- Email: [email protected]
Synthesis, SARs and therapeutic potential of HMG-CoA reductase inhibitors
442
TIPS - November 1987 [Vol. 8] practical realization of this goal has been advanced greatly by the recent discoveries of several novel fungal metabolites, which are potent inhibitors of HMG-CoA reductase.
Synthesis, SARs and therapeutic potential of HMGCoA reductase inhibitors Ta-Jyh Lee
Inhibitory activities of mevastatin lovastatin Mevastatin (Fig. 2,1a, formerly known as ML-236B (Ref. 8) and Compactin 9) was isolated independently by two different groups from the cultures of Penicillium citrinum and Penicillium brevicompactum and proved to be a remarkably potent HMG-CoA reductase inhibitor. Later, a closely related compound, lovastatin (Fig. 2,1b), formerly known as monacolin K (Refs 10, 11) and mevinolin 12 was isolated independently by two different groups from cultures of Monacus ruber and Aspergillus terreus. The subsequent isolation of a n u m b e r of related compounds has been reported (see Ref. 13). Mevastatin and lovastatin are clearly the most extensively investigated members of this family of compounds. The inhibition of HMG-CoA reductase by mevastatin and lovastatin is reversible and competitive with respect to HMG-CoA, the natural substrate. The Ki values of the dihydroxy acid forms of mevastatin and lovastatin (Fig. 2, 2a and 2b, which are the biologically active forms of mevastatin and lovastatin) are 1 nM and 0.6 riM, respectively. Neither compound affects other enzymes involved in cholesterol biosynthesis. In mammalian cells cultured in a medium containing LDL, the synthesis of other biologically important substances such as ubiquinone, dolichol and tRNA, which also are derived from mevalonate and re-
and
Elevated plasma levels of low-density lipoprotein cholesterol is a major risk factor for the development of coronary heart disease, the leading cause of death and disability in Western countries. Because most cholesterol in the body is synthesized de novo, the control of endogenous cholesterogenesis would be an attractive and potentially effective means of lowering plasma cholesterol levels. This approach has been validated by the recent discoveries of two novel fungal metabolites, mevastatin and Iovastatin. These compounds are potent inhibitors of HMG-CoA reductase, which regulates the rate-limiting step in the biosynthetic pathway of cholesterol. Ta-Jyh Lee discusses the rationale for the design and synthesis of several potent inhibitors related to mevastatin and Iovastatin, and the therapeutic potential of HMG-CoA reductase inhibitors. The atheromatous plaque or atheroma is formed in part from lipid deposits, primarily cholesteryl esters, in the inner walls of arteries. Growth of the atheroma leads to constriction of the arterial lumen and ultimately results in atherosclerosis and coronary heart disease (CHD), the leading cause of death and disability in Western countries. Epidemiological evidence 1-3 strongly indicates that hypercholesterolemia or more accurately, elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) - is a major risk factor for the development of CHD. These observations have stimulated intensive efforts directed towards the development of therapeutic agents for prevention and treatment of atherosclerosis based on the attenuation of plasma cholesterol levels (see Ref. 4). Results of the recently completed Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT)5'6 provide strong support for the rationale underlying this approach. This trial clearly demonstrated that the reduction of LDL-C through dietary modification and treatment with the bile acid sequesterant colestyramine, either alone or in combination, diminished the incidence of CHD morbidity and mortality in hypercholesterolemic men at high risk for CHD. Nevertheless, these mea-
-
sures often fail to lower elevated plasma LDL-C levels to the desired extent, particularly in patients with familial hypercholesterolemia. Because most cholesterol in the body is synthesized de novo, the control of endogenous cholesterogenesis would be an attractive and potentially more effective way to lower plasma cholesterol levels. Cholesterol is synthesized from acetyl-CoA via a series of more than twenty enzymatic reactions. The major rate-limiting step in this pathway is regulated by the activity of the enzyme 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC1.1.1.34) 7, which catalyses the conversion of HMG-CoA to mevalonic acid (Fig. 1). Therefore, for several decades, this enzyme has been considered to be a prime target for pharmacological intervention for the control of endogenous cholesterogenesis. The
Ta-]yh Lee is Senior Research Fellow in the Department of Medicinal Chemistry, Merck Sharp & Dohme Research Laboratories,West Point, PA 19486, USA. 1987, Elsevier Publications, Cambridge 0165 6147/87/$02.00
Acetate
Cholesterol
f
'I Multiple
J Multiple Steps CH3 HO~'~-COzH
Steps
CH3 HMG-CoA Reductase
HO~7"-CO2H
NADPH
k,, OH
SCoA
H MG -CoA
Fig. 1. Biosynthetic pathway for cholesterol.
Mevalonic Acid
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TIPS - N o v e m b e r 1987 [Vol. 8]
q u i r e d for cell growth, is not affected e v e n w h e n the activity of H M G - C o A reductase is severely s u p p r e s s e d (up to 98%) b y mevastatin 14,15. These findings strongly suggest that mevastatin and lovastatin are specific inhibitors of H M G - C o A reductase. H o w e v e r , of greatest interest is the finding that these two natural products are highly effective hypocholesterolemic agents in several animal spec i es 13 and h u m a n s 16-19. Not surprisingly, after the disclosure of these initial findings, efforts directed t o w a r d the synthesis of mevastatin, lovastatin and related analogs w e r e initiated in m a n y laboratories (see Ref. 20).
H
H
o .o o
HO~"CO2H O
l"Y. OH
-I-H20
--H20 a, R --
b, R=
H (Mevastatin) (Lovastatin)
OH 3
HO~O
HO~co2H A x
Before the discovery of lovastatin, an effort was initiated in our laboratories directed towards the d e v e l o p m e n t of totally synthetic H M G - C o A reductase inhibitors d e v o i d of the structural complexities of mevastatin. A m o d e l synthetic analog of mevastatin was formulated b a s e d largely on the following considerations. The structural similarity bet w e e n the d i h y d r o x y acid m o i e t y of structure 2 and H M G - C o A or mevalonic acid is readily recognized. H o w e v e r , since the Km value for H M G - C o A , the natural substrate of the enzyme, is approximately 10-5M (about 10 4 w e a k e r than the Ki values of m e v a s t a t i n and lovastatin), the s u b s t i t u t e d p o l y h y d r o n a p h t h y l moieties in mevastatin 21 and lovastatin m u s t also play an i m p o r t a n t role in the i n h i b i t i o n of the enzyme. Based on this line of r e a s o n i n g and the k n o w n facts about the H M G - C o A reductase-catalysed reduction of H M G - C o A to mevalonic acid, our early model of a synthetic analog of structure 2 is r e p r e s e n t e d b y the generalized structure 3. It consists of a dih y d r o x y acid m o i e t y similar to that of structure 2a and a lipophilic group linked b y either a saturated or an u n s a t u r a t e d 2-carbon bridge. Thus, structure 4, w h i c h is the c o r r e s p o n d i n g lactone form of structure 3, was the initial target• It should be n o t e d that the stereochemical requirements at positions C-4 and C-6 of lactone 4 were not k n o w n at this point. Although the chiralities of two steric centers in the lactone ring of mevastatin (cf.
3
A
A CH2CH2or CH= CH X-- Aromatics =
I
Totally synthetic analogs of mevastatin
W° I
x
HO~r'~O HO~ , . / ~ O
i
A
I
X cI
6
HO~co2Na
HO!~O!
~..o.
R" I,,,,~f6 R20 H r ,~
~CH
3
CH3" : 8
7
(SRI-62320) HOy"",,CO2Na
HO~T,,~ CO2Na o
~:.~~ OH3......
'-.y..OH
~,,.oa O
:
I~CH3 HO" ~
~ 10
(CS-514or Sq-31000) Fig. 2. Structures of mevastatin, Iovastatin and the corresponding ring-opened dihydroxy acid forms, and other analogs.
a a~
TIPS - November
TABLE I. In-vitro inhibitory activities of lactones 6 (Fig. 2) towards H M G - C o A reductase
Code •
Optical activity +
A
X
'. . . . . . . . .
a
+
when suitable aryl groups were placed at the 6-position of the 2,4dichlorophenyl ring in 6a(+). For example, a 1750-fold increase in potency attended the formal conversion of compound 6a(+) to 6d(+) (Table I). In view of the toxicity ascribed to chlorinated biphenyls, it was deemed desirable to find a suitable replacement for the chloro groups in 6d(+). The methyl group was considered to be a favorable bioisosteric replacement for the chloro group because of their comparable sizes and lipophilicities. Furthermore, aromatic methyl groups are susceptible to biooxidation and the resultant metabolite(s) is expected to be more readily eliminated from the body. Indeed, replacement of the chlorosubstituents in structure 6d(+) with methyl groups and further refinement ultimately afforded compound 6e(+), which has an inhibitory potency almost three times that of mevastatin. It is also noteworthy that a substantial decrease in potency occurs when structure 6e(+) is reduced to 6f(+). This result is contrary to earlier observations in the benzyl ether series and the prediction of an advantage in having a saturated or an unsaturated 2-carbon bridge connecting the lactone and the lipophilic groups remains precarious. Finally, the recent disclosure of research on compounds related to compound 7 (SRI62320) 2s appears to be particularly interesting. As evidence of the therapeutic usefulness of HMGCoA reductase inhibitors continues to mount, it is anticipated that research activities in search of new and novel inhibitors will be greatly intensified in the future.
Relative potency*
:+
+
:'++. "
t-CH=CH
+ - ........... ,i
.....
0.16 CI
C
+
CH2CH2
F-~-C.,O~-C+ T
38
CI
•' : ~ , +
d
+
t'CH=CH
c~
+:,
100
CI : •
::
i
e
.+
=
.:
+
.+
t-CH=CH
C
.
~'~'~1 ~ H
~
289
CH~
CH3
• All compounds in this table were tested after being converted to the sodium salts of the corresponding dihydroxy acids. The relative potency of the test compound was determined by compadng its I C ~ value with that of mevastatin, which was tested simultaneously and arbitrarily assigned a relative potency value of 100.
structure la) had been determined earlier 9, their critical importance to intrinsic inhibitory activity was yet to be ascertained. This issue was quickly resolved by determining that: (1) all activity resides in the t r a n s - d i a s t e r e o m e r 6a(+) (i.e. the c i s - d i a s t e r e o m e r 5(+) is inactive); and (2) only enantiomer 6a(+) is active 22. Similar results were observed subsequently in every compound series examined (see Table I), indicating that the chiralities of the two steric centers in structure 6 are critical in determining intrinsic HMG-CoA reductase inhibitory activity. Later, the finding that compound 6d(+) •possessed the same chirality in the lactone ring as that present in mevastatin was determined by Xray crystallography23 and further supported this conclusion. However, the weak intrinsic inhibitory potency of structure 6a(+) needed to be optimized. Attachment of either an arylmethoxy group (benzyl ether series) 24 or an aryl moiety (biphenyl series) 2s at the 6-position of the 2,4dichlorophenyl ring in compound 6a(+) dramatically enhanced potency. In the benzyl ether series,
1987 [ V o l . 8]
introduction of a 4-fluoro group on the benzyl moiety induces a remarkable increase in potency. Later, this substitution proved to be equally useful in other series. Another significant contribution towards the improvement of potency was observed when compound 6b(+) was hydrogenated to 6c(+). Resolution of 6c(+) yielded 6c(+), which had an inhibitory potency approaching that of mevastatin. Even more profound enhancement of potency was observed
TABLE II. In-vitro inhibitory activities of lactones 8 (Fig. 2) towards H M G - C o A reductase
Code
R~
R2
Relative potency*
O
b
H
CH,CH,C-~H
.......
d
®
H
F.~-c~
+++,
*See Table I.
254
CH~
-
119
+++,
TIPS - N o v e m b e r 1987 [Vol. 8]
445
Bile acid depletion
Untreated
HMG CoA Reductase inhibitor
Reductase inhibitor + bile acid depletion
Plasma
Liver
Intestine
(,
~
U
Fig. 3. Rationale for the therapeutic treatment of hypercholesterolemia.
Semisynthetic analogs of lovastatin Lovastatin (lb) 12 differs from mevastatin (la) structurally only by the presence of a 60c-methyl group and its relative potency is approximately twice that of mevastatin. Its discovery added a new direction to research in this a r e a delineation of the structural features of lovastatin responsible for its remarkable inhibitory activity and the use of this information to develop even more potent and efficacious inhibitors. Studies on the modification of the 2(s)-methylbutyryl moiety of lovastatin 26 yielded the following conclusions: • The presence of a side chain ester moiety is crucial to inhibitory potency (the potency of compound 8a is very weak). • The stereochemistry at the carbon 0c to the ester carbonyl group is not critical, since diastereomer 8b has the same potency as that of lovastatin (lb). • The spatial requirements of the acyl moiety are compatible with compact, branched aliphatic acyl groups. • Additional branching at the 0ccarbon of the acyl moiety enhances potency. In particular, the introduction of an additional methyl group on the carbon 0cto the ester carbonyl in lovastatin afforded structure 8c (MK-733) which, with an intrinsic potency more than six times that of mevastatin, is the most potent HMG-CoA reductase inhibitor reported to date. A series of side chain ether analogs of lovastatin were found to be mostly weaker inhibitors 27,
although compound 8d, the best in the series, shows a respectable level of potency. Introduction of a methyl group (0~-orientation) to the hydroxy-bearing carbon of the lactone in lovastatin provides homolog 8e (Ref. 27) in which the lactone moiety bears a close structural resemblance to HMG-CoA and mevalonic acid, the respective substrate and product of the reduction catalysed by HMG-CoA reductase (Fig. 1). Surprisingly, this modification is detrimental to activity. This was also true w h e n similar modifications were made in the synthetic analogs. Insertion of a methylene unit between the lactone hydroxy-bearing carbon and the carbonyl group in lovastatin afforded the carboxylate 9, which was devoid of intrinsic HMG-CoA reductase inhibitory activity. Finally, minimal decreases in potency were observed when either one or two double bonds in the hexalin moiety of lovastatin were hydrogenated, providing that the ring juncture of the resultant hydrogenation product is trans; when the ring juncture is cis, activity is markedly diminished. A most noteworthy finding has been the recent discovery of CS-514 (structure 10, also known as SQ31000) 28, a microbial oxidation product of mevastatin. CS-514 is currently undergoing clinical evaluation. The initial reports from investigations of this new HMGCoA reductase inhibitor show promise.
Therapeutic potential as hypocholesterolemic drugs One of the major recent developments in the prevention and
treatment of CHD is the understanding of how plasma cholesterol levels, especially LDL-C levels, are controlled. Based on the findings of M. S. Brown and J. L. Goldstein (see Ref. 29), a rationale for the therapeutic treatment of hypercholesterolemia is schematically presented in Fig. 3. A liver cell normally receives its cholesterol from: (1) uptake from circulating LDL-C via receptormediated endocytosis; (2) de-novo biosynthesis; and (3) uptake from chylomicron remnants. It then converts much of its cholesterol pool into bile acids. A large fraction of the bile acids secreted from the liver is returned to the liver through enterohepatic cycling. It is well established that the liver is the major site of LDL receptors and the production of LDL receptors is driven by the liver cell's d e m a n d for cholesterol. An effective therapeutic approach for lowering plasma LDL-C levels should be focused on ways to increase the production of L D L receptors in the liver. This is achievable by inhibition of the intestinal reabsorption of bile acids and/or inhibition of endogeneous cholesterol synthesis. Ultimately, when a new steady state is attained, the absolute amount of cholesterol entering the liver through the receptor pathway remains approximately the same as before, because the fall in plasma LDL levels is balanced by the increase in LDL receptors. But the important difference is that this delivery is now occurring at a lower plasma LDL level. In practice, the ingestion of bile acid resins (e.g. colestyramine and colestipol) causes plasma LDL
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T I P S - N o v e m b e r 1987 [Vol. 8]
levels to d e c r e a s e b y b l o c k i n g the r e a b s o r p t i o n of bile acids a n d t h e r e b y i n c r e a s i n g the m e t a b o l i s m of cholesterol. H o w e v e r , the effect of this t h e r a p y is n o t p r o f o u n d . It usually produces only about a 20% d r o p i n p l a s m a L D L - C levels 5"6. The second method, using HMGC o A r e d u c t a s e i n h i b i t o r s s u c h as m e v a s t a t i n 16 a n d l o v a s t a t i n 17-19 to i n h i b i t c h o l e s t e r o l s y n t h e s i s , is a m u c h m o r e effective a p p r o a c h for l o w e r i n g p l a s m a L D L levels. W h e n g i v e n as a single a g e n t (40 m g p e r day) to familial h y p e r c h o l e s t e r o l e m i c h e t e r o z y g o t e s , lovastatin d e c r e a s e s p l a s m a LDL levels b y 33% (from 361 + 13 m g c m -3 to 242 + 15 m g cm-3) 19. W h e n g i v e n t o g e t h e r w i t h colestipol, an e v e n greater d r o p (46%) in p l a s m a L D L levels w a s r e p o r t e d 19. T h e h y p o c h o l e s t e r o l e m i c effect of l o v a s t a t i n h a s b e e n a s c r i b e d to the i n c r e a s e d p r o d u c t i o n of L D L r e c e p t o r s res u i t i n g f r o m the i n h i b i t i o n of de-novo cholesterogenesis. Other s t u d i e s also s u g g e s t that the s y n t h e s i s of v e r y l o w d e n s i t y l i p o p r o t e i n (VLDL), a p r e c u r s o r of LDL, h a s also b e e n s u p p r e s s e d b y l o v a s t a t i n (D. R. I l l i n g w o r t h , pers. c o m m u n . ) . Recently, lovastatin w a s also f o u n d to be effective in t h e t r e a t m e n t of p a t i e n t s with nonfamilial hypercholestero l e m i a 3°. Specifically, p a t i e n t s receiving 40 m g t w i c e a d a y e x p e r i e n c e d a m e a n r e d u c t i o n in p l a s m a L D L levels of 40%. So far, c o n s i s tently good patient acceptance and a l o w i n c i d e n c e of side effects h a v e b e e n o b s e r v e d in all s t u d i e s w i t h l o v a s t a t i n (for d e t a i l e d d i s c u s s i o n , see Ref. 31). T h e i m p o r t a n t p r i n ciple t h a t h a s b e e n realized f r o m t h e s e s t u d i e s is t h a t s t i m u l a t i o n of L D L r e c e p t o r activity u l t i m a t e l y l o w e r s p l a s m a L D L - C levels w i t h o u t g r o s s l y altering cholesterol s u p p l y to cells. H M G - C o A r e d u c tase i n h i b i t o r s are p o w e r f u l m e a n s of t a k i n g a d v a n t a g e of this p r i n ciple in t h e effective r e d u c t i o n of p l a s m a L D L levels. []
[]
[]
E v i d e n c e r e l a t i n g p l a s m a cholesterol levels to a t h e r o s c l e r o s i s a n d CHD has become compelling. M e c h a n i s m s r e s p o n s i b l e for h i g h c o n c e n t r a t i o n s of LDL are b e c o m ing understood. The LDL receptor is r e c o g n i z e d as a n i m p o r t a n t elem e n t in t h e r e g u l a t i o n of p l a s m a L D L levels. T h e fact t h a t H M G -
CoA reductase inhibitors are h i g h l y effective h y p o c h o l e s t e r o l e m i c a g e n t s is n o w well e s t a b lished. T h e u s e of this n e w l y disc o v e r e d t h e r a p y to treat a fraction of the p o p u l a t i o n at h i g h risk of C H D b e c a u s e of e x t r e m e l y h i g h p l a s m a L D L levels (e.g. familial hypercholesterolemia patients) a p p e a r s to be w i d e l y a c c e p t e d b y the m e d i c a l c o m m u n i t y . Will HMG-CoA reductase inhibitors also b e c o m e the c h o i c e of t h e r a p i e s to treat m o d e r a t e hyperchole s t e r o l e m i a , w h i c h is a m u c h m o r e common health problem than familial h y p e r c h o l e s t e r o l e m i a ? Int e n s i v e efforts to e s t a b l i s h the l o n g - t e r m clinical s a f e t y of H M G CoA reductase inhibitors are clearly vital to this issue.
References 1 Kannel, W. B., Castelli, W. D., Gordon, T. and McNamara, P.M. (1971) Ann. Intern. Med. 74, 1-12 2 Keys, A. (1980) in Seven Countries: A Multivariate Analysis of Death and Coronary Heart Disease, pp. 1--381,
Harvard University Press 3 Stamler, J., Wentworth, D. and Neaton, J. D. (1986) J. Am. Med. Assoc. 256, 28232828 4 Prugh, J. D., Rooney, C~ S. and Smith, R.L. (1983) Annu. Rev. Med. Chem. 18, 161-170 5 LRC-CPPT (1984)J. Am. Med. Assoc. 251, 351-364 6 LRC-CPPT (1984)J. Am. Med. Assoc. 251, 365-374 7 Rodwell, V. W., Nordstrom, J. L. and Mitschelen, J. J. (1976) Adv. Lipid Res. 14, 1-74 8 Endo, A., Kuroda, M. and Tsujita, Y. (1976) J. Antibiot. 29, 1346-1348 9 Brown, A. G., Smale, T. C., King, T. J., Hasenkam, P. and Thompson, R.H. (1976) J. Chem. Soc. Perkin Trans. 1,11651170 10 Endo, A. (1979) J. Antibiot. 32, 852-854 11 Endo, A. (1980) J. Antibiot. 33, 334-336 12 Alberts, A. W., Chen, J., Kuron, G., Hunt, V., Huff, J., Hoffman, C., Rothrock, J., Lopez, J., Joshua, H., Harris, E., Pachett, A., Monaghan, R., Currie, S., Stapley, E., Albers-Schonberg, G., Hensen, O., Hirshfield, J., Hoogsteen, K.,
Liesch, J. and Springer, J. (1980) Proc. Natl Acad. Sci. USA 77, 3957--3961 13 Endo, A. (1985)J. Med. Chem. 28, 401-405 14 Faust, J. R., Brown, M. S. and Go!dstein, J. L. (1980) J. Biol. Chem. 255, 6546-6548 15 Brown, M. S. and Goldstein, J. L. (1980) J. Lipid Res. 21, 505-517 16 Mabuchi, H., Sakai, T., Sakai, Y., Yoshimura, A., Watanabe, A., Wakasugi, T., Koizumi,. J. and Tekeda, R. (1983) N. Engl. J. Med. 308, 609-613 17 Bilheimer, D. W., Grundy, S. M., Brown, M.S. and Goldstein, J.L. (1983) Proc. Natl Acad. Sci. USA 80, 4124-4128 18 lUingworth, D. R. and Sexton, G. J. (1984) J. Clin. Invest. 74, 1972-1978 19 Illingworth, D. R. (1984) Ann. Intern. Med. 101, 598-604 20 Rosen, T. and Heathcock, C. H. (1986) Tetrahedron 42, 4909---4951 21 Nakamura, C. E. and Abeles, R. H. (1985) Biochemistry 24, 1364-1376 22 Stokker, G. E., Hoffman, W. F., Alberts, A.W., Cragoe, Jr, E.J., Deana, A.A., Gilfillar~, J. L., Huff, J. W., NoveUo, F. C., Prug~, J. D., Smith, R. L. and Willard, A. I~:,(1985) J. Med. Chem. 28, 347-358 23 Stokl~er,G. E., Alberts, A. W., Anderson, P.S., Cragoe, Jr, E.J., Deana, A.A., Gilfillan, J. L., Hirshfield, J., Holtz, W. J., Hoffman, W. F., Huff, J. W., Lee, T.-J., Novello, F.C., Prugh, J.D., Rooney, C.S., Smith, R.L. and Willard, A.K. (1986) J. Med. Chem. 29, 170-181 24 Hoffman, W. F., Alberts, A. W., Cragoe,
Jr, E.J., Deana, A.A., Evans, B.E., Gilfillan, J. L., Gould, N. P., Huff, J. W., Novello, F. C., Prugh, J. D., Rittle, K. E., Smith, R. L., Stokker, G. E. and WiUard, A. K. (1986) J. Med. Chem. 29, 159-169 25 Engstrom, R. G., Weinstein, D. B., Kathawala, F. G., Scallen, T., Eskesen, J. B., Rucker, M. L. and Miserendino, R. (1986) IX International Symposium on Drugs Affecting Lipid Metabolism,
Florence, Italy, p. 26 (Abstr.) 26 Hoffman, W. F., Alberts, A. W., Anderson, P. S., Chen, J. S., Smith, R. L. and WiUard, A. K. (1986) J. Med. Chem. 29, 849-852 27 Lee, T.-J., Holtz, W. J. and Smith, R. L. (1982) J. Org. Chem. 47, 4750--4757 28 Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S. (1986) Biochim. Biophys. Acta 877, 50-60 29 Brown, M. S. and Goldstein, J. L. (1986) Science 232, 34-47 30 Lovastatin Study Group II (1986) J. Am. Med. Assoc. 256, 2829-2834 31 Tobert, J. A. (1987) Circulation 76, 534538
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TIPS - November 1987 [Vol. 8] practical realization of this goal has been advanced greatly by the recent discoveries of several novel fungal metabolites, which are potent inhibitors of HMG-CoA reductase.
Synthesis, SARs and therapeutic potential of HMGCoA reductase inhibitors Ta-Jyh Lee
Inhibitory activities of mevastatin lovastatin Mevastatin (Fig. 2,1a, formerly known as ML-236B (Ref. 8) and Compactin 9) was isolated independently by two different groups from the cultures of Penicillium citrinum and Penicillium brevicompactum and proved to be a remarkably potent HMG-CoA reductase inhibitor. Later, a closely related compound, lovastatin (Fig. 2,1b), formerly known as monacolin K (Refs 10, 11) and mevinolin 12 was isolated independently by two different groups from cultures of Monacus ruber and Aspergillus terreus. The subsequent isolation of a n u m b e r of related compounds has been reported (see Ref. 13). Mevastatin and lovastatin are clearly the most extensively investigated members of this family of compounds. The inhibition of HMG-CoA reductase by mevastatin and lovastatin is reversible and competitive with respect to HMG-CoA, the natural substrate. The Ki values of the dihydroxy acid forms of mevastatin and lovastatin (Fig. 2, 2a and 2b, which are the biologically active forms of mevastatin and lovastatin) are 1 nM and 0.6 riM, respectively. Neither compound affects other enzymes involved in cholesterol biosynthesis. In mammalian cells cultured in a medium containing LDL, the synthesis of other biologically important substances such as ubiquinone, dolichol and tRNA, which also are derived from mevalonate and re-
and
Elevated plasma levels of low-density lipoprotein cholesterol is a major risk factor for the development of coronary heart disease, the leading cause of death and disability in Western countries. Because most cholesterol in the body is synthesized de novo, the control of endogenous cholesterogenesis would be an attractive and potentially effective means of lowering plasma cholesterol levels. This approach has been validated by the recent discoveries of two novel fungal metabolites, mevastatin and Iovastatin. These compounds are potent inhibitors of HMG-CoA reductase, which regulates the rate-limiting step in the biosynthetic pathway of cholesterol. Ta-Jyh Lee discusses the rationale for the design and synthesis of several potent inhibitors related to mevastatin and Iovastatin, and the therapeutic potential of HMG-CoA reductase inhibitors. The atheromatous plaque or atheroma is formed in part from lipid deposits, primarily cholesteryl esters, in the inner walls of arteries. Growth of the atheroma leads to constriction of the arterial lumen and ultimately results in atherosclerosis and coronary heart disease (CHD), the leading cause of death and disability in Western countries. Epidemiological evidence 1-3 strongly indicates that hypercholesterolemia or more accurately, elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) - is a major risk factor for the development of CHD. These observations have stimulated intensive efforts directed towards the development of therapeutic agents for prevention and treatment of atherosclerosis based on the attenuation of plasma cholesterol levels (see Ref. 4). Results of the recently completed Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT)5'6 provide strong support for the rationale underlying this approach. This trial clearly demonstrated that the reduction of LDL-C through dietary modification and treatment with the bile acid sequesterant colestyramine, either alone or in combination, diminished the incidence of CHD morbidity and mortality in hypercholesterolemic men at high risk for CHD. Nevertheless, these mea-
-
sures often fail to lower elevated plasma LDL-C levels to the desired extent, particularly in patients with familial hypercholesterolemia. Because most cholesterol in the body is synthesized de novo, the control of endogenous cholesterogenesis would be an attractive and potentially more effective way to lower plasma cholesterol levels. Cholesterol is synthesized from acetyl-CoA via a series of more than twenty enzymatic reactions. The major rate-limiting step in this pathway is regulated by the activity of the enzyme 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC1.1.1.34) 7, which catalyses the conversion of HMG-CoA to mevalonic acid (Fig. 1). Therefore, for several decades, this enzyme has been considered to be a prime target for pharmacological intervention for the control of endogenous cholesterogenesis. The
Ta-]yh Lee is Senior Research Fellow in the Department of Medicinal Chemistry, Merck Sharp & Dohme Research Laboratories,West Point, PA 19486, USA. 1987, Elsevier Publications, Cambridge 0165 6147/87/$02.00
Acetate
Cholesterol
f
'I Multiple
J Multiple Steps CH3 HO~'~-COzH
Steps
CH3 HMG-CoA Reductase
HO~7"-CO2H
NADPH
k,, OH
SCoA
H MG -CoA
Fig. 1. Biosynthetic pathway for cholesterol.
Mevalonic Acid
443
TIPS - N o v e m b e r 1987 [Vol. 8]
q u i r e d for cell growth, is not affected e v e n w h e n the activity of H M G - C o A reductase is severely s u p p r e s s e d (up to 98%) b y mevastatin 14,15. These findings strongly suggest that mevastatin and lovastatin are specific inhibitors of H M G - C o A reductase. H o w e v e r , of greatest interest is the finding that these two natural products are highly effective hypocholesterolemic agents in several animal spec i es 13 and h u m a n s 16-19. Not surprisingly, after the disclosure of these initial findings, efforts directed t o w a r d the synthesis of mevastatin, lovastatin and related analogs w e r e initiated in m a n y laboratories (see Ref. 20).
H
H
o .o o
HO~"CO2H O
l"Y. OH
-I-H20
--H20 a, R --
b, R=
H (Mevastatin) (Lovastatin)
OH 3
HO~O
HO~co2H A x
Before the discovery of lovastatin, an effort was initiated in our laboratories directed towards the d e v e l o p m e n t of totally synthetic H M G - C o A reductase inhibitors d e v o i d of the structural complexities of mevastatin. A m o d e l synthetic analog of mevastatin was formulated b a s e d largely on the following considerations. The structural similarity bet w e e n the d i h y d r o x y acid m o i e t y of structure 2 and H M G - C o A or mevalonic acid is readily recognized. H o w e v e r , since the Km value for H M G - C o A , the natural substrate of the enzyme, is approximately 10-5M (about 10 4 w e a k e r than the Ki values of m e v a s t a t i n and lovastatin), the s u b s t i t u t e d p o l y h y d r o n a p h t h y l moieties in mevastatin 21 and lovastatin m u s t also play an i m p o r t a n t role in the i n h i b i t i o n of the enzyme. Based on this line of r e a s o n i n g and the k n o w n facts about the H M G - C o A reductase-catalysed reduction of H M G - C o A to mevalonic acid, our early model of a synthetic analog of structure 2 is r e p r e s e n t e d b y the generalized structure 3. It consists of a dih y d r o x y acid m o i e t y similar to that of structure 2a and a lipophilic group linked b y either a saturated or an u n s a t u r a t e d 2-carbon bridge. Thus, structure 4, w h i c h is the c o r r e s p o n d i n g lactone form of structure 3, was the initial target• It should be n o t e d that the stereochemical requirements at positions C-4 and C-6 of lactone 4 were not k n o w n at this point. Although the chiralities of two steric centers in the lactone ring of mevastatin (cf.
3
A
A CH2CH2or CH= CH X-- Aromatics =
I
Totally synthetic analogs of mevastatin
W° I
x
HO~r'~O HO~ , . / ~ O
i
A
I
X cI
6
HO~co2Na
HO!~O!
~..o.
R" I,,,,~f6 R20 H r ,~
~CH
3
CH3" : 8
7
(SRI-62320) HOy"",,CO2Na
HO~T,,~ CO2Na o
~:.~~ OH3......
'-.y..OH
~,,.oa O
:
I~CH3 HO" ~
~ 10
(CS-514or Sq-31000) Fig. 2. Structures of mevastatin, Iovastatin and the corresponding ring-opened dihydroxy acid forms, and other analogs.
a a~
TIPS - November
TABLE I. In-vitro inhibitory activities of lactones 6 (Fig. 2) towards H M G - C o A reductase
Code •
Optical activity +
A
X
'. . . . . . . . .
a
+
when suitable aryl groups were placed at the 6-position of the 2,4dichlorophenyl ring in 6a(+). For example, a 1750-fold increase in potency attended the formal conversion of compound 6a(+) to 6d(+) (Table I). In view of the toxicity ascribed to chlorinated biphenyls, it was deemed desirable to find a suitable replacement for the chloro groups in 6d(+). The methyl group was considered to be a favorable bioisosteric replacement for the chloro group because of their comparable sizes and lipophilicities. Furthermore, aromatic methyl groups are susceptible to biooxidation and the resultant metabolite(s) is expected to be more readily eliminated from the body. Indeed, replacement of the chlorosubstituents in structure 6d(+) with methyl groups and further refinement ultimately afforded compound 6e(+), which has an inhibitory potency almost three times that of mevastatin. It is also noteworthy that a substantial decrease in potency occurs when structure 6e(+) is reduced to 6f(+). This result is contrary to earlier observations in the benzyl ether series and the prediction of an advantage in having a saturated or an unsaturated 2-carbon bridge connecting the lactone and the lipophilic groups remains precarious. Finally, the recent disclosure of research on compounds related to compound 7 (SRI62320) 2s appears to be particularly interesting. As evidence of the therapeutic usefulness of HMGCoA reductase inhibitors continues to mount, it is anticipated that research activities in search of new and novel inhibitors will be greatly intensified in the future.
Relative potency*
:+
+
:'++. "
t-CH=CH
+ - ........... ,i
.....
0.16 CI
C
+
CH2CH2
F-~-C.,O~-C+ T
38
CI
•' : ~ , +
d
+
t'CH=CH
c~
+:,
100
CI : •
::
i
e
.+
=
.:
+
.+
t-CH=CH
C
.
~'~'~1 ~ H
~
289
CH~
CH3
• All compounds in this table were tested after being converted to the sodium salts of the corresponding dihydroxy acids. The relative potency of the test compound was determined by compadng its I C ~ value with that of mevastatin, which was tested simultaneously and arbitrarily assigned a relative potency value of 100.
structure la) had been determined earlier 9, their critical importance to intrinsic inhibitory activity was yet to be ascertained. This issue was quickly resolved by determining that: (1) all activity resides in the t r a n s - d i a s t e r e o m e r 6a(+) (i.e. the c i s - d i a s t e r e o m e r 5(+) is inactive); and (2) only enantiomer 6a(+) is active 22. Similar results were observed subsequently in every compound series examined (see Table I), indicating that the chiralities of the two steric centers in structure 6 are critical in determining intrinsic HMG-CoA reductase inhibitory activity. Later, the finding that compound 6d(+) •possessed the same chirality in the lactone ring as that present in mevastatin was determined by Xray crystallography23 and further supported this conclusion. However, the weak intrinsic inhibitory potency of structure 6a(+) needed to be optimized. Attachment of either an arylmethoxy group (benzyl ether series) 24 or an aryl moiety (biphenyl series) 2s at the 6-position of the 2,4dichlorophenyl ring in compound 6a(+) dramatically enhanced potency. In the benzyl ether series,
1987 [ V o l . 8]
introduction of a 4-fluoro group on the benzyl moiety induces a remarkable increase in potency. Later, this substitution proved to be equally useful in other series. Another significant contribution towards the improvement of potency was observed when compound 6b(+) was hydrogenated to 6c(+). Resolution of 6c(+) yielded 6c(+), which had an inhibitory potency approaching that of mevastatin. Even more profound enhancement of potency was observed
TABLE II. In-vitro inhibitory activities of lactones 8 (Fig. 2) towards H M G - C o A reductase
Code
R~
R2
Relative potency*
O
b
H
CH,CH,C-~H
.......
d
®
H
F.~-c~
+++,
*See Table I.
254
CH~
-
119
+++,
TIPS - N o v e m b e r 1987 [Vol. 8]
445
Bile acid depletion
Untreated
HMG CoA Reductase inhibitor
Reductase inhibitor + bile acid depletion
Plasma
Liver
Intestine
(,
~
U
Fig. 3. Rationale for the therapeutic treatment of hypercholesterolemia.
Semisynthetic analogs of lovastatin Lovastatin (lb) 12 differs from mevastatin (la) structurally only by the presence of a 60c-methyl group and its relative potency is approximately twice that of mevastatin. Its discovery added a new direction to research in this a r e a delineation of the structural features of lovastatin responsible for its remarkable inhibitory activity and the use of this information to develop even more potent and efficacious inhibitors. Studies on the modification of the 2(s)-methylbutyryl moiety of lovastatin 26 yielded the following conclusions: • The presence of a side chain ester moiety is crucial to inhibitory potency (the potency of compound 8a is very weak). • The stereochemistry at the carbon 0c to the ester carbonyl group is not critical, since diastereomer 8b has the same potency as that of lovastatin (lb). • The spatial requirements of the acyl moiety are compatible with compact, branched aliphatic acyl groups. • Additional branching at the 0ccarbon of the acyl moiety enhances potency. In particular, the introduction of an additional methyl group on the carbon 0cto the ester carbonyl in lovastatin afforded structure 8c (MK-733) which, with an intrinsic potency more than six times that of mevastatin, is the most potent HMG-CoA reductase inhibitor reported to date. A series of side chain ether analogs of lovastatin were found to be mostly weaker inhibitors 27,
although compound 8d, the best in the series, shows a respectable level of potency. Introduction of a methyl group (0~-orientation) to the hydroxy-bearing carbon of the lactone in lovastatin provides homolog 8e (Ref. 27) in which the lactone moiety bears a close structural resemblance to HMG-CoA and mevalonic acid, the respective substrate and product of the reduction catalysed by HMG-CoA reductase (Fig. 1). Surprisingly, this modification is detrimental to activity. This was also true w h e n similar modifications were made in the synthetic analogs. Insertion of a methylene unit between the lactone hydroxy-bearing carbon and the carbonyl group in lovastatin afforded the carboxylate 9, which was devoid of intrinsic HMG-CoA reductase inhibitory activity. Finally, minimal decreases in potency were observed when either one or two double bonds in the hexalin moiety of lovastatin were hydrogenated, providing that the ring juncture of the resultant hydrogenation product is trans; when the ring juncture is cis, activity is markedly diminished. A most noteworthy finding has been the recent discovery of CS-514 (structure 10, also known as SQ31000) 28, a microbial oxidation product of mevastatin. CS-514 is currently undergoing clinical evaluation. The initial reports from investigations of this new HMGCoA reductase inhibitor show promise.
Therapeutic potential as hypocholesterolemic drugs One of the major recent developments in the prevention and
treatment of CHD is the understanding of how plasma cholesterol levels, especially LDL-C levels, are controlled. Based on the findings of M. S. Brown and J. L. Goldstein (see Ref. 29), a rationale for the therapeutic treatment of hypercholesterolemia is schematically presented in Fig. 3. A liver cell normally receives its cholesterol from: (1) uptake from circulating LDL-C via receptormediated endocytosis; (2) de-novo biosynthesis; and (3) uptake from chylomicron remnants. It then converts much of its cholesterol pool into bile acids. A large fraction of the bile acids secreted from the liver is returned to the liver through enterohepatic cycling. It is well established that the liver is the major site of LDL receptors and the production of LDL receptors is driven by the liver cell's d e m a n d for cholesterol. An effective therapeutic approach for lowering plasma LDL-C levels should be focused on ways to increase the production of L D L receptors in the liver. This is achievable by inhibition of the intestinal reabsorption of bile acids and/or inhibition of endogeneous cholesterol synthesis. Ultimately, when a new steady state is attained, the absolute amount of cholesterol entering the liver through the receptor pathway remains approximately the same as before, because the fall in plasma LDL levels is balanced by the increase in LDL receptors. But the important difference is that this delivery is now occurring at a lower plasma LDL level. In practice, the ingestion of bile acid resins (e.g. colestyramine and colestipol) causes plasma LDL
446
T I P S - N o v e m b e r 1987 [Vol. 8]
levels to d e c r e a s e b y b l o c k i n g the r e a b s o r p t i o n of bile acids a n d t h e r e b y i n c r e a s i n g the m e t a b o l i s m of cholesterol. H o w e v e r , the effect of this t h e r a p y is n o t p r o f o u n d . It usually produces only about a 20% d r o p i n p l a s m a L D L - C levels 5"6. The second method, using HMGC o A r e d u c t a s e i n h i b i t o r s s u c h as m e v a s t a t i n 16 a n d l o v a s t a t i n 17-19 to i n h i b i t c h o l e s t e r o l s y n t h e s i s , is a m u c h m o r e effective a p p r o a c h for l o w e r i n g p l a s m a L D L levels. W h e n g i v e n as a single a g e n t (40 m g p e r day) to familial h y p e r c h o l e s t e r o l e m i c h e t e r o z y g o t e s , lovastatin d e c r e a s e s p l a s m a LDL levels b y 33% (from 361 + 13 m g c m -3 to 242 + 15 m g cm-3) 19. W h e n g i v e n t o g e t h e r w i t h colestipol, an e v e n greater d r o p (46%) in p l a s m a L D L levels w a s r e p o r t e d 19. T h e h y p o c h o l e s t e r o l e m i c effect of l o v a s t a t i n h a s b e e n a s c r i b e d to the i n c r e a s e d p r o d u c t i o n of L D L r e c e p t o r s res u i t i n g f r o m the i n h i b i t i o n of de-novo cholesterogenesis. Other s t u d i e s also s u g g e s t that the s y n t h e s i s of v e r y l o w d e n s i t y l i p o p r o t e i n (VLDL), a p r e c u r s o r of LDL, h a s also b e e n s u p p r e s s e d b y l o v a s t a t i n (D. R. I l l i n g w o r t h , pers. c o m m u n . ) . Recently, lovastatin w a s also f o u n d to be effective in t h e t r e a t m e n t of p a t i e n t s with nonfamilial hypercholestero l e m i a 3°. Specifically, p a t i e n t s receiving 40 m g t w i c e a d a y e x p e r i e n c e d a m e a n r e d u c t i o n in p l a s m a L D L levels of 40%. So far, c o n s i s tently good patient acceptance and a l o w i n c i d e n c e of side effects h a v e b e e n o b s e r v e d in all s t u d i e s w i t h l o v a s t a t i n (for d e t a i l e d d i s c u s s i o n , see Ref. 31). T h e i m p o r t a n t p r i n ciple t h a t h a s b e e n realized f r o m t h e s e s t u d i e s is t h a t s t i m u l a t i o n of L D L r e c e p t o r activity u l t i m a t e l y l o w e r s p l a s m a L D L - C levels w i t h o u t g r o s s l y altering cholesterol s u p p l y to cells. H M G - C o A r e d u c tase i n h i b i t o r s are p o w e r f u l m e a n s of t a k i n g a d v a n t a g e of this p r i n ciple in t h e effective r e d u c t i o n of p l a s m a L D L levels. []
[]
[]
E v i d e n c e r e l a t i n g p l a s m a cholesterol levels to a t h e r o s c l e r o s i s a n d CHD has become compelling. M e c h a n i s m s r e s p o n s i b l e for h i g h c o n c e n t r a t i o n s of LDL are b e c o m ing understood. The LDL receptor is r e c o g n i z e d as a n i m p o r t a n t elem e n t in t h e r e g u l a t i o n of p l a s m a L D L levels. T h e fact t h a t H M G -
CoA reductase inhibitors are h i g h l y effective h y p o c h o l e s t e r o l e m i c a g e n t s is n o w well e s t a b lished. T h e u s e of this n e w l y disc o v e r e d t h e r a p y to treat a fraction of the p o p u l a t i o n at h i g h risk of C H D b e c a u s e of e x t r e m e l y h i g h p l a s m a L D L levels (e.g. familial hypercholesterolemia patients) a p p e a r s to be w i d e l y a c c e p t e d b y the m e d i c a l c o m m u n i t y . Will HMG-CoA reductase inhibitors also b e c o m e the c h o i c e of t h e r a p i e s to treat m o d e r a t e hyperchole s t e r o l e m i a , w h i c h is a m u c h m o r e common health problem than familial h y p e r c h o l e s t e r o l e m i a ? Int e n s i v e efforts to e s t a b l i s h the l o n g - t e r m clinical s a f e t y of H M G CoA reductase inhibitors are clearly vital to this issue.
References 1 Kannel, W. B., Castelli, W. D., Gordon, T. and McNamara, P.M. (1971) Ann. Intern. Med. 74, 1-12 2 Keys, A. (1980) in Seven Countries: A Multivariate Analysis of Death and Coronary Heart Disease, pp. 1--381,
Harvard University Press 3 Stamler, J., Wentworth, D. and Neaton, J. D. (1986) J. Am. Med. Assoc. 256, 28232828 4 Prugh, J. D., Rooney, C~ S. and Smith, R.L. (1983) Annu. Rev. Med. Chem. 18, 161-170 5 LRC-CPPT (1984)J. Am. Med. Assoc. 251, 351-364 6 LRC-CPPT (1984)J. Am. Med. Assoc. 251, 365-374 7 Rodwell, V. W., Nordstrom, J. L. and Mitschelen, J. J. (1976) Adv. Lipid Res. 14, 1-74 8 Endo, A., Kuroda, M. and Tsujita, Y. (1976) J. Antibiot. 29, 1346-1348 9 Brown, A. G., Smale, T. C., King, T. J., Hasenkam, P. and Thompson, R.H. (1976) J. Chem. Soc. Perkin Trans. 1,11651170 10 Endo, A. (1979) J. Antibiot. 32, 852-854 11 Endo, A. (1980) J. Antibiot. 33, 334-336 12 Alberts, A. W., Chen, J., Kuron, G., Hunt, V., Huff, J., Hoffman, C., Rothrock, J., Lopez, J., Joshua, H., Harris, E., Pachett, A., Monaghan, R., Currie, S., Stapley, E., Albers-Schonberg, G., Hensen, O., Hirshfield, J., Hoogsteen, K.,
Liesch, J. and Springer, J. (1980) Proc. Natl Acad. Sci. USA 77, 3957--3961 13 Endo, A. (1985)J. Med. Chem. 28, 401-405 14 Faust, J. R., Brown, M. S. and Go!dstein, J. L. (1980) J. Biol. Chem. 255, 6546-6548 15 Brown, M. S. and Goldstein, J. L. (1980) J. Lipid Res. 21, 505-517 16 Mabuchi, H., Sakai, T., Sakai, Y., Yoshimura, A., Watanabe, A., Wakasugi, T., Koizumi,. J. and Tekeda, R. (1983) N. Engl. J. Med. 308, 609-613 17 Bilheimer, D. W., Grundy, S. M., Brown, M.S. and Goldstein, J.L. (1983) Proc. Natl Acad. Sci. USA 80, 4124-4128 18 lUingworth, D. R. and Sexton, G. J. (1984) J. Clin. Invest. 74, 1972-1978 19 Illingworth, D. R. (1984) Ann. Intern. Med. 101, 598-604 20 Rosen, T. and Heathcock, C. H. (1986) Tetrahedron 42, 4909---4951 21 Nakamura, C. E. and Abeles, R. H. (1985) Biochemistry 24, 1364-1376 22 Stokker, G. E., Hoffman, W. F., Alberts, A.W., Cragoe, Jr, E.J., Deana, A.A., Gilfillar~, J. L., Huff, J. W., NoveUo, F. C., Prug~, J. D., Smith, R. L. and Willard, A. I~:,(1985) J. Med. Chem. 28, 347-358 23 Stokl~er,G. E., Alberts, A. W., Anderson, P.S., Cragoe, Jr, E.J., Deana, A.A., Gilfillan, J. L., Hirshfield, J., Holtz, W. J., Hoffman, W. F., Huff, J. W., Lee, T.-J., Novello, F.C., Prugh, J.D., Rooney, C.S., Smith, R.L. and Willard, A.K. (1986) J. Med. Chem. 29, 170-181 24 Hoffman, W. F., Alberts, A. W., Cragoe,
Jr, E.J., Deana, A.A., Evans, B.E., Gilfillan, J. L., Gould, N. P., Huff, J. W., Novello, F. C., Prugh, J. D., Rittle, K. E., Smith, R. L., Stokker, G. E. and WiUard, A. K. (1986) J. Med. Chem. 29, 159-169 25 Engstrom, R. G., Weinstein, D. B., Kathawala, F. G., Scallen, T., Eskesen, J. B., Rucker, M. L. and Miserendino, R. (1986) IX International Symposium on Drugs Affecting Lipid Metabolism,
Florence, Italy, p. 26 (Abstr.) 26 Hoffman, W. F., Alberts, A. W., Anderson, P. S., Chen, J. S., Smith, R. L. and WiUard, A. K. (1986) J. Med. Chem. 29, 849-852 27 Lee, T.-J., Holtz, W. J. and Smith, R. L. (1982) J. Org. Chem. 47, 4750--4757 28 Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S. (1986) Biochim. Biophys. Acta 877, 50-60 29 Brown, M. S. and Goldstein, J. L. (1986) Science 232, 34-47 30 Lovastatin Study Group II (1986) J. Am. Med. Assoc. 256, 2829-2834 31 Tobert, J. A. (1987) Circulation 76, 534538
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