Inhibitors and stimulators of cholesterolgenesis enzymes. Structure

Aug 1, 1971 - Donald T. Witiak, Roger A. Parker, Mary E. Dempsey, Mary C. Ritter. J. Med. Chem. , 1971, 14 (8), pp 684–693. DOI: 10.1021/jm00290a005...
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Journal of illedicinal Chemistry, 1971, Vol. 14, h70.8

WITIAK,et al.

from inadequate ranges of x , c, and E R values. One should be careful to avoid the use of only those substituents n-hick lie on or near a straight line in Figures 1-3; i.e., those which are highly correlated. Acknowledgment.-The

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acknowledge helpful discussions with Professor Corwin Hansch. This study was supported by the I?.S. Army Medical Research & Development Command under Contract No. DADA-17-69-C-9106. The paper is Contribution No. 896 to the Army Research Program on Malaria.

Inhibitors and Stimulators of Cholesterolgenesis Enzymes. A Structure-Activity Study in Vitro of Amino and Selected N-Containing Analogs of 5a-Cholestane-3~,5~~,6~-triol~~-~ DONALD T. WITIAK,*ROGERA. P A R K E R , ~ ~ - ~ Division of ;Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43610

MARYE. DEMPSEY,AND MARYC. RITTERQ Department of Bzochemzstry, School of M edxzne, llnzverszty of Slznnesota, Manneapolzs, Jhnesota 55456 Received December 9, 1970 The atereoselective synthesib and biological evaluation zn vitro of the 3p-, sa-, 5,- and 6p-monoamino and 3p,6pdiamino analogs of 5a-cholestane-3~,5a,6p-triol and selected azido and oximino intermediates are discussed. Compounds were studied for their inhibitory action on acetate-b-14C and mevalonate-2-I4C incorporation into nonsaponifiable producta catalyzed by a rat liver homogenate preparation and for their inhibitory or stimulatory action on two semipurified liver enzymes, A7-sterol A6-dehydrogenase and A5>'-sterolA7-reductase. Some of our preliminary studies designed to probe the mechanism of action of three inhibitors and one stimulator of the A7 reductase enzyme are also described. The results suggest that the analogs exert their actions by direct effect on the microsomal enzyme and by altering the function of a sterol carrier protein (SCP) required for full activity of the enzyme.

Studies n-it'h oxo analogs and esters of 5,-cholest'ane3p,5a16p-triol (1) suggested t'he free 5a-OH function to be important for lowering serum cholest'erol levels in the cholesterol-fed hypercholesterolemic rabbit. Triol 1 also inhibits cholesterol biosynthesis in. v i t y o , causing accumulation of a previously undetected 2930 C atom int~ermediate.lc~* We anticipat'ed therefore that replacement of the sa-OH with a 5a-NH2 would render the compound x more potent inhibitor of cholesterol biosynthesis; z'.e., the SH, function, either protonated or unprotonat'ed, would bind strongly to a specific enzyme syst'em. In this regard, examination of Dreiding molecular models3 shows t'he topographical relationship betmu-eenthe 5a-KHZ and 3p-OH funct'ions of 2 to be similar to the relationship between the 4 a - X e arid 3p-OH groups of lanosterol, in which the A ring probably exists in a flattened chair c ~ n f o r m a t i o n . ~ Further, it is known that' removal of the 4a-Me represents the first' step in the enzymatic conversion of lanosterol to cholesterol.5 (1) is) U. T. JVitiak, R . .i. Parker, K. E. Connor, a n d D. .\I. Brahmanliar, 155th Xational Meeting of t h e American Chemical Society, San Francisco, Calif., April 1988, p X70; (b) D. T. Witiak, W. E. Connor, D . M . 13ralimankar, *IFYartman, . and R. Parker, J . Clin. Inrest., 47, 104 (1968); . Dempsey, M . C . Ritter, D. T. Witiak, and P r o c . I n t . Sump., 2, Springer-Verlag, New l!J70, p 290: ((1) abstracted in part from t h e dissertation presented by R. A . P., July 1969, t o t h e Graduate School of t h e Ohio State University; ( e ) Daw-Pitman-Moore Graduate Fellow 1965-1966; ( f ) U. S. Public Health S e r r i w Predoctoral Fellow (5-Fl-GM-29,392) 1 9 6 6 1 9 6 9 ; ( 8 ) U. S. Public llealtli Service Predoctoral Felloi~(l-FOl-GM42265) 1968-1970. ( 2 ) I>. T. IT-itiak, I t , A . Parker, D. R . Brann, M . E. Dempsey, h l . C. Ititter, I Y . E. Connor, and I). 11. Rrahmankar, J. .?fed. Chem., 14, 216 (lii71). t:i) ,\. Ilreiding, H e l r . Chim.Actn, 42,1330 (1959). (-1) (a) K ,1,. Allinger a n d 11..I DaRouge, , J . Amer. Chem. Soc., 84, 4561 (1962): (11) J. Lehn, .I. M . Levisalles, and G . Ourisson, Tetrahedron Lett., 882 (1!48lJ. 15) It. KaIiman, I %

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Figure 1.-Time courses of A?-sterol A5-dehydrogeiiase and A5,7-rterol A?-reductase in the absence of cholestaiie analogs: c-:~, A5-dehydrogeiiase activity [conversion of d7-choleslenol ( A ? ) to .+?-cholestadieuol (A5,?)] ; 0-0, A'-reductase activity, (A5,?)to ~holesterol-4-~~~(' coiiversiori of A5~7-cholestadienol-4-14C (As). Incubation and assay conditions were as given with the footnotes for Table 11, except that the substrate concentratiori f o r each time course was 64 p M .

placement of both of the 3p and rip OH groups bj, S H , renders the compound a potent inhibitor prior to formation of iionsaponifiable products. This suggests that the diamino compound should be evaluated in vivo since accumulation of undesirable sterols may be less of a problem than with triol 1. However, as discussed in a later section, the diamine does inhibit semipurified A5j7-sterolA7-reductase as well as stimulate semipurified A'-sterol As-dehydrogenase in oitro.'',li These t'wo enzymes exert' their catalytic action after squalene cyclization. The biological results obt'ained for the niono and diamino analogs (Table I) may be contrasted with the activity reported for certain amino compounds by Corey arid cowxkers." While 2,3-iminosqualene blocks cyclization of 2,3-oxidosqualene t o lanosterol, 3P-amino-8,25-lanostadierie arid 3,f~'-amino-8-lanostene show little inhihition. Counsel1 and c ~ n - o r k e r have s~~ also reported on 22,25-diazacholestan-3-olswhich block (lti) ILI. b:, L)cmpsej, A n n . IT. Y . Aciid.Sci., 148, ti31 (1968).

(17) ( a ) R l . C . Rittcr and M , E. Dempsey, Biochrm. B z o p h g s . R e s . Conrmun., 38, 921 (1970); (I)) 11. C. Ritter a n d bl. E . Dempsey, Circulation, 42, Supp?., 3 , 2 (1970); ( e ) R f . E. Dempsey, "Chemistry of Brain D e d o i J m e n t , " R . Paoletti and 11. Dai-ison, Ed., Plenum Press, S e \ v T o r k . N. Y . , 1QiO.

(18) 1,;. J. Corey, P. R . Ortiz de Montellano, I