United States Patent
`Billet
`
`[191
`
`[11] Patent Number:
`
`4,871,721
`
`{45] Date of Patent:
`
`Oct. 3, 1989
`
`[54] PI-IOSPHORUS-CONTAINING SQUALENE
`SYNTHETASE INHIBITORS
`
`[75]
`
`Inventor:
`
`Scott A. Billet, Ewing, NJ.
`
`[73] Assignee: E. R. Squibb & Sons, Iuc., Princeton,
`NJ.
`
`[21] Appl. No.: 141,744
`
`[22] Filed:
`
`Jan. 11, 1988
`
`A61K 31/66; C07F 9/40
`Int. Cl.‘
`[51]
`[52] US. Cl. .................................... 514/102; 558/ 134;
`558/155; 562/21
`260/302.4 P; 558/155;
`514/102
`
`[58] Field of Search
`
`[561
`
`References Cited
`PUBLICATIONS
`
`Poulter, C. D., et al., Bfasynrhesis of Isoprenafd Com-
`pounds. “Conversion of Famesyl Ryrophosphare to Squal-
`ene”, vol. I, Chapter 8, pp. 413-441, J. Wiley and Sons.
`I981.
`Faust, J. R. et 2:]. Pros. Nat. Acad Sci, U..S‘.A., “Sgualene
`Synrhetase Activity in Human Fibroblasts.‘ Regulation via
`the Low Dertsfty Ltpapmtein Receptor”,
`I979.
`76.
`5018-5022.
`de Montellano, P. Ortiz et al., J. Med. Chem, “Inhibi-
`tion of Sqnalene Synthetase by Famesyl Pyrophqsphate
`Analogues”, 1977, 2|), 243-249.
`Corey and Volante, J. Am. Chem. Cos. “Application of
`Unreactive Analogs of Terpenoid Pvrophosphates to
`Studies of Multistep Biosynthesis, Demonstration that
`‘Presqualene Pyrophosphate' is an Essential Intermedi-
`ate on the Path to Squalene”, £976, 93, 129 1-3.
`Sandifer, R. M. et al., I Am. Chem. Soc, 1982, 104-,
`7376—8. “Squaleue Synthetase, Inhibition by an Ammo-
`nium Analogue of a Carbocationic Intermediate in the
`Conversion of Presqualene Pyrophosphate to Squal-
`ene”.
`Bertolino, A. et al., Biochim Biophys. Aura, 1978, 530,
`17-23, “Polyisoprenoid Amphiphilic Compounds as
`Inhibitors of Squalene Synthesis and Other Microsomal
`Enzymes”.
`
`Poulter, C. D. et al., J. Org. Chem, 1986, 51, 4':'68-—47';'9,
`“Phosphorylation of Isoprenoid Alcohols”.
`Poulter, C. D. et al.,J'.A.CS., 1982 109, 5542, “Methane
`and Dtjfluoromethunediplzosphonute Analogues ofGerany:'
`Diphasphure: Hydr0Iys1‘s—Iner: Alters are Substrates".
`McClard. R. W. et al.. J’. Am. Chem. Soc, 1987. 109.
`5544-5545. “Novel Phosphonylphosphinyl (P—C—P-C)
`Analogues of Biochernically Interesting Diphosphates,
`Syntheses and Properties of P—C—P—C Analogues of
`Isopentenyl Diphosphate and Dimethylallyl Diphos-
`phate”.
`Capson, Phd Dissertation, Jun. 193?, Dept. of Med.
`Chem. Univ., Utah. Abstract, Table (1 Contents, pp. 16,
`17, 40-43, 48-51, Summary.
`
`Primary Exam:'rter—Anton H. Sutto
`Attorney, Agent, or F:'rm—Burton Rodney
`
`[57]
`
`ABSTRACT
`
`-Compounds whichare useful as inhibitors of cholesterol
`biosynthesis and thus as hypocholesterolemic agents are
`provided which have the structure
`
`‘Ff.’
`if
`CH3-FCH-Cl-I1-CH;-|C=CH-Q-Z--I|’——(|:—li*—0R
`CH3
`CH3
`0111 R3 on”
`
`wherein Q is ‘g‘(CH2)1"'(I:=CH'§' or a bond;
`CH3
`
`Z is —(CI-Iz),,— or —(CH2)p—CH=CH—(CH;n_),.,—-,
`whereinnis 1 to5;pisD, 1 or2;n1is0, 1 or2;
`R, R1 and R1“ are the same or different and are H,
`lower alkyl or a metal ion; and
`R2 and R3 may he the same or different and are H or
`halogen.
`New intermediates used in preparing the above com-
`pounds and method for preparing same, pharmaceutical
`compositions containing such compounds and a method
`for using such compounds to inhibit cholesterol biosyn-
`thesis are also provided.
`
`18 Claims, Nu Drawings
`
`Iofl8
`
`PENN EX. 2196
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`1
`
`PI-IOSPHORUS-OONTAINING SQUALENE
` ASE INHIBITORS
`
`4,871,721
`
`2
`synthetase enzyme. These substances retain the unstable
`allylic pyrosphosphate moiety of F-PP.
`TABLE A
`
`FIELD OF TI-IE INVENTION
`
`Z
`
`Y
`
`o
`
`o
`
`The present invention relates to new phosphorus-
`containing compounds which inhibit the activity of
`squslene synthetase and thus are useful in inhibithlg
`cholesterol biosynthesis, to hypocholesterolemic com-
`positions containing such compounds, to a method of
`using such compounds for inhibiting cholesterol biosyn-
`thesis, to new intermediates formed in the preparation
`of such compounds and to a method for preparing such
`compounds.
`BACKGROUND OF THE INVENTION
`
`Squalene synthetase is a microsomal enzyme which
`catalyzes the reductive dimerization of two molecules
`of farnesyl pyrophosphate (FPP) in the presence of
`nicotinamide adenine dinucleotide phosphate (reduced
`form) (NADPH) to form squalene (Poulter. C. 1).; kill-
`ing, I-I. C., in “Biosynthesis of Isoprenoid Compounds".
`Vol. I, Chapter 8, pp. 413-441. J‘. Wiley and Sons, 1981
`and references therein). "l"h.is enzyme is the first commit-
`ted step of the de novo cholesterol biosynthetic path-
`
`10
`
`15
`
`20
`
` o\%/0"‘!-...$__o_
`
`X
`0-
`0-
`
`No.
`x
`-Y
`z
`1
`CH3
`CH3
`H
`2
`H
`H
`H
`3
`C1115
`H
`H
`4-
`I
`H
`H
`5
`H.
`I
`H
`
`5
`CH3
`H
`SCH;
`
`JE Am. Chem. Soc 1976, 98,
`Corey and Volante,
`1291-3, have prepared FPP analog A and presqualene
`pyrophosphate (PSQ-PP) analog B as inhibitors of
`squalene biosynthesis. (Presqualene pyrophosphste is an
`intermediate in the conversion of FPP to squaleue).
`These inhibitors possess methylene groups in place of
`the allylic oxygen moiety of FPP and PSQ-PP, but still
`retain the chemically and enzymatically unstable pyro-
`phosphate linkage.
`
`0
`0
`X II 0 ll
`“P” “P-0-
`I
`I
`0-
`0-
`
`
`
`45
`
`50
`
`way. The selective inhibition of this step should allow
`the essential pathways to isopeuteuyl tRNA, ubiqui-
`ncne, and dolichol to proceed unimpeded. Squalene
`synthetase, along with HMG-CoA reductase has been
`shown to be down-regulated by receptor mediated
`LDL uptake (Faust, J. R.; Goldstein. J. L.; Brown. M.
`S. Proc. Nat. Acad. Sci. USA, 1979, '.-'6, 5018-5022), lend-
`ing credence to the proposal that inhibiting squalene
`synthetase will lead to an up-regulation of LDL recep-
`tor levels, as has been demonstrated for HMG-CoA
`reductase, and thus ultimately should be useful for the
`Lteatlnent and prevention of hypercholesterolemia and
`atheroschlerosis.
`One approach to inhibitors of squalene synthetase is
`to dgn analogs of the substrate FPP. It is clear from
`the literature that the pyrophosphate moiety is essential
`for binding to the enzyme. However, such pyrophos-
`phates are unsuitable as components of pharmacological
`agents due to their chcal and enzymatic liability
`towards allylic C-0 cleavage, as well as their suscepti-
`bility to metabolism by phosphatases.
`P. Ortiz de Montellano et al in 4'. Med. Chem. 1971',
`20, 243-249 describe the preparation of a series of sub-
`stituted terpenoid pyrophosphate (Table A), and have
`shown these to be competitive inhibitors of the squalene
`
`Poulter and co-workers have prepared cyclopropane
`C (Sandifer, R. M., et al., J’. Am. Chem. Soc. 1982, 104,
`7376-8) which in the presence of inorganic pyrophos-
`phate is an intermediate analog inhibitor of the enzyme
`squalene syntbetase.
`
`Me~
`
`Altman and co-workers. Bertolino, A., et al., Bio-
`chim. Biopbys. Acts. 1978, 530, 17-23, reported that
`farnesyl amine and related derivatives D inhibit squal-
`ene synthetase. but provide evidence that this inhibition
`
`2of18
`
`PENN EX. 2196
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4, 87 1,721
`
`3
`is non-specific and probably related to membrane dis-
`ruption.
`
` NHR 5
`
`R = H, CH2CH20H, CH2CH:OCH3
`
`_ D
`
`DESCRIPTION OF THE INVENTION
`
`In accordance with the present invention, there is
`provided phosphorus-containing compounds which
`inhibit the enzyme squalene synthetase and thus are
`useful as hy-pocholesterolemic agents and have the fol- 15
`lowing structure
`
`‘Vi.’
`if
`cr13—<i.:cH—cH2—cH;—«i,=cH—o—z—1|=—t|:—rl*—oR
`CH3
`CH3
`0111 R3 on"
`
`"
`
`20
`
`wherein Q is -§-(cH2);—t|:=cH-f- or a bond;
`CH3
`
`Z is e—(CH2),,-— or —-(CH;:_),—-CH=CH—(CI-I2),-,.-—,
`whereinnis1to5;pis0,1or2;rnis0,1or2.;
`R,R1 and Rlflmaybe thesameordiiferentandare H.
`lower alkyl or a metal ion; and
`R1 and R3 may be the same or different and are H or
`halogen.
`Hereinafter the moiety
`
`30
`
`35
`
`IC
`
`CH3—C-CH—CH2—CH2—(|.‘.=CH-
`H3
`CH3
`
`will be expressed as “X” in the strucuiral formulae set
`out below.
`
`40
`
`Thus, the following types of compounds are included
`Within the scope of the present invention.
`
`‘Ff.’
`it
`x—(cHg)g—<|:=cr-r—(cH-_a),.-1|’-<|:—1|*—on
`CH3
`cal
`113 01116
`
`R1 0
`0
`I
`II
`II
`x—{cHg),,—P-—c—P-on
`I
`I
`I
`onl R3 onla
`
`4
`dimethylpentyl, octyl, 2,2,4»-trimethylpentyl, nonyl,
`decyl, undecyl, dodecyl, the various branched chain
`isomers thereof, and the like. The lower alkyl or allryl
`group may be substituted with a substituent including a
`halo—substituent, such as F, Br, C1 or I or CF3, an alkoxy
`substituent, an aryl substituent, an alkyl-aryl substituent,
`at haloaryl substituent, a cycloalkyl substituent, an alkyl-
`cycloalkyl substituent, hydrory, and alkylamiuo substit-
`uent,
`an
`alkanoylamino
`substituent.
`an arylcar-
`bonylamiuo substituent, a nitro suhstituent, a cyano
`substituent, athio substitnent orsn alkylthio suhstituent.
`The term “cycloalkyl” as employed herein alone or
`as part of another group includes saturated cyclic hy-
`drocarbon groups containing 3 to 12 carbons, prefera-
`bly 3 to 8 carbons, which include cyclopropyl, cyclobu-
`tyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
`cyclodecyl and cyclododecyl, any of which groups may
`be substituted with l or 2 halogens, 1 or 2 lower alkyl
`groups, 1 or 2 alkoxy groups, 1 or 2 hydroxy groups, 1
`or 2 ailtylamino groups, 1 or 2 alkanolyarnino groups, 1
`or 2 arylcarbonylamino groups, 1 or 2 amino groups, 1
`or 2 nitro groups,
`1 or 2 cyano groups,
`1 or 2 thiol
`groups, and/or 1 2 alkylthio groups.
`The term “ary ” or “Ar” as employed herein refers to
`monocyciic or bicyclic aromatic groups containing
`from 6 to 10 carbons in the ring portion, such as pheuyl.
`naphthyl, substituted phenyl or substituted naphthyl
`wherein the substituent on either the phenyl or naph-
`thyl may be 1, 2 or 3 lower alkyl groups, halogens (Cl,
`Br or F), l, 2 or 3 lower alkory groups, 1, 2 or 3 hy-
`droxy groups, 1, 2 or 3 phenyl groups, 1, 2 or 3 al-
`kanoyloxy groups, 1, 2 or 3 benzoyloxy groups, 1, 2 or
`3 haioalkyl groups, 1, 2 or 3 halophenyl groups, 1, 2 or
`3 allyl groups, 1, 2 or 3 cycloslkylttlkyl groups, I, 2 or
`3 adamantylaikyl groups, 1, 2 or 3 alkylarnino groups, 1,
`2 or 3 alkanoylamino groups,
`1, 2 or
`3 ary1car-
`bonylamino groups, 1, 2 or 3 amino groups, 1, 2 or 3
`nitro groups, 1, 2 or 3 cyano groups, 1, 2 or 3 thiol
`groups, and/or 1, 2 or 3 slkylthio groups with the aryl
`group preferably containing 3 substituents.
`The term “aralkyl”, “a.ryl-alkyl" or “aryl-lower al-
`kyl" as used herein alone or as part of another group
`
`IA.
`
`IC.
`
`I1
`
`11 0
`
`0I
`
`I
`X—(CH2)g-$=CH—(l‘.:l-I-;_);—CI-[=13]-I-(CE-12}...-P-—'C—P-~OR
`CH3
`OR! R3 Okla
`
`"3';
`girl
`fl)
`.
`x—(CH2),—cH=cI-I-—(CH1),,.—1f—rf—if—oR
`0111
`113 on“
`
`ID.
`
`The term “lower alkyl” or “alkyl" as employed
`herein alone or as part of another group includes both
`straight and branched chain hydrocarbons, containing 1 65
`to 12 carbons in the normal chain, preferably 1 to 7
`carbons, such as methyl, ethyl, propyl, isopropyl, hutyl,
`t-butyi. isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-
`
`refers to lower alkyl groups as discussed above having
`an aryl subsitutent, such as benzyl.
`The term “lower aikoxy”, "aikoxy”, or "a.ryIoxy” or
`“aralkoxy” as employed herein alone or as part of an-
`
`3of18
`
`PENN EX. 2196
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`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`5
`other group includes any of the above lower alkyl,
`alkyl, aralkyl or aryl groups linked to an oxygen atom.
`The term “lower alkylthio”, “alkylt.hio", “arylthio”
`or "aralkylthio” as employed herein alone or as part of
`another group includes any of the above lower alkyl,
`alkyl, aralkyl or aryl groups linked to a. sulfur atom.
`The
`term “lower
`alkylaniino”,
`"a1kylan1ino”,
`“arylaniino”, “arylalkylamino” as employed herein
`alone or as part of another group includes any of the
`above lower alkyl, alkyl. aryl or arylalkyl groups linked
`to a nitrogen atom.
`The term "a]kanoy1” as used herein as part of another
`group refers to lower alkyl linked to a carbonyl group.
`The term “halogen” or “halo” as used herein refers to
`chlorine, bromine, fluorine, iodine and CF13. with chlo-
`rine or fluorine being preferred.
`The term. “metal ion" refers to alkali metal ions such
`as sodium. potassium or lithium and alkali earth metal
`ions such as magnesium.
`Preferred are those compounds of formula I which
`have the following structure
`
`5
`
`20
`
`R1 o
`o
`1
`[I
`II
`x—Q-—z—1=—c—1=—oR
`I
`I
`I
`0111 R3 0111*
`
`wherein Q is
`
`-v-CI-lq—cH2—ti:=CI~i-.
`CH3
`
`2 is —CH2CH;J,— or
`
`‘E-
`
`25
`
`so
`
`4,871,721
`
`6
`-continued
`
`‘F1 ii
`in’
`X---Q--Z----I|’------(|I:--}I’--OM%
`OM R3 OM
`(where M is a metal ion)
`IG.
`
`0 R2 0
`II
`I
`II
`X—Q—Z—E|'—(|J—!I’—OH
`OHR-°‘ on
`
`111.
`
`AU) Alternative Preparation of Compounds of
`'
`Invention
`
`(1) Derllkylation '
`*"““H
`'
`'?
`
`IF.
`
`III.
`
`B. Preparation of Starting Materials
`
`{l)W'hereZis(CHz),.andnis1to5orZis
`(cHz)p~cH=cH—(cH2),,,_ and p is o to 2 and rn is
`1 or 2
`
`K-Q—z—Ha1 + P(0a|Jo'l)3 ——>
`V
`VI
`(wherein I-lal is
`a halogen such
`as Cl. Br or 1)
`
`—CH.TeCH—;l?.5-’a.nd1?.3a.reeach Hor eachF; R,R1
`and R10 are OH or metal ions
`The compounds of formula I of the invention may be 40
`prepared according to the following reaction sequence
`and description thereof.
`
`ii’
`X--Q--Z—1|’--Oalkyl 3”“ H
`Oalkyl
`VII
`
`1
`
`if
`'3 X-Q-Z—I|’—0H
`Oalkyl
`II
`
`A. Preparation of Compounds of Invention
`
`(2) Where 2 is (CH1). and u is 2 to 5 or Z is —(CH;.
`)p—Cl'I==Cl'I--(CHz)m— and p is 0 to 2 and In is 2
`
`43
`
`f‘
`Oxaiyl Chloride
`x—Q—z—1|:—oH ii}09.4
`
`(where R‘! is alkyl)
`II
`
`(Add :31 Formation)
`
`i‘: ii
`Li®9C—P—oa1kyI
`1'13 nitvalkyl
`‘I?
`x—Q—z—i|=—c1 +L)
`0114
`(Phosphinyl-Phosphonnte
`. Formation)
`
`I11
`
`50
`
`55
`
`60
`
`0
`R‘ 0
`(1) Deaikylation
`"
`I
`ll
`Basic H drol as
`x_Q_z_1iI:(|3_}l:_Qa]uy .% 65
`0114 113 Oalkyl
`IIF.
`
`K-Q‘('-'-‘-H‘2.)n—l‘H3-1
`VA
`
`or
`
`+
`
`x—Q—(m{2)p—cH=cH—(cH1),._1—Hu1
`VB
`
`if
`.
`if
`I.i$9CH3—l|’—0aJkyl —-—> X—Q—Z'-fi'-Oalkyl —9
`Oalkyl
`Oalkyl
`
`IIIB
`
`v1_IA
`
`ii‘
`x—Q—z—1|=—oaIky1
`OH
`I]
`
`(wherein Hnl is a halogen such in Br or I}
`
`(3) Where 2 is (CI-I;)_.,——CI-I-_-CI*I—-(CI-lg),,.—, p is 0
`to 2 and m is o
`
`4of18
`
`PEl§iN EX. 2196
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,871,721
`
`112 o
`o
`I
`II
`II
`X-Q—Z—P—-C—P—Dalky1
`I
`I
`I
`0114 R3 Oalkyl
`
`X—Q—(CI-I1)P—CH0 +
`VIII
`
`HomenEmmons
`
`modification of Wittig 9
`
`0 I
`
`o
`I,
`aIkylO__‘ ||
`Oaikyl
`P-CH;—P,_____Oaikyl
`anyto’
`DC
`
`0 I
`
`I
`X—Q—(CH1}_g—CH=CIi-I“II’-Dulltyl —---)
`Oelkyl
`X
`
`0I
`
`I
`x—Q—z-1|=—osuy|
`on
`I!
`
`As seen in reaction sequence “A", compounds of
`Formula [may be prepared by treating monoacid II
`
`0 l
`
`l
`x—Q-z—1i--on
`or.‘
`
`(wherein R‘ is an alltyl group) in an aromatic solvent
`such as benzene or toluene, preferably containing die
`methylformamide, or other appropriate inert organic
`solvent, with oxalyl chloride, and then evaporating the
`reaction mixture to give acid chloride III
`
`0I
`
`I
`X—Q—Z—P—Cl
`ut
`
`Io
`
`To a stirred solution of an optionally substituted dial-
`ltyl methyl phosphonate
`
`R20
`I
`ll
`H—c—p—oauy1
`I3 I
`11 Oelkyl
`
`The lithium salt HIA will be employed in a molar ratio
`to acid chlorida III of within the range of from about
`1.8:! to about 2.5:l and preferably from about 2.0:l to
`about 2.4:l. Ester IF, in an inert organic solvent such a
`methylene chloride, may then be subjected to dealky1a-
`tion by treating with bromotrimethylsilane or iodo-
`trimethylsilane in the presence of 2,4,6—collidine or bis(-
`trimethyl)si1yltrifluoroacetamide and then treating with
`a strong inorganic base such as aqueous NaOH, KO!-I,
`Li0H or Mg(OI-D2, optionally in the presence of an
`alcohol such as methyl alchol, to form the salt IG which
`may be separted out by chromatography. Salt IG may
`be treated with a strong acid such as HCl to form acid
`1]-I.
`
`Intermediates II and III are novel compounds and
`thus form a part of the present invention.
`As seen in reaction sequence “B”, the starting materi-
`als II may be prepared as follows.
`As seen in reaction scheme B(l), where Z is (CH2),
`and 11 is 1 to 5 or Z is —(Cl-1:)p—Cl-I::CI-I—(CI-l2),,.—
`anclpiso,1or2andmislor2,startingmaterialIImay
`be prepared by treating halide V
`
`20
`
`25
`
`v
`
`with trialkyl phosphite VI
`
`v1
`
`X—Q-Z—Ha1
`
`1’(0aIkyl)3
`
`underan inert atmosphere. such as argon. at an elevated
`temperature of within the range of from about 120' to
`about 165' C. for a period of from about 1 to about 30
`hours to form the phosphonic ester compound VII
`
`45
`
`0
`II
`x—Q-—z—1|>—oaJky1.
`Oalltyl
`
`vii
`
`The reaction of phosphite VI and halide V is carried
`out employing a molar ratio of VIN of with.i.n the range
`of from about 2:1 to about 50:1.
`
`Phosphonic ester VII is subjected to basic hydrolysis
`such as by treatment with alkali metal hydroxide such as
`aqueous K01-I or N801-I, optionally in the presence of
`an alcohol such as methanol, ethanol or isopropauol
`under an inert atmosphere, such as argon, at reflux
`temperature to form the phosphonic acid II which is a
`novel compound and thus forms a part of the present
`invention.
`
`The starting halides V are either known or are pre-
`pared from farnesol or geraniol by conventional means.
`As seen in reaction scheme B(2), where in starting
`materialII.Zis(CHz)I.andnis2to5orZis—ECH2-
`);—CH=CI'I—(CH2}m—audpis0to2a:ndmis2,II
`may be prepared by treating halide VA or VB with the
`salt IIIB such as
`
`in an inert organic solvent such as tetrahydrofuran
`cooled to a temperature within the range of from about
`-90’ C. to about 0' C. is added a lithium source, such
`as 11-butyl lithium or lithium diisopropylamide in a. hex-
`ane or other inert organic solvent under an inert atmo-
`sphere such as argon to form the lithium salt IIIA.
`
`R20
`I
`II
`_
`L1$e(E—l'i‘—Oa1kyl
`113 Oalkyl
`
`INA
`
`The lithium salt [HA is maintained at a reduced tem-
`perature as described above and acid chloride III in an
`inert organic solvent such as tetrahydrofimln or ethyl
`ether is added to form the phosphinyl-phosphonate IF.
`
`65
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`

`
`9
`
`(Iii)
`9
`LigH1""l|’—Oa]kyl
`Oalkyl
`
`4,871,721
`
`HIE
`
`10
`
`_{cH2)2...(|:=CH_
`CH3
`
`5
`
`10
`
`in the presence of an inert organic solvent such as tetra-
`hydrofuran or ethyl ether at a reduced temperature of '
`within the range of from about —78° to about 0° C. for
`a period within the range of from about I to about 24
`hours, to form the phosphonic ester VIIA
`
`{III}
`X"'Q—Z"'I|’—0a.lk)'l
`Oalkyl
`
`VIIA
`
`which is subjected to basic hydrolysis as described
`above for scheme B0) to form phosphinic acid starting
`material H.
`The reaction of VA or VB and [[113 is carried out
`
`employing a molar ratio of VA or VI-3_:IIlB of about 1: l.
`The salt IIIB is formed by treating phosphonate HIB’
`
`0
`ll
`H--Cl-I3--P—CIaLkyl
`
`Oalkyl
`
`1113'
`
`30
`
`and p is O is farnceal, a known compound.
`As will be appreciated by one skilled in the art, other
`starting aldehydes and starting alcohols are either
`known in the art or prepared from geranoil or farnesol
`employing conventional chemical synthesis.
`The novel intermediates in accordance with the in-
`vention may be defined by the following formula:
`
`('1')
`CH3--(l'.‘=CH—CH;—CH3--(E=('JH--Q-Z--f--R5
`CH3
`CH3
`Oalkyl
`
`IV
`
`wherein R5 is OH or C1.
`The compounds of the present invention are inhibi-
`tors of squalene synthetase and thus are useful in inhibit-
`ing cholesterol biosynthesis and treating hypercho1es-
`terolernia and atherosclerosis. Inhibition of squalene
`synthetase may be measured by the following proce-
`dure.
`'
`Rat liver microsomal squalene synthetase activity is
`measured using farnesyl pyrophosphate as substrate and
`quantitating squalene synthesis using gas chromato-
`graphic analysis. The assay was developed by modify-
`ing conditions originally described by Agnew (Methods
`in Enzymology 1l0:357, 1985).
`
`Preparation" of Rat Liver Microsomes
`
`in an inert organic solvent such as tetrahydrofuran or
`ethyl ether under an inert atmosphere such as argon
`with a source of alkali metal such as n-butyllithium in an
`inert organic solvent such as hexane.
`The starting halides VA or VB are either known or
`prepared from geraniol or farnesol by conventional
`Illeflflfi.
`
`As seen in reaction scheme B(3), where in starting
`material II, 2 is —(CH2),—C!-I_—_CH—-(C11-1;),,.— and p
`isotozandrniso, Ilmaybepreparedby employing a
`Hornet-Ernruons modification of the Wittig reaction by
`treating aldehyde VIII
`
`VIII
`
`X—Q—~(CH2.
`)p—CHO
`
`with tetraalkyl methylenebisphosphonate [X in the
`presence of sodium hydride and an inert organic solvent
`such as tetrahydrofuran,
`toluene or ethyl ether at a
`temperature within the range of from about ——20° to
`about 25° C. for a period of from about 1 to about 24
`hours, to form phcsphonic ester X
`
`35
`
`40
`
`Livers are dissected from 2 or 3 decapitated Sprague
`Dawley ram and are quickly transferred to ice cold
`buffer (potassium phosphate, 0.05M, (pH 7.4); MgClz,
`0.004-M; EDTA, 0.00lM;
`and 2-mercaptoethanol
`0.01M) and rinsed thoroughly. The livers are minced in
`cold buffer (2.0 1111/3) and homogenized using a Potter-
`Elvejhem_hom.ogenizer. The homogenate is centrifuged
`at 5,000)(g, 10 minutes (4° C), and the supernatant
`poured through 2 layers of cheese cloth. The superna-
`tant is then centrifuged at 15,000)-(3 for 15 minutes (4‘'
`C.). Again the supernatant is filtered through 2 layers of
`cheese cloth, and centrifuged at third time at l00,000)<'g
`for 1.0 hour at 4° C. Following centrifugation the mi-
`crosomal pellet is resuspended in a volume of buffer
`equivalent to 1/5 the volume of the original homoge-
`so nate, and homogenized in a ground glass homogenizer.
`Aliquotted microsomes are frozen at -80” C., and re-
`tain activity for at least two months.
`
`4-5
`
`0
`x—Q—(cH;),—cn=-=cn—i|i—_oa1ky1
`Oallryl
`
`X
`
`55
`
`(a novel compound in accordance with the present
`invtion) which is subjected to basic hydrolysis as
`described in scheme B(l) to form phosphonic acid II.
`The reaction of aldehyde VIII and phosphonate IX is
`carried out employing a. molar ratio of VIII:IX of
`within the range of from about 1:1 to about 1:2.
`The aldehyde starting material VIII where Q is a
`bondandpis0,thatisgeranial.isaknowncompound.
`Where Q is
`
`60
`
`65
`
`Enzyme Assay
`Reaction Mixtures are prepared in 50 ml round bot-
`tom pyrex glass tubes with tight-fitting, teflon-lined,
`screw caps. Tubes are cooled to 4° C., and the following
`components are added in sequence:
`
`1.
`
`Pctamium phosphate buffer
`(0.275 M, pH 7.4)
`KF (55 n1M)
`2
`3. NADPH (5.0 mM. freshly prepared)
`-it H20 {or H20 + test compound)
`5. MgCl1 (27.5 n1N[)
`6 Microsomal Enzyme (0.48 mg
`micmscmsl protein in homogeni-
`zation huffer) (15 pl prep.
`4f23;'86)
`
`0.36 ml
`
`0.35 ml
`0.36 ml
`0.16 ml
`(1.36 ml
`
`0.10 ml
`
`6of18
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`
`11
`-continued
`1.8 ml
`
`4,871,721
`
`12
`-continued
`.
`
`fluslene Peal: Area
`X RR
`Docanue Peak Area
`‘RR = Ruponselhrio [Docas|ne.I"Squ:lene]
`‘RR = 0.56
`
`This mixture is equilibrated under N; at 4° C. for 5-15
`minutes. Reaction mixtures are then warmed to 30' C.,
`and the enzyme reaction
`by adding 0.2 ml of
`farnesyl pyrophosphate (21.9 uM) prepared in H20.
`Each tube is again overlayered with N2, and incubated
`at 30' C. for 60 minutes. The reaction is stopped by the
`addition of 1.0 ml KOH (40%). Ethanol (95%) (spectral
`grade) (1.0 ml) is added to each tube. Doeosane (5
`nmoles in hexane) isadded to each tube as an internal
`standard. The mixture is saponified at 65' C. for 30
`minutes. The tubes are cooled to room temperature and
`extracted twice with 10.0 ml spectral grade hexane.
`The upper organic phase fractions are pooled in glass
`20.0 ml scintillation vials and reduced in volume to =-
`1.0 ml under a stream of N2. The sample is then trans-
`ferred to acid-washed, conical bottom, glass (1.0 ml)
`microvials, and brought to dryness under N3. The resi-
`due is resuspended in 50 pl hexane (spectral grade), and
`these samples are spun at 1000 rpm at room temperature
`for 10 minutes. Following centrifugation approximately
`40 pl of supernatant is transferred to 100 pl acid-washed
`microvials with septa/crimp-top caps (compatible with
`the Hewlett-Packard GC auto injector).
`
`Gas Chromatography
`
`Two p.L of each sample is injected onto a fused silica
`megabore DB-17 column (15M><D.525 mm) (l&W Sci-
`entific) using a splitless mode of injection. Gas flow
`rates are listed below:
`
`Make up gas (helium)
`
`20 mlfmin
`
`Hydrogen
`Carrier (helium)
`Septum purge vent
`
`30 mlfmin.
`15 mlfmin.
`5 mlfmln.
`(Septuni purge oft‘ 0.00
`min, on at 0.5 min.)
`
`The injector temperature is 200' C., and the FID
`detector tperature is set at 270' C. Oven tperature ‘5
`is programmed through a two ramp sequence as fol-
`lows:
`Oven:
`
`time 10 minutes
`
`Initial tperature 130° C.,
`Ratnp one: 20‘ C./minute
`Second temperature 250' C., second time 10 minutes
`Ramp two: 20‘ C./minute
`Third tperature 260’ C., third time 10 minutes
`(Equilibration time 1.0 minute)
`Using this gas chromatographic system, docosane
`(internal standard) has a retention time of 3.6-3.7 min-
`utes, and squalene has a retention time of 14.7-14.9
`minutes. The amount of squalene in each reaction mix-
`ture is determined by obtaining the areas under the
`squalene and docosane peaks and using the following
`formula to calculate the amount of squalene (nmoles) in
`. the total reaction mixture.
`
`Squaiene (nmolesfruction = 5.0 (nmolu docasane X
`mixture)
`internal standard)
`
`Compounds Testing
`
`Compounds are dissolved in H20 and added to reac-
`tion mixtures prior to addition of farnesyl pyrophospate
`substrate. All reaction mixtures are run in duplicate, at
`several concentrations. Additionally, all compound I50
`values are derived from composite dose response data
`with the 95% confidence interval indicated.
`A further aspect of the present invention is a pharma-
`ceutical composition consisting of at least one of the
`compounds of Formula I in association with a pharma-
`ceutical vehicle or diluent. The pharmaceutical compo-
`sition can be formulated employing conventional solid
`or liquid vehicles or diluents and pharmaceutical addi-
`tives of a type appropriate to the mode of desired ad-
`ministration. The compounds can be administered to
`mammalian species including humans, monkeys, dogs,
`etc. by an oral route, for example, in the form of tablets,
`capsules. granules or powders, or they can be adminis-
`tered by a parenteral route in the form of injectahle
`preparations. The dose for adults is preferably between
`200 and 2,000 mg per day, which can be administered in
`a single dose or in the form of individual doses from 1-4
`times per day.
`A typical capsule for oral administration contains
`active ingredient (250 mg), lactose (75 mg) and magne-
`sium stearate (15 mg). The mixture is passed through a
`60 mesh sieve and packed into a No. 1 gelatin capsule.
`A typical injectible preparation is produced by ascep-
`tically placing 250 mg of sterile active ingredient into a
`vial, asceptically freeze-drying and sealing. For use, the
`contents of the vial are mixed with 2 ml of physiological
`saline, to produce an injectible preparation.
`The following Examples represent preferred embodi-
`ments of the present invention- Unless otherwise'indi-
`cated. all temperatures are expressed in degrees Centi-
`grade. Purification of final targets was often achieved
`by chromatography on Cl-lIP20P gel (referred to herein
`as I-IP-20), a highly porous styrene-divinyl benzene
`copolymer available from Mitsubishi Chemical Indus-
`tries. 31P NMR spectra were accumulated in the ‘H
`decoupled mode, employing 35% I-I3PO4 (6=0 ppm) as
`the external reference. For 1‘-‘F NMR spectra, CF3CCl3
`(8=I-32.6 ppm) was employed as an internal reference
`EXAMPLE 1
`
`(E,E}-[[Hydroxy(4,B,12-trimethyl-3,7,1l-trideea-
`trienyl)phosphiny1]met.hy1]phosphonic acid,
`tripotassiutn sale
`A. (E.E)-3,7, 1 l-Trimethyl-2,6, 10-do-decatrienyl
`bromide
`
`A solution of 1.00 g (4.5 mmol) of E.E-farnesol (Ald-
`rich, further purified by flash chromatography) in 10 ml
`of distilled ether at 0' C. under argon in the dark was
`treated dropwise with a solution of 195 u.L (2.05 mmol,
`0.45 eq.) of PB:-3 in 2 ml of ether. The resultant mixture
`was stirred at 0° C. for one hour, then quenched with
`water and separated. The organic phase was washed
`with 5 ml of H20, 5 ml of saturated Na!-ICO3, and 5 ml
`of brine, dried over Na2S04and evaporated to give 1.26
`
`7uf18
`
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`lPR20l5-01836
`
`

`
`13
`(98%) of crude bromide as a clear oil. TLC Silica (2.-8
`ethyl acetate:Hexarte) Rf=0.69 (decomposes).
`‘H NMR (CDCI3)
`6
`
`5.52 (t, 1H. J=8.5 Hz)
`5.08 (in, 21-1)
`4.01 (d, 21-1)
`1.9--2.2 (in, 3H)
`1.73 (s, 3H)
`1.68 (s, 31-1)
`1.60 (s, 61-I) ppm.
`
`4,871,721
`
`14
`
`5
`
`5.10 (m, 3H)
`3.72 (d, 3H. J= 10.6 Hz)
`2.30 (:11, 211)
`2.02 (In, an)
`1.77 (m, 211)
`1.68 (s, 3H)
`1.52 (s, 311)
`1.59 (5, 61-1) ppm.
`Mass Spec (CI—I-I20, + ions) m/e 315 (M+ H), 179.
`D.
`
`B.
`(E,E)-(4,8,12-Trimethyl-3,7,1 l-tridecatrieny1)pl1os-
`phonic acid, dimethyl ester
`To a stirred solution of 3.06 g (24.64 mmol) of di-
`methyl methylphospllonate in 50 ml of T!-[F under
`argon was added 14.6 ml (23.47 moi) of 1.60M n-
`butyllithium in hexane over 10 minutes to give a white
`suspension. After stirring for 30 minutes at -78” C.,
`6.36 g (22.32 mmol) of Part A bromide in 14 ml of THF
`was added over 10 minutes. The reaction was allowed
`to stir at -78° C. for one hour, followed by 0° C. for 45
`minutes, and was then quenched with eltcms methanol.
`Most of the solvent was evaporated and the residue was
`dissolved in ethyl acetate, washed with water and brine,
`dried M3804) and evaporated to provide 7.35 g of an
`oil. The crude material was flash chromatographed on
`350 g of silica gel, packed in 75:25 and eluted with 90:10
`ethyl acetate: petroleum ether, collecting 70 ml frac-
`tions. Fractions 23-72 yielded 5.78 g (72%) of pure title
`compound as a colorless oil.
`IR(CCI4) 2967,
`TLC Silica gel, (Et0Ac) Rf=0.2l
`2952, 2918, 2852, 14-49, 1382, 1250, 1223, 1185, 106.5,
`1039, 831 cm—1.
`$11 NMIR (CD013)
`5.10 (m, 3H)
`3.13-(d. 6H, J=1l Hz)
`2.23 (m, 2H)
`2.03 (111, 81-1)
`1.77 (m, 21-1)
`1.68 (a, 31-1)
`1.62 (s, 3H)
`1606. 6H) ppm-
`Mass Spec (CI-CH4/N20, + ions) m/e 309 (m+C31-15),
`_ 357 (M+C1H5), 329 (l\‘[+1-I).
`C
`(E,E)-(4,3,l2-Trimethyl-3,7,11-tridecatrie11yl)phos-
`phonic acid, monomethyl ester
`A stirred solution of 2.22 g (6.76 mmol) of Part 13
`compound in 10 ml of 1:1 methanohwater containing
`4.4 g (53.2 mmol) of potassium hydroxide was heated to
`65°—75° C. for 5.5 hours. The methanol was evaporated 55
`and the residue was stirred with dichioromethane/we
`ter and acidified by adding 10.0 g of solid Kl-IS04. The
`mixture was stirred until the phases were homogeneous.
`The organic layer was washed with dichloromethane.
`The combined organic extracts were washed with 1:1 50
`brine:water, dried (M3804) and evaporated to provide
`?l:{‘3'C3s(’i1l‘:';9‘(=g_§f1‘1:1°(‘:“1IfIT"C1)‘g‘P“°id 33 3931‘ Y°“°f °“-
`'
`'
`'
`3
`'°°n NH3'H20} Rf‘0'62
`IRIGCI4) 2918, 2853, 1449, 1229, 1193, 1056, 987
`cm—1
`1].] NMR (CDC]3)
`3
`
`15
`
`25
`
`30
`
`45
`
`50
`
`65
`
`12_22 (3, 11.1}
`
`(E,E)-[[Hydroxy(4,8,12—trimethy1)-3,7,11-trideca-
`trieny1)p11osphinyl]tuethyl]phosphonic acid, trimethyi
`ester
`
`To a stirred solution of 2.05 g (6.50 rrlmol) of Part C
`monoacid in 20 ml of benzene containing two drops of
`DMF was added 1.7 ml (19.50 mmol.) of oxalyl chloride
`over 10 minutes at room temperature. After 2.5 hours,
`the solution was evaporated and the residue was twice
`dissolved in benzene and evaporated to provide the acid
`chloride as an orange oil.
`To a stirred solution of 1.80 g (14.50 mmol) of di-
`methyl methylphosphonate in 30 ml of THF at —-78” C.
`was added 8.7 ml (14.0 mmol) of 1.6M n-butyllithium in
`hexane over five minutes under argon to provide a
`white suspension. After stirring for 15 minutes at —-78°
`C., the acid chloride described above was added in 13
`ml of THIF over five minutes. After one hour at —-78°
`C., the reaction was allowed to warm to 0'’ C. for one
`hour and was diluted with ether and quenched with
`10% H0]. The ether solution was separated, washed
`with water,
`saturated NaHOO3 and brine, dried
`(1113304) and evaporated to give 1.57 g of crude title
`compound. The combined aqueous layers were back
`extracted with CHzCl1 and the organic layer was
`washed with brine. dried (MgS0.1) and evaporated to
`provide an additional 1.10 g of crude title compound.
`Flash chromatography on 200 g of silica gel packed in
`2:98 and eluted with 4:96 CH30H:CH1(11 gave 565.1
`mg (18%) of slightly impure title compound followed
`by 1.79 g (58%) of pure title compound as a colorless
`oil.
`Rechromatography of the impure fractions gave an
`additional 225.6 mg (8%) of pure title compound.
`TLC Silica (5-.95 CH:-;0H:CH1Cl1) Rf=0.22. IR(CCl4)
`2955, 2917, 2853, 1450, 1259, 1240, 1184, 1166, 1064,
`1039, 845, 822, 781 cm'1.
`‘H NMR (CDCI3)
`3
`5_14 (11,, 31-1)
`33] ((1, 51-], 1:11 Hz)
`3-77(d.3H.J=1t15 Hz)
`2-4'0 15¢ 7-H: J-=21-1» 15-3 HZ)
`2-30 (m. 2H)
`2-91 (ma 10H)
`1-53 (3: 3H}
`1-53 (3. 33)
`_
`1-50 (51 53} PP“1-
`Mass Spec (CI-CH4/N20 + Ions) In/= 393 (M-|~C3Hs)»
`331 [M+C2H5)- 353 {M-I-H}
`E
`(E,E)-[I-Iydroxy(4,3,12-trimethyl-3,1,11-tridecatrieny1)-
`phosphinyl]methyl]phosphon.ic acid, tripotassium salt
`_
`_
`_ To a stirred solution of 1.475 g (3.51 mmol) of Part D
`title compound in 20 ml of CI-I2Cl2 at 0° C. under argon
`was added 0.93 ml (7.02 mmol) of 2,4.6:co1l1d1ne fol-
`lowed by 1.90 1111 (14.4 mmol) of bromotrlmethylsllame,
`
`8 of 18
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`
`

`
`4,871,721
`
`5
`
`16
`-
`1.39 (t, 6H, J -47.5 Hz) ppm.
`‘::C NMR (C