Chapter 7
Potent, Rationally Designed Inhibitors of Squalene Synthase 1
2
3
Scott A. Biller , Michael J. Sofia , Jeffrey W. Abt , Barbara DeLange, John K. Dickson, Jr., Cornelia Forster , Eric M . Gordon, Thomas Harrity, David R. Magnin, Joseph Marretta , Lois C. Rich, and Carl P. Ciosek, Jr. 3
4
1
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543
The first class of potent inhibitors of squalene synthase is described. The lead inhibitor, ether 10, was discovered as a result of rational design on the basis of a proposed mechanism for the enzymatic reaction. The ether oxygen was positioned to hydrogen bond with a putative active site acid catalyst and was found to make a substantial contribution to the inhibitor-enzyme binding energy. The structural requirements for potent inhibition are discussed.
W i t h the d i s c o v e r y o f the m e v i n i c a c i d f a m i l y of H M G - C o A r e d u c t a s e i n h i b i t o r s , the i n h i b i t i o n of de novo c h o l e s t e r o l b i o s y n t h e s i s h a s b e e n v a l i d a t e d as a m e t h o d for the t r e a t m e n t o f h y p e r c h o l e s t e r o l e m i a i n h u m a n s (1-3). T h r e e m e m b e r s o f this f a m i l y , l o v a s t a t i n , p r a v a s t a t i n a n d s i m v a s t a t i n , are c u r r e n t l y i n c l i n i c a l use. I n a d d i t i o n , a l a r g e n u m b e r o f s y n t h e t i c a n d s e m i s y n t h e t i c a n a l o g u e s h a v e b e e n r e p o r t e d a n d are i n v a r i o u s stages o f d e v e l o p m e n t (4-8). A l t h o u g h H M G - C o A r e d u c t a s e i n h i b i t o r s are safe a n d h i g h l y e f f i c a c i o u s d r u g s (2, 3), the s t u d y o f a l t e r n a t i v e targets i n the c h o l e s t e r o l b i o s y n t h e t i c p a t h w a y s h o u l d p r o v i d e a d d i t i o n a l i n s i g h t i n t o the m o d u l a t i o n of l i p i d h o m e o s t a s i s .
1
Corresponding authors Current address: Lilly Research Labs, Indianapolis, IN 46285 Current address: SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Current address: University of Illinois, Biotechnology Center B62-1, Urbana, IL 61801
2
3 4
0097-6156/92/0497-O065S06.00/0 © 1992 American Chemical Society
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
66
REGULATION OF ISOPENTENOID METABOLISM
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
W e p r o p o s e d (9-11) that the i n h i b i t i o n o f s q u a l e n e s y n t h a s e m a y h a v e a d v a n t a g e s o v e r i n t e r v e n t i o n e a r l i e r i n the c h o l e s t e r o l b i o s y n t h e t i c p a t h w a y ( F i g u r e 1). S q u a l e n e s y n t h a s e (12-14) c a t a l y z e s the r e d u c t i v e d i m e r i z a t i o n o f f a r n e s y l d i p h o s p h a t e (1, F P P ) to s q u a l e n e (2). T h i s e n z y m e o c c u p i e s a strategic site at the f i n a l b r a n c h p o i n t i n the p a t h w a y . S e l e c t i v e i n h i b i t i o n of s q u a l e n e s y n t h a s e s h o u l d not d i r e c t l y s u p p r e s s the p r o d u c t i o n o f the n o n - s t e r o l i s o p r e n e m e t a b o l i t e s i n c l u d i n g u b i q u i n o n e , d o l i c h o l , isopentenyl f - R N A and isoprenylated proteins. This chapter d e s c r i b e s o u r p r o g r e s s t o w a r d s the d e s i g n , s y n t h e s i s a n d e v a l u a t i o n o f s q u a l e n e s y n t h a s e i n h i b i t o r s (9-11, 15). O u r u l t i m a t e g o a l is the d i s c o v e r y o f a s u p e r i o r agent for treatment o f h y p e r c h o l e s t e r o l e m i a .
Acetyl CoA
^
HMG- a CoA
Cholesterol
a = HMG-CoA Reductase b = Squalene Synthase
Mevalonic Acid
Isopentenyl t-RNA
TOp
S q u a l e n e (2)
F P P (1)
Isoprenyl Proteins
j H
e
m
e
•
Dolichol
Ubiquinone A
F i g u r e 1. I s o p r e n e B i o s y n t h e s i s P a t h w a y : a n a b b r e v i a t e d r e p r e s e n t a t i o n of the b i o s y n t h e t i c routes to c h o l e s t e r o l a n d the n o n - s t e r o l i s o p r e n e s .
E n z y m o l o g y . S q u a l e n e s y n t h a s e is a m i c r o s o m a l p r o t e i n a n d i s t y p i c a l l y s t u d i e d as a c r u d e m i c r o s o m a l p r e p a r a t i o n f r o m l i v e r o r yeast (12-14). W e h a v e d e v e l o p e d a n assay for s q u a l e n e s y n t h a s e a c t i v i t y w h i c h u t i l i z e s the 100,000 χ g m i c r o s o m a l p e l l e t d e r i v e d f r o m rat l i v e r h o m o g e n a t e as a s o u r c e o f e n z y m e a n d gas c h r o m a t o g r a p h y to q u a n t i t a t e s q u a l e n e synthesis f r o m u n l a b e l e d F P P s u b s t r a t e (9, 10). T h e p r o d u c t i o n o f s q u a l e n e is m o r e c o m m o n l y m o n i t o r e d b y the i n c o r p o r a t i o n o f r a d i o l a b e l f r o m H - o r C F P P (12, 14). D i v a l e n t m e t a l i o n ( M g or M n ) a n d r e d u c e d p y r i d i n e n u c l e o t i d e ( N A D H o r N A D P H ) are r e q u i r e d for the e n z y m a t i c r e a c t i o n . 3
+ 2
1
4
+ 2
T h e p u r i f i c a t i o n o f s q u a l e n e s y n t h a s e i n s o l u b l e f o r m has b e e n a c h a l l e n g e . R e c e n t l y , R i l l i n g a n d c o w o r k e r s (16) s u c c e e d e d i n p u r i f y i n g s o l u b i l i z e d s q u a l e n e s y n t h a s e f r o m y e a s t to h o m o g e n e i t y . N o n i o n i c d e t e r g e n t s , m e t h a n o l a n d s u c r o s e w e r e f o u n d to e n h a n c e the s t a b i l i t y o f the s o l u b l e e n z y m e . T w o r e s e a r c h g r o u p s r e p o r t e d the c l o n i n g a n d e x p r e s s i o n o f the s q u a l e n e s y n t h a s e g e n e f r o m the yeast Saccharomyces cerevisiae (17, 18). T h e gene codes for a p r o t e i n o f 444 a m i n o a c i d s ( M = 51.7 k D ) . T h e C - t e r m i n a l h y d r o p h o b i c d o m a i n w a s i d e n t i f i e d as a l i k e l y m e m b r a n e s p a n n i n g r e g i o n w h i c h s e r v e s to a n c h o r the p r o t e i n i n the e n d o p l a s m i c r e t i c u l u m . S q u a l e n e s y n t h a s e a c t i v i t y is c o o r d i n a t e l y r e g u l a t e d (19) b y i n t r a c e l l u l a r s t e r o l c o n c e n t r a t i o n s (20-22), i n a m a n n e r s i m i l a r to H M G - C o A reductase. r
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
7. BILLER ET AL.
Inhibitors of Squalene Synthase
67
R e a c t i o n M e c h a n i s m . T h e s q u a l e n e s y n t h a s e r e a c t i o n is a t w o s t e p t r a n s f o r m a t i o n w h i c h p r o c e e d s t h r o u g h the i n t e r m e d i a t e c y c l o p r o p a n e , p r e s q u a l e n e d i p h o s p h a t e (3, S c h e m e 1) (23). T h e s o l u b l e , p u r i f i e d e n z y m e (16) a n d the r e c o m b i n a n t e n z y m e (17, 18) f r o m y e a s t p r o d u c e b o t h p r e s q u a l e n e d i p h o s p h a t e a n d s q u a l e n e , i n d i c a t i n g that the t w o t r a n s f o r m a t i o n s are c a t a l y z e d b y a s i n g l e p r o t e i n . I n the first step (12, 13), o n e e q u i v a l e n t o f F P P (the p r e n y l d o n o r ) u n d e r g o e s a loss o f the pro-Sh y d r o g e n a n d i n o r g a n i c d i p h o s p h a t e (PPi) f r o m C - l , c o n c o m i t a n t w i t h the i n s e r t i o n o f C - l i n t o the C - 2 / C - 3 a l k e n e o f the s e c o n d e q u i v a l e n t o f F P P (the p r e n y l a c c e p t o r ) to f o r m 3. I n the s u b s e q u e n t s t e p (12, 13, 24), presqualene diphosphate undergoes a reductive rearrangement i n w h i c h a s e c o n d m o l e c u l e o f P P i is released a n d N A D ( P ) H is c o n s u m e d to f o r m the s y m m e t r i c a l d i m e r , squalene. T h e d o n o r a n d acceptor b i n d i n g sites e x h i b i t i n d e p e n d e n t s e l e c t i v i t y for the acceptance o f m o d i f i e d F P P a n a l o g u e s as substrates, i n d i c a t i n g that the t w o sites are d i s t i n c t (25, 26).
It has b e e n p r o p o s e d that the i n i t i a l process i n v o l v e d i n b o t h steps o f the s q u a l e n e s y n t h a s e r e a c t i o n is h e t e r o l y s i s o f the C - O P P i b o n d to f o r m a n i o n p a i r ( S c h e m e 2) (12, 24, 27). I n the case o f s t e p 1, a l l y l c a t i o n 4 is p r o d u c e d , w h e r e a s i n the case o f step 2, the c y c l o p r o p y l c a r b i n y l c a t i o n 5 is f o r m e d . T h i s p r o p o s a l is s u p p o r t e d b y c h e m i c a l m o d e l s t u d i e s for step 2 as w e l l as e l e g a n t i n h i b i t i o n s t u d i e s i n v o l v i n g m i m i c s o f i o n p a i r 5 (12, 24, 27). S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e that the c l o s e l y r e l a t e d p r e n y l transferase r e a c t i o n c a t a l y z e d b y F P P s y n t h a s e p r o c e e d s via a l l y l i o n p a i r s a n a l o g o u s to 4 (27-31). I n a d d i t i o n , h e t e r o l y s i s o f d i p h o s p h a t e esters to f o r m s t a b i l i z e d c a r b o n i u m i o n s is a d o c u m e n t e d aspect o f the i n t r i n s i c c h e m i s t r y o f t h i s f u n c t i o n a l g r o u p (32). T h e s t u d i e s d e s c r i b e d i n t h i s c h a p t e r center o n the i n i t i a l process o f i o n p a i r f o r m a t i o n . S p a c e d o e s n o t
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
68
REGULATION OF ISOPENTENOID METABOLISM
allow for discussion of the fascinating mechanistic transformations subsequent to the initial step and the reader is referred to several key reviews (12-14, 24, 27).
Scheme 2 R' /
Ρ
Ρ
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
Ο
,0-
ι
I
Η
ο
ο
I
I
Ο
Me
Mg • 2
Λ
ρ -
Mg • 2 Step 2
Stepl
Ο Ρ
tu
Ρ^
ι
-ο.
R'
Ο I
.ΡMg*
Ο €Ηο
+
ο
I
I
-Ο
.0
Me
2
D i p h o s p h a t e Surrogates In the d e s i g n o f i n h i b i t o r s o f s q u a l e n e s y n t h a s e b a s e d o n the s t r u c t u r e o f s u b s t r a t e 1, as w e l l as r e l a t e d t r a n s i t i o n states a n d i n t e r m e d i a t e s , stable s u r r o g a t e s for the d i p h o s p h a t e m o i e t y h a v e to b e d e v i s e d . A l t h o u g h the d i p h o s p h a t e is c r u c i a l for b i n d i n g a n d c a t a l y s i s , i t is u n s u i t a b l e as a n i n h i b i t o r c o m p o n e n t d u e to its c h e m i c a l ( h i g h a n d l o w p H ) (32) a n d biological (phosphatases) lability. W e have evaluated a n u m b e r of p o t e n t i a l d i p h o s p h a t e s u r r o g a t e s (9-11), a n d c o n c e n t r a t e h e r e o n the ( p h o s p h i n y l m e t h y l ) p h o s p h o n a t e ( P M P ) m o i e t y (9, 15). P a r e n t P M P 6a is s i m p l y the r e s u l t o f r e p l a c i n g b o t h the r e a c t i v e a l l y l i c a n d a n h y d r i d e o x y g e n s o f F P P w i t h C H 2 (9). T h i s m o d i f i c a t i o n affords a stable m o l e c u l e w h i c h is u n a b l e to u n d e r g o the i n i t i a l step o f the s q u a l e n e s y n t h a s e r e a c t i o n . A priori, it w a s d i f f i c u l t to p r e d i c t w h e t h e r e i t h e r o f these t w o o x y g e n s w a s c r u c i a l for i n t e r a c t i o n w i t h the e n z y m e . U p o n e v a l u a t i o n i n the rat l i v e r m i c r o s o m a l s q u a l e n e s y n t h a s e a s s a y , 6a p r o v e d to b e a n effective i n h i b i t o r of the e n z y m e , I50 = 31.5 μ Μ ( T a b l e 1). L i n e w e a v e r - B u r k a n a l y s i s i n d i c a t e s that 6a is c o m p e t i t i v e w i t h respect to F P P w i t h K j = 10 μ Μ ( F i g u r e 2A). C o m p a r i s o n w i t h the M i c h a e l i s c o n s t a n t for F P P u n d e r o u r assay c o n d i t i o n s ( a p p a r e n t K = 12.7 μ Μ ) reveals that 6a a n d F P P b i n d to the e n z y m e w i t h s i m i l a r a f f i n i t y . W e i n f e r f r o m t h i s t h a t n e i t h e r the a n h y d r i d e n o r a l l y l i c o x y g e n s o f F P P are critical for substrate b i n d i n g . m
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
7. BILLERETAL.
69
Inhibitors of Squalene Synthase
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
R
T a b l e 1. I n h i b i t i o n o f S q u a l e n e S y n t h a s e b y C o m p o u n d s 6, 7 a n d 8* Cpd Cpd ΐ5ο (μΜ) 6a 7a 31.5 6b 12.2 7b 6c 8a -40 % @ 600 *For assay conditions, see references 9 and 10.
I 0 (μΜ) 29.9 15.5 42 5
T h e r e s u l t s of o u r i n i t i a l s t u d y o n P M P i n h i b i t o r s r e l a t e d to 6a are p r e s e n t e d i n T a b l e 1. 1 , 3 - D i e n y l P M P 6 b e x h i b i t e d i m p r o v e d a c t i v i t y r e l a t i v e to 6a, s u g g e s t i n g that a v i n y l g r o u p m a y be a s u p e r i o r substitute for the a l l y l i c C - O b o n d . T h e d i f l u o r o - P M P s u r r o g a t e of 7a a n d 7b w a s i n t e n d e d to m o r e c l o s e l y m i m i c the d i p h o s p h a t e of F P P w i t h respect to p K v a l u e s (33) , t h e r e b y f a v o r i n g the f u l l y i o n i z e d f o r m at p h y s i o l o g i c a l p H . T h e e q u i v a l e n t i n h i b i t o r y p o t e n c i e s o f the c o r r e s p o n d i n g P C H 2 P (6a, 6 b ) a n d P C F 2 P ( 7 a , 7 b ) a n a l o g u e s i n d i c a t e that b o t h 6 a n d 7 c a n a c h i e v e the p r o t o n a t i o n state n e c e s s a r y for b i n d i n g to the e n z y m e . T h e g e r a n y l d e r i v a t i v e 6c is at least 20-fold less potent t h a n its f a r n e s y l c o u n t e r p a r t 6 b . T h i s is c o n s i s t e n t w i t h r e p o r t s that g e r a n y l d i p h o s p h a t e is n o t a substrate (34) a n d is a p o o r i n h i b i t o r (35) for s q u a l e n e synthase. I n h i b i t o r 8a (36), a n a
1/(Π>ΡμΜ)
1/ΕΡΡμΜΐ
F i g u r e 2. L i n e w e a v e r - B u r k a n a l y s i s o f the i n h i b i t i o n o f rat l i v e r m i c r o s o m a l s q u a l e n e synthase b y 6a ( F i g u r e 2 A ) a n d 10 ( F i g u r e 2 B ) .
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
70
REGULATION OF ISOPENTENOID METABOLISM
a n a l o g u e w h i c h retains the a n h y d r i d e l i n k a g e , possesses a c t i v i t y c o m p a r a b l e to 6 a , p r o v i d i n g further e v i d e n c e that the a n h y d r i d e o x y g e n is n o t e s s e n t i a l . I n r e l a t e d w o r k (37), P o u l t e r , M c C l a r d a n d c o w o r k e r s d e m o n s t r a t e that the P M P a n a l o g u e s o f i s o p e n t e n y l - a n d d i m e t h y l a l l y l d i p h o s p h a t e are i n h i b i t o r s o f the p r e n y l transferase, F P P s y n t h a s e . R a t i o n a l D e s i g n of Potent P M P Inhibitors
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
R a t i o n a l e . P M P i n h i b i t o r s 6a,b a n d 7a,b b i n d to s q u a l e n e s y n t h a s e w i t h a n a f f i n i t y w h i c h is c o m p a r a b l e to that o f the substrate F P P a n d t h u s r e p r e s e n t a s t a r t i n g p o i n t for f u r t h e r s t u d i e s . A s a b a s i s for r a t i o n a l d e s i g n , w e f o c u s e d o n a h y p o t h e t i c a l m o d e l for F P P b i n d i n g to the d o n o r site o f the e n z y m e a n d s u b s e q u e n t i o n - p a i r f o r m a t i o n ( F i g u r e 3). W e e n v i s i o n that
F i g u r e 3. H y p o t h e t i c a l m o d e l of the d o n o r site o f s q u a l e n e s y n t h a s e , b o u n d to: (A) s u b s t r a t e F P P , (B) the a l l y l c a t i o n - d i p h o s p h a t e i o n p a i r 4 a n d ( C ) e t h e r i n h i b i t o r 10. T h e a c t i v e s i t e f u n c t i o n A - H r e p r e s e n t s the p u t a t i v e a c i d catalyst a n d X r e p r e s e n t s c a t i o n i c s i d e chain(s) w h i c h i o n p a i r w i t h the p h o s p h a t e g r o u p s . +
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
7.
BILLER ET AL.
71
Inhibitors of Squalene Synthase
F P P i s b o u n d to the d o n o r site o f s q u a l e n e s y n t h a s e via h y d r o p h o b i c i n t e r a c t i o n s w i t h the i s o p r e n e s u b u n i t a n d i o n i c i n t e r a c t i o n s w i t h the highly charged diphosphate moiety (Figure 3A). W e propose two m e c h a n i s m s b y w h i c h the e n z y m e m i g h t c a t a l y z e the g e n e r a t i o n o f i o n p a i r 4 f r o m e n z y m e b o u n d 1. F i r s t , w e e x p e c t that the e n z y m e interacts m o r e s t r o n g l y w i t h the t w o fragments f o r m e d i n the c o n v e r s i o n o f 1 to 4 as the d i s t a n c e b e t w e e n t h e m a p p r o a c h e s the transition-state s e p a r a t i o n . T h e e n z y m e m i g h t a c c o m p l i s h this b y p o s i t i o n i n g the i s o p r e n e a n d d i p h o s p h a t e b i n d i n g p o c k e t s s u c h that o p t i m a l b i n d i n g e n e r g i e s are a c h i e v e d at the t r a n s i t i o n - s t a t e d i s t a n c e . I n this m a n n e r , the a c t i v e site s e r v e s to stretch the C - O P P j b o n d . S e c o n d , w e p r o p o s e that s q u a l e n e s y n t h a s e u t i l i z e s an a c t i v e site a c i d catalyst to p r o m o t e i o n - p a i r f o r m a t i o n ( A - H i n F i g u r e 3). T h i s a c i d is e x p e c t e d to interact w e a k l y w i t h substrate 1 d u e to the p o o r b a s i c i t y of the ester o x y g e n , b u t m u c h m o r e s t r o n g l y as the b a s i c p h o s p h a t e a n i o n o f 4 is f o r m e d . I d e n t i c a l a r g u m e n t s c a n be a p p l i e d to the f o r m a t i o n o f i o n - p a i r 5 f r o m 3. F o l l o w i n g the a b o v e a n a l y s i s , w e a t t e m p t e d to create m o r e p o t e n t P M P i n h i b i t o r s b y : (1) i n c r e a s i n g the s e p a r a t i o n b e t w e e n the i s o p r e n y l s u b u n i t a n d the P M P s u r r o g a t e i n o r d e r to a p p r o x i m a t e t h e i o n p a i r s e p a r a t i o n a n d / o r (2) i n c o r p o r a t i n g f u n c t i o n s i n the l i n k i n g c h a i n to i n t e r a c t w i t h the p u t a t i v e a c t i v e site a c i d . T h e f o r m e r m a y be d i f f i c u l t to a c c o m p l i s h , s i n c e w e are r e s t r i c t e d to n o r m a l c o v a l e n t b o n d l e n g t h s , a l i m i t a t i o n n o t s h a r e d b y t r a n s i t i o n states. R e s u l t s . L i n k e r h o m o l o g 6 d (9), a n a t t e m p t to a p p r o x i m a t e the t r a n s i t i o n state s e p a r a t i o n b e t w e e n the f r a g m e n t s , is a c t u a l l y a s o m e w h a t w e a k e r i n h i b i t o r of the e n z y m e than 6a ( T a b l e 2). I n c o r p o r a t i o n o f a p h o s p h o n a t e ester o x y g e n a t o m i n the l i n k i n g c h a i n to interact w i t h the p u t a t i v e a c t i v e site a c i d ( e x a m p l e 9) results i n a 4 - f o l d increase i n a c t i v i t y r e l a t i v e to 6 d . T h i s m o d e s t i m p r o v e m e n t is n o t s u r p r i s i n g , s i n c e a n ester o x y g e n , l i k e that of F P P itself, is expected to f o r m a r e l a t i v e l y w e a k h y d r o g e n b o n d w i t h T a b l e 2. L i n k e r M o d i f i c a t i o n s o f the P M P I n h i b i t o r s * v
Ο
Ο _
o- oCpd
Linker X
ΐ5θ(μΜ)
Cpd
Linker X
I50 ( μ Μ ) 0.15
31.5
12
-CH OCH -
6d
-CH CH -
67.0
13
-CH CH OCH -
79.3
9
-CH 0
16.0
14
-NH +CH -
16.0
10
-OCH -
0.05
15
-SCH -
122
6a
-CH 2
2
2
2
2
2
2
2
2
2
2
2
2
11 -OCH CH 2.3 *For assay conditions, sec references 9 and 10. 2
2
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
REGULATION OF ISOPENTENOID METABOLISM
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
72
the a c i d m o i e t y . W h e n a m o r e b a s i c e t h e r o x y g e n w a s i n s e r t e d i n the l i n k i n g c h a i n (example 10), a p r o f o u n d increase i n i n h i b i t o r y p o t e n c y r e s u l t e d (15). E t h e r 10 (I50 = 0.05 μ Μ ) is 1 3 4 0 - f o l d m o r e p o t e n t t h a n its c a r b o n isostere 6 d as a n i n h i b i t o r o f s q u a l e n e s y n t h a s e ! I n h i b i t o r 10 w a s d e m o n s t r a t e d to be c o m p e t i t i v e w i t h respect to F P P , K j = 0.037 μ Μ ( F i g u r e 2B). F u r t h e r m o d i f i c a t i o n s o f the l i n k i n g r e g i o n (15) w e r e a i m e d at r e v e a l i n g s t r u c t u r a l features that are i m p o r t a n t for i n h i b i t i o n ( T a b l e 2). T h e p o s i t i o n of the ether o x y g e n r e l a t i v e to the p h o s p h i n i c a c i d is c r i t i c a l . I n s e r t i o n o f a n a d d i t i o n a l C H 2 b e t w e e n the e t h e r a n d p h o s p h i n i c a c i d results i n a 4 6 - f o l d loss i n a c t i v i t y ( e x a m p l e 11), w h e r e a s h o m o l o g a t i o n o n the o t h e r s i d e o f the ether ( e x a m p l e 12) leads to just a 3 - f o l d loss. F u r t h e r h o m o l o g a t i o n o f 12 to 13, h o w e v e r , is h i g h l y d e l e t e r i o u s . C o n s i s t e n t w i t h the r e q u i r e m e n t f o r a h y d r o g e n b o n d a c c e p t o r is t h e u n i m p r e s s i v e i n h i b i t o r y p o t e n c i e s o f a m m o n i u m i o n 14, w h i c h is a n Η - b o n d d o n o r , a n d thioether 15, w h i c h is a p o o r Η - b o n d acceptor. Enzyme-Inhibitor B i n d i n g Energy It w o u l d be u s e f u l for future w o r k i n the area o f m o l e c u l a r r e c o g n i t i o n to a r r i v e at a q u a n t i t a t i v e a c c o u n t i n g o f the c o n t r i b u t i o n m a d e b y the e t h e r o x y g e n to e n z y m e - i n h i b i t o r i n t e r a c t i o n s . In o r d e r to a c c o m p l i s h t h i s , w e c o m p a r e ether 10 to its c a r b o n isostere 6 d ( F i g u r e 4). T h e difference b e t w e e n the e n z y m e b i n d i n g energies of 10 a n d 6 d i n a q u e o u s s o l u t i o n ( A A G t e r ) is c a l c u l a t e d to b e 4.3 k c a l / m o l u t i l i z i n g t h e e x p r e s s i o n : A A G t e r = - R T l n [ l 5 o ( 1 0 ) / l 5 o ( 6 d ) ] . T h e d i f f e r e n t i a l b i n d i n g e n e r g y i n the a b s e n c e o f s o l v e n t ( A A G ) (38) is a m o r e m e a n i n g f u l m e a s u r e o f the d i f f e r e n c e b e t w e e n the t w o i n h i b i t o r s , s i n c e , b y f a c t o r i n g o u t s o l v a t i o n , A A G is s o l e l y a f u n c t i o n o f the i n t e r a c t i o n s w i t h i n the E-I c o m p l e x . U s i n g the t h e r m o d y n a m i c c y c l e i n F i g u r e 4 as s u g g e s t e d b y the w o r k of Bartlett (38-41), the e x p r e s s i o n i n e q 1 for A A G t r is d e r i v e d . S o l v i n g for AAGgas affords e q 2, s i n c e A A G 1 V - E is z e r o . T o f u r t h e r s i m p l i f y t h i s e x p r e s s i o n (38), w e m a k e the a s s u m p t i o n that the t e r m AAGSO1V-E-I is a l s o z e r o , s i n c e i t is l i k e l y that w a t e r is e x c l u d e d f r o m the b i n d i n g site o f b o t h E - I c o m p l e x e s . If a n y t h i n g , t h i s a p p r o x i m a t i o n s h o u l d l e a d to a n underestimation of A A G , s i n c e the s o l v a t i o n e n e r g y o f E - 6 d h a s the p o t e n t i a l to be l a r g e r t h a n that of E-10 i n the e v e n t that w a t e r is accessible to the a c t i v e site f u n c t i o n a l i t y (eg. the p u t a t i v e a c i d c a t a l y s t ) w h i c h i s satisfied b y the ether i n the case o f E - 1 0 . T h u s , A A G is a p p r o x i m a t e d as the s u m o f A A G r (4.3 k c a l / m o l ) a n d A A G 1 V - I , w h e r e A A G I - I i s t h e difference b e t w e e n the s o l v a t i o n energies o f 10 a n d 6 d . w a
w a
g a s
g a s
w a
e
S 0
g a s
g a s
w a t e
S 0
S O
V
A c c o r d i n g to the e m p i r i c a l c o r r e l a t i o n s o f H i n e (42), a n ether o x y g e n c o n t r i b u t e s 4.2 k c a l / m o l m o r e t h a n a C H 2 to the s o l v a t i o n e n e r g y . T h u s , AAG for 10 r e l a t i v e to 6 d is e s t i m a t e d to b e 8.5 k c a l / m o l . I n w a t e r , a p p r o x i m a t e l y h a l f o f this e n e r g y is e x p e n d e d f o r the d e s o l v a t i o n o f the e t h e r f u n c t i o n . A s s u m i n g that 10 a n d 6 d b i n d to s q u a l e n e s y n t h a s e i n the s a m e m a n n e r , w e s p e c u l a t e that t h i s l a r g e d i f f e r e n t i a l b i n d i n g e n e r g y is d u e to h y d r o g e n b o n d i n g o f the ether o x y g e n to a n active-site a c i d catalyst g a s
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
7. BILLER ET AL.
Inhibitors of Squalene Synthase
73
as i l l u s t r a t e d i n F i g u r e 3 - C . T h e ether o x y g e n i s o f i n t e r m e d i a t e b a s i c i t y r e l a t i v e to the w e a k l y b a s i c p h o s p h a t e ester o x y g e n i n F i g u r e 3 - A a n d the s t r o n g l y b a s i c p h o s p h a t e a n i o n i n F i g u r e 3 - B , a n d c a n b e c o n s i d e r e d to b e a m i m i c o f a species a l o n g the r e a c t i o n c o o r d i n a t e f r o m A to B . L a r g e A A G h a v e b e e n o b s e r v e d p r e v i o u s l y for i s o s t e r i c f u n c t i o n a l g r o u p r e p l a c e m e n t s i n i n h i b i t o r s o f t h e r m o l y s i n (38, 39) a n d n u c l e o s i d e d e a m i n a s e s (40, 41).
g a s
A G water
F + τ ^water ^ water
E - I water
1
- A G solv-I
- A G solv-E
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
E AAG*
= A A G
g
a
s
-AAG
+
s o l v
A G gas
*gas _
E
A G solv-E-I
- A A G solv-I + A A G
A A G
A A G
E - I gas
g a s
+
AAG
AAG
gas = water + s o l v - I " solv-E-I = 4.3 + 4 . 2 + 0 = 8.5 k c a l / m o l
s o l v
e(
( l
. . E
2
I
(eq D
)
F i g u r e 4. T h e r m o d y n a m i c c y c l e is u t i l i z e d to estimate the d i f f e r e n t i a l b i n d i n g e n e r g y o f 10 r e l a t i v e to 6 d i n the absence o f s o l v e n t ( A A G
g a s
).
A n a l t e r n a t i v e e x p l a n a t i o n for the l a r g e d i f f e r e n t i a l b i n d i n g e n e r g y is a n i n d i r e c t effect o f the ether o n the i n t e r a c t i o n o f t h e P M P s u r r o g a t e w i t h t h e e n z y m e . If a n y t h i n g , the e l e c t r o n w i t h d r a w i n g c a p a b i l i t i e s o f the e t h e r w o u l d b e e x p e c t e d to decrease the b i n d i n g e n e r g y o f t h e adjacent p h o s p h i n i c a c i d g r o u p (38, 43). A g a i n , t h i s w o u l d l e a d t o a n u n d e r e s t i m a t i o n o f the c o n t r i b u t i o n m a d e b y the ether. Further Structure-Activity Studies M o d i f i c a t i o n s o f the P M P D i p h o s p h a t e Surrogate. Studies were undertaken to d e t e r m i n e w h e t h e r the c o m p l e t e t r i a c i d P M P s u r r o g a t e w a s e s s e n t i a l for potent i n h i b i t i o n . T h e strong interaction offered b y the ether does not c o m p e n s a t e f o r t h e loss o f either p h o s p h o r u s m o i e t y , as e v i d e n c e d b y the p o o r a c t i v i t y e x h i b i t e d b y 16 a n d 17 ( T a b l e 3). F u r t h e r a t t e m p t s t o r e d u c e the t r i a c i d P M P s u r r o g a t e o f 10 to a d i a c i d r e s u l t e d i n c o n s i d e r a b l e loss o f p o t e n c y as e v i d e n c e d b y e x a m p l e s 18 - 23. I n the case o f p h o s p h i n y l acetate
24, Iso = 102 μ Μ
25, X = C H , 1 2
5 0
= 31.8 u M
26, X = 0 , 1 ^ = 8.7 μ Μ
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
74
REGULATION OF ISOPENTENOID METABOLISM T a b l e 3. M o d i f i c a t i o n s o f the P M P D i p h o s p h a t e S u r r o g a t e *
Cpd
10
X
C
0
0
II
II
00
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
16
ξ-Ρ-000
e
0
0
II
II
ξ— P s ^ P - O M e '
19
C
1
Cpd
0.05
20
C
1
II
0 0
II
H
\—P^P-OCH 0 -
94
3
75
*For assay condirions, see references 9 and 10. 2 1 , the ether a n a l o g u e is 8-fold m o r e p o t e n t t h a n the c o r r e s p o n d i n g ester l i n k e d c o m p o u n d 24; h o w e v e r , this is a m i n o r i m p r o v e m e n t c o m p a r e d to the s a m e m o d i f i c a t i o n i n the P M P series (9 vs 10). P h o s p h i n y l f o r m a t e 22 is a c t u a l l y less p o t e n t t h a n isosteres 2 5 a n d 26(11), s u g g e s t i n g that the p h o s p h i n y l formates b i n d e i t h e r to a different site o n the e n z y m e (eg, the acceptor site), o r to the same site b u t i n a different m a n n e r . M o d i f i c a t i o n o f the I s o p r e n e S u b u n i t . T h e results o f o u r s t u d i e s o n the m o d i f i c a t i o n o f the f a r n e s y l s u b u n i t o f 10 are p r e s e n t e d i n T a b l e 4. B e g i n n i n g w i t h the t e r m i n a l i s o p r e n e u n i t , r e d u c t i o n o f the o l e f i n ( e x a m p l e 27) a n d further r e m o v a l o f a C H 3 g r o u p ( e x a m p l e 28) b o t h l e d to > 10-fold loss i n p o t e n c y r e l a t i v e to 10. T h e s e s a m e m o d i f i c a t i o n s to F P P itself h a v e b e e n r e p o r t e d to afford g o o d substrates for s q u a l e n e s y n t h a s e (26, 34). D e l e t i o n o f the entire t e r m i n a l i s o p r e n e u n i t ( g e r a n y l a n a l o g u e 29) effects a n a l m o s t c o m p l e t e l o s s o f i n h i b i t o r y a c t i v i t y . T h i s is c o n s i s t e n t w i t h o u r p r i o r o b s e r v a t i o n w i t h g e r a n y l P M P 6c (vide supra), a n d c o n f i r m s the r e q u i r e m e n t for a c o m p l e t e f a r n e s y l c h a i n - l e n g t h for s t r o n g e n z y m e inhibitor binding. T h e e n z y m e is m o r e tolerant of s t r u c t u r a l v a r i a t i o n s i n the i s o p r e n e u n i t p r o x i m a l to the ether. R e m o v a l o f the v i n y l i c m e t h y l g r o u p ( e x a m p l e
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
7. BILLER ET AL.
IS
Inhibitors of Squalene Synthase
30) , h o m o l o g a t i o n to a n e t h y l g r o u p ( e x a m p l e 33) a n d r e p l a c e m e n t w i t h c h l o r i d e ( e x a m p l e 34) a l l r e s u l t i n m i n i m a l loss o f p o t e n c y . W h e n the first t w o m o d i f i c a t i o n s are m a d e to F P P itself, the r e s u l t i n g a l l y l i c d i p h o s p h a t e s a r e r e p o r t e d to b e e x t r e m e l y p o o r s u b s t r a t e s f o r the e n z y m e (25, 26). S u r p r i s i n g l y , r e p l a c i n g the p r o x i m a l (E)-olefin w i t h a n a c e t y l e n e ( e x a m p l e 31) o r a ( Z ) - o l e f i n ( e x a m p l e 32) l e a d s to c o m p l e t e r e t e n t i o n o f i n h i b i t o r y a c t i v i t y . M o l e c u l a r m o d e l i n g studies suggest that b o t h 31 a n d 32 c a n attain c o n f o r m a t i o n s that o v e r l a p w i t h 10 at b o t h the t e r m i n a l i s o p r e n e u n i t a n d the e t h e r - P M P p o r t i o n s of the m o l e c u l e , w h i l e b r i d g i n g the t w o t e r m i n i via d i f f e r e n t r o u t e s . T h e s e r e s u l t s i n d i c a t e that t h e r e is a n e l e m e n t o f s t r u c t u r a l f l e x i b i l i t y i n the i n t e r i o r r e g i o n o f the i n h i b i t o r .
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
T a b l e 4. M o d i f i c a t i o n s o f the I s o p r e n y l S u b u n i t * Ο II
ο II
.P.
ο- ο Cpd
R
οR
ΙδοίμΜ)
Ι50 (μΜ)
Cpd
10
0.05
33
0.078
27
0.53
34
0.107
28
0.59
35
0.155
29
>300
36
0.315
30
0.17
37
0.218
31
0.066
38
0.412
32
0.047
39
0.178
*For assay conditions, see references 9 and 10.
S u m m a r y o f S A R S t u d i e s . T h e results of o u r S A R studies o n P M P i n h i b i t o r s o f s q u a l e n e s y n t h a s e are s u m m a r i z e d i n F i g u r e 5. T h e r e q u i r e m e n t s for p o t e n t e n z y m e i n h i b i t i o n are: (1) a n intact, t r i a c i d P M P s u r r o g a t e , (2) a n ether o x y g e n adjacent to the P M P s u r r o g a t e , a n d (3) a f a r n e s y l - l e n g t h i s o p r e n e s u b u n i t . A c e r t a i n degree o f s t r u c t u r a l f l e x i b i l i t y is a l l o w e d i n the i s o p r e n e s u b u n i t that is p r o x i m a l to the ether.
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
76
REGULATION OF ISOPENTENOID METABOLISM
Diphosphate Surrogate (PMP)
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
-triacid
required-
Required Terminal Isoprene F i g u r e 5. A n a t o m y o f a S q u a l e n e S y n t h a s e I n h i b i t o r F u r t h e r B i o l o g i c a l E v a l u a t i o n o f 10 The i n h i b i t i o n of squalene synthase b y inhibitor 10 i n h u m a n liver a n d yeast m i c r o s o m e s w a s c o m p a r e d to that i n rat l i v e r m i c r o s o m e s u n d e r o u r s t a n d a r d assay c o n d i t i o n s (9, 10) ( T a b l e 5). A l t h o u g h the results f r o m b o t h m a m m a l i a n sources w e r e n e a r l y i d e n t i c a l , 1 0 is m u c h less effective against the yeast e n z y m e . T h e a p p a r e n t K v a l u e s for F P P i n the rat (12.7 μ Μ ) a n d yeast (25 μ Μ ) m i c r o s o m a l systems are s i m i l a r . U p o n e x p r e s s i n g the r e l a t i v e p o t e n c y for the t w o e n z y m e p r e p a r a t i o n s as ( K m / K i ) t / ( K m / K i ) y a s t / d e m o n s t r a t e d to be 16-fold m o r e active against the rat vs the yeast e n z y m e . T h i s m a y be a n i n d i c a t i o n that the a c t i v e sites o f the yeast a n d m a m m a l i a n e n z y m e s are s i g n i f i c a n t l y d i f f e r e n t . I n a d d i t i o n , 10 i n h i b i t s c h o l e s t e r o l b i o s y n t h e s i s f r o m C - a c e t a t e i n w h o l e , f r e s h l y - i s o l a t e d h e p a t o c y t e s (I50 = 2.7 μ Μ ) . T h e p a r e n t P M P a n a l o g u e 6a is i n a c t i v e i n the w h o l e cell assay at u p to 100 μ Μ (11). m
1 0
r a
e
14
T a b l e 5. Effect of 10 o n S q u a l e n e B i o s y n t h e s i s i n M i c r o s o m e s a n d o n Cholesterol Biosynthesis i n Rat Hepatocytes Assay
Ki 150 ( μ Μ ) 0.037 0.05 0.038 — 1.18 2.6 Yeast M i c r o s o m e s ' 2.7 W h o l e Rat H e p a t o c y t e s ^ — (a) For standard assay conditions, see ref s 9 and 10. (b) From frozen human liver. (c) Recombinant, overexpressed enzyme (17). (d) For assay, see ref. 11. 3
Rat L i v e r M i c r o s o m e s H u m a n Liver Microsomes ' 3
3
1 5
0
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
i s
7.
BILLER ET AL.
77
Inhibitors of Squalene Synthase
S y n t h e s i s o f I n h i b i t o r 10
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
T h e s y n t h e t i c r o u t e to e t h e r - P M P 10 ( S c h e m e 3) is g e n e r a l a n d w a s u t i l i z e d to p r e p a r e m o s t o f the i n h i b i t o r s d e s c r i b e d h e r e i n . T w o m e t h o d s for the p r e p a r a t i o n o f a l k o x y p h o s p h o n a t e 41 f r o m f a r n e s o l 40 w e r e e v a l u a t e d . R h o d i u m - c a t a l y z e d c a r b e n e i n s e r t i o n (44) o f d i a z o m e t h y l p h o s p h o n a t e 42 (45) p r o v i d e d d i m e t h y l ester 4 1 a i n m o d e s t y i e l d . T h e r e a c t i o n o f the l i t h i u m a l k o x i d e d e r i v e d f r o m f a r n e s o l w i t h triflate 43 (46) a f f o r d e d the c o r r e s p o n d i n g d i i s o p r o p y l ester 4 1 b i n 92 % y i e l d . T r i f l a t e 43 is p r e p a r e d f r o m d i i s o p r o p y l p h o s p h i t e i n t w o steps ( p a r a f o r m a l d e h y d e , E t 3 N , 100 ° C (47); t r i f l u o r o m e t h a n e s u l f o n i c a n h y d r i d e , i - P r 2 N E t , ether, -78 to 0 ° C , 52 % o v e r a l l ) . E i t h e r 41a o r 4 1 b c a n be u s e d to c o m p l e t e the s y n t h e s i s f o l l o w i n g o u r p r e v i o u s l y r e p o r t e d m e t h o d o l o g y f o r the p r e p a r a t i o n o f P M P i n h i b i t o r s (48). I n the case of 4 1 b , c o n v e r s i o n to a c i d c h l o r i d e 4 4 w a s Scheme 3
42
43
(a) 0.2 equiv Rh (OAc) , 2 cquiv 42, PhH, RT, 36 %. (b) BuLi, THF, -78 °C; 43, 0 °C, 92 %. (c) KOH, H 0 , i-PrOH, 100 °C; TMSNEt C H C 1 , oxalyl chloride, cat DMF, C H C 1 . (d) LiCH PO(OMe) , THF, -78 °C, 79% overall from 41b. (e) TMSBr, C H C 1 2,4,6-collidine; KOH, H Q , 93 %. 2
2
2
2
2/
2
2
2
2/
2
2
2
2
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
78
REGULATION OF ISOPENTENOID METABOLISM
followed by coupling with the l i t h i u m anion of dimethyl m e t h y l p h o s p h o n a t e t o p r o v i d e P M P t r i e s t e r 4 5 (79 % y i e l d f r o m 4 1 b ) . B r o m o t r i m e t h y l s i l a n e (49) p r o m o t e d d e e s t e r i f i c a t i o n g a v e i n h i b i t o r 10 i n 93 % y i e l d .
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
Conclusion In s u m m a r y , o u r s t u d i e s h a v e l e d to the first f a m i l y o f p o t e n t i n h i b i t o r s o f s q u a l e n e s y n t h a s e . I n h i b i t o r 10 w a s d i s c o v e r e d as t h e r e s u l t o f r a t i o n a l d e s i g n o n the basis o f a p r o p o s e d m e c h a n i s m for the e n z y m a t i c catalysis o f s q u a l e n e b i o s y n t h e s i s . T h i s s t u d y r e v e a l e d a s u r p r i s i n g l y large c o n t r i b u t i o n o f a n e t h e r o x y g e n to the i n h i b i t o r - e n z y m e b i n d i n g e n e r g y . W e s p e c u l a t e that this o x y g e n is e n g a g e d i n a h y d r o g e n b o n d i n g i n t e r a c t i o n w i t h a k e y a c t i v e site a c i d c a t a l y s t . F u r t h e r s t r u c t u r e - a c t i v i t y s t u d i e s r e v e a l that a t r i a c i d d i p h o s p h a t e s u r r o g a t e a n d a f a r n e s y l - l e n g t h i s o p r e n y l s u b u n i t are r e q u i r e d for o p t i m a l i n h i b i t o r y a c t i v i t y , a n d that a d e g r e e o f s t r u c t u r a l f l e x i b i l i t y is t o l e r a t e d i n the i s o p r e n y l p o r t i o n o f the i n h i b i t o r . A c k n o w l e d g e m e n t s . T h e a u t h o r s w o u l d l i k e to t h a n k D r s . P h i l i p M . S h e r , W i l l i a m H . K o s t e r a n d R i c h a r d E . G r e g g for t h e i r h e l p f u l c o m m e n t s o n this m a n u s c r i p t , D r . V a l S. G o o d f e l l o w for assistance w i t h t h e m o l e c u l a r m o d e l i n g s t u d i e s , a n d D r . Jeffrey M . H o e g o f t h e N a t i o n a l I n s t i t u t e s o f H e a l t h f o r a s a m p l e o f h u m a n l i v e r tissue.
Literature Cited (1) Roth, B. D.; Sliskovic, D. R.; Trivedi, Β. K. Annu. Rep. Med. Chem. 1989, 24, 147-156. (2) Grundy, S. M . New EnglandJ.Med. 1988, 319, 24-33. (3) Hoeg, J. M.; Brewer, H . B., Jr. J. Am. Med. Assoc. 1987, 258, 3532-3536. (4) Karanewsky, D. S.; Badia, M. C.; Ciosek, C. P., Jr.; Robl, J. Α.; Sofia, M . J.; Simpkins, L. M.; DeLange, B.; Harrity, T. W.; Biller, S. Α.; Gordon, Ε. M . J. Med. Chem. 1990, 33, 2952-2956. (5) Sit, S. Y.; Parker, R. Α.; Motoc, I.; Han, W.; Balasubramanian, N.; Catt, J. D.; Brown, P. J.; Harte, W. E.; Thompson, M . D.; Wright, J. J. J. Med. Chem. 1990, 33, 2982-2999. (6) Beck, G.; Kesseler, K.; Baader, E.; Bartmann, W.; Bergmann, Α.; Granzer, E.; Jendralla, H.; Kerekjarto, B. v.; Krause, R.; Paulus, E.; Schubert, W.; Wess, G. J. Med. Chem. 1990, 33, 52-60. (7) Roth, B. D.; Ortwine, D. F.; Hoefle, M . L.; Stratton, C. D.; Sliskovic, D. R.; Wilson, M . W.; Newton, R. S. J. Med. Chem. 1990, 33, 21-31. (8) Lee, T.-J. Trends Pharm. Sci. 1987, 8, 442-446. (9) Biller, S. Α.; Forster, C.; Gordon, E. M.; Harrity, T.; Scott, W. Α.; Ciosek, C. P., Jr. J. Med. Chem. 1988, 31, 1869-1871. (10) Biller, S. Α.; Forster, C.; Gordon, Ε. M.; Harrity, T.; Rich, L. C.; Marretta, J.; Scott, W. Α.; Ciosek, C. P., Jr. In Drugs Affecting Lipid Metabolism, X: Proceedings of the Xth International Symposium,
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
7. BILLER ET AL.
(11) (12)
(13) (14) (15)
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
(16) (17) (18)
(19) (20) (21) (22)
(23) (24) (25) (26) (27) (28)
(29) (30) (31)
(32) (33) (34)
Inhibitors of Squalene Synthase
79
Gotto, A. M., Jr., Smith, L. C., Eds.; Elsevier: Amsterdam, 1990; pp 213-216. Biller, S. Α.; Forster, C.; Gordon, E. M.; Harrity, T.; Rich, L. C.; Marretta, J.; Ciosek, C. P., Jr. J. Med. Chem. 1991, 34, 1912-1914. Poulter, C. D.; Rilling, H . C. In Biosynthesis of Isoprenoid Compounds; Porter, J. W.; Spurgeon, S. L. Eds.; Wiley: NY, 1981; Vol. 1, Chapter 8. Popják, G.; Agnew, W. S. Mol. Cell. Biochem. 1979, 27, 97-116. Agnew, W. S. Methods Enzymol. 1985, 110, 359-373. Biller, S. Α.; Sofia, M. J.; DeLange, B.; Forster, C.; Gordon, E. M.; Harrity, T.; Rich, L. C.; Ciosek, C. P., Jr. J. Am. Chem. Soc. 1991, 113, 8522-8524. Sasiak. K.; Rilling, H . C. Arch. Biochem. Biophys. 1988, 260, 622-627. Jennings, S. M.; Tsay, Y. H.; Fisch, T. M.; Robinson, G. W. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6038-6042. Händler, B.; Soltis, D.; Movva, R.; Kocher, H . P.; Ha, S.; Kathawala, F.; Nemecek, G. Abstract of Papers, 201st National Meeting of the American Chemical Society, Atlanta, GA. American Chemical Society: Washington, DC, 1991; BIOT 92. Goldstein, J. L.; Brown, M. S. Nature 1990, 343, 425-430. Faust, J. R.; Goldstein, J. L.; Brown, M . S. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 5018-5022. Cohen, L. H.; Miert, E. v.; Griffioen, M . Biochim. Biophys. Acta 1989, 1002, 69-72. Cohen, L. H.; Griffioen, A. M.; Wanders, R. J. Α.; Van Roermund, C. W. T.; Huysman, C. M. G.; Princen, H . M . G. Biochem. Biophys. Res. Commun. 1986, 138, 335-341. Epstein, W. W.; Rilling, H . C. J. Biol. Chem. 1970, 245, 4597-4605. Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77. Ortiz de Montellano, P. R.; Wei, J. S.; Vinson, W. Α.; Castillo, R.; Boparai, A. S. Biochemistry 1977, 16, 2680-2685. Washburn, W. N.; Kow, R. Tetrahedron Lett. 1977, 18, 1555-1558. Rilling, H . C. Biochem. Soc. Trans., 1985, 13, 997-1003. Poulter, C. D.; Argyle, J. C.; Mash, Ε. Α.; Laskovics, P. L.; Wiggins, P. L.; King, C. R. Pr. Nauk. Inst. Chem. Org. Fiz. Politech. Wroclaw 1981, 22, 149-162. Poulter, C. D.; Wiggins, P. L.; Le, A. T. J. Am. Chem. Soc. 1981, 103, 3926-3927. Mash, Ε. Α.; Gurria, G. M.; Poulter, C. D J. Am. Chem. Soc. 1981, 103, 3927-3929. Poulter, C. D.; Rilling, H. C. In Biosynthesis of Isoprenoid Compounds; Porter, J. W.; Spurgeon, S. L. Eds.; Wiley: NY, 1981; Vol. 1, Chapter 4. Tidd, Β. K. J. Chem. Soc (B) 1971, 1168-1176. Blackburn, G.M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin Trans. 1 1984, 1119-1125. Koyama, T.; Ogura, K.; Seto, S. Biochim. Biophys. Acta 1980, 617, 218-224.
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007
80
REGULATION OF ISOPENTENOID METABOLISM
(35) Ortiz de Montellano, P. R.; Wei, J. S.; Castillo, R.; Hsu, C. K.; Boparai, A. S. J. Med. Chem. 1977, 20, 243-249. (36) Corey, E. J.; Volante, R. P. J. Am. Chem. Soc. 1976, 98, 1291-1293. (37) McClard, R. W.; Fujita, T. S.; Stremler, Κ. E.; Poulter, C. D.J.Am. Chem. Soc. 1987, 109, 5544-5545. (38) Morgan, B. P.; Schlotz, J. M.; Ballinger, M . D.; Zipkin, I. D.; Bartlett, P. A. J. Am. Chem. Soc. 1991, 113, 297-307. (39) Bartlett, P. Α.; Marlowe, C. K. Science 1987, 235, 569-571. (40) Kati, W. M.; Wolfenden, R. Biochemistry 1989, 28, 7919-7927. (41) Frick, L.; Yang, C.; Marquez, V. Ε.; Wolfenden, R. Biochemistry 1989, 28, 9423-9430. (42) Hine, J.; Mookerjee, P. K. J. Org. Chem. 1975, 40, 292-298. (43) Grobelny, D.; Goli, U. B.; Galardy, R. E. Biochemistry 1989, 28, 49484951. (44) Doyle, M . P. Chem. Rev. 1986, 86, 919-939. (45) Seyferth, D.; Marmor, R. S.; Hubert, P. H . J. Org. Chem. 1971, 36, 1379-1385. (46) Phillion, D. P.; Andrew, S. S. Tetrahedron Lett. 1986, 27, 1477-1480. (47) Kluge, A. F. Organic Synthesis 1985, 64, 80-83. (48) Biller, S. Α.; Forster, C. Tetrahedron 1990, 46, 6645-6658. (49) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M . C. Tetrahedron Lett. 1977, 155-158. RECEIVED January 6, 1992
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.