I” '
`
`
`
`
`5f"‘§§z213n
`I1IIIIIIIIi/EfllflflflIQTIIIIIIIIIIIIIIII
`
`
`
`
`
`
`SERIAL
`
`‘
`J
`I
`
`
`
`
`
`ifs?
`4‘i.»¥1i$*i‘5~?'i
`an-m.-~
`
`EXAMRJER
`zirnl-nnu—._..un...._—n._--.-.«
`
`,
`suacmss
`Gaoumxnrunn
`r...;3 ,.
`1:/'1")/“?=3
`13’
`,
`,
`4."-'»“§",”_§‘§’E,".3A;2
`
`-"‘ ‘
`‘
`"
`‘
`»:LiL£
`3."!
`
`
`
`F‘..i'l.~*.-§,1,—;.4.1'-:3’,-lip
`tJ.r"1"-""*“-‘5{=‘:”’(‘1C 53_ZUK,I’.il'f:*§§?::I-SH1’A‘W
`5’-’
`Y{).':‘.'~I[.~—I‘{£€-‘.}
`F‘JJ{<>\‘-.«J"+‘;
`3
`J~\i’.-‘X4;
`2-IE?h)3-H’.
`Iw‘ASA'-(Er
`F*;'IA-‘~‘«-.?"s_-»‘r1_r
`.:-:‘.’.«fi. flfsufml
`-»
`N
`o
`"
`E
`3HI_..,V.‘)-§:A_F,MCH{'
`JApA,\;
`=.s.‘.,',"i}({
`;;,j7,:'-«.“.—‘.t\p
`:-'=H19.*-\:)‘<A-;"IA=.HIr
`JAPAN.
`2':
`
`.
`
`It
`
`-
`
`’
`
`>'
`
`E..-
`
`..
`
`"
`t
`
`
`
`
`4n:coNT1Nu][NG DATA*t**ifi-itiitittwt-ktikt
`\l‘:i??£F1T:'D
`IAEL; A.°°L~a'
`13
`M
`«I1?-‘~i
`~.!F
`
`t
`.
`'.,?/333,7)? -J3/1-2/.53
`
`....?'q‘.&.
`
`J
`
`S:NI"
`'':'.-'l’;;_'_
`
`_‘
`
`:._-I
`
`A
`
`‘..
`.
`
`‘I
`
`
`
`
`STATE OR SHEETS
`7r"?‘o‘r€E“'~'rT’"'"
`FILING FEE
`
`RECEIVED
`SOCKET N0?
`coumnv DRWGS.
`cumws
`
`
`
`
`‘
`
`
`
`m,_
`
`__
`
`__
`
`________
`
`__
`
`_ ______
`
`_~___‘ J
`
`.
`
`LLS. DEPT. of COMM.~Pat. 5: TM O?fIce~- I-"i'O43§L (rev. E0-78)
`
`AS
`FILED
`
`
`
`B...
`
`
`
`E ‘yes
`.
`yes
`
`"\ /I
`tn.
`
`Ho
`I
`CI no
`
`Examif r'sinltIa 5
`
`Foreign priority claimed
`as use 119 conamons met
`Verified and
`
`m
`inin
`i:
`
`A°""°W'°d99¢
`
`DO <
`
`EI
`.’:I-
`
`PARTS OF APPLICATION
`FILED SEPARATELY
`
`NOTICE OF ALLOWANCE MAILED
`
`Lsawrck L. §>mw:m
`xi./'
`#2 is
`,
`c;_7KOL~g,‘,.\_, C;K
`V
`
`,/
`/9” ‘ " “ "
`‘*’ Primary Examiner
`
`ISSUE CLASSIFICATION
`ISSUE
`V
`Subclass
`
`
`
`‘
`
`Class
`
`
`.
`'°‘-""/J" I
`
`Label
`Area
`
`iorm PTO436
`iev. 5/89
`
`'
`
`W
`
`' .
`
`I
`
`
`
`WARNING: The intormation disclosod herein may be restrictetjfilllnodthorized disclosure may be
`
`, 181 and 368.
`prohibited by the United States Code Title 35. Sections 1
`Possession outside the u.s. Patent & Trademark Ottice‘ 3 restricted to authorized employees
`and contractors only.
`
`Sawai Ex 1003
`
`Page 1 of 175
`
`

`

`PATENT APPLICATILN SERIAL NO.
`
`[H ($31092
`
`U.S. DEPARTMENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`FEE RECORD SHEET
`
`u‘.;-'\;*
`
`L.“
`
`_
`
`.v-‘--_-~'\~'
`...
`___
`‘rL..-"~'.u5.~"ri u,/mwvr.‘
`
`.L
`
`.'.".;.
`‘
`
`PTO-1556
`(S/87)
`
`Sawai Ex 1003
`
`Page 2 of 175
`
`

`

`_
`
`HllllllllmIN||lI|m|H||fl||H|J||||“W
`
`U.S. PATENT APPLICATION
`
`07/631.092
`
`I2/19/90
`RULE 60
`
`APPLICANT
`
`JAPAN; MIKIO SUZUKI, FUNABASHI-SHI,
`YOSHIHIRO FUJIKAWA, FUNABASH|—SHl,
`JAPAN; HIROSHI
`IWASAKI, FUNABASHI-SH1,
`JAPAN; METSUAKI SAKASHITA,
`SHIRAOKA-MACHI.
`JAPAN; MASAKI KITAHARA, SHIRAOKA-MACHI,
`JAPAN.
`
`**CQNT|NU|NQ BATA*kk*#*fik***kfifi***kk*k
`
`VERIFIED
`
`THIS APPLN IS A CON OF
`
`07/233,752 O8/19/88
`
`7'~‘*F ORE IGN/PCT APPL I CAT | ON59:2’:fo'::‘<:'n‘::'::‘:ink)‘:
`VERIFIED
`
`.‘
`A
`COUNTRY
`
`_'
`DRAWING
`
`-
`
`' L
`CLAIMS
`
`_,
`CLAIM
`
`_
`RECEIVED
`
`,
`
`JPX
`
`0
`
`2
`
`5
`
`630.00
`
`h9—1h6-0—CON
`
`ADDRESS
`
`OBLON, SPIVAC, Nc CLELLAND,
`MAIER 5 NEUSTADT
`FOURTH FLOOR
`1755 JEFFERSON DAVIS HIGHWAY
`ARLINGTON, VA
`22202
`
`QU|NOL|NE{TYPE MEVALONOACTONES
`
`the Uni§eq States
`This is to certify that annexed hereto 1s a true copy From the records of
`Patent and Trademark Office of
`the application as origina11y filed which is 1dent1f1ed above.
`
`the
`By authority of
`COMMISSIONER OF PATENTS AND TRADEMARKS
`
`Date
`
`Certifying Officer
`
`Sawai Ex 1003
`
`Page 3 of 175
`
`

`

`lll $31092
`
`
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Docket No. 49-146-0 CONT
`
`‘*-
`
`HONORABLE COMMISSIONER OF PATENTS 8: TRADEMARKS
`WASHINGTON, D.C. 20231
`
`SIR: This is a request for filing a
`
`IE Continuation
`
`application under 37 C.F.R. 1.60,
`
`D Division
`
`of copending prior application Serial No.07/233,752 filed on_P;U§_U_3.T 191.1988.
`YOSHIHIRO FUJIKAWA ET AL
`"‘"°
`for QUINOLINE TYPE NOmflONEStitle of invention
`Enclosed is a copy of the prior application as originally filed and an affidavit or declaration verify-
`ing it as a true copy.
`
`__..__..._
`
`of
`
`1.
`
`2. D Small Entity Status of this application has been established by a Verified Statement submitted in
`the parent application.
`
`3.
`
`The filing fee is calculated below:
`
`CLAlMS AS FILED, LESS ANY CLAIMS CANCELLED BY AMENDMENT BELOW
`
`.1 —-3 Total Filing Fee .
`
`Fee
`Basic
`Rats $630.00
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. ..= $630
`
`Total Claims .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`independent Claims.
`
`.1 -20
`
`.
`
`.
`
`.
`
`.
`
`4. D The Commissioner is hereby authorized to charge any fees which may be required for the papers
`being filed herewith and for which no check is enclosed herewith, or credit any overpayment to
`Account No. __1§'_0_0_3_9_
`. A duplicate copy of this sheet is enclosed.
`
`5. El A check i_n the amount of $ ...5_3.Q;Q0_
`
`_ is enclosed.
`
`a. Kl Cancel Claims _?.;9..amL.L1;4n_L_,L___ .
`
`7. Q Amend the specification by inserting before the first line the sentence:
`This is a L continuation,
`division, of application Serial No. .97/233E_2.___, filed
`on AUGUST 19, 1988
`
`8. D New Drawings are enclosed.
`
`9. D The prior application is assigned to: __..__.-._
`
`
`
`Sawai Ex 1003
`
`Page 4 of 175
`
`

`

`10.
`
`11.
`
`12.
`
`The Power of Attorney in the prior application is to: Norman F. Oblon, Reg. No. 24,618; Marvin
`J. Spivak, Reg. No. 24,913; C.
`lrvin McC|el|and, Reg. No. 21,124; Gregory J. Maier, Reg. No.
`25,599; Arthur 1. Neustadt, Reg. No. 24,854; Robert C. Miller, Reg. No. 25,357; Richard D. Kelly,
`Reg. No. 27,757; James D. Hamilton, Reg. No. 28,421; Eckhard H. Kuesters, Reg. No. 28,870;
`Robert T. Pous, Reg. No. 29,099; Charles L. Gholz, Reg. No. 26,395; Vincent J. Sunderdick,
`Reg. No. 29,004; William E. Beaumont, Reg. No. 30,996; Steve B. Kelber, Reg. No. 30,073; Stuart
`D. Dwork, Reg. No. 31,103; Robert F. Gnuse, Reg. No. 27,295;and Jean-Paul Lavalleye, Reg. No.
`31,451, all of OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C., Fourth Floor, 1755
`Jefferson Davis Highway, Arlington, Virginia 22202.
`A
`(Copy enclosed)
`a. [:1] The power appears in the original papers of the prior application.
`b. E] Since the power does not appear in the original papers, a copy of the power in the prior
`application is enclosed.
`
`c. D Recognize as associate attorney and address all future communications to:
`
`_;;£ .;.j_:? ?
`name, registration number and address
`
`A Preliminary Amendment is enclosed.
`
`Priority under §l20 is enclosed as well as Declaration of Steven
`B. Kelber. White Advance Serial Number Postal Card (postage pre—
`
`Paid) attached -
`
`Respectfully submitted,
`
`OB LON, SPIVAK, MCCLELLAND,
`MAIER & NEUSTADT, P.C.
`
`--
`
`Nonnan F . Oblon
`
`Attorney of Record
`Registration No. 241618
`Steven B. Kelber
`Attorney of Record
`Registration No. 30,073
`
`FOURTH FLOOR
`1755 JEFFERSON DAVIS HIGHWAY
`ARLINGTON, VIRGINIA 22202
`(703) 521-5940
`
`4/89
`
`Sawai Ex 1003
`
`Page 5 of 175
`
`

`

`,
`
`A
`
`i
`
`m 631092
`
`
`
`Our Ref.: NC-ll5
`
`f§
`M
`.
`\f
`%
`
`_ 1 _
`,g“Jfi
`gU.iN,oL1N§ TYPE MEVALQNOLACTONES
`,{‘x>
`‘>/"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.
`V
`
`"ms
`
`10
`
`Some fermentation metabolic products such as
`
`compactine, CS—5l4, 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—5l4 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
`
`Sawai Ex 1003
`
`Page 6 of 175
`
`

`

`/1:
`
`l0
`
`l5
`
`20
`
`_ 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
`
`disclosed in the following literatures:
`
`WPI ACC NO. 84-l5867S, 86—028274, 86-098816,
`
`86-332070, 87-124519, 87-220987, 88-07781, 88-008460,
`
`88+O9l798 and 88-ll2505.
`
`The present inventors have found that mevalonolactone
`
`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
`
`accomplished on the basis of this discovery.
`
`The novel mevalonolactone derivatives of the present
`
`invention are represented by the following formula I:
`
`R3
`
`R‘
`
`R6
`
`R2
`
`Y-Z
`
`(I)
`
`it RI?
`
`N
`
`R5
`
`wherein R1, R2, R3, R4 and R6 are independently hydrogen,
`
`Cl_6 alkyl, C3_6 cycloalkyl, Cl_3 alkoxy, n-butoxy,
`i-butoxy, sec—butoxy, R7R8N— (wherein R7 and R8 are
`
`25
`
`independently hydrogen or Cl_3 alkyl),
`
`trifluoromethyl,
`
`trifluoromethoxy, difluoromethoxy, fluoro, chloro, bromo,
`
`Sawai Ex 1003
`
`Page 7 of 175
`
`

`

`_ 3 _
`
`19
`
`4
`
`trimethylsilyloxy,
`phenyl, phenoxy, benzyloxy, hydroxy,
`diphenyl-t-butylsilyloxy, hydroxymethyl or —O(CH2)£ORl9
`(wherein R
`is hydrogen or Cl_3 alkyl, and 2
`is l,
`2 or
`3); or when located at the ortho position to each other,
`R1 and R2, or R3 and R
`togetherwfornj—CH=CH-CH=CH-; or
`when located at the ortho position to each other, R1 and
`35‘ r‘«-LK
`R2 togetherfiform ~OC(Rl5)(Rl6
`15 and R16 are
`r
`independently hydrogen or Cl_3 alkyl); Y is —CH
`2-!
`-CHZCHZ-,
`-CH=CH-,
`-CH2~CH=CH- or -CH=CH-CH2-; and Z
`2
`-Q-CHZWCHZ-CO2Rl
`,
`
`)O-
`
`(wherein R
`
`i5
`
`R11
`
`H0
`
`0
`
`f
`
`RI’?
`
`.
`
`I
`
`R
`
`3
`
`Otsp//A\\7$?0
`L\.\//O
`
`;
`
`C0zR‘°
`
`Rll
`I
`0
`\/(/\L’?%
`/,
`
`;
`
`0
`
`/J
`
`4 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 C
`14
`
`l2
`
`is hydrogen or R
`
`l_3 alkyl;
`(wherein R14 is physiologically
`R
`hydrolyzable alkyl or M (wherein M is NH
`sodium,
`
`potassium,
`
`l/2 calcium or a hydrate of lower alkylamine,
`
`diylower alkylamine or tri-lower alkylamine)):
`
`two R13 are
`
`4 I
`
`5
`
`10
`
`15
`
`s
`
`20
`
`25
`
`30
`
`independently primary or secondary Cl_6 alkyl; or two R13
`2)3-; Rl7 and R18 are
`together form —(CH2)2- or -(CH
`5
`
`independently hydrogen or Cl_3
`_ hydrogen, Cl_6 alkyl, C2_3 alkenyl, C3_6 cycloalkyl,
`9
`.<§3{R (wherein R9 is hydrogen, Cl_4 alkyl, Cl_3
`
`alkyl; and R
`
`is
`
`~_.-
`
`I
`
`Sawai Ex 1003
`
`Page 8 of 175
`
`

`

`_ 4 -
`
`alkoxy, fluoro, chloro, bromo or trifluoromethyl),
`
`phenyl-(CH2)m-
`
`(wherein m is l,
`
`2 or 3),
`
`-(CH2)nCH(CH3)-phenyl or phenyl-(CH2)nCH(CH3)~ (wherein n
`is 0,
`l or 2).
`
`Various substituents in the formula I will be
`
`described in detail with reference to specific examples.
`
`However, it should be understood that the present
`
`invention is by no means restricted by such specific
`
`examples.
`
`l0
`
`Cl_6 alkyl for R1, R2, R3, R4, R6 and R9 includes,
`
`for
`
`example, methyl, ethyl, n—propyl,
`
`i-propyl, n-butyl,
`
`i—butyl, sec—butyl and t—butyl. Cl_3 alkoxy for R1, R2,
`R3, R4 and R6 includes, for example, methoxy, ethoxy,
`
`15
`
`n—propoxy and i~propoxy.
`Cl_3 alkyl for R11 includes, for example, methyl,
`
`ethyl, n-propyl and i—propyl.
`
`Cl_3 alkyl for R13 includes, for example, methyl,
`
`ethyl, n-propyl and i—propyl.
`Alkyl for R14 includes, for example, methyl, ethyl,
`
`20
`
`n-propyl,
`
`i-propyl, n-butyl and i—butyl.
`
`M is a metal capable of forming a pharmaceutically
`
`acceptable salt, and it includes,
`
`for example, sodium and
`
`potassium.
`
`trimethylamine).
`
`Cl_6 alkyl for R5 includes,
`
`for example, methyl,
`
`‘““£
`
`Sawai Ex 1003
`
`Page 9 of 175
`
`

`

`-5-
`
`i-propyl, n—butyl,
`ethyl, n-propyl,
`t—butyl, n—pentyl and n-hexyl.
`
`i—butyl, sec—butyl,
`
`C3_6 cycloalkyl for R5 includes, for example,
`cyclopropyl, cyclobutyl,
`
`cyclopentyl and cyclohexyl.
`5 includes, for example, vinyl and
`
`C2_3 alkenyl for R
`
`i—propenyl.
`
`Phenyl-(CH2)m— for R5 includes, for example, benzyl,
`B—phenylethyl and y-phenylpropyl.
`
`10
`
`Phenyl-(CH2)nCH(CH3)- for R5 includes, for example,
`a-phenylethyl and a—benzylethyl.
`C1_3 alkyl for R7 and R8 includes, for example,
`methyl, ethyl, n-propyl and i—propyl.
`
`Further,
`
`l5 optical isomers.
`
`20
`
`falling outside the definition of —co2Rl2 of the
`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
`-CO2Rl2 moiety is -COZH) are equivalent to the compounds
`of the present
`invention.
`
`25
`
`Now, preferred substituents of the compounds of the
`
`present invention will be described.
`
`Sawai Ex 1003
`
`Page 10 of 175
`
`

`

`_-v
`
`_6_
`
`5
`
`10
`
`15
`
`In the following preferred, more preferred still
`further perferred and most preferred examples,
`the
`numerals for the positions of the substituents indicate
`the positions on the quinoline ring.
`For example, N’
`shown by e.g. 1' or 2'
`indicates the position of the
`substituent on the phenyl substituted at the 4-position of
`the quinoline ring (the carbon connected to the quinoline
`ring is designated as 1').
`The meanings of the respective
`substituents are the same as the above—mentioned meanings.
`Preferred substituents for R1, R2 and R6 are hydrogen,
`fluoro, chloro, bromo, Cl_3 alkyl, Cl_3 alkoxy, C 3-6
`cycloalkyl, dimethylamino, hydroxy, hydroxymethyl,
`hydroxyethyl,
`trifluoromethyl,
`trifluoromethoxy,
`difluoromethoxy, phenoxy and benzyloxy.
`Further, when R6 is hydrogen, it is preferred that R1
`and R2 together form methylenedioxy.
`As preferred examples for R3 and R4, when R4
`3
`hydrogen, R
`is hydrogen, 3'—fluoro, 3'-chloro, 3'-methyl,
`4'-methyl, 4'-chloro and 4'—fluoro.
`
`is
`
`20
`
`Other preferred combinations of R3 and R4 include
`3'-methyl-4'—chloro, 3',5'-dichloro, 3',S'—difluoro,
`3',5'-dimethyl and 3'-methyl-4'-fluoro.
`
`Preferred examples for R5 include primary and
`
`secondary Cl_6 alkyl and C3_6 cycloalkyl.
`Preferred examples for Y include —CH2-CH2— and
`
`25
`
`-CH=CH- .
`
`Preferred examples for Z include
`
`V“-‘-g
`
`Sawai Ex 1003
`
`Page 11 of 175
`
`

`

`-CH(OH)CH2CH2(OH)CH
`_
`13
`CH(OH)CH2C(OR
`)2CH2CO2R
`
`2CO2R
`
`12
`
`,
`12
`
`.
`
`—CH(OH)CH C(O)CH cozalz and
`2
`2
`
`Now, more preferred substituents of the compounds of
`
`the present
`
`invention will be described.
`
`10
`
`As more preferred examples for R1, R2 and R6, when
`both R2 and R6 are hydrogen, R1 is hydrogen, 5—fluoro,
`6-fluoro, 7-fluoro, 8—fluoro, 5-chloro, 6—chloro,
`
`7-chloro, 8-chloro, S—bromo, 6—bromo, 7-bromo, 8—bromo,
`
`15
`
`5—methyl, 6—methyl, 7-methyl, 8-methyl, 5—methoxy,
`6-methoxy, 7-methoxy, 8—methoxy, 5-trifluoromethyl,
`
`6—trifluoromethyl, 7—trifluoromethyl, 8—trifluoromethyl,
`
`6-trifluoromethoxy, 6—difluoromethoxy, 8-hydroxyethyl,
`
`C*Ҥg
`
`5-hydroxy, 6—hydroxy, 7—hydroxy, 8—hydroxy, 6-ethyl,
`
`6—n-butyl and 7—dimethylamino.
`when R6 is hydrogen, R1 and R2 together represent
`
`20
`
`6—chloro-8-methyl, 6—bromo-7—methoxy, 6—methyl—7-chloro,
`
`6—ohloro—8-hydroxy, 5-methyl—2—hydroxy,
`Gsnethoxy-7—chloro, 6-chloro—7—methoxy,
`6-hydroxy—7—chloro, 6—chloro—7—hydroxy, 6—chloro—8—bromo,
`
`25
`
`5—chloro-6—hydroxy, 6—bromo—8—chloro, 6—bromo—8—hydroxy,
`
`5-methyl-8-chloro, 7-hydroxy~8—chloro, 6—bromo—8—hydroxy,
`
`6—methoxy—7-methyl, 6—chloro—8-bromo, 6—methyl—8—bromo,
`
`Sawai Ex 1003
`
`Page 12 of 175
`
`

`

`.3.
`
`6,7—difluoro, 6,8-difluoro, 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.
`I
`when R1
`R2 and R6
`
`are not hydrogen,
`they together
`represent 5,7-dimethoxy—8—hydroxy, 5,8—dichloro-6—hydroxy,
`6,7,8—trimethoxy, 6,7,8—trimethyl, 6,7,8-trichloro,
`S—fluoro—6,8—dibromo or 5-chloro-6,8—dibromo.
`As more preferred examples for R3 and R4, when R3 is
`hydrogen, R4
`is hydrogen, 4'—methyl, 4'-chloro or
`10 4'-fluoro. when both R3 and R4
`
`are not hydrogen,
`they
`together represent 3',5'—dimethyl or 3'—methyl-4'-fluoro.
`As more preferred examples for R5,
`the above-mentioned
`preferred examples of R5 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
`
`....4‘
`
`As
`
`20
`
`, when both R2 and R6 are
`is hydrogen, 6—methyl, 6—ethyl,
`hydrogen, R
`6—trifluoromethyl, 6—hydroxy, 6-methoxy, 6-chloro,
`6—bromo, 6-n—butyl and 7-dimethylamino.
`‘,When only R6 is hydrogen, R1 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 R
`
`and R4,
`
` —
`
`Sawai Ex 1003
`
`Page 13 of 175
`
`

`

`._9_.
`
`when R3 is hydrogen, R4 is hydrogen, 4'—chloro or
`4'-fluoro, or R3 and R4 together represent
`
`3'-methyl-4'-fluoro.
`
`Still further preferred examples for R5 include ethyl,
`n-propyl,
`i-propyl and cyclopropyl.
`
`Still further preferred examples for Y include
`
`(E)*-CH=CH-.
`
`As still further preferred examples for Z,
`the
`above—mentioned preferred example for Z may be mentioned.
`Now,
`the most preferred substituents for the compounds
`of the present
`invention will be described.
`
`10
`
`the most preferred examples for R1, R2 and R6, when
`As
`both R2 and R6 are hydrogen, R1 is hydrogen, 6-methyl or
`
`6-chloro.
`
`when only R6 is hydrogen, R1 and R2 together
`
`represent,
`
`for example, 6,7—dimethoxy.
`
`the most preferred examples for R3 and R4, R3 is
`As
`hydrogen and R4 is hydrogen, 4'—chloro or 4'—fluoro.
`
`20
`
`The most preferred examples for R5 include i-propyl
`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
`
`0*“‘*l
`
`Sawai Ex 1003
`
`Page 14 of 175
`
`

`

`carboxylic acids.
`
`(E)—3,5-dihydroxy—7—[4'—(4"-fluorophenyl)-2'-
`(a)
`(l"-methylethyl)—quinolin—3'—yl]-hept-6-enoic acid
`
`10
`
`(E)-3,5-dihydroxy-7-[4'-(4"-fluorophenyl)-2'-
`(b)
`(l"~methylethyl)-6'-chloro-quinolin—3'—yl]-hept-6—enoic
`acid
`
`(E)-3,5-dihydroxy-7—[4'-(4"-fluorophenyl)-2'-
`(C)
`(l"—methylethyl)-6'-methyl—quinolin~3'—yl]—hept—6—enoic
`acid
`
`15
`
`(d)
`(E)-3,5-dihydroxy—7—[4'-(4"-fluorophenyl)-2'-
`(l"-methylethyl)-6',7'—dimethoxy—quinolin—3'—yl]—hept—6—
`enoic acid
`
`(E)—3,5—dihydroxy—7—[4'-(4"-fluorophenyl)-2'-
`(e)
`cyclopropyl-quinolin-3'-yl]-hept—6—enoic acid
`
`20
`
`(f)
`
`(E)-3,5~dihydroxy—7—[4'—(4"-fluorophenyl)—2’—
`
`cyclopropyl-6'-chloro-quinolin-3'
`2
`
`—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—dihydroxy-7-[4'-(4"—fluorophenyl)~2'—
`
`cyclopropyl—6',7’-dimethoxy—quinolin—3'-y1]—hept-6—enoic
`acid
`
`VA-«g
`
`Sawai Ex 1003
`
`Page 15 of 175
`
`

`

`_ll_.
`
`(E)-3,5-dihydroxy—7—[4'-(4"—chlorophenyl)-2'-
`(i)
`(l"—methylethyl)-quinolin-3'—yl}-hept—6—enoic acid
`
`(E)-3,5-dihydroxy-7-[4'-(4"-chlorophenyl)-2'-
`(j)
`(l"-methylethyl)—6'—chloro—quinolin~3'~yl]—hept-6-enoic
`acid
`
`(k)
`
`(E)-3,5-dihydroxy-7—[4'-(4"—chlorophenyl)-2'-
`
`(l"—methylethyl)—6'~methyl—quinolin—3'-yl]-hept-6-enoic
`acid
`
`(1)
`
`(E)-3,5-dihydroxy—7—[4'-(4"-chlorophenyl)~2'—
`
`(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
`
`(n)
`
`(E)*3,5-dihydroxy—7—[4'-(4"—chlorophenyl)—2'—
`
`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'-
`
`l0
`
`15
`
`cyclopropyl~6'7'—dimethoxy—quinolin—3'—yl]-hept-6-enoic
`acid
`
`20
`
`(q)
`(E)-3,5-dihydroxy-7—[4'-phenyl-2'-(l"-
`mathylethyl)—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'~(l"-
`
`Sawai Ex 1003
`
`Page 16 of 175
`
`

`

`_l2_.
`
`methylethyl)-6',7’-dimethoxy-quinolin-3'-yl]—hept-6—enoic
`
`acid
`
`5
`
`l0
`
`(E)-3,5-dihydroxy—7—[4'—phenyl-2'—cyclopropyl—
`(U)
`quinolin-3'-yl]-hept-6—enoic acid
`(V)
`(E)-3,5-dihydroxy-7—[4'~phenyl—2'—cyclopropyl—6'—
`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-
`6',7'—dimethoxy—quinolin-3'—yl]—hept—6—enoic acid
`(Y)
`(E)-3,5-dihydroxy—7—[4'-(4"—fluorophenyl)-2'-
`(l"-methylethyl)—6'-methoxy—quinolin-3'-yl]-hept-6-enoic
`
`acid
`
`(2)
`
`(E)-3,5-dihydroxy-7—[4'—(4"—fluorophenyl)—2'-
`cyclopropyl¥6'—methoxy-quinolin-3'—yl]-hept—6—enoic acid
`
`15
`
`Sawai Ex 1003
`
`Page 17 of 175
`
`

`

`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 18 of 175Page 18 of 175
`
`

`

`
`
`-14 —-14 —
`
`
`
`C0zR'?C0zR'?
`
`
`
`«(Alto«(Alto
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 19 of 175Page 19 of 175
`
`
`
`
`
`

`

`
`
`-15 --15 -
`
`
`
`
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 20 of 175Page 20 of 175
`
`

`

`
`
`‘l6"'‘l6"'
`
`
`
`
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 21 of 175Page 21 of 175
`
`

`

`
`
`
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 22 of 175Page 22 of 175
`
`

`

`-18-
`
`6
`
`2
`3
`4
`5
`In the above reaction scheme, R1, R , R , R , R , R
`and R12 are as defined above with respect to the formula
`21
`22
`
`I, and R
`
`and R
`
`alkyl such as methyl, ethyl, n-propyl,
`n-butyl.
`
`independently represent C
`
`lower
`
`l-4
`i—propyl or
`
`Step A represents a reduction reaction of the ester to
`
`a primary alcohol.
`
`Such reduction reaction can be
`
`conducted by using various metal hydrides, preferably
`
`diisobutylaluminium hydride,
`
`in a solvent such as
`
`l0
`
`15
`
`tetrahydrofuran or toluene at a temperature of from -20 to
`20°C, preferably from -10 to 10°C.
`
`Step B represents an oxidation reaction of the primary
`alcohol
`to an aldehyde, which can be conducted by using
`various oxidizing agents. Preferably,
`the reaction can be
`
`conducted by using pyridinium chlorochromate in methylene
`chloride at.a temperature of from O to 25°C, or by using
`
`oxalyl chloride, dimethyl sulfoxide and a tertiary amine
`
`such as triethylamine (Swern oxidation), or by using a
`
`D-~4A~‘
`
`sulfur trioxide pyridine complex.
`
`20
`
`Step C represents a synthesis of a
`
`3—ethoxy—l—hydroxy~2—propene derivative, which can be
`
`prepared by reacting a compound V to lithium compound
`
`which has been preliminarily formed by treating
`
`cis—l—ethoxy-2-(tri—n—butylstannyl)ethylene with butyl
`
`25
`
`lithium in tetrahydrofuran.
`
`As
`
`the reaction temperature, it is preferred to employ
`
`a low temperature at a level of from -60 to -78°C.
`
`Sawai Ex 1003
`
`Page 23 of 175
`
`

`

`_ 19 _
`
`Step D represents a synthesis of an enal by acidic
`
`hydrolysis.
`
`As
`
`the acid catalyst, it is preferred to
`
`employ p-toluene sulfonic acid, hydrochloric acid or
`
`sulfuric acid, and the reaction may be conducted in a
`
`solvent mixture of water and tetrahydrofuran or ethanol at
`
`a temperature of from 10 to 25°C.
`
`The
`
`3—ethoxy-l—hydroxy—2—propene derivative obtained in Step C
`can be used in Step D without purification i.e. by simply
`
`removing tetra—n-butyl tin formed simultaneously.
`
`l0
`
`Step E represents a double anion condensation reaction
`
`between the enal III and an acetoacetate.
`
`Such
`
`condensation reaction is preferably conducted by using
`
`sodium hydride and n~butyl
`
`lithium as the base in
`
`tetrahydrofuran at a temperature of from -80 to 0°C,
`preferably from -30 to -10°C.
`
`15
`
`Step F represents a reduction reaction of the carbonyl
`
`group, which can be conudcted by using a metal hydride,
`
`preferably sodium borohydride in ethanol at a temperature
`
`of from -10 to 25°C, preferably from -10 to 5°C.
`
`Further,
`
`the reduction reaction may be conducted by
`
`using zinc borohydride in dry ethyl ether or dry
`
`tetrahydrofuran at a temperature of -100 to 25°C,
`preferably from -80 to -50°C.
`
`Step G is a step for hydrolyzing the ester.
`
`The
`
`hydrolysis can be conducted by using an equimolar amount
`
`of a base, preferably potassium hydroxide or sodium
`
`hydroxide,
`
`in a solvent mixture of water and methanol or
`
`20
`
`25
`
`Sawai Ex 1003
`
`Page 24 of 175
`
`

`

`-20..
`
`5
`
`10
`
`Further
`
`such as carbodiimide, preferably a water soluble
`
`carbodiimide such as
`
`N-cyclohexyl—N'—[2'-(methylmorpholinium)ethylIcarbodiimide
`15 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
`conducted by using a catalytic amount of palladium-carbon
`
`20
`
`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
`
`Sawai Ex 1003
`
`Page 25 of 175
`
`

`

`-21..
`
`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 —l5°C.
`
`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
`
`temperature of from -10 to 10°C, preferably from -l0 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
`
`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
`
`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
`
`tetrahydrofuran, benzene or
`solvent such as ethyl ether,
`toluene at a temperature of from 20 to 80°C, preferably
`
`l0
`
`15
`
`20
`
`25
`
`from 40 to 80°C.
`
`In addition to the compounds disclosed in Examples
`
`given hereinafter, compounds of the formulas I-2 and I-5
`
`Sawai Ex 1003
`
`Page 26 of 175
`
`

`

`-22-
`
`given in Table l can be prepared by the process of the
`
`present invention.
`
`In Table 1,
`
`i- means iso, sec« means
`
`secondary and c— means cyclo. Likewise, Me means methyl,
`
`Et means ethyl, Pr means propyl, Bu means butyl, Pent
`
`means pentyl, Hex means hexyl and Ph means phenyl.
`
`>.._\{
`
`Sawai Ex 1003
`
`Page 27 of 175
`
`

`

`Tablel
`
`_ 23 _
`
`/EH
`4
`3
`R\/\< R»: \l
`R6
`'
`M00212‘
`[Q/J;
`R = \/\\¢\/..l\/‘I
`“:3-‘C73
`R‘ /‘v”\.v”‘~ R5
`
`2
`
`I-2(R'==H)
`1- 5 (R‘3=.'\Ja)
`
`
`
`R‘
`
`R’
`
`R“
`
`R‘
`
`R5
`
`R“
`
`6-Ofie
`6-Ofle
`6-Br
`6-"Ia
`7-'0P’le
`6-3:‘
`6.7
`
`(K
`
`H
`
`H
`H
`
`6-C2
`6—CZ
`5-0CHzPh
`H
`H
`
`6-CZ
`
`H
`H
`H
`8-He
`8—0fle
`H
`
`H
`
`H
`H
`
`H
`H
`H
`H
`H
`
`H
`
`H
`4-}?
`4-F
`4-F
`41-F
`2-?
`
`4-F
`4-F
`
`H
`H
`H
`H
`H
`H
`
`H
`H
`
`H
`4-Ph
`4-PhCHz H
`
`4-17
`4-17
`-i"F
`4-F
`4*F
`
`4-‘F
`
`H
`H
`H
`H
`H
`
`H
`
`i—Pr
`i-Pr
`1—P'
`1-Pr
`1-Pr
`i-Pr
`
`H
`H
`H
`H
`H
`H
`
`1-91»
`1?.)
`
`1—Pr
`1—Pr
`
`H
`H
`
`H
`H
`
`c—Pr
`H
`sec-Bu
`H
`1 Pr
`H
`;—Bu
`H
`a. Pen: H
`
`g Pent H
`
`6—?1ezN
`
`H
`
`4-17
`
`H
`
`H
`
`i-Pr
`
`SawaiEx1003
`
`Page 28 of 175
`
`

`

`
`
`C“PI‘C“PI‘
`
`
`
`HH
`
`H H
`H H
`
`
`
`c-Hexc-Hex
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 29 of 175Page 29 of 175
`
`
`
`-24--24-
`
`

`

`
`
`-25--25-
`
`
`RR
`
`HH
`
`442442
`
`HH
`
`4-c24-c2
`
`4-F4-F
`
`HH
`
`4—c24—c2
`
`HH
`
`4—c24—c2
`
`4-}?4-}?
`
`
`
`HH
`
`
`
`4.024.02
`
`
`
`HH
`
`
`
`4-c.a.4-c.a.
`
`
`6—.‘1e6—.‘1e
`
`6-He6-He
`
`6-Me6-Me
`
`5-He5-He
`
`6-Me6-Me
`
`(3-0,?(3-0,?
`
`s—c2s—c2
`
`5—c25—c2
`
`54225422
`
`s—c2s—c2
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`H4H4
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`R‘R‘
`
`i-Pri-Pr
`
`1-9:1-9:
`
`c P»c P»
`
`c—Prc—Pr
`
`c-Prc-Pr
`
`1-Pr1-Pr
`
`1—Pr1—Pr
`
`c-Prc-Pr
`
`c-Prc-Pr
`
`c-Prc-Pr
`
`
`
`i—Pri—Pr
`
`
`
`1.9:1.9:
`
`
`
`c-P*'c-P*'
`
`
`
`c-Prc-Pr
`
`
`R°R°
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`HH
`
`HH
`
`HH
`
`HH
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`HH
`
`
`
`
`
`
`
`HH
`HH
`4-F4-F
`HH
`c-Prc-Pr
`HH
`
` # #
`
`
`
`Sawai Ex 1003Sawai Ex 1003
`
`
`
`Page 30 of 175Page 30 of 175
`
`

`

`- 25 _
`
`Thus,
`the compounds
`invention are useful as curing agents
`of the present
`against hyperlipidemia, hyperlipoproteinemia and
`atheroscleosis.
`
`20
`
`suitable pharmaceutically acceptable carrier including a
`binder such as hydroxypropyl cellulose, syrup, gum arabic,
`gelatin, sorbitol,
`tragacanth gum, polyvinyl pyrrolidone
`
`Sawai Ex 1003
`
`Page 31 of 175
`
`

`

`‘J
`
`-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
`
`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
`
`suppository formulated by using oily base material such as
`
`cacao butter, polyethylene glycol,
`
`lanolin or fatty acid
`
`triglyceride, a transdermal therapeutic base formulated by
`
`using liquid paraffin, white Vaseline,
`
`a higher alcohol,
`
`Macrogol ointment, hydrophilic ointment or hydfO‘9el base
`
`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
`
`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
`
`gastraintestinal tract.
`
`The daily dose of the compound of the formula I is
`
`l0
`
`15
`
`20
`
`25
`
`5&4‘
`
`Sawai Ex 1003
`
`Page 32 of 175
`
`

`

`. "'0
`
`10
`
`15
`
`-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
`
`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
`
`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 Preparation Examples and Formulation
`
`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.; fiuroda, M., et. al., Biochim.
`
`Sawai Ex 1003
`
`Page 33 of 175
`
`

`

`- 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 37°C in 200
`
`pl of the reaction mixture containing ATP;
`
`1 mM,
`
`5 Glutathione;
`
`6 mM, G1ucose—l—phosphate; 10 mM, NAD; 0.25
`
`mM, NADP; 0.25 mM, COA; 0.04 mM and 0.2 mM [2-l4C]sodium
`
`acetate (0.2 pCi) with 4 pl 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
`14
`
`and incorporated
`
`C radioactivity was counted.
`
`Inhibitory activity of compounds was indicated with IC50.
`
`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 (PBS) 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 pl of test compound solution dissolved in
`
`25 water or DMSO were added.
`
`0.2 pCi of
`
`[2
`
`_14
`
`Clsodium
`
`acetate (20 pl) was added at O hr(B-1) or 4 hrs(B—2) after
`
`addition of compounds. After 4 hrs further incubation
`14
`with [2— Clsodium acetate, medium was removed and cells
`
`Sawai Ex 1003
`
`Page 34 of 175
`
`

`

`_30_
`
`were washed with phosphate buffered saline(PBS) chilled at
`
`