United States Patent
`
`[191
`
`[111 Patent Number:
`
`4,686,237
`
`Anderson
`
`[45] Date of Patent:
`
`Aug. 11, 198_'?
`
`[54] ERYTHRO-(E)-7—[3'-C1_3ALKYL-I'-(3",5"-
`DIMETHYLPHENYL)NAPHTH-2’-YL]-3,5-
`DIHYDROXYHEPT-I5-ENOIC ACIDS AND
`DERIVATIVES THEREOF
`
`Primary Exc1mt'm:'r——Glennon H. I-Iollrah
`Assistant Exa.=m'ner—Dara L. Dinner
`Attorney. Agent. or Ft'rm—Cierald D. Sharkin; Richard
`E. Vila: Melvyn M. Kassenoff
`
`[75]
`
`Inventor:
`
`Paul L. Anderson, Randolph, N.J.
`
`[57]
`
`ABSTRACT
`
`[T3] Assignce:
`
`Santloz Pharrnaeentils Corp., E.
`Hanover, NJ.
`
`Compounds of the formula
`
`[21] Appl. No.: 831.394
`
`[22] Filed:
`
`Feb. 19, 1986
`
`Related US. Application Data
`
`[63]
`
`Continuation of Ser. No. 633.809. Jul. 24, 1984. aban-
`cloned.
`
`[51]
`
`Int. Cl.‘
`
`CUTD 309/30; 007C 69/613;
`AGIK 31/365; A6lK 31/225
`514/532; 560/56;
`562/466; 549/292; 514/557; 514/324
`[53] Field of Search
`549/289, 274, 292, 291,
`549/415, 417; 560/104. 56. 59, 119; 514/451,
`532, 557; 562/501, 466
`
`[52] US. Cl.
`
`wherein
`
`R1 is C1_3alky‘l, and
`Z is
`
`[5 6]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`—CH-Cl-l3—C'l-i—CH3—CO0R1 or
`I
`I
`OH
`OH
`
`C 2
`H
`
`,0}!
`H‘
`‘"‘r|:1uL1
`1mIc/
`0 H C / CH3
`II0
`
`,
`
`wherein R’? is hydrogen, Rg or M,
`wherein
`
`R5 is a physiologically acceptable and hydrolyzable
`ester group, and
`M is a pharmaceutically acceptable cation,
`the use thereof for inhibiting cholesterol biosynthesis
`and lowering the blood cholesterol level and, therefore,
`in the treatment of hyperlipoproteinernia and athero-
`sclerosis. pharmaceutical compositions comprising such
`compounds and processes for and intermediates in the
`synthesis of such compounds.
`
`I5 Claims. No Drawings
`
`9/19T6 Endo et al. ..................... .. 260/343.5
`3.983.140
`-l/1980 Mitsui et al.
`.... .. 424/2".-'9
`4,l98.425
`
`4,248,839 2/I981
`.... .. 424/308
`4,255,444 3/I98]
`.... .. 424/279
`4,303,378 12/I981
`.... .. 542./44]
`4.351.844 9/1932
`.... .. 424/279
`4-.36l,5l5 11/'198_2
`.... .. 549K292
`4.375,-1-'.u"5
`3/1983
`.... .. 424/279
`4,376,863
`3/1933
`.... .. 549/“Z92
`4,387.3‘-‘l-2
`5,/1933
`.... .. 560./l I9
`-I-.4-40.927
`4/1984
`.. 549/292
`4-,4'i'4,9Tl 10/1934 Wareing ............................ .. 549./114
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`-’l-/1983 Belgium ............................ .. 549/192
`03954-1-5
`0038061 ID/1981 European Pat. Off.
`.
`OTHER PUBLICATIONS
`
`Derwent Abstracts 20213 D/12, JP S6—'!Tr'5 (l/27/8]]
`Sankyo KK.
`I-Iulchcr, Arch. Biochem. Biophys. 146, 422-427 (1971).
`Saw et al., Chem. Pharm. Bull. 23, 1509-1525 (1980).
`Singer et al., Proc. Soc. Exp. Biol. Med. 102, 3'!0—3';'3
`(1959).
`
`Iuflr-I
`
`PENN EX. 2131
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`1
`
`4,686,237
`
`2
`
`ERYTI-IRO-(E)—7-[3’-C 1_;ALKYL-1'- 3",5"-DIME-
`THYLPHENYDNAPHTH-2'-YL]-3,5-DIHYDROIL
`YHEPT-6-ENOIC ACIDS AND DERIVATIVES
`THEREOF
`
`This application is a continuation of application Ser.
`No. 63 3,809, filed July 24, 1984 and now abandoned.
`This invention relates to compounds of the formula
`
`CH3
`
`/I
`
`I
`
`H
`
`CH;
`
`\
`
`c=c
`
`gfi 2
`
`3 R1
`
`2
`
`,
`
`H
`
`/’
`
`\
`
`wherein
`R1 is C1_3alky1, and
`Z is
`
`-CH—CH3—CH—CHg—COIIJR1 Br
`is
`L’
`on
`OH
`
`OH.
`CH2
`H
`15111 C/ “cfifiin
`I
`I‘
`OXC/CH2
`ll0
`
`(ll
`
`(90
`
`(bl
`
`wherein R1 is hydrogen, R3 or M,
`wherein
`
`R3 is a physiologically acceptable and hydrolyzable
`ester group, and
`M is a phannaceutically acceptable cation,
`processes for and intermediates in the synthesis thereof,
`pharmaceutical compositions comprising a compound
`of Formula I and the use of the compounds of Fonnula
`I for inhibiting cholesterol biosynthesis and lowering
`the blood cholesterol level and, therefore, in the treat-
`ment of hyperlipoproteinemia and atherosclerosis.
`By the term “physiologically acceptable and hydro-
`lyzablc ester group” is meant a group which, together
`with the -—COO— radical to which it is attached, forms
`an ester group which is physiologically acceptable and
`hydrolyzable under physiological conditions to yield a
`compound of Formula I wherein R1 is hydrogen and an
`alcohol which itself is physiologically acceptable, i.e.,
`non-toxic at the desired dosage level, and which, prefer-
`ably, is free of centers of asymmetry. Examples of such
`groups are C1_3alkyl, n-butyl, i-butyl, t-butyl and ben-
`zyl, collectively referred to as Rs’.
`As is self—evident to those in the art, each compound
`of Formula I (and every subscope and species thereof)
`has two centers of asymmetry (the two carbon atoms
`bearing the hydroxy groups in the group of Formula a
`and the carbon atom bearing the hydroxy group and the
`carbon atom having the free valence in the group of
`Formula b) and, therefore, there are two stereoisomeric
`forms (enantiomers) of each compound (a racemaie),
`provided that R-1 does not contain any center of asym-
`metry. The two stereoisomers of
`the compounds
`wherein Z is a group of Formula a are the 3R,5S and
`3S,5R isomers and the two stereoisomers of the com-
`
`pounds wherein Z is a group of Formula b are the 4R,6S
`and 4-S,6R isomers, both the individual stereoisomers
`and the raoemates being within the scope of this inven-
`tion. When R1 contains one or more centers of asymme-
`try, there are four or more stereoisomers. Since it
`is
`preferred that R1 not contain a center of asymmetry and
`for reasons of simplicity any additional stereoisomers
`resulting from the presence of one or more centers of
`asymmetry in R1 will be ignored, it being assumed that
`R1 is free of centers of asymmetry.
`The preferred stereoisomer of each compound
`wherein Z is a group of Formula a is the 3R,5S isomer
`and the preferred stereoisomer of each compound
`wherein Z is a group of Formula b is the 4R,6S isomer.
`These preferences also apply to the compounds of For-
`mula I having more than two centers of asymmetry and
`represent the preferred configurations of the indicated
`positions.
`R1 is preferably methyl or isopropyl.
`R1 is preferably R1’, where R1’ is hydrogen, C1-3alkyl
`or M, more preferably R1", where R1" is hydrogen,
`C1.2alkyl or M, and most preferably M, especially so-
`dium. M is preferably M’ and most preferably sodium.
`R11 is preferably R3‘, where R3’ is C1-3alkyl, n-butyl,
`i-butyl, t-butyl or benzyl, more preferably C1-3alkyl and
`most preferably C1_2alkyl.
`_
`M is preferably free from centers of asymmetry and is
`more preferably M‘, i.e., sodium, potassium or ammo-
`nium. and most preferably sodium. For simplicity, each
`of the formulae in which M appears (in the specification
`and the claims) has been written as if M were monova—
`lent and, preferably, it is. However, it may also be diva-
`lent or trivalent and, when it is, it balances the charge of
`two or three carboxy groups, respectively.
`The preferred compounds of Formula I wherein Z is
`a group of Formula a are those
`(i) wherein R1 is R1’,
`(ii) of (i) wherein R1 is R1",
`(iii) of (ii) wherein R1 is M,
`(iv) of (iii) wherein R-1 is M’,
`(v) of (iv) wherein R1 is sodium, and
`(vi)—(x) of (i)—(v) wherein R1 is methyl or isopropyl.
`Groups (i)—(x) embrace the 3R,SS-3S,5R racemates
`and the 3R,5S and 3S,5R enantiomers, of which the
`racemates and the 3R,5S enantiomers are preferred.
`The preferred compounds of Formula I wherein Z is
`a group of Formula b are those wherein R1 is methyl or
`isopropyl. This group embraces the 4R,6S-4S,6R race-
`mates and the R,6S and 4S,6R enantiomers, of which
`the racernates and the R,6S enantiomers are preferred.
`The compounds of Formula I wherein R1 is methyl,
`and Z is a group of Formula a wherein R1 is hydrogen,
`ethyl or or a group of Formula b are synthesized as set
`forth in Examples 1-5.
`The compounds of Formula I wherein R1 is ethyl,
`and Z is a group of Formula a wherein R1 is hydrogen,
`ethyl or sodium or a group of Formula b may be synthe-
`sized by the processes of Examples 1-5 with the follow-
`ing additional step: The compound of Formula CCXLII
`is converted to the corresponding compound having an
`ethyl group in the 3-position of the naphthalene ring by
`the procedure utilized to convert
`the compound of
`Formula CCXLI
`to the
`compound of Formula
`OCXLII. i.e., the procedure of Step 8 of Example 1.
`Otherwise, the synthesis is the same.
`The compounds of Formula I wherein R1 is isopro-
`pyl, and Z is a group of Formula a wherein R1 is hydro-
`
`ID
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`PENN EX. 2131
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,686,237
`
`3
`gen, ethyl or sodium or a group of Formula b may be
`synthesized by the processes of Example A, Steps 9-13
`of Example 1 and Examples 2-5. The corresponding
`compounds of Formula I wherein R1 is n-C1_3alkyl may
`be synthesized analogously utilizing in Step 8 of Exam-
`ple A the corresponding n-Cpsallrylmagnesium chlo-
`ride or bromide in lieu of isopropylmagnesium chloride.
`The compounds of Formula I wherein Z is a group of
`Formula a wherein R7 is any other significance of Rs‘
`may be synthesized by the processes of the preceding
`three paragraphs _utilizing appropriate starting materi-
`als.
`
`The compounds of Formula I wherein Z is a group of
`Formula a wherein R7 is Rs may be synthesized by
`reacting the corresponding compound wherein Z is a
`group of Formula b with at least 2, e. g., 2-10, preferably
`2.05-2.5, moles of a compound of
`the formula
`M2G3e0Rg, wherein M2 is sodium or potassium, per
`mole of the compound wherein Z is a group of Formula
`b, at 0°—'.~'0“ C., preferably 20°—50° C., for 2-12 hours, in
`an inert organic solvent, preferably an inert ether sol-
`vent such as tetrahydrofuran or, if the corresponding
`alcohol of the formula Rs—0H is a liquid at the desired
`reaction temperature, preferably said corresponding
`alcohol. By the term “corresponding alcohol” is meant
`that R3 in the compound of the formula MgEBe0Rs and
`in the alcohol of the formula R3—0H is the same. The
`compounds of Formula I wherein Z is a group of For-
`mula a wherein R7 is Rs may also be prepared by esteri-
`fying the corresponding compound wherein R-,1 is hy-
`drogen with an alcohol of the formula Rs—0H. Conve-
`niently,
`the compound wherein R7 is hydrogen is
`treated with a large excess of the alcohol of the formula
`Rs——0H (e.g., 2-50 moles per mole of the compound
`wherein R7 is hydrogen) at 2D°—40° C. for 2-12 hours in
`the presence of a catalytic amount of an acid such as
`p-toluenesulfonic acid. The excess alcohol serves as the
`solvent. The reaction may also be run in an inert organic
`solvent, e. g., an ether such as tetrahydrofuran, and must
`be run in such a solvent if the alcohol of the formula
`R3—OH is not a liquid at the desired reaction tempera-
`"Hire.
`
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`4
`example a compound of Formula I wherein Z is a group
`of Formula b, may be esterified with an optically pure
`carboxylic acid having at least one center of asymmetry
`to form -a mixture of diastereoisomeric esters or it may
`be reacted with an optically pure trisubstituted silyl
`halide having an asymmetric silicon atom, for example
`(—)-.-1-naphthylplnenylmethylchlorosilane (Sommer et
`al., J. Am. Chem. Soc. 80, 3271 (1958).), to form a mix-
`ture of two diastereoisomeric silyloxy compounds,
`which mixture may be separated by conventional tech-
`niques. After separation, the optically pure salts. am-
`ides, esters or silyloxy compounds are reconverted to
`the corresponding carboxy group- or hydroxy group-
`oontaining compounds with retention of optical purity.
`For example,
`(-—)-ct-naphthylphenylmethylsilyl and
`other silyl groups may be cleaved with a fluoride rea-
`gent, for example, tetra-n-butylammonium fluoride in
`an anhydrous inert organic medium containing glacial
`acetic acid, preferably tetrahydrofuran containing 1-2
`moles, preferably 1.2-1.5 moles, of glacial acetic acid
`per mole of the fluoride compound. The reaction tem-
`perature is suitably 20°—60° C., preferably 20°—30° C.,
`and the reaction time is suitably 8-24 hours, particularly
`when the reaction temperature is 20°—30° C Approxi-
`mately 1-5 moles, preferably 2-4 moles, of fluoride
`reagent per mole of the silyl group-containing com-
`pound are utilized. The reaction mixture should be
`acidic at the time the fluoride reagent is added to maxi-
`mine production of the desired product.
`Most of the processes described above are described
`in greater detail in my application Ser. No. 06/570,584,
`filed Jan. 13, 1984 and now abandoned and titled Naph-
`thalene And Tetrahydronaphthalcne Derivatives Of
`Mevalonolactone and Derivatives Thereof. Where the
`reaction conditions set forth in said application differ
`from those set forth herein, the reaction conditions set
`forth in said application may also be utilized for the
`compounds of this specification. Said application, par-
`ticularly pages 15-67, 109-113 and 125-151, is hereby
`incorporated by reference.
`Ser. No.
`application
`Also described in
`said
`06/570,584 are processes by which the 4R,6S isomers of
`the compounds of Formula I wherein Z is a group of
`Formula b and the 3R.5S isomers of the compounds of
`Formula I wherein Z is a group of Formula a may be
`synthesized.
`All of the reagents and reactants the synthesis -of
`which is not described in this specification are either
`known or are synthesizable by processes analogous to
`those described in the literature for similar known com-
`pounds.
`The product of each reaction may, if desired, be puri-
`fied by conventional techniques such as recrystalliza-
`tion (if a solid), column chromatography, preparative
`thin layer chromatography, gas chromatography (if
`sufficiently volatile), fractional distillation under high
`vacuum (if sufficiently volatile) or high pressure liquid
`chromatography. Often, however, the crude product of
`one reaction may be employed in the following reaction
`without purification.
`Since any compound of Formula I wherein Z is a
`group of Formula a wherein R7 is a cation other than M
`may be converted into the corresponding compounds
`wherein R1 is hydrogen, M or Rs by acidification op-
`tionally followed by neutralization or esterification, ion
`exchange, etc., the compounds of Formula I wherein Z
`is a group of Formula a and R7 is a pharmaceutically
`unacceptable cation are also within the scope of this
`
`The compounds of Formula I wherein Z is a group of
`Formula a wherein R1 is M may be synthesized by neu-
`tralizing the corresponding compounds wherein R7 is
`hydrogen with 0.95—I, preferably 0.96-0.99, equivalent
`of a base of the formula M99011 per mole of the
`compound wherein R7 is hydrogen at 0°—25° C., prefer-
`ably 2t}°—25' C., for 1-10 minutes. in an inert aqueous .
`50
`organic solvent, for example a mixture of water and a
`C;_galkanol. As should be evident from what is set forth
`above, the formula MGBGOH embraces bases of the
`formulae M@1(e0H)2 and Me3(e0H)3.
`The processes described above yield racemates.
`However, techniques for separating a racemate into its
`two optically active enantiomers are known. For exam-
`ple, a racemic compound having a carboxylic acid
`group may be reacted with an optically pure organic
`base having at least one center of asymmetry to form a
`mixture of diastereoisomeric salts or amides that may be
`separated by fractional crystallization, column chroma-
`tography, preparative thin layer chromatography, high
`pressure liquid chromatography, etc. or it may be re-
`acted with an optically pure alcohol having at least one
`center of asymmetry to form a mixture of diastercoiso-
`meric esters which may be separated by conventional
`techniques such as those set forth above. On the other
`hand, a racemic compound having a hydroxy group, for
`
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`PENN EX. £131
`CFAD V. UPENN
`lPR20l5-01836
`
`

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`4,686,231’
`
`5
`invention inasmuch as they are useful as intermediates.
`However, such compounds are not compounds of For-
`mula I as utilized in this specification, except where
`indicated to the contrary.
`Besides having the utility set forth below, every com-
`pound of Formula I is useful as an intermediate in the
`synthesis of one or more other compounds of Formula
`I utilizing the reactions set forth in this specification,
`e. g., those of Examples 2-5.
`Also within the scope of this invention are the inter-
`mediates of Formulae CCXLLCCXLVI and the com-
`
`pounds corresponding to those of Formulae CCXLlI-
`—CCXLVI but having a C;_3alkyl group in the 3-posi-
`tion of the naphthalene ring in lieu of the methyl group
`and those corresponding to that of Formula CCXLVI
`but having, in lieu, of the ethyl group, a different signifi-
`cance of R3‘ and, optionally, in lieu of the methyl group
`in the 3-position of the naphthalene ring, a C2_3a|kyl
`group.
`The compounds of Formula I are competitive inhibi-
`tors of 3-hydroxy-3-methylglutaryl
`coenzyme A
`(HMG-CoA) reductase,
`the rate limiting enzyme in
`cholesterol biosynthesis. and, therefore, they are inhibi-
`tors of cholesterol biosynthesis. Consequently, they are
`useful for lowering the blood cholesterol level in ani-
`mals, e.g., mammals, especially larger primates, and,
`therefore, as hypolipoproteinemic and a.ntiatheroscle-
`rotic agents. The biological activity of the compounds
`of Formula I may be demonstrated in the following
`three tests:
`Test A. In Vitro Microsomal Assay of HMG-CoA
`Reductase Inhibition:
`
`200 it]. aliquots (l.08—l .50 mg/ml.} of rat liver micro-
`somal suspensions, freshly prepared from male Sprague-
`Dawley rats (l50—225 g. body weight), in Buffer A with
`l0 mmol. dithiothreitol are incubated with 10 pl. of a
`solution of the test substance in dimethylacetamide and
`assayed for HMG-CoA reduotase activity as described
`in Ackcrmarl et al.. J . Lipid Res. 18, 403-413 (1977). In
`the assay the microsomes are the source of the HMG-
`CoA reductase enzyme which catalyzes the reduction
`of HMG-COA to mevalonate. The assay employs a
`chloroform extraction
`to
`separate
`the product,
`[l4C}mevaIonolactone, formed by the HMG-CoA re-
`ductase reaction from the substrate, [14C}HMG-CoA.
`[3H]mevalonolactone is added as an internal reference.
`Inhibition of HMG-CoA reductase is calculated from
`the decrease in specific activity ([‘4C/31-flmevalonate)
`of test groups compared to controls.
`Test B. In Vitro Cell Culture Cholesterol Biosynthe-
`sis Screen:
`The cell culture is prepared as follows: Stock mono-
`layer cultures of the FuSAH rat hepatorna cell line
`(originally obtained from G. Rothblat; see Rothblat,
`Lipids 9, 526-535 (1974)) are routinely maintained in
`Eag|e’s Minimum Essential Medium (EMEM) supple-
`mented with 10% fetal bovine serum (FBS) in 75 cm}
`tissue culture flasks. For these studies, when the cul-
`tures reach confluence, they are removed by mild enzy-
`matic treatment with 0.25% trypsin in Hanks’ balanced
`salt solution (without calcium and magnesium). After
`centrifugation of the cell suspension and aspiration of
`the enzymatic solution, the cell pellet is resuspended in
`an appropriate volume of media for seeding into 60 mm.
`tissue culture dishes. The cultures are incubated at 3?"
`C. in an atmosphere of high humidity and 5% carbon
`dioxide. When the cultures are confluent (approxi-
`mately 5 days), they are ready for use. The culture
`
`6
`media is aspirated from the dishes and replaced with 3
`ml. of EMEM supplemented with 5 mg./ml. of delipi-
`dized serum protein (DLSP) prepared by the method of
`Rothblat et al., In Vitro 12, 554-557 (1976). Replace-
`ment of the FBS with DLSP has been shown to stimu-
`late the incorporation of 1‘iC]acetate into sterol by re-
`moving the exogenous sterol supplied by the FBS,
`thereby requiring the cells to synthesize sterol. En-
`hanced 3=hydroxy-3-methylglutaryl Coenzyme A re-
`ductase (HMG-CoA reductase) actiVily is measurable
`in the cells in response to the lack of exogenous sterol.
`Following approximately 24 hours incubation at 3'?” C.
`in the DLSP supplemented media, the assay is initiated
`by the addition of 3;.tCi of [”C]acetate and the test
`substance solubilized in dimethylsulfoxide (DMSO) or
`distilled water. Solvent controls and compactin-treated
`controls are always prepared. Triplicate 60 mm. tissue
`culture dishes are run for each group. After 3 hours
`incubation at 37° C., the cultures are examined micro-
`scopically using an inverted phase contrast microscope.
`Notations are made of any morphological changes
`which may have occurred in the cultures. The media is
`aspirated and the cell layer is gently washed twice with
`0.9% sodium chloride solution (saline). The cell layer is
`then harvested in 3 ml. of 0.9% saline by gentle scraping
`with a rubber policeman and transferred to a clean glass
`tube with Teflon lined cap. The dishes are rinsed with 3
`ml. of 0.9% saline and rescraped. and the cells are com-
`bined with the first harvest. The tubes are centrifuged at
`1500 r.p.m. for 10 minutes in an IEC PR-J centrifuge.
`and the supernatant is aspirated.
`The cells are then extracted as follows: One ml. of
`l00% ethanol is added to the cell pellet followed by
`sonication for 10 seconds with a “LO" setting of 50 on
`a Bronwell Biosonik IV. One hundred l. are taken for
`protein determination. One ml. of 15% potassium hy-
`droxide (KOH) is added, and the samples are thor-
`oughly vortexed. Saponiiication is accomplished by
`heating the ethanol-KOI-I treated samples at 60° C., for
`60 minutes in a water bath. Following dilution of the
`samples with 2 ml. of distilled water, they are extracted
`three times with 7 ml. of petroleum ether. The petro-
`leum ether extracts are then washed three times with 2
`ml. of distilled water and finally taken to dryness under
`a stream of nitrogen.
`The obtained samples are then analyzed by thin layer
`chromatography (TLC) as follows: Residues from the
`petroleum ether extraction are taken up in a small vol-
`ume of hexane and spotted on silica gel 60 TLC plates
`(E. Merck). Development of the plates is carried out in
`a three phase solvent system consisting of 150 parts by
`volume hexane: 50 parts by volume diethyl ether: 5
`parts by volume glacial acetic acid. Visualization is
`accomplished in an iodine vapor chamber. The plates
`are divided into five sections such that each section
`contains the molecules having the following approxi-
`mate Rf values: section l—-0-0.4, section 2—0.4—[].5S,
`section 3-—0.55—0.?', section 4—D.7—0.9 and section
`5—0.9—l.fJ. Section 2 contains the non-saponifiable ste-
`rols. The five sections of the TLC plates are scraped
`into scintillation vials. Blanks are also prepared from
`scrapings of chromatographed non-labelled standards.
`ACS ® scintillation cocktail is added, and the radioac-
`tivity is determined in a liquid scintillation spectrome-
`ter. [”C]hexadecane standards are used to determine
`counting efficiencies. The total protein content of the
`samples is determined employing the Bio-Rad Protein
`Assay System.
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`PENN EX. 2131
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,686,237
`
`7
`The results are reported as disintegrations per minute
`per mg. protein (d.p.rn./mg. protein) for each of the five
`TLC sections. Mean d.p.m./mg. protein 1 standard
`error of the mean are calculated, and drug treated
`means are compared for percentage change (%A) and
`statistical significance with solvent control means. TLC
`section 2 data is taken as a measure of HMG-CoA re-
`ductase activity inhibition.
`Test C: In Vivo Cholesterol Biosynthesis Inhibition
`Test: In vivo studies utilize male Wistar Royal Hart rats
`weighing l50:20 g. which have been kept for 7-10
`days on an altered light cycle (6:30 A.M.-6:30 P.M.
`dark) housed two per cage and fed powdered Purina
`Rat Chow and water ad libitum. Three hours before the
`diurnal maximum of cholesterol synthesis at mid-dark,
`the rats are administered the test substance dissolved or
`as a suspension in 0.5% carboxymethylcellulose in a
`volume of l ml./100 g. body weight. Controls receive
`vehicle alone. One hour after receiving the test sub-
`stance, the rats are injected intraperitoneally with about
`25 p,Ci/100 g. body weight-of sodium [l-”C]acetate 1-3
`mCi/mmol. Two hours after mid-dark, blood samples
`are obtained under sodium hexobarbitol anesthesia and
`the serum is separated by centrifugation.
`Serum samples are saponified and neutralized, and
`the 3,8-hydroxysterols are precipitated with digitonin
`basically as described in Sperry et al., J. Biol. Chem.
`18?, 97 (1950). The [14C}digitonides are then counted by
`liquid scintillation spectrometry. After correcting for
`efficiencies, the results are calculated in nCi (nanocn-
`ries) of sterol formed per 100 ml. of serum. Inhibition of
`sterol synthesis is calculated from the reduction in the
`uCi of sterols formed from test groups compared to
`controls.
`The following results were obtained:
`
`Test A:
`Example I
`IC5o = 0.08 ttmolar
`Example 2
`IC5u -—-. 0.0] p-molar
`Example 4
`IC5u = 0.33 pzmolar
`Cornpactin
`IC5u = 0.1"?’ ptrnolar
`Mevinolirl
`IC5g = 0.14 ptmolar
`IC5o is the concentration of the test substance in
`the assay system calculated to produce a 50%
`inhibition of HMG-CoA reductase activity.
`Example I
`-35% at 0.5 mgfkg.
`Example 2
`E1350 = 0.3 rt1g.}'kg.
`Example 4
`-20% at 0.5 mg./kg.
`Compactin
`ED5o = 3.5 rt1g.fkg.
`
`Mefinolin ED5o = 0.41 mg./kg.
`
`Test C:
`
`the compounds of Formula I
`As set forth above,
`{including each and every subgroup thereof set forth in
`the specification and/or the claims) inhibit cholesterol
`biosynthesis and are useful for lowering the blood cho-
`lesterol
`level
`in animals, particularly mammals and
`more particularly larger primates, and,
`therefore, as
`hypolipoproteinemic and antiatherosclerotic agents.
`The compounds of Formula I may be formulated into
`conventional pharmaceutical compositions and admin-
`istered by conventional modes of administration. The
`compounds of each and every subgroup thereof in the
`specification and/or claims may likewise be formulated
`into conventional pharmaceutical compositions.
`The compounds of Formula I may be combined with
`one or more pharmaceutically acceptable carriers and,
`optionally, one or more other conventional pharmaceu-
`tical adjuvants and administered orally in the form of
`tablets, dispersible powders, granules, capsules, elixirs,
`suspensions and the like or parenterally in the form of
`sterile injectable solutions or suspensions. The composi-
`
`8
`tions may be prepared by conventional means. The
`preferred pharmaceutical compositions from the stand-
`point of ease of preparation and administration are solid
`compositions, particularly tablets and capsules.
`The precise dosage of the compound of Formula I to
`be employed for inhibiting cholesterol biosynthesis de-
`pends upon several factors including the host, the na-
`ture and the severity of the condition being treated, the
`mode of administration and the particular compound
`employed. However, in general, satisfactory inhibition
`or reduction of cholesterol biosynthesis (i.e., satisfac-
`tory reduction of the blood cholesterol level and satis-
`factory treatment of hyperlipoproteinemia and athero-
`sclerosis) is achieved when a compound of Formula I is
`administered orally at a daily dosage of 0.0l—l0G
`mg./kg. body weight, e.g., 0111-20 mg./kg. body
`weight for the compound of Example 2, or, for most
`larger primates, a daily dosage of 1-1000 mg. and suit-
`ably 1-150 mg., e.g., 5-100 mg., for the compound of
`Example 2.
`'
`The daily dosage is usually divided into two to four
`equal portions or administered in sustained release form.
`Usually, a small dosage is administered initially, and the
`dosage is gradually increased until the optimal dosage
`for the host under treatment is determined. For adminis-
`tration by injection, a dosage somewhat
`lower than
`would be used for oral administration of the same com-
`pound to the same host having the same condition is
`usually employed. However, the above dosages are also
`typically used for i.v. administration.
`A typical dosage unit for oral administration may
`contain 05-500 mg. of a compound of Formula 1. Pre-
`ferred dosage units contain 0.5 to '15 mg. of a compound
`of Formula I, for example, 1-50 mg. of the compound of
`Example 2.
`The compounds of Formula I (including those of
`each and every subgroup thereof) may be formulated
`into such pharmaceutical compositions containing an
`amount of the active substance that is effective for in-
`hibiting cholesterol biosynthesis, such compositions in
`unit dosage form and such compositions comprising a '
`solid pharmaceutically acceptable carrier.
`A representative formulation prepared by conven-
`tional techniques for encapsulation in a hard gelatin
`capsule is:
`
`
`Compound of Formula 1. e.g., the compound of
`Example 2
`224 mg.
`Corn starch
`
`Magnesium stesrate 1 mg.
`
`25 mg.
`
`A representative formulation suitable for preparing
`tablets by conventional means is:
`
`
`Compound of Formula I, e.g., the compound of
`Example 2
`5 mg.
`Polyvinylpyrrolidone USP
`I24 mg.
`Powdered lactose
`I0 mg.
`Corn starch
`
`Magnesium stearate 1 mg.
`
`10 mg.
`
`The following examples show representative com-
`pounds encompassed by this invention and their synthe-
`sis. However, it should be understood that they are for
`purposes of illustration only.
`
`10
`
`15
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`.5ofl4
`
`PENN EX. 2131
`
`CFAD V. UPENN
`
`lPR20l5-01836
`
`

`
`9
`
`4,686,237
`
`10
`
`EXAMPLE 1
`erythro-(E)-3,5-dihydroxy-?~[l'-(3",5"-dime—
`Ethyl
`thylphenyl)-3-methylnaphth-2'-yl]hept-6-enoate
`
`CH3 0 CH3
`H\
`00
`
`/EH-CH3—EH—CH2-‘COOCQH5
`C=C.\OI-l
`OH
`H
`on;
`
`(CCXL)
`
`OH
`
`so
`tccxvll)
`
`'
`
`I‘1]NaOH 9
`(2)110
`
`OCH;
`
`00 COOCH’
`tccxvul)
`OCH3
`
`COOH cI—-co—co-cl ;
`
`[CCXDO
`
`OCH;
`
`NH;-C(CH3)3—CH20H
`3 C00 ""'o-—-%
`(ccxx)
`
`OCH3
`
`(CCXXI)
`
`OCH3
`
`(CCXX II)
`
`N
`
`O
`
`CH3
`CH3.HC|
`
`s0c1
`
`E29315,
`
`CH3
`
`CH
`
`_
`:3” Br Mg
`-%—>
`
`OCH3
`
`N
`
`0
`
`tccx’-um
`CH1
`' 0
`
`CH3
`
`N
`
`CH3
`CH]
`
`in n_BuLi
`00 I" we
`0
`[ccx1_1}
`
`CH
`
`3
`
`O
`
`Cficontinued
`.1-
`CH,
`CH; CH I, A
`-—’%
`
`N
`
`
`
`19
`(l}I.iBH4
`(2)2N.HCI 9
`
`(ccxurx)
`
`0
`
`<5‘-3”")
`CH3 O CH3
`H
`
`CH0 (1)
`
`'-‘\
`
`051’-H5
`
`C=C/
`\
`/
`CH3 -Carlo‘
`" E
`2
`
`CH0
`(l}CH:.—O0--CH3—C00CgH5+
`LDA
`(2) Compound CCXLV’
`:"“3'
`
`\ = /'
`'3
`*3
`08 \I_[
`cu}
`cccxur)
`
`
`
`CH1
`
`cH—cH —c—-CH —CO0C H
`/l
`2
`H
`2
`2
`c=c or:
`o
`('1 B(C-Hl +
`\H
`O1 9 53
`
`s
`
`CH3 0 CH3
`H\ H /EH—CHg'-EH""‘CH2—COOC3H5
`C—C\0H
`on
`H
`
`CH3
`
`(ccx L)
`
`Step 1
`Methyl 1-methoxy-2-naphthoate (Compound
`ccxvm)
`
`5
`
`‘O
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`Over a 15 minute period, 149 g. (2.? moles) of ground
`potassium hydroxide is added to 180 g. (0.96 mole) of
`60 1-hydroxy-2-naphthoic acid in 2.5 1. of din1ethylform-
`amide (during the course of which the temperature rises
`to 32” C.}. The reaction mixture is stirred at 20°—25° C.
`for 16 hours and at 50° C. for 2 hours and cooled to 40°
`C. 38.7 g. (2.7 rnolcs)‘ofrr1ethyl iodide is added, and the
`55 reaction mixture 15 st1rred_at 40 -59"
`for 4 hours. 4 l.
`of saturated sodtum chloride solution 13 added. and the
`mixture is extracted with diethyl ether. The diethyl
`ether extract is dried over anhydrous sodium sulfate and
`
`6 Bf”
`
`PENN EX. 213i
`CFAD V. UPENN
`lPR2015-01836
`
`

`
`4,686,237
`
`11
`evaporated at reduced pressure to obtain the crude
`product as a light brown oil (255 g.).
`
`5
`
`10
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`Step 2
`
`1-Methoxy-2-naphthoic acid (Compound CCXIX)
`Over a 15-20 minute period, 750 ml. of 2N. sodium
`hydroxide (1 .5 moles) is added to 255 g. (£0.96 mole) of
`crude Compound CCXVIII in 2.5 l. of methanol stirred
`at 20°—25° C., and the reaction mixture is stirred at
`20°—25“ C. for 16 hours. 5 l. of water is added, and the
`mixture is extracted with diethyl ether (to recover any
`unreacted starting material). The aqueous phase is acidi-
`fied with 2N. hydrochloric acid and extracted with
`methylene chloride. The methylene chloride extract is
`dried over anhydrous sodium sulfate and evaporated at
`reduced pressure, and the residue is crystallized from
`ethyl acetate/petroleum ether. The solids are collected
`by filtration, washed with petroleum ether and vacuum
`dried at 40“ C. to obtain the white crystalline product
`(164 g. (84% Steps 1 and 2 combined». m.p. 124°—l27°
`C.
`
`Step 3
`
`1-Methoxy-2—naphthoyl chloride (Compound CCXX)
`Over a 30 minute period, 20] g. (1.58 moles) of oxalyl
`chloride is added to 160 g. (0.79 mole) of Compound
`CCXIX in 1 1. of dry‘toluene stirred at 60° C. under
`nitrogen. (Compound CCXIX slowly dissolves with
`moderate foaming during the addition.) The reaction
`mixture is refluxed for 2 hours under nitrogen and con-
`centrated to a volume of 600 ml. using a Dean-Stark
`trap. 300 ml. of dry toluene is added in small portions
`while the volume is gradually reduced. The reaction
`mixture is evaporated to dryness at reduced pressure to
`obtain the crude product.
`
`Step 4
`1-Methoxy-2-naphthoic acid
`N-1, 1-dimethyl-2-hydroxyethylamide (Com pound
`CCXXI)
`
`The crude product of Step 3 in 300 ml. of dry methy-
`lene chloride is added, over a 1 hour period, to 239.9 g.
`(1.89 moles) of 2-amino—2—methy]-l-propanol in 1.5 1. of
`methylene chloride stirred at 0°—l0" C. under nitrogen.
`The reaction mixture is allowed to wan-n to 20"—25" C.
`and is stirred at this temperature for 16 hours, the reac-
`tion mixture being maintained under nitrogen through-
`out. .Water is added, and the mixture is extracted with
`methylene chloride. The methylene chloride extract is
`washed with 2N. hydrochloric acid, washed with 10%
`sodium bicarbonate solution, dried over anhydrous
`sodium sulfate and evaporated at reduced pres