Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 1 of 18
`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 1 of 18
`
`EXHIBIT 1
`EXHIBIT 1
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 2 of 18
`
`1111111111111111111111114111(1018 1,1911111111111111111111111111111
`
`United States Patent [19]
`Fujikawa et al.
`
`[n] Patent Number:
`[45] Date of Patent:
`
`5,856,336
`Jan. 5, 1999
`
`[54] QUINOLINE TYPE MEVALONOLACTONES
`
`[75] Inventors: Yoshihiro Fujikawa; Mikio Suzuki;
`Hiroshi Iwasaki, all of Funabashi;
`Mitsuaki Sakashita; Masaki Kitahara,
`both of Shiraoka-machi, all of Japan
`
`[73] Assignee: Nissan Chemical Industries Ltd.,
`Tokyo, Japan
`
`[21] Appl. No.: 883,398
`
`[22] Filed:
`
`May 15, 1992
`
`Related U.S. Application Data
`
`[62] Division of Ser. No. 631,092, Dec. 19, 1990, which is a
`continuation of Ser. No. 233,752, Aug. 19, 1988.
`
`[30]
`
`Foreign Application Priority Data
`
`Aug. 20, 1987 [JP] Japan 62-207224
`Jan. 26, 1988 [JP] Japan 63-15585
`Aug. 3, 1988 [JP] Japan 63-193606
`
`[51] Int. Cl.' A61K 31/47; CO7D 215/12
`[52] U.S. Cl. 514/311; 546/173
`[58] Field of Search 546/173; 514/311
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`Primary Examiner--Laura L. Stockton
`Attorney, Agent, or Firm—Oblon, Spivak, McClelland,
`Maier & Neustadt, P.C.
`
`[57]
`
`ABSTRACT
`
`A compound of the formula
`
`[A]
`
`0 .1/2C a
`Z= —CH( 0 H)—C —CH(0 H)—C
`have HMG—CoA inhibiting effects, making them use-
`ful as inhibitors of cholesterol biosynthesis. The com-
`pound may be prepared as a pharmaceutical for reduc-
`ing hyperlipidemia, hyperlipoproteinemia or
`atherosclerosis.
`
`5,753,675 5/1998 Wattanasin 514/311
`
`2 Claims, No Drawings
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 3 of 18
`
`1
`QUINOLINE TYPE MEVALONOLACTONES
`
`5,856,336
`
`R
`
`HO
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`This is a division, of application Ser. No. 07/631,092,
`filed on Dec. 19, 1990, which is a continuation of 07/233,
`752, filed Aug. 19, 1988.
`The present invention relates to novel mcvalonolactoncs
`having a quinoline ring, processes for their production,
`pharmaceutical compositions containing 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.
`Some fermentation metabolic products such as
`compactine, CS-514, Mevinolin or semi-synthetic deriva-
`tives or fully synthetic derivatives thereof are known to be
`inhibitors against HMG-CoA reductase which is a rate
`limiting enzyme for cholesterol biosynthesis. (A. Endo J.
`Med Chem., 28(4) 401 (1985))
`CS-514 and Mevinolin have been clinically proved to he
`potentially useful anti-hyperlipoproteinemic agents, and
`they are considered to be effective for curing or preventing
`diseases of coronary artery sclerosis or atherosclerosis.
`(IXth Int. Symp. Drugs Affect. Lipid Metab., 1986, p30,
`p31, p66)
`However, with respect to fully synthetic derivatives,
`particularly hetero aromatic derivatives of inhibitors against
`HMG-CoAreductase, limited information is 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
`derivatives having a quinoline ring, the corresponding dihy-
`droxy 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:
`
`(0
`
`R4
`
`• Y
`
`-Z
`
`R6
`xz
`
`wherein R„ R,, R,, R4 and R6 are independently hydrogen,
`C,.6 alkyl, C3.6 cycloalkyl, C2,3 alkoxy, n-butoxy, i-butoxy,
`sec-butoxy, R7125N- (wherein 127 and R8 are independently 55
`hydrogen or
`alkyl), trifluoromethyl, trifluoromethoxy,
`difluoromethoxy, fluoro, chloro, bromo, phenyl, phenoxy,
`benzyloxy, hydroxy, trimethylsilyloxy, diphenyl-t-
`butylsilyloxy, hydroxymethyl or -0(CH2),01219 (wherein
`1219 is hydrogen or C,_, alkyl, and 1 is 1, 2 or 3); or when 60
`located at the ortho position to each other, I21 and R2, or R3
`and R4 together form -CH=CH-CH=CH-; or when
`located at the ortho position to each other, R1 and R2
`together form -0C(1215)(R16)0-(wherein1215 and RI' are
`independently hydrogen or C,_, alkyl); Y is -CH2-, 65
`-CH2CH2-, -CI-I=CH-, -CH2-CH=CH- or
`-C11.-----CH-CII2-; and Z is -Q-CH2WCH2-0O212.12,
`
`2
`
`0 O/\ 0
`
`0
`
`° R
`
`"
`
`0
`
`>r,
`
`R"
`
`Rts
`
`(wherein Q is -C(0)-, -C(01213),- or -CH(OH)-;
`W is -C(0)-, -C(01213)2- or
`C(1211)(OH)-; Ril is
`hydrogen or C,_, alkyl; R12 is hydrogen or R'4 (wherein 1214
`is physiologically hydrolyzable alkyl or M (wherein M is
`NH4, sodium, potassium, 1/2, calcium or a hydrate of lower
`alkylamine, di-lower alkylamine or tri-lower alkylaminc));
`two 1213 are independently primary or secondary C1 _6 alkyl;
`or two R'3 together form -(CH,)- or -(CH,),-; R17
`and I218 are independently hydrogen or C,_„ alkyl; and 125 is
`hydrogen, C,_, alkyl, C6,3 alkenyl, C3_6 cycloalkyl,
`
`9
`
`(wherein I29 is hydrogen, C.,_4 alkyl, C,_, alkoxy, fluoro,
`chloro, bromo or trifluoromethyl), phenyl-(CH2)„,-
`(wherein m is 1, 2 or 3), -(CH2)„CH(CH3)-phenyl or
`phenyl-(C1-12)„CH(CH3)- (wherein n is 0, 1 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.
`C,_, alkyl for R1, R2, R3, R4, Fe and R9 includes, for
`example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
`sec-butyl and t-butyl. C1 _, alkoxy for 121, R2, R3,124 and 126
`includes, for example, methoxy, ethoxy, n-propoxy and
`i-propoxy.
`C2.3 alkyl for 12" includes, for example, methyl, ethyl,
`n-propyl and i-propyl.
`C2.3 alkyl for R' includes, for example, methyl, ethyl,
`n-propyl and i-propyl.
`Alkyl for R" includes, for example, methyl, ethyl,
`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.
`CO2M includes, for example, -CO2NI-I4 and -0O21-I.
`(primary to tertiary lower alkylamine such as
`trimethylamine).
`C,_6 alkyl for R5 includes, for example, methyl, 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
`i-propenyl.
`Phenyl-(CH,)„,- for R5 includes, for example, benzyl,
`(3-phenylethyl and y-phenylpropyl.
`Phenyl-(CII2)„CII(CH3)- for R5 includes, for example,
`a-phenylethyl and a-benzylethyl.
`C1.3 alkyl for 127 and 128 includes, for example, methyl,
`ethyl, n-propyl and i-propyl.
`Further, these compoundsmay have at least one or two
`asymmetric carbon atoms and may have at least two to four
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 4 of 18
`
`5,856,336
`
`5
`
`10
`
`15
`
`3
`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 fall-
`ing outside the definition of -00,1212 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 -CO,R' moiety is -00,11) are
`equivalent to the compounds of the present invention.
`Now, preferred substituents of the compounds of the
`present invention will be described.
`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, C1.3 alkyl, C1_3 alkoxy, C3_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 124 is
`hydrogen, R3 is hydrogen, 3'-fluoro, 3'-chloro, 3'-methyl,
`4'-methyl, 4'-chloro and 4'-fluoro.
`Other preferred combinations of R3 and R4 include
`3'-methyl-4'-chloro, 3',5'-dichloro, 3',5'-difluoro, 3',5'-
`dimethyl and 3'-methyl-4'-fitioro.
`Preferred examples for R5 include primary and secondary
`C1_6 alkyl and C.3..6 cycloalkyl.
`Preferred examples for Y include -CH2--CH,-- and 35
`-CH=CH-.
`Preferred examples for Z include
`
`20
`
`25
`
`30
`
`Ito
`
`0
`
`o
`
`0
`
`°
`
`40
`
`-CH(OH)CH2CI-12(OH)CH,CO,R12, -CH(OH)CH,C(0) 45
`CH,CO2R12 and -CH(OH)CH2C(01213)2CH,CO2R12.
`Now, more preferred substituents of the compounds of the
`present invention will be described.
`As more preferred examples for R1, R2 and R6, when both
`R2 and R6 are hydrogen, R1 is hydrogen, 5-fluoro, 6-fluoro, 50
`7-fluoro, 8-fluoro, 5-chloro, 6-chloro, 7-chloro, 8-chloro,
`5-bromo, 6-bromo, 7-bromo, 8-bromo, 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, 55
`6-difiuoromethoxy, 8-hydroxyethyl, 5-hydroxy, 6-hydroxy,
`7-hydroxy, 8-hydroxy, 6-ethyl, 6-n-butyl and
`7-dimethylamino.
`When R6 is hydrogen, RI and R2 together represent
`6-chloro-8-methyl, 6-bromo-7-methoxy, 6-methyl-7-chloro, 60
`6-chloro-8-hydroxy, 5-methyl-2-hydroxy, 6-methoxy-7-
`chloro, 6-chloro-7-methoxy, 6-hydroxy-7-chloro, 6-chloro-
`7-hydroxy, 6-chloro-8-bromo, 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- as
`methyl, 6-chloro-8-bromo, 6-methyl-8-bromo, 6,7-difluoro,
`6,8-difluoro, 6,7-methylenedioxy, 6,8-dichloro, 5,8-
`
`4
`dimethyl, 6,8-dimethyl, 6,7-dimethoxy, 6,7-diethoxy, 6,7-
`dibromo or 6,8-dibromo.
`When R', 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,
`5-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 4'-fluoro.
`When both R3 and re 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, -C1-12-CF12- and (E)-
`-CH---CH---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 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, R' and R2 represent 6,8-
`dichloro, 5,8-dimethyl, 6,8-dimethyl, 6,7-dimethoxy, 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, 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.
`As the most preferred examples for R', R2 and R6, when
`both R2 and R6 are hydrogen, R' is hydrogen, 6-methyl or
`6-chloro.
`When only R6 is hydrogen, R 1 and 122 together represent,
`for example, 6,7-dimethoxy.
`As the most preferred examples for R3 and R4, R3 is
`hydrogen and R4 is hydrogen, 4'-chloro or 4'-fluoro.
`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
`present invention will be presented. The following com-
`pounds (a) to (z) are shown in the form of carboxylic acids.
`However, the present invention include not only the com-
`pounds in the form of carboxylic acids but also the corre-
`sponding lactones formed by the condensation of the car-
`boxylic acids with hydroxy at the 5-position, and sodium
`salts and lower alkyl esters (such as methyl, ethyl, i-propyl
`and n-propyl esters) of the carboxylic acids, which can be
`physiologically hydrolyzed to the carboxylic acids.
`(a) (E)-3,5-clihydroxy-744'-(4"-fluoropheny1)-2'-(1"-
`methylethyl)-quinolin-3'-yfthept-6-enoic acid
`(b) (E)-3,5-dihydroxy-744'-(4"-fluoropheny1)-2'-(1"-
`methylethyl)-6'-chloro-quinolin-3'-yll-hept-6-enoic acid
`(c) (E)-3,5-dihydroxy-744'-(4"-fluorophenyl)-2'-(1"-
`methylethyl)-6'-methyl-quinolin-3'-y1]-hept-6-enoic acid
`(d) (E)-3,5-dihydroxy-744'-(4"-fluoropheny1)-2'-
`(1"methylethyl)-6',7'-dimethoxy-quinolin-3'-yll-hept-6-
`enoic acid
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 5 of 18
`
`5,856,336
`
`is
`
`20
`
`5
`(e) (E)-3,5-dihydroxy-744'-(4"-fluoropheny1)-2'-
`cyclopropyl-quinolin-3'-yll-hept-6-enoic acid
`(f) (E)-3,5-clihydroxy-744'-(4"-fluoropheny1)-2'-
`cyclopropyl-6'-chloro-quinolin-3'-y1]-hept-6-enoic acid
`(g) (E)-3,5-dihydroxy-744'-(4"-lluoropheny1)-2'- s
`cyclopropy1-6'-methyl-quinolin-3'-y1]-hept-6-enoic acid
`(h) (E)-3,5-dihydroxy-744'-(4"-fluoropheny1)-2'-
`cyclopropy1-6',7'-dirnethoxy-qu
`acid
`(i) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'41"- to
`methylethyl)-quinolin-3'-y1]-hept-6-enoic acid
`(j) (E)-3,5-dihydroxy-744.-(4"-chloropheny1)-2'-(1"-
`tnethylethyl)-6'-chloro-quinolin-3Ly1]-hept-6-enoic acid
`
`(k) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'-(1" -
`methylethyl)-6'-methyl-quinolin-3'-y1]-hept-6-enoic acid
`(1) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'-(1"-
`methylethyl)-6',7'-dimethoxy-quinolin-3'-y1]-hept-6-enoic
`acid
`(m) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'-
`cyclopropyl-quinolin-3'-y1]-hept-6-enoic acid
`(n) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'-
`cyclopropyl-6'-chloro-quinolin-3'-yThept-6-enoic acid
`(o) (E)-3,5-dihydroxy-744'-(4"-chloropheny1)-2'-
`cyclopropyl-6'-methyl-quinolin-3'-yfthept-6-enoic acid
`(p) (E)-3,5-dihydroxy-7[4'44"-chloropheny1)-2'- 25
`cyclopropy1-6'7'-dimethoxy-quinoliri-3'-y1]-hept-6-enoic
`acid
`(q) (E)-3,5-clihydroxy-744-'-pheny1-2;-(1"-methylethyl)-
`quinolin-3'-y1]-hept-6-enoic acid
`(r) (E)-3,5-dihydroxy-7[4'-pheny1-2'-(1"-methylethyl)- 30
`6'-chloro-quinolin-3'-y1]-hept-6-enoic acid
`(s) (E)-3,5-dihydroxy-744'-pheny1-2'-(1"-methylethyl)-
`6'-methyl-quinolin-Y-A-hept-6-enoic acid
`(t) (E)-3,5-dihydroxy-744'-pheny1-2'-( 1"-methylethyl)-
`6',7'-dimethoxy-quinolin-3'-y11-hept-6-enoic acid
`(u) (E)-3,5-dihydroxy-744'-pheny1-2'-cyclopropyl-
`quinolin-3'-y1]-hept-6-enoic acid
`(v) (E)-3,5-dihydroxy-744'-pheny1-2'-cyclopropy1-6'-
`chloro-quinolin-3'-yll-hept-6-enoic acid
`(w) (E)-3,5-clihydroxy-744Lphenyl-2'-cyclopropy1-6'- 40
`methyl-quinolin-3'-y1]-hept-6-enoic acid
`(x) (E)-3,5-dihydroxy-744Lphenyl-2'-cyclopropy1-6',7-
`dimethoxy-quinolin-3'-y1]-hept-6-enoic acid
`(y) (E)-3,5-dihydroxy-7-[4'-(4"-iluoropheny1)-2'-(1"-
`methylethyl)-6'-methoxy-quinolin-3.-y1]-hept-6-enoic acid 45
`(z) (E)-3,5-dihydroxy-744'-(4"-lluoropheny1)-2'-
`cyclopropyl-6'-methoxy-quinolin-3'-y1]-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.
`
`35
`
`SO
`
`6
`-continued
`
`CH2OH
`
`B
`
`R3
`
`VI
`
`Ra
`
`R6
`
`R3
`
`121
`
`R6
`
`R2
`
`IV
`
`123
`
`R
`
`R6
`
`CHO
`
`D
`
`E
`
`ut
`
`CO212,2
`
`A
`
`VII
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 6 of 18
`
`5,856,336
`
`7
`-continued
`R4
`
`011
`
`CO2RI2
`
`1-1
`
`R4
`
`OH
`
`CO21212
`
`8
`-continued
`R4
`
`CO2R22
`
`VIII
`
`Rh
`Zz
`
`R
`
`RI
`
`R3
`
`IX
`
`R4
`
`M
`
`C1120H
`
`CHO
`
`5
`
`10
`
`15
`
`20
`
`25
`
`3S
`
`RI
`
`III
`
`R3
`
`R4
`
`OH
`
`co2R12
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`R3
`
`011
`
`co,RI2
`
`In the above reaction scheme, 121, R2, R3, R4, R5, R6 and
`Ri2 are as defined above with respect to the formula 1, and
`
`H
`
`J
`
`K
`
`R2
`
`RI
`
`R2
`
`R
`
`1-4
`
`V
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 7 of 18
`
`5,856,336
`
`9
`R21 and R22 independently represent C1_4 lower alkyl such
`as methyl, ethyl, n-propyl, i-propyl or n-butyl.
`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 diisobutylalu- s
`minium hydride, in a solvent such as tetrahydrofilran 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 10
`various oxidizing agents. Preferably, the reaction can be
`conducted by using pyridinium chlorochromate in methyl-
`ene chloride at a temperature of from 0° to 25 ° C., or by
`using oxalyl chloride, climethyl sulfoxide and a tertiary
`amine such as triethylamine (Swern oxidation), or by using 15
`a sulfur trioxide pyridine complex.
`Step C represents a synthesis of a 3-ethoxy-1-hydroxy-
`2-propene derivative, which can be prepared by reacting a
`compound V to lithium compound which has been prelimi-
`narily formed by treating cis-1-ethoxy-2-(tri-n-butylstannyl) 20
`ethylene with butyl lithium in tetrahydrofuran.
`As the reaction temperature, it is preferred to employ a
`low temperature at a level of from -60° to -78° C.
`Step D represents a synthesis of an enal by acidic hydroly-
`sis. As the acid catalyst, it is preferred to employ p-toluene 25
`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-1-hydroxy-2-propene derivative
`obtained in Step C can be used in Step D without purification 30
`i.e. by simply removing tetra-n-butyl tin formed simulta-
`neously.
`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 35
`and n-butyl lithium as the base in tetrahydrofuran at a
`temperature of from -80° to 0° C., preferably from -30° to
`-10° C.
`Step F represents a reduction reaction of the carbonyl
`group, which can be conudctecl by using a metal hydride, 40
`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 borohydrkie in dry ethyl ether or dry tetrahydrofuran at
`a temperature of -100° to 25° C., preferably from -80° to 45
`-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 ethanol at a 50
`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
`dehydration reaction of the free hydroxy acid 1-2. The
`dehydration reaction can be conducted in benzene or toluene
`under reflux while removing the resulting water or by adding
`a suitable dehydrating agent such as molecular sieve.
`Further, the dehydration reaction may be conducted in dry
`methylene chloride by using a lactone-forming agent such as
`carbodiimide, preferably a water soluble carbodiimide such
`as N-cyclohexyl-N'-[2'-(methylmorpholinium)ethyl]
`carbodiimide 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 quino-
`line ring. This hydrogenation reaction can be conducted by
`using a catalytic amount of palladium-carbon or rhodium-
`
`65
`
`55
`
`60
`
`10
`carbon in a solvent such as methanol, ethanol, tetrahydro-
`furan 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
`a,13-unsaturated carboxylic acid ester, whereby a trans-form
`a,(3-unsaturated carboxylic acid ester can be obtained by a
`so-called Horner-Wittig reaction by using an alkoxycarbo-
`nylmethyl phosphonate. The reaction is conducted by using
`sodium hydride or potassium t-butoxicle as the base in dry
`tetrahydrofuran at a temperature of from -30° to 0° C.,
`preferably from -20° to -15° C.
`Step L represents a reduction reaction of the a,(3-
`unsaturated carboxylic acid ester to an allyl alcohol. This
`reduction reaction can be conducted by using various metal
`hydrides, preferably diisobutylaluminiumhydrkle, in a sol-
`vent such as dry tetrahydrofuran or toluene at a temperature
`of from -10° to 10° C., 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 active man-
`ganese 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,(3-unsaturated ketone by the selective oxidation of the
`dihydroxy carboxylic acid ester. This reaction can be con-
`ducted by using activated manganese dioxide in a solvent
`such as ethyl ether, tetrahydrofuran, benzene or toluene at a
`temperature of from 20° to 80° C., preferably from 40° to
`80° C.
`In addition to the compounds disclosed in Examples given
`hereinafter, compounds of the formulas 1-2 and 1-5 given in
`Table 1 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.
`
`TABLE 1
`
`1-2 (102.--
`1_5 (R12= Na)
`
`R'
`
`6-0Ntle
`6-0Me
`6-Br
`6-Me
`7-OMe
`6-Br
`
`6,7
`
`11
`
`11
`8-Me
`8-0Me
`
`R'
`
`1-1
`4-F
`4-F
`4-0
`4-F
`2-F
`
`4-F
`
`FI
`
`1-1
`II
`
`II
`
`i-Pr
`i-Pr
`i-Pr
`i-Pr
`i-Pr
`i-Pr
`
`i-Pr
`
`FT
`H
`
`11
`1-1
`H
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 8 of 18
`
`5,856,336
`
`12
`They may be formulated into various suitable formula-
`tions 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
`5 hydrolyzable and acceptable esters or lactones, or pharma-
`ceutically acceptable salts.
`The pharmaceutical composition of the present invention
`is preferably administered orally in the form of the com-
`pound of the present invention per se or in the form of
`10 powders, granules, tablets or capsules formulated by mixing
`the compound of the present invention with a suitable
`pharmaceutically acceptable carrier including a binder such
`as hydroxypropyl cellulose, syrup, gum arable, gelatin,
`sorbitol, tragacanth gum, polyvinyl pyrrolidone or CMC-Ca,
`15 an excipient such as lactose, sugar, corn starch, calcium
`phosphate, sorbitol, glycine or crystal cellulose powder, a
`R° 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, polyethyl-
`ene 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 hydro-gel base material, an injection formula-
`tion formulated by using one or more materials selected
`from the group consisting of polyethylene glycol, hydro-gel
`30 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 gas-
`trointestinal tract.
`'The daily dose of the compound of the formula I is from
`0 05 to 500 mg, preferably from 0.5 to 50 mg for an adult.
`-
`40
`It is administered from once to three times per day. The close
`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 inven-
`tion 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 pharmaco-
`logical 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.
`
`20
`
`25
`
`35
`
`45
`
`50
`
`11
`
`TABLE 1-continued
`
`R3 R3 OH
`
`CO2102
`
`OH
`
`R2
`
`H
`
`R3
`
`4-F
`
`R6 R5
`
`126
`
`R2
`
`RI
`
`12`
`
`H
`
`II
`II
`4-Ph
`II
`i-Pr
`II
`II
`4-PhCII2 H
`i-Pr
`6-CI
`H
`4-F
`H c-Pr
`6-CI
`H
`4-F
`H
`sec-Bu
`6-0CH2Ph H
`4-F
`H
`i-Pr
`H
`4-F
`H
`H
`i-Bu
`H
`H
`4-F
`FI
`c-Pent
`6-Cl
`H
`4-F
`H c-Pent
`6-Me2INI
`II
`4-F
`II
`i-Pr
`6-Me
`4-F
`II
`II
`c-Pr
`6-i-Pr
`II
`4-F
`II
`i-Pr
`7-Me
`H
`4-F
`H
`c-Pr
`6-OMe
`H
`4-F
`H
`c-Pr
`6-Br
`4-F
`H
`H
`c-Pr
`6-i-Pr
`F1
`4-F
`II
`c-Pr
`6-CI
`8-CI
`4-F
`Il
`c-Pr
`5-F
`6-Br
`4-F
`H i-Pr
`6-OMe
`4-F
`7-OMe
`II
`i-Pr
`6-Me
`7-Me
`4-F
`H
`i-P1
`6-CI
`7-CI
`4-F
`FI
`i-Pr
`H
`H
`4-F
`H
`c-Bu
`FI
`H
`4-F
`1-1
`c-Hex
`6-OMe
`7-OMe
`H
`H
`i-Pr
`6-OMe
`7-OMe
`4-CI
`I-I
`i-Pr
`6-0Me
`7-OMe H
`H c-Pr
`6-0Me
`7-OMe 4-CI
`H
`c-Pr
`6-0Me
`7-OMe
`4-F
`H
`c-Pr
`6-Me
`II
`H
`H
`i-Pr
`6-Me
`H
`4-Cl
`H
`i-Pr
`6-Mc
`II
`H
`1-I
`c-Pr
`6-Mc
`II
`4-CI
`II
`c-Pr
`6-Me
`II
`4-F
`II
`c-Pr
`6-CI
`H
`H
`H
`i-P1
`6-Cl
`II
`4-CI
`li
`i-Pr
`6-CI
`H
`H
`H c-Pr
`6-CI
`H
`4-Cl
`H c-Pr
`6-CI
`II
`4-F
`H c-Pr
`II
`H
`II
`H i-Pr
`II
`II
`4-CI
`Fl
`i-Pr
`H
`II
`H
`H c-Pr
`1-1
`H
`4-Cl
`I-1
`c-Pr
`H
`H
`4-F
`H c-Pr
`
`1-2 (R 12= H)
`1-5 (R'2— Na)
`
`II
`II
`EI
`H
`H
`H
`II
`H
`II
`II
`1-1
`II
`H
`II
`14
`II
`8-Br
`8-0Me
`8-Me
`8-CI
`H
`I-1
`H
`II
`I-I
`H
`II
`H
`H
`II
`II
`II
`Ii
`H
`II
`H
`II
`LI
`II
`II
`H
`II
`
`Further, pharmaceutically acceptable salts such as potas-
`sium salts or esters such as ethyl esters or methyl esters of
`these compounds can be prepared in the same manner.
`The compounds of the present invention exhibit high
`inhibitory activities against the cholesterol biosynthesis
`wherein IIMG-CoAreductase 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 cholesterol
`in blood as lipoprotein. Thus, the compounds of the present
`invention are useful as curing agents against hyperlipidemia,
`hyperlipoproteinemia and atheroscleosis.
`
`55
`
`PHARMACOLOGICAL TEST EXAMPLES
`Test A: Inhibition of cholesterol biosynthesis from acetate in
`vitro
`Enzyme solution was prepared from liver of male Wistar
`rat billialy cannulated and discharged bile for over 24 hours.
`60 Liver was cut out at mid-dark and microsome and superna-
`tant fraction which was precipitable with 40-80% of satu-
`ration of ammonium sulfate (sup fraction) were prepared
`from liver homogenate according to the modified method of
`Knauss et. al.; Kuroda, M., et. al., Biochim. Biophys. Acta,
`65 489, 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 20O p1 of the
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 9 of 18
`
`5,856,336
`
`14
`
`TABLE 2
`
`Inhibitory activities by Test A
`
`10
`
`
`(Compounds of theinvention])
`present
`
`1-13
`1-51
`1-52
`I-53
`(Reference compounds)
`
`Mevinolin
`CS-514
`
`[so (molar concentration)
`
`1.25 x 10-7
`
`1.0 x 10-6
`7.1 x 10-6
`1.9 x 10'
`
`1.4 x 10-6
`9.0 x 10-9
`
`In Table 2-2, the relative activities are shown based on the
`activities of CS-514 being evaluated to be 1.
`
`TABLE 2-2
`
`Relative activities by Test A
`
`Relative activities
`
`1.75
`2.25
`037
`3.21
`0.76
`
`OH
`
`CH3
`
`13
`reaction mixture containing ATP; 1 mM, Glutathione; 6 mM,
`Glucose-l-phosphate; 10 mM, NAD; 0.25 mM, NADP; 0.25
`mM, CoA; 0.04 mM and 0.2 mM [2-14C]sodium acetate (0.2
`,tiCi) with 4 id of test compound solution dissolved in water
`or dimethyl sulfoxide. "co stop reaction and saponify, 1. ml of 5 Compound
`15% Et0II-K011 was added to the reactions and heated at
`75° C. for 1 hour. Nonsaponifiable lipids were extracted with
`petroleum ether and incorporated 14C radioactivity was
`counted. Inhibitory activity of compounds was indicated
`with IC50.
`Test B: Inhibition of cholesterol biosynthesis in 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 15
`(PBS) at 37° C., 5% CO, until cells were confluent for about
`7 days. 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 20
`assay and 10 ,t41 of test compound solution dissolved in water
`or DMSO were added. 0.2 p.Ci of [2-14C]sodium acetate (20
`,eel) was added at O hr(B-1) or 4 hrs(B-2) after addition of
`compounds. After 4 hrs further incubation with [2-14C]
`Compound
`sodium acetate, medium was removed and cells were 25
`washed with phosphate buffered saline(PBS) chilled at 4° C.
`(Compounds of the
`Cells were scraped with rubber policeman and collected to
`present invention)
`tubes with PBS and digested with 0.2 ml of 0.5N KOH at
`37° C. Aliquot of digestion was used for protein analysis and
`remaining was saponified with 1 ml of 15% EtOH-KOH at 30
`75° C. for 1 hour. Nonsaponifiable lipids were extracted with
`petroleum ether and 14C radioactivity was counted. Counts
`were revised by cell protein and indicated with DPM/mg
`protein. Inhibitory activity of compounds was indicated with
`1050.
`Test C: Inhibition of cholesterol biosynthesis in vivo
`Male Sprague-Dawley 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 40
`PM-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 0.02-0.2 mg/kg
`body weight (0.4 m1/100 g body weight), were dissolved in as
`water or suspended or in 0.5% methyl cellulose and orally
`administered at 2-3 hours before mid-dark (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 sample administration, rats were
`injected intraperitoneally with 10 itCi of [2-14C]sodium
`acetate at volume of 0.2 ml per one. 2 Hours later, blood
`samples were obtained and serum were separated immedi-
`ately. Total lipids were extracted according to the method of
`Folch et al. and saponified with Et0H-KOH. Nonsaponifi-
`able lipids were extracted with petroleum ether and radio
`activity 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 60
`that in control group.
`With respect to the compounds of the present 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 65
`results are shown in Tables, 2, 2-2, 3 and 3-2. Further, the
`results of the measurements by Test C are also presented.
`
`1-16
`1-116
`
`11--112107
`1-522
`
`35
`
`Structures of reference compounds:
`
`(1) Mevinolin
`
`H3c
`
`CH3
`
`fl3c.*
`
`(2) CS-514
`
`50
`
`55
`
`H3c
`
`

`

`Case 1:14-cv-02758-PAC Document 63-1 Filed 05/08/15 Page 10 of 18
`
`5,856,336
`
`15
`
`TABLE