Journal of Medicinal Chemistry
`
`0 Copyright 1985 by the American Chemical Society
`
`Volume 28, Number 4
`
`April 1985
`
`Perspective
`
`Compactin (ML-236B) and Related Compounds as Potential Cholesterol-Lowering
`Agents That Inhibit HMG-CoA Reductase
`
`to compactin (named monacolin K) was isolated from the
`fungus Monascus ruber by EndoS3p4 The same compound
`(named mevinolin) was also isolated by Alberts et al, from
`Aspergillus terreus.6 In addition to these products, several
`related metabolites were isolated from cultures of these
`fungi, which include dihydrocompactin from P. citrinum?
`dihydromevinolin from A. terreus,I monacolin J and L
`from M. ruber? and dihydromonacolin L and monacolin
`X from a mutant strain of M. r ~ b e r . ~ All these metabo-
`lites are structurally related to each other (Figure 1) and
`are specific inhibitors of HMG-CoA reductase.
`Several active compounds have also been derived from
`either compactin or monacolin K by microbial conversion
`(Figure 2). 3/3-Hydroxycompactin, 6a-hydroxyisocom-
`pactin and 3-hydroxymonacolin K are produced by grow-
`ing the fungus Mucos hiemalis in a culture medium that
`contains, in addition to nutrients, compactin and mona-
`Colin K, respectively.1° Compactin is also converted to
`3a-hydroxycompactin by Syncephalastrum nigricansll
`and to 6a-hydroxyisocompatin by Absidia coerulea.12
`8a-Hydroxycompactin and 8a-hydroxymonacolin K are
`derived from compactin and monacolin K, respectively by
`growing Schizophyllum commune.13 Phosphorylated
`
`Akira Endo
`Department of Agricultural and Biological Chemistry, Tokyo Noko University, Fuchu-shi, Tokyo 183, Japan.
`Received July 31, 1984
`<
`One of the major causes of death in the U.S. and other
`developed countries is coronary heart disease. Approxi-
`mately 800 000 Americans die of it a year, amounting to
`40% of all deaths. Coronary heart disease actually is a
`wide assortment of diseases. The basic manifestation of
`many of them is atherosclerosis, caused when fatty*deposit.a
`called plaque build ,up on the inner walls of arteries.
`Cholesterol is a major. component of the atherosclerotic
`plaque. Many scientists believe that a high level of cho-
`lesterol in the blood is a major contributor to (he devel-
`opment of atherosclerosis. Since in humans the greater
`part of the cholesterol in the body is synthesized de novo,
`mostly in the liver, the search for drugs to inhibit chole-
`sterol biosynthesis has long been pursured as a means to
`lower the level of plasma cholesterol and so help to prevent
`and treat atherosclerosis.
`Cholesterol is synthesized from acetyl-coA via a series
`of more than 20 enzymatic reactions, This synthetic
`pathway is mainly regulated by the activity of the enzyme
`3-hydroxy-3-methylglutaryl (HMG-CoA) redyctase, which
`catalyzes the reduction of HMG-CoA to mevalonate. In
`many tissues, changes in the activity of this enzyme are
`closely related to changes in the overall rate of cholesterol
`synthesis over a wide range of physiological conditions.
`This enzyme is, therefore, a prime target for pharmaco-
`logical intervention.
`Endo and his associates at Sankyo Co. (Tokyo), who had
`tested 8000 strains of microorganisms for their ability to
`produce an inhibitor of sterol synthesis in vitro, first
`discovered three active compounds, designated ML-236A,
`ML-236B, and ML-236C, in the culture broth of the fungus
`Penicillium citrinum.' The main compound ML-236B
`(generic name: mevastatin) is identical to one later isolated
`from Penicillium breuicompactum as an antifungal agent
`named compactin.2 ML-236B has been shown to be a
`specific inhibitor of HMG-CoA reductase and highly ef-
`fective in lowering plasma cholesterol levels in animals and
`men.
`Isolation and Chemistry of Compactin (ML-236B)
`Related Compounds. Another active compound related
`
`(3) A. Endo, J. Antibiot., 32, 852 (1979).
`(4) A. Endo, J. Antibiot., 33, 334 (1980).
`(5) A. W. Alberta, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoff-
`man, J. Rothrock, J. Lopez, H. Joshua, E. Harris, A. Pachett,
`R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonberg, 0.
`Hensen, J. Hirshfield, K. Hoogsteen, J. Liesch, and J. Springer,
`Proc. Natl. Acad. Sci. U.S.A., 77, 3957 (1980).
`(6) T. Y. K. Lam, V. P. Gullo, R. T. Goegelman, D. Jorn, L.
`Huang, C. DeRiso, R. L. Monaghan, and L. Putter, J. Anti-
`biot., 34, 614 (1981).
`(7) G. Albers-Schonberg, H. Joshua, M. B. Lopez, 0. D. Hensens,
`J. P. Springer, J. Chen, S. Ostrove, C. H. Hoffman, A. W.
`Alberts, and A. A. Pachett, J. Antibiot., 34, 507 (1981).
`(8) A. Endo, K. Hasumi, and S. Negishi, J. Antibiot., in press.
`(9) A. Endo, K. Hasumi, T. Nakamura, M. Kunishima, and M.
`Masuda, J. Antibiot., in press.
`(10) N. Serizawa, K. Nakagawa, K. Hamano, Y. Tsujita, A. Tera-
`hara, and H. Kuwano, J. Antibiot., 36, 604 (1983).
`(11) N. Serizawa, K. Nakagawa, K. Hamano, Y. Tsujita, A. Tera-
`hara, and H. Kuwano, J. Antibiot., 36, 608 (1983).
`(12) N. Serizawa, K. Nakagawa, Y. Teujita, A. Terahara, H. Ku-
`wano, and M. Tanaka, J. Antibiot., 36, 918 (1983).
`(13) H. Yamashita, S. Tsubokawa, and A. Endo, J. Antibiot., in
`press.
`
`0 1985 American Chemical Society
`
`(1) A. Endo, M. Kuroda, and Y: Tsujita, J. Antibiot., 29, 1346
`(1976).
`(2) A. G. Brown, T. C. Smale, T. J. King, R. Hasenkamp, and R.
`H. Thompson, J. Chem. SOC., Perkin Trans. 1 1165 (1976).
`
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`

`
`402 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 4
`
`Hoqo
`
`ML - 236A( 2
`ML-236B(Compactin)(50)
`ML-236C(10)
`Monocolin J ( 4 )
`
`R l R2
`H OH
`0
`H 0
`H
`H
`CH, OH
`0
`
`
`
`H O o O
`
`Monacolin K(Mevinolin)(100) CH3 0
`Monacolin L(15)
`CH, H
`
`Monocolin X(20)
`
`CH, O
`
`v
`
`
`
`R l Rz
`
`Dihydrocornpact~n(SO)
`
`H 0%
`
`Dihydromevinolin(l00)
`Dihydromonacoltn L(20)
`
`CH3 0%
`CH, H
`
`Figure 1. Compactin (ML236B) related compounds of microbial
`origin. Numbers in the parentheses represent relative activity
`to inhibit rat liver HMG-CoA reductase.
`derivatives (5’-phosphocompactin acid and 5’-phospho-
`monacolin K acid) are produced by several fungal strains.14
`Recently new microorganisms producing compactin have
`been isolated by Endo et al. (unpublished data), which
`include Paecilomyces sp. and Hypomyces chrysospermus.
`These fungal strains are particularly suited for the pro-
`duction of compactin, since no mycotoxin has been isolated
`from culture broth of these fungi. Other producers, Pen-
`icillium citrinum and P. brevicompactum, are known to
`produce mycotoxin^.'^ Compactin-related compounds are
`produced as the water-soluble acid form which is converted
`to the water-insoluble lactone form by acidification or
`drying except for the 5’-phosphorylated derivatives (Figure
`3) * Mechanism for HMG-CoA Reductase Inhibition.
`The inhibition of HMG-CoA reductase by compactin and
`related compounds is reversible.16J7 As can be expected
`from the structure of their acid forms, the inhibition by
`these compounds is competitive with respect to HMG-CoA.
`The Ki value for the acid form of compactin, which is
`determined from the partially purified rat liver enzyme,
`
`is - 1 X lo4 M, while under the same conditions, the K,
`value for HMG-CoA is - 10” M.17 Thus, the affinity of
`
`HMG-CoA reductase for compactin is 10 000-fold higher
`than its affinity for the natural substrate HMG-CoA,
`showing compactin to be a highly potent inhibitor.
`Compactin does not affect other enzymes involved in
`cholesterol biosynthesis.’* In addition, almost all studies
`on compactin with cultured cells and intact animals suggest
`that reductase is the only enzyme that is inhibited by
`compactin (see below).
`Structure-Activity Relationships at Enzyme Level.
`Structural similarity between HMG-CoA and compactin-
`related compounds suggests that the active center of these
`agents in the inhibition of HMG-CoA reductase is at the
`&lactone moiety of the molecules. This hypothesis is
`supported by the data that inhibitory activity of compactin
`is reduced to l/loo or less by acetylation of the hydroxy
`
`(14) A. Endo and H. Yamashita, J. Antibiot., in press.
`(15) A. Ciegler, S. Kadis, and S: H. Ajl, Eds., “Microbial Toxins”,
`Academic Press, New York, 1971, Vol. 5.
`(16) A. Endo, M. Kuroda, and K. Tanzawa, FEBS Lett., 72, 323
`(1976).
`(17) K. Tanzawa and A. Endo, Eur. J. Biochem., 98, 195 (1979).
`(18) A. Endo, Y. Tsujita, M. Kurda, and K. Tanzawa, Eur. J. Bio-
`chem., 77, 31 (1977).
`
`Perspective
`group at either C3’ or C5, (unpublished data) and that
`5‘-phosphocompactin acid and 5’-phosphomonacolin K
`and 1/20 of compactin and monacolin K in the
`acid are
`inhibitory activity, re~pective1y.l~
`Other portions of compactin molecule also seem to be
`involved in inhibitory activity (Figure 1). Among them,
`the cu-methylbutyrate ester plays a significant role, since
`analogues that lack such an ester (ML-236A and monacolin
`J) are ‘ / 2 5 in the activity, as compared with their respective
`counterparts (compactin and monacolin K).
`The decalin ring of compactin-related compounds is
`essential to the inhibitory activity. This is shown by the
`data that HMG is more than 106-fold less active than
`compa~tin.’~ Dihydrocompactin, dihydromevinolin, and
`dihydromonacolin L are comparable in the activity to
`compactin, monacolin K, and monacolin L, respecti~ely.6J~~
`Monacolin K analogues that have a methyl group at C3
`are twice as active as their respective compactin analogues
`(Figure l), indicating a contribution of the methyl radical
`to potency. However, hydroxylation at C,, CB, or C6 gives
`no significant effect.lD-13
`Inhibition of Sterol Synthesis in Cultured Cells
`and in Animals. Compactin significantly inhibits cho-
`lesterol biosynthesis in a variety of cultured animal and
`human cells at as low as nM (loe9 M) concentration.20v21
`In cultured human skin fibroblasts, inhibition of sterol
`synthesis from [14C]acetate is 50% at 1 nM, 80% at 10 nM,
`90% at 100 nM, 95% at 1 pM, and 100% at 10 pM, re-
`spectively.20 Under these conditions, sterol synthesis from
`[14C]mevalonate and fatty acid synthesis from [14C]acetate
`are not significantly affected.
`The inhibition of HMG-CoA reductase by compactin
`results in the reduction of mevalonate production. As
`shown in Figure 4, in addition to cholesterol, ubquinones
`and dolichols which are involved in electron transport in
`the mitochondria and glycoprotein synthesis are also de-
`rived from mevalonate. In human skin fibroblasts which
`are grown in the presence of LDL (low-density lipo-
`protein)-cholesterol, compactin has no detectable effects
`on the synthesis of these two isoprenoids at 10 nM, a
`concentration that causes over 50% inhibition of sterol
`synthesis (unpublished data). When HMG-CoA reductase
`is partially suppressed by compactin, cells must have some
`way of diverting the small amounts of synthesized meva-
`lonate preferentially into these crucial nonsterol products.
`At higher concentrations where sterol synthesis is re-
`duced by over go%, compactin inhibits cell growth?O This
`inhibition can be overcome and cells can grow normally
`if a small amount of mevalonate is added to the culture
`medium. The compactin inhibition of growth and its re-
`versal by mevalonate have been reported in other cultured
`
`cells and in animal~.~~-~O These findings strongly suggest
`
`(19) M. S. Brown, J. R. Faust, J. L. Goldstein, I. Kaneko, and A.
`Endo, J. Biol. Chem., 253, 1121 (1978).
`(20) I. Kaneko, Y. Hazama-Shimasa, and A. Endo, Eur. J. Bio-
`chem., 87, 313 (1978).
`(21) A. Endo, Trends Biochem. Sci., 6, 10 (1981).
`(22) V. Quesney-Huneeus, M. H. Wiley, and M. D. Siperstein, Proc.
`Nutl. Acad. Sci. U.S.A., 76, 5056 (1979).
`(23) A. J. R. Habenicht, J. A. Glomset, and R. Ross, J. Biol. Chem.,
`255, 5134 (1980).
`(24) S. L. Perkins, S. F. Leiden, and J. D. Stubbs, Biochim. Bio-
`phys. Acta, 711, 83 (1982).
`(25) J. A. Cuthbert and P. E. Lipsky, J. Immunol., 124,2240 (1980).
`(26) D. D. Carson and W. J. Lennarz, Proc. Nutl. Acud. Sci. U.S.A.,
`76, 5709 (1979).
`(27) D. H. Minsker, J. S. MacDonald, R. T. Robertson, and D. L.
`Bokelman, Teratology, 28, 449 (1983).
`(28) J. Ryan, E. C. Hardeman, A. Endo, and R. D. Simoni, J. Biol.
`Chem., 256,6762 (1981).
`
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`

`
`Perspective
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 4 403
`
`HO 0
`
`0
`
`
`
`HO -0
`
`Ho
`
`COOH
`
`RI=H
`
`,Rz=H Cornpactin
`
`R,=OH .R2=H ea-13-Hydroxy-
`cornpactin
`
`6-Hydroxy- iso-
`compactin
`
`5'- Phosphocompactin
`acid
`
`R,.H
`
`Hoc:H
`
`n
`
`A
`B
`Figure 3. HMG-CoA (A) and the acid form of compactin (B).
`
`Acetyl-coA
`
`Mevalonate
`
`+ +
`HMG -CoA 1 HMG -CoA Reductase
`1 +
`- -
`1 + +
`+
`1 +
`
`,R2=OH 3-Hydroxy-
`cornpactin
`Figure 2. Compactin (ML-236B) related compounds produced by microbial conversion.
`the feeding of compactin
`the rat,34 mouse,34 and
`does not decrease plasma cholesterol.
`In the rat, the inhibition of hepatic cholesterol synthesis
`by compactin administration is accompanied by a large (5-
`to 10-fold) increase in HMG-CoA reductase activity in the
`liver.34 In addition, compactin causes in the rat a signif-
`icant decrease in the fecal elimination of bile acids which
`are synthesized from cholesterol in the liver.
`
`In the dog and m0nkey,3~'~~ compactin produces a rapid
`reduction of plasma cholesterol levels at a daily dose of
`10-20 mg/kg. Plasma triglyceride is not consistently
`lowered. Of the plasma lipoproteins, LDL, which are
`known to be atherogenic, are reduced, but HDL (high-
`density lipoproteins), antiatherogenic lipoproteins, are not
`affected. Compactin stimulates hepatic HMG-CoA re-
`ductase in the dog, but the increase is of considerably lesser
`magnitude than in the rat (unpublished data). In addition,
`fecal excretion of bile acids is, also unlike in the rat, not
`reduced but rather slightly elevated by compactin both in
`
`the dog and m ~ n k e y . ~ ~ ~ ~ ~ These data suggest that the lack
`of hypocholesterolemic effect in the rat is due, at least
`partly, to the large increase in hepatic HMG-CoA reduc-
`tase activity and to the decrease in the bile acid excretion.
`Metabolic disposition of compactin in the rat has not yet
`been reported.
`In normal rabbits, compactin and mevinolin reduce
`significantly plasma cholesterol levels at a daily dose of
`Essentially the same
`5 and 2 mg/kg, r e ~ p e c t i v e l y . ~ ~ ? ~ ~
`activity is obtained in rabbits with inborn hyperlipidemia
`(called WHHL rabbits).33
`In healthy persons, compactin and mevinolin are well
`tolerated and exert a rapid and profound cholesterol-low-
`ering effect at a dose of 5-10 m g / d a ~ . ~ ~ a ~ Of the various
`
`types of hypercholesterolemia, FH (familial hypercho-
`lesterolemia) characterized by a marked increase in plasma
`LDL, are notably resistant to drug therapy. Hyperli-
`pidemia is present in such persons and cardiovascular
`disease often, but not always, occurs prematurely. In
`
`lsopentenyl
`Pyrophosphate
`
`lsopentenyl
`Adenine
`
`Dolichol-
`
`Farnesyl
`-pyrophosphate-
`
`-Coenzyme
`
`A
`
`Cholesterol
`Figure 4. Synthetic pathway for cholesterol, ubiquinones, and
`dolichols. Compactin inhibits conversion of HMG-CoA to me-
`valonate catalyzed by HMG-CoA reductase.
`that compactin is a specific inhibitor of HMG-CoA re-
`ductase.
`Hypocholesterolemic Activity. When given orally,
`compactin is effective in lowering plasma cholesterol levels
`in man and some animal species, notably the chicken,21
`rabbit,31
`and monkey.33 In other species, such as
`
`(29) D. C. Cohen, S. L. Massoglia, and D. Gospodarowicz, J. Biol.
`Chem., 257,9429 (1982).
`(30) D. C. Cohen, S. L. Massoglia, and D. Gospodarowicz, J. Biol.
`Chem., 257, 11106 (1982).
`(31) Y. Watanabe, T. Ito, M. Saeki, M. Kuroda, K. Tanzawa, M.
`Mochizuki, Y. Tsujita, and M. Arai, Atherosclerosis, 38, 27
`(1981).
`
`~
`
`~
`
`(32) Y. Tsujita, M. Kuroda, K. Tanzawa, N. Kitano, and A. Endo,
`Atherosclerosis, 32, 307 (1979).
`(33) M. Kuroda, Y. Tsujita, K. Tanzawa, and A. Endo, Lipids, 14,
`585 (1979).
`(34) A. Endo, Y. Tsujita, M. Kuroda, and K. Tanzawa, Biochim.
`Biophys. Acta, 575, 266 (1979).
`(35) P. A. Kroon, K. M. Hand, J. W. Huff, and A. W. Alberts,
`Atherosclerosis, 44, 41 (1982).
`(36) H. Shigematsu, Y. Hata, M. Yamamoto, T. Oikawa, Y. Ya-
`mauchi, N. Nakaya, and Y. Goto, Geriatr. Med., 17, 1564
`(1979).
`(37) J. A. Tobert, G. D. Bell, J. Birtwell, I. James, W. R. Kukovetz,
`J. S. Pryor, A. Buntinx, I. B. Holmes, Y. S. Chao, and J. A.
`Bologese, J. Clin. Invest., 69, 913 (1982).
`
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`
`404 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 4
`
`Perspective
`lation and are responsible for the transport and delivery
`of cholesterol as well as triglyceride to extrahepatic tissues
`(Figure 5).43p44 In the circulation, VLDL becomes a sub-
`strate for lipoprotein lipase. As lipolysis occurs, VLDL
`become smaller and form remnants called IDL (interme-
`diate-density lipoproteins) (Figure 5). In man, IDL serve
`as the precursors of LDL, which are the major carrier of
`cholesterol to extrahepatic tissues.
`LDL in the circulation are taken up by extrahepatic cells
`by a LDL receptor-mediated process.43 This process in-
`volves the binding of LDL to specific, high-affinity binding
`sites located on the surface of plasma membranes. Once
`binding has occurred, the LDL-receptor complex is in-
`ternalized by endocytosis and digested by lysosomal en-
`Figure 5. Biosynthesis and metabolism for cholesterol and li-
`zymes to liberate free cholesterol. This cholesterol is
`poproteins and possible mechanisms for action of compactin.
`Arrows in the parentheses represent increase (7) or decrease (3.)
`utilized as an important structural component for cell
`membranes and in several tissues as the precursor for the
`by compactin.
`synthesis of steroid hormones.
`patients with heterozygous FH, Yamamoto et al. obtained
`An additional pathway for LDL metabolism involves a
`a 27% decrease in plasma cholesterol after 4-8 weeks of
`lower affinity process associated with scavenger cells or
`compactin treatment at a dose of 60-100 m g / d a ~ . ~ ~
`macrophages of the reticuloendothelial system (Figure 5).
`Plasma cholesterol started to decrease within 2 weeks of
`In man, one-third to two-thirds of the plasma LDL is
`treatment. According to Mabuchi et al.,39 a 29% decrease
`cleared by the high-affinity receptor-mediated process, and
`in plasma cholesterol is obtained in heterozygous FH pa-
`the remainder is degraded by the scavenger cells.
`tienb treated with compactin for 24 weeks at a dose of
`Although HDL are initially formed in the liver and in-
`30-60 mg/day. The reduced cholesterol levels is sustained
`testine, they become modified in the circulation by in-
`during the period of drug treatment and returns to the
`teraction with the other lipoproteins and extrahepatic cells.
`pretreatment concentrations after terminating drug
`It has been proposed that HDL are involved in removing
`therapy. LDL are the only lipoprotein fractions that are
`cholesterol from extrahepatic cells. The HDL-cholesterol
`are then transferred to IDL via a plasma exchange protein.
`decreased by compactin treatment; HDL are slightly ele-
`vated. Plasma triglyceride levels are not significantly
`As mentioned, compactin does lower plasma LDL-
`lowered.
`cholesterol levels both in animals and man but does not
`A much greater decrease in plasma LDL levels can be
`show a significant effect on HDL and IDL. There are two
`obtained by the combination of compactin and chole-
`possible mechanisms for lowering plasma LDL levels by
`styramine, bile acid binding resin, than can be produced
`a HMG-CoA reductase inhibitor, namely, decreasing LDL
`by either agent a l 0 n e . 4 ~ ~ ~ ~ While the reduction of LDL-
`synthesis and increasing its clearance from the circulation.
`associated cholesterol in patients with heterozygous FH
`The inhibition of cholesterol synthesis in the liver by
`is 28% by cholestyramine (12 g daily), that obtained by
`compactin is expected to decrease hepatic cholesterol
`content, which would be partly compensated by the de-
`the combination of cholestyramine (12 g daily) and com-
`pactin (30 mg daily) is as high as 53%.40 This combination
`crease in VLDL synthesis and thus in its transport to the
`circulation. The reduced supply of plasma VLDL, in turn,
`seems to be the most effective therapy of hypercholest-
`erolemia so far reported. The combination effect of these
`would induce the decrease in IDL and then LDL synthesis.
`This hypothesis is supported by the data that mevinolin
`two drugs could be due to the inhibition by compactin of
`treatment of the dog produces a significant decrease in the
`the increased cholesterol synthesis caused by cholestyr-
`synthetic rate for plasma LDL.45 In FH heterozygotes
`amine treatment, since cholestyramine is known to induce
`studied by Bilheimer et al.,& however, the LDL synthesis
`hepatic HMG-CoA reductase activity in animals.% Some
`does not change significantly by mevinolin treatment. The
`FH patients in Japan have been on the compactin treat-
`inhibition of VLDL and IDL synthesis by compactin or
`ment for over 2 years with no serious side effects. Com-
`mevinolin and of VLDL supply to the circulation has not
`pactin is much more effective in patients with hypercho-
`yet been shown and remains to be studied (Figure 5).
`lesterolemia other than FH; a significant cholesterol-low-
`ering activity is obtained at a dose of 5-10
`Secondly, the decreased content of hepatic cholesterol
`Compactin Effects in VLDL and LDL Metabolism.
`due to compactin inhibition is expected to be partly com-
`pensated by stimulating the hepatic uptake and degra-
`The greater part of the daily supply of body cholesterol
`dation of plasma LDL. This hypothesis is supported by
`is endogenously provided by synthesis, mainly in the liver,
`studies both in the dog and man!5$46 In the dog, mevinolin
`where the endogenous cholesterol is used in VLDL syn-
`raises the number of hepatic LDL receptors, which, in turn,
`thesis as a structural component. VLDL enter the circu-
`enhances receptor-dependent uptake and degradation of
`plasma LDL (Figure 5). The receptor-independent
`clearance by scavenger cells does not change by mevinolin
`
`(38) A. Yamamoto, H. Sudo, and A. Endo, Atherosclerosis, 36,259
`(1980).
`(39) H. Mabuchi, T. Haba, R. Tatami, S. Miyamoto, Y. Sakai, T.
`Wakasugi, A. Watanabe, J. Koizumi, and R. Takeda, N. Engl.
`J. Med., 306,478 (1981).
`(40) H. Mabuchi, T. Sakai, Y. Sakai, A. Yoshimura, A. Watanabe,
`T. Wakasugi, J. Koizumi, and R. Takeda, N. Engl. J. Med.,
`308, 609 (1983).
`(41) A. Yamamoto, T. Yamamura, S. Yokoyama, H. Sudo, and Y.
`Matsuzawa, Znt. J. Clin. Pharmacol. Ther. Toxicol., 22, 493
`(1984).
`(42) Y. Hata, H. Shigematsu, T. Oikawa, M. Yamamoto, Y. Ya-
`mauchi, and Y. Goto, Geriatr. Med., 18, 104 (1980).
`
`(43) M. S. Brown, P. T. Kovanen, and J. L. Goldstein, Science, 212,
`. ,
`628 (1981).
`(44) R. W. Mahley and T. L. Innerarity, Biochirn. Biophys. Acta,
`737, 197 (1983).
`(45) P. T. Kovanen, D. W. Bilheimer, J. L. Goldstein, J. J. Jaram-
`illo, and M. S. Brown, Proc. Natl. Acad. Sci. U.S.A., 78, 1194
`(1981).
`(46) D. W. Bilheimer, S. M. Grundy, M. S. Brown, and J. L. Gold-
`stein, Proc. Natl. Acad. Sei. U.S.A., 80, 4124 (1983).
`
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`
`J. Med. Chem. 1985,28,405-407
`
`405
`
`treatment. The receptor-dependent clearance of plasma
`LDL is also significantly increased in FH heterozygotes
`by mevinolin treatment.46 Thus, LDL lowering in these
`patients can be explained by the increased LDL receptor
`activity.
`Conclusion. In the Japanese population, as well as the
`European and American populations, FH occurs at a fre-
`quency of 1 in 500. It is closely associated with premature
`atherosclerotic heart disease. The increased levels of LDL
`in FH is believed to accelerate atherosclerosis.
`F H is a disorder characterized by the defect in LDL
`receptor, which causes a decrease in LDL uptake by the
`cell. The decreased LDL uptake, in turn, results in higher
`levels of both plasma LDL and cellular HMG-CoA re-
`ductase activity. Thus, treatment should increase LDL
`catabolism by stimulating the LDL pathway. As men-
`tioned, treatment with compactin (or mevinolin) is ideal
`in FH in that it reduces plasma LDL levels by stimulating
`
`the LDL receptor-mediated catabolism.
`Of the analogues related to compactin, the acid form of
`3@-hydroxycompactin (CS-514) (Figure 2), which is com-
`parable to compactin in both in vitro and in vivo activities,
`has recently been reported to be superior to the parent
`compound in safety.47 Compactin-related compounds
`have not been widely used in the treatment of hypercho-
`lesterolemia. Yet the studies with these agents have es-
`tablished a general principle: a competitive inhibitor of
`HMG-CoA reductase can reduce LDL levels in plasma by
`increasing LDL receptor without depleting vital body
`stores of cholesterol.
`Registry No. HMG-CoA reductase, 9028-35-7; compactin,
`73573-88-3; cholesterol, 57-88-5.
`
`(47) S. Fujii, M. Arai, Y. Tsujita, and M. Tanaka, presented at the
`16th General Meeting of the Japanese Society of Atheroscler-
`osis (Tokyo), July 22-23, 1984.
`
`Communications to the Editor
`
`Novel Photoaffinity Label for the Dopamine Dz
`Receptor: Synthesis of
`4-Azido-5-iodo-2-methoxy-N-[
`1-(phenylmethy1)-4-
`piperidinyllbenzamide (Iodoazidoclebopride, IAC)
`and the Corresponding '2SI-Labeled Analogue
`(12SIAC)
`Sir:
`Dopamine agonist elecited behaviors (rotation, psycho-
`tomimetic actions, antiparkinsonian action, stereotypy, and
`locomotion) appear to be mediated by dopamine D, re-
`ceptors in the brain.'
`Although the dopamine Dz receptor has been solubilized
`by several laboratories,2 attempts to isolate and purify the
`protein by affinity chromatography3 or photoaffinity la-
`beling4i5 have been relatively unsuccessful.6~7 Alkylating
`type irreversible ligands have been developed (i.e.,
`[3H]-N-(chloroethyl)norapomorphine, phenoxybenzamine,
`N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, flu-
`penthixyl chloride), but have been too low in receptor
`affinity and/or selectivity to be of value in dopamine D2
`receptor is~lation.~J'-'~ The molecular characterization
`of dopamine Dz receptors has been hampered by the lack
`of specific photoaffinity probes which can be used to co-
`valently label these sites.
`
`(1) Seeman, P. Pharmacol. Rev. 1980,32,229-313.
`(2) Madras, B. K.; Davis, A.; Seeman, P. Eur. J. Pharmacol. 1982;
`78, 431.
`(3) Ramwani, J.; Mishra, R. K. Fed. Proc., Fed. Am. SOC. Exp.
`Biol. 1982, 41, 1325.
`(4) Nishikori, K.; Noshiro, 0.; Sano, K.; Maeno, H. J. Biol. Chem.
`1980,255,10909-10915.
`(5) Thermos, K.; Murphy, R. B.; Schuster, D. I. Biochem. Biophys.
`Res. Commun. 1982,1-6, 1469-1477.
`(6) Davis, B.; Abood, L.; Tometako, A. M. Life Sci. 1980,26,85-88.
`(7) Testylier, G.; Daveloose, D.; Letterrier, F.; Buchman, 0.; Shi-
`moni, M. Photochem. Photobiol. 1984,39, 273.
`(8) Guan, J. H.; Neumeyer, J. L.; Filer, C. N.; Ahern, D. G.; Lilly,
`.
`L.; Watanabe, M.; Grigoriadis, D.; Seeman, P. J. Med. Chem.
`1984, 27, 806.
`(9) Hamblin, M. W.; Creese, I. Life Sci. 1983, 32, 2247.
`(10) Schuster, D. I.; Holden, W. L.; Narula, A. P. S.; Murphy, R. B.
`Eur. J. Pharmacol. 1982, 77, 313-316.
`
`We report here the synthesis of a series of substituted
`benzamides (Ib-Ig) and in particular Ig, an agent, useful
`as a photoaffinity label, which selectively and irreversibly
`labels dopamine Dz receptors upon light irradiation.
`0
`
`Ia, R, = C1; R, = NH,;clebopride
`Ib, R, = C1; R, = N,; azidoclebopride (AC)
`IC, R, = H; R, = NH,;.
`Id, R, = I ; R, = NH,; iodoclebopride
`Ie, R, = I ; R, = N,; iodoazidoclebopride (IAC)
`I f , R, = 1251; R, = NH,; [1251]iodoclebopride
`Ig, R, = lZ5I; R, = N,; [1251]iodoazidoclebopride ( IfSIAC)
`Since clebopride (Ia) was reported as a selective D, re-
`ceptor antag~nist,'l-~~ we selected the substituted benz-
`amide as a ligand that has inherent affinity for the binding
`site of D, receptors and incorporated an azido group as a
`photosensitive functional group, replacing the amino group
`in Ia. When Ib was photoactivated with light, it was ca-
`pable of forming a covalent bond at or near the binding
`~ i t e . ' ~ J ~ The association with the recognition site will
`ordinarily be reversible until photolysis is initiated; the
`covalent linkage thus formed between the photoprobe and
`the binding site will thus facilitate the characterization and
`isolation of the dopamine D, receptor.
`Clebopride (Ia) when reacted with sodium nitrite and
`concentrated hydrochloric acid formed the intermediate
`diazoniuni salt which was treated with an aqueous solution
`
`(11) Elliott, P. N. C.; Jenner, P.; Huizing, G.; Marsden, G. D.;
`Miller, R. Neuropharmacology 1977, 16, 333.
`(12) Fleminger, S.; Van de Waterbeemed, H.; Rupniak, N. M. J.;
`Revill, C.; Testa, B.; Jenner, P.; Marsden, C. D. J. Pharm.
`Pharmacol. 1983,35, 363.
`(13) Kebabian, J. W.; Caine, D. B. Nature (London) 1979,277,93.
`(14) Niznik, H. B.; Guan, J. H.; Neumeyer, J. L.; Seeman, P. Eur.
`J. Pharmacol. 1984,104, 389.
`(16) Niznik, H. B.; Guan, J. H.; Neumeyer, J. L.; Seeman, P. Mol.
`Pharmacol., in press.
`
`0022-2623/85/1828-0405$01.50/0 Q 1985 American Chemical Society
`
`NCI Exhibit 2024
`Page 5 of 5