Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 1 of 97
`
`Kowa Company, Ltd. et al. v. Aurobindo Pharma Limited et al.,
`Civil Action No. 14-CV-2497 (PAC) (and related cases)
`
`
`
`
`
`
`
`Exhibit 5 to Declaration of Thomas R. Burns,
`dated May 8, 2015, in support of Defendants’
`Joint Opening Claim Construction Brief
`
`
`
`Kowa Company, Ltd. et al. v. Aurobindo Pharma Limited et al.,
`Civil Action No. 14-CV-2497 (PAC) (and related cases)
`
`
`
`
`
`
`
`Exhibit 5 to Declaration of Thomas R. Burns,
`dated May 8, 2015, in support of Defendants’
`Joint Opening Claim Construction Brief
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 2 of 97
`Filed 05/08/15 Page 2 of 97
`
`
`
`
`PN 7458293
`
`||l1L|"'||:||'|_l
`
`lll'IllLllIl!|‘_..
`
`.JJJIIIL'IMLIII ILIIII I'Ilmlflll
`
`!
`
`'.
`
`" IIJ. .!
`
`||||| Illlll illlll
`
`1 UNITED STATES OFADIERICA
`
`mmnmmdmmflmemmewema
`
`‘
`
`UNITED STATES DEPARTMENT OF COMMERCE
`
`United States Patent and Trademark Office
`
` February 12, 2014
`
`
`
`RECORDS OF THIS OFFICE OF THE FILE WRAPPER AND CONTENTS
`
`OF:
`
`THIS IS TO CERTIFY THAT ANNEXED IS A TRUE COPY FROM THE
`
` APPLICATION NUMBER: 07/233, 752
`
`FILING DATE: August I 9, 1983
`
`
`
`By Authority of the
`
`
`
`
`
`MYLAN Pitav 011237
`
`
`
`
`
`
`
`#724,,
`
`
`
`Under Secretary of Commerce for Intellectual Property
`and Director of the United States Patent and Trademark Office
`
`M. TARVER
`
`Certifying Officer
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 3 of 97
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`
`
`
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`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 4 of 97
`Case 1:14-cy-02758-PAC Document 62-5 Filed 05/08/15 Page4of 97 ‘
`
`PATENT AP?LICATION SERIAL NO.
`
`I
`
`233752
`
`U.S. DEPARTMENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`FEE RECORD SHEET
`
`080
`
`OBXEEXBB
`
`233?52
`
`1 101
`
`$30.Q0 EK
`
`b
`
`PTO—1556
`(5/87)
`
`
`MYLAN(Pitav)011239
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 5 of 97
`Case 1:14-Cv-02758-PAC Document 62-5 Filed 05/08/15 Page 5 Of 97
`
`XI: NTS AND TRADEMARKS
`
`DOCS
`
`49—111—0
`
`V
`
`>
`
`THE COMMI
`
`WASHING
`SIR:
`
`
`
`OR FILING IS THE PATENT APPLICATION OF
`\
`THANSMITT
`INVENTOHIS) IHIRO FUJIKAWA ET AL
`
`FOR
`
`QUINOLINE TYPE MEVALONOLACTONES
`
`ENCLOSED ARE
`
`[3 .__,___ SHEETS OF DRAWINGS.
`
`[j A CERTIFIED COPY OF A___,.____.______ APPLICATION.
`
`
`|:] AN ASSIGNMENT OF THE INVENTION TO
`
`
`'xwg.» —
`
`D A VERIFIED STATEMENT TO ESTABLISH SMALL ENTITY STATUS UNDER 37 CFR 1.27.
`
`LIST OF INVENTORS' NAMES AND ADDRESSES, NOTICE OF PRIORITY
`WHITE ADVANCE SERIAL NUMBER POSTCARD -
`
`SMALL ENTITY
`
`OTHER THAN A
`SMALL ENTITY
`
`THE FILING FEE IS CALCULATED BELOW
`(COL. 1)
`_ (COL. 2)
`
`
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`
`
`
`
`
`I '
`
`-
`
`
`
`IF THE DIFFERENCE IN COL. 1 IS LESS THAN ZERO,
`ENTER “0" IN COL. 2
`
`
`
`
`
`
`
`1500:”
`E] PLEASE CHARGE MY DEPOSIT ACCOUNT NO.
`OF _______. . A DUPLICATE COPY OF THIS SHEET Is ENCLOSED.
`
`IN THE AMOUNT
`
`THE COMMISSIONER IS HEREBY'AUTHOHIZED TO CHARGE ANY ADDITIONAL FEES WHICH
`MAY BE REQUIRED, 'OR CREDIT ANY OVERPAYMENT TO ACCOUNT NO. ___15~_0_0_30_ .A
`DUPLICATE COPY OF THIS SHEET IS ENCLOSED.
`
`A CHECK IN THE AMOUNT OF __6_3_0_-_QQ__ TO COVER THE FILING FEE Is ENCLOSED.
`
`CRYSTAL SQUARE FIVE—SUITE 400
`1755 s. JEFFERSON DAVIS HIGHWAY
`ARLINGTON, VIRGINIA 22202
`7036215940
`
`OBLON, FISHER, SPIVAK,
`McCL
`AND AND MAIER, P.C.
`
`
`
`Norman F. 'Oblon
`ATTORNEY OF RECORD
`REGISTRATION NO.
`24 , 61 8
`
`SamUel H. _Blech
`Reglstratlon No:
`
`32,082
`
`
`MYLAN(Pitav)011240
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 6 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 6 of 97
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`
`
`
`
`.-
`
`/
`
`Wfl
`
`
`Our Ref.: NC-llS
`
`ur)
`
`_,_
`
`
`QUINOLINE TYPE MEVALONOLACTONES
`M .
`The present invention relates to novel
`
`mevalonolactones having a quinoline ring, processes for
`
`their production, pharmaceutical compositions containing
`
`5
`
`them and their pharmaceutical uses particularly as
`
`anti-hyperlipidemic, hypolipoproteinemic and
`
`anti—atherosclerotic agents, and intermediates useful for
`
`their production and processes for the production of such
`
`intermediates.
`
`10
`
`Some fermentation metabolic products such as
`
`compactine, CS—Sl4, Mevinolin or semi—synthetic
`
`derivatives or fully synthetic derivatives thereof are
`
`known to be inhibitors against HMG—CoA reductase which is
`
`a rate limiting enzyme for cholesterol biosynthesis.
`
`(A.
`
`15
`
`Endo J. Med Chem., 28(4) 401 (1985))
`CS+514 and Mevinolin have been clinically proved to be
`
`potentially useful anti-hyperlipoproteinemic agents, and
`
`they are considered to be effective for curing or
`
`preventing diseases of coronary artery sclerosis or
`
`20
`
`atherosclerosis.
`
`(IXth Int. Symp. Drugs Affect. Lipid
`
`
`MYLAN(Pitav)011241
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 7 of 97
`Case 1:14-CV-02758-PAC Document 62-5 Filed 05/08/15 Page 7 Of 97
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`
`
`_ 2 _
`
`' Metab., 1986, p30, p31, p66)
`
`However, with respect to fully synthetic derivatives,
`
`particularly hetero aromatic derivatives of inhibitors
`
`against HMG—CoA reductase,
`
`limited information is
`
`5
`
`. disclosed in the following literatures:
`
`WPI ACC NO. 84—158675, 86-028274, 86—098816,
`
`86-332070, 87-124519, 87-220987, 88-07781, 88—008460,
`
`88-091798 and 88-112505.
`
`The present inventors have found that mevalonolactone
`
`lO derivatives having a quinoline ring,
`
`the corresponding
`
`dihydroxy carboxylic acids and salts and esters thereof
`
`have high.inhibitory activities against cholesterol
`
`biosynthesis wherein HMG-CoA reductase acts as a rate
`
`limiting enzyme.
`
`The present invention has been
`
`15
`
`accomplished on the basis of this discovery.
`
`The novel mevalonolactone derivatives of the present
`
`invention are represented by the following formula I:
`
`3
`R \
`
`4
`
`R
`
`20
`
`R6
`
`Hz
`
`y-z
`
`(1)
`
`RI/
`
`N
`
`R5
`
`3
`2
`» wherein R1, R , R , R
`
`4’
`
`6
`
`and R
`
`are independently hydrogen,
`
`alkyl, C3_6 cycloalkyl, Cl_3 alkoxy, n-butoxy,
`C
`1—6
`i—butoxy, sec—butoxy, R7R8N— (wherein R7 and R8 are
`
`25
`
`independently hydrogen or Cl_3 alkyl),
`
`trifluoromethyl,
`
`trifluoromethoxy, difluoromethoxy, fluoro, chloro, bromo,
`
`
`MYLAN(Pitav)011242
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 8 of 97
`Page 8 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15
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`_3_.
`
`_ phenyl, phenoxy, benzyloxy, hydroxy,
`
`trimethylsilyloxy,
`
`diphenyl—t—butylsilyloxy, hydroxymethyl or —O(CH
`19
`
`2) £012
`is hydrogen or Cl_3 alkyl, and l is l,
`2 or
`
`19
`
`(wherein R
`
`3); or when located at the ortho position to each other,
`flfl/Mfl/f
`R1 and R2, or R3 and R4 togetheabform —CH=CH-CH=CH—; or
`when located
`the ortho position to each other, R1 and
`2
`fl
`/””4/%7i5
`l6
`”
`15
`16
`and R
`togetheaLform —OC(R
`)(R )0-
`(wherein R
`R
`independently hydrogen or Cl_3 alkyl); Y is —CH2—,
`-CH2CH2-,
`-CH=CH-,
`-CH2—CH=CH- or —CH=CH—CH2—; and Z is
`12
`'
`
`-Q-CH2WCH2—CO2R
`
`,
`
`are
`
`R11
`/
`
`m
`
`,\\<;?0
`
`l
`
`//0
`
`T
`
`R17
`
`I
`
`0
`
`R11
`
`//\\%/,0
`l
`‘r\V//0
`I
`
`T
`0>l/\f/\COZR‘2
`
`R18,
`0\
`
`/
`
`\7//
`or
`-C(ORl3)2— or -CH(OH)-; w is —C(O)-,
`(wherein Q is -C(O)—,
`—C(ORl3)2— or -C(Rll)(OH)-; R11 is hydrogen or Cl_3 alkyl;
`R
`is hydrogen or R
`(wherein Rl4 is physiologically
`
`12
`
`14
`
`hydrolyzable alkyl or M (wherein M is NH
`
`4/
`
`sodium,
`
`potassium, 1/2 calcium or a hydrate of lower alkylamine,
`
`di—lower alkylamine or tri—lower alkylamine));
`
`two R13
`
`are
`
`independently primary or secondary Cl_6 alkyl; or two R13
`together form —(CH2)2— or ~(CH2)3—; R17 and R18 are
`independently hydrOgen or Cl_3 alkyl; and R5 is
`
`hydrogen,'Cl_6 alkyl, C2__3 alkenyl, C3_6 cycloalkyl,
`9
`'
`"i
`_<§3{R (wherein R
`15 hydrogen, Cl_4 alkyl, Cl_3
`
`.
`
`9
`
`,
`
`,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN(Pitav)011243
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 9 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 9 of 97
`
`_5_
`
`ethyl, n-propyl,
`
`i~propyl, n—butyl,
`
`i—butyl, sec—butyl,
`
`t—butyl, n-pentyl and n—hexyl.
`
`C3-6
`
`cycloalkyl for R5 includes, for example,
`
`cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.-
`C2_3 alkenyl for R5 includes, for example, vinyl and
`
`5
`
`i—propenyl.
`
`Phenyl-(CH2)m- for R
`
`5
`
`.
`includes, for example, benzyl,
`
`B-phenylethyl and y—phenylpropyl.
`5
`
`Phenyl+(CH2)nCH(CH3)— for R
`
`includes, for example,
`
`10 a-phenylethyl and a—benzylethyl.
`8
`
`C
`
`1—3
`
`alkyl for R7 and R
`
`includes, for example,
`
`methyl, ethyl, n¥propyl and i-propyl.
`
`Further,
`
`these compoundsnwy have at least one or two
`
`asymmetric carbon atoms and may have at least two to four
`
`15 optical isomers.
`
`The compounds of the formula I_include
`
`all of these Optical
`
`isomers and all of the mixtures
`
`thereof.
`
`Among compounds having carboxylic acid moieties
`12
`
`falling outside the definition of -C02R
`
`of the
`
`20
`
`carboxylic acid moiety of substituent Z of the compounds
`
`of the present invention,
`
`those which undergo
`
`physiological hydrolysis, after intake,
`
`to produce the
`
`corresponding carboxylic acids (compounds wherein the
`—CO Rl2 moiety is —C02H) are equivalent to the compounds
`2
`
`25
`
`of the present invention.‘
`
`Now, preferred substituents of the compounds of the
`
`present invention will be described.
`
`
`MYLAN(Pitav)011244
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 10 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 10 of 97
`
`
`
`
`
`_ 6 _
`
`In the following preferred, more preferred still
`
`further perferred and most preferred examples,
`
`the
`
`numerals for the positions of the substituents indicate
`
`For example, N'
`the positions on the quinoline ring.
`shown by e.g. l' or 2"indicates the position of the
`
`5
`
`substituent on the phenyl substituted at the 4—position of'
`
`the quinoline ring (the carbon connected to the quinoline
`
`ring is designated as l').
`
`The meanings of the respective
`
`substituents are the same as the above—mentioned meanings.
`Preferred subStituents for R1, R2 and R6 are hydrogen,
`
`10
`
`fluoro, chloro, bromo, Cl_3 alkyl, Cl_3 alkoxy, C3_6
`cycloalkyl, dimethylamino, hydroxy, hydroxymethyl,
`
`hydroxyethyl,
`
`trifluoromethyl,
`
`trifluoromethoxy,
`
`difluoromethoxy, phenoxy and benzyloxy.
`
`15
`
`Further, when R6 is hydrogen, it is preferred that R1
`and R2 together form methylenedioxy.
`3
`
`and R4, when R
`
`4
`
`is
`
`As preferred examples for R
`3
`
`hydrogen, R-
`
`is hydrogen, 3'-fluoro, 3'—chloro, 3'—methyl,
`
`4'-methyl, 4'—chloro and 4’—fluoro.
`
`.20
`
`Other preferred combinations of R
`
`3 and R4 include
`
`3'-methyl-4'—chloro, 3',5'-dichloro, 3',5'—difluoro,
`
`3',5'—dimethyl and 3'-methyl-4'-fluoro.
`
`Preferred examples for R5 include primary and
`
`secondary Cl_6 alkyl and C3_6 cycloalkyl.
`
`25
`
`Preferred examples for Y include -CH2-CH2— and
`‘CH=CH—.
`
`Preferred examples for Z include
`
`MYLAN(Pitav)011245
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 11 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 11 of 97
`
`_.8_
`
`' 6,7—difluoro, 6,8edifluoro, 6,7—methylenedioxy,
`
`6,8—dichloro, 5,8-dimethyl, 6,8-dimethyl, 6,7-dimethoxy,
`
`6,7—diethoxy, 6,7-dibromo or 6,8—dibromo.
`l
`2
`6
`When R , R
`
`are not hydrogen,
`
`and R
`
`they together
`
`5
`
`represent 5,7—dimethoxy—8-hydroxy, 5,8-dichloro-6—hydroxy,
`
`6,7,8-trimethoxy, 6,7,8-trimethyl, 6,7,8—trichloro,
`
`5—fluoro—6,8—dibromo or 5—chloro—6,8—dibromo.
`3
`4
`and R , when R
`
`As more preferred examples for R
`
`3
`
`is
`
`hydrogen, R4 is hydrogen, 4'—methyl, 4'—chloro or
`3
`
`10 4'-fluoro. When both R
`
`and R4 are not hydrogen,
`
`they
`
`together represent 3',5'-dimethyl or 3'-methyl—4'—fluoro.
`5
`As more preferred examples for R ,
`
`the above-mentioned
`
`preferred examples of R
`
`5 may be mentioned.
`
`As preferred examples for Y, —CH2—CH2— and (E)——CH=CH—
`
`15 may be mentioned. As more preferred examples for Z,
`
`the
`
`above preferred examples for Z may be mentioned.
`
`Now, still further preferred substituents of the
`
`compounds of the present invention will be described.
`
`As
`
`examples for R1, R2 and R6, when both R2 and R6 are
`
`_20
`
`hydrogen, R1 is hydrogen, 6-methyl, 6—ethyl,
`6—trifluoromethyl, 6—hydroxy, 6—methoxy, 6—chloro,
`
`6—bromo, 6-n-butyl and 7—dimethylamino.
`
`when only R6 is hydrogen, Rl and-R2 represent
`6,8—dichloro, 5,8—dimethyl, 6,8—dimethyl, 6,7—dimethoxy,
`
`25 6,7-diethoxy, 6,7—dibromo, 6,8-dibromo, 6,7-difluoro and
`
`6,8—difluoro.->
`
`_As still further preferred examples for R3 and R4,
`
`
`MYLAN(Pitav)011246
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 12 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 12 of 97
`
`
`
`when R3 is hydrogen, R4 is hydrogen, 4'—chloro or
`
`4'—fluoro, or R3 and R4 together represent
`
`3'wmethyl-4f—fluoro.
`
`Still further preferred examples for R
`
`5
`
`include ethyl,
`
`5
`
`n-propyl,
`
`i-propyl and cyclopropyl.
`
`Still further preferred examples for Y include
`
`(E)--CH=CH~.
`
`As still further preferred examples foer, the
`
`above-mentioned preferred example for Z may be mentioned.
`
`10
`
`Now,
`
`the most preferred substituents for the compounds
`
`*
`
`I
`
`of the present invention will be described.
`
`As the most preferred examples for R1, R2 and R6, when
`both R2 and R6 are hydrogen, R1 is hydrogen, 6—methyl or
`
`6—chloro.
`
`.
`
`15
`
`-
`
`When only R
`
`6
`
`.
`is hydrogen, R
`
`l
`
`2
`
`and R
`
`together
`
`represent, for example, 6,7-dimethoxy.
`
`As the most preferred examples for R
`
`3 and R4, R3 is
`
`hydrogen and R4 is hydrogen, 4'-chloro or 4'—fluoro.
`
`The most preferred examples for R5 include i—propyl
`
`.20
`
`and cyclopropyl.
`
`The most preferred example for Y may be
`
`(E)-'CH=CH—.
`
`As the most preferred examples for Z,
`
`the
`
`above—mentioned preferred examples for Z may be mentioned.
`
`Now, particularly preferred specific compounds of the
`
`25 present invention will be presented.
`
`The following
`
`compounds
`
`(a)
`
`to (z) are shown in the form of carboxylic
`
`acids. However,
`
`the present invention include not only
`
`
`MYLAN(Pitav)011247
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 13 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 13 of 97
`
`
`
`
`
`_ lo _
`
`the compounds in the form of carboxylic acids but also the
`
`corresponding lactones formed by the condensation of the
`
`carboxylic acids with hydroxy at the 5-position, and
`
`sodium salts and loweh alkyl esters (such as methyl,
`
`5
`
`i—propyl and n—propyl esters) of the carboxylic
`ethyl,
`acids, which can be physiologically hydrolyaed to the
`
`carboxylic acids.
`
`(a)
`
`(E)—3,5-dihydroxy—7—[4'—(4"—fluorophenyl)-2'—
`
`(l"-methylethyl)-quinolin—3'—yl]—hept-6—enoic acid
`
`10
`
`(b)
`
`(E)—3,5-dihydroxy-7-[4'—(4"-fluorophenyl)-2'-
`
`(l"-methylethyl)—6'-chloro—quinolin-3'—yl]—hept—6—enoic
`acid
`I
`I
`(E)-3,5—dihydtoxy—7-[4'—(4"-fluorophenyl)—2'—
`
`(c)
`
`(1"-methylethyl)—6'—methyl-quinolin—3'-yl]~hept—6—enoic
`
`15
`
`acid
`
`I
`
`I
`
`(d)
`
`(E)-3,5-dihydroxy—7-[4'-(4"-fluorophenyl]—2'-
`
`(l"—methylethyl)-6',7'-dimethoxy—quinolin—3'-yl]—hept—6—
`
`enoic acid
`
`(e)
`
`(E)—3,5-dihydroxy—7—[4'-(4"-fluorophenyl)-2'—
`
`V20
`
`cyclopropyl-quinolin—B'—yl]-hept~6—enoic acid
`
`(f)
`
`(E)-3,5-dihydroxy-7—[4'-(4"-fluorophenyl)—2'—
`
`cyclopropyl—G'—chloro—quinolin—3'-yl]—hept-6—enoic acid
`(9)
`(E)—3,5-dihydroxy—7—[4‘-(4"—fluorophenyl)—2'—
`
`Cyclopropyl—6'—methyl-quinolin—3'-yl]-hept-6—enoic acid
`
`25
`
`(h)
`
`(E)-3,5-dihydroxyj7-[4‘—(4"-fluorophenyl)-2'-
`
`cyclopropyl—S',7'—dimethoxy—quinolin—3'—yl]—hept—6—enoic
`
`acid
`
`MYLAN(Pitav)011248
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 14 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 14 of 97
`
`_ll_.
`
`(i)
`
`(E)—3,5-dihydroxy-7-[4'—(4"-chlorophenyl)-2'—
`
`(l"-methylethyl)—quinolin—3'—yl]—hept-6—enoic acid
`
`(j)
`
`(E)—3,5-dihydroxy-7—[4'—(4"—chlorophenyl)—2'-
`
`(l"—methylethyl)—6'—chloro—quinolin—3'—yl]—hept—6—enoich
`
`5
`
`acid
`
`(k)
`
`(E)-3,5-dihydroxy—7—[4'—(4"-chlorophenyl)-2'-
`
`(l"-methylethyl)—6'—methyl—quinolin—3'-yl]-hept—65enoic
`
`acid
`
`(1)
`
`(E)—3,5-dihydroxy-7—[4'-(4"-chlorophenyl)-2'-
`
`lO
`
`(l"-methylethyl)—6',7'-dimethoxy—quinolin—3‘—yl]—hept—6—
`
`enoic acid
`
`(m)
`
`(E)—3,5-dihydroxy-7—[4'—(4"—chlorophenyl)-2'-
`
`cyclopropyl—quinolin-3'-yl]-hept-6—enoic acid
`
`(H)
`
`(E)-3,5-dihydroxy—7-[4'-(4"—chlorophenyl)-2'—
`
`15
`
`cyclopropyl-6'—chloro—quinolin—3'—yl]—hept—6—enoic acid
`
`(0)
`
`(E)—3,5—dihydroxy—7—[4'—(4"—chlorophenyl)—2'-
`
`cyclopropyl-6'—methyl—quinolin-3'-yl]-hept—6—enoic acid
`
`(p)
`
`(E)-3,5—dihydroxy-7-[4'—(4"—chlorophenyl)-2'-
`
`cyclopropyl—6'7'—dimethoxy-quinolin—3"yll-hept-6—enoic
`acid
`I
`
`20
`
`(q)
`
`(E)-3,5-dihydroxy—7—[4'—phenyl-2'-(l"—
`
`methylethyl)—quinolin—3'—yl]—hept—6—enoic acid
`
`(r)
`
`(E)-3,5-dihydroxy-7—[4'-phenyl—2'—(l"—
`
`methylethyl)—6'—chloro—quinolin—3'—yl]—hept—6—enoic acid
`
`25
`
`(s)
`
`(E)—3,5—dihydroxy—7—[4'—phenyl—2'—(l"—
`
`methylethyl)—6'—methyl—quinolin—3'—yl]—hept—6—enoic acid
`
`(t)
`
`(E)-3,5—dihydroxy-7-[4'-phenyl-2'-(15'-
`
`
`MYLAN(Pitav)011249
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 15 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 15 of 97
`
`-12..
`
`methylethyl)—6',7'—dimethoxy—quinolin-3'—yl]—hept—6—enoic
`
`acid
`
`(u)
`
`(E)-3,5-dihydroxy—7—[4’—phenyl-2'—cyclopropyl—
`
`quinolin-3'—yl]—hept-6-enoic acid
`(v)
`(E)-3,5—dihydroxy—7~[4'-phenyl—2'—cyclopropyl—6'L
`
`5
`
`chloro—quinolin—3'-yl]—hept-6—enoic acid
`
`(W)
`
`(E)-3,5—dihydroxy-7-[4'-phenyl—2'-cyclopropyl—6'—
`
`methyl-quinolin-3‘—yl]—hept—6—enoic acid
`
`(X)
`
`(E)—3,5—dihydroxy-7-[4'-phenyl-2'-cyclopropyl—
`
`lO 6',7'—dimethoxy—quinolin-3'—yl]—hept—6—enoic acid
`
`(y)
`
`(E)—3,5—dihydroxy—7—[4'-(4"—fluorophenyl)-2‘—
`
`‘(1"-methylethyl)—6'—methoxy—quinolin—3’—yl]—hept-6—enoio
`
`acid
`
`(2)
`
`(E)—3,5-dihydroxy~7—[4'—(4"-fluorophenyl)—2'-
`
`15
`
`cyclopropyl-6'—methoxy~quinolin—3'—yl]—hept—6—enoic acid
`
`The mevalonolactones of the formula I can be prepared
`
`by the following reaction scheme.
`
`The enal III can also
`
`be prepared by processes K, L and M.
`
`MYLAN(Pitav)011250
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 16 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 16 of 97
`
`..13 .,'
`
`R3
`
`R‘
`
`R3
`
`R‘
`
`2|
`
`002R
`
`R5
`
`O
`N
`
`VII
`
`A
`
`E
`
`RA
`
`1
`
`R
`
`R2
`
`'
`
`CH20II
`
`R5
`
`B
`
`E-
`
`O
`N
`
`VI
`
`R3
`
`R‘
`
`R3‘
`
`R“
`
`R5
`
`R5
`
`Rl
`
`R‘
`
`R2
`
`R'
`
`we
`C
`
`R6
`
`R2
`
`R‘
`
`.
`
`CHO
`
`R5
`
`O
`N
`
`V
`
`O
`N
`
`IV
`
`/
`
`\
`
`(\0“
`0"
`
`‘R5
`
`———-—————>
`D
`
`
`MYLAN(Pitav)011251
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 17 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 17 of 97
`
`_/'
`
`f- ‘
`
`_14_.
`
`
`
`
`0H
`
`(IOz'R'2
`
`
`MYLAN(Pitav)011252
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 18 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 18 of 97
`
`-15 _
`
`R‘
`
`R"
`
`
`MYLAN(Pitav)011253
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 19 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 19 of 97
`
`-16-
`
`R3
`
`R5
`
`R2
`
`CHO
`
`
`MYLAN(Pitav)011254
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 20 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 20 of 97
`
`-17-
`
`on
`
`R‘ b/cozn'z
`I
`
`o
`
`R5
`
`ON
`
`1—6
`
`
`
`com[2
`
`R“
`
`N
`
`9
`
`12.,
`
`R2
`RI
`
`
`MYLAN(Pitav)011255
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 21 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 21 of 97
`
`
`
`-20..
`
`ethanol at a temperature of from 10 to 25°C.
`
`The free
`
`acid hereby obtained may be converted to a salt with a
`
`suitable base.
`
`‘Step H is a step for forming a mevalonolactone by the
`
`5
`
`dehydration reaction of the free hydroxy acid 1—2.
`dehydration reaction can be conducted in benzene or
`
`The
`
`toluene under reflux while removing the resulting water or
`
`by adding a suitable dehydrating agent such as molecular
`
`Sieve.
`
`10
`
`Further,
`
`the dehydration reaction may be conducted in
`
`dry methylene chloride by using a lactcne—forming agent
`
`such as carbodiimide, preferably a water soluble
`
`carbodiimide such as
`
`N—cyclohexyl-N'-[2'-(methylmorpholinium)ethyllcarbodiimide
`l5 p-toluene sulfonate at a temperature of from 10 to 35°C,
`preferably from 20 to 25°C.
`
`Step J-represents a reaction for hydrogenating the
`
`double bond connecting the mevalonolactone moiety and the
`
`quinoline ring. This hydrogenation reaction can be
`
`20
`
`conducted by using a catalytic amount of palladium-carbon
`
`or rhodium—carbon in a solvent such as methanol, ethanol,
`
`tetrahydrofuran or acetonitrile at a temperature of from 0
`
`to 50°C, preferably from 10 to 25°C.
`
`Step K represents a reaction for the synthesis of an
`
`25
`
`a,B—unsaturated carboxylic acid ester, whereby a
`
`trans—form a,B-unsaturated carboxylic acid ester can be
`
`obtained by a so—called Horner—Wittig reaction by using an
`
`MYLAN(Pitav)011256
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 22 of 97
`Case 1:14-cv-02758—PAC Document 62-5 Filed 05/08/15 Page 22 of 97
`
`-2l_
`
`alkoxycarbonylmethyl phosphonate.
`
`The reaction is
`
`conducted by using sodium hydride or potassium t—butoxide
`
`as the base in dry tetrahydrofuran at a temperature of
`
`from -30 to 0°C, preferably from —20 to —150C.
`
`5
`
`Step L represents a reduction reaction of the
`
`a,B-unsaturated carboxylic acid ester to an allyl alcohol.
`
`This reduction reaction can be conducted by using various
`
`metal hydrides, preferably diisobutylaluminiumhydride,
`
`in
`
`a solvent such as dry tetrahydrofuran or toluene at a
`
`10
`
`temperature of from -lO to 100C, preferably from —10 to
`
`0°C.
`
`Step M represents an oxidation reaction of the allyl
`
`alcohol
`
`to an enal. This oxidation reaction can be
`
`conducted by using various oxidizing agents, particularly
`
`15 active manganese dioxide,
`
`in a solvent such as
`
`tetrahydrofuran, acetone, ethyl ether or ethyl acetate at
`
`a temperatrue of from 0 to 100°C, preferably from 15 to
`
`50°C.
`
`Step N represents a reaction for the synthesis of an
`
`20
`
`a,B—unsaturated ketone by the selective oxidation of the
`
`dihydroxy carboxylic acid ester. This reaction can be
`
`conducted by using activated manganese dioxide in a
`
`solvent such as ethyl ether,
`
`tetrahydrofuran, benzene or
`
`toluene at a temperature of from 20 to 809C, preferably
`
`25
`
`from 40 to 80°C.
`
`In addition to the compounds disclosed in Examples
`
`given hereinafter, compounds of the formulas 1—2 and 1-5
`
`
`MYLAN(Pitav)011257
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 23 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 23 of 97
`
`_22_
`
`given in Table 1 can be prepared by the process of the
`
`present invention.
`
`In Table l,
`
`1— means iso,
`
`sec— means
`
`secondary and c— means cycle. Likewise, Me means methyl,
`
`Et means ethyl, Pr means propyl, Bu means butyl, Pent
`
`5
`
`means pentyl, Hex means hexyl and Ph means phenyl.
`
`
`MYLAN(Pitav)011258
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 24 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 24 of 97
`
`Tablel
`
`_ 23 _
`
`R3
`
`R‘1
`
`R6
`
`I
`Rage/K»
`121%GCle
`
`OH
`
`0H
`
`COlez
`
`£2 ’3‘ Efiiifli)
`
`
`
`R“
`
`R"
`
`R“
`
`R‘
`
`R2
`
`R3
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`i-Pr
`
`i-Pr
`
`i—Pr
`
`i—Pr
`
`H
`
`H
`
`H
`
`H
`
`i—Pr
`
`-H
`
`i-Pr
`
`H
`
`6-0He
`
`6—0Me
`
`G-Br
`
`‘
`
`H»
`
`H
`
`H
`
`H
`
`4-F
`
`4—F
`
`6—Me
`
`8—Me
`
`4-F
`
`7—0Me
`
`8—0Me 4"F
`
`S-Br
`
`H
`
`2-F
`
`6,7
`
`H
`
`H
`i—Pr
`\3 H
`
`i-Pr
`
`4-PhCHz H
`
`i—Pr
`
`H
`
`H
`
`H
`
`64C£
`
`6-C2
`
`6-OCH2Ph
`
`H
`
`6-62
`
`6-H82N
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`4-F
`4-F
`
`4-Ph
`
`H
`H
`
`H
`
`4- F
`
`4— F
`
`4- F
`
`4- F
`
`4- F
`
`4— F
`
`4— F
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`c-Pr
`
`sec‘Bu
`
`1-Pr
`
`i-Bu
`
`C—Pent H
`
`c—Pent
`
`i—Pr
`
`H
`
`
`MYLAN(Pitav)011259
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 25 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15
`Page 25 of 97
`
`-24-
`
`R 3
`
`4'- F
`
`4'- F
`
`4-F
`
`4— F
`
`4-F
`
`4— F
`
`4- F
`
`4 - F
`
`4 - F
`
`4- F
`
`4-'F
`
`4- F
`
`4— F
`
`6—He
`
`6-i—Pr
`
`7-Me
`
`6-0He
`
`6eBr
`
`6—i-Pr
`
`6-02
`
`5-F
`
`6‘0Me
`
`6-Me
`
`6-02
`
`6-0Me
`
`7-0He
`
`H
`
`6-0Me
`
`7-0Me
`
`4-09
`
`6—0He
`
`7-0He
`
`H
`
`6-0He
`
`T-OMe
`
`4-02
`
`6—0He
`
`4-F
`T-OHe
`
`Z
`
`LEEDSEZBEE
`
`RS
`
`c-Pr
`
`i-Pr
`
`c-Pr
`
`c-Pr
`
`¢-Pr
`
`c-Pr
`
`c—Pr
`
`i—Pr
`
`8—Br
`
`i—Pr
`
`i-Pr
`
`8-0Me
`
`8-Me
`
`i—Pr
`
`8-Cfl
`
`c-Bu
`
`c-Hex
`
`i-Pr
`
`i—Pr
`
`c—Pr
`
`c—Pr
`
`c-Pr
`
`:ECGDZECGZE
`
`MYLAN(Pitav)011260
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 26 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 26 of 97
`
`-25-
`
`R‘
`
`R2
`
`. R3
`
`Rd
`
`Ra
`
`Ra
`
`S—Me
`
`B-Me
`
`S-Me
`
`6-Me
`
`5-02
`
`6-H
`
`6‘01;
`
`6-02
`
`6-02
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`4-C£
`
`H
`
`4—H
`
`4-1:
`
`H
`
`4-H
`
`H
`
`4-02
`
`4-F
`
`H
`
`4—6;;
`
`H
`
`4-sz
`
`4-F
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`i—Pr
`
`c—Pr
`
`c-Pr
`
`c-Pr‘
`
`1-Pr
`
`1-Pr
`
`c-Pr
`
`c-pr
`
`c-Pr
`
`1-Pr
`
`1—Pr
`
`c-Pr
`
`c-Pr
`
`C-Pr
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`MYLAN(Pitav)011261
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 27 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 27 of 97
`
`
`
`-26—
`
`Further, pharmaceutically acceptable salts such as
`
`potassium salts or esters such as ethyl esters or methyl
`
`esters of these compounds can be prepared in the same
`manner.
`
`5
`
`The compounds of the present invention exhibit high
`
`inhibitory activities against the cholesterol biosynthesis
`
`wherein HMG—CoA reductase acts as a rate limiting enzyme,
`
`as shown by the test results given hereinafter, and thus
`
`are capable of suppressing or reducing the amount of
`
`10
`
`the compounds
`cholesterol in blood as lipoprotein. uThus,
`of the present invention are useful as curing agents
`
`against hyperlipidemia, hyperlipoproteinemia and
`
`atheroscleosis.
`
`They may be formulated into various suitable
`
`15
`
`formulations depending upon the manner of the
`
`administration.
`
`The compounds of the present invention
`
`may be administered in the form of free acids or in the
`
`form of physiologically hydrolyzable and acceptable esters
`
`or lactones, or pharmaceutically acceptable salts.
`
`20
`
`The pharmaceutical composition of the present
`invention is preferably administered orally in the form of
`the compound of the present
`invention per se or in the
`
`form of powders, granules,
`
`tablets or capsules formulated
`
`by mixing the compound of the present inVention with a
`
`25
`
`suitable pharmaceutically acceptable carrier including a
`
`binder such as hydroxypropyl cellulose, syrup, gum arabic,
`
`gelatin, sorbitol,
`
`tragacanth gum, polyvinyl pyrrolidone
`
`MYLAN(Pitav)011262
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 28 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 28 of 97
`
`
`
`-27..
`
`or CMC—Ca, an excipient such as lactose, sugar, corn
`
`starch, calcium phosphate, sorbitol, glycine or crystal
`
`cellulose powder, a lubricant such as magnesium stearate,
`
`talk, polyethylene glycol or silica, and a disintegrator
`
`5
`
`such as potato starch.
`
`However,
`
`the pharmaceutical composition of the present
`
`invention is not limited to such oral administration and
`
`it is applicable for parenteral administration.
`
`For
`
`example, it may be administered in the form of e.g. a
`
`10
`
`suppository formulated by using oily base material such as
`
`lanolin or fatty acid
`cacao butter, polyethylene glycol,
`triglyceride, a transdermal therapeutic base formulated by
`
`using liquid paraffin, white vaseline, a higher alcohol,
`
`Macrogol ointment, hydrophilic ointment or hydro—gel base
`
`15 material, an injection formulation formulated by using one
`
`or more materials selected from the group consisting of
`
`polyethylene glycol, hydro-gel base material, distilled
`
`water, distilled water for injection and excipient such as
`
`lactose or corn starch, or a formulation for
`
`20
`
`administration through mucous membranes such as an ocular
`
`mucous membrane,
`
`a nasal mucous membrane and an oral
`
`mucous membrane.
`
`Further,
`
`the compounds of the present
`
`invention may be
`
`combined with basic ion—exchange resins which are capable
`of binding bile acids and yet not being absorbed in
`
`25
`
`gastraintestinal tract.
`
`The daily dose of the compound of the formula I is
`
`MYLAN(Pitav)011263
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 29 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 29 of 97
`
`-28—
`
`from 0.05 to 500 mg, preferably from 0.5 to 50 mg for an
`
`adult.
`
`It is administered from once to three times per
`
`day.
`
`The dose may of course be varied depending upon the
`
`age,
`
`the weight or the condition of illness of the
`
`5
`
`patient.
`
`The compounds of the formulas II to VII are novel, and
`
`they are important
`
`intermediates for the preparation of
`
`the compounds of the formula I. Accordingly,
`
`the present
`
`invention relates also to the compounds of the formulas II
`
`10
`
`to VII and the processes for their production.
`
`Now,
`
`the present invention will be described in
`
`further detail with reference to Test Examples for the
`
`pharmacological activities of the compounds of the present
`
`invention, their Preparatioanxamples and Formulation
`
`15 Examples. However, it should be understood that the
`
`present invention is by no means restricted by such
`
`specific Examples.
`
`PHARMACOLOGICAL TEST EXAMPLES
`
`Test A:
`
`Inhibition of cholesterol biosynthesis from
`
`20
`
`acetate in vitro
`
`Enzyme solution was prepared from liver of male Wistar
`
`rat billialy cannulated and discharged bile for over 24
`
`hours. Liver was cut out at mid-dark and microsome and
`
`supernatant fraction which was precipitable with 40-80% of
`
`25
`
`saturation of ammonium sulfate (sup fraction) were
`
`prepared from liver homogenate according to the modified
`
`method of Knauss et. al.; Kuroda, M., et. al., Biochim.
`
`
`MYLAN(Pitav)011264
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 30 of 97
`Case 1:14-cv-02758—PAC Document 62-5 Filed 05/08/15 Page 30 of 97
`
`-29..
`
`Biophys. Acta, 489, 119 (1977).
`
`For assay of cholesterol
`
`biosynthesis, microsome (0.1 mg protein) and sup fraction
`
`(1.0 mg protein) were incubated for 2 hours at 370C in 200
`
`pl of the reaction mixture containing ATP;
`
`1 mM,
`
`5 Glutathione;
`
`6 mM, Glucose—l-phosphate; 10 mM, NAD; 0.25
`
`mM, NADP; 0.25 mM, CoA; 0.04 mM and 0.2 mM [2—l4clsodium
`
`acetate (0.2 uCi) with 4 ul of test compound solution
`
`dissolved in water or dimethyl sulfoxide.
`
`To stop
`
`reaction and saponify,
`
`1 ml of 15% EtOH—KOH was added to
`
`10-
`
`the reactions and heated at 75°C for 1 hour.
`
`Nonsaponifiable lipids were extracted with petroleum ether
`
`and incorporated l4C radioactivity was counted.
`
`Inhibitory activity of compounds was indicated with ICSO.
`
`Test B:
`
`Inhibition of cholesterol biosynthesis in
`
`15
`
`culture cells
`
`Hep G2 cells at over 5th passage were seeded to 12
`
`well plates and incubated with Dulbecco's modified Eagle
`
`(DME) medium containing 10% of fetal bovine serum (FBS) at
`
`37°C;
`
`5% CO2 until cells were confluent for about 7 days.
`
`20 Cells were exposed to the DME medium containing 5% of
`
`lipoprotein deficient serum (LpDS) prepared by
`
`ultracentrifugation method for over 24 hours: Medium was
`
`changed to 0.5 ml of fresh 5% LpDS containing DME before
`
`assay and 10 ul of test compound solution dissolved in
`
`25 water or DMSO were added.
`
`0.2 uCi of
`
`[2—14 Clsodium
`
`acetate (20 ul) was added at O hr(B-l) or 4 hrs(B—2) after
`
`addition of compounds. After 4 hrs further incubation
`
`with [2-14C]sodium acetate, medium was removed and cells
`
`MYLAN(Pitav)011265
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 31 of 97
`Case 1:14-cv-02758—PAC Document 62-5 Filed 05/08/15 Page 31 of 97
`
`
`
`-30-
`
`were washed with phosphate buffered saline(PBS) chilled at-
`
`4OC. Cells were scraped with rubber policeman and
`
`collected to tubes with 9133 and digested with 0.2 ml of
`
`0.5 N KOH at 370C. Aliquot of digestion was used for
`
`5
`
`protein analysis and remaining was saponified with 1 ml of
`
`15% EtOH—KOH at 75°C for 1 hour. Nonsaponifiable lipids
`
`were extracted with petroleum ether and 14 C radioactivity
`
`was counted. Counts were revised by cell protein and
`
`indicated with DPM/mg protein.
`
`Inhibitory activity of
`
`10
`
`compounds was indicated with IC50.
`
`Test C:
`
`Inhibition of cholesterol biosynthesis in vivo
`
`Male Sprague—Dawléy rats weighing about 150 g were fed
`
`normal Purina chow diet and water ad libitum, and exposed
`
`to 12 hours light/12 hours dark lighting pattern (2:00 PM
`
`15
`
`— 2:00 AM dark) prior to use for in vivo inhibition test
`of cholesterol biosynthesis. Animals were separated
`
`groups consisting of five rats as to be average mean body
`
`weight
`
`in each groups. Test compounds at dosage of
`
`0.02-O.2 mg/kg body weight
`
`(0.4 ml/100 g body weight),
`
`20 were dissolved in water or suspended or in 0.5% methyl
`
`cellulose and orally administered at 2—3 hours before
`
`midedark (8:00 PM), while cholesterol biosynthesis reaches
`
`to maximum in rats.
`
`As control, rats were orally
`
`administered only water or vehicle. At 90 minutes after
`
`25
`
`sample administration, rats were injected
`14
`[2- C]sodium acetate at
`
`intraperitoneally with 10 uCi of
`
`volume of 0.2 ml per one.
`
`2 Hours later, blood samples
`
`MYLAN(Pitav)011266
`
`
`
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page 32 of 97
`Case 1:14-cv-02758-PAC Document 62-5 Filed 05/08/15 Page32>of
`
`-31—
`
`were obtained and serum were separated immediately. Total
`
`lipids were extracted according to the method of Folch et
`al. and saponified with EtOH—KOH. NonSaponifiable lipids
`
`were extracted with petroleum ether and radio activity
`
`5
`
`incorporated into nonsaponifiable lipids was counted.
`
`Inhibitory activity was indicated as percent decrease
`
`of counts in testing groups (DPM/2 ml serum/2 hours)
`
`from
`
`that in control group.
`With respect to the compounds of the present
`
`10
`
`invention,
`
`the inhibitory activities against the
`
`cholesterol biosynthesis in which HMG—CoA reductase serves
`
`as a rate limiting enzyme, were measured by the above Test
`
`A and B.
`
`The results are shown in Tables, 2, 2—2,
`
`3
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