6
Downloaded via MACQUARIE UNIV on February 13, 2019 at 10:16:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Bioorganotin Chemistry: Stereo- and Situselectivity in the Monooxygenase Enzyme Reactions of Cyclohexyltin Compounds RICHARD H. FISH, JOHN E. CASIDA, and ELLA C. KIMMEL Pesticide Chemistry and Toxicology Laboratory, College of Natural Resources, Wellman Hall, University of California, Berkeley, CA 94720 The in vitro reactions of organotin compounds with mono oxygenase enzymes utilizing rat liver microsomes were previously studied with tributyltin derivatives (1a,b). The results from that study confirmed carbon-hydroxylation as the primary bio chemical reaction occurring with these compounds. Furthermore, the tin-carbon sigma electrons were implicated in the possible stabilization of carbon free radicals generated on the α and β carbon atoms to the tin atom. Additionally, by using several criteria, we established that the metabolism of these tributyltin derivatives involved the interesting and biologically important cytochrome P-450 dependent monooxygenase enzyme system (1a). Recent studies on this system have concluded that a l l the available evidence points to a heme-iron-monooxygen complex which converts carbon-hydrogen bonds to carbon-hydroxyl bonds (2), Figure 1. The reaction has been shown to be highly stereospecific 3+ 3+ Fe
+
RH
- R H
>**' .RH
3+
2+ 2+
Fe " 1
[+o
- R O H
+ Fe:= Q 1 i
RH
2
2+ Fe0
+2H+ -H 0
_RH
2
2
RH
Figure 1. Mechanism of Cytochrome P-450 en zyme hydroxyhtion reaction
(3); however, surprisingly few investigations have been concerned with the cyclohexyl ring system (4). Our work i n this area was logically extended to cyclohexyltin compounds for several reasons. F i r s t l y , these compounds are used as agricultural miticides on 0-8412-0461-6/78/47-082-082$05.00/0 © 1978 American Chemical Society Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
FISH E T A L .
Bioorganotin Chemistry
83
food crops and a study o f t h e i r monooxygenase enzyme r e a c t i o n s would be important f o r b i o l o g i c a l and t o x i c o l o g i c a l reasons. Secondly, we wanted t o a s c e r t a i n the s t e r e o as w e l l as s i t u s e l e c t i v i t i e s o f these r e a c t i o n s w i t h a p p r o p r i a t e c y c l o h e x y l t i n model compounds, s i n c e t h i s aspect, as f a r as we c o u l d e s t a b l i s h w i t h t h e cytochrome P-U50 monooxygenase enzyme system, has n o t been e l u c i d a t e d i n a d e f i n i t i v e manner. We decided i n i t i a l l y (j?) t o study c y c l o h e x y l t r i p h e n y l t i n , ^ because we found i n an e a r l i e r i n v e s t i g a t i o n ( l b ) t h a t the t r i p h e n y l t i n d e r i v a t i v e s were not h y d r o x y l a t e d under i n v i t r o r e a c t i o n c o n d i t i o n s . More i m p o r t a n t l y , the s y n t h e s i s o f potent i a l m e t a b o l i t e s , which w i t h the c y c l o h e x y l system i n v o l v e s t h e c i s - and t r a n s - h y d r o x y c y c l o h e x y l t i n isomers i n the 2,3> and *Jp o s i t i o n s ( t r i p h e n y l t i n being p o s i t i o n l ) , would be more conveni e n t u s i n g the t r i p h e n y l t i n group as a s y n t h e t i c handle (£.). A d d i t i o n a l l y , the e x t e n s i o n t o c y c l o h e x y l d i p h e n y l t i n a c e t a t e , J^, would o n l y i n v o l v e e l e c t r o p h i l i c cleavage o f a phenyl group i n order t o prepare p o t e n t i a l m e t a b o l i t e s f o r t h i s model substrate ( 7 ) . A d i s c u s s i o n o f the s t e r e o and s i t u s e l e c t i v i t y i n v o l v e d i n the P-lj-50 monooxygenase r e a c t i o n s o f 1 and and the consequence o f t h e i r conformation a t the a c t i v e sTte w i l l be presented. Results The p r e p a r a t i o n o f [ l - ^ C ] c y c l o h e x y l t r i p h e n y l t i n , j ^ , was r e a d i l y accomplished by the r e a c t i o n o f [ l - ^ l e y c l o h e x y l magnesium bromide w i t h t r i p h e n y l t i n c h l o r i d e i n 3 3 $ y i e l d w i t h a s p e c i f i c a c t i v i t y o f 1 . 2 6 mCi/mmole (Eq l ) . The use o f [ C ] 1 4
h /
-
=
y f ^ B r
h»
1) Mg/THF 2>(©)snCI
/
^
- ^ 7 ^ S n ( ^
(1) 1
l a b e l l e d o r g a n o t i n s u b s t r a t e s i s mandatory i n these metabolism s t u d i e s , s i n c e the amount of metabolism i s g e n e r a l l y t o t h e extent o f 10% o f t h e s t a r t i n g r a d i o l a b e l l e d s u b s t r a t e . Thus, compound 1 ( 0 . 0 5 pmole) was incubated w i t h our source o f c y t o chrome P - Ç 5 0 , r a t l i v e r microsomes ( l a ) ( 1 0 . 6 mg p r o t e i n ) , f o r 1 h r a t 3 7 ° i n 2 . 0 ml phosphate b u f f e r (pH 7·*0 c o n t a i n i n g ΝΑΌΡΗ (2 Mmole) the e s s e n t i a l c o f a c t o r . A f t e r chloroform e x t r a c t i o n , we u t i l i z e d t h i n l a y e r chromatography (TLC) t o separate the m e t a b o l i t e s and l i q u i d s c i n t i l l a t i o n counting t o q u a n t i f y them. They were i d e n t i f i e d by a combination o f TLC cochromatography, p r e p a r a t i v e TLC i n c o n j u n c t i o n w i t h 360 MHz % FT nmr s p e c t r o scopy and by s p e c i f i c degradation r e a c t i o n s (Eq 2 ) . The
Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
84
ORGANOMETALS
•Sn(®)
pH 7.4,
Ih
A N D ORGANOMETALLOIDS
3
H
I
g (85.6%)
(2) 3 (6.5%)
4 (3.0%)
•Sn(®)
6 (1.9%)
3
+
5 (1.6%)
/ ^ ^ S n ( © )
3
7 (1.4%)
percentages i n Eq 2 represent normalized values o f i d e n t i f i e d m e t a b o l i t e s and account f o r 8 $ o f s t a r t i n g s u b s t r a t e , ^ The remainder was 1 ( 7 0 $ ) and u n i d e n t i f i e d m a t e r i a l s ( 2 2 $ ) . One important aspect of t h i s type o f work i s the a b i l i t y t o synthesize p o t e n t i a l m e t a b o l i t e s and t o understand t h e i r subsequent chemistry. This f a c i l i t a t e s t h e i r u l t i m a t e i d e n t i f i c a t i o n and allows t h e use o f cochromatography as one c r i t e r i o n f o r t h i s purpose. A l l the m e t a b o l i t e s , 2,-£, were separated from one another u s i n g n e u t r a l TLC s o l v e n t systems (5) and then cochromatographed w i t h s y n t h e t i c standards ( 6 j j ) . Furthermore, compound 2 was p u r i f i e d by p r e p a r a t i v e TLC and a 360 MHz % FT nmr spectrum was obtained ( 1 3 , 6 0 0 acquisitions,CDC1-> TMS) conf i r m i n g t h a t the major m e t a b o l i t e ( 8 5 . 6 $ ) was trans-4-hydroxyc y c l o h e x y l t r i p h e n y l t i n , 2 . The nmr spectrum showed t h e a x i a l methine proton on the carbon (ck) bearing the h y d r o x y l group as a 9 u n e m u l t i p l e t ( 3 . 5 8 ppm, J - J = 11.1 Hz; J - J = h.O Hz) c o n s i s t e n t w i t h a spectrum or the a u t h e n t i c compound 2 ( 6 , 7 ) · We were a b l e t o detect the corresponding c i s isomer o f ^15y t h i s nmr technique; however, none was observed i n the nmr spectrum of metabolite 2 . The c i l f - and trans-3-hydroxy m e t a b o l i t e s , ^ and j ^ , were i d e n t i f i e d o n l y by TLC cochromatography w i t h a u t h e n t i c compounds, because o f the low amounts produced i n the b i o l o g i c a l o x i d a t i o n r e a c t i o n . I n t h i s regard, we are c o n f i d e n t o f t h e i r assigned s t r u c t u r e s , s i n c e both isomers are r e a d i l y separable by t h i s technique. M e t a b o l i t e 5 was i d e n t i f i e d by TLC cochromatography and by a s p e c i f i c degradation r e a c t i o n . The m e t a b o l i t e mixture was a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d and TLC a n a l y s i s showed the disappearance o f m e t a b o l i t e 5 . Experiments w i t h a u t h e n t i c ^ r e v e a l e d t h a t t h i s trans-2-Kydroxy compound undergoes a f a c i l e 1 , 2 - d e o x y s t a n n y l a t i o n r e a c t i o n g i v i n g cyclohexene, t r i p h e n y l t i n a c e t a t e and water. In agreement w i t h t h i s , we assume t h a t m e t a b o l i t e 5 r e a c t e d s i m i l a r l y under a c i d i c c o n d i t i o n s (Eq 3 ) · a
x
a x
e q
Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
FISH E T A L .
Bioorganotin Chemistry
85
The t r a n s m e t a b o l i t e , but not t h e corresponding c i s isomer o f 5, can form eycloiiexene, s i n c e a t r a n s d i a x i a l con formation ( I ) i s needed f o r r e a c t i o n t o take p l a c e . The
(+OH
2
I
corresponding c i s-methyl e t h e r , a model f o r t h e c i s - a l c o h o l we were not able t o s y n t h e s i z e , d i d not r e a c t even a f t e r t h r e e days w i t h g l a c i a l a c e t i c a c i d (Eq k).
^ H
CH
3
S n
(@),
HOAc Ίί^
hs.
3days
Η
_ ( @ ) SnOAc + CH OH /
+
Λ
3
(4)
Η
These r e s u l t s a r e a l s o c o n s i s t e n t w i t h those found w i t h t h e corresponding c i s - and t r a i i s - 2 - h y d r o x y c y c l o h e x y l t r i m e t h y l s i l i c o n d e r i v a t i v e s under weakly a c i d c o n d i t i o n s ( 8 ) . A c c o r d i n g l y , any cis-2-hydroxy m e t a b o l i t e t h a t might form would have been detected and was not. We a l s o found t h a t t h e ketones and J£ from t h e alcohols and ^ were produced, and v e r i f i e d t h i s r e s u l t by TIC cochromatography, but again, we a r e c o n f i d e n t o f t h e i r assigned s t r u c t u r e s u s i n g t h i s technique. Compound j ^ , 1 - h y d r o x y c y c l o h e x y l t r i p h e n y l t i n , a m e t a b o l i t e t h a t might a l s o be formed, c o u l d not be detected because o f i t s probable low c o n c e n t r a t i o n and experimental d i f f i c u l t i e s a s s o c i ated w i t h q u a n t i f y i n g i t . We found t h a t 1 - h y d r o x y a l k y l t i n d e r i v a t i v e s undergo e l e c t r o p h i l i c cleavage r e a c t i o n s o f t h e t i n carbon bond b e a r i n g t h e h y d r o x y l group w i t h h y d r o c h l o r i c a c i d t o g i v e t h e a l c o h o l and t h e corresponding d e a l k y l a t e d t i n d e r i v a t i v e ( l a ) . Consequently, r e a c t i o n o f j3 i f present w i t h hydro c h l o r i c a c i d would provide [ l - M2 ]cycïohexanol and t r i p h e n y l t i n c h l o r i d e (Eq 5)· I n c o n t r o l experiments w i t h [ l - ^ C l c y c l o h e x a nol,determined as i t s phenylcarbamate d e r i v a t i v e , we c o u l d detect l e v e l s o f t h i s d e r i v a t i v e down t o 2$ but not lower. S i m i l a r experiments w i t h t h e r e a c t i o n mixture gave no d e t e c t a b l e phenylcarbamate o f [ l - ^ C Jcyclohexanol and thus we r a t i o n a l i z e d 1
Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
ORGANOMETALS
86
£ = J - H m
3
JSL
+
M
AND
ORGANOMETALLOIDS
S N C (
3
8 |©-N=C=0
(5)
t h a t < 2 $ o r none o f 8 was formed. The study o f [ l - J ^ C ] c y c l o h e x y l d i p h e n y l t i n a c e t a t e , in those monooxygenase enzyme r e a c t i o n s was accomplished by conv e r t i n g compounds J^-J^, t o t h e i r corresponding c y c l o h e x y l d i p h e n y l t i n bromides. For example, ^ r e a c t e d w i t h bromine i n i s o propanol/chloroform t o g i v e an e x c e l l e n t y i e l d o f [ l - l ^ C ] c y c l o h e x y l d l p h e n y l t i n bromide (Eq 6 ) . P r e p a r a t i v e TLC u s i n g d i i s o -
p r o p y l e t h e r / a c e t i c a c i d (^9:1) converted t h e bromide t o t h e acetate, as analyzed by 90 MHz hi nmr spectroscopy. Reaction o f £ w i t t i * r a t l i v e r microsomes, as w i t h 1 , gave t h e f o l l o w i n g r e s u l t s a f t e r a c i d i f i c a t i o n o f the r e a c t i o n mixture and e x t r a c t i o n w i t h chloroform (Eq 7 ) . The percentages i n Eq 7 represent
(7)
normalized v a l u e s o f i d e n t i f i e d m e t a b o l i t e s which account f o r 1 0 $ o f s t a r t i n g compound, 9 . The remaining m a t e r i a l s were (82$) as w e l l as 8 $ u n i d e n t i f i e d compounds.
Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
6.
FISH E T A L .
Bioorganotin Chemistry
87
The corresponding ketones from and although a v a i l a b l e , were not p o s i t i v e l y i d e n t i f i e d as m e t a b o l i t e s because o f i n t e r f e r i n g compounds and i n c o n s i s t e n t cochromatography r e s u l t s , r e s p e c t i v e l y , upon TLC a n a l y s i s . We a l s o analyzed f o r [ l - ^ ! ] c y c l o h e x a n o l , as p r e v i o u s l y d e s c r i b e d f o r 1-hydroxycyclohexylt r i p h e n y l t i n , j ^ , but were not a b l e t o detect t h i s compound as i t s phenylcarbamate due t o experimental d i f f i c u l t i e s . Thus, l - h y d r o x y c y c l o h e x y l d i i ) h e n y l t i n acetate was not determined u s i n g t h i s method. Compounds and J £ were i d e n t i f i e d by TLC cochromatography, but u n f o r t u n a t e l y , s e p a r a t i o n o f both the c i s - and t r a n s - 3 or -U-hydroxyl isomers was not s u c c e s s f u l by TLC and t h e r e f o r e no assignments c o u l d be made. The t r a n s stereochemistry o f was a s c e r t a i n e d , as w i t h J^, by r e a c t i o n w i t h g l a c i a l a c e t i c a c i d . Since the TLC a n a l y s i s o f compounds ^ j ) - ^ was performed i n a c i d i c s o l v e n t s , m e t a b o l i t e 10 c o u l d not be d i r e c t l y analyzed as was done w i t h m e t a b o l i t e 5· tïônsequently, t r a p p i n g o f the [ l - ^ C l c y c l o h e x e n e was e s s e n t i a l * * f o r q u a n t i f i c a t i o n o f JJjh T h i s was accomplished by r e a c t i o n o f the [ l - ^ C J c y c l o h e x e n e w i t h mercuric a c e t a t e i n methanol t o g i v e the oxymercuration product, 3^, which c o u l d be r eery s t a l i i zed t o constant s p e c i f i c a c t i v i t y and q u a n t i f i e d (Eq 8 ) . 1
13 Discussion Cytochrome P-^50 monooxygenase enzyme r e a c t i o n s have been w i d e l y s t u d i e d ; however, the use o f monosubstituted c y c l o h e x y l d e r i v a t i v e s as s u b s t r a t e s has r e c e i v e d o n l y l i m i t e d a t t e n t i o n . One such i n v i t r o study (k) concerned methylcyclohexane, ijjb and i t was shown t h a t P-^50 enzyme h y d r o x y l a t i o n gave the
14 f o l l o w i n g s i t u s e l e c t i v i t y on a per hydrogen b a s i s , i . e . , C^ : C : C : C o f 5 : 5 : 1 : 1 1 . No stereochemical l
Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
ORGANOMETALS
88
A N D ORGANOMETALLOIDS
assignments were made, u n f o r t u n a t e l y , t h u s , t h i s important aspect cannot be compared t o our r e s u l t s . In c o n t r a s t t o the observed s i t u s e l e c t i v i t y f o r j ^ , compound ] ^ presents a d r a m a t i c a l l y d i f f e r e n t r e s u l t . Thus, t h e s i t u s e l e c t i v i t y f o r 1 on a per hydrogen b a s i s f o r : C-d : C£ : C± i s 1 0 9 : 7 : 1 Τ 0 . T h i s s t r i k i n g s i t u s e l e c t i v i t y f o r 1 i s a l s o complemented by a h i g h degree o f s t e r e o s e l e c t i v i t y f o r predominantly e q u a t o r i a l h y d r o x y l a t e d m e t a b o l i t e s , w i t h compounds 2> ^ and representing 95$ o f the m e t a b o l i t e s formed and g i v i n g an e q u a t o r i a l / a x i a l r a t i o o f 59· While s t e r i c e f f e c t s c o u l d be invoked t o e x p l a i n the s i t u s e l e c t i v i t y d i f f e r e n c e s between ] ^ an
