Japan. J . Pharmacol.

34, 4 3 5 - 4 4 0 (1984) 435

Structure-Affinity Relationships Between Several New

Benzodiazepine Derivatives and H-Diazepam Receptor Sites 3

Takeshi S H I B U Y A * - * * , Richard F I E L D * * , Yasuo W A T A N A B E * * ,

Katsuhiko S A T O * and Bernard S A L A F S K Y * *
"Department of Pharmacology, Tokyo Medical College, Tokyo 1 60, Japan
" D e p a r t m e n t of Biomedical Sciences, University of Illinois College of Medicine at
Rockford, Rockford, Illinois 6 1 1 0 7 - 1 8 9 7 , U.S.A.

Accepted December 23, 1983

A b s t r a c t — S e v e r a l n e w benzodiazepines w e r e studied w i t h respect t o their ability t o

b i n d specifically t o benzodiazepine receptor sites in rat cerebral cortex m e m b r a n e
f r a c t i o n . The IC50 values of n e w benzodiazepines w e r e c o m p a r e d t o t h a t of
3
diazepam. A g r o u p of t r i a z o l o - [ 1 , 4 ] benzodiazepines displaced H - d i a z e p a m very
effectively. The most p o t e n t of this g r o u p w a s b r o t i z o l a m . Its p o t e n c y w a s a b o u t
ten times higher t h a n t h a t of diazepam. In this study, c a m a z e p a m , w h i c h differs
f r o m diazepam in its C-3 s u b s t i t u t i o n , had the l o w e s t affinity t o the benzodiazepine
receptor site. This p o t e n c y w a s a b o u t 0.006 t h a t of diazepam. C M 7 1 1 6 b o u n d
w i t h the highest affinity to the benzodiazepine receptor site a m o n g the metabolites
of C M 691 2. The length of the side chain at the C-3 p o s i t i o n of this c o m p o u n d is
shorter than t h a t of the other metabolites of C M 6 9 1 2 . These results indicated t h a t
the long side chain at the C-3 position m i g h t i n h i b i t a close interaction b e t w e e n the
receptor site and the substrate m o l e c u l e , thereby leading to l o w - a f f i n i t y b i n d i n g .

W e have already reported t h a t benzodiaze­ the b i n d i n g affinity of several benzodiazepine

pine derivatives affect the rat brain m o n o ­
amine levels ( 1 , 2) and t h a t relationships
b e t w e e n s t r u c t u r e - a c t i v i t y and m o n o a m i n e
levels exist. Recently, h i g h affinity b e n z o d i ­
azepine receptors in rat brain have been
discovered ( 3 , 4) and partially characterized
( 5 ) . Benzodiazepines, in general, share phar­
m a c o l o g i c a l activity due to their a n x i o l y t i c
activity and their depressive effect u p o n the
central nervous system. S t r u c t u r e - a c t i v i t y
relationships have been d e v e l o p e d for the
benzodiazepines. S u b s t i t u t i o n patterns ( 6 ) ,
l i p o p h i l i c i t y ( 7 ) , molecular structure by x - r a y
diffraction ( 8 ) , and b i n d i n g characteristics
( 3 , 4 , 9) have all been used to correlate
specific benzodiazepine drugs t o their phar­
m a c o l o g i c a l p o t e n c y and effects. A s these
results and others ( 1 0 ) suggest, there is a
strong correlation b e t w e e n in vitro b i n d i n g
3
i n h i b i t i o n of H - b e n z o d i a z e p i n e s and in v i v o
tests of benzodiazepine p o t e n c y , w h i c h are
indicative of anxiolytic, a n t i c o n v u l s a n t , or
sedative properties. In this report, w e s t u d i e d
3
derivatives t o H - d i a z e p a m b i n d i n g sites in
rat cortex, and w e discuss the relationship
3
b e t w e e n structure and affinity for the H -
diazepam b i n d i n g sites.
Materials a n d M e t h o d s
M e m b r a n e preparation: M a l e S p r a g u e -
D a w l e y rats w e i g h i n g 2 0 0 - 2 5 0 g w e r e
decapitated, and the brain r e m o v e d rapidly,
dissected into several regions o n ice, and
frozen at - 8 0 ° C . For this assay, frozen rat
brain cortex tissue w a s h o m o g e n i z e d in 10
v o l u m e s of i c e - c o l d 0.32 M sucrose w i t h a
Polytron homogenizer (Brinkman Ins.:
setting 6, 5 sec, 3 X ; 3 0 0 m g = 3 . 0 0 m l ) . The
h o m o g e n a t e w a s c e n t r i f u g e d at 1,000 χ g
( 3 , 5 0 0 P R M . J A - 2 0 rotor, B e c k m a n M o d e l
J - 2 1 C) for 1 0 min at a c o n s t a n t temperature
of 4 ° C . After c e n t r i f u g a t i o n , the resulting
supernatant w a s recentrifuged at 3 0 , 0 0 0 χ g
( 1 9 , 6 5 0 R P M , J A - 2 0 rotor) for 30 min at
4 ° C . The resultant m e m b r a n e pellet w a s
resuspended in i c e - c o l d 50 m M Tris-HCI

Copyright 1984. Production and Hosting by Elseiver B.V. On behalf of Japanese Pharmacological Society.
This is an open access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/ ).
 436 T. Shibuya et al.
buffer ( p H = 7 . 4 at 4 ° C ) and c e n t r i f u g e d (3.0
ml buffer, 3 0 , 0 0 0 χ g ) . A f t e r c e n t r i f u g a t i o n ,
the pellet w a s frozen at - 8 0 ° C . for 1 - 3 0 days
before use.
B i n d i n g assay: The frozen P 2 pellet
prepared previously w a s resuspended in
3.0 ml of Tris-HCI buffer ( p H = 7 . 4 at 4 ° C ) .
Various c o n c e n t r a t i o n s of drugs ( 5 0 β\) w e r e
placed into labelled t u b e s , f o l l o w e d by
a d d i t i o n of 50 β\ of protein ( - 2 5 0 ßg) and
e n o u g h Tris-HCI buffer so t h a t the final
i n c u b a t i o n v o l u m e of each t u b e , after a d d i t i o n
of the radioisotope, w o u l d be 0 . 5 0 0 m l .
A f t e r preparing the tubes, 50 ß\ of 16 n M
3
H - d i a z e p a m (specific activity = 8 6 . 6 G i /
m m o l . N e w England Nuclear) w a s added to
each t u b e at 3 0 sec intervals. After i n c u b a t i o n
f o r 30 m i n in an i c e - w a t e r bath ( ~ 4 ° C ) , 2 x 5
ml of i c e - c o l d Tris-HCI buffer ( 5 0 m M ,
p H = 7 . 4 ) w a s added to the incubation
mixture immediately, and the entire c o n t e n t s
w e r e v a c u u m filtered using a M i l i p o r e filter
apparatus on W h a t m a n G F / B glass fiber
filters, w h i c h had been p r e w e t t e d w i t h 3.0 ml
o f buffer. The filters w e r e t h e n i m m e d i a t e l y
w a s h e d w i t h an additional 2 x 5 ml of i c e - c o l d
buffer. After filtration and d r y i n g for 10 m i n ,
the filters w e r e placed into p o l y e t h y l e n e
scintillation vials ( K i m b l e ) . To each vial w a s
added 10.0 ml of A q u a s o l II ( N e w E n g l a n d .
N u c l e a r ) , f o l l o w e d by moderate shaking for
15 m i n . The radioactivity b o u n d t o each
filter w a s c o u n t e d by l i q u i d scintillation
spectrometry (Searle, Delta 3 0 0 ) for 1 0 m i n
per sample, at a c o u n t i n g efficiency of
40-45%.
3
Specific b i n d i n g of H - d i a z e p a m was
o b t a i n e d by s u b t r a c t i n g the degree of n o n -
3
specific b i n d i n g ( H - d i a z e p . a m b i n d i n g w h i c h
occurred in the presence of 3 ßM diazepam)
3 3
f r o m the total b i n d i n g of H - d i a z e p a m ( H -
diazepam b i n d i n g in the absence of unlabelled
diazepam).
The 50 percent i n h i b i t o r y c o n c e n t r a t i o n
( I C 5 0 ) w a s calculated by log probit analysis.
A n a l i q u o t of the resuspended P pellet 2
w a s used to measure protein c o n c e n t r a t i o n
a c c o r d i n g t o the m e t h o d of L o w r y et al. ( 1 1 ) .
M o s t of the benzodiazepine derivatives
w e r e dissolved in e t h a n o l , and these 1 0 ~ M 2
s o l u t i o n s w e r e stocked at 2 °C until e x p e r i m e n t
w i t h i n one w e e k . Brotizolam and estazolam
w e r e dissolved in m e t h a n o l . C M 6 9 1 3 w a s
freshly dissolved in 50 m M p h o s p h a t e buffer
( p H 7 . 4 ) . This d r u g , w h i c h is a metabolite of
C M 6 9 1 2 , is unstable in acidic or alkaline
s o l u t i o n s and the use of p h o s p h a t e buffer
circumvents possible decarboxylation to
C M 7 1 1 6 , w h i c h c o u l d o c c u r in acidic or
alkaline media.
Results
Structures of the benzodiazepines i n -
vestigated are presented in Fig. 1 .
Figure 2 demonstrates the i n h i b i t o r y effects
of several benzodiazepine derivatives on the
3
H - d i a z e p a m b i n d i n g sites in rat cortex.
IC50 values w e r e calculated for each
benzodiazepine and t h e y are presented in
Table 1 . Linear regression coefficients of all
test drugs were aproximately equal,
i n d i c a t i n g t h a t the same receptor was
involved.
Three t r i a z o l a - [ 1 , 4 ] benzodiazepines w e r e
3
s t u d i e d . A l l three displaced H - d i a z e p a m very
effectively; the m o s t p o t e n t in the g r o u p of
benzodiazepines tested w a s b r o t i z o l a m , w h i c h
3
b o u n d w i t h the highest affinity t o the H -
diazepam b i n d i n g site. The p o t e n c y of
brotizolam w a s a b o u t ten times higher t h a n
t h a t of diazepam (Fig. 1 , Table 1 ).
Of the t y p i c a l benzodiazepine derivatives,
3
lorazepam had the highest affinity t o the H -
diazepam b i n d i n g site, having a p o t e n c y t h a t
w a s three times higher t h a n t h a t of diazepam.
In contrast, t h e p o t e n c y of camazepam w a s
a b o u t 0.006 t h a t of diazepam, and this value
w a s the l o w e s t in the g r o u p of benzodiaze-
pines tested. On the other h a n d , the p o t e n c y
of temazepam w h i c h is one of metabolites of
camazepam w a s a b o u t 7 4 times higher t h a n
t h a t of camazepam. B o t h c l o b a z a m , w h i c h
is a [ 1 , 5] benzodiazepine, and C M 6 9 1 3 ,
w h i c h is a metabolite of C M 6 9 1 2 , w e r e
3
w e a k l y b o u n d t o the H - d i a z e p a m b i n d i n g
site. The p o t e n c y of each w a s a b o u t 0.025
and 0.017 t h a t of diazepam, respectively.
Discussion
Several benzodiazepines w e r e s t u d i e d w i t h
respect to their ability t o b i n d specifically to
benzodiazepine receptor sites i n rat cerebral
cortex tissue. In this report, w e s t u d i e d 3
major different types o f benzodiazepine
 Structure-Activity Relationships of Benzodiazepine to Binding Sites 437

Benzodiazepine 1 subs 2 subs 3 subs 7 subs

Lorazepam H CI OH Cl
CM-7116 H F H Cl
Diazepam CH 3 H H Cl
Nitrazepam H H H N0 2

Flurazepam CH CH N(C2H5)2
2 2
F H Cl
Temazepam CH 3 H OH Cl
CM-6912 H F C0 C H
2 2 5 Cl
CM-6913 H F C0 H 2 Cl
Camazepam CH 3 H OCON(C H 2 3 ) 2 Cl

Brotizolam(WE-94l) R =H. Estazolam Clobazam

R=CH , 3 Alprazolam

Fig. 1. Structures of benzodiazepines investigated

derivatives: triazolo [ 1 , 4 ] benzodiazepines, been d e m o n s t r a t e d ( 1 5 ) .

[ 1 , 5 ] benzodiazepines, and t y p i c a l [ 1 , 4 ]
benzodiazepines ( F i g . 1 ) .
Brotizolam, w h i c h is one of the triazolo [ 1 ,
4 ] benzodiazepines, w a s the strongest dis-
3
placer at the H - d i a z e p a m b i n d i n g site
(Table 1 ). This d r u g is a n e w t h i e n o d i a z e p i n e
and has been reported t o be a p o t e n t
h y p n o t i c - s e d a t i v e in man ( 1 2 - 1 4 ) . A n o t h e r
derivatives of this t y p e , alprazolam ( T U S - 1 ) ,
also effectively displaced the H - d i a z e p a m3
b i n d i n g site. This p o t e n c y w a s a b o u t 3 times
higher t h a n t h a t of diazepam. Interesting
a n x i o l y t i c activity for alprazolam has already
A m o n g the c o m p o u n d s t e s t e d , differences
in b i n d i n g affinity can be related to structural
differences. C M - 7 1 1 6 and flurazepam are
very similar in structure, differing o n l y in
their p o s i t i o n 1 s u b s t i t u t i o n ( F i g . 1 ) . C M -
7 1 1 6 binds nearly five times more s t r o n g l y
t h a n does flurazepam (Table 1 ) ; the amine
side chain f r o m p o s i t i o n 1 in flurazepam most
likely is p r o t o n a t e d at p h y s i o l o g i c a l p H and
t h u s w o u l d affect the l i p o p h y i l i c i t y of the
molecule as c o m p a r e d to the more neutral
molecule C M - 7 1 1 6 .
The h i g h b i n d i n g affinity of lorazepam, as
438 T. Shibuya et al.

U N L A B E L E D LIGAND CONCENTRATION (MOLAR)

3
Fig. 2. Inhibition of H - d i a z e p a m binding in rat cortex by a series of benzodiazepine derivatives. Assays
3
contained approximately 2 5 0 ,ug of synaptosomal membrane protein and 1—6 μ H - d i a z e p a m in 50 m M
Tris-buffer ( p H 7.4 at 0 ° C ) . Each point represents a mean of between 3 - 4 experiments.

3
Table 1. Inhibition of specific H - d i a z e p a m binding (1.6 n M ) to rat brain cortex membranes by b e n ­
zodiazepines

Benzodiazepine IC50 ( n M ) r* Relative potency**

Brotizolam (WE-941 ) 0.6±0.1 0.993 9.50

Lorazepam 1.8±0.4 0.989 3.17
CM-7116 2.1 ± 0 . 6 0.990 2,71
Alprazolam ( T U S - 1 ) 2.9±0.4 0.994 1.97
Diazepam 5.7±0.3 0.996 1.00
Nitrazepam 6.2±0.5 0.990 0.92
Estazolam 6.3±0.7 0.996 0.90
Flurazepam 9.9±.1.3 0.993 0.58
Temazepam 12.2±0.8 0.994 0.47
CM-6912 28.9±0.4 0.995 0.20
Clobazam 225±4 0.995 0.025
CM-6913 331 ± 5 2 0.978 0.017
Camazepam 901 ± 6 2 0.991 0.006
3
Specific H - d i a z e p a m binding to the crude synaptosomal preparation f r o m rat cerebral cortex w a s
assayed as described in the text. Inhibition of specific binding by benzodiazepines w a s determined
at four separate concentrations (in triplicate). The 50 percent inhibitory concentration (IC50) w a s calcu­
lated by log probit analysis. The IC50 values represented are the means of three experiments ± S . E . M .
"Linear regression coefficient from log probit analysis. " C o m p a r e d to unlabelled diazepam.
 Structure-Activity Relationships of Benzodiazepine to Binding Sites
c o m p a r e d t o t h a t of t e m a z e p a m , can be
explained by the added significance of a halo
g r o u p in the 2 ' p o s i t i o n and by the f a c t t h a t
the N-1 position is u n s u b s t i t u t e d in lorazepam
(Table 1 ) . Camazepam, w h i c h is a precursor
of t e m a z e p a m , possesses b i n d i n g w i t h the
l o w e s t affinity t o the benzodiazepine receptor
site. Camazepam differs f r o m diazepam in
its C-3 s u b s t i t u t i o n ; camazepam and its long
carb mate side chain at the C-3 p o s i t i o n
m i g h t i n h i b i t close interaction of the receptor
site and the substrate m o l e c u l e , thereby
leading t o l o w - a f f i n i t y b i n d i n g . It has been
d o c u m e n t e d t h a t t h e benzodiazepine receptor
site is stereoselective for chiral C-3 p o s i t i o n
enantiomers ( 3 ) ; it can be p o s t u l a t e d , t h e n ,
t h a t the molecular p o s i t i o n i n g of the s u b -
strate in the receptor has very specific
g e o m e t r i c requirements and t h a t for overall
effective b i n d i n g , a small s u b s t i t u e n t (if a n y )
m u s t be at the C-3 p o s i t i o n t o maximize t h e
b i n d i n g interaction.
Of the t w o metabolites of C M - 6 9 1 2 tested,
C M - 6 9 1 3 , and C M - 7 1 1 6 , b o t h have an F-
s u b s t i t u t i o n at the C-2 p o s i t i o n . Steric
effects at p o s i t i o n 3 m i g h t explain w h y C M -
6 9 1 2 and C M - 6 9 1 3 bind w i t h l o w e r affinity
t h a n C M - 7 1 1 6 (Table 1 ) ; h o w e v e r , C M -
691 3 binds m o s t w e a k l y of the three possibly
due t o its free carboxyl g r o u p , w h i c h w o u l d
be in the ionized f o r m at p h y s i o l o g i c a l p H .
F o l l o w i n g ester hydrolysis and d e c a r b o x y -
l a t i o n , C M - 6 9 1 2Ns c o n v e r t e d to its metabolite
C M - 7 1 1 6 , a d r u g w h i c h binds w i t h very h i g h
affinity to the benzodiazepine receptor site.
One atypical benzodiazepine derivative w a s
s t u d i e d , c l o b a z a m , a [ 1 , 5 ] benzodiazepine.
This drug e x h i b i t e d very w e a k binding
affinity, 1 / 4 0 t h t h a t of diazepam (Table 1 ) .
Clobazam differs f r o m the others most
n o t a b l y in the geometric c o n f o r m a t i o n of its
s e v e n - m e m b e r e d ring and the presence of
t w o carbonyl g r o u p s at positions 2 and 4.
A l t h o u g h it is n o t possible to correlate
directly binding affinities of the new
benzodiazepines investigated w i t h their as-
sociated p h a r m a c o l o g i c a l activity, s t r u c t u r e -
activity relationships d o c u m e n t that of those
benzodiazepines studied ( 3 , 4 , 9, 1 0 ) (e.g.,
diazepam, nitrazepam, f l u r a z e p a m ) , there is a
relationship b e t w e e n a d r u g ' s b i n d i n g affinity
and its p o t e n c y in v i v o , s u g g e s t i n g there
439
w o u l d be a similar correlation in t h e series of
n e w benzodiazepines under i n v e s t i g a t i o n .
These f i n d i n g s are s u p p o r t e d f r o m data in
a separate study w h e r e a n u m b e r of drugs
used in the present investigation were
e x a m i n e d w i t h respect t o preventing p e n -
tylenetetrazol i n d u c e d c o n v u l s i o n s . A g o o d
positive correlation b e t w e e n these data and
3
the IC50 values on H - d i a z e p a m b i n d i n g
sites in rat cortex w a s n o t e d .
Using the t e c h n i q u e of receptor b i n d i n g ,
one can potentially f i n d n e w and better drugs
based u p o n their ability to b i n d w i t h t h e
appropriate affinity t o a specific receptor.
A d d i t i o n a l l y , by investigating b i n d i n g differ-
ences a m o n g drugs and h o w the structure of
each drug m i g h t affect the affinity of the
receptor site for the substrate m o l e c u l e , the
receptor protein may be more f u l l y char-
acterized.
Finally, using the t e c h n i q u e of receptor
b i n d i n g , c o u p l e d w i t h animal t e s t i n g , it may
be possible t o distinguish in a given drug
w h i c h moiety(ies) of the molecule are
responsible for specific p h a r m a c o l o g i c effects.
Potentially, o n e c o u l d t h e n f i n d a n e w
benzodiazepine w i t h any degree of b i n d i n g
affinity w h i c h demonstrates the desired
p h a r m a c o l o g i c a l profile.
References
1 Shibuya, T., Sato, K., Matsuda, H., Nishimori,
T., Hayashi, M., Nomura, K. and Chen, P.C.:
Effects of benzodiazepines on brain monoamines.
Japan. J . Pharmacol. 26, Supp. 102P (1976)
2 Shibuya, T., Sato, K. and Salafsky, B.: S i m u l -
taneous measurement of biogenic amines and
related c o m p o u n d s by high performance liquid
chromatography ( H P L C ) . Int. J . Clin. Pharmacol.
20, 2 9 7 - 3 0 1 (1982)
3 Möhler, H. and Okada, T.: Benzodiazepine
receptor: Demonstration in the central nervous
system. Science 198, 8 4 9 - 8 5 1 (1977)
4 Squires, R.F. and Braestrup, G.: Benzodiazepine
receptors in rat brain. Nature 266, 7 3 2 - 7 3 4 ,
(1977)
3
5 Möhler, H. and Okada, T.: Properties of H -
diazepam binding to benzodiazepine receptors in
rat cerebral cortex. Life Sei. 20, 2 1 0 1 - 2 1 1 0
(1977)
6 Sternbach, L.H.: Chemistry of 1,4-benzodiaze-
pines and some aspects of the structure-activity
relationship. In The Benzodiazepines, Edited by
440 T. Shibuya et al.
Garattini, S., Mussini, E. and Randall, L O . , p.
4 4 3 - 4 6 1 , Raven Press, N e w York (1973)
7 Biagi, G.L., Barbara, A . M . , Guerra, M.C.,
Babbini, M., Gaiardi, M., Bartoletti, M . and
Borea, P.A.: Values and structure activity
relationship of benzodiazepines. J . M e d . Chem.
23, 1 9 3 - 2 0 1 (1980)
8 Borea, P.A.: Structure-activity relationship in 1,4
benzodiazepines. Boll. Soc. Ital. Biol. Sper. 57,
6 2 8 - 6 3 1 (1981)
9 Tallman, J.F., Paul, S.M., Skolnick, P. and
Gallager, D.W.: Receptors for the age of anxiety:
Pharmacology of the benzodiazepines. Science
207, 2 7 4 - 2 8 1 (1980)
10 Speth, R.C., J o h n s o n , R.W., Regan, J . , Reisine,
T., Kobayashi, R.M., Bresolin, N., Roeske, W.R.
and Yamamura, H.I.: The benzodiazepine receptor
of mammalian brain. Fed. Proc. 39, 3 0 3 2 - 3 0 3 8
(1980)
11 L o w r y , O.H., Rosebrough, N.S., Farr, A . L and
Randall, R.J.: Protein measurement w i t h the
Folin phenol reagent. J . Biol. Chem. 193, 2 6 5 ¬
275 (1951)
12 Fink, M. and I r w i n , P.: Pharmacoelectro-
encephalographic study of brotizolam, a novel
hypnotic. Clin. Pharmacol. Ther. 30, 3 3 6 - 3 4 2
(1981)
13 Nicholson, A . N . , Stone, B.M. and Pascoe, P.A.:
Studies on sleep and performance w i t h a triazolo-
1.4-thienodiazepam (brotizolam). Br. J . Clin.
Pharmacol. 10, 7 5 - 8 1 ( 1 9 8 0 )
14 Saletu, B., Grunberger, J . and Stollberger, I.:
Confirmation of pharmaco-EEG predictions c o n -
cerning pharmacodynamic properties of an
anxiolytic sedative by sleep studies. A d v . Biol.
Psychiat. 6, 1 2 6 - 1 4 2 (1981)
15 Itil, T . M . , Polvan, N., Egilmez, S., Saletu, B. and
Marasa, J . : Anxiolytic effects of a n e w triazoio-
benzodiazepine. Curr. Ther. Res. Clin. Exp. 15,
6 0 3 - 6 1 5 (1973)