Micro Labs Exhibit 1035
`Micro Labs v. Santen Pharm. and Asahi Glass
`IPR2017-01434
`
`

`

`Page 2
`
`5,296,504
`
`OTHER PUBLICATIONS
`
`Camras C. B., Bito L. Z. (1981). Reduction of intraocu-
`lar pressure in normal and glaucomatous primate (Aotus
`trivirgatus) eyes by topically applied prostaglandin F2a.
`“Curr Eye Res” 1:205-209.
`Camras, C. B., Podos S. M., Rosenthal J. S., Lee P. Y.,
`Sever-in C. H. (1987a). Mulitple dosing of prostaglandin
`F2, or epinephrine on cynomolgus monkey eyes.
`I.
`Aqueous humor dynamics. Invest Ophthalmol Vis Sci
`28:463—469.
`
`Camras C. B., Bhuyan K. C., Podos S. M., Bhuyan D.
`K. Master R. W. P. (1987b) “Multiple Dosing of Prosta-
`glandin an or epinephrine on cynomologus mokey
`eyes. 11. Slitlamp biomicrosCOPY, aqueous humor analy-
`sis, and flourescein angiography.” Invest. Ophthalmol.
`Vis Sci 28:921-926.
`
`Camras C. B., Siebold E. C., Lustgarten J. S., Serle J.
`B., Frisch S. C., Podos S. M., Bito L. Z. (1988) “Reduc-
`tion of IOP by prostaglandin F2“ —l—isopropyl ester
`topically applied in glaucoma patients”, Ophthalmology
`95(Supp.) 129 [This article is referrenced in the specifi-
`cation, and was raised as prior art in an opposition to
`Australian patent application 625096, which is related
`to the instant application. However, we have been un-
`able to locate a copy of this article].
`Crawford K., Kaufman P. L. and True Gable, B. A.
`(1987), “Pilocarpine antagonizes PGan -induced ocu-
`lar hypotension: Evidence for enhancement of uveos-
`cleral outflow of PGFZQ”. Invest. Ophthalmol. Vis. Sci.
`(Supp) ARVO Abstracts 11.
`Crawford, K. and Kaufman, P. L., (1987) “Pilocarpine
`Antagonizes Prostaglandin an—Induced Ocular Hypo-
`tension in Monkeys” Arch. Ophthamology 105 1112.
`Flach A. J., Elisason J. A. (1988), “Topical prostaglan-
`din Ez effects on normal human intraocular pressure”. J.
`Ocu. Pharmacal. 4:13—18.
`
`Gabelt, B. and Kaufman, P. L., (1989) “Prostaglandin
`an Increases Uvelscleral Outflow in the Cynomologus
`Monkey”, Exp. Eye Res. 49 389—402.
`Giuffré G. (1985). “The effects of prostaglandin F20, in
`the human eye.” Graefes Arch Clin. Exp. Ophthalmol.
`222:139-141.
`
`17--
`“Metabolism of
`(1975)
`E.,
`Granstrom,
`Phenyl—l8,19,20—Trinor—Prostaglandin F2“ in the Cy-
`
`nomolgus Monkey and the Human Female”, Prostaglan-
`dins 9:19—45.
`
`Kaufman P. L. (1986). “Effects of intracamerally in-
`fused prostaglandins on outflow facility in cynomolgus
`monkey eyes with intact or retrodisplaced ciliary mus-
`cle.” Exp. Eye Res. 43:819—827.
`Kerstetter J. R., Brubaker R. R, Wilson S. E., Kullers-
`trand L. J. (1988). “Prostaglandin an —l—isopropylester
`lower intraocular pressure without decreasing aqueous
`humor flow.” Am. J. Ophthalmol. 105230—34.
`Kirk—Othmar “Encyclopedia of Chemical Technol-
`ogy”, 3d Ed. Supplement vol. 711-752 (1984).
`Lee, P.—Y., Shae H., XZu L., Qu C.-K. (1988). “The
`effect of prostaglandin an on intraocular pressure in
`normotensive human subjects.” Invest. Ophthalmol. Vis.
`Sci. 29:1474-1477.
`
`Miller, “Biological Activities of 17—phenyl—18,19,20—
`trinorprostaglandins”, Prostaglandins 9 9-18 (1975).
`Nilsson, S. F. E., Stjernschantz J. and Bill A. .(1987)
`“PGFZa increase uveoscleral outflow”, Invest. Ophthal-
`mol Vis Sci. Suppl. 284.
`Villumsen J. Alm A (1989) “Prostaglandin an isopro-
`pylester eye drops. Effects in normal human eyes.” Br.
`J. Ophthalmol. 73:419—426.
`Yankee, Chem Abst. 88:62048x, 1978.
`Woodward et al., “Prostaglandin F2a Effects on IOP
`Negatively Correlate with Classical PGan —Receptor
`Stimulation” Presented in Eightly International Con-
`gress of Eye Research in San Francisco Sep. 4-8, 1988.
`Woodward et al. (1989) “Prostaglandin F2a Effects on
`Intraocular Pressure Negatively Correlate with FP—Re-
`ceptor Stimulation” Invest. Ophthal. 30 (8) 1838—1842.
`Kirk—Othmar “Encyclopedia of Chemical Technol-
`' ogy”, 3d Ed. Supplement vol. 711—752 (1984).
`Lee, P—Y, Shae H, XZu, L, Qu C—K (1988). “The effect
`of prostaglandin F20, on intraocular pressure in normo-
`tensive human subjects.” Invest. Ophthalmol. Vis. Sci.
`29:1474—1477.
`
`Miller, “Biological Activities of l7—pheny1—18,l9,20—-
`trinorprostaglandins”, Prostaglandins 9 9—18 (1975).
`Nilsson SFE, Stjernschantz J. and Bill A.
`(1987)
`“PGan increase uveoscleral outflow.” Invest. Ophthal-
`mol Vis. Sci. Suppl. 284.
`Villumsen J. Alm A (1989) “Prostaglandin an isopro-
`pylester eye drops. Effects in normal human eyes.” Br.
`J. Ophthalmol. 73:419—426.
`Yankee, Chem. Abst. 88:62048x, 1978.
`
`Micro Labs Exhibit 1035-2
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`Micro Labs Exhibit 1035-2
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`

`5,296,504
`
`Page 3
`
`OTHER PUBLICATIONS
`
`Woodward et a1., “Prostaglandin an Effects of IOP
`Negatively Correlate with Classical PGan-Receptor
`Stimuluation” Presented in Eightly International Con-
`gress of Eye Research in San Francisco Sep. 4—8, 1988.
`Woodward et a1. (1989) “Prostaglandin an Effects on
`Intraocular Pressure Negatively Correlate with FP-Re-
`ceptor Stimulation” Invest. Ophthal. 30(8) 1838-1842.
`Camras C. B., Bito L. Z. and Eakins K. E. (1977): “Re-
`duction of intraocular pressure by prostaglandins ap-
`plied to the eyes of concious rabbits”, Invest Ophthal-
`mol Vis. Sic, 1621125.
`Kerstetter J. R., Brubaker R. E, Wilson S. E. and Kull-
`erstrand L.
`(1988) “Prostaglandin F2a—1-Isopropy-
`lester Lowers Intracolor Pressure Without Decreasing
`Aqueous Humor Flow”. Am.
`J. Ophthalmology
`105:30—34.
`
`Bito, Baroody and Miranda (1987): “Eicosanoids as a
`new class of ocular Hypotensive agents. The apparent
`therapeutic advantage of derived Prostaglandins of the
`A and B type as compared with primary prostaglandins
`of the E, F and D types”, Experimental Eye Research,
`44:825.
`Villumser, J., Alm A., Soderstrom M., (1989) “Prosta-
`glandin FZa—isopropylester Eye Drops: Effect on Intra-
`ocular Pressure in Open Angle Glaucoma”. Brit. J.
`Ophthalmology 73, 975—79.
`Ritch, Shields and Krupin (1989): “The Glaucomas”, C.
`V. Mosby, p. 561.
`
`Supplement to Investigative Ophthalmology and Visual
`Science 22 39 (1982), L. Z. Bito, A. Draga, J. Blanco,
`and C. B. Camras, “Maintenance of Reduced Intraocu-
`lar Pressure (IOP) for several months by topical appli-
`cation of prostaglandin (PG) E1 ti Eyes of Trained
`Cats”.
`
`Starr, M. S. (1971) “Further studies on the effect of
`prostaglandin on intraocular pressure in the rabit”, Exp.
`.. Eye, Res. 11:170.
`Kass, M. A., Posos, S. M., Moses, R. A., and Becker B.
`(1972): “Prostaglandin E1 and aqueous humor dynam-
`‘ ics”, Invest Ophthalmol. 11:1022.
`Villumsen J. and Alm A. (1987): “The effect of prosta-
`glandin F2a eye drops in open angle glaucoma”, Invest
`’ Opthalmol. Vis. Sci. 28:378.
`Wang R—F, Cmamras C. B., Lee P—Y, Podos S. M. and
`Bito L. Z. (1987) “The ocular hypotensive effects of
`Prostaglandins F2a isopropyl ester and A2 in glautoma-
`tous monkeys” Invest Ophthalmol Vis Sci ARVO Supl.
`28:266.
`
`Kass M. A., Mandel] A1, Goldbert I, Paine J. M. and
`Becker B (1979) “Dipivefrin and epinephrine treatment
`of elevated intraocular pressure: A comparative study”,
`Arch Ophthalmol. 97:1865.
`
`Goldberg, 1., Kolker A. E., Kass M. A. and Becker B.
`(1980) “Dipivefrin: current concepts”, Australia J. Oph-
`thalmol., 8:147.
`
`Micro Labs Exhibit 1035-3
`
`Micro Labs Exhibit 1035-3
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`

`

`1
`
`5,296,504
`
`2
`is to lower either the flow (F) or the resistance (R)
`which, according to formula (1) above, will result in a
`reduced IOP; alternatively to increase the flow via the
`uveoscleral route which according to formula (2) also
`gives a reduced pressure. Cholinergic agonists, for in~
`stance pilocarpine,
`reduce the intraocular pressure
`mainly by increasing the outflow through Schlemm’s
`canal.
`Prostaglandins, which recently have met an increas-
`ing interest as IOP-lowering substances may be active in
`that they will cause an increase in the uveoscleral out-
`flow (Crawford et a1, 1987, and Nilsson et al, 1987).
`They do not appear, however to have any effect on the
`formation of aqueous humor or on the conventional
`outflow through Schlemm’s canal (Crawford et al,
`1987).
`The use of prostaglandins and their derivatives is
`described for instance in US. Pat. No. 4,599,353 and EP
`871037149, and by Bito LZ et a1 (1983), Camras CB et
`a1 (1981, 1987a, 1987b, 1988), Giuffré G (1985), Kauf-
`man PL (1986), Kersetter JR et al (1988), Lee P-Y et al
`(1988) and Villumsen J et al (1989).
`With respect to the practical usefulness of some of the
`previously described prostaglandins and derivatives, as
`suitable drugs for treating glaucoma or ocular hyperten-
`sion, a limiting factor is their property of causing super-
`ficial irritation and vasodilation in the conjunctive. It is
`probable, moreover, that prostaglandins have an irritant
`effect on the sensory nerves of the cornea. Thus local
`side effects will arise in the eye already when the
`amounts of prostaglandin administered are quite smal-
`l—that is, already when the doses are lower than those
`that would be desirable for achieving maximum pres-
`sure reduction. It has thus been found, for instance, that
`for this reason it is clinically impossible to use PGan-l-
`isopropyl ester in the amount that would give maximum
`pressure reduction. Prostaglandins, being naturally oc-
`curring autacoids, are very potent pharmacologically
`and affect both sensory nerves and smooth muscle of
`the blood vessels. Since the effects caused by adminis-
`trations of PGFZa and its esters to the eye, comprise in
`addition to pressure reduction also irritation and hyper-
`emia (increased blood flow), the doses currently practi-
`cable in clinical tests are necessarily very low. The
`irritation experienced when PGan or its esters are
`applied, consists mainly in a feeling of grittiness or of
`having a foreign body in one’s eye, this being usually
`accompanied by increased lacrimation.
`We have now, found that a solution to the problems
`discussed above is the use of certain derivatives of pros-
`taglandins A, B, D, E and F, in which the omega chain
`has been modified with the common feature of contain-
`ing a ring structure, for the treatment of glaucoma or
`ocular hypertension.
`The prostaglandin derivatives have the general struc-
`ture
`
`PROSTAGLANDIN DERIVATIVES FOR THE
`TREATMENT OF GLAUCOMA OR OCULAR
`HYPERTENSION
`
`This application is a continuation of application Ser.
`No. 07/469,442, filed on Apr. 10, 1990, now abandoned.
`The invention is concerned with the use of prosta-
`glandin derivatives of PGA, PGB, PGD, PGE and
`PGF, in which the omega chain has been modified with
`the common feature of containing a ring structure, for
`the treatment of glaucoma or ocular hypertension. The
`invention relates also to ophthalmic compositions, con-
`taining an active amount of these prostaglandin deriva-
`tives, and the manufacture of such compositions.
`Glaucoma is an eye disorder characterized by in-
`creased intraocular pressure, excavation of the optic
`nerve head and gradual loss of the visual field. An ab-
`normally high intraocular pressure is commonly known
`to be detrimental to the eye, and there are clear indica-
`tions that, 'in glaucoma patients, this probably is the
`most important factor causing degenerative changes in
`the retina. The pathophysiological mechanism of open
`angle glaucoma is, however, still unknown. Unless
`treated successfully glaucoma will lead to blindness
`sooner or later, its course towards that stage is typically
`slow with progressive loss cf the vision.
`The intraocular pressure, IOP (abbr. of intraocular
`pressure) can be defined as according to the formula:
`
`10P=P9+FXR
`
`(1)
`
`where Fe is the episcleral venous pressure, generally
`regarded as being around 9 mm Hg, F the flow of aque-
`ous humor, and R the resistance to outflow of aqueous
`humor through the trabecular meshwork and adjacent
`tissue into Schlemm’s canal.
`Besides passing through Schlemm’s, canal aqueous
`humor might also pass through the ciliary muscle into
`the suprachoroidal space and finally leave the eye
`through sclera. This uveoscleral route has been de-
`scribed for instance by Bill (1975). The pressure gradi-
`ent in this case is insignificant compared to the gradient
`over the interior wall of Schlemm’s canal and adjacent
`tissue in the former case. The flow limiting step along
`the uveoscleral route is assumed to be the flow from the
`anterior chamber into the suprachoraidal space.
`A more complete formula is given by:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`10P=Pe+(Fr—Fu)XR
`
`(2)
`
`50
`
`55
`
`where P9 and R are defined as above, F, is the total
`outflow of aqueous humor and F“ is the fraction passing
`via the uveoscleral route.
`IOP in human beings is normally in the range of
`12—22 mm Hg. At higher values, for instance over 22
`mm Hg, there is a risk that the eye may be affected. In
`one particular form of glaucoma, low tension glaucoma,
`damage may occur at intraocular pressure levels other—
`wise regarded as physiologically normal. The reason
`for this could be that the eye in these individuals is
`unusually sensitive to pressure. The opposite situation is
`also known, that some individuals may exhibit an abnor-
`mally high intraocular pressure without any manifest
`defects in the visual field or optic nerve head. Such
`conditions are usually referred to as ocular hyperten-
`sron.
`
`Glaucoma treatments can be given by means of
`drugs, laser or surgery. In drug treatment, the purpose
`
`alpha chain
`
`omega chain
`
`65
`
`wherein A represents the alicyclic ring Cg—Cu and the
`bonds between the ring and the side chains represent the
`various isomers. In PGA, PGB, PGD, PGE and PGF
`A has the formula
`
`Micro Labs Exhibit 1035-4
`
`Micro Labs Exhibit 1035-4
`
`

`

`\\
`
`PGA
`
`\\
`
`
`
`PGE
`
`
`
`on
`PGF
`
`II
`
`10
`
`15
`
`III
`
`20
`
`IV
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`The invention is based on the use of derivatives char-
`acterized by their omega chain and various modifica-
`tions of the alpha chain is therefore possible still using
`the inventive concept. The alpha chain could typically
`be the naturally occuring alpha chain, which is esteri-
`fled to the structure
`
`WCOO R1
`
`in which R1 is an alkyl group, preferably with 1—10
`carbon, especially 1—6 atoms, for instance metyl, ethyl,
`propyl, isopropyl, butyl, isobutyl, neopentyl or benzyl
`or a derivative giving the final substance equivalent
`properties as a glaucoma agent. The chain could prefer-
`ably be a C6—C1o chain which might be saturated or
`unsaturated having one or more double bonds, and
`allenes, or a triple bond and the chain might contain one
`or more substituents such as alkyl groups, alicyclic
`rings, or aromatic rings with or without hetero atoms.
`The omega chain is defined by the following formula:
`
`(15-24)
`(14)
`(13)
`c BC —D
`
`—R2
`
`wherein
`
`5,296,504
`
`4
`C is a carbon atom (the number is indicated within
`parenthesis);
`'
`B is a single bond, a double bond or a triple bond;
`D is a chain with l-lO, preferably 2—8, and especially
`2—5, and particularly 3 carbon atoms, optionally
`interrupted by preferably not more than two hetero
`atoms (0, S, or N), the substituent on each carbon
`atom being H, alkyl groups, preferably lower alkyl
`groups within 1-5 carbon atoms, a carbonyl group,
`or a hydroxyl group, whereby the substituent on
`C15 preferably being a carbonyl group, or (R)—OH
`or (S)—OH; each chain D containing preferably
`not more than three hydroxyl groups or not more
`than three carbonyl groups;
`R2 is a ring structure such as a phenyl group which is
`unsubstituted or has at least one substituent se-
`lected from C1-C5 alkyl groups, C1-C4 alkoxy
`groups,
`trifluoromethyl groups, C1-C3 aliphatic
`acylamino groups, nitro groups, halogen atoms,
`and phenyl group; or an aromatic heterocyclic
`group having 5—6 ring atoms, like thiazol, imidaz-
`ole, pyrrolidine, thiophene and oxazole; or a cyclo-
`alkane or a cyclcalkene with 3-7 carbon atoms in
`the ring, optionally substituted with lower alkyl
`groups with 1—5 carbon atoms.
`Some examples on derivatives which were evaluated
`are the following (for structure information see Table
`I):
`16-phenyl-17,l8,19,20tetranor-PGF2a-iwpropy-
`(l)
`lester
`
`(2) 17-phenyl-1 8, l9,ZO-trinor-PGan-isopropylester
`(3)
`15-dehydro-17-phenyl-18,19,20-trinor-PGF2a-iso-
`propylester
`(4)
`lS-phenoxy-l7,18,19,20-tetranor-PGan-isopropy-
`lester
`
`(5) l7-phenyl-18,19,20-trinor-PGE2- isopropylester
`(6) 13,14—dihydro-l7-phenyl-18,19,20-trinor-PGA2- iso- ~
`propylester
`(7)
`1S-(R)-17-phenyl-18,19,20—trinor-PGan-isopropy-
`lester
`
`l6-[4-(methoxy)-phenyl]-l7,18,19,20-tetranor-
`(8)
`PGan-isopropylester
`(9) 13,l4-dihydro-17-phenyl-18,19,20-tn'nor-PGF2a-iso-
`propylester
`(10) lS-phenyl- l 9,20vdinor~PGF2a-isopropylester
`(20) 19-phenyl-20-nor-PGan-isopropylester
`The most preferred derivatives at present are those in
`which the omega chain of the prostaglandin has the 18,
`l9,20~trinor form, and especially the l7-phenyl analogs,
`such as the lS-(R)—, 15-dehydro and 13,14—dihydro—17-
`phenyl-18,19,ZO-trinor forms. Such derivatives are rep-
`resented by (3), (6), (7) and (9) in the formulas given in
`Table I.
`In the formula given above the most preferred struc—
`ture at present is accordingly obtained when the prosta-
`glandin is a derivative of PGA, PGD, PGE or PGF,
`eSpecially of PGAz, PGDz, PGEz and PGan
`B is a single bond or a double bond;
`D is a carbon chain with 2—5, especially 3 atoms; C15
`having a carbonyl or (S)—OH substituent and
`C16—C19 having lower alkyl substituents, or prefer-
`ably H;
`R2 is a phenyl ring optionally having substituents
`selected among alkyl and alkoxy groups.
`The invention thus relates to the use of certain deriva-
`tives of PGA, PGB, PGD, PGE and PGF for the treat-
`ment of glaucoma or ocular hypertension. Among these
`
`Micro Labs Exhibit 1035-5
`
`Micro Labs Exhibit 1035-5
`
`

`

`5,296,504
`
`6
`
`5
`derivatives defined above it has been found that some
`are irritating or otherwise not optimal, and in certain
`cases not even useful due to adverse effects and these
`are excluded in that the group of prostaglandin deriva-
`tives defined above is limited to therapeutically effec-
`tive and physiologically acceptable derivatives. So is for
`instance (1) 16-pheny1-17,18,19,20-tetranor-PGF2a-iso-
`propyl ester irritating while this can be eliminated by
`substituting the phenyl ring with a methoxy group giv-
`ing formula (8) which represents a therapeutically more
`useful compound.
`The method for treating glaucoma or ocular hyper-
`tension consists in contacting an effective intraocular
`pressure reducing amount of a composition, as afore-
`said, with the eye in order to reduce the eye pessure and
`to maintain said pressure on a reduced level. The com-
`position contains 0.1—30 pg, especially 1-10 pg, per
`application of the active substance i.e. a therapeutically
`active and physiologically acceptable derivative from
`the group defined above; the treatment may advanta-
`geously be carried out in that one drop of the composi-
`tion, corresponding to about 30 11.1,
`is administered
`about 1 to 2 times per day to the patient’s eye. This
`therapy is applicable both to human beings and to ani-
`mals.
`The invention further relates to the use of therapeuti-
`cally active and physiologically acceptable prostaglan-
`din derivatives from the group defined above for the
`preparation of an ophthalmological composition for the
`treatment of glaucoma or ocular hypertension.
`The prostaglandin derivative is mixed with an oph-
`thalmologically compatible vehicle known per se. The
`vehicle which may be employed for preparing composi—
`tions of this invention comprises aqueous solutions as
`e.g. physiological salines, oil solutions or ointments.
`The vehicle furthermore may contain ophthalmologi-
`cally compatible preservatives such as e.g. benzalko-
`nium chloride, surfactants like e.g. polysorbate 80, lipo-
`somes or polymers, for example methyl cellulose, poly-
`vinyl alcohol, polyvinyl pyrrolidone and hyaluronic
`acid; these may be used for increasing the viscosity.
`Furthermore, it is also possible to use soluble or insolu-
`ble drug inserts when the drug is to be administered.
`The invention is also related to ophthalmological
`compositions for topical treatment of glaucoma or ocu-
`lar hypertension which comprise an effective intra ocu-
`lar pressure reducing amount of a prostaglandin deriva-
`tive as defined above and an ophthalmologically com-
`patible carrier, the‘effective amount comprising a dose
`of about 0.1—30 p. in about 10-50 11 of the composition.
`In the experiments carried out in this investigation the
`active compound, in an amount, varying with potency
`of the drug, from 30 ug to 300 pg/ml was dissolved in
`a sterilized aqueous solution (saline 0.9%) containing
`0.5% polysorbate-80 as solubilizing agent.
`The invention is illustrated by means of the following
`non-limitative examples.
`SYNTHESIS OF PROSTAGLANDIN
`DERIVATIVES
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`mg (0.07 mmol) of isopropyltriflate (freshly prepared)
`was added. This solution was allowed to stand at — 10“
`C. for 15 min and was then slowly warmed to room
`temperature. When the esterification was complete
`according to TLC (usually after 3—4 h at room tempera-
`ture) the solvent was removed in vacuo. The residue
`was diluted with 20 ml ethylacetate, washed with 2 X10
`ml 5% sodium hydrogencarbonate and 2X10 ml 3%
`citric acid. The organic layer was dried over unhydrous
`sodium sulfate. The solvent was removed in vacuo and
`the residue was purified by column chromatography on
`silica gel-60 using ethyl acetate: aceton 2:1 as eluent.
`The title compound was obtained as a colourless oily
`substance (71% yield).
`
`
`Nuclear Magnetic Resonance spectrum (CDClg) - ppm: 8
`1.2
`(6H d)
`3.3
`(1H q)
`2.85
`(2H d)
`5.0
`(1H m)
`3.35
`(1H 111)
`53-57
`(41-1 in)
`4.15
`(1H t)
`7.15—7.35
`(51-! in)
`
`EXAMPLE 2
`
`Preparation of l7-phenyl-18,19,20- trinor
`PGF2a-isopropyl ester (2)
`
`A 50 ml round bottom flask equipped with a magnetic
`stirring bar was charged whith 20 mg (0.05 mmol) 17-
`phenyl-18,19,20-trinor PGan (Cayman Chemicals), 6
`ml acetone, 39.2 mg (0.25 mmol) DBU and 42.5 mg
`(0.25 mmol) isopropyl iodide. The solution was allowed
`to stand at room temperature for 24 h, the solvent was
`removed in vacuo and the residue was diluted with 30
`m1 of ethyl acetate, washed twice with 10 ml 5% sodi-
`umhydragen carbonate and 10 m1 3% citric acid. The
`solvent was removed in vacuo, and the crude product
`was chromatographed on silica gel-60 using ethyl ace—
`tate: acetone 2:1 as eluent. The title compound (2) was
`obtained as an oily substance (65% yield).
`
`
`Nuclear Magnetic Resonance spectrum (CDC13) - ppm: 8
`1.2
`(6m)
`4.9
`(1H m)
`3.9
`(1H m)
`5.4—5.6
`(4H m)
`4.1
`(1H !)
`7.1—7.3
`(5H m)
`
`4.2
`(1H m)
`
`EXAMPLE 3
`
`Preparation of l5-dehydro-l7-pheny1-18,19,20—trinor
`PGan-isopropyl ester (3)
`
`20.9 mg (0.092 mmol) DDQ was added to a solution
`of 10 mg (0.023 mmol) 17-phenyl-18,19,20 trinor
`PGFZQ-isopropyl ester (2) in 8 ml dioxane. The reaction
`mixture immediately turned brown, the reaction mix-
`ture was stirred at room temperature for 24 h. The
`precipitate formed was filtered, washed with 10 m1
`ethyl acetate, the filtrate was diluted with 10 ml ethy-
`lacetate washed with 2X 10 ml water, 2X10 m1 NaOH
`IM and 20 ml brine. The organic layer was dried on
`unhydrous sodium sulfate and the solvent was removed
`in vacuo, the residue was purified by column chroma-
`tography on silica gel using ethyl acetate: ether 1:1 as
`eluent. The title compound (3) was obtained as a colour-
`less oily substance (76% yield).
`
`Micro Labs Exhibit 1035-6
`
`EXAMPLE 1
`
`Preparation of l6-phenyl-17,18,19,20-tetranor
`PGan-isopropyl ester (1)
`
`A 50 ml round bottom flask equipped with a magnetic
`stirring bar w’as charged with 17.5 mg (0.04 mmol)
`16-phenyl-l7,18,19,20-tetranor PGan (Cayman Chemi-
`cal), 5 m1 CH2C12,30.2 mg (0.23 mmol) diisopropyle-
`thylamine. This solution was stirred at — 10° C. and 13.5
`
`65
`
`Micro Labs Exhibit 1035-6
`
`

`

`5,296,504
`
`8
`
`EXAMPLE 7
`
`
`
`Nuclear Magnetic Resonance spectrum (CDCl3), - ppm: 5
`1.2
`(6H d)
`5.4
`(2H m)
`4.0
`(1H m)
`6.2
`(1H d)
`4.2
`(1H m)
`6.7
`(111 q)
`5.0
`(1H m)
`7.15—7.35
`(SH m)
`
`EXAMPLE 4
`
`Preparation of l6-phenoxy-l7,18,l9,20 -tetranor
`PGan-isopropyl ester(4)
`
`Following a procedure similar to that described in
`example 2 using 20 mg (0.051 mmol)
`l6—phenoxy-
`17,18,19,20 -tetranor PGF;,¢JL (Cayman Chemicals). The
`title compound (4) was an oily substance (53.2% yield).
`
`
`
`Nuclear Magnetic Resonance spectrum (CDCl3) - ppm: 5
`1.2
`(6H d)
`5.4
`(2H m)
`3.9
`(3H m)
`5.7
`(2H m)
`4.2
`(1H m)
`6.9
`(3H m)
`4.5
`(1H m)
`7.3
`(2H m)
`
`5.0
`(1H m)
`
`EXAMPLE 5
`
`Preparation of 17-phenyl-18,19,20-trinor
`PGEz-isopropyl ester (5)
`
`Following a procedure similar to that described in
`example 2 using 10 mg (0.026 mmol) 17-phenyl-
`18,19,20- trinor PGEz (Cayman Chemicals). The crude
`product was purified by column chromatography an
`silica gel-6O using ether as eluent. The title compound
`(5) was an oily substance (38.9% yield).
`
`Nuclear Magnetic Resonance spectrum (CDClg) - ppm: '6
`1.2
`(61-1 d)
`5.3
`(2H m)
`3.94.1
`(2H m)
`5.6
`(2H m)
`
`4.9
`(1H m)
`7.2
`(5H m)
`
`EXAMPLE 6
`
`Preparation of 13,14-dihydro-17-pheny1-18,19,20-trinor
`PGAz-isopropyl ester (6)
`
`Following a procedure similar to that described in
`example 2 using 10 mg (0.026 mmol) 13,14-dihydro-17-
`phenyl PGAz (Cayman Chemicals). The crude product
`was chromatographed on silica gel-60 using ether as
`eluent.
`
`
`
`Nuclear Magnetic Resonance spectrum (CDCl3) - ppm: 5
`
`1.2
`4.35
`5.0
`
`(6H d)
`(1H m)
`(1H m)
`
`5.4
`7.3
`
`(2H m)
`(5H m)
`
`Preparation of 15-(R)-17-phenyl-18,19,20-trinor
`PGan-isopropyl ester (7) (Table II)
`
`7.1 Preparation of
`1-(S)—2-oxa-3-oxo-6-(R)-(3-oxo-5-phenyl-1-trans-pen-
`tenyl)-7-(R)-(4-phenylbenzoyloxy)-cis-bicyclo [3,3,0]
`octane (13)
`
`18 g (0.05 mol) alcohol (11), 32 g (0.15 mol) DCC,
`39.1 g (0.5 mol) DMSO (newly distilled from CaHz) and
`30 ml DME were charged to a 200 ml flask under nitro-
`gen. Orthophosphoric acid was added in one portion,
`and an exothermic reaction occured. The reaction mix-
`ture was stirred mechanically at room temperature for
`2h, and the resultant precipitate was filtered and washed
`with DME. The filtrate (12) can be used directly for
`Emmon condensation reaction.
`
`To a suspension of 1.2 g (0.04 mol) NaH (80%
`washed with n-pentane to remove mineral oil) in 100 ml
`DME under nitrogen was added dropwise 12.3 g
`(0.048) dimethyl-Z-oxo-4-phenyl-butyl-phosphonate in
`30 ml DME. The mixture was stirred mechanically for
`1h at room temperature, then cooled to — 10° C. and a
`solution of the crude aldehyde (12) was added in drop-
`wise. After 15 min at 0° C. and 1h at room temperature
`the reaction mixture was neutralized with glacial acetic
`acid, the solvent was removed under vaccum, and to
`the residue was added 100 ml ethyl acetate, washed
`with 50 ml water and 50 m1 brine. The organic layer
`was dried over unhydrous sodium sulfate. The solvent
`was removed in vacuo and the resulting white precipi-
`tate filtered and washed with cold ether. The title com-
`
`pound (13) was obtained as a crystalline substance mp
`134.5—135.5 (53%gyie1d).
`
`7.2 Preparation of
`1-(S)-2-oxa-30x0-6-(R)-[3-(R,S)-hydroxy-4-phenyl-1—
`trans-pentenyl]-7-(R)-(4-phenylbenzoyloxy) cis-bicyclo
`[3,3,0]octane (14)
`
`10 g (0.021 mol) enone (l3) and 3.1 g (0,008 mol)
`cerouschloride heptahydrate in 50 ml methanol and 20
`ml CH2C12 were charged to a 200 ml round bottom flask
`equipped with a magnetic stirring bar and was cooled to
`-—78° C. under nitrogen. Sodium borohydride was
`added in small portions, after 30 min the reaction mix-
`ture was quenched by addition of saturuted NH4C1, and
`extracted with 2X 50 ml ethyl acetate. The extracts
`were dried and concentrated to leave a colourless cil
`(98% yield).
`'
`
`7.3 Preparation of
`l-(S)-2-oxa-3-oxo-6-(R)-[3-(R,S)—hydroxy-4-phenyl-l-
`trans-pentenyl]-7-(R)-hydroxy-cis-bicyclo-[3,3,0]
`octane (15)
`
`To a solution of 9.8 g (0.02 mol) ketal (14) in 100 ml
`absolute methanol was added 1.7 (0.012 mol) potassium
`carbonate. The mixture was stirred with a magnetic bar,
`at room temperature after 3 h. The mixture was neutral-
`ized with 40 m1 HCl 1M, and extracted with 2x50 ml
`ethyl acetate. The extracts were then dried on unhy—
`drous sodium sulfate and concentrated. The crude prod—
`uct was chromatographed on silica gel using ethyl ace-
`tate: acetone as eluent. The title compound (15) was
`obtained as an oily substance (85% yield).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`50
`
`55
`
`60
`
`65
`
`Micro Labs Exhibit 1035-7
`
`Micro Labs Exhibit 1035-7
`
`

`

`9
`
`5,296,504
`
`7.4 Preparation of
`1-(S)-2-oxa-3-hydroxy-6-(R)-[3-(R,S)-hydroxy-4~phe-
`nyl-l-trans-pentenyl ]-7-(R)-hydroxy-cis-bicyclo[3,3,0]
`(16)
`To a solution of 3 g(0.011 mol) lactone (15) in 60 ml
`unhydrous THF, stirred magnetically and cooled to
`-—78° C., 4.5 g (0.0315 mol) DIBAL-H in toluene was
`added dropwise. After 2h the reaction mixture was
`quenched by addition of 75 ml methanol. The mixture
`was filtered,the filtrate was concentrated in vacuo and
`the residue was chromatographed on silica gel-60 using
`ethyl acetate: acetone 1:1 as eluent. The title compound
`(16) was obtained as a semisolid substance (78% yield).
`
`7.5 Preparation of 15-(R,S)-l7-phenyl-18,19,20-trinor
`PGF2a(l7)
`‘
`
`2.5 g (25 mmol) sodium methyl sulfinylmethide in
`DMSO (freshly prepared from sodium anhydride and
`DMSO) was added dropwise to a solution of 5.6 g (12.6
`mmol) 4-caboxybutyl triphenyl-phosphonium bromide
`in 12 ml DMSO. To the resultant red solution of the
`ylide was added dropwise a solution of the 1.2 g (4.2
`mmol) hemiacetal (16) in 13 ml DMSO, and the mixture
`was stirred for 1h. The reaction mixture was diluted
`with 10 g ice and 10 ml water and extracted with 2 X 50
`m1 ethyl acetate, whereafter the aqueous layer was
`cooled, acidified with HCl 1M and extracted with ethyl
`acetate, and then the organic layer was dried and con-
`centrated. The resulting crude product was a colourless
`substance. The purity of the title compound (17) was
`estimated by TLC on silica gel using ethyl acetate:
`acetone: acetic acid l:1:0.2 v/v/v as eluent.
`
`7.6 Preparation of l5-(R)-l7-phenyl-18,19,20- trinor
`PGFZQ-isopropyl ester (7)
`
`The crude product (17) was esterified following a
`procedure similar to that described in example 2 the
`product was purified by column chromatography on
`silica gel-60 using ethyl acetate as eluent and the result-
`ing mixture of C15 epirneric alcohol were separated.
`The title compound (7) was obtained as a colourless
`oily substance (46% yield).
`
`
`Nuclear Magnetic Resonance spectrum (CDC13), - ppm: 5
`1.2
`(6H m)
`5.4
`(21-1 in)
`3.9
`(1H m)
`5.6
`(2H m)
`4.15
`(2H m)
`7.2
`(5H m)
`
`4.95
`(1H m)
`
`EXAMPLE 8
`
`Preparation of
`l6-[4—(methoxy)phenyl]-l7,18, 19,20-tetranor
`PGFZa-isopropyl ester (8)
`
`Following a procedure similar to that described in
`example 7 with modified step 7-2, the aldehyde 12 de—
`scribed in step 7-2 was reacted with dimethyl-Z-oxo-3-
`[4-(methoxy)phenyl]-propylphosphonate and was puri-
`fied by column chromatography on silica gel-60 using
`ethyl acetate: toluene 1:1 as eluent. A colourless oily
`substance was obtained (57% yield).
`The
`title
`compound
`l6-[4-(methoxy)phenyl]-
`17,18,19,20—tetranor PGan-isopropyl ester (8) was ob-
`tained as an oily substance, and purified by column
`chromatography on silica gel-60 using ethyl acetate as
`eluent (46% yield).
`'
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`10
`
`
`Nuclear Magnetic Resonance spectrum (CDC13) - ppm: 6
`1.2
`(6H d)
`5.0
`(1H m)
`2.8
`(2H d)
`5.4
`(2H m)
`3.75
`(3H S)
`5.6
`(21-! n1)
`3.9
`(1H m)
`6.8
`(2H d)
`4.15
`(1H m)
`7.2
`(2H d)
`
`4.3
`(1H m)
`
`EXAMPLE 9
`
`Preparation of l3,14~dihydro-17~phenyl-l8,19,20—trinor
`PGan-isopropyl ester (9)
`
`Following a procedure similar to that described in
`example 7, with minor modification, 5 g (0.018 mol)
`enone (13) in 100 ml THF was reduced using 2.03 g
`10% pd/c under hydrogen atmosphere. After comple-
`tion of the reaction (as determined by TLC on silica gel
`using ethylacetate: toluene 1:1 as eluent) the mixture
`was filtered on celite. The filtrate was concentrated in
`vacuo and an oily substance was obtained (86% yield).
`The final product 13,l4-dihydro-l7-phenyl-18,19,20-
`trinor PGan-isopropyl ester containing a mixture of
`C15 epirneric alcohols were separated by preparative
`liquid chromatography using 40% CH3CN in water
`v/v as eluent.
`
`Nuclear Magnetic Resonance spectrum (CDC13) - ppm: 8
`1.2
`(6H d)
`5.0
`(1H m)
`3.6
`(1H m)
`5.4
`(2H m)
`3.9
`(1H m)
`7.2
`(5H m)
`
`4.15
`(1H m)
`
`EXAMPLE 10
`
`Preparation of 18-pheny1-19,20-trinor PGan-isopropyl
`ester (10)
`
`Following a procedure similar to that described in
`example (7) with modified step 7-2. The aldehyde (12)
`described in 7-2 was reacted with dimethyl-Z-oxo-S-
`phenyl pentyl phosphonate gave a crystalline substance
`trans-enone lactone (67% yield).
`The final product lS-phenyl-l9,20-dinor PGan-iso-
`propyl ester (10) was purified by column chromatogra-
`phy on silica gel—60 using ethyl acetate as eluent gave a
`colourless oil (41% yield).
`
`>
`
`(1H m)
`5.0
`(6H d)
`1.2
`(2H m)
`5.4
`(1H m)
`3.95
`4.10
`(1H m)
`5.6
`(2H q)
`
`4.20
`(1H m)
`7.2
`(5H m)
`
`EXAMPLE 11
`
`Preparation of l9-phenyl-20-nor-PGF2a-isopropyl ester
`(20)
`
`Following a procedure similar to that described in
`example (7) with modified step (7-2).
`The aldehyde (12) described in (7-2) was reacted with
`dimethyl-Z-oxo-6-phenyl-hexylphosphonate gave a co-
`lourless oi