uro ean aen
`
`P t
`
`ice
`
`tOff
`
`® Publication number:
`
`0
`
`p
`
`Office européen des brevets
`
`E
`
`@
`
`. ~0)
`
`®
`
`
`
`@ A 15-deoxyprostaglandin derivative of general formula (I) and an intraocuIar pressure depressant containing
`said derivative or a pharmaceutically acceptable salt thereof as the active ingredient. In formula (I), (a) is a five-
`membered ring selected from among (b),
`(c),
`(d),
`(
`),
`(f); R1
`represents hydrogen or
`lower alkyl; and R2
`represents Cs to 012 alkyl, Cs to C12 alkenyl or Cs to 012 alkadienyl, provided that a compound of formula (II)
`(wherein R' represents H or CH3) is excluded.
`
`EUROPEAN PATENT APPLICATION
`published in accordance with Art.
`158(3) EPC
`
`® Application number: 91905484.1
`
`@ Int. 01.5: C07C 405/00, A61K 31/557
`
`@ Date of filing: 07.03.91
`
`International application number:
`PCT/JP91/00305
`
`International publication number:
`WO 91/13869 (19.09.91 91/22)
`
`Priority: 08.03.90 JP 57476/90
`
`Date of publication of application:
`26.02.92 Bulletin 92/09
`
`Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IT LI LU NL SE
`
`@ 15-DEOXYPROSTAGLANDIN DERIVATIVE.
`
`EP0471856A1
`
`® Applicant: SHIONOGI & 00., LTD.
`1-8, Doshomachi 3-chome Chuo-ku
`Osaka 541(JP)
`
`@ Inventor: KISHI, Morio, 31-26,
`Katagihara-takodencho
`NishikyO-ku
`Kyoto-shi, Kyoto 615(JP)
`Inventor: TAKAHASHI, Kimio, 1-115,
`Furukawa-cho 3-chome
`
`Nishinomiya-shi
`Hyogo 663(JP)
`Inventor: KAWADA, Kenji, C22-107, 3-1,
`Shinsenrinishi-machi
`
`Toyonaka-shi
`Osaka 565(JP)
`Inventor: GOH, Yasumasa, 5-8, Yayoigaoka
`4-chome
`Sanda-shi
`
`Hyogo 669-13(JP)
`
`Representative: Hardisty, David Robert et al
`BOULT, WADE & TENNANT 27 Furnival Street
`London EC4A IPQ(GB)
`
`
`
`Rank Xerox (UK) Business Services
`
`Micro Labs Exhibit 1004
`
`Micro Labs Exhibit 1004
`
`

`

`EP 0 471 856 A1
`
`CE:CH-CH(CH;)3C00R‘
`
`<1
`
`R2
`
`'fifiéiaid
`
`
`
`<9:
`
`30.,
`
`‘.
`
`/
`
`no
`
`(13)
`
`0 (c)
`
`‘0'.
`
`(d5
`
`0 (e)
`
`(f)
`
`H0“
`
`(m
`
`Micro Labs Exhibit 1004-2
`
`Micro Labs Exhibit 1004-2
`
`

`

`EP 0 471 856 A1
`
`The present invention relates to 15-deoxyprostaglandin derivatives and to pharmaceutical composition
`containing the same.
`Prostaglandins are a class of physiologically active substances which are derived from eicosapolyenoic
`acid such as arachidonic acid through biosynthetic pathway in animal tissues and have, as a fundamental
`chemical structure, prostanoic acid of the formula:
`
`5
`
`es\/\/\/COOH
`
`a chain
`
`cu chain
`
`Prostaglandins are produced through biosynthesis in various tissues, and classified into several families.
`Thus, prostaglandins are categorized into A - J groups depending on the position of oxgen atom attached to
`the 5 membered ring moiety and the position of a double bond in the ring moiety. Alternatively,
`prostaglandins can be categorized into 3 groups depending on the number of bouble bonds in the side
`chains. As a result, prostaglandins are designated as PGAz, PGE1, PGFZQ, according to both categoriza-
`tions.
`
`Prostaglandins possess as a whole diverse bioactivities, which include, for example, vasodilator activity,
`platlet aggregation-inhibiting activity, uterotonic activity, gastrointestinal motility-promoting activity, etc.
`Futher, some prostaglandins have intraocular pressure-reducing activity. For example, Japanese Patent
`Publication (kokai) No. 1418/1984 describes that PGFZQ has high intraocular pressure-reducing activity and
`that 15-keto-PGF2a has the same activity although it is less potent. However, these natural prostaglandins
`are chemically and biologically labile, and are easily subject to metabolic degradation because they contain
`in the chemical structure a labile ally alcohol moiety comprising a double bond between 13 and 14 positions
`and a hydroxy group at 15 position in to chain.
`13,14-Dihydro-15-ketoprostaglandin which is a metabolic product of prostaglandins has been known as
`a compound which does not contain the labile moiety and has been known to be a useful compound having
`the intraocular pressure-reducing activity.
`The inventors of the invention have found new useful compounds by screening a large amount of
`prostaglandin derivatives which are stable and capable of being chemically synthesized.
`Thus, the present invention provides 15-deoxyprostaglandin derivatives of the formula (I):
`
`
`
`CH2CH=CH(CH2)3C00R1
`
`is a 5 membered ring which is selected from a group consisting of
`
`Micro Labs Exhibit 1004-3
`
`Micro Labs Exhibit 1004-3
`
`

`

`EP 0 471 856 A1
`
`Fi1 is hydrogen or lower alkyl;
`R2 is 06'012 alkyl, 05-012 alkenyl or 05-012 alkadienyl;
`provided that the compound of the following formula is excluded:
`
`
`
`the invention provides a new formulation which is useful as an intraocular
`As another embodiment,
`pressure-reducing agent. The inventors of the invention found that derivatives of conventional prostaglan-
`dins which are derived from said conventional prostaglandins by deleting the hydroxy group at 15-position
`are more stable, particularly in liquid phase, than the conventional prostaglandins, and that they show the
`intraocular pressure-reducing activity. Thus,
`the invention provides a new use of these derivatives.
`In
`particular,
`the compounds of the invention as described above have a significant
`intraocular pressure-
`reducing activity, while they do not produce any side effects such as hyperemia of conjunctiva, and initial
`increase in intraocular pressure which are often observed in known prostaglandins. Accordingly, the 15-
`deoxyprostaglandins of the invention may be a therapeutical agent useful for treating an ocular disease, in
`particular glaucoma, which is assumed to be caused by increased intaocular pressure.
`The term "15-deoxyprostaglandin derivatives" used in the specification and the claims refer to any
`derivatives from natural prostaglandins (preferably, PGAl, PGAz, PGB1, PGBz, PGE1, PGEz, PGEa, PGFM,
`PGFZQ, PGFaa, PGJ1, or PGJ2), which lack the hydroxy group at 15 position in the to chain. Said to chain
`may be saturated or unsaturated, and may have one or more double and/or triple bonds. Further,
`the
`double bonds may be E form, Z form or a mixture thereof. Preferably, however, the a chain has Z form and
`the to chain has E form. Further, the derivatives of the invention include those in which the terminal carbon
`
`wherein R' is hydrogen or methyl;
`or pharmaceutically acceptable salts thereof. The term "Cs-012 alkyl" in the definition of R2 refers to hexyl,
`heptyl, octyl, nonyl, decyl, undecyl, dodecyl. R2 may be an unsaturated haydrocarbon chain containing one
`or more double bonds, with Cs-C1o alkenyl or C8-C1o alkadienyl being preferred. Specific examples of the
`unsaturated hydrocarbon chain are 1-hexenyl, 2—hexenyl, 1,3-hexadienyl, 1-heptenyl, 2—heptenyl, 1,3-
`heptadienyl, 1-octenyl, 2—octenyl, 1,3-octadienyl, 1-nonenyl, 2—nonenyl, 1,3-nonadienyl, 1-decenyl, 2—de-
`cenyl, 1,3-decadienyl, 1-undecenyl, 2-undecenyl, 1,3-undecadienyl, 1-dodecenyl, 2-dodecenyl, and 1,3-
`dodecadienyl.
`Further, 15-deoxyprostaglandin derivatives of the above foumula in which
`
`are also preferred.
`15-Deoxyprostaglandin derivatives of
`thereof.
`
`the formula (I)
`
`include all of
`
`the stereoisomers and mixture
`
`in the to chain (20 position) is bound to a lower alkyl or a lower alkenyl. The derivatives may have 2 - 4
`
`Micro Labs Exhibit 1004-4
`
`Micro Labs Exhibit 1004-4
`
`

`

`EP 0 471 856 A1
`
`asymmetric carbon atoms. The invention includes all of the optical isomers and the mixture thereof.
`Among the 15-deoxyprostaglandin derivatives of
`the invention which are useful as an intraocular
`pressure-reducing agent, preferred derivatives are those having the formula (I):
`
`CH 2CH=CH(CH z) 3C00R1
`
`is a 5-membered ring selected from
`
`Fl1 is hydrogen or lower alkyl;
`R2 iS Cs-C12 alkyl, C5-C12 alkenyl or Cs-C12 alkadienyl;
`or pharmaceutically acceptable salts thereof.
`Most preferred derivatives are (52,13E,98,11R)—9,11-dihydroxy-5,13-prostadienoic acid of the formula:
`
`
`
`or pharmaceutically acceptable salts or esters thereof. Among the above preferred derivatives, a sodium
`salt of (52,13E,98,11R)-9,11-dihydroxy-5,13-prostadienoic acid is especially preferred in the light of solubil-
`ity in water.
`It should be noted that (52,13E,98,11R)—9,11-dihydroxy-5,13-prostadienoic acid of the formula:
`
`“0W CO 2 H
`
`and methyl ester thereof have been described in Gorman, Proc., Natl., Acad., Sci, U.S.A. 74 vol.,§,4007—
`
`Micro Labs Exhibit 1004-5
`
`Micro Labs Exhibit 1004-5
`
`

`

`EP 0 471 856 A1
`
`in which R is hydroxy-protecting group, R2 is the same meaning as defined above is reduced with a metal
`hydride to form a lactol of the formula (III):
`
`4011. However, Gorman neither describes nor suggests the use of the compounds as an intraocular
`pressure-reducing agent.
`The carboxy moiety in the above-noted preferred compounds may be either a free carboxylic acid or a
`pharmaceutically acceptable salt or ester thereof. The salt may be, for example, an alkali metal salt such as
`a lithium salt, a sodium salt, or a potassium salt; an alkaline earth metal salt such as a calcium salt; an
`ammonium salt;
`an
`organic
`base
`salt
`such
`as
`triethylamine,
`2-aminobutane,
`tertbutylamine,
`diisopropylethylamine, n-butylmethylamine, n-butyldimethylamine,
`tri-n-butylamine, dicyclohexylamine, N-
`isopropylcyclohexylamine,
`tromethamine, furfurylamine, benzylamine, methylbenzylamine, dibenzylamine,
`N,N,-dimethylbenzylamine, 2—chlorobenzylamine, 4-methoxybenzylamine, 1-naphthylmethylamine, diphenyl-
`benzylamine,
`triphenylamine,
`1-naphtylamine,
`1-aminoanthracene,
`2-aminoanthracene,
`de-
`hydroabietylamine, N-methylmorpholine or pyridine; an amino acid salt such as a lysine or arginine salt.
`Examples of the ester are a lower alkyl ester, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
`tert-butyl or pentyl ester. The lower alkyl ester moiety may be substituted with an optional group.
`The compounds of the invention can be prepared by the following process:
`A lactone of the formula (IV):
`
`
`
`in which R and R2 are the same meaning as above.
`The resultant lactol
`(III)
`is then reacted with an ylide in the condition of Wittig reaction to yield the
`compound of the formula (II):
`
`\CHZCH=CH(CH2)3C00H
`
`\
`
`in which R and R2 are the same meaning as above.
`Following deprotection and optional esterification or salification of the compound, 15-deoxyprostaglandin
`derivatives of the type of PGF2,, can be obtained. The hydroxy-protecting group may be selected from
`
`Micro Labs Exhibit 1004-6
`
`Micro Labs Exhibit 1004-6
`
`

`

`EP 0 471 856 A1
`
`various protecting groups which are described in Protective Groups in Organic Synthesis, T.W.Greene,
`John Wiley & Sons, Inc., New York, p. 10, 1981. Examples of the protecting group are those forming an
`alkyl ether, such as methyl, methoxymethyl, methylthiomethyl, 2-methoxyethoxymethyl, tetrahydropyranyl,
`1-ethoxyethyl, benzyl, and p-methoxybenzyl;
`those forming a silyl ether, such as triethylsilyl,
`t-butyl-
`dimethylsilyl, and t-butyldiphenylsilyl; those forming an ester, such as acetyl, benzoyl, p-methylbenzoyl, and
`o-methoxybenzoyl.
`is prepared from the aldehyde analogue which is obtained by
`The lactone (IV) as a starting material,
`oxidating commercially available Corey lactone, using the process described below:
`
`(i) Synthesis of compounds (IV) wherein R2 is 1-alkenyl
`
`The compounds (IV) can be prepared by reacting the above-noted aldehyde and an ylide prepared
`from an alkyltriphenylphosphonium halide in the condition of Wittig reaction.
`If desired, cis-trans isomeriza-
`tion reaction may be conducted on the resultant compounds.
`
`The above aldehyde is reacted with a formylalkylenetriphenylphosphorane or an ylide obtainable from
`an alkyltriphenylphosphonium halide having a protected hydroxy in the condition of Wittig reaction.
`If
`desired, catalytic reduction or cis-trans isomerization reaction can be conducted.
`In the case that the ylide having a protected hydroxy is used, deprotection and oxidation are carried out to
`form formyl compounds. A desired compound (IV) can be prepared by treating the resultant aldehyde thus
`obtained according to the process (i).
`15-Deoxyprostaglandin derivatives of the type of PGD2, PGE2, PGJ2 or PGA2 can be prepared by using
`the derivatives of the type of PGFZQ as a starting material. The outline of the process is shown in the
`scheme shown below. The details are illustrated in the working examples hereinafter described.
`
`
`
`(ii) Synthesis of compounds (IV) wherein R2 is alkyl
`
`The compounds (IV) can be prepared by catalytic reduction of the lactone (IV) obtained in the above
`process (i).
`
`(iii) Synthesis of compounds (IV) wherein R2 is unsaturated alkyl other than 1-alkenyl.
`
`Micro Labs Exhibit 1004-7
`
`Micro Labs Exhibit 1004-7
`
`

`

`EP 0 471 856 A1
`
`COMPOUDS (I I)
`
`l Protection
`
`Hon,
`
`CHZCH =CH(CH2) 3000123
`
`HOS A
`/
`O‘CHECH = CH(CH2) 30001?3
`
`6
`
`R2
`
`R3 : protecting group
`
`oxidation
`
`\ 0
`31
`
`6‘,
`H0‘
`
`R2
`
`CHZCH = CH(CH2) 3C00R3
`
`and
`
`110,,
`
`R 2
`
`0
`
`
`
`The 15-deoxyprostaglandin derivatives of the invention can be administered topically or systemically
`using known procedures for administration. The derivatives of the invention can be formulated into a dosage
`form which is suitable for oral,
`intraarterial,
`intravenous,
`intracutaneous,
`intramuscular,
`intrarectal, or
`ophthalmic administration.
`Recently, the inventors have found that the compounds in which the carboxy moiety is a lower alkyl
`ester are preferred in terms of pharmacological activity. However, esterification of the carboxylic acid results
`
`CD deprotection
`® optional esterification
`and/or salification
`
`CD deprotection
`(2 optional esterification
`and/or salification
`
`oCHzCH: CH(CH2)3CO0R1
`
`‘Rz
`
`(lb)
`
`0 \
`
`Ho“
`
`H0,,,’
`
`;
`
`‘
`
`oCH2CH:CH(CH:;)3000131
`
`(1c)
`
`dehydration
`l
`
`dehydration
`l
`
`SCHgCH =CH(CH2) 3C00R3
`
`O
`
`R2
`
`0
`
`¢CH2CH =CH(CH2)3COOR3
`
`Q
`
`R2
`
`(D deprotection
`® optional esterification
`and/or salification
`
`(D deprotection
`(2) optional esterification
`and/or salification
`
`€CH2CH=CH(CH2)3COORI
`
`@
`
`R2
`
`0
`
`°CH2CH=CH(CH2)3COOR1
`(Id) Q
`
`R2
`
`(1e)
`
`Micro Labs Exhibit 1004-8
`
`Micro Labs Exhibit 1004-8
`
`

`

`EP 0 471 856 A1
`
`the
`in preparing a formulation for use as an eye drop,
`in decrease of water-solubility. Accordingly,
`compound in the form of an ester is preferably formulated into an oily formulation, or the solubility of the
`compound may be improved either by addition of any surfactant to the formulation or introduction of any
`hydrophilic group (for example hydroxy or lower alkoxy such as methoxy) into the ester moiety of the
`compound.
`The dose of the compounds of the invention varys depending on purpose of the treatment and effect,
`method for administration, or age and body weight of a particular patient. Typically,
`in the case of oral
`administration,
`the dose is about 200 ug/kg/day - about 20 mg/kg/day, preferablly about 1 mg/kg/day -
`about 10 mg/kg/day, but not limited to them. In the case of ophthalmic application for treating glaucoma, the
`dose is about 0.01 ug - about 1000ug/eye/day, preferablly about 0.1 (Lg - about 200ug/eye/day, and this
`dose may be divided into 1 - 5 portions in application.
`
`Examples
`
`The following detailed examples are presented by way of illustration of certain specific embodiments of
`the invention. The examples are representative only and should not be construed as limiting the invention in
`any respect.
`
`Example 1
`
`Preparation
`(compound 2)
`
`of
`
`(1S,6R,7R)—2-oxa-3-oxo-6-[(1Z)-octenyl]—7-tetrahydropyranyloxy-E—bicyclom.3.0]octane
`
`
`
`Heptyltriphenylphosphonium bromide (3.78 g, 8.58 mmole) was suspended in tetrahydrofuran (23 ml),
`and potassium tert-butoxide (0.873 g, 7.79 mmole) was added thereto under ice-cooling. The mixture was
`stirred at the same temperature for 10 minutes. The dark orange mixture thus obtained was cooled to -
`78° C and 10 ml of a solution of (1S,6R,7R)-2-oxa-3-oxo-6-formyl-7-tetrahydropyranyloxy-cis—bicyclo[3.3.0]—
`octane (compound 1) (0.99 g, 3.89 mmole) in tetrahydrofuran, which had been prepared fro—m Corey lactone
`according to E.J.Co_rey, H. Shirahama, H. Yamamoto, S. Terashima, A. Venkateswarlu and T. K. Schaaf, J.
`Am. Chem.Soc., 93, 1490 (1971), was added thereto. The ice bath was removed, and the reaction mixture
`was allowed to wa—rm up to room temparature over 30 minutes while stirring. After stirred for additional one
`hour at
`room temparature, an aqueous solution of ammonium chloride was added. The mixture was
`concentrated in vacuo, and the residue was extracted with ether - ethyl acetate (2 : 1). The extract was
`washed with diuted hydrochloric acid, aqueous sodium bicarbonate and brine successively, and then dried
`over anhydrous magnescium sulfate, and the solvent was evaporated off. The resultant brown semi-solid
`material (3.1 g) was chromatographed over silica gel (39 g), eluting with 1
`: 7 -
`1
`: 3 of ethyl acetate : n-
`hexane, to yield 600 mg of a crude product. The product was purified with Lobar column (ethyl acetate : n-
`hexane = 1
`: 3) to yield 484 mg of the title compound 2 as a colorless oil (yield 37%).
`NMR (CDClg); 5 0.88 (t, J =7Hz, 3H), 1.1-3.05 (m, 22H),—3.50 (m, 1H), 3.8-4.1 (m, 2H), 4.67 (m, 1H), 5.05 (m,
`2H), 5.50 (m, 1H),
`IR (CHCls); 1765cm‘1.
`
`Example 2
`
`Preparation
`(compound 3)
`
`of
`
`(1S,6R,7R)—2-oxa-3-oxo-6-[(1E)-octenyl]-7-tetrahydropyranyloxy-cis—bicyclo[3.3.0]octane
`—
`
`1 mmole) was dissolved in benzene (15 ml), and 5-
`The compound 2 obtained in Example 1 (336 mg,
`mercapto-1-methyltetrazole disulfide (115 mg, 0.5 mmole) and a,a'-azobis-isobutyronitrile (16 mg, 0.1
`mmole) were added thereto, and the mixture was refluxed with stirring for 10 hours. The reaction mixture
`was washed with aqueous sodium carbonate and then with brine, and dried over anhydrous sodium sulfate,
`and the solvent was evaporated off. The resultant colorless oil (400 mg) was chromatographed over silica
`gel (10 g), eluting with 1:7 - 1:5 of ethyl acetate:n-hexane, to yield a curde product (260 mg). The product
`was further purified with Lobar column (ethyl acetate : n-hexane = 1:3) to yield 202 mg of the title
`compound 3 as a colorless oil (yield 60%).
`NMR (CDCE); 5 0.88 (t, J=7Hz, 3H), 1.1-2.8 (m, 22H), 3.50 (m, 1H), 3.7-4.2 (m, 2H), 4.68 (m, 1H), 4.95 (m,
`1H), 5.25 (m, 1H), 5.50 (m, 1H),
`IR (CHCls); 1765cm‘1.
`
`Micro Labs Exhibit 1004-9
`
`Micro Labs Exhibit 1004-9
`
`

`

`EP 0 471 856 A1
`
`Example 3
`
`Preparation of (5Z,13E,9S,11R)-9-hydroxy-11-tetrahydropyranyloxy-5,13-prostadienoic acid (compound 5)
`
`1 mmole) was dissolved in toluene (12 ml), and 1 M
`The compound 3 obtained above (336 mg,
`diisobutyl aluminum hydride in toluene (1.1 ml, 1.1 mmole) was added thereto at -78° C. The solution was
`stirred at the same temperature for one hour, and then aqueous ammonium chloride was added thereto,
`and the temperature was allowed to warm to room temperature. The reaction mixture was extracted with
`ethyl acetate, and the extract was washed with brine and dried over anhydrous sodium sulfate, and then the
`solvent was evaporated off. The residue (340 mg) was chromatographed over silica gel (3.4 g), eluting with
`1
`: 2 of ethyl acetate:n-hexane, to yield lactol 4 (314 mg, 93%) as a colorless oil.
`IR (CHCls); 3610, 3390
`cm“.
`
`suspended in
`(1.219 g, 2.76 mmole) was
`After 4-carboxybutyltriphenylphosphonium bromide
`tetrahydrofuran (15 ml), a potassium tert-butoxide (680 mg, 6.07 mmole) was added thereto under ice
`cooling, and the mixture was stirred at the same temperature for 10 minutes. The resultant red mixture was
`cooled to -30°C, and 6 ml of a solution of
`the lactol 4 obtained above (310 mg, 0.92 mmole)
`in
`tetrahydrofuran was added thereto. After removing the cooling bath, the reaction mixture was allowed to
`warm to room temperature with stirring, and stirred for additional one hour. To the resultant yellow mixture
`was added a solution of ammonium chloride in water, and the mixture was concentrated in vacuo. An ice
`
`water was added to the residue, and the mixture was acidified with diluted hydrochloric—acid to pH 4-5
`under cooling, and extracted with ethyl acetate. The ethyl acetate layer was extracted with aqueous sodium
`carbonate, and the water phase was acidified again to pH 4-5, and then extracted with a mixture of
`methylene chloride and ethyl acetate (2:1). After the extract was dried over anhydrous magnesium sulfate,
`the solvent was evaporated off to yield 790 mg of a semicrystalline product. To the product was added
`ethyl ether, and the dissolved portion was filtered and the solvent was evaporated off to yield 288 mg of a
`crude product. The product was chromatographed over silicic acid (8.6 g), eluting with 1:10 - 1:3 of ethyl
`acetate : n-hexane, to yield 234 mg of the title compound as a colorless oil (yield 60%).
`NMR (CDCls); 6 0.88 (t, J=7Hz, 3H), 1.2-2.5 (m, 30H), 3.45 (m, 1H), 3.85 (m, 1H), 4.07 (m, 2H), 4.69 (m,
`1H), 5.2-5.6 (m, 4H).
`IR (CHClg); 3500, 2700-2400 (broad), 1705 cm“.
`
`
`
`Example 4
`
`Preparation of (5Z,13E,9S,11R)-9,11-dihydroxy-5,13-prostadienoic acid (compound 6)
`
`The compound 5 obtained above (205 mg, 0.485 mmole) was dissolved in 4 ml of a mixture of acetic
`acid, water, and tetrahydrofuran (65:35:10). The mixture was stirred under heating at 40-45° C for one and
`half hours, and then evaporated to dryness in vacuo. The residue (184 mg) was chromatographed over
`silicic acid (5.59), eluting with 1:5 - 1:1 of ethyTacetate : n-hexane, to yield 149 mg of the title compound 6
`as a colorless oil (yield 91%).
`_
`NMR (CDClg); 6 0.88 (t, J=7Hz,3H), 1.2-2.5 (m, 22H), 3.94 (m, 1H), 4.17 (m, 1H), 4.0-4.8 (broad ,3H), 5.2-
`5.6 (m, 4H).
`IR (CHClg); 3400, 2700-2400(broad), 1705 cm“.
`
`Example 5
`
`Preparation of (5Z,13E,9S,11R)-9,11-dihydroxy-5,13-prostadienoic acid metyl ester (compound 7)
`
`The compound 6 obtained obove (104 mg, 0.307 mmole) was dissolved in acetonitrile (5 ml), and 1,8-
`diazabicyclo[5.4.0]—77undecene (70 LL], 0.46 mmole) was added thereto. The mixture was stirred at room
`temperature for 15 minuites. Then, methyl
`iodide (0.19 ml, 3 mmole) was added thereto and the mixture
`was stirred at room temperature for five hours, and then concentrated in vacuo. To the residue was added
`water, and the mixture was extracted with ethyl acetate. The extract w§s washed successively with diluted
`hydrochloric acid, aqueous sodium sulfite, and brine, and then dried over anhydrous magnesium sulfate.
`After the solvent was evaporated off, 113 mg portion of the residue was purified with Lobar colum (ethyl
`acetate : n-hexane = 1:1), to yield 103 mg of the title compound 7 as a colorless oil (yield 95%).
`NMR (CDClg); 5 0.88(t, J =7Hz, 3H), 1.2-2.4 (m, 24H), 3.67 (s, SHX 3.92 (m, 1H), 4.17 (m, 1H), 5.18-5.61 (m,
`4H).
`
`Micro Labs Exhibit 1004-10
`
`Micro Labs Exhibit 1004-10
`
`

`

`EP 0 471 856 A1
`
`IR (CHCls); 3450, 1720cm-1.
`
`Example 6
`
`Preparation of (52,13E,98,11R)-9,11-dihydroxy-5,13-prostadienoic acid isopropyl ester (compound 0)
`
`The title compound 8 was obtained as a colorless oil (36 mg, 95%), following a procedure similar to that
`disclosed in Example 5_except that the compound 6 obtained above (33 mg, 0.1 mmole) was dissolved in
`acetonitrile (1 ml), and 1,8—diazabicyclo[5.4.0]—7—undecene (40 ul, 0.3 mmole) and then isopropyl
`iodide
`(0.19 ul, 1 mmole) were added thereto.
`NMR (CDClg); 5 0.88 (t, J=7Hz, 3H), 1.22 (d, J=6Hz, 6H), 1.25-2.35 (m, 24H), 3.92 (m, 1H), 4.17 (m, 1H),
`5.00 (sept, J =6Hz, 1H), 5.18-5.60 (m,4H).
`IR (CHClg); 3450, 1720cm‘1.
`
`Example 7
`
`Test for demonstrating stability of the compound 6 [The formation of (52,13E,98,11R)—9,11-dihydroxy-5,13-
`prostadienoic acid (compound 0)]
`_
`
`The compound 7 obtained above (73 mg, 0.207 mmole) was dissolved in 2 ml of methanol. One ml of 1
`N aqueous pottasium hydroxide solution was added thereto, and the mixture was refluxed with stirring for
`one hour. After concentrating the solution to remove the methanol, the residue was dissolved in water and
`washed with ethyl acetate. The aqueous solution was acidified with adding diluted hydrochloric acid under
`cooling and extracted with ethyl acetate. The extract was washed with brine, and dried over anhydrous
`magnesium sulfate. Then, the solvent was evaporated off to give 70 mg of a crude product. The product
`was chromatographed over silicic acid (2 g), eluting with 1:1 of ethyl acetate : n-hexane, to give the title
`compound (68 mg, 97 %) as a colorless oil.
`The above reaction is illustrated in the following scheme.
`
`
`
`1N KOH
`—%
`MeOH
`
`Example 8
`
`Preparation
`(compound a)
`
`of
`
`(18,6R,7R)—2—oxa-3-oxo-6-[(1Z)-decenyl]-7-tetrahydropyranyloxy-cis-bicyclo[3.3.0]octane
`—
`
`Nonyltriphenylphosphonium bromide (3.85 g, 8.20 mmole) was suspended in 25 ml of tetrahydrofuran,
`and 0.836 g (7.46 mmole) of potassium tert-butoxide was added, and the mixture was stirred at the same
`temperature for 10 minutes. The deep orange mixture thus obtained was cooled to -78° C, and 10 ml of a
`solution of the compound 1 obtained above (0.95 g, 3.74 mmole) in tetrahydrofuran was added thereto.
`
`11
`
`Micro Labs Exhibit 1004-11
`
`Micro Labs Exhibit 1004-11
`
`

`

`EP 0 471 856 A1
`
`Then, the reaction mixture was treated following a procedure similar to that discolsed in Example 1, to yield
`the title compound 2' (573 mg, 42 %) as a colorless oil.
`NMR (CDCIg); 6 0.88 (t, J =7H2, 3H), 1.1-3.05 (m, 26H), 3.50 (m, 1H), 3.8-4.1 (m, 2H), 4.68 (m, 1H), 5.05 (m,
`2H), 5.50 (m, 1H).
`IR (CHCI3); 1765cm‘1.
`
`Example 9
`
`Preparation
`(compound i)
`
`of
`
`(18,6R,7R)—2-oxa-3-oxo-6-[(1E)-decenyI]-7-tetrahydropyranyloxy-cis-bicyclo[3.3.0]octane
`—
`
`The compounds 2' obtained above (566 mg, 1.55 mmole) was dissolved in 30 ml of benzene, and 5-
`mercapto-1-methyltet§zole disulfide (180 mg, 0.78 mmole) and a,a'-azobis-isobutyronitriIe (25 mg, 0.15
`mmole) were added thereto, and the mixture was refluxed with stirring for 2.5 hours. Then, the reaction
`mixture was treated following a procedure similar to that of Example 2, to yield the compound 3' (383 mg,
`67%) as a colorless oil.
`i
`NMR (CDCIg); 6 0.88(t, J=7Hz, 3H), 1.1-2.85 (m, 26H), 3.50 (m, 1H), 3.75-4.15 (m, 2H), 4.68 (m, 1H), 4.97
`(m, 1H), 5.25 (m, 1H), 5.50 (m, 1H).
`IR (CHCls); 1765cm-1.
`
`Example 10
`
`Preparation
`
`of
`
`(5Z,13E,98,11R)-20-ethyl-9-hydroxy-11-tetrahydropyrany|oxy-5,13-prostadienoic
`
`acid
`
`(compound 5')
`
`The compound 3' obtained above (398 mg, 1.09 mmole) was dissolved in 14 ml of toluene, and 1.2 ml
`of 1 M solution of dWsobutyI aluminum hydride (1.2 mmole) in toluene was added thereto at -78° C. Then,
`the reaction mixture was treated following the procedure similar to that of Example 3, to yield the compound
`4' (395 mg, 98%) as a colorless oil.
`W8 (CHClg); 3600, 3390 cm“.
`
`
`
`4-Carboxybutyltriphenylphosphonium bromide (1.409 g, 3.18 mmole) was suspended in tetrahydrofuran
`(15 ml), and potassium tert—butoxide (780 mg, 6.99 mmole) was added under ice-cooling, and the mixture
`was stirred at the same temperature for 10 minutes. The red mixture thus obtained was cooled to -30° C,
`and to the mixture was added 10 ml of a solution of the lactol compound 4' obtained above (388 mg, 1.06
`mmole) in tetrahydrofuran. Then, the reaction mixture was treated following—the procedure similar to that of
`Example 3, to yield the title compound 5' (304 mg, 64 °/o) as a colorless oil.
`NMR (CDCla); 5 0.88(t, J=7Hz, 3H), 112.5 (m, 34H), 3.45 (m, 1H), 3.85 (m, 1H), 4.07 (m, 2H), 4.70 (m,
`1H), 5.2 -5.6 (m, 4H).
`IR (CHClg); 3512, 2700-2400 (broad), 1708 cm“.
`
`Example 11
`
`Preparation of (5Z,13E,9$,11R)-20-ethyI-9,11-dihydroxy-5,13-prostadienoic acid (compound E)
`
`The compound 5' obtained above (297 mg, 0.66 mmole) was dissolved in 6 ml of a mixture of acetic
`acid, water, and tetrahydrofuran (65:35:10), and the solution was heated with stirring at 40-45°C for two
`hours. Then, the reaction mixture was treated following the procedure similar to that of Example 4, to yield
`the title compound 6' (194 mg, 80 %) as a colorless oil.
`NMR (CDCI3); 5 0.88 (t, J=7Hz, 3H), 1.2-2.4 (m, 26H), 3.93 (m, 1H), 4.18 (m, 1H), 3.4-4.5 (broad, 3H), 5.15
`-5.60 (m, 4H).
`IR (CHClg); 3400, 2700-2400 (broad), 1705 cm“.
`
`Example 12
`
`Preparation of (5Z,13E,98,11R)-20-ethyl-9,11-dihydroxy-5,13-prostadienoic acid isopropyl ester (compound
`8')
`
`Micro Labs Exhibit 1004-12
`
`Micro Labs Exhibit 1004-12
`
`

`

`EP 0 471 856 A1
`
`The compound 6' obtained above (37 mg, 0.1 mmole) was dissolved in acetonitrile (1ml), and 1,8-
`diazabicyclo[5.4.0]—73ndecene(75 MI, 0.5 mmole) and isopropyl iodide (0.29ml, 3mmole) were successively
`added thereto. Then, the reaction mixture was treated following the procedure similar to that of Example 5,
`to yield the title compound 8' (35 mg, 85 %) as a colorless oil.
`NMR (CDCig); 6 0.88 (t, J=_7Hz, 3H), 1.22 (d, J=6Hz, 6H), 1.25-2.40 (m, 28H), 3.92 (m, 1H), 4.18 (m, 1H),
`5.01 (sept, J =6Hz, 1H), 5.15-5.60 (m,4H).
`IR (CHCls); 3500, 1715cm‘l.
`The comprehensive scheme illustrating the reactions described in Examples 1-12 is shown below.
`
`
`
`Micro Labs Exhibit 1004-13
`
`Micro Labs Exhibit 1004-13
`
`

`

`EP 0 471 856 A1
`
`Reaction scheme for preparation of
`
`[113—136
`
`(Examples 1-12)
`
`Br—
`Ph3P+(CH2)n+1CHg
`t—BuOK
`THF
`
`CH0
`
`’3
`
`5
`
`(CH2)DCH3
`
`2
`
`Y
`
`fi
`
`)IN\
`
`\IS
`CH3
`AIBN
`CeHe
`
`———————————+
`i-BUZMH
`toluene
`
`Br
`
`Ph3P+(CH2)4C02H
`
`————————9
`t—B 0K
`“
`THF
`
`'
`
`<<::]:;/E::i/\\//\‘C02H
`
`//
`
`(CH2)nCH3
`
`¢
`
`A603
`
`a
`H O,THF
`2
`
`THPO
`
`1
`
`n
`
`n
`
`M61
`
`DBU
`CH3CN
`
`e
`
`Hq
`"4,
`
`e
`
`H0
`
`(or i-PrI) <:::[;::::f/\\//\\cozk
`
`
`
`(1S,6R,7R)-2-oxa-3-oxo-6-octyl-7-tetrahydropyranyloxy-cis—bicyclo[3.3.0]octane (compound
`—
`
`H0”
`I,’
`
`v
`
`¢\\;=;/A\V/»\\coz
`
`Example 13
`
`Preparation of
`9)
`
`/
`
`(CH2)nCH3
`
`8 "=5
`=isopropy1
`
`R=isopropy1
`
`Micro Labs Exhibit 1004-14
`
`Micro Labs Exhibit 1004-14
`
`

`

`EP 0 471 856 A1
`
`The compound 2 obtained above (405 mg, 1.20 mmole) was dissolved in methanol (10 ml), and 5%
`paladium - carbon _(80 mg) was added thereto, and the mixture was stirred under hydrogen at one
`atmospheric pressure for one hour. After the mixture was filtered and the filtrate was evaporated,
`the
`residue (405 mg) was chromatographed over silica gel (4 g), eluting with 1:10 - 1:3 of ethyl acetate : n-
`hexane, to yield the title compound 9 (393 mg, 97 %) as a colorless oil.
`NMR (CDClg); 6 0.88 (t, J=7Hz, 3H_), 1.1-2.9 (m, 26H), 3.50 (m, 1H), 3.75-4.15 (m, 2H), 4.66 (m, 1H), 5.01
`(m, 1H).
`IR (CHCls); 1764 cm“.
`
`Example 14
`
`Preparation of (52,98,11R)-9-hydroxy-11-tetrahydropyranyloxy-5-prostenoic acid (compound 11)
`
`The compound 9 obtained above (378 mg, 1.12 mmole) was dissolved in toluene (12 ml), and 1 M
`solution of diisobutyl_aluminum hydride in toluene (1.23 ml, 1.23 mmole) was added thereto at -78° G. Then,
`the reaction mixture was treated following the procedure similar to that of Example 3, to yield lactol 10 (370
`mg, 97 %) as a colorless oiI.
`—
`IR (CHCl3); 3594, 3384 cm‘1.
`4-Carboxybutyltriphenylphosphonium bromide (1.427 g, 3.21 mmole) was suspended in tetrahydrofuran
`(15 ml), and potassium tert—butoxide (794 mg, 7.06 mmole) was added thereto under ice cooling, and the
`mixture was stirred at the same temperature for 10 minutes. The red mixture thus obtained was cooled to
`-30°C, and to the mixture was added 7 ml of a solution of the lactol 10 obtained above (365 mg, 1.07
`mmole) in tetrahydrofuran. Then, the reaction mixture was treated followirfi the procedure similar to that of
`Example 3, to yield the title compound 11 (317 mg, 70 %) as a colorless oil.
`NMR (CDClg); 6 0.88 (t, J=7Hz, 3H), 17245 (m, 34H), 3.50 (m, 1H), 3.85 (m, 1H), 4.06 (m, 2H), 4.69 (m,
`1H), 5.3-5.6 (m, 2H).
`IR (CHCa); 3512, 2700-2400 (broad), 1708 cm“.
`
`
`
`Example 15
`
`Preparation of (5Z,9S,11R)-9,11-dihydroxy-5-prostenoic acid E
`
`The compound 11 obtained above (220 mg, 0.518 mmole) was dissolved in 4 ml of a mixture of acetic
`acid-water-tetrahydrcWJran (65:35:10) and the solution was heated with stirring at 40-45° C for one and half
`hours. Then, the reaction mixture was treated following the procedure similar to that of Example 4, to yield
`the title compound 12 (156 mg, 88 %) as a colorless oil.
`NMR (CDCIs); 5 0.886, J=7Hz, 3H), 1.1-2.45 (m, 26H), 3.96 (m, 1H), 4.18 (m, 1H), 3.6-4.9 (broad, 3H), 5.3-
`5.6 (m,2H).
`IR (CHCIg); 3664, 3432, 2700-2400 (broad), 1707 cm“.
`
`Example 16
`
`Preparation of (5Z,9S,11R)-9,11-dihydroxy-5-prostenoic acid isopropyl ester (compound E)
`
`The compound 12 obtained above (80 mg, 0.235 mmole) was dissolved in acetonitrile (3 ml), and 1,8-
`diazabicyclo[5.4.0]—7$ndecene (0.21 ml, 1.4 mmole) and then isopropyl iodide (0.69 ml, 7.0 mmole) were
`added thereto. Then, the reaction mixture was treated following the procedure similar to that of Example 5,
`to yield the title compound 13 (82 mg, 91 %) as a colorless oil.
`NMR (CDClg); 6 0.88 (t, J=—7Hz, 3H), 1.22 (d, J=6Hz, 6H), 12-245 (m, 28H), 3.95 (m, 1H), 4.18 (m, 1H),
`5.01 (sept, J =6Hz, 1H), 5.3-5.6 (m, 2H).
`IR (CHCls); 3512, 1716 cm“.
`The comprehensive scheme illustrating the reactions described in Examples 13-16 is shown below.
`
`Micro Labs Exhibit 1004-15
`
`Micro Labs Exhibit 1004-15
`
`

`

`EP 0 471 856 A1
`
`Reaction scheme for preparation of saturated PG
`
`(Examples 13~16)
`
`H2
`Pd—C
`
`,
`
`‘
`
`(CH2)5CH3
`
`/
`
`i-BU2A2H
`
`t