ®) is a promising new ant m eopl ast t c agent active against various kinds of tumors, e.g. lymphatic, testaeular and small cell lung c a r c i n o m a [1]. C o m p o u n d Ia m a semasynthetic podophyllotoxan derivative It bears a phenolic group m ring whach is absent m the p a r e n t c o m p o u n d , p o d o p h y l l o t o x m (lb). This p h e n o h c group is believed to be partly responsible for tts specific an tmeo p l as t l c activity [2,3], p r o b a b l y revolving the f o r m a t i o n of radicals [4], by o x i d a u v e e nz ym at i c systems like c y t o c h r o m e P-450. T h e mechanism of _~ acuon, chnical p h a r m a c o l o g y and m e t a b o h s m have recently been intensively studied and atteL-qpts have been m a d e to identify its metabolites [5-7]. Emlier we described [8] a sensattve high p e r f o r m a n c e liqmd c h r o m a t o g r a p h i c m e t h o d for the d e t e r m i n a t i o n of tl~s c o m p o u n d (Ia) and its possible m e t a b o h t e s m biologacal flmds. This m e t h o d was based on the use of an electrochemical det ect or revolving a centered injection against a glassy c a r b o n mdacator electrode. Neither m ',his study [8] n o r in a n o t h e r p a p e r menUoning the possibthty of eleetroaetlvity of c o m p o u n d Ia [9] was attention paid to the n a t u r e of the electrochemzcal process 0 0 2 2 - 0 7 2 8 / 8 5 / $ 0 3 30

© 1985 Elsevier S e q u o l a S.A

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In the present mvesngatlon, the m e c h a m s m of electro-oxidation of the 2,6-dlm e t h o x y p h e n o l group of etoposlde (Ia) at a glassy c a r b o n electrode was investigated and the pr oduc t ~dentafled. A n underst andi ng of this m e c h a m s m is essentml for opttmazatton of the p e r f o r m a n c e of the electrochemacal det ect or for H P L C [8], as well as for a deeper mslght in the pharmacological b~oact~vation pattern. T h e r e are, namely, strong indications of parallels between such patterns and m e c h a m s m s of electrochemical oxadat~on and reductions [10,11]. EX PER/MEN

TAL

Apparatus Cychc voltamnaetnc curves were recorded on a Bruker A C sweep m o d u l e in e o m b m a t t o n w~th a M e t r o h m Polarecord E 506, eqtupped with a P A R R E 0074 X - Y recorder ( E G and G Co.).

319 A glassy c a r b o n w o r k i n g e l e c t r o d e ( M e t r o h m E A 2 8 6 / 1 "I-okay) w a s used f o r the cyclic v o l t a m m e t r i c e x p e r i m e n t s . T h e e l e c t r o d e w a s cleavexl b y p o h s t u n g f o r 30 s w i t h AI zO3 p o w d e r , 0.3 ~ m size ( M e t r o h m E A 1086), a n d r i n s i n g w a t e r , a f t e r e a c h e x p e r i m e n t A n A g / A g C I w i r e p l a c e d in 3 M K C I s o l u t i o n s a t u r a t e d w i t h A g C I w a s u s e d as the r e f e r e n c e e l e c t r o d e Electrical c o n t a c t w i t h the s o l u t i o n to b e s t u d i e d w a s e s t a b h s h e d via a V t c o r ® glass t i p p e d j a c k e t . A Pt w i r e w a s u s e d as dae a u x t h a r y electrode C o n t r o l l e d p o t e n t i a l electrolysis v a t h a Pt g a u z e e l e c t r o d e (120 c m 2) w a s c a n n e d o u t in a n electrolysis cell u s i n g a M e t r o h m E 506 p o l a r o g r a p h as the p o t e n t i a l o f + 8 5 0 m V vs. A g / A g C I r e f e r e n c e e l e c t r o d e . T h e c u r r e n t w a s r e c o r d e d w i t h a K a p p & Z o n e n f l a t b e d B D - 4 1 y - t r e c o r d e r . A n e.uxlhary Pt w i r e e l e c t r o d e w a s p l a c e d in a glass t u b e filled w i t h 1 M N a N O j s o l u t i o n a n d c o n t a c t w i t h the s o l u t i o n w a s e s t a b h s h e d b y a V1cor ® glass tapped j a c k e t T h e electrolysis w a s c a r r i e d o u t m 5 ml o f a 0.1 m M s o l u t i o n o f e t o p o s l d e ( I a ) m 0.2 AI K H 2 P O a s o l u t i o n ( p H 4.6). T h e p H o f tillS u n b u f f e r e d s o l u t i o n r e m a i n e d c o n s t a n t m the c o u r s e o f electrolysis. A f t e r c o m p l e t e electrolysis, the s o l u t i o n w a s e x t r a c t e d twice with 15 m l o f C H C ! 3 T h e o r g a n i c s o l v e n t w a s chstdled o f f u n d e r N 2 at 2 0 ° C . T h e c o u r s e o f electrolysis w a s f o l l o w e d s p e c t r o m e t n c a l l y ; the s p e c t r a o f the e l e c t r o l y z e d s o l u t i o n f r o m 200 to 500 n m w e r e r e c o r d e d e v e r y 3 - 5 r a m . T h e p H v a l u e s w e r e m e a s u r e d w i t h a R a d i o m e t e r P H M 64 r e s e a r c h p H m e t e r e q m p p e d wath a n I n g o l d L O T - 4 0 1 c o m b i n e d g l a s s - r e f e r e n c e e l e c t r o d e . T h e U V spectra were recorded using a double beam Shtmadzu spectrophotometer UV-200 with a 1 c m s~hca cell. I R s p e c t r a w e r e m e a s u r e d w i t h J o u a n - J a s c o I R A - g r a t i n g i n f r a - r e d s p e c t r o p h o t o m e t e r , u s i n g K B r for p r e p a r a t a o n o f the disc. M a s s spectrometry Tills w a s c a r n e d o u t w i t h a K r a t o s M S - 8 0 ( K r a t o s , M a n c h e s t e r , U . K ) m a s s s p e c t r o m e t e r m c o m b i n a t i o n w i t h a C a r l o E r b a 4160 gas c h r o m a t o g r a p h . T h e s a m p l e w a s i n t r o d u c e d m t o the ion s o u r c e ( 2 5 0 ° C ) b y a d i r e c t inlet p r o b e . A n e l e c t r o n e n e r g y o f 70 eV a n d a n ionizing c u r r e n t o f 100 /_tA w a s used. T h e d a t a o b t a i n e d w e r e p r o c e s s e d with a D S 55 c o m p u t e r s y s t e m c o n n e c t e d to the G C - M S Chemicals a n d solutions E t o p o s l d e w a s k i n d l y s u p p l i e d b y Bristol M y e r s B.V. ( B u s s u m , T h e N e t h e r l a n d s ) . T h e p u r i t y w a s 98.2% a c c o r d i n g to t h e m a n u f a c t u r e r M e t h a n o l ( " z u r A n a l y s e " , M e r c k ) w a s u s e d as received. O t h e r c h e r m c a l s u s e d w e r e o f a n a l y t i c a l g r a d e . All s o l u t i o n s w e r e p r e p a r e d w i t h d e t o n t z e d water. A 1 × 1 0 - 3 M s t o c k solutaon o f c o m p o u n d I a m m e t h a n o l k e p t at 4 ° C w a s f o u n d to b e stable, a t least f o r 4 w e e k s T h e c o m p o s i t i o n s o f the a q u e o u s b u f f e r s w e r e the s a m e as t h o s e used m o u r p r e v i o u s p u b h c a t i o n [12]

320

goltammetrtc experiments T h e b l a n k c u r r e n t v o l t a g e c u r v e w a s o b t a i n e d w i t h 1 ml o f the s u p p o r t i n g electrolyte, c o n s m t m g o f 0 1 m l o f m e t h a n o l a n d 0.9 m l o f b u f f e r . T o 0.9 m l o f b u f f e r , 0.1 ml o f a freshly p r e p a r e d s t o c k s o l u t i o n o f c o m p o u n d I a in m e t h a n o l w a s a d d e d . T h e v o l t a m m e t r i c c u r v e o f the r e s u l t i n g s o l u t i o n w a s r e c o r d e d a f t e r m i x i n g , u s m g a s c a n r a t e o f 50 m V s - ~ T h e s o l u u o n s at p H > 8 w e r e d e a e r a t e d b y a s t r e a m of mtrogen

Determmatton of p K S p e c t r a o f a 1 × 10 - 4 M s o l u t i o n o f c o m p o u n d l a m 0.05 M b o r a t e b u f f e r s c o n t a i n i n g 4% o f m e t h a n o l w e r e r e c o r d e d a n d the l o m c s t r e n g t h w a s k e p t to p = 0.150 .Aarb y the a d d i t i o n of K C I . F r o m the inflexion o f the p l o t o f a b s o r b a n c e as a f u n c t i o n o f p H , a p K a o f 9.70 was f o u n d . T t u s c o m p a r e s well w i t h the v a l u e o f 9.98 r e p o r t e d for 2 - m e t h o x y p h e n o l [13]. RESU L"IS

Cychc ooltammetry T h e o x i d a t i o n o f e t o p o s l d e ( I a ) at a glassy c a r b o n e l e c t r o d e results in t w o a n o d i c v o l t a m m e t r l c p e a k s , ! ! a n d llt ( F i g 1A). A t p H 2 5 - 1 4 , the h e i g h t s o f these t w o p e a k s c o r r e s p o n d to an overall u p t a k e o f t w o electrons, a n d their r a t i o m e r g e s to 1 . 1 ( T a b l e 1, Fig 2). A t p H < 2 5 ( F i g 3A), the two a n o d i c p e a k s o v e r l a p a n d their total height (tl + ~n) c o r r e s p o n d s to a t w o - e l e c t r o n process. T l u s o v e r l a p is c a u s e d b y the fact that with d e c r e a s i n g p H the p e a k p o t e n t m l o f q is shifted ( b e t w e e n p H 2.5 a n d 9.6) b y 57 m V / p H , w h e r e a s t h a t o f p e a k h~ at p H < 8 r e m a i n s p r a c u c a l l y p H i n d e p e n d e n t T h e p e a k p o t e n t i a l o f the c o m b i n e d p e a k 11 +IlI at p H < 2 5 is also p H - i n d e p e n d e n t . T h e potentaal o f p e a k t I b e c o m e s p H - l n d e p e n d e n t at p H > 10 (Fig. 3A). A t p H > - 11, the height o f p e a k t n d e c r e a s e s w i t h i n c r e a s i n g p H . T h e a p p a r e n t v a n a U o n s o f the p o t e n t i a l o f p e a k ttj at p H > 9 are d u e to specific b u f f e r e f f e c t s B o t h p e a k c u r r e n t s t I a n d t u h a v e b e e n p r o v e d to b e a l i n e a r f u n c t i o n o f t h e c o n c e n t r a t i o n o f the c o m p o u n d I a b e t w e e n 5 × 10 -5 a n d 1 × 10 - 3 m a n d o f the s q u a r e r o o t o f the s c a n r a t e f r o m 1.4 to 22 4 m V s -l/z. The p o t e n t i a l s o f b o t h p e a k s i x a n d zn w e r e f o u n d to b e i n d e p e n d e n t o f the c o n c e n t r a L t o n o f I a a n d o f the s c a n r a t e o v e r the w h o l e r a n g e studied. R e v e r s a l o f the potentaal s c a n at 0.8 V results m a c a t h o d i c r e d u c t i o n p e a k tl a n d a_•other p e a k , tnt, at m o r e n e g a t i v e potentaals (Fig. 1A) A s e c o n d r e v e r s a l o f the d t r e c t i o n o f the p o t e n t i a l scan at - - 0 . 2 V results in the f o r m a t i o n o f a n e w anoclJc p e a k , tin. T h e d e p e n d e n c e o f the p e a k p o t e n t i a l o f ii o n p H ( F i g 3B) closely r e s e m b l e s t h a t o f q (Ftg 2A) a n d their d i f f e r e n c e r e m a i n s a b o u t 60 m V . T h e p H - d e p e n d e n t s e c t i o n

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324 f o u n d for t] o f p H 3 - i 0 s h o w s a s l o p e o f 57 m V / p H F u r t h e r m o r e , the p H d e p e n d e n c e o f p e a k t~n ~s stmalar to t h a t o f qll a n d the s l o p e o f the p H - d e p e n d e n t s e c u o n is e q u a l to 58 m V / p H f o r b o t h p e a k s R e v e r s a l o f the p o t e n t i a l s c a n at + 0 . 4 5 V (Fig. 1B) results o n l y in a r e d u e u o n p e a k l~ w h e r e a s p e a k t m d o e s n o t a p p e a r . A s e c o n d r e v e r s a l at - 0 . 2 V yields o n l y p e a k t I a n d n o p e a k l~u. U n d e r n o c o n d i t i o n s , u p to scan r a t e s o f "1500 m V s -x, w a s a c a t h o d l c p e a k o b s e r v e d c o r r e s p o n d a n g to the arlochc p r o c e s s o c c u r r i n g at p e a k t H.

Potential step cychc voitammetry (PSCV) A t t e m p t s to use p o t e n t m l s t e p c y c h c v o l t a m m e t r y (PSCV) [14,15] to e v a l u a t e the r a t e c o n s t a n t o f the e l e c t r o c h e r m c a l r e a c t i o n d e a c u v a t i n g the p r o d u c t o f the t w o e l e c t r o n o x a d a u o n p r o c e s s w e r e t h w a r t e d b y the a b s e n c e o f the r e d u c t x o n p e a k t~, even at the baghest s c a n r a t e s accessible

Controlled potenttal electrolysts O x a d a u o n at + 0.85 V at p H 4.6 at a Pt g a u z e e l e c t r o d e p r o d u c e d a species w h i c h m c y c h c v o l t a m m e t r y at p H 4 0 o n a ~ a s s y c a r b o n e l e c t r o d e g a v e o n l y p e a k s t m a n d tin (F~g 4) T h e p o t e n t i a l s of the c a t h o d i c a n d a n o d i c p e a k s o b t a i n e d a f t e r p r e p a r a t i v e electrolysis d i f f e r e d slightly ( b y 0.05 V) f r o m t h o s e o b t a i n e d b y cyclic voha,-rm~etry o f the p a r e n t c o m p o u n d l a (Fig. 1). T h e p r o d u c t o f electrolysis gives a b s o r p t i o n m a x i m a at 365 a n d 470 n m (Fig. 5, c u r v e 8) m adchtaon to a r o m a t a e a b s o r p t a o n m the 280 n m range, w i n c h is also o b s e r v e c for the p a r e n t c o m p o u n d la ( F i g 5, c u r v e I) T h e a b s o r p t a o n b a n d s at 365 a n d 470 n m s h o w a g r a d u a l i n c r e a s e m the c o u r s e o f electrolysis ( c u r v e s 2 - 8 , Fig. 5). C o m p a r i s o n o f the I R s p e c t r a s h o w s t h a t the b a n d at 3400 c m - I c o r r e s p o n d i n g

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o

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H3CO ~ o O (rr) T h e q u m o l d s t r u c t u r e (II) was f u r t h e r s u p p o r t e d b y the e l e c t r o n - i m p a c t mass s p e c t r a o f the electrolysis p r o d u c t II a n d the c o m p o u n d f o r m e d b y ~ts s f l y l a u o n with N,O-b~s(trimethylsflyl)-tnfluoroacetamade In the mass s p e c t r u m o f electrolysis p r o d u c t II (Fig 6A), the m o l e c u l a r p e a k at m / e 572 ~s rmssmg b u t the s p e c t r u m shows the p r e s e n c e o f an m n at m / e 574. T h i s c o r r e s p o n d s to the formalaon o f a n ( M + 2) + ion, c h a r a c t e n s u c o f o r t h o q u m o n e s [16], resulting f r o m the h y d r o q u m o n e f o r m e d b y the r e d u c U o n m the s o u r c e m the p r e s e n c e o f traces o f moisture. T h e ion p e a k at rn/e 366 c o r r e s p o n d s to the ( M - 106) + 1on f o r m e d f r o m the q u m o n e b y the loss o f the glycoside moiety. T h e r a t m o f the p r o b a b t h t y o f the f o r m a t m n o f the ion with rn/e 574 to that o f the 1on with role 366 d e c r e a s e d with the increasing residue tame o f the s a m p l e m the ion source, whach c o r r e s p o n d s to a n increase m the e x t e n t o f p y r o l y t i c d e c o m p o s f l a o n . T h e highest p e a k m the mass s p e c t r u m o f the sflylated e!ectrolys~s p r o d u c t (Fig. 6B) at m / e 862 c o r r e s p o n d s to an o r t h o q u m o n e derivative bemang f o u r sflyl g r o u p s T l u s results f r o m s t l y l a u o n o f the two h y d r o x y l g r o u p s m the g l y c o s i d e moxety a n d two sflyl g r o u p s wbach reacted wath the h y d r o x y l g r o u p s resulting m the re,ductaon o f the o r t h o q u m o n e DISCUSSION

T h e parallel b e h a v i o u r o f the E o - p H plots for p e a k s t I a n d zl mdacates t h a t o x i d a t i o n o f etopos~de (la) o c c u r s m a reversible o n e - e l e c t r o n p r o c e s s 0 i ) - T h e

327 p r o d u c t formed is reduced m wave i[. O n the ot her hand, the transfer of the second electron c o r r e s p o n d i n g to the oxidation wave ! n occurs irrevers,bly. T h e prmaary electrolysis p r o d u c t undergoes a rapia conversion into the o r t h o q u i n o n e , wbach is revermbly reduced in peak qu- T h e rate of the conversion us bagh since e v e n at the tughest rate of scanning used (1.5 V / s ) , no mchcation of a cat hodi c r e d u c t i o n ,s observed T h e conversion is f'trst o rder m the p r t m a r y electro!ysls product , as is indicated by the practical i n d e p e n d e n c e of the ratao l,/l~ of the c o n c e n t r a t i o n of ,~.topos~de (II). T h e q u m o i d structure of the p r o d u c t of conversion is s u p p o r t e d by ,,he parallel be ha vi our of the E p - p H plots for peaks III and III' (Fig. 3) and by the IR and mass spectra of the oxidation product. T h e oxidation in peak ~I at p H > 2 occurs m the p h e n o l a t e form which Is m ore easdy oyachzed, as are other conjugate bases. H e n c e at p H > pKa (9.7), where the p h e n o l a t e form p r e d o m i n a t e s m the bulk of the solutaon, the peak p o t e n u a l Ep~ ~s p H - m d e p e n d e n t (Table 1, Fig. 3A). T h e b.near d e p e n d e n c e of f p l at p H 2 - 9 . 7 mchcates that the a c i d - b a s e e q m h b n u m between the phenol at e ~on and the phenol f o r m us established rapidly. T h e p H i n d e p e n d e n c e of Ep,, (Table 1, Fag. 3A) indicates the absence of a p r o t o n transfer between the two electron transfers. T h e irreverslbthty of the second electron-uptake us at least partly caused by the rapid conversions of the p r i m a r y electrolysis product. T h e p r o d u c t of tiros fast conversion is an o r t h o q u m o n e ; this reaclaon must hence revolve solvolytac elimination o f methanol. Such a course of oxadataon of phenol us not very c o m m o n . T h e major p a t h w a y of the electro-oxadatxon of phenols is the f o r m a t i o n o f duners [14,17-20] T h e m i nor p a t h w a y yielding o r t h o q u m o n e s has, nevertheless, been r e p o r t e d for s o me simple q u m o n e s bem"ing a substatuent at position 4 to the h y d r o x y l groups, m additaon to an adjacent m e t h o x y [21] or p h e n o x y group [19]. F o r etoposide (Ia), the presence of rings A, B and C at position 4 relative to the phenol group prevents the f o r m a t i o n of dLmers or quinone-mettudes and favours solvolyuc f o r m a u o n of an orthoqumone Thus, the behaxaour of etoposlde at p H > 2 can be d e s c n b e d by the m e c h a m s m : R

R

ON

-I-

I-I "I"

"~"

e--

(1)

0

R

~co~ 0 0

oc~ 0 0

(2)

328

R

R

(3) CHAD/

~'~ ~ , , ' ' ~ ( ~ ,

CH3

0

0 e

R

Ri l

'~ i-4j:o-

-t- OH e

.~L C H 3 0 H

- "~""~o~--- c H 3

(4)

CH 0

O ®

The formauon of peaks tm and t~n in the cychc voltammetry of the product of conversion--the orthoquinone--,s then attributed to processes (5)-(7):

R

'=

(5) H3CO

0e

0

0 e

R

R

i

H3CO O

4-

H -I-

O e

OH

R

R +

H3CO

H+

!

PKr;~

(7)

Oe OH

OH

The plots of Ep,,,-pH and E~I,[-pH indicate that pK., and pKr_. are larger than 11 (the comphcated pattern shown m Fig. 3B at pH > 11 is due to nucleopbalic attacks of hydroxide ions on the qumone nng). The increase m the total height i, + ,, at pH < 2 indicates that the phenolic form is oxad~zed by a larger number of electrons, probably four The peak potential

329

c o r r e s p o n d i n g to ti+ n m this p H range is p H - m d e p e n d e n t , ,_ndlcating that the first electron transfer on the phenolic form ~s not preceded by an acad-base e q u t h b n u m . T h e potential of the hr s t electron transfer on p h e n o l a t e in current q and that of the electro-cxadation of the phenolic form in t~+H at p H 1 - 2 is so close that it ~s tmpossible to observe the formation of two separate peaks ( q and q + H ) and the replacement of -'i by tl+ n with decreasing p H , but the peaks m thas p H range are broader, indicating two overlapping processes. Also, the extension o f the p H - m d e p e n d e n t segment of the E p - p H plot (F~g 3A) mchcates that a p H < 2 the oxadatmn o f the p h enol at e ~s replaced by that of a n o t h e r species. T h e decrease of peak /ix at p H > 11 is p r o b a b l y due to a nucleoptuhc attack of h y d r o x i d e ions on the rachcal zwltterion f o r m e d m reactmn (2). T h e increase m current t~ at p H > 13 is p r o b a b l y due to a d e g r a d a t i o n p r o d u c t of the q u m o i d species. ACKNOWLEDGEMENTS

Ttus work was s u p p o r t e d m part by Z W O ('Nederlandse o r g a m s a u e voor Z m v e r Wetenschappeh.lk Onderzoek) by means of a vis~_ting sc~enttst a p p o i n t m e n t (P. Zu man ) , which Is gratefully acknowledged. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 lg 19 20 21

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