Spectroelectrochemical Examination of the ... - ACS Publications

19

Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch019

Spectroelectrochemical Examination of the Reactions of Chlorpromazine Cation Radical with Physiological Nucleophiles J. S. MAYAUSKY, H. Y. CHENG, P. H. SACKETT, and R. L. McCREERY The Ohio State University, Department of Chemistry, Columbus, OH 43210

Previously, the reactions of cation radicals derived from phenothiazine drugs were examined using conventional spectrophotometry and liquid chromatography. The time scale of the techniques limited the pH range to 5 and below, where the reactions were slow enough to monitor accurately. In the present work, the pH range was extended to 4 to 9 using a reflective spectroelectrochemical technique. Reactions of chlorpromazine cation radical with acetate, phosphate, and citrate buffer were examined as a function of pH and buffer concentration. The observed rate was critically dependent on which buffer species were present and on the degree of protonation for polyprotic buffers. The dianions of phosphate and citrate had rates that were 62 and 330 times faster than the monoanions, all else being equal. This rate difference may be due to the ability of the buffer to simultaneously interact with two radicals, thus promoting charge transfer.

T j r u g s based o n the phenothiazine nucleus, particularly chlor p r o m a z i n e ( C P Z ) , h a v e b e c o m e the major class o f a n t i p s y c h o t i c s for t h e t r e a t m e n t o f m e n t a l d i s o r d e r s , i n c l u d i n g s c h i z o p h r e n i a . T h e i m p a c t o f the existence o f these drugs o n the treatment o f m e n t a l i l l ness h a s b e e n e n o r m o u s , w i t h t h e n e e d for i n s t i t u t i o n a l c a r e o f t e n b e i n g e l i m i n a t e d through proper d r u g therapy. T h e c l i n i c a l impor0065-2393/82/0201-0443$06.00/0 © 1982 A m e r i c a n C h e m i c a l Society

Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


tance o f the p h e n o t h i a z i n e s has s p a w n e d a large n u m b e r o f research efforts o n t h e i r c h e m i s t r y , p h a r m a c o l o g y , a n a l y s i s , a n d m e t a b o l i s m . S e v e r a l r e v i e w s o f this w o r k have a p p e a r e d , b o t h i n m e d i c a l a n d i n c h e m i c a l p u b l i c a t i o n s (1-3). O f p a r t i c u l a r r e l e v a n c e to the present d i s c u s s i o n are i n v e s t i g a t i o n s of the cation r a d i c a l o f C P Z a n d related drugs f o r m e d u p o n t h e re m o v a l o f one electron from ther i n g system. T h e cation radical a n d other o x i d i z e d forms h a v e b e e n e x a m i n e d i n some d e t a i l from b o t h b i o l o g i c a l a n d strictly c h e m i c a l standpoints. T h e interest i n the r a d i c a l s t e m s f r o m t h r e e g e n e r a l p o i n t s . F i r s t , C P Z f o r m s a c a t i o n r a d i c a l (4,5) t h a t i s s i m i l a r t o t h o s e o f p o l y n u c l e a r a r o m a t i c s (e.g., a n t h r a c e n e a n d t h i a n t h r e n e ) , a n d is t h e r e f o r e a n e x a m p l e o f a c l a s s o f w i d e l y s t u d i e d h e t e r o c y c l i c r a d i c a l ions. T h e characteristics a n d reactions o f these m o l e c u l e s have been s t u d i e d i n some detail, but questions r e m a i n a b o u t w h a t factors affect r a d i c a l b e h a v i o r . U n l i k e d i p h e n y l a n t h r a c e n e a n d thianthrene, the 10-substituted phenothiazines form relatively s t a b l e r a d i c a l s i n a q u e o u s s o l u t i o n s , b u t t h e r e a s o n s for t h i s s t a b i l i t y are not clear. S e c o n d , the cation r a d i c a l o f C P Z is b e l i e v e d t o b e a k e y i n t e r m e d i a t e i n t h e m e t a b o l i s m o f t h e d r u g ( 6 , 7). R a d i c a l i o n p a t h w a y s have b e e n p r o p o s e d i n the formation o f t w o o f the three major metabolites, the sulfoxide a n d r i n g h y d r o x y l a t e d species, a n d a r a d i c a l h a s b e e n d e t e c t e d b y E S R i n p a t i e n t s b e i n g t r e a t e d w i t h C P Z (8). T h i r d , t h e C P Z r a d i c a l ( C P Z * ) h a s a v a r i e t y o f effects o n b i o l o g i c a l systems, i n c l u d i n g e n z y m e s (9), D N A (10), a n d m e m b r a n e s ( J J ) . CPZ m a y b e r e s p o n s i b l e for s o m e o f t h e s i d e effects o f C P Z treat m e n t , o r e v e n for t h e d e s i r e d a n t i p s y c h o t i c effect (4, 6,12). I n a n y c a s e , t h e r a d i c a l is v e r y l i k e l y t o b e f o r m e d i n v i v o , a n d its b e h a v i o r is l i k e l y to b e i m p o r t a n t t o t h e m e t a b o l i s m a n d a c t i v i t y o f t h e p a r e n t d r u g . +

+

U n t i l 1975, t h e great majority o f w o r k o n p h e n o t h i a z i n e r a d i c a l i o n s w a s c a r r i e d o u t i n s t r o n g a q u e o u s a c i d s (e.g., 4 M H 2 S O 4 ) t o stabilize t h e r a d i c a l e n o u g h to obtain spectroscopic or k i n e t i c data (13). M o s t w o r k e r s c o n c l u d e d t h a t t h e d e c a y o f C P Z w a s s e c o n d o r d e r a n d r e s u l t e d from disproportionation o f the r a d i c a l to C P Z a n d C P Z sulfoxide. H o w e v e r , the kinetics were n o t straightforward, a n d the stability o f the r a d i c a l was h i g h l y d e p e n d e n t on the s o l u t i o n e n v i r o n +


19.

MAYAUSKY ET AL.

Chlorpromazine Cation Radical Reactions

m e n t for r e a s o n s t h a t w e r e 10-phenylphenothiazine

not clear. A n excellent examination o f

cation radical i n acetonitrile a n d p y r i d i n e

r e v e a l e d that the r a d i c a l d i d not disproportionate

b u t was attacked

d i r e c t l y b y a v a i l a b l e n u c l e o p h i l e s (14). T h i s result indicates that the p r e v i o u s w o r k o n r a d i c a l ions o f p h e n o t h i a z i n e drugs i n a q u e o u s s o l u tion warrants

reexamination.

I n a d d i t i o n to w o r k s p e c i f i c a l l y r e l a t e d to p h e n o t h i a z i n e other

h e t e r o c y c l i c r a d i c a l ions

have

been

studied

drugs,

extensively i n

n o n a q u e o u s m e d i a . T h e vast majority i n d i c a t e that the r a d i c a l ions +

react a c c o r d i n g to Reactions 1 - 3 , w h e r e A

is t h e i n i t i a l r a d i c a l i o n a n d


Ζ is a n u c l e o p h i l e . +

A AZ+

+

+ Z ^ ΑΖ ·

+ A

AZ

2 +

+

(1)

2

AZ + + A

(2)

products

(3)

A disproportionation, Reactions 4 - 6 , 2

2Α+· - * A + A + 2

A ~ + Ζ -+ A Z AZ

2 +

(4)

2 +

(5)

- » products

(6)

w o u l d l e a d to t h e s a m e o v e r a l l s t o i c h i o m e t r y , b u t t h e r e a c t i v e s p e c i e s for t h e d i s p r o p o r t i o n a t i o n c a s e is a d i c a t i o n r a t h e r t h a n a c a t i o n r a d i c a l . G i v e n this b a c k g r o u n d , o u r laboratory i n v e s t i g a t e d the k i n e t i c s a n d m e c h a n i s m o f the reactions o f p h e n o t h i a z i n e radicals i n a q u e o u s solution i n the p H range 2 - 7 . T h e radicals were d e r i v e d from chlor p r o m a z i n e a n d other c l i n i c a l l y important phenothiazines, a n d the o t h e r s o l u t i o n c o n s t i t u e n t s w e r e r e s t r i c t e d to p h y s i o l o g i c a l l y o c c u r r i n g m a t e r i a l s . T h e o b j e c t i v e s o f t h i s a p p r o a c h w e r e as f o l l o w s : (1) d e t e r m i n a t i o n o f t h e p r o d u c t d i s t r i b u t i o n o f c a t i o n r a d i c a l r e a c t i o n s as a f u n c t i o n o f m e d i u m , p H , a n d r a d i c a l s t r u c t u r e ; (2) o b s e r v a t i o n o f t h e rate o f r a d i c a l d e c a y o v e r a w i d e r a n g e o f c o n d i t i o n s ; a n d (3) e s t a b l i s h m e n t o f a r e a c t i o n m e c h a n i s m for r a d i c a l d e c a y a n d c l a r i f i c a t i o n o f h o w r a d i c a l s t r u c t u r e o r r e a c t i o n m e d i u m affects t h i s m e c h a n i s m . P r e v i o u s reports demonstrated that the products o f a pheno t h i a z i n e cation r a d i c a l reaction i n a q u e o u s solution are h i g h l y de p e n d e n t on b o t h the structure o f the radical a n d o n the identity o f n u c l e o p h i l e s i n the reaction m e d i u m . F o r C P Z i n p h o s p h a t e or carb o x y l a t e buffers, 1 m o l o f r a d i c a l r e a c t s to f o r m 0 . 5 m o l e a c h o f C P Z a n d c h l o r p r o m a z i n e s u l f o x i d e ( 1 5 ) . I f a n a m i n e is p r e s e n t i n t h e m e d i u m , particularly an amine w i t h a l o w ρ Κ « , r i n g hydroxylation occurs a n d t h e y i e l d o f s u l f o x i d e is s u b s t a n t i a l l y b e l o w 5 0 % ( 1 6 ) . I f a +


445

BIOLOGICAL REDOX COMPONENTS

446


p i p e r a z i n e r i n g is i n c l u d e d i n t h e s i d e c h a i n (as i n fluphenazine), h y d r o x y l a t i o n is o b s e r v e d r e g a r d l e s s o f t h e b u f f e r e m p l o y e d (17). F i n a l l y , s u b s t i t u t i o n at t h e 2 - p o s i t i o n d o e s n o t affect p r o d u c t d i s t r i b u t i o n , n o r d o e s d e m e t h y l a t i o n o f t h e C P Z s i d e - c h a i n a m i n e (18). T h e results p e r m i t the c o n c l u s i o n that the r a d i c a l w i l l either b e h y d r o x y l a t e d or f o r m s u l f o x i d e , d e p e n d i n g o n t h e p r e s e n c e o f n u c l e o p h i l i c , d e p r o t o n a t e d a m i n e s . I n C P Z , the s i d e - c h a i n n i t r o g e n ρ Κ α is h i g h (9.30) a n d is u n l i k e l y to b e i n v o l v e d i n n u c l e o p h i l i c a t t a c k . T h e r e f o r e , t h e i m p o r t a n t n u c l e o p h i l e s are b u f f e r c o m p o n e n t s a n d o n l y s u l f o x i d e a n d p a r e n t c o m p o u n d a r e o b s e r v e d as p r o d u c t s i n p h o s p h a t e , c i t r a t e , or a c e t a t e m e d i u m . H o w e v e r , w h e n n u c l e o p h i l i c a m i n e s are p r e s e n t , e i t h e r as a b u f f e r c o m p o n e n t [ e . g . , N - m o r p h o l i n o e t h a n e s u l f o n i c a c i d ( M E S ) , ρ Κ α = 6.2] or i n t h e r a d i c a l ' s o w n s i d e c h a i n (e.g., fluphenazine, p ^ = 3.9), h y d r o x y l a t i o n is o b s e r v e d (17). F o r a n a m i n e to a t t a c k t h e r a d i c a l a n d c a u s e h y d r o x y l a t i o n its ρ Κ α m u s t b e l o w e n o u g h so a s u f f i c i e n t f r a c t i o n o f t h e a m i n e is d e p r o t o n a t e d to c o m p e t e w i t h other, n o n a m i n e n u c l e o p h i l e s . T h e details o f the hy d r o x y l a t i o n p r o c e s s are n o t k n o w n , b u t t h e s u l f o x i d e a n d h y d r o x y l a t e d d e r i v a t i v e s a r e t w o o f the t h r e e m a j o r classes o f m e t a b o l i c p r o d u c t s i n vivo. B e c a u s e the s t o i c h i o m e t r y o f the r e a c t i o n o f the r a d i c a l i n c a r b o x y l a t e o r p h o s p h a t e buffers w a s e s t a b l i s h e d q u a n t i t a t i v e l y , t h e k i n e t i c s a n d m e c h a n i s m s o f the d e c a y o f s e v e r a l r a d i c a l s w e r e e x a m i n e d i n d e t a i l i n t h e s e m e d i a (15, 16, 18). T h e o v e r a l l r e a c t i o n is g i v e n b y E q u a t i o n 7, w h e r e C P Z O r e p r e s e n t s C P Z s u l f o x i d e . S y n t h e t i c r a d i c a l perchlorate salt ( p r e p a r e d e l e c t r o c h e m i c a l l y ) w a s d i s s o l v e d i n a p p r o priate s o l u t i o n , a n d the d e c a y 2CPZ+ + H 0 -» C P Z + C P Z O + 2 H

+

(7)

2

+

o f C P Z ' w a s m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y at 5 2 5 n m . C a r e f u l analysis o f the d e c a y k i n e t i c s c o n f i r m e d that the reaction was s e c o n d order, b u t that it d i d not i n v o l v e a disproportionation. T h e buffer a n i o n a c t e d as a c a t a l y s t , w i t h t h e m e c h a n i s m b e i n g r e p r e s e n t e d b y R e a c t i o n s 8 - 1 0 , w h e r e B " is b u f f e r a n i o n . CPZ+ +

B - ^ ( C P Z B ) -

(CPZB)- + CPZ+ d=± (CPZB) k. i_9

+

(8) + CPZ

(9)

9

H 0 + (CPZB)

+

CPZO + HB + H

2

T h e r a t e l a w for t h i s r e a c t i o n is g i v e n b y E q u a t i o n l l

+

(10) 1

1

The involvement of water in Reaction 10 dictates that the rate law involve water as a reagent. For the sake of clarity, k will be assumed to include the water term. So wherever k appears, it in fact equals k[ [H 0]. 10

10

0

2


19.

MAYAUSKY ET AL.


d[CPZ+] _ -2K UIQ[B-][CPZ^] dt k. [CPZ] + k 8

9

447

2 N U

N A

;

10

T h e o r i g i n a l r e p o r t s s h o u l d b e c o n s u l t e d for t h e d e t a i l s o f t h i s r e a c t i o n but several important points deserve comment. First, a disproportiona t i o n o f r a d i c a l t o d i c a t i o n w a s r u l e d o u t for a l l t h e s y s t e m s s t u d i e d , a n d the m e c h a n i s m is consistent w i t h reactions o f other r a d i c a l ions i n n o n a q u e o u s m e d i a (14, 19). S e c o n d , t h e buffer n u c l e o p h i l e greatly accelerates the reaction relative to that i n p l a i n water. T h e half-life o f C P Z * i n w a t e r a t p H 7 ( u s i n g a p H stat w i t h n o buffer) i s 4 m i n ( 1 6 ) , a n d t h e half-life i n p H 7 p h o s p h a t e buffer is a p p r o x i m a t e l y 100 m s . T h e r e f o r e , w a t e r is a p o o r n u c l e o p h i l e for t h i s r e a c t i o n , a n d t h e r a d i c a l w i l l b e a t t a c k e d b y e v e n w e a k e r n u c l e o p h i l e s s u c h as p h o s p h a t e s a n d carboxylates i f they are present. T h i r d , protonation o f B ~ r e d u c e s t h e free B " c o n c e n t r a t i o n , s l o w i n g t h e r a t e at l o w e r p H . I n t h e w o r k p r e s e n t e d so far, t h e p H r a n g e w a s l i m i t e d t o t h e r e g i o n f r o m 2 t o 5 , b e c a u s e t h e r a t e at h i g h e r p H w a s t o o fast t o m o n i t o r b y c o n v e n t i o n a l s p e c t r o p h o t o m e t r y . T h e p H d e p e n d e n c e results f r o m p r o t o n loss i n R e a c t i o n 8. B e c a u s e R e a c t i o n 8 is i n e q u i l i b r i u m , t h e c a s e w h e r e H P 0 ~ attacks cannot b e d i s t i n g u i s h e d k i n e t i c a l l y from t h e case w h e r e H P 0 ~ attacks w i t h loss o f a p r o t o n . F o u r t h , t h e r a d i c a l forms a covalent adduct w i t h the n u c l e o p h i l e , w h i c h e v e n t u a l l y hydrolyzes to r e g e n e r a t e b u f f e r . T h u s , t h e m o s t l i k e l y fate o f t h e r a d i c a l i n a p h y s i o l o g i c a l e n v i r o n m e n t is a t t a c k b y b i o l o g i c a l l y o c c u r r i n g n u c l e o p h i l e s , p e r h a p s p e p t i d e s f r o m r e c e p t o r sites. F i f t h , s u b s t i t u t i o n o f C P Z a t t h e 2-position changes t h e rate i n a p r e d i c t a b l e fashion p r o v i d e d t h e m e c h a n i s m is t h e s a m e (18). E l e c t r o n - w i t h d r a w i n g substituents i n c r e a s e t h e rate o f r e a c t i o n b e c a u s e t h e r a d i c a l b e c o m e s m o r e e l e c trophilic.


+

2

4

2

4

T h e m a i n objective o f the n e w results presented i n the f o l l o w i n g section was to extend the k i n e t i c analysis o f radical decay to the p H r a n g e 2 - 9 . I n a d d i t i o n to t h e g r e a t e r p h y s i o l o g i c a l i m p o r t a n c e o f t h e s e r e s u l t s , t h e factors a f f e c t i n g n u c l e o p h i l e r e a c t i v i t y c a n b e e l u c i d a t e d .

Experimental +

T h e t e c h n i q u e used to determine the reaction rate o f C P Z * decay i n buffers o f near neutral p H was a c o m b i n a t i o n o f the approach u s e d b y B l o u n t (20) a n d a recently d e v e l o p e d reflective spectroelectrochemical t e c h n i q u e (21) . A light b e a m reflected off a glassy carbon electrode i m m e r s e d i n solu tion was used to monitor C P Z * generated at the electrode ( X = 525 nm). T h e absorbance vs. t i m e transient o c c u r r i n g after the b e g i n n i n g o f elec trolysis was recorded i n the absence a n d i n the presence o f a n u c l e o p h i l e . I n a l l cases where n u c l e o p h i l e was absent, the A vs. t plot was linear, as ex p e c t e d for an u n c o m p l i c a t e d diffusion-controlled generation o f chromophore. +

m a x

1/2



448

In the presence o f a n u c l e o p h i l e (phosphate, citrate, or acetate) the absorbance was smaller than the u n c o m p l i c a t e d case, because the C P Z " generated at the electrode reacted to form u n c o l o r e d products ( C P Z + C P Z O ) . T h e ratio o f the absorbance w i t h reaction to that without reaction w i l l be referred to as n o r m a l i z e d absorbance, A . T h e shape o f an A vs. t i m e (or l o g t) plot was characteristic o f the rate a n d m e c h a n i s m o f the reaction be tween C P Z a n d n u c l e o p h i l e . Comparisons o f experimental and theoretical A vs. l o g t plots a l l o w e d determination o f observed rate constants for C P Z " / n u c l e o p h i l e reactions h a v i n g t i m e scales i n the 10-m s to 10-s range. W h e n C P Z is generated from r e d u c e d C P Z at an electrode, the reaction sequence is the same as that i n Equations 8 - 1 0 , except that Reaction 8 is preceded b y the one-electron oxidation o f C P Z . T h e decay o f C P Z ' after electrogeneration was s i m u l a t e d w i t h well-established finite difference methods, u s i n g the fact that the observed quantity is the integral of the C P Z concentration throughout the diffusion layer (20). W h e n the rate l a w ( E q u a tion 12) is converted to a finite difference form one obtains +

n

n

+

n

+

+

+


+

AFRj àt

_ -2UER/ " FCj + X

,

m

U

;

where FRj is the fractional radical concentration (relative to b u l k C P Z ) ; FCj is the fractional C P Z concentration (relative to b u l k ) ; k is the observed rate constant, e q u a l to l Q K f c [B~] where K = k /k- ; a n d X equals k /k- [CPZ] . W o r k i n g curves of A vs. l o g (k bst) were calculated based on E q u a tion 12, w i t h the absorbance b e i n g d e t e r m i n e d from the integrated concen tration i n a l l boxes. T h e v a l u e o f k /k„ , a n d therefore X , was calculated from homogeneous k i n e t i c data obtained at l o w e r p H for a l l three nucleo philes e x a m i n e d here (15, 16). In a l l cases, the absorbance of the reflected b e a m was m o n i t o r e d at 525 n m after a potential step corresponding to diffusion-controlled generation o f C P Z . T h e C P Z cation radical was the o n l y solution component that absorbed light at this w a v e l e n g t h . Since C P Z a n d s i m i l a r drugs adsorb significantly on s o l i d electrodes, it was necessary to a d d 5 0 % b y v o l u m e o f methanol to the aqueous buffers to e l i m i n a t e adsorption. It was verified that the stoichiometry of the reaction was unchanged i n this m e d i u m . Spectrophotometric examina tion o f the decay kinetics i n 5 0 % m e t h a n o l - H 0 , u s i n g the approach d e s c r i b e d p r e v i o u s l y (15), r e v e a l e d that the m e c h a n i s m was q u a l i t a t i v e l y unchanged from p u r e l y aqueous solution. Plots o f l/k - [ C P Z ] (15) revealed that the value k /k- i n 5 0 % methanol was 2.8 χ 10~ for citrate at p H 2.46, 43 χ 1 0 " for phosphate at p H 3.14, a n d 0 for acetate ( p H 3.4). These values are some what larger than those i n water, a n d w i l l b e used i n a l l subsequent calcula tions. T h e pKa values o f the buffers e m p l o y e d were d e t e r m i n e d i n 5 0 % methanol b y p H titration a n d were as follows: acetate pKa, 5.20; phosphate p K j , 3.30; phosphate p K , 7.65; phosphate p K , 11.75; citrate pK = 3.70; citrate p K , 5.30; citrate pK^, 6.85. T h e use o f 5 0 % methanol solutions for both the titration a n d k i n e t i c experiments ensures that species distributions can b e accurately calculated from these p K values. H o w e v e r , 100% aqueous buffers were used for meter calibrations i n this w o r k so that p H and pK values are internally consistent but shifted slightly i n the absolute sense. I n a l l experi ments the ionic strength was adjusted to 0.2 M w i t h N a C l . K i n e t i c runs were conducted at ambient temperature, 23 ± 1°C, w i t h the total buffer concentra tion at least a n order o f magnitude greater than the b u l k C P Z concentration. obs

9

10

9

9

9

10

9

b u l k

n

n

l0

9

+

2

v s

obs

3

l0

3

9

2

3

t

2

a

a


MAYAUSKY ET AL.

19.

Chlorpromazine

Cation

Radical

Reactions

449

Results T y p i c a l a b s o r b a n c e v s . t i m e t r a n s i e n t s for t h e e l e c t r o g e n e r a t i o n o f +

C P Z * a r e s h o w n i n F i g u r e 1. I n t h e a b s e n c e o f a n u c l e o p h i l e t h e 1/2

c u r v e has the t

b e h a v i o r ( C u r v e 1) e x p e c t e d for a s t a b l e e l e c t r o g e n -

erated chromophore. W h e n phosphate

is p r e s e n t , t h e a b s o r b a n c e

is

+

l o w e r d u e to d e c a y o f e l e c t r o g e n e r a t e d

C P Z * . T h e d e c a y rate i n

c r e a s e s w i t h p H as s h o w n i n C u r v e s 2 - 4 . F i g u r e 2 c o m p a r e s p l o t s o f n o r m a l i z e d a b s o r b a n c e v s . l o g kt for s e v e r a l k i n e t i c c a s e s w i t h

the

e x p e r i m e n t a l p o i n t s for p h o s p h a t e b u f f e r . C u r v e 3 s h o w s t h e w o r k i n g


c u r v e for a first-order r e a c t i o n , as w o u l d b e o b s e r v e d i f R e a c t i o n 8 w e r e rate l i m i t i n g . C u r v e 2 is t h e c a s e for X = 0 , w h i c h c o r r e s p o n d s R e a c t i o n s 8 a n d 9 i n e q u i l i b r i u m , w i t h R e a c t i o n 10 rate

to

limiting. +

C u r v e 2 w o u l d a l s o b e o b s e r v e d for a d i s p r o p o r t i o n a t i o n o f C P Z " to C P Z and C P Z

+ 2

2

f o l l o w e d b y r e a c t i o n o f C P Z + . C u r v e 1 is a p l o t for X

= 8.3, t h e v a l u e c a l c u l a t e d f r o m t h e k /k10

9

r a t i o for p h o s p h a t e d e t e r

m i n e d from c o n v e n t i o n a l s p e c t r o p h o t o m e t r i c k i n e t i c s . T h e p o i n t s are e x p e r i m e n t a l r e s u l t s for s e v e r a l p H v a l u e s i n p h o s p h a t e b u f f e r , w h i c h

ι 1

» 2

ι 3

I 4

I 5

I 6

I 7

I 8

1 9

T i m e (s) Figure 1. Experimental absorbance vs. time curves (single runs) for 5.0 m M C P Z in 50% MeOHIH 0 solutions. The potential step was from 0 to +0.8 V vs. S C E , the observation wavelength was 525 nm, and the input beam angle was 3.2°. The medium for Curve 1 was 0.2 M NaCl; Curves 2, 3, and 4 were 0.02 F phosphate buffer at pH 3.0, 5.0, and 7.0, respectively. 2


1 10



450

log (kt) Figure 2. Comparison of theoretical vs. observed normalized ab sorbance vs. log (k t). Curve 1 was calculated from Equation 13 with X = 8.3, and Curve 2 was calculated from the same equation with X = 0. Curve 3 was calculated for a reaction that was first order in C P Z ' . Points are experimental values in conditions corresponding to the theoretical Curve 1 (0.02 F phosphate buffer, X = 8.3), at various pH values. Key: O , pH 4.20; X, pH 5.45; Δ , pH 6.4; • , pH 7.12; and · , pH 7.41. o6e

+

w e r e fit to C u r v e 1 b y a d j u s t i n g &o . T h e b u l k C P Z c o n c e n t r a t i o n u s e d e x p e r i m e n t a l l y w a s t h a t u s e d to c a l c u l a t e t h e X v a l u e a p p r o p r i a t e to C u r v e 1. T h u s , o n c e a n e x p e r i m e n t a l A v s . t t r a n s i e n t is a v a i l a b l e , k m a y b e c a l c u l a t e d b y c o m p a r i s o n w i t h C u r v e 1, o r o n e l i k e i t c o r r e s p o n d i n g to t h e X v a l u e a p p r o p r i a t e t o t h e b u f f e r e m p l o y e d . T h e p r o c e d u r e i l l u s t r a t e d i n F i g u r e 2 w a s u s e d to d e t e r m i n e o b s e r v e d rate c o n s t a n t s for r e a c t i o n s o f C P Z " w i t h c i t r a t e , p h o s p h a t e , a n d a c e t a t e buffers as a f u n c t i o n o f p H . F o r cases w h e r e Χ Φ 0 ( c i t r a t e a n d phosphate), the v a l u e K k c o u l d b e c a l c u l a t e d from k , X , a n d the b u l k C P Z c o n c e n t r a t i o n . F o r the case w h e r e X = 0 (acetate) o n l y the o v e r a l l c o n s t a n t Κ Κ^ c o u l d b e d e t e r m i n e d . V a l u e s for fc bs are l i s t e d i n T a b l e I for t h e t h r e e n u c l e o p h i l e s e x a m i n e d . O b s e r v e d r a t e c o n stants are p r e s e n t e d i n g r a p h i c a l f o r m i n F i g u r e 3 o v e r t h e p H r a n g e 4 to 9. A b o v e p H 9 C P Z w i l l n o t d i s s o l v e s i g n i f i c a n t l y a n d m e a n i n g f u l results w e r e not o b t a i n e d . F i g u r e 4 s h o w s the d e p e n d e n c e o f k on t o t a l b u f f e r c o n c e n t r a t i o n at p H 7. bs

n

obs

+

8

8

ί0

9

ohs

0

obs


19.

MAYAUSKY ET AL.

Table L


pH


O b s e r v e d S p e c t r o e l e c t r o c h e m i c a l R a t e C o n s t a n t s for CPZ+ Decay

Acetate

Phosphate

Citrate

(0.2 F )

(0.025 F )

(0.005 F )

1

KtoiM-'s' )

3.96 4.45 5.0 5.5 6.0 6.5 7.0 7.5

0.029 0.076 0.197 0.337 0.425 0.495 0.562 0.510

pH

pH 4.30 4.40 4.88 5.00 5.25 5.75 6.5 6.9 7.0 7.5 7.7 7.75 8.00 8.20 8.70 9.1

0.23 0.230 0.343 0.439 0.697 1.10 5.04 17.2 20.3 44.9 67.0 67.7 75.5 75.7 80.4 80.7

3.61 4.05 4.50 5.05 5.53 6.00 6.40 6.60 6.92 7.26 7.80

kobs 0.035 0.238 0.579 1.49 2.79 4.95 5.01 6.09 6.24 6.16 6.18

Discussion T h e agreement between theory a n d experiment s h o w n i n F i g u r e 2 indicates that s p e c t r o e l e c t r o c h e m i c a l results are consistent w i t h the m e c h a n i s m o f Reactions 8 - 1 0 . T h e s p e c t r o e l e c t r o c h e m i c a l response c a n b e u s e d to d i s c r i m i n a t e b e t w e e n first- a n d s e c o n d - o r d e r r e a c t i o n s , b u t p r e v i o u s w o r k e r s d e m o n s t r a t e d that the response is not p a r t i c u l a r l y s e n s i t i v e t o less o b v i o u s c h a n g e s i n k i n e t i c p a r a m e t e r s , s u c h as the r a t e - d e t e r m i n i n g step. I n the present m e t h o d , h o w e v e r , a d d i t i o n a l c h e c k s besides c u r v e shape are a v a i l a b l e to establish the v a l i d i t y o f the r e s u l t s . A g r e e m e n t w i t h s p e c t r o p h o t o m e t r i c d a t a o b t a i n e d at l o w e r p H v a l u e s s u p p o r t s t h e c o n c l u s i o n t h a t t h e m e c h a n i s m at p H 7 i s t h e s a m e as t h a t at l o w e r p H v a l u e s . T h u s , t h e s p e c t r o e l e c t r o c h e m i c a l e x p e r i m e n t is n o t b e i n g u s e d t o d e t e r m i n e a m e c h a n i s m , b u t t o a l l o w m e a surements i n a shorter t i m e d o m a i n . T h e results s h o w n i n F i g u r e 2 d o not b y t h e m s e l v e s establish the m e c h a n i s m , b u t c o m b i n e d w i t h data from other m e t h o d s , i n d i c a t e that the o b s e r v e d rate constants are m e a n i n g f u l n u m b e r s r e f l e c t i n g rates o f r e a c t i o n . A d d i t i o n a l s u p p o r t f o r t h e v a l i d i t y o f t h e s p e c t r o e l e c t r o c h e m i c a l r e s u l t s is p r o v i d e d b y F i g ure 4. T h i s figure demonstrates the e x p e c t e d first-order d e p e n d e n c e o n


451


452


Figure 3. Observed rate constants vs. pH for citrate, phosphate, and acetate buffers (ionic strength = 0.2). Key: · , 0.025 F phosphate; O , 0.005 F citrate; and A, 0.2 F acetate.


19.

MAYAUSKY ET AL.

Chlorpromazine Cation Radical Reactions 4 5 3

b u f f e r ( n u c l e o p h i l e ) c o n c e n t r a t i o n a n d t h e l i n e a r i t y , at v e r y l o w t o t a l c o n c e n t r a t i o n , reflects the c a t a l y t i c n a t u r e o f the buffer. K i n e t i c results o b t a i n e d o v e r the w i d e p H range p e r m i t several conclusions about the active form o f the n u c l e o p h i l e i n v o l v e d i n the r e a c t i o n . W i t h a c e t a t e i o n , t h e l o g k v s . p H p l o t ( F i g u r e 3) s h o w s a b r e a k at t h e pK for a c e t i c a c i d a n d a p H i n d e p e n d e n t r e g i o n a b o v e this v a l u e . W h e n K K k is c a l c u l a t e d f r o m k b y d i v i d i n g b y free a c e t a t e c o n c e n t r a t i o n , t h e v a l u e is c o n s t a n t to 1 0 % o v e r t h e p H r a n g e 4 - 8 . T h e c o n s t a n t v a l u e o f KgK k c a l c u l a t e d for t h i s r a n g e i n d i c a t e s t h a t t h e p H effect o n k is c a u s e d s o l e l y b y c h a n g e s i n free a c e t a t e concentration, a n d that the entire d e c a y c a n b e e x p l a i n e d b y n u c l e o p h i l i c a t t a c k o f C P Z ' b y a c e t a t e i o n . E v e n at p H 7, r e a c t i o n s o f o t h e r n u c l e o p h i l e s , s u c h as w a t e r , h y d r o x i d e , or t h e C P Z s i d e c h a i n , are u n d e t e c t a b l e c o m p a r e d to t h e a c e t a t e r e a c t i o n . ohs

a

8

9

10

ohs

9

10

ohs


+

Q u a l i t a t i v e l y s i m i l a r b e h a v i o r w a s o b s e r v e d for p h o s p h a t e , w i t h t h e rate a b o v e p K b e i n g p H i n d e p e n d e n t . I n t h e p H r a n g e 8 - 9 , H P 0 ~ is c l e a r l y t h e a c t i v e f o r m o f t o t a l p h o s p h a t e a n d P 0 " p r o v i d e s n o o b s e r v a b l e c o n t r i b u t i o n . I n t h e p H r e g i o n 6 - 7 , the o b s e r v e d r a t e reflects t h e v a r i a t i o n i n H P 0 ~ c o n c e n t r a t i o n w i t h p H , a n d is c o n s i s tent w i t h Η Ρ 0 " attack o f C P Z + w i t h o u t p r o t o n loss. B e l o w p H 6 the rates are t o o fast to b e a c c o u n t e d for s o l e l y b y H P 0 " , so H P 0 m u s t c o n t r i b u t e t o t h e rate. T h e o b s e r v e d rate is t h e r e f o r e g o v e r n e d b y 2

3

4

4

2

4

2

4

2

4

2

4

+ *ΗΡΟ'-[ΗΡ0-] 2

fcobsCr = W o - [ H P 0 - ] 4

2

4

4

(13)

4

and

2

Kbs

= « 1 ^ H P 0 - + «2&ΗΡθ4 2

w h e r e C is t o t a l p h o s p h a t e

(14)

4

c o n c e n t r a t i o n ; a a n d a are f r a c t i o n a l

T

t

2

2

concentrations o f H P 0 ~ a n d H P 0 ~ , respectively; k 2

4

- is t h e v a l u e

H2P04

2

o f KsK k 9

4

i0

for attack b y H P 0 ~ ( w i t h o u t d e p r o t o n a t i o n ) ; a n d & H P O - is 2

the v a l u e o f K K k 8

9

10

4

4

for a t t a c k b y H P 0

2 4

" . The value of K po H

2

4

was

d e t e r m i n e d f r o m t h e p H i n d e p e n d e n t r e g i o n a b o v e p H 7, a n d K p o H 2

4

w a s c a l c u l a t e d f r o m t h e rates i n t h e p H r e g i o n 3 - 5 . F o r t h e e n t i r e p H r a n g e f r o m 3 to 8, E q u a t i o n 14 w a s v a l i d to w i t h i n 1 5 % , i n d i c a t i n g t h a t a n y r e a c t i o n s d u e to H P 0 3

4

or P 0

3 4

" are n e g l i g i b l e . T h e k i n e t i c d a t a

for t h e t w o p h o s p h a t e s p e c i e s are i n c l u d e d i n T a b l e I I . T h e d i s t i n c t i o n b e t w e e n a r a t e for H P 0 ~ a n d o n e for 2

HP0

4

s h o u l d b e c l a r i f i e d at t h i s p o i n t . T h e c a s e w h e r e H P 0 2

2 4

2 4

~

~ attacks w i t h

p r o t o n l o s s is k i n e t i c a l l y i n d i s t i n g u i s h a b l e f r o m a t t a c k b y H P 0

2 4

~ , be-



454 Table II.

D e r i v e d C o n s t a n t s for V a r i o u s N u c l e o p h i l i c S p e c i e s

Nucleophile

K K k g

CH3COOH

2

~0 2.3 x 7.4 x ~0 4.9 x 3.0 x 4.3 x

4

2

3

2

2


3

10 10 10 10 10

1 0

~0 2.5 ~0 9.7 3200 ~0 14 860 1200

—

CH3COOH3PO4 H P0 HPO4 H Cit H CitHCit Cit -

9

3

5

3

5

5

c a u s e R e a c t i o n 8 is i n e q u i l i b r i u m . F o r t h e s a k e o f c l a r i t y , t h e p r o c e s s w i l l b e r e f e r r e d to as a t t a c k b y H P O 4 " , a n d its r a t e c o n s t a n t is k 2-. H o w e v e r , w h e n H P 0 ~ a t t a c k s w i t h o u t p r o t o n loss ( a n a l o g o u s to t h e a c e t a t e r e a c t i o n ) t h e a p p r o p r i a t e c o n s t a n t is k - . Therefore, the v a l u e s o f K k i n T a b l e I I for H P O 4 " a n d H P 0 ~ r e p r e s e n t s i m i l a r r e a c tions that differ b y the a d d i t i o n a l p r o t o n on the p h o s p h a t e g r o u p , w h i c h is n o t l o s t d u r i n g R e a c t i o n 8. 2

H P 0 4

2

4

H

2

P

0

4

2

B

2

9

4

T h e p H d e p e n d e n c e o f the citrate reaction c a n b e a n a l y z e d i n a s i m i l a r f a s h i o n e x c e p t t h a t t h r e e d i s t i n c t c i t r a t e s p e c i e s c o n t r i b u t e d to t h e d e c a y rate. T h e o b s e r v e d rate is a c c u r a t e l y d e s c r i b e d b y E q u a t i o n 16 w h e r e a t e r m s Khs

=

OLlkuzCxt-

+

«2^HCit2-

+

« fc 3

C

I

T

3-

(16)

a n d k terms h a v e m e a n i n g s a n a l o g o u s t o the p h o s p h a t e case. T h e e n t i r e p H p r o f i l e c a n b e d e s c r i b e d b y t h i s e x p r e s s i o n to w i t h i n 1 0 % , a n d t h e v a l u e s o f rate c o n s t a n t s for p a r t i c u l a r s p e c i e s a r e g i v e n i n Table II. T h e v a l u e s o f d e r i v e d constants i n T a b l e II are d i r e c t i n d i c a t i o n s o f t h e o v e r a l l rates o f r e a c t i o n o f i n d i v i d u a l n u c l e o p h i l i c s p e c i e s after r e m o v a l o f t h e effects o f p H a n d c o n c e n t r a t i o n . S e v e r a l i m p o r t a n t o b s e r v a t i o n s c a n b e m a d e b y c o m p a r i n g t h e v a l u e s o f K%K ki for t h e s i x species e x a m i n e d . First, the m o n o a n i o n i c n u c l e o p h i l e s ( C H s C O O , H P 0 " , a n d H C i t ~ h a v e rates t h a t a r e c o m p a r a b l e , v a r y i n g b y o n l y a factor o f 5 d e s p i t e s i g n i f i c a n t c h a n g e s i n s t r u c t u r e . T h e r e f o r e , t h e l a r g e rate d i f f e r e n c e b e t w e e n c i t r a t e a n d a c e t a t e buffers is d u e to the more h i g h l y i o n i z e d forms o f citrate. S e c o n d , r e m o v a l o f a s e c o n d p r o t o n (as i n H P 0 " ) g r e a t l y a c c e l e r a t e s t h e r a t e , b y a factor o f 3 3 0 for p h o s p h a t e a n d 6 2 for c i t r a t e . T h i r d , r e m o v a l o f t h e t h i r d p r o t o n f r o m c i t r a t e c a u s e s a s m a l l i n c r e a s e i n rate o f a b o u t 4 0 % . F i n a l l y , t h e r a t e i n c r e a s e f r o m s i n g l y to d o u b l y i o n i z e d p h o s p h a t e a n d c i t r a t e s p e c i e s is d u e to t h e K k t e r m , a n d t h e r e f o r e r e s u l t s f r o m c h a n g e s i n t h efirstt w o reactions o f the three-step process. 9

9

-

2

4

2

2

4

8

9


19.

MAYAUSKY ET AL.

Chlorpromazine Cation Radical Reactions 3

Because the rate difference between C i t " a n d H C i t

2 -

455

is fairly

small, the rate acceleration with deprotonation probably is not simply an electrostatic effect. R e m o v a l o f protons may aid the oxidation of the adduct, w h i c h increases the value of k , or the greater negative charge 9

may influence other steps i n the mechanism. H o w e v e r , the small dif ference i n rate between C i t

3 -

2

a n d H C i t " indicates that the influence of

electrostatic charge is minor a n d u n l i k e l y to account for the large dif ference between the singly a n d d o u b l y ionized nucleophiles. A more l i k e l y explanation for the rate difference between singly and d o u b l y deprotonated nucleophiles can be p r o v i d e d after consider


ing the intermediate formed b y Reaction 8. For H P 0

2 4

2

~ and H C i t " ,

this intermediate possesses a second n u c l e o p h i l i c site. W h e n a second +

molecule o f C P Z ,

necessary as an o x i d i z i n g agent i n Reaction 9,

approaches, it can b o n d to the intermediate through the available site. T h e proximity of the two radicals can then accelerate the charge trans fer of Reaction 9. W h e n the intermediate contains Η Ρ θ 4 ~ or H C i t ~ , 2

2

3

no free site is available and no facilitation can occur. T h e C i t " species provides two extra sites when only one is necessary, resulting i n only a 2

modest rate enhancement over that for H C i t " . Another possibility for the rate difference between singly a n d t

d o u b l y i o n i z e d nucleophiles is variation i n k /kl0

9

w i t h p H . Previous

work (15) showed this value to be constant w i t h p H i n a l i m i t e d range in the region of p H 2 - 3 . W i t h the w i d e range of p H e m p l o y e d i n the present work, proton loss may occur i n Reaction 9, m a k i n g the ratio k /kw

9

(and therefore X) p H dependent. T h i s possibility is presently

b e i n g investigated with stop-flow techniques. In conclusion, the kinetics of the decay of chlorpromazine cation radical d e p e n d on p H i n a fairly complex fashion over the p H range from 3 to 8. E a c h buffer component has a different rate of reaction with +

C P Z , a n d the variation in overall rate with p H results from changes in the distribution o f buffer species. W h e n the n u c l e o p h i l e has two n u c l e o p h i l i c sites, the rate of reaction is greatly enhanced over the case where only one site is available.

Acknowledgments T h i s work was supported b y grants from N I M H (28412) a n d N S F ( C H E - 7 8 2 8 0 6 8 ) . T h e authors thank Robert E n g e l b a c h for preliminary work on the kinetic simulations.

Literature Cited 1. Bodea, C . ; Silbert, I. Adv. in Heterocycl. Chem. 1968, 9, 321. 2. Forrest, I.; Usdin, E . "Psycho Therapeutic Drugs;" Dekker: New York, 1977. 3. Usdin, E . ; Eckert, H . ; Forrest, I. Devel. in Neuroscience 1980, 7.



456


4. Piette, L. H.; Bulow, G.; Yamazaki, F. Biochim. Biophys. Acta 1964, 88, 120. 5. Merkle, F. H.; Discher, C. A.J.Pharm. Sci. 1964, 53, 620. 6. Forrest, I. S.; Green, D. E.J. Forensic Sci. 1972, 17, 592. 7. Forrest and Usdin, op. cit. pp. 709-719. 8. Forrest, I. S.; Forrest, F.; Berger, M. Biochem. Biophys. Acta 1958, 29, 441. 9. Akera, T.; Brody, T. Biochem. Pharmacol. 1972, 21, 1403. 10. Ohnishi, S.; McConnell,H.J.Am. Chem. Soc. 1965, 87, 2243. 11. Akera, T.; Kee, C. Y.; Brody, T. Biochem. Pharm. 1976, 25, 1751. 12. Godey, G. M.; Keyser, H.; Setchell, F. Nature (London) 1969, 223, 80. 13. Levy, L.; Tozer, T.; Tuck, D.; Loveland, D. J. Med. Chem. 1972, 15, 989. 14. Evans, J. F.; Lenhard, J. R.; Blount, H . N.J. Org. Chem. 1977, 42, 983. 15. Cheng, H. Y.; Sackett, P. H.; McCreery, R.L.J.Am. Chem. Soc. 1978, 100, 962. 16. Cheng, H. Y.; Sackett, P. H.; McCreery, R.L.J.Med. Chem. 1978, 21, 948. 17. Sackett, P. H.; Mayausky, J. S.; Smith, T. M.; Kalus, S.; McCreery, R. L., J. Med. Chem. 1981, 24, 1342. 18. Sackett, P. H.; McCreery, R.L.J.Med. Chem. 1979, 22, 1447. 19. Evans, J. F.; Blount, H. N.J.Am. Chem. Soc. 1978, 100, 4191. 20. Blount, H. N.J. Electroanal. Chem. 1973, 42, 271. 21. Skully, J.; McCreery, R. L. Anal. Chem. 1980, 52, 1885. RECEIVED for review June 2, 1981. ACCEPTED August 24, 1981.


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