- Email: [email protected]
The relationship between adrenergic neurone blocking activity and local anaesthetic activity in a series of guanidine derivatives
EUROPEAN JOURNAL OF PHARMACOLOGY 1 (1967) 200-209. NORTH-HOLLAND PUBL. COMP., AMSTERDAM
THE RELATIONSHIP BETWEEN ADRENERGIC NEURONE BLOCKING ACTIVITY AND LOCAL ANAESTHETIC ACTIVITY IN A SERIES OF GUANIDINE DERIVATIVES M. J. RAND * a n d J. W I L S O N * Department of Pharmacology, School of Pharmacy, University of London, London, U.K. Accepted 15 F e b r u a r y 1967
M . J . R A N D and J. WILSON, The relationship between adrenergic neurone blocking activity and local anaesthetic activity in a series of guanidine derivatives, European J. P h a r m a c o l . 1 (1967) 200-209. The a d r e n e r g i c neurone blocking potencies of a n u m b e r of guanidine d e r i v a t i v e s were a s s e s s e d using t h r e e isolated sympathetically innervated organs: rabbit ileum, guinea pig vas deferens and the a r t e r y segment from the r a b b i t ' s ear. The most suitable of these p r e p a r a t i o n s for making quantitative comp a r i s o n s was the isolated a r t e r y segment, and the r e s u l t s so obtained were used as the basis for a study of s t r u c t u r e - a c t i v i t y relationships. Some of these compounds were also tested for local a n a e s thetic activity using the guinea pig weal and mouse tail methods. It was found that the local anaesthetic activity of these compounds was unrelated to t h e i r a d r e n e r g i c neurone blocking activity. Adrenergic neurone blocking drug Guanidine d e r i v a t i v e s Guanethidine
1. I N T R O D U C T I O N Since bretylium and guanethidine were found to b e e f f e c t i v e in t h e t r e a t m e n t of h y p e r t e n s i o n ( B o u r n e t al., 1959; P a g e a n d D u s t a n , 1959), a l a r g e n u m b e r of a d r e n e r g i c n e u r o n e b l o c k i n g drugs has been synthesized. Structure-activity r e l a t i o n s h i p s i n g r o u p s of t h e s e c o m p o u n d s h a v e b e e n s t u d i e d b y S c h l i t t l e r et al. (1962); S h o r t e t al. (1963) a n d C o p p (1964). T h e s e s t u d i e s u s e d , as a criterion of a c t i v i t y of t h e c o m p o u n d s , either their antihypertensive action (Schlittler et al., 1962), o r r e l a x a t i o n of t h e n i c t i t a t i n g m e m b r a n e in t h e c o n s c i o u s c a t ( S h o r t e t al., 1963; Copp, 1964). T h e u s e of s u c h p h a r m a c o l o g i c a l tests in studying structure-activity relationships i s o p e n to t h e o b j e c t i o n t h a t t h e d r u g s a r e a c t i n g on c o m p l e x s y s t e m s , w h e r e n u m e r o u s f a c t o r s i n a d d i t i o n to a d r e n e r g i c n e u r o n e b l o c k a d e m a y * P r e s e n t a d d r e s s : Department of Pharmacology, Univ e r s i t y of Melbourne, Parkville, N. 2., Victoria, Australia.
Local anaesthetic Procaine Structure-activity relationships
play a part. The isolated arterial segment from t h e r a b b i t e a r (De l a L a n d e a n d R a n d , 1965) i s a c o n v e n i e n t a n d s i m p l e p r e p a r a t i o n to u s e i n t h e determination of a d r e n e r g i c n e u r o n e b l o c k i n g p o t e n c y . In t h e e x p e r i m e n t s described here it w a s u s e d to i n v e s t i g a t e s t r u c t u r e - a c t i v i t y relat i o n s h i p s in a s e r i e s of g u a n i d i n e d e r i v a t i v e s . The mechanism by which adrenergic neurone b l o c k i n g d r u g s p r e v e n t t h e r e l e a s e of n o r a d r e n a l ine at the sympathetic nerve endings is unk n o w n . B o u r a e t al. (1960) r e p o r t e d t h a t f o l l o w i n g a s u b c u t a n e o u s i n j e c t i o n of b r e t y l i u m i n t h e c a t , l a r g e c o n c e n t r a t i o n s of t h e d r u g w e r e f o u n d in the sympathetic ganglia and postganglionic n e r v e s , w h i l e o n l y s m a l l a m o u n t s w e r e f o u n d in other nervous structures. They suggested that t h e c o n c e n t r a t i o n s of b r e t y l i u m w h i c h a c c u m u lated in the sympathetic nerve endings were s i m i l a r to t h e c o n c e n t r a t i o n s r e q u i r e d to b l o c k c o n d u c t i o n w h e n a p p l i e d l o c a l l y to n e r v e t r u n k s . T h e a d r e n e r g i c n e u r o n e b l o c k i n g a c t i o n of b r e tylium could therefore be explained by its local a n a e s t h e t i c a c t i o n c a u s i n g a b l o c k a d e of c o n d u c -
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA tion in the nerve terminals. However, the experiments described here demonstrated that the loc a l a n a e s t h e t i c a c t i v i t y of t h e g u a n i d i n e c o m p o u n d s w a s u n r e l a t e d to t h e i r e f f e c t i v e n e s s a s adrenergic neurone blocking drugs. 2. M E T H O D S . E X P E R I M E N T S ORGANS
USING I S O L A T E D
2.1. Rabbit ear artery T h e p r e p a r a t i o n d e s c r i b e d b y De l a L a n d e a n d R a n d (1965) w a s u s e d . R a b b i t s w e i g h i n g 2 - 3 k g w e r e k i l l e d b y a b l o w on t h e b a c k of t h e n e c k . T h e s k i n w a s r e m o v e d f r o m t h e e a r in t h e r e g i o n of t h e c e n t r a l a r t e r y a n d t h e a r t e r y w a s d i s s e c t ed free from the surrounding tissues. The artery was cannulated with fine polythene tubing and a l e n g t h of 3 - 4 c m w a s e x c i s e d . T h e a r t e r y s e g m e n t w a s t h e n p e r f u s e d w i t h M c E w e n ' s (1956) s o l u t i o n a t 3 7 o c a n d t h e r e s e r v o i r of M c E w e n ' s s o l u t i o n w a s g a s s e d w i t h 5% c a r b o n d i o x i d e i n o x y g e n . T h e flow of t h e p e r f u s i o n f l u i d w a s maintained with a constant volume roller pump a t a r a t e of 5 m l / m i n . T h e p e r f u s i o n p r e s s u r e was measured with a mercury manometer. The periarterial sympathetic nerves were stimulated b y m e a n s of b i p o l a r p l a t i n u m r i n g e l e c t r o d e s p l a c e d on t h e u p p e r e n d of t h e v e s s e l . S t i m u l a t i o n w a s a t a f r e q u e n c y of 10 p u l s e s / s e c f o r 1015 s e c e v e r y 4 r a i n , u s i n g 1 m s e c p u l s e s a n d s u pramaximal v o l t a g e . I n j e c t i o n s of d r u g s w e r e made into the perfusion fluid just before it reached the cannula. Vasoconstrictor responses were recorded as rises in perfusion pressure. B l o c k i n g d r u g s w e r e i n f u s e d b y m e a n s of a Palmer slow injection apparatus and their conc e n t r a t i o n s a r e e x p r e s s e d a s g g / m l of t h e p e r f u s i o n fluid. A l l i n j e c t i o n s a n d i n f u s i o n s of d r u g s w e r e m a d e up in M c E w e n ' s s o l u t i o n . I n j e c t i o n s h a d a m a x i m u m v o l u m e of 0.2 m l a n d i n f u s i o n s w e r e m a d e a t t h e r a t e of 0.1 to 0.2 m l / m i n . 2.2. Rabbil ileum S e g m e n t s of r a b b i t i l e u m w i t h t h e i r s y m p a t h e t i c n e r v e s w e r e p r e p a r e d b y t h e m e t h o d of F i n k l e m a n (1930). A l e n g t h of i l e u m 2 - 3 c m l o n g w a s s e t up i n a 50 m l b a t h c o n t a i n i n g M c E w e n ' s s o l u t i o n a t 3 7 o c , a n d m o v e m e n t s of t h e i l e u m were recorded with an isotonic, frontal-writing lever. The periarterial nerves were stimulated w i t h b i p o l a r p l a t i n u m e l e c t r o d e s a t 20 p u l s e s / s e c using 2 msec pulses at supramaximal voltage for 20-30 sec every 4 min.
T h e v a s d e f e r e n s w a s s e t up in a 50 m l b a t h c o n t a i n i n g M c E w e n ' s (1956) s o l u t i o n g a s s e d w i t h 5% carbon dioxide in oxygen at 32oc. The hypogastric nerve was stimulated with bipolar platinum e l e c t r o d e s f o r 10 s e c e v e r y 2 - 4 r a i n at 1 0 - 2 0 pulses/sec using 2 msec pulses at supramaximal voltage. 2.4. T e s t s f o r local anaesthetic activity Local anaesthetic activity was investigated u s i n g t h e m e t h o d s of B i i l b r i n g a n d W a j d a (1945) a n d B i a n c h i (1956). In t h e m e t h o d of B t i l b r i n g a n d W a j d a t h e l o s s of s e n s a t i o n i n t h e s k i n of t h e g u i n e a p i g f o l l o w i n g a n i n t r a d e r m a l i n j e c t i o n of a l o c a l a n a e s t h e t i c d r u g i s a s s e s s e d . T h e f l a n k s of t h e g u i n e a p i g s w e r e d e p i l a t e d on t h e day b e f o r e t h e t e s t s w e r e c a r r i e d out. T h e d r u g s w e r e d i s s o l v e d i n n o r m a l s a l i n e a n d a v o l u m e of 0.25 m l i n j e c t e d . T w o i n j e c t i o n s of e a c h of two c o n c e n t r a t i o n s of t h e d r u g a n d two i n j e c t i o n s of n o r m a l s a l i n e w e r e g i v e n to e a c h g u i n e a pig. T h r e e guinea pigs were used for each test and the solut i o n s w e r e i n j e c t e d in r a n d o m o r d e r . Light p r i c k i n g of p a r t of t h e f l a n k o u t s i d e t h e a n a e s Table 1 Concentrations of some a d r e n e r g i c neurone blocking drugs which reduced the r e s p o n s e s of the isolated r a b bit ileum and guinea pig vas deferens to sympathetic stimulation by 50% in 10 min.
//NH NH-R-NH-C \NH 2 Equiactive concentrations Drug
EM310
-R-
-(CH2) 2-
Rabbit ileum
Guinea pig vas deferens
10-20 /£g/ml
5-10 /Xg/ml
1- 2 /~g/ml
2- 5 # g / m l
25 # g / m l
10-20 //g/ml
100 /xg/ml
15-20 /2g/ml
20 # g / m l
5-10 /xg / m l
100 g g / m t
25-50 /xg / m l
1- 2 # g / m l
5-10 /ag/ml
CH3
EM311
J
-CH-CH 2CH3
J
EM 313
-CH-CH 2
l
CH3
(~2H5 EM336
-CH-CH 2 EM 257
CH 3
I
-CH2-CHEM 97
2.3. Guinea p i g vas deferens T h e p r e p a r a t i o n of H u k o v i ~ (1961) w a s u s e d .
201
Guanethidine
-(ell2) 3-
202
M. J. RAND and J. WILSON
thetized areas produced a characteristic cont r a c t i o n of t h e s k i n . T h e d e g r e e of a n a e s t h e s i a p r o d u c e d by e a c h c o n c e n t r a t i o n of d r u g w a s a s s e s s e d b y n o t i n g t h e n u m b e r of t i m e s t h e a n i m a l s f a i l e d to r e s p o n d o u t of a t o t a l of 36 t e s t s . Bianchi's method uses the response of a m o u s e to a n a r t e r y c l i p a t t a c h e d to t h e t a i l . G r o u p s of t e n m i c e w e r e u s e d f o r e a c h t e s t . T h e d r u g s w e r e m a d e up i n n o r m a l s a l i n e c o n t a i n i n g 10 t t g / m l of a d r e n a l i n e . A v o l u m e of 0.1 m l of the solution was injected intradermally 1.5-2 cm f r o m t h e b a s e of t h e t a i l of e a c h m o u s e . T h e c l i p w a s a t t a c h e d d i s t a l to t h e s i t e of i n j e c t i o n a t 15 m i n i n t e r v a l s a n d t h e d e g r e e of a n a e s t h e s i a w a s Table 2 Equiactive concentrations of a n u m b e r of g'uanidine derivatives. The concentrations shown reduced the r e s p o n s e s of isolated a r t e r i a l segments to sympathetic stimulation by 50% in about 20 min.
@ Drug
NH_Ra_NH.C~ NH \NHR b
-R a-
-R b
Concentration producing 50% blockade in 20 rain
-H
2 #g/ml
EM 311
-H
1 #g/ml
-H
1- 2 # g / m l
-H
4 ~g/ml
-CH-CH 2CH 3
I
-CH2-CHCIt 3 EM 313 - ? - C H 2-
t a k e n a s t h e p r o p o r t i o n of m i c e f a i l i n g to a t t e m p t to r e m o v e t h e clip.
2.5. Drugs used
EM 31( -CH2-CH 2-
EM 257
Fig. 1. Isolated rabbit ileum. Effect of EM 311 (2 / l g / ml) on the r e s p o n s e s to stimulation of the l u m b a r sympathetic n e r v e s (at the dots) for 30 see in every 4 min, using 2 msee pulses at a frequency of 2 0 / s e e and sup r a m a x i m a l voltage, and to 0.1 / l g / m l noradrenaline (Nor).
T h e new d r u g s w h o s e a c t i v i t y i s r e p o r t e d in t h i s p a p e r a r e cyclohexylamino-alkyl-guanidines ( E M 9 7 , 257, 310, 311, 313, 326 a n d 3 3 6 ) a n d bis-alkyl-aminoethylguanidines ( E M 65 a n d 66); t h e i r f o r m u l a e a r e s h o w n in t a b l e s 1 a n d 2. T h e s e c o m p o u n d s w e r e p r e p a r e d by D r . W. J. C. Dyke at the Evans Medical Research Laboratories, Liverpool. They were supplied as sulphates. Other drugs used were bretylium tosylate, guanethidine sulphate, guanoxan sulphate, bethanidine sulphate, procaine hydrochloride and dexamphetamine sulphate. Doses and concentrations referred to i n t h e t e x t a r e in t e r m s of these salts. Noradrenaline was used as the bitartrate and the amounts are expressed as the base.
CH 3 EM326
-CH 3
20 p.g/ml
3. R E S U L T S
-CH-CH 2
3.1. Rabbit ileum EM 336
-H
15-20 ~g/ml
-H
> 40 # g / m l
-CH-CH 2EM 97
Drug
-CH2-CH2-CH 2
(R)2-N-CH2-CH2-NH-C~ NH NH 2 Concentration producing R50% blockade in 20 min
EM 65
C2H 5-
6- 8 # g / m l
EM 66
CH 3-
> 40 # g / m l
S t i m u l a t i o n of t h e p e r i a r t e r i a l sympathetic n e r v e s o r t h e a d d i t i o n of n o r a d r e n a l i n e i n h i b i t e d the pendular movements and produced relaxation of t h e i s o l a t e d s e g m e n t of r a b b i t i l e u m . T h e a c t i o n s of t h e c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s o n t h e s e r e s p o n s e s c l o s e l y r e s e m b l e d t h o s e of b r e tylium and guanethidine. They caused a progress i v e r e d u c t i o n i n t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n a n d f o r e a c h d r u g t h e r a t e of d e v e l o p m e n t of b l o c k a d e d e p e n d e d on t h e c o n c e n t r a tion in the bath. For this reason the drugs were c o m p a r e d b y f i n d i n g t h e c o n c e n t r a t i o n of e a c h d r u g w h i c h r e d u c e d s y m p a t h e t i c i n h i b i t i o n by 50% i n 10 m i n .
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA T a b l e 1 s h o w s t h e r e l a t i v e p o t e n c i e s of s o m e of t h e c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s in b l o c k i n g t h e r e s p o n s e s of t h e r a b b i t i l e u m to s t i m u l a t i o n of t h e s y m p a t h e t i c n e r v e s , c o m p a r e d w i t h g u a n e t h i d i n e . T h e s e c o m p o u n d s r a n g e d in p o t e n c y f r o m E M 311, w h i c h w a s a s a c t i v e a s g u a n e t h i d i n e , to E M 97, w h i c h o n l y s l o w l y b l o c k e d t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n i n a c o n c e n t r a t i o n of 0.1 m g / m l . T h e b l o c k a d e w a s very persistent and complete reversal was diffic u l t to p r o d u c e , e v e n w i t h f r e q u e n t w a s h i n g o v e r a n u m b e r of h o u r s . W i t h o n l y o n e e x c e p t i o n , t h e m i n i m u m c o n c e n t r a t i o n s of t h e s e d r u g s w h i c h c o m p l e t e l y b l o c k e d t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n d i d n o t r e d u c e t h e r e s p o n s e to n o r a d r e n a l i n e (fig. 1). H o w e v e r , E M 97 r e d u c e d t h e r e s p o n s e s to n o r a d r e n a l i n e b y a b o u t 50% in t h e
203
t f mm Hg
Fig. 3. P e r f u s e d a r t e r i a l segments. Responses to per i a r t e r i a l stimulation at 10 p u l s e s / s e c for 15 sec in every 4 min, using 1 msec pulses and s u p r a m a x i m a l voltage. The r e c o r d s were obtained using t h r e e different p r e p a r a t i o n s and show the effect of i n c r e a s i n g the concentration of guanethidine (0.125, 0.5 and 2 /Xg/ml) on the rate of reduction of the r e s p o n s e s . c o n c e n t r a t i o n t h a t b l o c k e d t h e r e s p o n s e s to s y m pathetic stimulation. T h e b l o c k i n g e f f e c t s of t h e s e d r u g s w e r e a n t a g o n i z e d b y d e x a m p h e t a m i n e . B l o c k a d e of s y m pathetic responses could be prevented by previo u s l y a d d i n g d e x a m p h e t a m i n e to t h e b a t h (fig. 2), and once the blockade was established it could be reversed by dexamphetamine.
Fig. 2. Isolated r a b b i t ileum. Responses to s t i m u l a tion of the l u m b a r sympathetic n e r v e s (at the dots) for 30 sec in every 4 rain using 2 msec pulses at a f r e quency of 2 0 / s e e and s u p r a m a x i m a l voltage. The r e cords show the action of EM 310 (20 //g/ml) on adjacent segments of ileum in simultaneous e x p e r i m e n t s . In the p r e s e n c e of dexamphetamine (0.5 p g / m l ) the blockade of the sympathetic r e s p o n s e s failed to develop.
3.2. Guinea p i g vas deferens T h e a d r e n e r g i c n e u r o n e b l o c k i n g a c t i v i t y of a n u m b e r of t h e g u a n i d i n e c o m p o u n d s w a s a l s o d e termined using the isolated vas deferens. The guinea pig vas deferens and the rabbit ileum p r e p a r a t i o n a r e f r e q u e n t l y u s e d to a s s e s s t h e p o t e n c y of a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s a n d i t w a s t h o u g h t to b e of i n t e r e s t to c o m p a r e t h e r e s u l t s u s i n g t h e two m e t h o d s ( t a b l e 1). 3.3. Rabbit ea r a r t e r y A s s h o w n b y De l a L a n d e a n d R a n d (1965), bretylium and guanethidine completely blocked t h e r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n of t h e i s o l a t e d a r t e r y p r e p a r a t i o n . T h e r e w a s no r e s i d -
204
M . J . RAND and J. WILSON
u a l r e s p o n s e a t t r i b u t a b l e to d i r e c t m u s c l e s t i m u l a t i o n in t h e s e e x p e r i m e n t s . A s w i t h the r a b b i t i l e u m and the g u i n e a p i g v a s d e f e r e n s , the r a t e of d e v e l o p m e n t of b l o c k a d e w a s r e l a t e d to the c o n c e n t r a t i o n of the drug. T h e e f f e c t of i n c r e a s ing the c o n c e n t r a t i o n of g u a n e t h i d i n e i s shown in fig. 3. U s i n g t h i s p r e p a r a t i o n , d r u g s w e r e c o m p a r e d by finding the c o n c e n t r a t i o n w h i c h r e d u c e d the r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n by 50% in 20 min. F o r b r e t y l i u m and g u a n e t h i d i n e , t h e s e Wequiactive c o n c e n t r a t i o n s " w e r e 1-2 t z g / m l , r e spectively. M o s t of the a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s u s e d in t h e s e e x p e r i m e n t s p o t e n t i a t e d n o r a d r e n -
aline. After infusing guanethidine, bretylium, b e t h a n i d i n e , E M 310, E M 311, E M 313 o r E M 257, the r e s p o n s e s to n o r a d r e n a l i n e w e r e inc r e a s e d by 10-140%. G u a n o x a n (0.5-1 t z g / m l ) , h o w e v e r , r e d u c e d the r e s p o n s e s to n o r a d r e n a l i n e (fig. 4). D e x a m p h e t a m i n e w a s found to be e f f e c t i v e in r e v e r s i n g the b l o c k i n g a c t i o n s of g u a n e t h i d i n e , b r e t y l i u m , b e t h a n i d i n e , g u a n o x a n and the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s on t h i s p r e p a r a t i o n . T h e r e s u l t shown in fig. 5 is t y p i c a l . M o r e than 60 m i n a f t e r s t o p p i n g an i n f u s i o n of b e t h a n i d i n e (1 t z g / m l ) , the r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n w e r e s h o w i n g only s l i g h t r e c o v e r y . T h e
Nor
r,rv~
Hq
.o3T
T!
5ng lOng
5ng lOng
Fig. 4. Effects of EM 257 (8 tig/ml) and guanoxan (1 ttg/ml) on the responses of isolated artery segments to periarterial stimulation and to noradrenaline (Nor). The preparations were stimulated for 15 sec every 4 min using 1 msec pulses at 10/sec and supramaximal voltage.
60"Jmin Fig. 5. Responses of the isolated artery segment to p e r i a r t e r i a l stimulation at a frequency of 10/sec for 15 sec every 4 min, using 2 msec pulses and supramaximal voltage. In the first panel, the blocking action of bethanidine (1 /ig/ml) is shown, and in the second the blockade was r e v e r s e d by dexamphetamine (0.5/ig/ml). The duration of infusion is indicated by the white horizontal bars and vertical strokes.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA
In EM 65 and EM 66, the hydrogen and c y c l o hexyl ring on the a m i n o - n i t r o g e n w e r e r e p l a c e d by two m e t h y l groups (EM 66) or ethyl groups (EM 65). EM 65 was a f a i r l y a c t i v e a d r e n e r g i c blocking drug, w h e r e a s EM 66 was p r a c t i c a l l y inactive.
introduction of d e x a m p h e t a m i n e (0.5 p g / m l ) into the p e r f u s i o n fluid f o r 10 min p r o d u c e d a r a p i d and c o m p l e t e r e v e r s a l of the blockade. Table 2 shows the group of guanidine c o m pounds whose p o t e n c i e s w e r e a s s e s s e d using the p e r f u s e d a r t e r y p r e p a r a t i o n . In all of the c o m pounds, with the ex c e p t io n of EM 97, the a m i n o n i t r o g e n and the guanidine group a r e s e p a r a t e d by an ethylene link. Most of the m e m b e r s of the group differ in the alkyl groups attached to this link, the c y c l o h e x y l r i n g and guanidine group r e maining u n a l t e r e d . The m o s t potent compounds in the s e r i e s a r e EM 310, in which the ethylene link is unsubstituted, and EM 257 and EM 311, which have a m e t h y l group on C1 and C2 r e s p e c tively. The introduction of the methyl group slightly i n c r e a s e s a c ti v i ty , EM 311 being r a t h e r m o r e a c t i v e than EM 257. A second methyl group on C 2 (EM 313) c a u s e s a slight fall in activity. R e p l a c e m e n t of the methyl group on EM 311 by an ethyl group c o n s i d e r a b l y r e d u c e d a c t i v i t y (EM 336), as did the introduction of a methyl group onto the guanidine p o r t i o n of the m o l e c u l e (in EM 326). When the length of the alkyl chain linking the c y c l o h e x y l r i n g to the guanidine group was i n c r e a s e d to t h r e e c a r b o n a t o m s , the r e sulting compound (EM 97) had v e r y little a d r e n e r g i c n eu r o n e blocking activity.
3.4. Local anaesthe/ic effecls of adrenergic neu-
rone blocking d~'ugs 3.4.1. Guinea pig weal method The l o cal a n a e s t h e t i c act i o n s of guanethidine, EM 311, E M 3 3 6 and E M 9 7 w e r e c o m p a r e d by i n t r a d e r m a l i n j ect i o n in the guinea pig. T h e s e compounds w e r e s e l e c t e d f o r i n v e s t i g a t i o n b e cau se although c l o s e l y r e l a t e d s t r u c t u r a l l y , they differ c o n s i d e r a b l y in a d r e n e r g i c neurone blocking activity. EM 311 is a potent a d r e n e r g i c neurone blocking drug while EM 336 is much l e s s a c t i v e and EM 97 is a l m o s t inactive. Each drug was i n j e c t e d into two s i t e s on each of t h r e e guinea pigs and each site was t e s t e d by p r i c k i n g six t i m e s . The d e g r e e of a n a e s t h e s i a was taken as the n u m b e r of t i m e s the guinea pigs f a i l e d to r e s p o n d out of the total of 36 t e s t s . The r e s u l t s a r e shown in fig. 6. The local a n a e s t h e t i c potency of the drugs d e c r e a s e d in the following o r d e r : EM 336 > EM 311 > EM 97 = guanethidine. The action of t h ese compounds as l o cal a n a e s -
EM311
Guanethidine 1%
100'
205
i%
ioo
~
75'
75
50'
50
25
25~
r. Q)
0
0
3"0
6o
¢o
12o
15"o
leo
o
3b
6b
9b
i~;o
15"o
105
t-
<
EM97
e3
~, 100.
100'
I~ 75,
75
5o, 25,
50"
3'o
go
,pb
rain
i~
lS'O
la6
O/ o
/
EM336 0.57.
3O min
Fig. 6. Local anaesthetic effect of intradermal injections of adrenergic neurone blocking drugs in the guinea pig. Each point represents the number of times the guinea pig failed to respond the pricking in the injected area, out of a total of 36 tests, expressed as a percentage.
206
M.J. RAND and J. WILSON
t h e t i c s w a s slow i n o n s e t b u t l o n g in d u r a t i o n . T h e d r u g s u s u a l l y took f r o m 1 5 - 3 0 m i n to r e a c h t h e p e a k of t h e i r a c t i o n , b u t in s o m e c a s e s , n o t a b l y w i t h t h e l o w e r c o n c e n t r a t i o n s of E M 311, t h e full e f f e c t w a s not s e e n u n t i l a b o u t 60 m i n a f t e r i n j e c t i o n . One p e r c e n t s o l u t i o n s of g u a n e t h i dine o r E M 311, o r a 0.5% s o l u t i o n of E M 3 3 6 , p r o d u c e d a c o m p l e t e l o c a l a n a e s t h e s i a w i t h i n 30 min after injection and this was still virtually c o m p l e t e a f t e r 180 m i n .
Table 3 Degree of anaesthesia produced by subcutaneous injection of various concentrations of EM 311, EM 336 and guanethidine in the m o u s e ' s tail. The figures show the proportion of each group of ten mice failing to respond to an a r t e r y clip ~laced on the tail.
3.4.2. M o u s e t a i l m e t h o d T h e m e t h o d of B i a n c h i (1956) w a s u s e d to c o m p a r e t h e l o c a l a n a e s t h e t i c p o t e n c y of g u a n e t h i d i n e , E M 311 a n d E M 336. V a r i o u s c o n c e n t r a t i o n s of t h e d r u g s w e r e i n j e c t e d s u b c u t a n e o u s l y into t h e t a i l s of g r o u p s of m i c e . E a c h m o u s e w a s t e s t e d p e r i o d i c a l l y by p l a c i n g a n a r t e r y c l i p on the t a i l , d i s t a l to t h e s i t e of i n j e c t i o n , a n d the d e g r e e of a n a e s t h e s i a w a s t a k e n a s t h e p r o p o r t i o n of t h e g r o u p f a i l i n g to a t t e m p t to r e m o v e t h e c l i p . T h e r e s u l t s a r e s h o w n in t a b l e 3. In t h i s t e s t , E M 311 a n d E M 336, in c o n c e n t r a t i o n s of 0 . 5 - 1 . 5 % , h a d v e r y s i m i l a r l o c a l a n aesthetic activity, and guanethidine was rather less potent. As was found using the guinea pig weal method, t h e o n s e t of l o c a l a n a e s t h e t i c a c t i o n of t h e s e d r u g s w a s slow. T h e m a x i m u m e f f e c t w a s not obtained until from 30-45 min after injecting the drug. This is shown for various concentrations of g u a n e t h i d i n e in fig. 7.
Concentration
EM 311
EM 336
Guanethidine
0.5%
50%
50%
30%
0.75%
60%
60%
50%
1%
70%
70%
60%
1.5%
80%
90%
2%
s0% 100%
-
stimulation, is s h o w n in fig. 8. Procaine (100 p g / m l ) s l i g h t l y r e d u c e d t h e r e s p o n s e s , a n d w h e n t h e c o n c e n t r a t i o n w a s i n c r e a s e d to 200 pg/ ml, the responses were completely blocked. Unl i k e the g r a d u a l , p r o g r e s s i v e b l o c k a d e p r o d u c e d by the adrenergic neurone blocking drugs, the b l o c k a d e p r o d u c e d by p r o c a i n e w a s a l m o s t i m m e d i a t e in o n s e t . W h e n t h e i n f u s i o n of p r o c a i n e w a s s t o p p e d , t h e r e s p o n s e s r e t u r n e d to t h e i r i n i tial height very rapidly. T h e b l o c k i n g a c t i o n of p r o c a i n e on t h e i s o l a t e d g u i n e a p i g v a s d e f e r e n s w a s s i m i l a r to t h a t on t h e a r t e r y s e g m e n t . T h e r e s p o n s e s of t h e v a s d e f e r e n s to h y p o g a s t r i c n e r v e s t i m u l a t i o n w e r e s l i g h t l y r e d u c e d by 40 p g / m l of p r o c a i n e , w h i l e a c o n c e n t r a t i o n of 80 p g / m l r a p i d l y a n d c o m pletely blocked the responses, T h i s b l o c k a d e c o u l d not b e r e v e r s e d by d e x amphetamine (0.5-5 pg/ml), but the responses r a p i d l y r e c o v e r e d on w a s h i n g t h e b a t h .
3.5. Action of procaine on sympathelic nerves T h e e f f e c t of p r o c a i n e on t h e r e s p o n s e of t h e i s o l a t e d a r t e r y s e g m e n t to s y m p a t h e t i c n e r v e
2~. 100"
15
30
45
60
rnin
Fig. 7. Local anaesthetic effect of g~anethidine using the mouse tail method. Each point r e p r e s e n t s the n u m b e r of mice in a group of ten. failing to respond to an a r t e r y clip placed on the tail. The mice were tested at 15 rain i n t e r vals after subcutaneous injection of 0.5, 1 or 2% g~anethidine into the tail.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA
L
i
I
i
I l
L Proc
207
lOO u g m ;
--
,
I
2oougn !
Fig. 8. R e s p o n s e s of the isolated a r t e r y s e g m e n t f r o m the r a b b i t ' s e a r to p e r i a r t e r i a l s t i m u l a t i o n . The p r e p a r a t i o n w a s s t i m u l a t e d at 10 p u l s e s / s e c for 15 sec in e v e r y 4 min, u s i n g 1 m s e c p u l s e s and s u p r a m a x i m a l voltage. The r e c o r d s h o w s the effect of p r o c a i n e (100 and 200 ~ g / m l } , the d u r a t i o n of infusion being indicated by the h o r i z o n t a l b a r s .
4. DISCUSSION The cyclohexylaminoalkylguanidines were v e r y s i m i l a r in t h e i r a c t i o n s on isolated, s y m p a t h e t i c a l l y i n n e r v a t e d o r g a n s , to b r e t y l i u m and guanethidine. They p r o d u c e d a p r o g r e s s i v e r e duction in the r e s p o n s e s to s y m p a t h e t i c n e r v e stimulation. The blockade was v e r y p e r s i s t e n t but could be r e v e r s e d by d e x a m p h e t a m i n e . With the e x cep t i o n of EM 97, c o n c e n t r a t i o n s of th e s e compounds which blo c k e d the r e s p o n s e s to s y m pathetic n e r v e s t i m u l a t i o n , p o t e n ti a t e d n o r a d r e n aline. The r e l a t i v e p o t e n c i e s of t h e s e compounds in blocking the s y m p a t h e t i c n e r v e s to the rabbit il eum d i f f e r e d f r o m t h e i r r e l a t i v e p o t e n c i e s on the h y p o g a s t r i c n e r v e to the v a s d e f e r e n s . Thus, guanethidine and EM 311 w e r e l e s s potent, and EM 97 and EM 336 c o n s i d e r a b l y m o r e potent in blocking s y m p a t h e t i c r e s p o n s e s of the vas d e f e r ens than those of the rabbit ileum. S jS s t r a n d (1962) supposed that the h y p o g a s t r i c n e r v e to the va s d e f e r e n s contains a p r o p o r t i o n of p r e g a n g l i onic s y m p a t h e t i c f i b r e s , and this was s u p p o r t e d by the work of B i r m i n g h a m and Wilson (1963). It is t h e r e f o r e p o s s i b l e that the r e d u c t i o n in the s y m p a t h e t i c r e s p o n s e s of the v a s d e f e r e n s by the l e s s potent guanidines was due to ganglion blockade r a t h e r than to a d r e n e r g i c neurone blockade. The v a l u e s fo r the r e l a t i v e a d r e n e r g i c neur o n e blocking a c t i v i t i e s of the guanidine c o m pounds using the a r t e r y s e g m e n t did not always a g r e e with those found when using the other two i s o l a t e d o rg an s . A notable e x a m p l e is EM 313, which was f a i r l y a c t i v e on the a r t e r y p r e p a r a t i o n although r a t h e r i n a c t i v e in blocking the s y m p a thetic n e r v e s to the r a b b i t ileum. When a d m i n i s t e r e d to c o n s c i o u s r a t s , EM 313 was found to be a highly a c t i v e a n t i h y p e r t e n s i v e agent (Wilson, 1966). It s e e m s p r o b a b l e that an i s o l a t e d v a s c u -
l a r p r e p a r a t i o n is a m o r e a p p r o p r i a t e t e s t object to use in s c r e e n i n g drugs for a d r e n e r g i c neurone blocking activity. In a r e c e n t r e v i e w , Bo u r a and G r e e n (1965) s t a t e d that drugs with a d r e n e r g i c n eu r o n e blocking a c t i v i t y c o n s i s t e d of t h r e e units: a highly b a s i c group, such as q u a t e r n a r y a m m o n i u m , guanidine or a m i d o x i m e , joined through a connecting chain to a ring s t r u c t u r e which may contain a second, m o r e weakly b asi c group. Most of the compounds in the guanidine s e r i e s u s e d conf o r m e d to this pattern. The, a m i n o - n i t r o g e n in the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s c o r r e s p o n d s to the n i t r o g e n in the e i g h t - m e m b e r e d ring of guanethidine. The pKa v a l u e s for EM 310, EM 311, EM 313 and guanethidine w e r e c l o s e l y s i m i l a r ; pKa 1 was 8.25 to 8.6 and pKa2 was 11.3 to 11.4 ( p e r s o n a l c o m m u n i c a t i o n , W . J . C . Dyke). S c h l i t t l e r et al. (1962) found, using a s e r i e s of h e t e r o c y c l i c guanidines and a m i d o x i m e s , that substitution on the ethylene link r e d u c e d activity. With the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s , the p r e s e n c e of one m e t h y l substituent on the e t h y l ene chain i n c r e a s e d a d r e n e r g i c neurone blocking activity, but with f u r t h e r substitution in the chain a c t i v i t y fell. Costa et al. (1962) studied a s e r i e s of c o m pounds in which the guanidine group was sep ar a t e d f r o m a benzene r i n g by one, two or t h r e e c a r b o n atoms. They found that those compounds having a t w o - c a r b o n chain had " g u a n e t h i d i n e like" a c t i v i t y in that they depleted the content of c a t e c h o l a m i n e s in the heart. T h o s e compounds which had a single carbon link had " b r e t y l i u m like" activity and p r e v e n t e d the depletion of c a t e c h o l a m i n e s by guanethidine. When the length of the chain was i n c r e a s e d to t h r e e carbon a t o m s , the a c t i v i t y was lost (cf. EM 97). The s t r u c t u r a l r e q u i r e m e n t s for o p t i m al a c t i v i t y in the c o m pounds having single c a r b o n links d i f f e r e d s o m e -
208
M.J. RAND and J. WILSON
w h a t f r o m t h o s e of t h e c o m p o u n d s w i t h a n e t h y l e n e l i n k a g e . T h u s , w i t h t h e E M s e r i e s of g u a n i dines and with the ethylguanidine derivatives s t u d i e d b y S c h l i t t l e r e t al. (1962) a n d b y C o s t a e t al. (1962), t h e i n t r o d u c t i o n of m e t h y l g r o u p s i n t o the guanidine portion considerably reduced activity. However, in the methyl guanidine derivat i v e s ( C o s t a et al., 1962; Copp, 1964) m e t h y l s u b s t i t u t i o n in t h e g u a n i d i n e g r o u p r e s u l t e d in increased potency. A l t h o u g h t h e r i n g s t r u c t u r e w a s s a i d to b e necessary for adrenergic neurone blocking act i v i t y ( S c h l i t t l e r e t al., 1962), d i e t h y l a m i n o e t h y l g u a n i d i n e ( E M 65) w a s f o u n d to b e o n l y s l i g h t l y less active than cyclohexylaminoethylguanidine ( E M 310). T h e d i m e t h y l a n a l o g u e ( E M 66) w a s f o u n d to b e i n a c t i v e . S i m i l a r f i n d i n g s to t h e s e w i t h E M 65 a n d E M 66 w e r e r e p o r t e d b y S h o r t e t al. (1963) u s i n g t h e n i c t i t a t i n g m e m b r a n e of t h e conscious cat. G u a n e t h i d i n e , E M 311, E M 336 a n d E M 97 w e r e found, u s i n g t h e B ~ i l b r i n g a n d W a j d a (1945) g u i n e a p i g w e a l t e c h n i q u e , to b e f a i r l y p o t e n t l o cal anaesthetics with a prolonged action. The compound with the most powerful local anaest h e t i c a c t i o n w a s E M 336. A 0.25% s o l u t i o n of E M 336 w a s a s a c t i v e a s a 0.5% s o l u t i o n of E M 311 a n d a l m o s t a s a c t i v e a s a 1% s o l u t i o n of g u a n e t h i d i n e . T h e r e l a t i v e p o t e n c i e s of E M 311, E M 336 a n d g u a n e t h i d i n e , a s d e t e r m i n e d b y t h e B i a n c h i (1956) m o u s e t a i l t e c h n i q u e , d i f f e r e d s l i g h t l y from those found using the guinea pig weal method. E M 311 w a s a s a c t i v e a s E M 336, a n d b o t h compounds were a little more potent than guanethidine. F r o m t h e s e r e s u l t s it m a y b e s e e n t h a t t h e r e i s no d i r e c t r e l a t i o n s h i p b e t w e e n t h e l o c a l a n a e s t h e t i c a c t i v i t y of t h e s e d r u g s a n d t h e i r p o t e n cy a s a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s . B o y d e t al. (1961) i n v e s t i g a t e d t h e l o c a l a n a e s t h e t i c a c t i v i t y of b r e t y l i u m , u s i n g the g u i n e a p i g w e a l m e t h o d , a n d f o u n d t h a t t h e a c t i o n of b r e t y l i u m d i f f e r e d f r o m t h a t of p r o c a i n e in b e i n g s l o w in o n s e t a n d m u c h m o r e p r o l o n g e d . In t h e experiments described here, guanethidine, EM 311, E M 336 a n d E M 97 w e r e f o u n d to h a v e a s i m i l a r t i m e c o u r s e of a c t i o n to t h a t d e s c r i b e d b y B o y d e t al. (1961) f o r b r e t y l i u m . S i m i l a r l y , u s i n g t h e m o u s e t a i l m e t h o d , t h e p e a k of a c t i v i t y f o r e a c h c o n c e n t r a t i o n of g u a n e t h i d i n e , E M 311 a n d E M 336 w a s not r e a c h e d u n t i l f r o m 3 0 - 4 5 min after injecting the drug. This suggests that the adrenergic neurone blocking drugs differ from procaine in the manner in which they produce their local anaesthetic effect. T h e b l o c k i n g a c t i o n of p r o c a i n e on t h e r e -
s p o n s e s of t h e i s o l a t e d p e r f u s e d a r t e r y f r o m t h e r a b b i t ' s e a r d i f f e r e d m a r k e d l y f r o m t h a t of t h e adrenergic neurone blocking drugs. The minim u m c o n c e n t r a t i o n of p r o c a i n e w h i c h b l o c k e d t h e r e s p o n s e s of t h e a r t e r y s e g m e n t to s y m p a t h e t i c nerve stimulation was about one thousand times t h e m i n i m u m b l o c k i n g c o n c e n t r a t i o n of g u a n e t h i dine on t h i s p r e p a r a t i o n . T h i s w a s r a t h e r s u r p r i s i n g s i n c e l o c a l a n a e s t h e t i c s a r e r e p u t e d to be quite active in blocking vasoconstrictor n e r v e s ( H i r s c h f e l d e r a n d B i e t e r , 1932). T h e o n s e t of b l o c k a d e b y p r o c a i n e w a s a l m o s t i m m e d i ate and recovery was also very rapid after stopp i n g t h e i n f u s i o n of t h e d r u g . S i m i l a r r e s u l t s w e r e r e p o r t e d b y B o y d e t al. (1961) a n d in t h i s paper, using the guinea pig vas deferens. Anothe r p o i n t of d i f f e r e n c e b e t w e e n t h e b l o c k a d e of sympathetic nerves by procaine and by the adr e n e r g i c n e u r o n e b l o c k i n g d r u g s , w a s the e f f e c t of d e x a m p h e t a m i n e on t h e b l o c k a d e . T h e b l o c k a d e of t h e r e s p o n s e s of t h e r a b b i t e a r a r t e r y to s y m p a t h e t i c n e r v e s t i m u l a t i o n b y a n y of t h e a d renergic neurone blocking drugs investigated, was reversed by dexamphetamine. The blockade produced by procaine however was unaffected by d e x a m p h e t a m i n e in c o n c e n t r a t i o n s up to 5 p g / m l . The experiments described therefore demons t r a t e a n u m b e r of d i f f e r e n c e s b e t w e e n t h e a d renergic neurone blocking drugs and procaine, b o t h in t h e i r l o c a l a n a e s t h e t i c e f f e c t s a n d i n their sympathetic blocking actions. These res u l t s s u g g e s t t h a t t h e l o c a l a n a e s t h e t i c e f f e c t s of a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s a r e not r e l a t e d to t h e i r b l o c k i n g a c t i o n a t s y m p a t h e t i c nerve endings.
ACKNOWLEDGEMENTS We a r e grateful to Dr. E. M. Glaser, who was Medical D i r e c t o r of Evans Medical at the time when these e x p e r i m e n t s were c a r r i e d out, for his support, and to Dr. W. J. C. Dyke who synthesized the compounds. P a r t of the work reported here was c a r r i e d out during the tenure of an Evans Medical R e s e a r c h Scholarship by J. Wilson.
REFERENCES Bianchi, C., 1956, A simple new quantitative method for testing local anaesthetics, Brit. J. P h a r m a c o l . Chemother. 11, 104. Birmingham, A.T. and A. B. Wilson, 1963, P r e g a n g l i onic and postganglionic stimulation of the guinea pig isolated vas deferens preparation. Brit. J. P h a r m a col. Chemother. 21, 569.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA Boura, A . L . A . , F . C . C o p p , W.G. Duneombe, A . F . Green and A. MeCoubrey, 1960, The s e l e c t i v e a c c u mulation of b r e t y l i u m in s y m p a t h e t i c ganglia and t h e i r postganglionic n e r v e s , Brit. J. P h a r m a e o l . C h e m o t h e r . 15, 265. Boura, A . L . A . and A . F . Green, 1965, A d r e n e r g i c neurone blocking a g e n t s , Ann. Rev. P h a r m a c o l . 5, 183. Boura, A.L.A., A.F. Green, A.McCoubrey, D.R.Laurence, R. Moulton and M.L.Rosenheim, 1959, Darenthin. Hypotensive agent of a new type, Lancet 2, 17. Boyd, H., V. Chang and M . J . ' R a n d , 1961, The local a n a e s t h e t i c activity of b r e t y l i u m in relation to the a c tion in blocking s y m p a t h e t i c r e s p o n s e s , Arch. Int. P h a r m a c o d y n . ThOr. 131, 10. Biilbring, E. and I. Wajda, 1945, Biological c o m p a r i son of local a n a e s t h e t i c s , J. P h a r m a e o l . Exp. Ther. 85, 78. Copp, F.C., 1964, A d r e n e r g i c neurone blocking a g e n t s , in: Advances in Drug R e s e a r c h , Vol. 1, eds. N . J . H a r p e r and A. B. S i m m o n d s (Academic P r e s s , London). Costa, E., R . K u n t z m a n , G . L . G e s s a and B. B. Brodie, 1962, S t r u c t u r a l r e q u i r e m e n t s for b r e t y l i u m and guanethidine-like activity in a s e r i e s of g~anidine d e r i v a t i v e s , Life Sci. Oxford 3, 75. De la Lande, I.S. and M . J . Rand, 1965, A s i m p l e i s o lated n e r v e - b l o o d v e s s e l p r e p a r a t i o n , Aust. J. Exp. Biol. Med. Sei. 43, 639.
209
Finkleman, B., 1930, On the n a t u r e of inhibition in the intestine, J. Physiol. 70, 145. H i r s c h f e l d e r , A.D. and R.N. Bieter, 1932, Local ana e s t h e t i c s , Physiol. Rev. 12, 190. Hukovid, S., 1961, R e s p o n s e s of the isolated s y m p a thetie n e r v e - d u c t u s d e f e r e n s p r e p a r a t i o n of the guinea pig, Brit. J. P h a r m a c o l . C h e m o t h e r . 16, 188. McEwen, L . M . , 1956, The effect on the isolated rabbit h e a r t of vagal s t i m u l a t i o n and its modification by cocaine, h e x a m e t h o n i u m and ouabain, J. Physiol. 131, 678. Page, I.H. and H . P . D u s t a n , 1959, A new, potent ant i h y p e r t e n s i v e drug, J. Am. Med. A s s . 170. 1265. Schlittler, E., J. Druey and A . M a r x e r . 1962, Guanidines and a m i d o x i m e s , in: P r o g r e s s in Drug Res e a r c h . Vol. 4, ed. E . J u e k e r (Birkh~user, Basel) p. 341. Short, J . H . , U. B i e r m a e h e r , D . A . D u n n i g a n and T.D. Leth, 1963, Sympathetic n e r v o u s s y s t e m blocking agents. D e r i v a t i v e s of guanidine and related cornpounds, J. Med. Chem. 6, 275. SjSstrand, N.O., 1962, Inhibition by ganglionic blocking a g e n t s of the m o t o r r e s p o n s e of the isolated guinea pig v a s d e f e r e n s to h y p o g a s t r i e nerve s t i m u lation, Acta P h y s i o l . Seand. 54, 306. Wilson, J., 1966, The p h a r m a c o l o g y of hypotensive guanidine compounds, P h . D . T h e s i s , U n i v e r s i t y of London.
THE RELATIONSHIP BETWEEN ADRENERGIC NEURONE BLOCKING ACTIVITY AND LOCAL ANAESTHETIC ACTIVITY IN A SERIES OF GUANIDINE DERIVATIVES M. J. RAND * a n d J. W I L S O N * Department of Pharmacology, School of Pharmacy, University of London, London, U.K. Accepted 15 F e b r u a r y 1967
M . J . R A N D and J. WILSON, The relationship between adrenergic neurone blocking activity and local anaesthetic activity in a series of guanidine derivatives, European J. P h a r m a c o l . 1 (1967) 200-209. The a d r e n e r g i c neurone blocking potencies of a n u m b e r of guanidine d e r i v a t i v e s were a s s e s s e d using t h r e e isolated sympathetically innervated organs: rabbit ileum, guinea pig vas deferens and the a r t e r y segment from the r a b b i t ' s ear. The most suitable of these p r e p a r a t i o n s for making quantitative comp a r i s o n s was the isolated a r t e r y segment, and the r e s u l t s so obtained were used as the basis for a study of s t r u c t u r e - a c t i v i t y relationships. Some of these compounds were also tested for local a n a e s thetic activity using the guinea pig weal and mouse tail methods. It was found that the local anaesthetic activity of these compounds was unrelated to t h e i r a d r e n e r g i c neurone blocking activity. Adrenergic neurone blocking drug Guanidine d e r i v a t i v e s Guanethidine
1. I N T R O D U C T I O N Since bretylium and guanethidine were found to b e e f f e c t i v e in t h e t r e a t m e n t of h y p e r t e n s i o n ( B o u r n e t al., 1959; P a g e a n d D u s t a n , 1959), a l a r g e n u m b e r of a d r e n e r g i c n e u r o n e b l o c k i n g drugs has been synthesized. Structure-activity r e l a t i o n s h i p s i n g r o u p s of t h e s e c o m p o u n d s h a v e b e e n s t u d i e d b y S c h l i t t l e r et al. (1962); S h o r t e t al. (1963) a n d C o p p (1964). T h e s e s t u d i e s u s e d , as a criterion of a c t i v i t y of t h e c o m p o u n d s , either their antihypertensive action (Schlittler et al., 1962), o r r e l a x a t i o n of t h e n i c t i t a t i n g m e m b r a n e in t h e c o n s c i o u s c a t ( S h o r t e t al., 1963; Copp, 1964). T h e u s e of s u c h p h a r m a c o l o g i c a l tests in studying structure-activity relationships i s o p e n to t h e o b j e c t i o n t h a t t h e d r u g s a r e a c t i n g on c o m p l e x s y s t e m s , w h e r e n u m e r o u s f a c t o r s i n a d d i t i o n to a d r e n e r g i c n e u r o n e b l o c k a d e m a y * P r e s e n t a d d r e s s : Department of Pharmacology, Univ e r s i t y of Melbourne, Parkville, N. 2., Victoria, Australia.
Local anaesthetic Procaine Structure-activity relationships
play a part. The isolated arterial segment from t h e r a b b i t e a r (De l a L a n d e a n d R a n d , 1965) i s a c o n v e n i e n t a n d s i m p l e p r e p a r a t i o n to u s e i n t h e determination of a d r e n e r g i c n e u r o n e b l o c k i n g p o t e n c y . In t h e e x p e r i m e n t s described here it w a s u s e d to i n v e s t i g a t e s t r u c t u r e - a c t i v i t y relat i o n s h i p s in a s e r i e s of g u a n i d i n e d e r i v a t i v e s . The mechanism by which adrenergic neurone b l o c k i n g d r u g s p r e v e n t t h e r e l e a s e of n o r a d r e n a l ine at the sympathetic nerve endings is unk n o w n . B o u r a e t al. (1960) r e p o r t e d t h a t f o l l o w i n g a s u b c u t a n e o u s i n j e c t i o n of b r e t y l i u m i n t h e c a t , l a r g e c o n c e n t r a t i o n s of t h e d r u g w e r e f o u n d in the sympathetic ganglia and postganglionic n e r v e s , w h i l e o n l y s m a l l a m o u n t s w e r e f o u n d in other nervous structures. They suggested that t h e c o n c e n t r a t i o n s of b r e t y l i u m w h i c h a c c u m u lated in the sympathetic nerve endings were s i m i l a r to t h e c o n c e n t r a t i o n s r e q u i r e d to b l o c k c o n d u c t i o n w h e n a p p l i e d l o c a l l y to n e r v e t r u n k s . T h e a d r e n e r g i c n e u r o n e b l o c k i n g a c t i o n of b r e tylium could therefore be explained by its local a n a e s t h e t i c a c t i o n c a u s i n g a b l o c k a d e of c o n d u c -
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA tion in the nerve terminals. However, the experiments described here demonstrated that the loc a l a n a e s t h e t i c a c t i v i t y of t h e g u a n i d i n e c o m p o u n d s w a s u n r e l a t e d to t h e i r e f f e c t i v e n e s s a s adrenergic neurone blocking drugs. 2. M E T H O D S . E X P E R I M E N T S ORGANS
USING I S O L A T E D
2.1. Rabbit ear artery T h e p r e p a r a t i o n d e s c r i b e d b y De l a L a n d e a n d R a n d (1965) w a s u s e d . R a b b i t s w e i g h i n g 2 - 3 k g w e r e k i l l e d b y a b l o w on t h e b a c k of t h e n e c k . T h e s k i n w a s r e m o v e d f r o m t h e e a r in t h e r e g i o n of t h e c e n t r a l a r t e r y a n d t h e a r t e r y w a s d i s s e c t ed free from the surrounding tissues. The artery was cannulated with fine polythene tubing and a l e n g t h of 3 - 4 c m w a s e x c i s e d . T h e a r t e r y s e g m e n t w a s t h e n p e r f u s e d w i t h M c E w e n ' s (1956) s o l u t i o n a t 3 7 o c a n d t h e r e s e r v o i r of M c E w e n ' s s o l u t i o n w a s g a s s e d w i t h 5% c a r b o n d i o x i d e i n o x y g e n . T h e flow of t h e p e r f u s i o n f l u i d w a s maintained with a constant volume roller pump a t a r a t e of 5 m l / m i n . T h e p e r f u s i o n p r e s s u r e was measured with a mercury manometer. The periarterial sympathetic nerves were stimulated b y m e a n s of b i p o l a r p l a t i n u m r i n g e l e c t r o d e s p l a c e d on t h e u p p e r e n d of t h e v e s s e l . S t i m u l a t i o n w a s a t a f r e q u e n c y of 10 p u l s e s / s e c f o r 1015 s e c e v e r y 4 r a i n , u s i n g 1 m s e c p u l s e s a n d s u pramaximal v o l t a g e . I n j e c t i o n s of d r u g s w e r e made into the perfusion fluid just before it reached the cannula. Vasoconstrictor responses were recorded as rises in perfusion pressure. B l o c k i n g d r u g s w e r e i n f u s e d b y m e a n s of a Palmer slow injection apparatus and their conc e n t r a t i o n s a r e e x p r e s s e d a s g g / m l of t h e p e r f u s i o n fluid. A l l i n j e c t i o n s a n d i n f u s i o n s of d r u g s w e r e m a d e up in M c E w e n ' s s o l u t i o n . I n j e c t i o n s h a d a m a x i m u m v o l u m e of 0.2 m l a n d i n f u s i o n s w e r e m a d e a t t h e r a t e of 0.1 to 0.2 m l / m i n . 2.2. Rabbil ileum S e g m e n t s of r a b b i t i l e u m w i t h t h e i r s y m p a t h e t i c n e r v e s w e r e p r e p a r e d b y t h e m e t h o d of F i n k l e m a n (1930). A l e n g t h of i l e u m 2 - 3 c m l o n g w a s s e t up i n a 50 m l b a t h c o n t a i n i n g M c E w e n ' s s o l u t i o n a t 3 7 o c , a n d m o v e m e n t s of t h e i l e u m were recorded with an isotonic, frontal-writing lever. The periarterial nerves were stimulated w i t h b i p o l a r p l a t i n u m e l e c t r o d e s a t 20 p u l s e s / s e c using 2 msec pulses at supramaximal voltage for 20-30 sec every 4 min.
T h e v a s d e f e r e n s w a s s e t up in a 50 m l b a t h c o n t a i n i n g M c E w e n ' s (1956) s o l u t i o n g a s s e d w i t h 5% carbon dioxide in oxygen at 32oc. The hypogastric nerve was stimulated with bipolar platinum e l e c t r o d e s f o r 10 s e c e v e r y 2 - 4 r a i n at 1 0 - 2 0 pulses/sec using 2 msec pulses at supramaximal voltage. 2.4. T e s t s f o r local anaesthetic activity Local anaesthetic activity was investigated u s i n g t h e m e t h o d s of B i i l b r i n g a n d W a j d a (1945) a n d B i a n c h i (1956). In t h e m e t h o d of B t i l b r i n g a n d W a j d a t h e l o s s of s e n s a t i o n i n t h e s k i n of t h e g u i n e a p i g f o l l o w i n g a n i n t r a d e r m a l i n j e c t i o n of a l o c a l a n a e s t h e t i c d r u g i s a s s e s s e d . T h e f l a n k s of t h e g u i n e a p i g s w e r e d e p i l a t e d on t h e day b e f o r e t h e t e s t s w e r e c a r r i e d out. T h e d r u g s w e r e d i s s o l v e d i n n o r m a l s a l i n e a n d a v o l u m e of 0.25 m l i n j e c t e d . T w o i n j e c t i o n s of e a c h of two c o n c e n t r a t i o n s of t h e d r u g a n d two i n j e c t i o n s of n o r m a l s a l i n e w e r e g i v e n to e a c h g u i n e a pig. T h r e e guinea pigs were used for each test and the solut i o n s w e r e i n j e c t e d in r a n d o m o r d e r . Light p r i c k i n g of p a r t of t h e f l a n k o u t s i d e t h e a n a e s Table 1 Concentrations of some a d r e n e r g i c neurone blocking drugs which reduced the r e s p o n s e s of the isolated r a b bit ileum and guinea pig vas deferens to sympathetic stimulation by 50% in 10 min.
//NH NH-R-NH-C \NH 2 Equiactive concentrations Drug
EM310
-R-
-(CH2) 2-
Rabbit ileum
Guinea pig vas deferens
10-20 /£g/ml
5-10 /Xg/ml
1- 2 /~g/ml
2- 5 # g / m l
25 # g / m l
10-20 //g/ml
100 /xg/ml
15-20 /2g/ml
20 # g / m l
5-10 /xg / m l
100 g g / m t
25-50 /xg / m l
1- 2 # g / m l
5-10 /ag/ml
CH3
EM311
J
-CH-CH 2CH3
J
EM 313
-CH-CH 2
l
CH3
(~2H5 EM336
-CH-CH 2 EM 257
CH 3
I
-CH2-CHEM 97
2.3. Guinea p i g vas deferens T h e p r e p a r a t i o n of H u k o v i ~ (1961) w a s u s e d .
201
Guanethidine
-(ell2) 3-
202
M. J. RAND and J. WILSON
thetized areas produced a characteristic cont r a c t i o n of t h e s k i n . T h e d e g r e e of a n a e s t h e s i a p r o d u c e d by e a c h c o n c e n t r a t i o n of d r u g w a s a s s e s s e d b y n o t i n g t h e n u m b e r of t i m e s t h e a n i m a l s f a i l e d to r e s p o n d o u t of a t o t a l of 36 t e s t s . Bianchi's method uses the response of a m o u s e to a n a r t e r y c l i p a t t a c h e d to t h e t a i l . G r o u p s of t e n m i c e w e r e u s e d f o r e a c h t e s t . T h e d r u g s w e r e m a d e up i n n o r m a l s a l i n e c o n t a i n i n g 10 t t g / m l of a d r e n a l i n e . A v o l u m e of 0.1 m l of the solution was injected intradermally 1.5-2 cm f r o m t h e b a s e of t h e t a i l of e a c h m o u s e . T h e c l i p w a s a t t a c h e d d i s t a l to t h e s i t e of i n j e c t i o n a t 15 m i n i n t e r v a l s a n d t h e d e g r e e of a n a e s t h e s i a w a s Table 2 Equiactive concentrations of a n u m b e r of g'uanidine derivatives. The concentrations shown reduced the r e s p o n s e s of isolated a r t e r i a l segments to sympathetic stimulation by 50% in about 20 min.
@ Drug
NH_Ra_NH.C~ NH \NHR b
-R a-
-R b
Concentration producing 50% blockade in 20 rain
-H
2 #g/ml
EM 311
-H
1 #g/ml
-H
1- 2 # g / m l
-H
4 ~g/ml
-CH-CH 2CH 3
I
-CH2-CHCIt 3 EM 313 - ? - C H 2-
t a k e n a s t h e p r o p o r t i o n of m i c e f a i l i n g to a t t e m p t to r e m o v e t h e clip.
2.5. Drugs used
EM 31( -CH2-CH 2-
EM 257
Fig. 1. Isolated rabbit ileum. Effect of EM 311 (2 / l g / ml) on the r e s p o n s e s to stimulation of the l u m b a r sympathetic n e r v e s (at the dots) for 30 see in every 4 min, using 2 msee pulses at a frequency of 2 0 / s e e and sup r a m a x i m a l voltage, and to 0.1 / l g / m l noradrenaline (Nor).
T h e new d r u g s w h o s e a c t i v i t y i s r e p o r t e d in t h i s p a p e r a r e cyclohexylamino-alkyl-guanidines ( E M 9 7 , 257, 310, 311, 313, 326 a n d 3 3 6 ) a n d bis-alkyl-aminoethylguanidines ( E M 65 a n d 66); t h e i r f o r m u l a e a r e s h o w n in t a b l e s 1 a n d 2. T h e s e c o m p o u n d s w e r e p r e p a r e d by D r . W. J. C. Dyke at the Evans Medical Research Laboratories, Liverpool. They were supplied as sulphates. Other drugs used were bretylium tosylate, guanethidine sulphate, guanoxan sulphate, bethanidine sulphate, procaine hydrochloride and dexamphetamine sulphate. Doses and concentrations referred to i n t h e t e x t a r e in t e r m s of these salts. Noradrenaline was used as the bitartrate and the amounts are expressed as the base.
CH 3 EM326
-CH 3
20 p.g/ml
3. R E S U L T S
-CH-CH 2
3.1. Rabbit ileum EM 336
-H
15-20 ~g/ml
-H
> 40 # g / m l
-CH-CH 2EM 97
Drug
-CH2-CH2-CH 2
(R)2-N-CH2-CH2-NH-C~ NH NH 2 Concentration producing R50% blockade in 20 min
EM 65
C2H 5-
6- 8 # g / m l
EM 66
CH 3-
> 40 # g / m l
S t i m u l a t i o n of t h e p e r i a r t e r i a l sympathetic n e r v e s o r t h e a d d i t i o n of n o r a d r e n a l i n e i n h i b i t e d the pendular movements and produced relaxation of t h e i s o l a t e d s e g m e n t of r a b b i t i l e u m . T h e a c t i o n s of t h e c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s o n t h e s e r e s p o n s e s c l o s e l y r e s e m b l e d t h o s e of b r e tylium and guanethidine. They caused a progress i v e r e d u c t i o n i n t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n a n d f o r e a c h d r u g t h e r a t e of d e v e l o p m e n t of b l o c k a d e d e p e n d e d on t h e c o n c e n t r a tion in the bath. For this reason the drugs were c o m p a r e d b y f i n d i n g t h e c o n c e n t r a t i o n of e a c h d r u g w h i c h r e d u c e d s y m p a t h e t i c i n h i b i t i o n by 50% i n 10 m i n .
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA T a b l e 1 s h o w s t h e r e l a t i v e p o t e n c i e s of s o m e of t h e c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s in b l o c k i n g t h e r e s p o n s e s of t h e r a b b i t i l e u m to s t i m u l a t i o n of t h e s y m p a t h e t i c n e r v e s , c o m p a r e d w i t h g u a n e t h i d i n e . T h e s e c o m p o u n d s r a n g e d in p o t e n c y f r o m E M 311, w h i c h w a s a s a c t i v e a s g u a n e t h i d i n e , to E M 97, w h i c h o n l y s l o w l y b l o c k e d t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n i n a c o n c e n t r a t i o n of 0.1 m g / m l . T h e b l o c k a d e w a s very persistent and complete reversal was diffic u l t to p r o d u c e , e v e n w i t h f r e q u e n t w a s h i n g o v e r a n u m b e r of h o u r s . W i t h o n l y o n e e x c e p t i o n , t h e m i n i m u m c o n c e n t r a t i o n s of t h e s e d r u g s w h i c h c o m p l e t e l y b l o c k e d t h e r e s p o n s e s to s y m p a t h e t i c s t i m u l a t i o n d i d n o t r e d u c e t h e r e s p o n s e to n o r a d r e n a l i n e (fig. 1). H o w e v e r , E M 97 r e d u c e d t h e r e s p o n s e s to n o r a d r e n a l i n e b y a b o u t 50% in t h e
203
t f mm Hg
Fig. 3. P e r f u s e d a r t e r i a l segments. Responses to per i a r t e r i a l stimulation at 10 p u l s e s / s e c for 15 sec in every 4 min, using 1 msec pulses and s u p r a m a x i m a l voltage. The r e c o r d s were obtained using t h r e e different p r e p a r a t i o n s and show the effect of i n c r e a s i n g the concentration of guanethidine (0.125, 0.5 and 2 /Xg/ml) on the rate of reduction of the r e s p o n s e s . c o n c e n t r a t i o n t h a t b l o c k e d t h e r e s p o n s e s to s y m pathetic stimulation. T h e b l o c k i n g e f f e c t s of t h e s e d r u g s w e r e a n t a g o n i z e d b y d e x a m p h e t a m i n e . B l o c k a d e of s y m pathetic responses could be prevented by previo u s l y a d d i n g d e x a m p h e t a m i n e to t h e b a t h (fig. 2), and once the blockade was established it could be reversed by dexamphetamine.
Fig. 2. Isolated r a b b i t ileum. Responses to s t i m u l a tion of the l u m b a r sympathetic n e r v e s (at the dots) for 30 sec in every 4 rain using 2 msec pulses at a f r e quency of 2 0 / s e e and s u p r a m a x i m a l voltage. The r e cords show the action of EM 310 (20 //g/ml) on adjacent segments of ileum in simultaneous e x p e r i m e n t s . In the p r e s e n c e of dexamphetamine (0.5 p g / m l ) the blockade of the sympathetic r e s p o n s e s failed to develop.
3.2. Guinea p i g vas deferens T h e a d r e n e r g i c n e u r o n e b l o c k i n g a c t i v i t y of a n u m b e r of t h e g u a n i d i n e c o m p o u n d s w a s a l s o d e termined using the isolated vas deferens. The guinea pig vas deferens and the rabbit ileum p r e p a r a t i o n a r e f r e q u e n t l y u s e d to a s s e s s t h e p o t e n c y of a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s a n d i t w a s t h o u g h t to b e of i n t e r e s t to c o m p a r e t h e r e s u l t s u s i n g t h e two m e t h o d s ( t a b l e 1). 3.3. Rabbit ea r a r t e r y A s s h o w n b y De l a L a n d e a n d R a n d (1965), bretylium and guanethidine completely blocked t h e r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n of t h e i s o l a t e d a r t e r y p r e p a r a t i o n . T h e r e w a s no r e s i d -
204
M . J . RAND and J. WILSON
u a l r e s p o n s e a t t r i b u t a b l e to d i r e c t m u s c l e s t i m u l a t i o n in t h e s e e x p e r i m e n t s . A s w i t h the r a b b i t i l e u m and the g u i n e a p i g v a s d e f e r e n s , the r a t e of d e v e l o p m e n t of b l o c k a d e w a s r e l a t e d to the c o n c e n t r a t i o n of the drug. T h e e f f e c t of i n c r e a s ing the c o n c e n t r a t i o n of g u a n e t h i d i n e i s shown in fig. 3. U s i n g t h i s p r e p a r a t i o n , d r u g s w e r e c o m p a r e d by finding the c o n c e n t r a t i o n w h i c h r e d u c e d the r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n by 50% in 20 min. F o r b r e t y l i u m and g u a n e t h i d i n e , t h e s e Wequiactive c o n c e n t r a t i o n s " w e r e 1-2 t z g / m l , r e spectively. M o s t of the a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s u s e d in t h e s e e x p e r i m e n t s p o t e n t i a t e d n o r a d r e n -
aline. After infusing guanethidine, bretylium, b e t h a n i d i n e , E M 310, E M 311, E M 313 o r E M 257, the r e s p o n s e s to n o r a d r e n a l i n e w e r e inc r e a s e d by 10-140%. G u a n o x a n (0.5-1 t z g / m l ) , h o w e v e r , r e d u c e d the r e s p o n s e s to n o r a d r e n a l i n e (fig. 4). D e x a m p h e t a m i n e w a s found to be e f f e c t i v e in r e v e r s i n g the b l o c k i n g a c t i o n s of g u a n e t h i d i n e , b r e t y l i u m , b e t h a n i d i n e , g u a n o x a n and the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s on t h i s p r e p a r a t i o n . T h e r e s u l t shown in fig. 5 is t y p i c a l . M o r e than 60 m i n a f t e r s t o p p i n g an i n f u s i o n of b e t h a n i d i n e (1 t z g / m l ) , the r e s p o n s e s to p e r i a r t e r i a l s t i m u l a t i o n w e r e s h o w i n g only s l i g h t r e c o v e r y . T h e
Nor
r,rv~
Hq
.o3T
T!
5ng lOng
5ng lOng
Fig. 4. Effects of EM 257 (8 tig/ml) and guanoxan (1 ttg/ml) on the responses of isolated artery segments to periarterial stimulation and to noradrenaline (Nor). The preparations were stimulated for 15 sec every 4 min using 1 msec pulses at 10/sec and supramaximal voltage.
60"Jmin Fig. 5. Responses of the isolated artery segment to p e r i a r t e r i a l stimulation at a frequency of 10/sec for 15 sec every 4 min, using 2 msec pulses and supramaximal voltage. In the first panel, the blocking action of bethanidine (1 /ig/ml) is shown, and in the second the blockade was r e v e r s e d by dexamphetamine (0.5/ig/ml). The duration of infusion is indicated by the white horizontal bars and vertical strokes.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA
In EM 65 and EM 66, the hydrogen and c y c l o hexyl ring on the a m i n o - n i t r o g e n w e r e r e p l a c e d by two m e t h y l groups (EM 66) or ethyl groups (EM 65). EM 65 was a f a i r l y a c t i v e a d r e n e r g i c blocking drug, w h e r e a s EM 66 was p r a c t i c a l l y inactive.
introduction of d e x a m p h e t a m i n e (0.5 p g / m l ) into the p e r f u s i o n fluid f o r 10 min p r o d u c e d a r a p i d and c o m p l e t e r e v e r s a l of the blockade. Table 2 shows the group of guanidine c o m pounds whose p o t e n c i e s w e r e a s s e s s e d using the p e r f u s e d a r t e r y p r e p a r a t i o n . In all of the c o m pounds, with the ex c e p t io n of EM 97, the a m i n o n i t r o g e n and the guanidine group a r e s e p a r a t e d by an ethylene link. Most of the m e m b e r s of the group differ in the alkyl groups attached to this link, the c y c l o h e x y l r i n g and guanidine group r e maining u n a l t e r e d . The m o s t potent compounds in the s e r i e s a r e EM 310, in which the ethylene link is unsubstituted, and EM 257 and EM 311, which have a m e t h y l group on C1 and C2 r e s p e c tively. The introduction of the methyl group slightly i n c r e a s e s a c ti v i ty , EM 311 being r a t h e r m o r e a c t i v e than EM 257. A second methyl group on C 2 (EM 313) c a u s e s a slight fall in activity. R e p l a c e m e n t of the methyl group on EM 311 by an ethyl group c o n s i d e r a b l y r e d u c e d a c t i v i t y (EM 336), as did the introduction of a methyl group onto the guanidine p o r t i o n of the m o l e c u l e (in EM 326). When the length of the alkyl chain linking the c y c l o h e x y l r i n g to the guanidine group was i n c r e a s e d to t h r e e c a r b o n a t o m s , the r e sulting compound (EM 97) had v e r y little a d r e n e r g i c n eu r o n e blocking activity.
3.4. Local anaesthe/ic effecls of adrenergic neu-
rone blocking d~'ugs 3.4.1. Guinea pig weal method The l o cal a n a e s t h e t i c act i o n s of guanethidine, EM 311, E M 3 3 6 and E M 9 7 w e r e c o m p a r e d by i n t r a d e r m a l i n j ect i o n in the guinea pig. T h e s e compounds w e r e s e l e c t e d f o r i n v e s t i g a t i o n b e cau se although c l o s e l y r e l a t e d s t r u c t u r a l l y , they differ c o n s i d e r a b l y in a d r e n e r g i c neurone blocking activity. EM 311 is a potent a d r e n e r g i c neurone blocking drug while EM 336 is much l e s s a c t i v e and EM 97 is a l m o s t inactive. Each drug was i n j e c t e d into two s i t e s on each of t h r e e guinea pigs and each site was t e s t e d by p r i c k i n g six t i m e s . The d e g r e e of a n a e s t h e s i a was taken as the n u m b e r of t i m e s the guinea pigs f a i l e d to r e s p o n d out of the total of 36 t e s t s . The r e s u l t s a r e shown in fig. 6. The local a n a e s t h e t i c potency of the drugs d e c r e a s e d in the following o r d e r : EM 336 > EM 311 > EM 97 = guanethidine. The action of t h ese compounds as l o cal a n a e s -
EM311
Guanethidine 1%
100'
205
i%
ioo
~
75'
75
50'
50
25
25~
r. Q)
0
0
3"0
6o
¢o
12o
15"o
leo
o
3b
6b
9b
i~;o
15"o
105
t-
<
EM97
e3
~, 100.
100'
I~ 75,
75
5o, 25,
50"
3'o
go
,pb
rain
i~
lS'O
la6
O/ o
/
EM336 0.57.
3O min
Fig. 6. Local anaesthetic effect of intradermal injections of adrenergic neurone blocking drugs in the guinea pig. Each point represents the number of times the guinea pig failed to respond the pricking in the injected area, out of a total of 36 tests, expressed as a percentage.
206
M.J. RAND and J. WILSON
t h e t i c s w a s slow i n o n s e t b u t l o n g in d u r a t i o n . T h e d r u g s u s u a l l y took f r o m 1 5 - 3 0 m i n to r e a c h t h e p e a k of t h e i r a c t i o n , b u t in s o m e c a s e s , n o t a b l y w i t h t h e l o w e r c o n c e n t r a t i o n s of E M 311, t h e full e f f e c t w a s not s e e n u n t i l a b o u t 60 m i n a f t e r i n j e c t i o n . One p e r c e n t s o l u t i o n s of g u a n e t h i dine o r E M 311, o r a 0.5% s o l u t i o n of E M 3 3 6 , p r o d u c e d a c o m p l e t e l o c a l a n a e s t h e s i a w i t h i n 30 min after injection and this was still virtually c o m p l e t e a f t e r 180 m i n .
Table 3 Degree of anaesthesia produced by subcutaneous injection of various concentrations of EM 311, EM 336 and guanethidine in the m o u s e ' s tail. The figures show the proportion of each group of ten mice failing to respond to an a r t e r y clip ~laced on the tail.
3.4.2. M o u s e t a i l m e t h o d T h e m e t h o d of B i a n c h i (1956) w a s u s e d to c o m p a r e t h e l o c a l a n a e s t h e t i c p o t e n c y of g u a n e t h i d i n e , E M 311 a n d E M 336. V a r i o u s c o n c e n t r a t i o n s of t h e d r u g s w e r e i n j e c t e d s u b c u t a n e o u s l y into t h e t a i l s of g r o u p s of m i c e . E a c h m o u s e w a s t e s t e d p e r i o d i c a l l y by p l a c i n g a n a r t e r y c l i p on the t a i l , d i s t a l to t h e s i t e of i n j e c t i o n , a n d the d e g r e e of a n a e s t h e s i a w a s t a k e n a s t h e p r o p o r t i o n of t h e g r o u p f a i l i n g to a t t e m p t to r e m o v e t h e c l i p . T h e r e s u l t s a r e s h o w n in t a b l e 3. In t h i s t e s t , E M 311 a n d E M 336, in c o n c e n t r a t i o n s of 0 . 5 - 1 . 5 % , h a d v e r y s i m i l a r l o c a l a n aesthetic activity, and guanethidine was rather less potent. As was found using the guinea pig weal method, t h e o n s e t of l o c a l a n a e s t h e t i c a c t i o n of t h e s e d r u g s w a s slow. T h e m a x i m u m e f f e c t w a s not obtained until from 30-45 min after injecting the drug. This is shown for various concentrations of g u a n e t h i d i n e in fig. 7.
Concentration
EM 311
EM 336
Guanethidine
0.5%
50%
50%
30%
0.75%
60%
60%
50%
1%
70%
70%
60%
1.5%
80%
90%
2%
s0% 100%
-
stimulation, is s h o w n in fig. 8. Procaine (100 p g / m l ) s l i g h t l y r e d u c e d t h e r e s p o n s e s , a n d w h e n t h e c o n c e n t r a t i o n w a s i n c r e a s e d to 200 pg/ ml, the responses were completely blocked. Unl i k e the g r a d u a l , p r o g r e s s i v e b l o c k a d e p r o d u c e d by the adrenergic neurone blocking drugs, the b l o c k a d e p r o d u c e d by p r o c a i n e w a s a l m o s t i m m e d i a t e in o n s e t . W h e n t h e i n f u s i o n of p r o c a i n e w a s s t o p p e d , t h e r e s p o n s e s r e t u r n e d to t h e i r i n i tial height very rapidly. T h e b l o c k i n g a c t i o n of p r o c a i n e on t h e i s o l a t e d g u i n e a p i g v a s d e f e r e n s w a s s i m i l a r to t h a t on t h e a r t e r y s e g m e n t . T h e r e s p o n s e s of t h e v a s d e f e r e n s to h y p o g a s t r i c n e r v e s t i m u l a t i o n w e r e s l i g h t l y r e d u c e d by 40 p g / m l of p r o c a i n e , w h i l e a c o n c e n t r a t i o n of 80 p g / m l r a p i d l y a n d c o m pletely blocked the responses, T h i s b l o c k a d e c o u l d not b e r e v e r s e d by d e x amphetamine (0.5-5 pg/ml), but the responses r a p i d l y r e c o v e r e d on w a s h i n g t h e b a t h .
3.5. Action of procaine on sympathelic nerves T h e e f f e c t of p r o c a i n e on t h e r e s p o n s e of t h e i s o l a t e d a r t e r y s e g m e n t to s y m p a t h e t i c n e r v e
2~. 100"
15
30
45
60
rnin
Fig. 7. Local anaesthetic effect of g~anethidine using the mouse tail method. Each point r e p r e s e n t s the n u m b e r of mice in a group of ten. failing to respond to an a r t e r y clip placed on the tail. The mice were tested at 15 rain i n t e r vals after subcutaneous injection of 0.5, 1 or 2% g~anethidine into the tail.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA
L
i
I
i
I l
L Proc
207
lOO u g m ;
--
,
I
2oougn !
Fig. 8. R e s p o n s e s of the isolated a r t e r y s e g m e n t f r o m the r a b b i t ' s e a r to p e r i a r t e r i a l s t i m u l a t i o n . The p r e p a r a t i o n w a s s t i m u l a t e d at 10 p u l s e s / s e c for 15 sec in e v e r y 4 min, u s i n g 1 m s e c p u l s e s and s u p r a m a x i m a l voltage. The r e c o r d s h o w s the effect of p r o c a i n e (100 and 200 ~ g / m l } , the d u r a t i o n of infusion being indicated by the h o r i z o n t a l b a r s .
4. DISCUSSION The cyclohexylaminoalkylguanidines were v e r y s i m i l a r in t h e i r a c t i o n s on isolated, s y m p a t h e t i c a l l y i n n e r v a t e d o r g a n s , to b r e t y l i u m and guanethidine. They p r o d u c e d a p r o g r e s s i v e r e duction in the r e s p o n s e s to s y m p a t h e t i c n e r v e stimulation. The blockade was v e r y p e r s i s t e n t but could be r e v e r s e d by d e x a m p h e t a m i n e . With the e x cep t i o n of EM 97, c o n c e n t r a t i o n s of th e s e compounds which blo c k e d the r e s p o n s e s to s y m pathetic n e r v e s t i m u l a t i o n , p o t e n ti a t e d n o r a d r e n aline. The r e l a t i v e p o t e n c i e s of t h e s e compounds in blocking the s y m p a t h e t i c n e r v e s to the rabbit il eum d i f f e r e d f r o m t h e i r r e l a t i v e p o t e n c i e s on the h y p o g a s t r i c n e r v e to the v a s d e f e r e n s . Thus, guanethidine and EM 311 w e r e l e s s potent, and EM 97 and EM 336 c o n s i d e r a b l y m o r e potent in blocking s y m p a t h e t i c r e s p o n s e s of the vas d e f e r ens than those of the rabbit ileum. S jS s t r a n d (1962) supposed that the h y p o g a s t r i c n e r v e to the va s d e f e r e n s contains a p r o p o r t i o n of p r e g a n g l i onic s y m p a t h e t i c f i b r e s , and this was s u p p o r t e d by the work of B i r m i n g h a m and Wilson (1963). It is t h e r e f o r e p o s s i b l e that the r e d u c t i o n in the s y m p a t h e t i c r e s p o n s e s of the v a s d e f e r e n s by the l e s s potent guanidines was due to ganglion blockade r a t h e r than to a d r e n e r g i c neurone blockade. The v a l u e s fo r the r e l a t i v e a d r e n e r g i c neur o n e blocking a c t i v i t i e s of the guanidine c o m pounds using the a r t e r y s e g m e n t did not always a g r e e with those found when using the other two i s o l a t e d o rg an s . A notable e x a m p l e is EM 313, which was f a i r l y a c t i v e on the a r t e r y p r e p a r a t i o n although r a t h e r i n a c t i v e in blocking the s y m p a thetic n e r v e s to the r a b b i t ileum. When a d m i n i s t e r e d to c o n s c i o u s r a t s , EM 313 was found to be a highly a c t i v e a n t i h y p e r t e n s i v e agent (Wilson, 1966). It s e e m s p r o b a b l e that an i s o l a t e d v a s c u -
l a r p r e p a r a t i o n is a m o r e a p p r o p r i a t e t e s t object to use in s c r e e n i n g drugs for a d r e n e r g i c neurone blocking activity. In a r e c e n t r e v i e w , Bo u r a and G r e e n (1965) s t a t e d that drugs with a d r e n e r g i c n eu r o n e blocking a c t i v i t y c o n s i s t e d of t h r e e units: a highly b a s i c group, such as q u a t e r n a r y a m m o n i u m , guanidine or a m i d o x i m e , joined through a connecting chain to a ring s t r u c t u r e which may contain a second, m o r e weakly b asi c group. Most of the compounds in the guanidine s e r i e s u s e d conf o r m e d to this pattern. The, a m i n o - n i t r o g e n in the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s c o r r e s p o n d s to the n i t r o g e n in the e i g h t - m e m b e r e d ring of guanethidine. The pKa v a l u e s for EM 310, EM 311, EM 313 and guanethidine w e r e c l o s e l y s i m i l a r ; pKa 1 was 8.25 to 8.6 and pKa2 was 11.3 to 11.4 ( p e r s o n a l c o m m u n i c a t i o n , W . J . C . Dyke). S c h l i t t l e r et al. (1962) found, using a s e r i e s of h e t e r o c y c l i c guanidines and a m i d o x i m e s , that substitution on the ethylene link r e d u c e d activity. With the c y c l o h e x y l a m i n o a l k y l g u a n i d i n e s , the p r e s e n c e of one m e t h y l substituent on the e t h y l ene chain i n c r e a s e d a d r e n e r g i c neurone blocking activity, but with f u r t h e r substitution in the chain a c t i v i t y fell. Costa et al. (1962) studied a s e r i e s of c o m pounds in which the guanidine group was sep ar a t e d f r o m a benzene r i n g by one, two or t h r e e c a r b o n atoms. They found that those compounds having a t w o - c a r b o n chain had " g u a n e t h i d i n e like" a c t i v i t y in that they depleted the content of c a t e c h o l a m i n e s in the heart. T h o s e compounds which had a single carbon link had " b r e t y l i u m like" activity and p r e v e n t e d the depletion of c a t e c h o l a m i n e s by guanethidine. When the length of the chain was i n c r e a s e d to t h r e e carbon a t o m s , the a c t i v i t y was lost (cf. EM 97). The s t r u c t u r a l r e q u i r e m e n t s for o p t i m al a c t i v i t y in the c o m pounds having single c a r b o n links d i f f e r e d s o m e -
208
M.J. RAND and J. WILSON
w h a t f r o m t h o s e of t h e c o m p o u n d s w i t h a n e t h y l e n e l i n k a g e . T h u s , w i t h t h e E M s e r i e s of g u a n i dines and with the ethylguanidine derivatives s t u d i e d b y S c h l i t t l e r e t al. (1962) a n d b y C o s t a e t al. (1962), t h e i n t r o d u c t i o n of m e t h y l g r o u p s i n t o the guanidine portion considerably reduced activity. However, in the methyl guanidine derivat i v e s ( C o s t a et al., 1962; Copp, 1964) m e t h y l s u b s t i t u t i o n in t h e g u a n i d i n e g r o u p r e s u l t e d in increased potency. A l t h o u g h t h e r i n g s t r u c t u r e w a s s a i d to b e necessary for adrenergic neurone blocking act i v i t y ( S c h l i t t l e r e t al., 1962), d i e t h y l a m i n o e t h y l g u a n i d i n e ( E M 65) w a s f o u n d to b e o n l y s l i g h t l y less active than cyclohexylaminoethylguanidine ( E M 310). T h e d i m e t h y l a n a l o g u e ( E M 66) w a s f o u n d to b e i n a c t i v e . S i m i l a r f i n d i n g s to t h e s e w i t h E M 65 a n d E M 66 w e r e r e p o r t e d b y S h o r t e t al. (1963) u s i n g t h e n i c t i t a t i n g m e m b r a n e of t h e conscious cat. G u a n e t h i d i n e , E M 311, E M 336 a n d E M 97 w e r e found, u s i n g t h e B ~ i l b r i n g a n d W a j d a (1945) g u i n e a p i g w e a l t e c h n i q u e , to b e f a i r l y p o t e n t l o cal anaesthetics with a prolonged action. The compound with the most powerful local anaest h e t i c a c t i o n w a s E M 336. A 0.25% s o l u t i o n of E M 336 w a s a s a c t i v e a s a 0.5% s o l u t i o n of E M 311 a n d a l m o s t a s a c t i v e a s a 1% s o l u t i o n of g u a n e t h i d i n e . T h e r e l a t i v e p o t e n c i e s of E M 311, E M 336 a n d g u a n e t h i d i n e , a s d e t e r m i n e d b y t h e B i a n c h i (1956) m o u s e t a i l t e c h n i q u e , d i f f e r e d s l i g h t l y from those found using the guinea pig weal method. E M 311 w a s a s a c t i v e a s E M 336, a n d b o t h compounds were a little more potent than guanethidine. F r o m t h e s e r e s u l t s it m a y b e s e e n t h a t t h e r e i s no d i r e c t r e l a t i o n s h i p b e t w e e n t h e l o c a l a n a e s t h e t i c a c t i v i t y of t h e s e d r u g s a n d t h e i r p o t e n cy a s a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s . B o y d e t al. (1961) i n v e s t i g a t e d t h e l o c a l a n a e s t h e t i c a c t i v i t y of b r e t y l i u m , u s i n g the g u i n e a p i g w e a l m e t h o d , a n d f o u n d t h a t t h e a c t i o n of b r e t y l i u m d i f f e r e d f r o m t h a t of p r o c a i n e in b e i n g s l o w in o n s e t a n d m u c h m o r e p r o l o n g e d . In t h e experiments described here, guanethidine, EM 311, E M 336 a n d E M 97 w e r e f o u n d to h a v e a s i m i l a r t i m e c o u r s e of a c t i o n to t h a t d e s c r i b e d b y B o y d e t al. (1961) f o r b r e t y l i u m . S i m i l a r l y , u s i n g t h e m o u s e t a i l m e t h o d , t h e p e a k of a c t i v i t y f o r e a c h c o n c e n t r a t i o n of g u a n e t h i d i n e , E M 311 a n d E M 336 w a s not r e a c h e d u n t i l f r o m 3 0 - 4 5 min after injecting the drug. This suggests that the adrenergic neurone blocking drugs differ from procaine in the manner in which they produce their local anaesthetic effect. T h e b l o c k i n g a c t i o n of p r o c a i n e on t h e r e -
s p o n s e s of t h e i s o l a t e d p e r f u s e d a r t e r y f r o m t h e r a b b i t ' s e a r d i f f e r e d m a r k e d l y f r o m t h a t of t h e adrenergic neurone blocking drugs. The minim u m c o n c e n t r a t i o n of p r o c a i n e w h i c h b l o c k e d t h e r e s p o n s e s of t h e a r t e r y s e g m e n t to s y m p a t h e t i c nerve stimulation was about one thousand times t h e m i n i m u m b l o c k i n g c o n c e n t r a t i o n of g u a n e t h i dine on t h i s p r e p a r a t i o n . T h i s w a s r a t h e r s u r p r i s i n g s i n c e l o c a l a n a e s t h e t i c s a r e r e p u t e d to be quite active in blocking vasoconstrictor n e r v e s ( H i r s c h f e l d e r a n d B i e t e r , 1932). T h e o n s e t of b l o c k a d e b y p r o c a i n e w a s a l m o s t i m m e d i ate and recovery was also very rapid after stopp i n g t h e i n f u s i o n of t h e d r u g . S i m i l a r r e s u l t s w e r e r e p o r t e d b y B o y d e t al. (1961) a n d in t h i s paper, using the guinea pig vas deferens. Anothe r p o i n t of d i f f e r e n c e b e t w e e n t h e b l o c k a d e of sympathetic nerves by procaine and by the adr e n e r g i c n e u r o n e b l o c k i n g d r u g s , w a s the e f f e c t of d e x a m p h e t a m i n e on t h e b l o c k a d e . T h e b l o c k a d e of t h e r e s p o n s e s of t h e r a b b i t e a r a r t e r y to s y m p a t h e t i c n e r v e s t i m u l a t i o n b y a n y of t h e a d renergic neurone blocking drugs investigated, was reversed by dexamphetamine. The blockade produced by procaine however was unaffected by d e x a m p h e t a m i n e in c o n c e n t r a t i o n s up to 5 p g / m l . The experiments described therefore demons t r a t e a n u m b e r of d i f f e r e n c e s b e t w e e n t h e a d renergic neurone blocking drugs and procaine, b o t h in t h e i r l o c a l a n a e s t h e t i c e f f e c t s a n d i n their sympathetic blocking actions. These res u l t s s u g g e s t t h a t t h e l o c a l a n a e s t h e t i c e f f e c t s of a d r e n e r g i c n e u r o n e b l o c k i n g d r u g s a r e not r e l a t e d to t h e i r b l o c k i n g a c t i o n a t s y m p a t h e t i c nerve endings.
ACKNOWLEDGEMENTS We a r e grateful to Dr. E. M. Glaser, who was Medical D i r e c t o r of Evans Medical at the time when these e x p e r i m e n t s were c a r r i e d out, for his support, and to Dr. W. J. C. Dyke who synthesized the compounds. P a r t of the work reported here was c a r r i e d out during the tenure of an Evans Medical R e s e a r c h Scholarship by J. Wilson.
REFERENCES Bianchi, C., 1956, A simple new quantitative method for testing local anaesthetics, Brit. J. P h a r m a c o l . Chemother. 11, 104. Birmingham, A.T. and A. B. Wilson, 1963, P r e g a n g l i onic and postganglionic stimulation of the guinea pig isolated vas deferens preparation. Brit. J. P h a r m a col. Chemother. 21, 569.
ADRENERGIC NEURONE BLOCKADE AND LOCAL ANAESTHESIA Boura, A . L . A . , F . C . C o p p , W.G. Duneombe, A . F . Green and A. MeCoubrey, 1960, The s e l e c t i v e a c c u mulation of b r e t y l i u m in s y m p a t h e t i c ganglia and t h e i r postganglionic n e r v e s , Brit. J. P h a r m a e o l . C h e m o t h e r . 15, 265. Boura, A . L . A . and A . F . Green, 1965, A d r e n e r g i c neurone blocking a g e n t s , Ann. Rev. P h a r m a c o l . 5, 183. Boura, A.L.A., A.F. Green, A.McCoubrey, D.R.Laurence, R. Moulton and M.L.Rosenheim, 1959, Darenthin. Hypotensive agent of a new type, Lancet 2, 17. Boyd, H., V. Chang and M . J . ' R a n d , 1961, The local a n a e s t h e t i c activity of b r e t y l i u m in relation to the a c tion in blocking s y m p a t h e t i c r e s p o n s e s , Arch. Int. P h a r m a c o d y n . ThOr. 131, 10. Biilbring, E. and I. Wajda, 1945, Biological c o m p a r i son of local a n a e s t h e t i c s , J. P h a r m a e o l . Exp. Ther. 85, 78. Copp, F.C., 1964, A d r e n e r g i c neurone blocking a g e n t s , in: Advances in Drug R e s e a r c h , Vol. 1, eds. N . J . H a r p e r and A. B. S i m m o n d s (Academic P r e s s , London). Costa, E., R . K u n t z m a n , G . L . G e s s a and B. B. Brodie, 1962, S t r u c t u r a l r e q u i r e m e n t s for b r e t y l i u m and guanethidine-like activity in a s e r i e s of g~anidine d e r i v a t i v e s , Life Sci. Oxford 3, 75. De la Lande, I.S. and M . J . Rand, 1965, A s i m p l e i s o lated n e r v e - b l o o d v e s s e l p r e p a r a t i o n , Aust. J. Exp. Biol. Med. Sei. 43, 639.
209
Finkleman, B., 1930, On the n a t u r e of inhibition in the intestine, J. Physiol. 70, 145. H i r s c h f e l d e r , A.D. and R.N. Bieter, 1932, Local ana e s t h e t i c s , Physiol. Rev. 12, 190. Hukovid, S., 1961, R e s p o n s e s of the isolated s y m p a thetie n e r v e - d u c t u s d e f e r e n s p r e p a r a t i o n of the guinea pig, Brit. J. P h a r m a c o l . C h e m o t h e r . 16, 188. McEwen, L . M . , 1956, The effect on the isolated rabbit h e a r t of vagal s t i m u l a t i o n and its modification by cocaine, h e x a m e t h o n i u m and ouabain, J. Physiol. 131, 678. Page, I.H. and H . P . D u s t a n , 1959, A new, potent ant i h y p e r t e n s i v e drug, J. Am. Med. A s s . 170. 1265. Schlittler, E., J. Druey and A . M a r x e r . 1962, Guanidines and a m i d o x i m e s , in: P r o g r e s s in Drug Res e a r c h . Vol. 4, ed. E . J u e k e r (Birkh~user, Basel) p. 341. Short, J . H . , U. B i e r m a e h e r , D . A . D u n n i g a n and T.D. Leth, 1963, Sympathetic n e r v o u s s y s t e m blocking agents. D e r i v a t i v e s of guanidine and related cornpounds, J. Med. Chem. 6, 275. SjSstrand, N.O., 1962, Inhibition by ganglionic blocking a g e n t s of the m o t o r r e s p o n s e of the isolated guinea pig v a s d e f e r e n s to h y p o g a s t r i e nerve s t i m u lation, Acta P h y s i o l . Seand. 54, 306. Wilson, J., 1966, The p h a r m a c o l o g y of hypotensive guanidine compounds, P h . D . T h e s i s , U n i v e r s i t y of London.