`United States Patent
`4,924,024
`[11] Patent Number:
`[45] Date of Patent:
`Biller
`May 3, 1990
`
`ane and Difluoromethanediphosphonate Analogues of
`Geranyl Diphosphate: Hydro1ysis—Inert Alternate Sub-
`strates".
`McC1a.rd. R. W., et al., I Am. Chem. Soc, 1987, 109,
`5544-5545, “Novel Phosphonylphosphinyl {P~C«P—C)
`Analogues of Biochemically Interesting Diphosphates,
`Syntheses and Properties of P—C-P—C Analogues of
`Isophentenyl Diphosphate and Dimethylallyl Diphos-
`pha ”.
`'
`Capson, T. L., PhD Dissertation, Jun. 1937, Dept. of
`Medicinal Chemistry, The University of Utah, Ab-
`stract, Tablc of Contents, pp. 16, 17, 40-43, 48-51, Sum-
`mary.
`
`Primary Examz'rter——Anton H. Sutto
`Attorney, Agent. or Firm—Bu1'ton Rodney
`
`[57]
`
`ABSTRACT
`
`Compounds which are useful as inhibitors of cholesterol
`biosynthesis and thus as hypocholesterolemic agents are
`provided which have the structure
`
`CH3—(|:.=CH.—CH3"CH.;_—lC=CH—
`CH3
`CH3
`
`in’
`
`‘ii 1’
`li’—GR
`oal R3 or.“
`
`wherein
`Q is
`
`‘i'(CH2)2—§3=CH'i'
`
`CH3
`
`3‘
`
`or a bond;
`Z is ———(CH2),,— or —(CH2)p—CH=CH—(CH1)m—,
`whereinnis 1 to 5; pisfl. 1 or2; mist}, 1 ml;
`R, R1 and R1“ are the same or different and are H, lower
`alkyl or a metal ion; and
`R2 and R3 may be 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.
`
`3.C1aims, No Drawings
`
`[54] PHOSPI-IORUS-CONTAINING SQUALENE
`SYNTHETASE INHIBITORS, NEW
`INTER.MEDIATES AND METHOD
`
`[75]
`
`Inventor:
`
`Scott A. Biller, Ewing, NJ.
`
`[73] Assignee:
`
`E. R. Squibb & Sons, Inc., Princeton,
`NJ.
`
`[2]] Appl. No.: 359,606
`
`[22] Filed:
`
`Jun. 1, 1989
`
`Related U.S. Application Data
`
`[62]
`
`Division of Ser. No. 141,744, Jan. 11. 1988. Pat. No.
`4,871,721.
`
`
`
`. . . .. C071?‘ 9/33; CUTF 9/42
`Int. Cl.5 . . . . . .
`[51]
`............ .. 558/202; 558/207
`[52] U.S. Cl.
`[53] Field of Search .............................. .. 558/202, 207
`
`
`
`[56]
`
`References Cited
`PUBLICATIONS
`
`Arald et al., “Current Abstracts of Chemistry”, vol.
`103, Issue 1199, (1986), No. 394994.
`Poulter, C. D. et al., Biosynrlrestis of Isoprenoid Com-
`pounds. “Conversion of Farnesyl Pyrophosphate to
`Squalene”, vol. 1, Chapter 8, pp. 413-441, J. Wiley and
`Sons. 1981.
`Faust, I. R., eta1., Proc. Nat. Acad. Sci, USA, "Squalene
`Synthetase Activity in Human Fibroblasts: Regulation
`via the Low Density Lipoprotein Receptor”, 1979, 76,
`5018-5022.
`de Montellano, P. Ortiz, et al., J. Med. Chem. “Inhibi-
`tion of Squalene Synthetase by Famesvl Pyrophosphate
`Analogues", 1977, 20, 243-249.
`Corey and Volante, J. Am. Chem. Soc, “Application of
`Unreactive Analogs of Ter-penoid Pyrophosphates to
`Studies of Multistep Biosynthesis, Demonstration that
`‘Presqualeue Pyrophosphate’ is an Essential Intermedi-
`ate on the Path to Squalene", 1976, 98, l29l~3.
`Sandifier, R. M. et al.,
`JE Am. Chem. Soc. 1982, 104,
`7376-8, “Squalene Synthetase, Inhibition by an Ammo-
`nium Analogue of a Carbocationic Intermeidate in the
`Conversion of Presqualene Pyrophosphate to Squal-
`eucli-
`Bertolino, A., et al., Bfocfiim Biuphys. Acza., 1978, 530,
`l7~23, “Polyisoprenoid Amphiphilic Compounds as
`Inhibitors of Squalene Synthesis and Other Microsomal
`Enzymes“.
`Poulter. C. D. et al., J. Org. Chem, 1986, 51, 4768-4779,
`“Phosphorylation of Isoprenoid Alcohols”.
`Poulter, C. D., et al., .£A.C.S., 1987, 109, 5542, “Meth-
`
`Ioflfi
`
`PENN EX. 2197
`
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`1
`
`4,924,024
`
`2
`TABLE A-continued
`
`0 El) 0 if
`Y
`2
`\|4\/\|4\/l\|//k/ ‘-~P/
`‘--P___0_
`x
`,I,_
`4,-
`2.
`
`No.
`
`X
`
`Y
`
`5
`
`10
`
`IS
`
`20
`
`25
`
`30
`
`45
`
`30
`
`S5
`
`PI-IOSPHORUS-CONTAINING SQUALENE
`SYNTI-[ETASE INHIBITORS, NEW
`INTERMLEDIATES AND METHOD
`
`This is a division of application Ser. No. 141,744, filed
`Jan. 11, 1988, now US. Pat. No. 4,871,721.
`FIELD OF THE INVENTION
`
`The present invention relates to new phosphorus-
`containing compounds which inhibit
`the activity of
`squalene synthetase and thus are useful
`in inhibiting
`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
`forrn) (NADPH) to form squalene (Poulter, C. D.; Rill-
`ing, H. C., in “Biosynthesis of lsoprenoid Compounds”,
`Vol. I, Chapter 8, pp. 413441, J. Wiley and Sons, 1981
`and references therein). This enzyme is the first commit-
`ted step of the de novo cholesterol biosynthetic path-
`way. The selective inhibition of this step should allow
`the essential pathways to isopentenyl IRNA, ubiqui-
`none, 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. Ami Sci‘. USA. 1919, 16, 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
`treatment and prevention of hypercholesterolemia and
`atheroschlerosis.
`One approach to inhibitors of squalene synthetase is
`to design 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 chemical and enzymatic lability
`towards allylic C-O cleavage, as well as their suscepti-
`bility to metabolism by phosphatases.
`P. Ortiz de Montellano et al in J. Med‘. Chem, 1977,
`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
`synthetase enzyme. These substances retain the unstable
`allylic pyrophosphate moiety of FPP.
`TABLE A
`
`
`Z
`
`Y
`
`' o
`
`o
`
` /oklf/0\g_o_I
`
`X
`0..
`O._
`
`No.
`X
`Y
`Z .
`I
`CH3
`CH3
`H
`2
`H
`H
`H
`3
`C2H5
`H.
`H
`4
`I
`H.
`H
`S
`H
`I
`H
`
`IS
`
`Cl-13
`
`H
`
`SCH}
`
`J’. Am. Chem. Soc. 1976, 93.
`Corey and Volante,
`1291-3, have prepared FPP analog A and presqualene
`pyrophosphate (PSQ-PP) analog B as inhibitors of
`squalene biosynthesis. (Presqualene pyrophosphate is an
`intermediate in the conversion of FPP to squalene).
`These inhibitors possess methylene groups in place of
`the allylic oxygen moiety of FPP and PSQ-PP, but still
`retain the chemically and enzyrnatically unstable pyro-
`phosphate linkage.
`
`O
`X [I
`-~tP/
`I
`0*
`
`CI
`
`0
`ll
`-~..P_O_
`J
`o-
`
`,
`
`X = CH}:
`B
`PSQ—PP K = O
`
`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 synthetase.
`
`Altman and co-workers, Bertolino, A, et al., Biochim.
`Brbphyr. Acts. 1978, 530, 17-23, reported that farnesyl
`amine and related derivatives D inhibit squalene synthe-
`taste, but provide evidence that this inhibition is non-
`specific and probably related to membrane disruption.
`
` NH—R D
`R = H. CH2C‘II'_;_Ol-I, CI-I1CH30Cl-I3
`
`DESCRIPTION OF THE INVENTION
`
`In accordance with the present invention, there is
`provided phosphonis-containing compounds which
`inhibit the enzyme squalene synthetase and thus are
`useful as hypocholesterolemic agents and have the fol-
`lowing structure
`
`2uf16
`
`PENN EX. 2197
`
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`4,924,024
`
`15
`
`20
`
`25
`
`30
`
`if
`‘P2
`if
`en,-—<b=cH—cn;—CH2-—p=cH—Q—z—-1=—tp—1r-—oR
`CH3
`CH3
`R3 on”
`on’
`
`I
`
`wherein
`Q is
`
`-ii-(CH2):-?=cH%
`CH3
`
`or a bond;
`Z is —[CHg),,-- or —(CH2),,—CH=Cl-I—(CH2),,,—,
`w11ereinnislto5;pis0,lor2;misO,lor2;
`R, R‘ and R1“ may be the same or different and are H,
`lower alkyl or a metal ion; and
`R3 and R3 may be the same or different and are H or
`halogen.
`Hereinafter the moiety
`
`CH3—C=CH—Cl'l1—CH}_—C=CH—
`
`IC
`
`H3
`
`CH3
`
`will be expressed as “X” in the structural formulae set
`out below.
`
`Thus, the following types of compounds are included
`within the scope of the present invention.
`
`'0' $2 1'};
`X-Q-CH2j7(|',‘=CH-(-CH;-)-,;P—C—l]’—0R
`CH3
`1:3 0111"
`onl
`
`0 R2 0
`ll
`1
`II
`X-(-CH1-);P—C-ll’-OR
`R3 ow
`on!
`
`lol
`llgl
`(“J
`X-(-CH2-)7$=CH-(-CH2-)_;CH=CH-(-CH2?-,,,P—fi3—l;’—OR
`CH3
`R3 01:10
`on‘
`
`W IRE (II)
`x-{-CH2-),;cH=cH-(-CH2-),-,,p—c-P-03
`l
`R3 (i_)R|a
`03.1
`
`IA.
`
`35
`
`IB.
`
`45
`
`IQ
`
`ID.
`
`SS
`
`The term “low alkyl” or "alkyl” as employed herein
`alone or as part of another group includes both straight
`and branched chain hydrocarbons, containing 1 to 12
`carbons in the normal chain, preferably 1 to ‘l carbons,
`such as methyl, ethyl, propyl. isopropyl, butyl, t-butyl,
`isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimetl1yl-
`pentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, un-
`decyl, dodecyl,
`the various branched chain isomers
`thereof, and the like. The lower alkyl or alkyl group
`may be substituted with a substituent including a halo- 65
`substituent, such as F, Br, C1 or I or CF3, an alkoxy
`substituent, an aryl substituent, an alkyl-aryl substituent,
`a haloaryl substituent, at cycloalkyl substituent, an alkyl-
`
`60
`
`4
`cycloalkyl substituent, hyclroxy, and alkylamino substit-
`uent,
`an
`alkanoylamjno
`substituent,
`an
`arylcar-
`bonylamino substituent,
`at nitro substituent, a cyano
`substituent, a thio substitueut or an alkylthio substituent.
`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 lower alkoxy groups,
`I or 2 hydroxy
`groups, 1 or 2 alkylarnino groups, 1 or 2 alkanoylarnino
`groups, 1 or 2 arylcarbonylamino groups,
`I or 2 amino
`groups, 1 or 2 nitro groups, 1 or 2 cyano groups, 1 or 2
`thiol groups, and/or 1 or 2 alkylthio groups.
`The term “aryl” or “Ar” as employed herein refers to
`rnonocyclic or bicyclic aromatic groups containing
`from 6 to 10 carbons in the ring portion, such as phenyl,
`naphthyl. substituted phenyl or substituted naphthyl
`wherein the substituent on either the phenyl or naph-
`tliyl may be 1, 2 or 3 lower alkyl groups, halogens (Cl,
`Br or F), 1, 2 or 3 lower alkoxy groups, 1, 2 or 3 hy-
`droxy groups, 1, 2 or 3 phenyl groups, 1. 2 or 3 al-
`kanoyloxy group, 1, 2 or 3 benzoyloxy groups, 1, 2 or 3
`haloalkyl groups, 1. 2 or 3 halophenyl groups, 1, 2 or 3
`allyl groups. 1, 2 or 3 cycloalkylalltyl groups, 1, 2 or 3
`adamantylalkyl groups, 1, 2 or 3 alkylamino groups, 1, 2
`or 3 alkanoylamino groups, 1. 2 or 3 arylcarbonylamino
`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 alkylthio groups with the aryl group preferably
`containing 3 substituents.
`The term “aralkyl", “aryl—alkyl" or “aryl-lower al-
`kyl" as used herein alone or as part of another group
`refers to lower alkyl groups as discussed above having
`an aryl substituent, such as benzyl.
`The term “lower alkoxy", “alkoxy“, or “aryloxy" or
`“araIkoxy" as employed herein alone or as part of an-
`other group includes any of the above lower alkyl,
`alkyl, aralkyl or aryl groups linked to an oxygen atom.
`The term “lower alkylthio”, “alkyltliio”. “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
`alkylamino”,
`“allcylan-lino”,
`“arylan1ino", “arylalkylarnino" 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 “alkanoyl” 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 CF3, 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
`
`2
`
`if
`1|‘
`|C|)
`X—Q-Z-'P—C—P-OR
`Ilia
`,|3R:a
`
`OR‘
`
`.
`
`E
`
`3 uf16-
`
`PENN Ex. 2197
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`wherein Q is
`
`5
`
`4,924,024
`
`6
`
`-CH2-CH2-¢|:=CH-.
`CH3
`
`.
`
`if
`x-Q-z—Ha1 + P(Oa1kyl}3 ex—Q—z-1|=—oa;ky1
`Oalkyl
`V
`VI
`VII
`
`5
`
`Z is —CHgCI-12- or —CH=CH—‘ R3 and R3 are each
`’
`‘
`H or each F; R, R1 and R10’ are OH or metal was.
`The compounds of formula I of the invention may be 10
`prepared according to the following reaction sequence
`and description thereof.
`A. Preparation of Compounds of invention
`
`15
`
`(wherein Ha] '5
`a halogen such
`as Cl, Br 01'!)
`
`vn 335*“ “"31
`
`‘“
`
`ii?
`X—Q—Z—P—0H
`(3.)film
`I)
`
`Clxalyl Chloride
`if
`.
`.
`1
`.
`IS 2 to 5 or Z15 —(cH2.
`X-Q-Z-1|’-OH2 (2) Where z1s(cHg),, and 11
`OR‘
`]p—CH=CI-I——-(CH2),,.— and p is D to 2 and in is 2
`2
`(where 114 is alkyl)
`11
`
`(Acid c1 Formation)
`
`D
`
`R2 0
`I
`ll
`Li$eC—P—0alkyl
`£3 t'.|)a.l|tyl
`1|“;
`
`.5
`
`15"
`x_Q_z_l|}_C1 +
`on‘
`
`111
`
`(Phosp11iJ1ylvPlIaspllonalc
`' Formation)
`
`4'
`
`X—Q—(CH;:_),,_ I-Ha]
`VA
`0‘
`25 X-Q-{CH;J_,—CH=CH—(CH2)m_.1—Hal
`VB
`(wherein He] Is a halogen such as Br or I)
`O
`1|
`I..1€99cHg—P—oa1ky1—)
`IOalkyl
`1113
`
`30
`
`_
`(1) Dealkylation
`fl)
`*1‘:
`if
`x_Q_z_[>_(13_1|:..0aJky1 > 35
`OR
`4&3 Oalkvl
`
`]1:.'_
`
`40
`
`fin
`(I?
`x—o—z—'1|»—oa1ky1—-+x—Q—z—fi*—oaIky|
`Oalkyi
`OH
`VIIR
`TI
`
`1'8 (fl;
`if
`X_Q_Z__E|,__(|:_li,_0M ,a;c_'1d%
`cm R3 01»:
`
`(3) Where Z is (CH2)p—CH=CH—(CH;),,.—, p is 0 to
`2 and In is 0
`
`(where M is a metal ion)
`[G1
`
`45 xHQ—(CH2)P_CHO 4'
`VIII
`
`0 113 0
`H
`I
`II
`x—Q— 2-9-—c—p—o1«1
`I
`I
`I
`on R3 OH
`IH.
`
`so
`
`o 0a1k)~'1H°mET'EmF“°f's
`aikyio 0
`11/
`m°‘““?*".'°“
`\11
`P—CH1—P
`
`_...:s%.0fWith
`/
`\
`Ix
`
`Oalkyl
`
`a]kylD
`
`t
`A 1 A1
`Iii“)
`
`t’
`erna we
`
`P
`
`t"
`repara Ion 0
`
`fCo
`
`d
`mpoun s o
`
`I
`
`f
`
`nven
`
`-
`
`55
`
`(1) Dcalkylalion
`
`(2) Acid Hydrolysis
`
`”
`IF‘
`IH‘
`_
`_
`_
`B. Preparation of Starting Materials
`to 5 or Z is
`1
`(1) Where Z is (CH2), and I1
`is
`(CH2)P--CH=CH—(CI-I2),,,— and p is 0 to 2 and in
`is 1 or 2
`
`60
`
`65
`
`.
`(II)
`X_Q_(CH2),__CH=CH_l;_oa1kyl%
`Oalkyl
`X
`
`tifi}
`X—Q-Z-—l]3—0A1kyl
`OH
`1;
`
`As seen in reaction sequence “A", compounds of
`Formula I may be prepared by treating monoacid II
`
`4 of 16
`
`PENN EX. 2197
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`4,924,024
`
`8
`As seen in reaction scheme B(l), where Z is (CH2),,
`and n is 1 to 5 or Z is —(CHg)p—CH:CH—(CH;)m—
`and p is 0, l or 2 and m is 1 or 2, starting material Il may
`be prepared by treating halide V
`
`X—Q—Z—-1-Ia]
`
`V
`
`VI
`
`in an aromatic solvent such as benzene or toluene. pref-
`erably containing dimethylformamidc, or other appro-
`priate inert organic solvent, with oxalyl chloride, and
`then evaporating the reaction mixture to give acid chlo-
`ride III
`
`10
`
`with trialkyl phosphite VI
`
`P(Oalkyl)3
`
`[I]
`
`15
`
`under an 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
`
`if
`X—Q—-Z-P—C1
`
`Io
`
`n‘
`
`ii
`X—Q—Z—£|’--OH
`0114
`(wherein R‘ is an alkyl group)
`
`‘in’
`K—Q"'Z—li“—Oalkyl.
`Oalkyl
`
`VII
`
`25
`
`The reaction of phosphite VI and halide V is carried
`out employing a molar ratio of VI:V of within the range
`of from about 2:1 to about 50:].
`
`To a stirred solution of an optionally substituted dial-
`kyl methyl phosphonate
`
`20
`
`R2 0
`I
`ll
`H—(|J--Ii-‘—0alltyl
`R3 Oalkyl
`
`um‘
`
`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 n-butyl lithium or lithium diisopropylaniicle in a hex-
`‘ane or other inert organic solvent under an inert atmo-
`sphere such as argon to form the lithium salt IIIA
`
`Phosphonic ester VII is subjected to basic hydrolysis
`such as by treatment with alkali metal hydroxide such as
`aqueous KOH or Na0H. optionally in the presence of
`an alcohol such as methanol, ethanol or isopropanol
`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
`material ll, 2. is (CH3), and n is 2 to 5 or Z is -{—CH2-
`)9-—~CH=CH—(Cl-I2-§—,,, and p is 0 to 2 and m is 2, Il
`may be prepared by treating halide VA or VB with the
`salt IIIB such as
`
`if
`Li9eCHz—li'-'0a1ky1
`Oalkyi
`
`IIIB
`
`in the presence of an inert organic solvent such as tetra-
`hydrcfuran 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 1 to about 24
`hours, to form the phosphonic ester VIIA
`
`if
`X—Q--Z—li’—Oalky1
`Oalkyl
`
`VIIA
`
`which is subjected to basic hydrolysis as described
`above for scheme B{1) to form phosphinic acid starting
`material 11.
`
`65
`
`The reaction of VA or VB and lllB is carried out
`employing a molar ratio of VA or VB:IllB of about 1:1.
`The salt IIIB is formed by treating phosphonate llIB'
`
`R2 0
`I
`ll
`Li&9e¢|:—r;—oaJky1
`R3 Oalkyl
`
`“IA
`
`35
`
`The lithium salt IIIA is maintained at a reduced tem-
`perature as described above and acid chloride III in an
`inert organic solvent such as tetrahydrofuran or ethyl
`ether is added to form the phosphinyl-phosphonate IF.
`
`R2 0
`0
`1
`ll
`ll
`x—Q—z—i|>—ti:—1|=—oa1ky1.
`on‘ R3 Oalkyl
`
`45
`
`IF
`
`50
`
`The lithium salt IIIA will be employed in a molar ratio
`to acid chloride III of within the range of from about
`13:1 to about 25:1 and preferably from about 2.0:l to
`about 2.4:l. Ester IF, in an inert organic solvent such as
`methylene chloride, may then be subjected to dealkyla-
`tion by ‘treating with brorootrimethylsilane or iodo-
`trimethylsilane in the presence of 2,4,6-collidine or bis(-
`trin'1ethyl)silyltrifluoroacetamide and then treating with
`a strong inorganic base such as aqueous NaOH, KOH,
`LiOH or Mg{OH)g, optionally in the presence of an
`alcohol such as methyl alcohol, to form the salt IG
`which may be separated out by chromatography. Salt
`IG may be treated with a strong acid such as HCl to
`form acid IH.
`
`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 Il may be prepared as follows.
`
`5ofl6
`
`PENN EX. 2197 '
`
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`4.924.024
`
`10
`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 ll0:357, 1985).
`Preparation of Rat Liver Microsomes:
`Livers are dissected from 2 or 3 decapitated Sprague
`Dawley rats and are quickly transferred to ice cold
`buffer (potassium phosphate, 0.05 M. (pH 7.4); MgCl;-,
`0.004 M; EDTA,-0.001 M; and 2-mercaptoethanol 0.01
`M) and rinsed thoroughly. The livers are minced in cold
`buifer (2.0 1111/S) and homogenized using a Potter-
`Elvejhem homogenizer. 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 l5.000>< g For 15 minutes (4°
`). Again the supernatant is filtered through 2 layers of
`cheese cloth, and centrifuged a 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 bufier
`equivalent to 1/5 the volume of the original homoge-
`nate, and homogenized in a ground glass homogenizer.
`Aliquotted rnicrosomes are frozen at -80” C., and re-
`tain activity for at least two months.
`Enzyme Assay:
`Reaction Mixtures are prepared in 50 in] 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:
`
`
`H18‘
`
`0I
`
`I
`H--CH;-1|’-Oalkyl
`Oalkyl
`
`in an inert organic solvent such as tetrahydrofnran 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
`IBCZIIIS.
`
`I0
`
`As seen in reaction scheme I-](3), where in starting
`material IL 2 is —(CHg)_,—CI-I=CI-I—-(CH2)m— and p
`is 0 to 2 and m is 0, II may be prepared by employing a
`Horner-Emmons modification of the Witti g reaction by
`treating aldehyde VIII
`
`X—Q—{CH2)p—C‘I-IO
`
`20
`
`V111
`
`25
`
`3'0
`
`35
`
`40
`
`45
`
`50
`
`SS
`
`with tetraalkyl methylenebisphosphonate IX in the
`presence of sodium hydride and an inert organic solvent
`such as tetrahydrofuran. toluene or ethyl ether at a
`temperature within the ra.nge of from about -20‘ to
`about 25° C. for a period of from about 1 to about 24
`hours, to form phosphonic ester X
`
`ol
`
`l
`x—Q—(cH2)p~CH=cn- ll’-Oallryl
`Oalkyl
`
`(a novel compound in accordance with the present
`invention) which is subjected to basic hydrolysis as
`described in scheme EU) to form phosphonic acid II.
`The reaction of aldehyde VIII and phosphonate IX is
`carried out employing a molar ratio of VIII:[X of
`within the range of from about 1:1 to about 1:2.
`The aldehyde starting material VIII where Q is a
`bond and p is O, that is geranial, is a known compound.
`Where Q is
`
`-(C-‘H2)2—$‘—"CH—
`CH3
`
`and p is O is farnesal, 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 geraniol or farnesol
`employing conventional chemical synthesis.
`The novel intermediates in accordance with the in-
`vention may be defined by the following formula:
`
`(Iii)
`CH3—(|3=CH—CHg-CHg—C!".=CI-I—Q—Z— il“-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 l:Iypercholes-
`terolemia and atherosclerosis. Inhibition of squalene
`synthetase may be measured by the following proce-
`dure.
`
`65
`
`0.36 1111
`
`1. Potassium phosphate buffer
`0.215 M. pH 1.4)
`2. KIF (55 mild)
`3.
`'NAD'PH (5.0 111M, freshly prepared)
`4. H20 [or H10 + test compound)
`5. MgCi2 (27.5 mM)
`6. Microsomal Enzyme (0.48 mg
`microsomai protein in homogeni-
`ration buffer) (l5 pl prep.
`#13/86)
`1.3 ml
`
`
`0.36 ml
`0.36 ml
`0.16 ml
`0.36 ml
`0.20 ml
`
`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 initiated by adding 0.2 ml of
`farnesyl pyrophosphate (21.9 nlfvi) prepared in I-I20.
`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. Docosane (5
`nmoles in hexane) is added 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 mi scintillation vials and reduced in volume to =1.0
`ml under a stream of N2. The sample is then transferred
`to acid-washed, conical bottom, glass (1.0 ml) microvi-
`als, and brought to dryness under N3. The residue is
`resuspended in 50 pl hexane (spectral grade), and these
`samples are spun at l000 rpm at room temperature for
`10 minutes. Following centrifugation approximately 40
`,u.l of supernatant is transferred to 100 l.Ll acid-washed
`microvials with septa/crimp-top caps (compatible with
`the Hewlett-Packard GC auto injector).
`Gas Chromatography:
`
`6uf16
`
`PENN EX. 2197
`
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`4,924,024
`
`11
`Two p.L of each sample is injected onto a fused silica
`megabore DB-l7 column (15 MXO.S25 mm) (J&W
`Scientific) using a splitless mode of injection. Gas flow
`rates are listed below:
`
`
`Make up gas (helium)
`20 mlfmin.
`Air
`400 ml/rnin.
`Hydrogen
`30 ml/min.
`Carrier (helium)
`is mlfmin.
`septum purge vent
`5 ml./min.
`(Septum purge off 0.00
`min, on at 0.5 min.)
`
`The injector temperature is 200° C., and the FID
`detector temperature is set at 270° C. Oven temperature
`is programmed through a two ramp sequence as fol-
`lows:
`-
`Oven:
`Initial temperature 180“ C., initial time 10 minutes
`Ramp one: 20“ C./minute
`Second temperature 250“ C., second time 10 minutes
`Ramp two: 20° C./minute
`Third temperature 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 obtain.i.ng 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.
`
`20
`
`25
`
`30
`
`Squslene (nmolesfreaction mixture) =
`
`35
`
`(X)
`
`5.0 {nmoles docasane internal standard)
`S unlene Peak Area
`Docasane Peak Area
`‘RR -1 Response Ratio {Docasaneisqunlenel
`‘RR = 0.56
`
`X RR‘
`
`12
`sium stearate (15 mg). The mixture is passed through a
`60 mesh sieve and packed into a No.
`l 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. Purificatiou of final targets was often achieved
`by chromatography on Cl-IP20? gel (referred to herein
`as HP-20), a highly porous styrene-divinyl benzene
`copolyrner available from Mitsubishi Chemical Indus-
`tries. 31P NMR spectra were accumulated in the ‘H
`decoupled mode, employing 85% H3PO4 (3-=0 ppm) as
`the external reference. For 19F NMR spectra, CF3CCl3
`(5=82.6 ppm) was employed as an internal reference.
`EXAMPLE 1
`
`(EB)-[[Hydroxy(4,8,12-trimethyl-3,7,1 l-trideca-
`trienyl)phosphir1yl]methyllphosphonic acid,
`tripotassium salt
`
`A. (E,E)-3,7,1 l-Trimethyl-2,6, 10-dodecatrienyl
`bromide
`
`A solution of 1.00 g (4.5 mmol) of E,E-famesol (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 pl. (2.05 mmol,
`0.45 eq.) of P131‘; 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 H10, 5 ml of saturated Nal-[C03, and 5 ml
`of brine, dried over Na2S0.1. and evaporated to give 1.26
`(93%) of crude bromide as a clear oil. TLC Silica (2:8
`ethyl acetate:Hexane) Rf=0.69 (decomposes).
`‘H NMR (CDCI3), 85.52 (t, 1H, J=8.5 Hz). 5.08 (in,
`2H), 4.01 (CI, 2H), 1.9-2.2 (m, 3H),
`l.'.r'3 (s, 3H), 1.63 (5,
`31-1"), 1.60 (5, 61-1) ppm.
`
`B.
`
`Compounds Testing:
`Compounds are dissolved in H10 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 com-
`postion can be formulated employing conventional
`solid or liquid vehicles or diluents and pharmaceutical
`additives of a type appropriate to the mode of desired
`administration. Tlie 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 injectable
`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 I-4
`times per day.
`A typical capsule for oral administration contains‘
`active ingredient (250 mg), lactose ("I5 mg) and magne-
`
`45
`
`50
`
`55
`
`65
`
`(E,I-3)-(-1,8,12-Trimethyl-3,7,1 1-tridecatrienyl)phos-
`phonic acid. dimethyl ester
`
`To a stirred solution of 3.06 g (24.64 mmol) of di-
`methyl rnethylphosphonate in 50 ml of THF under
`argon was added 14.6 ml (23.47 mmol) of 1.60 M 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 excess methanol.
`Most of the solvent was evaporated and the residue was
`dissolved in ethyl acetate, washed with water and brine,
`dried (MgS04) 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.
`TLC Silica gel (Et0Ac) Rf=0.2l
`IR(CCl4) 2967, 2952, 2918. 2852, 1449, 1332, 1250,
`1223, 1185, 1065, 1039, 831 cm-1.
`‘H NMR (CDCI3), 86 5.10 (in, 3H), 3.73 (d, 6H,
`J: 11 Hz), 2.28 (m, 2H), 2.03 (in, 8H), 1.77 (m, 2H), 1.68
`(5, 31-1), 1.62 (s, 3H), 1.60 (s, 6H) ppm.
`
`7uf16
`
`PENN EX. 2197
`
`CFAD V. UPENN
`lPR20l5-01836
`
`
`
`13
`+ions]
`(C1-CI-141930,
`Spec
`Mass
`(m+C3H5), 357 (M4-C21-I5), 329 (M+I-I).
`C.
`
`4,924,024
`
`m/e
`
`369
`
`14
`+ions) m/e
`(CI-C1-I4/N10,
`Spec
`Mass
`(M+C3Hs). 331 (M+C-.-I-Is). 353 (M+H)-
`E.
`
`393
`
`5
`
`10
`
`(E,E)-(4,8,12-Trimethyl-3,7,1 1-tridecatt'ienyl)pl1os-
`phonic acid. monomethyl ester
`
`A stirred solution of 2.22 g (6.76 mmol) of Part B
`compound in 10 ml of 1:1 rnethanolwvater containing
`4.4 g (68.2 mmol) of potassium hydroxide was heated to
`65"—75" C. for 5.5 hours. The methanol was evaporated
`and the residue was stirred with dichloromethane/wa-
`ter and acidified by adding 10.0 g of solid KHS04. 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
`brine:water, dried (Mgsi) and evaporated to provide
`2.13 g (100%) of the title monoacid as a pale yellow oil.
`TLC Silica
`(6:3:l
`n-C3H-;OH:con NI-13:1-I20)
`Rf=0.62
`
`IR(CCl4) 2918, 2853, 1449, 1229, 1193, 1056, 987
`cm-
`1H NMR(CDC13), 5 12.22 (s, 1H), 5.10 (m, 3H), 3.72
`(d, 3H, .l=l0.6 Hz). 2.30 (m, 2H), 2.02 (111, 81-1’), 1.77 (m,
`2H), 1.68 (s, 31-1), 1.62 (s, 3H), 1.59 (s, 6H) ppm.
`Mass Spec (CI-H20, -1-ions) m/e 315 (M+H), 179.
`D.
`
`25
`
`(E,E)-[[Hydroxy(4,8, 12-trirnethyl)-3,7,1 l-trideca-
`trieny1)phosphiny1]methy1]phosphon.ic acid, trimethyl
`ester
`
`30
`
`35
`
`40
`
`To a stirred solution of 2.05 g (6.50 mmol) of Part C
`monoacid in 20 ml of benzene containing two drops of
`DMF was added 1.7 ml (19.50 rnmo1)ofoxaly1 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 rnethylphoaphonate in 30 ml ofT1-IF at -78" C.
`was added 8.7 1111 (14.0 mmol) of 1.6 M 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 THF 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% HC1. The ether solution was separated, washed
`with water,
`saturated NaHCO3 and brine, dried
`(MgSO4) and evaporated to give 1.57 g of crude title
`compound. The combined aqueous layers were back
`extracted with CH2Cl2 and the organic layer was
`washed with brine, dried (MgSO4) 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:CHzCIg gave 565.1
`mg (18%) of slightly impure title compound followed
`by 1.79 g (5 8%) 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 CH30H:CI-l2CL3) Rf=0.22.
`1R(CCl4) 2955, 2917, 2853, 1450, 1259, 1240, 11