Drugs of the Future 1992, 17(8) : 691-704
`Copyright PRODS SCIENCE
`
`Correlates in Pharmacostructures
`
`Phenyl substituted prostaglandin analogs for glaucoma
`treatment
`
`Johan Stjernschantz and Bahram Resul
`Glaucoma Research Laboratories, Kabi Pharmacia
`Ophthalmics, S-75182 Uppsala, Sweden.
`
`CONTENTS
`
`Introduction
`General method for synthesis of phenyl substituted
`PGF2alpha analogues
`17-Phenyl substituted PGF2alpha analogues
`Structure-activity relationships of 17-phenyl substituted
`PGF2alpha- ie analogues
`Latanoprost - A new drug candidate for glaucoma
`treatment
`Preclinical studies
`Clinical studies
`Variation of length of phenyl substituted omega chain
`Structure-activity relationships
`Effects of substituents on the phenyl ring
`Structure-activity relationships
`Importance of ring structure on the omega chain
`Other phenyl substituted prostaglandin analogues
`Conclusions
`Acknowledgements
`References
`
`691
`
`692
`694
`
`694
`
`698
`698
`699
`700
`700
`701
`701
`701
`702
`702
`702
`702
`
`Introduction
`
`In the eye prostaglandins have generally been associated
`with inflammation. This misconception goes back to the late
`1960s and 1970s and was, to a large extent, due to studies
`designed to prove the inflammatory role of prostaglandins
`in the eye . One problem with these studies was that large
`quantities of prostaglandins were administered to the eye,
`and usually by direct injection . Another problem was that in
`many of the studies, rabbits were used as experimental ani-
`mals . The rabbit eye is prone to reacting to a variety of stimu-
`li with increased blood flow and disruption of the
`blood-aqueous barrier in the anterior uvea . This protective
`mechansim of the rabbit eye (1) is in sharp contrast to pri-
`mate and human eyes, which generally are much less sensi-
`tive to trauma . It is conceivable that endogenous prosta-
`glandins may play an important role in this protective
`mechanism of the rabbit eye .
`The first study to demonstrate a clear-cut reduction in in-
`traocular pressure (IOP) after topical administration of pros-
`taglandins was that of Camras et a!. (2) . In this study a bi-
`phasic response in IOP could be obtained with small doses
`
`of prostaglandins, e .g ., prostaglandin F2alphe, (PGF2alpha) 1
`(Scheme 1) ; first an increase and then a sustained de-
`crease . A topical dose of 5 mcg induced only a decrease in
`IOP (2) . Unfortunately, however, this study was performed
`in rabbits, a species exhibiting marked tachyphylaxis to
`prostaglandins, and is therefore not representative for the
`human and primate eye in which prostaglandins lower IOP
`by another mechanism of action . In subsequent studies it
`has been demonstrated that prostaglandins indeed reduce
`IOP in primates and cats as well as in dogs (3-10) .
`The most relevant animal model with respect to the hu-
`man eye is the monkey eye and mechanism studies per-
`formed with prostaglandins in monkeys will therefore be de-
`scribed . Several independent studies clearly indicate that
`the main mechanism of action to reduce IOP, at least of
`PGF2alpha and its isopropyl ester, is increased uveoscleral
`outflow of aqueous humor (11-14) . Aqueous humor is pro-
`duced in the ciliary processes behind the iris . It then flows
`through the pupil from the posterior chamber into the ante-
`rior chamber between the iris and the cornea (Fig . 1) . Nor-
`mally, most of the aqueous humor exits the eye through the
`trabecular meshwork and Schlemm's canal situated in the
`chamber angle . Schlemm's canal is directly connected to
`episcleral veins outside the eye. However, part of the
`aqueous humor bypasses this exit route and leaves the eye
`through the so-called uveoscleral outflow pathway (15) . In
`this pathway aqueous humor percolates through the ciliary
`muscle from the anterior chamber to enter into the supraci-
`liary and suprachoroidal spaces from which the fluid rela-
`tively easily can pass out from the eye through the sclera
`(Fig . 1) . The main resistance in this pathway is constituted
`by the ciliary muscle .
`In glaucoma the drainage of aqueous humor from the an-
`terior chamber is obstructed in the trabecular meshwork
`and/or the tissue adjacent to Schlemm's canal . Thus, if part
`of the fluid could be shunted out from the eye through anoth-
`er route this would be very attractive from a pathophysiolog-
`ical and clinical point of view. In fact, theoretically, since the
`pressure gradient forcing fluid into the uveoscleral outflow
`pathway is very small, if all aqueous humor were to exit the
`eye through this route an IOP close to the episcleral venous
`pressure would ensue . Such a pressure level, around 10
`mmHg, would be very desirable in glaucoma management .
`
`Micro Labs Exhibit 1015
`
`

`

`692
`
`Phenyl substituted PG analogs for glaucoma treatment
`
`Trabecular
`meshwork
`
`Ciliary
`processes
`
`Cornea
`
`Fig . 1 . Schematic picture of the anterior segment of the eye . The
`aqueous humor dynamics in the anterior segment determines the
`intraocular pressure together with the pressure in the blood ves-
`sels outside the eye . Aqueous humor is produced in the ciliary
`processes (1) . It flows through the pupil into the anterior chamber
`(2), through the trabecular meshwork into Schlemm's canal and
`out into the blood vessels on the surface of the eye (3) . Part of the
`aqueous humor exits through the uveoscleral pathway, traversing
`the ciliary muscle to enter into the supraciliary and suprachoroidal
`spaces, from where the fluid can leave the eye through the sclera
`(4 and 5, respectively) .
`
`PGF2alpha and PGF2alpha isopropyl ester (PGF2alphaie) 2
`(Scheme 1) have been shown effectively to reduce IOP both
`in normotensive healthy volunteers and in patients suffering
`from ocular hypertension or open angle glaucoma (16-23) .
`However, both PGF2alpha and PGF2alpha-ie cause pro-
`nounced local side effects when applied topically on the eye .
`A diester prodrug, 15-propionate-PGF2 al pha -ie, was not
`found to significantly improve the therapeutic index of
`PGF2alpha in the eye of human volunteers (24) . These side
`effects comprise superficial irritation, mostly experienced as
`a grittyness or foreign body sensation and conjunctival hy-
`peremia lasting for several hours (25) . Because of the side
`effects it has not been possible to develop PGF 2,lpha or an
`esterified prodrug of PGF2alpha to a useful drug for glaucoma
`treatment in spite of the very good IOP lowering effect of this
`prostaglandin . It should be stressed, however, that
`PGF2a ,p ,,a and PGF2e1ph,-ie have never been found to induce
`any intraocular side effects, and therefore from a clinical
`point of view, this class of drugs probably would be accept-
`able as long as the superficial ocular side effect profile Is im-
`proved.
`
`Attempts were made to reduce the local side effects of
`prostaglandins by a prodrug concept through esterification
`of different parts of the molecule . Esterification increases li-
`pophilicity of the molecule and thus the bioavailability in the
`eye . The sites of esterification of PGF2alpha used for the pro-
`drug concept are illustrated in Scheme 1 . These prodrugs
`of PGF2alpha were prepared in the early 1980s. Unfortunate-
`ly, the prod rugs did not significantly increase the therapeutic
`index of PGF2alpha in the eye . However, substituting part of
`the omega chain with a phenyl ring (Scheme 2) has been
`shown to change the pharmacological profile of PGF2alpha
`dramatically with respect to the side effects in the eye
`(26-29) .
`
`General method for synthesis of phenyl substituted
`PGF2alpha analogues
`
`The omega chains of the phenyl substituted PGF2alpha
`analogues were synthesized from the appropriate phospho-
`ranes (30-32) or phosphonates (33) as key reagents . Three
`general routes were utilized as outlined in Scheme 3 .
`The acyl triphenylphosphorane I (Scheme 3) was pre-
`pared by addition of an aryl halide to lithiotriphenyl phosphi-
`noacetonide (method A) or by reaction of methyl triphenyl-
`phosphonium bromide with aryl acid ester using potassium
`t. butoxide (method B) . The reactive dimethyl (2-oxoalkyl)
`phosphonates were prepared by reaction of aryl halide and
`dimethyl (2- oxopropyl) phosphonate in THF using n-BuLi
`(method C) . These precursors were prepared in 55-60%
`yield .
`The phenyl substituted PGF2alpha analogues were pre-
`pared from a commercially available bicyclic lactone (34,
`35) corresponding to formula III as outlined in Scheme 4 .
`The primary alcohol of lactone III was oxidized to aldehyde
`IV using dimethyl sulfoxide (DMSO) and dicyclohexylcarbo-
`diimide (DCC) in the presence of anhydrous phosphoric
`acid in dimethoxyethylene (DME) (36-38) . The crude aide-
`hyde IV was reacted with the appropriate acyl phosphorane
`or acyl phosphonate I, II (Scheme 3) using a method de-
`scribed by Emmon-Horner (39,40) affording alpha, beta un-
`saturated ketone V. The resulting enone V was treated with
`lithium tri sec butylboro hydride (lithium selectride) (41) at
`-120°C/-130°C, furnishing 70-75% S isomer Vla over R iso-
`mer Vlb . Sodium borohydride and cerium chloride (41) were
`also used but with lower stereoselectivity . The isomers were
`separated by column chromatography on silica gel using tol-
`uene : AcOEt 2 :1 as eluent. The phenyl benzoyl group was
`removed by using powdered potassium carbonate in meth-
`anol to give an 80% yield of the diol . The product was puri-
`fied by column chromatography on silica gel using AcOEt as
`eluent . The diol was treated with diisobutyl aluminium hy-
`dride (DIBAL) (42) in dry THF at -78°C to afford lactol (triol)
`VII in 75-80% yield . The triol VII underwent Wittig reaction
`with 4-carboxy butyl triphenylphosphonium bromide and
`Kot.Bu in THF furnishing the phenyl PGF 2a acid VIII . This
`was further reacted without isolation with isopropyl iodide
`(Ipri) and DBU in acetone (43) to give the corresponding es-
`ter in about 50% yield . The 15-allylic alcohol of the phenyl
`PGF 2. Iph , ester IX was oxidized with 2,3-dichloro-5,6-dicya-
`nobenzoquinone (DDQ) (44, 45) in dioxane to give the
`
`Micro Labs Exhibit 1015-2
`
`
`
`

`

`Drugs Fut 1992, 17(8)
`
`693
`
`desired 15-keto phenyl PGF 2a,pha analogue X in about 80%
`yield .
`13,14-dihydro phenyl PGF 2awp,, a analogues were synthe-
`sized as outlined in Scheme 5 . The trans allylic double bond
`of compound VI was reduced under hydrogen atmosphere
`using pd-c as a catalyst in the presence of sodium nitrite (46)
`affording compound XI in quantitative yield . The product XI
`was isolated and reacted subsequently following a proce-
`
`dure described above to give the desired product XII
`(Schemes 4 and 5) . The 9,11 dihydroxyl groups of the phe-
`nyl PGF2aipha analogue XII were protected with benzene bo-
`ronic acid (47) to give 9,11-phenyl boronate, which was fur-
`ther
`reacted
`without isolation with
`pyridinium
`chlorochromate (PCC) adsorbed on alumina (48) in CH2 CI 2
`to give the 15-keto analogue XI II . This was treated with hy-
`drogen peroxide to deprotect the 9,11-phenyl boronate,
`
`Micro Labs Exhibit 1015-3
`
`

`

`694
`
`Phenyl substituted PG analogs for glaucoma treatment
`
`Scheme 3
`
`METHOD A
`
`P =CH000H2-(CH,)n
`3
`1
`
`METHOD C
`
`(CH3O)2PO-CHpCOCH3 + X(CH2)n
`
`1-NaH
`"
`2-n-BuLi
`
`(CHAIPO -CH2cOCH1(CH2)n
`11
`
`Dose (µg)
`
`Fig, 2 . Miotic effect of 17-phenyl substituted PGF20iphe ie analogues in cat eyes 3 hours after topical application (maximum effect) .
`PGF2aipha-ie included for comparison (n = 6) .
`
`Micro Labs Exhibit 1015-4
`
`
`

`

`Drugs Fut 1992, 17(8)
`
`695
`
`DCC, DMSO
`
`N,P04
`
`tr1PPB
`Iv
`
`1- K,CO,ikOH
`
`2- DIBAUTHF
`
`(CH,)n
`
`VI
`
`a RIrOH R2.--H
`b RI=H R2=OH
`
`acylphosphonate or
`acylphosphorane
`
`NiGH4, C80 61 14,0
`
`(CHZ)
`
`0
`
`(CH,)n
`
`VII
`
`Ph,P,CH{CHt,000N
`
`OH
`
`000 d oxana
`
`(CNyn
`
`R,
`
`IX
`
`a R1--OH R2=H
`
`b RI=H RUM
`
`X
`
`giving the desired product, the 15-keto phenyl PGF2a1pha
`analogue XIV in good yield .
`The analogues were identified with 13C and 1 H NMR and
`the purity was determined with HPLC . All analogues were
`used as isopropyl esters to enhance bioavailability in the
`eye .
`
`17-Phenyl substituted PGF2alpha analogues
`
`Structure-activity relationships of 17-phenyl substituted
`PGF2a pr,a-ie analogues
`
`The test compounds were administered topically on the
`eye in aqueous solution . All the 17-phenyl substituted
`PGF2alpha ie analogues (Scheme 2) exhibited marked and
`dose dependent miotic (pupillary constrictive) effect in the
`cat (Fig . 2) . The horizontal pupillary diameter of the exper-
`imental eye was compared with that of the contralateral con-
`trol eye treated with vehicle only. In fact, some of the phenyl
`substituted analogues such as compound 5 (Scheme 2)
`were more potent than PGF2alpha (administered as the iso-
`propyl ester),which is endogenous in the eye (Fig . 2) . These
`results seem to be in fairly good agreement with those of
`previously reported studies with 17-phenyl-1 8,19,20-trinor-
`prostaglandins in other biological systems (49) . In spite of
`
`the fact that PGF2aiphaie is very irritative in the cat eye, none
`of the phenyl substituted PGF2alpha-ie analogues caused
`any ocular irritation as judged from the behavior of the ani-
`mals as well as from the degree of lid closure after topical
`administration of the compunds (Table I) . The marked miotic
`effect of these compounds in combination with the total lack
`of irritative effect strongly suggests that substitution of part
`of the omega chain with an aromatic ring structure either
`causes conformational alteration in the molecule or im-
`poses a steric hindrance, which enables a discrimination
`between different prostaglandin receptor subtypes .
`The IOP reducing effect of the new phenyl substituted PG
`analogues was investigated in cynomolgus monkeys using
`pneumatonometry. The pneumatonometer was calibrated
`for IOP measurement in monkeys using the closed stopcock
`method (50) . Again the experimental eye was treated topi-
`cally with the test compound while the contralateral eye re-
`ceived the vehicle only. It has to be emphasized that the IOP
`of the normotensive cynomolgus monkey is usually low, of-
`ten around 10-14 mmHg, and consequently only pressure
`reductions of a few mmHg can be obtained . In spite of this,
`many of the analogues caused a clear-cut reduction in IOP
`(Fig . 3) . Particularly analogues 5, 8 and 11 were effective .
`These compounds were roughly equipotent with PGF 2e ie .
`Least reduction was caused by the 15-epimers with the
`
`Micro Labs Exhibit 1015-5
`
`
`

`

`696
`
`Phenyl substituted PG analogs for glaucoma treatment
`
`Table 1. Maximum irritative and hyperemic responses of 17-phe-
`nyl trinor PGF2a1pha -ie analogues . PGF2alp l a -ie is included for
`comparison. All compounds were applied topically. The dose in
`cats was 1 mcg and in rabbits 0 .5 mcg (n = 6; Mean +/- SEM) .
`
`Irritation in the
`cat eye
`(0-3)
`
`Ocular surface
`hyperemia in
`rabbits (0 - 4)
`
`2 .7+/-0.2
`
`0 .0+/-0.0
`
`0.0+/-0 .0
`
`0.04-0 .0
`
`0.0+/-0 .0
`
`0.04-0 .0
`
`0.04-0 .0
`
`0.0+/-0 .0
`
`2 .9+/-0 .2
`
`1 .54-0 .3
`
`1 .1+/-0 .3
`
`1 .4+/-0 .2+
`
`1 .3+/-0 .1*
`
`0 .7 +/- 0 .1"
`
`0 .3+/-0 .3+
`
`0 .6+/-0 .3
`
`Compound No.
`
`PGF2aIPha ie
`5
`
`6
`
`7
`
`a
`
`9
`
`10
`
`11
`"pose 1 .0 mcg; +n = 3 .
`
`15-OH group in the upward position 6, 9 and by the
`15-keto-17-phenyl-PGF2ep,,,a -ie analogues 7, 10 (Fig . 3) .
`
`The log P values of the test compounds were computed
`using PACO program V 2 :10 (Chemodata-Computer Che-
`mie GmbH) . Log P values of compounds 2 and 8 were deter-
`mined experimentally using octanol/phosphate buffer (pH
`7 .4) and were found consistently to be about 1 log unit small-
`er than the computed values, indicating that the computed
`values give a satisfactory approximation of the true log P
`value . As can be seen from Table II the differences in log P
`values between compounds 5 to 11 were too small to ac-
`count for significant differences in bioavailability, and thus
`the differences obtained in biologic activity in the eye must
`be considered to reflect the inherent properties of the ana-
`logues tested .
`Repeated administration of compound 11 in laser treated
`ocular hypertensive monkeys caused a sustained reduction
`in IOP throughout the treatment period (51) . Suprisingly,
`even if these analogues reduce IOP in monkeys they have
`very little effect on the IOP in cats (28) or rabbits (unpub-
`lished results) .
`The effects of the phenyl substituted prostaglandin ana-
`logues on the conjunctival blood vessels have been studied
`in the rabbit eye . Rabbits were treated topically on the eye
`with the prostaglandin analogues, and surface (mostly con-
`junctival and episcleral) hyperemia was documented by col-
`
`Micro Labs Exhibit 1015-6
`
`
`
`
`

`

`Drugs Fut 1992, 17(8)
`
`697
`
`PGF2a- 1e
`
`7
`
`8
`
`9
`
`10
`
`11
`
`***
`
`T
`
`**
`
`T
`
`***
`
`y
`
`EEmyM O
`
`.
`
`L<
`
`O VQ 6
`
`C
`
`CO O d
`
`Fig . 3 . Maximum intraocular pressure reducing effect of 1 mcg of 17-phenyl substituted PGF2aipha-ie analogues in conscious cynomolgus
`monkeys after topical application . PGF2aipha-ie included for comparison (n = 6 ; Mean +/- SEM) . 'p < 0 .05, • • p a 0 .01 and ***p < 0 .001 .
`
`Table ll: Calculated log P values of the test compounds .
`
`OH
`
`COOCH(CH.0 A
`
`Compound
`No .
`
`5,6
`7
`8, 9,11
`10
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`2
`
`X
`
`DS
`DB
`SB
`SB
`SB
`DB
`D8
`DB
`D8
`DB
`DB
`DB
`DB
`DB
`DB
`DB
`DB
`
`X ~ Y-(CH2)n
`
`Y
`
`C-OH
`C=O
`C-OH
`C=O
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`C-OH
`PGF2a,,
`
`n
`
`2
`2
`2
`2
`0
`1
`3
`4
`5
`6
`9
`2
`2
`2
`2
`2
`2
`isopropyl ester
`
`R
`
`H
`H
`H
`H
`H
`H
`H
`H
`H
`H
`H
`2-CH3
`4-CH3
`3-OCH3
`4-OCH3
`4-CF3
`4-F
`
`Log P'
`
`5 .33
`5 .00
`5 .57
`4 .87
`4 .59
`4 .82
`5 .81
`6 .33
`6.85
`7.39
`9 .01
`5.64
`5 .68
`5.20
`5.18
`6.24
`5.39
`5.47
`
`*Log P was calculated with PACO program V 2 :10 (Chemodata-Computer Chemie GmbH) according to R .F. Rekker and C. Hansch. SB
`= Single Bond ; DB = Double bond .
`
`Micro Labs Exhibit 1015-7
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`698
`
`Phenyl substituted PG analogs for glaucoma treatment
`
`or photographs . The experimental eye received the test
`compound while the contralateral eye served as a control re-
`ceiving the vehicle only . The photographs were evaluated
`on an arbitrary scale from 0-4 . As can be seen in Table I, all
`phenyl substituted PGF2aIPhaie analogues (Scheme 2) in-
`duced clearly less hyperemia than PGF2a1pha-ie . The ana-
`logues exhibiting least conjunctival hyperemia were gener-
`allythose exhibiting least pharmacologic activity such as the
`earlier mentioned 15-OH epimers 6,9 and the 15-keto 7, 10
`17-phenyl substituted prostaglandin analogues (Table I) .
`
`Latanoprost - A new drug candidate for glaucoma
`treatment
`
`13,14-dihy-
`PhXA41 ;
`name :
`(code
`Latanoprost,
`dro-17-phenyl-18,19,20-trnor- PGF2aipha isopropyl ester ;
`Scheme 6) 8 is the pure 15R epimer of compound 11
`(PhXA34), which is a mixture of the 15R and 15S epimers
`in approximately equimolar proportion. Since the 15Sepim-
`er exerts similar biologic activity to the 15R epimer but is
`much weaker, exerting only about 10% of the activity of the
`15Repimer (29) several of the pharmacological and clinical
`studies with PhXA41 have been carried out using the epim-
`eric mixture (PhXA34) . In this context it is pertinent to men-
`
`Scheme 6
`
`OH
`
`/v `COOCH(CH3) 2
`
`OH
`
`OH
`
`LATANOPROST
`
`tion that the doses of PhXA34 used correspond roughly to
`50% of equivalent doses of PhXA41, The reason for using
`PhXA34 in these first studies was that this substance origi-
`nally was the drug candidate for a new prostaglandin based
`antiglaucoma drug .
`
`Preclinical studies
`
`Pharmacodynamic studies performed in monkeys have
`demonstrated that the main mechanism of action of latano-
`prost to reduce IOP is by increasing the uveoscleral outflow,
`and no or very little effect has been seen on the conventional
`outflow of aqueous humor through Schlemm's canal (14) .
`Of importance is that no negative, effect was seen on the
`aqueous humor production (14) . Thus, the reduction of IOP
`seen in primate eyes after topical administration of latano-
`prost is not due to a decrease in aqueous humor production,
`as is the case with beta-adrenergic antagonists .
`The effects of latanoprost on the ocular microcirculation
`have been carefully studied . Experiments performed with
`the radiolabelled microsphere technique (52) indicate that
`the acute effects of topically administered latanoprost in pri-
`mate eyes are very modest, and the only tissues in which a
`slight increase in blood flow could be detected were the an-
`terior portion of the sclera and the ciliary body (14) . In the
`same experiments the effect of latanoprost on capillary per-
`meability was also determined using 125 1 albumin, 13 '1 albu-
`min and 51 Cr labelled erythrocytes according to well estab-
`lished techniques (53, 54) . Topical administration of
`latanoprost in primate eyes had no effect on capillary per-
`meability in any of the ocular tissues (14) . Thus, these ex-
`periments show that latanoprost has negligible effects on
`the microcirculation in the eye . This is in contrast to
`PGF2alpha ie, which has been demonstrated to substantially
`increase the blood flow of the anterior segment upon topical
`application in monkeys (55) .
`The prostaglandin receptor profile of latanoprost has
`been worked out in vitro usirig a receptor classification sys-
`tem previously described (56) . In these experiments the free
`
`Table ill : EC-50 values of latanoprost 8 in different prostaglandin receptor systems compared with the specific ligands.
`
`Specific ligand
`
`Receptor
`
`FP
`
`EP,
`
`EP2
`
`EP,
`
`DP and IP
`
`TP
`
`Latanoprost EC-50
`(moles/I)
`
`3 .6 x 10 .9
`
`1 .1 x 10' 5
`
`3 .6 x 10 -
`
`1 .1 x 10-4
`
`3 .4 x 10-3
`
`>1 .0 x 10-3
`
`Substance
`
`PGF2aipha
`
`PGE 2
`
`PGE 2
`
`Sulprostone
`PGE 1
`
`BW245C
`PG12`
`
`U-46619"
`
`"Carbaprostacyclin used as stable ligand ; "Stable T,,A2 analogue.
`
`EC-50
`(moles/1)
`
`6 .7 x 10-s
`
`3 .3 x 10$
`
`1 .5 x 10'7
`
`1 .8 x 10 -10
`2 .5 x 10' 9
`
`4 .7 x 10-8
`4 .7 x 10' 9
`
`3 .1 x 10 -6
`
`Micro Labs Exhibit 1015-8
`
`

`

`Drugs Fut 1992, 17(8)
`
`Scheme 7
`
`699
`
`Name
`
`COMPOUND
`No
`
`15-PHENYL PGF
`16-PHENYL PGF
`17-PHENYL PGF
`18-PHENYL PGF
`19-PHENYL PGF
`20-PHENYL PGF
`20-METHYLENE-
`PHENYL PGF
`20-(4-PHENYL-
`BUTYL PGF
`
`1 3
`
`14
`1 5
`16
`
`0
`
`2
`
`4
`5
`6
`
`9
`
`'The compound was- used as 13,14-dihydro-15-phenyl-pentanor
`PGF2a-i9
`
`acid instead of the isopropyl ester has been used . Latano-
`prost has high affinity and selectivity for PGF2a,pha (FP) re-
`ceptors as demonstrated in Table 111 (57) . The affinity for
`EP2, EP3, DP, IP and TP receptors is very low compared
`with the specific ligands . However, the affinity for EP1 recep-
`tors is somewhat greater (57) . These results indicate that
`FP receptors most likely are important in the mechanism
`leading to increased uveoscleral outflow and reduced IOP
`in primate and human eyes . Even if the site of action prob-
`ably is the ciliary muscle and/or adjacent tissues, it is not ful-
`ly clear which cells mediate the effect .
`
`Clinical studies
`
`In the clinical studies published so far mostly PhXA34 11
`has been used . It should be recalled that PhXA34 contains
`about 50% PhXA41 . Several Phase II clinical trials are pres-
`ently in progress with latanoprost . In a first Phase I study the
`ocular effects of PhXA34 were investigated in healthy hu-
`man volunteers . PhXA34 was found to potently reduce IOP
`in a dose-dependent way with few side effects when applied
`topically on the eye (58) . The concentrations tested were
`0 .003%, 0 .01 % and 0 .03% . The only side effect observed
`was a conjunctival hyperemia which occurred with the high-
`est concentration and which was much less pronounced
`than that generally seen with PGF 2aip ha-ie . Repeated admin-
`istration of the highest concentration, once daily for a total
`of 7 days in healthy volunteers caused a sustained reduction
`
`in IOP throughout the treatment period (58) . There was no
`effect on the formation of aqueous humor as studied with
`fluorophotometry (58) .
`In a masked, placebo-controlled dose-finding study in pa-
`tients with ocular hypertension a dose of approximately 3
`mcg of PhXA34 per application corresponding to a concen-
`tration of 0 .01% was found to be close to optimal with re-
`spect to maximum IOP reduction and a minimum of con-
`junctival hyperemia (59) . This dose reduced IOP by an
`average of 30% from an initial pressure of around 25 mmHg
`without significant conjunctival hyperemia (59) . In another
`masked placebo-controlled study ocular hypertensive pa-
`tients were treated with 0 .003% and 0 .01 % PhXA34 twice
`daily for 6 days (60) . Both concentrations significantly re-
`duced IOP with few side effects. The maximum reduction in
`IOP with the higher concentration was 10 mmHg from an ini-
`tial pressure level of about 25 mmHg (60) .
`The longest duration of continuous treatment with latano-
`prost so far is 1 month (61) . In this recent masked, placebo
`controlled multicenter study it was shown that ocular hyper-
`tensive patients treated with latanoprost exhibited reduced
`IOP throughout the treatment period (61) . Thus, all clinical
`studies performed so far indicate that latanoprost (including
`PhXA34) effectively reduces IOP with markedly improved
`side effect profile compared to PGF 2a1 ,ha and its isopropyl
`ester. The duration of action of latanoprost in the eye is long,
`and a dose regimen of once a day application may be suffi-
`cient.
`
`Micro Labs Exhibit 1015-9
`
`∎
`

`

`700
`
`Phenyl substituted PG analogs for glaucoma treatment
`
`Variation of length of phenyl substituted omega chain
`
`As part of the structure-activity program in this
`project a series of phenyl substituted prostaglandin ana-
`logues with the omega chain length varying from 15-phenyl-
`pentanor-PGF2alPha ie to 20(4phenyl-butyl)-PGF2a 1p,,-ie
`(Scheme 7) were synthetized and investigated for biologic
`activity in the eye . The synthetic pathway of these com-
`pounds is outlined in Scheme 4 .
`
`Structure-activity relationships
`
`The purpose of these experiments was to study the influ-
`ence of omega chain length on the potency and specificity
`of the phenyl substituted PGF2a1Pha-ie analogues . The main
`emphasis has been on investigating general biologic activity
`and sensory irritative effect of the compounds . Thus, the cat
`eye has been used as a model, because exact measure-
`ments can be made on the miotic response reflecting gener-
`al biologic activity and the cat eye is a satisfactory model for
`the sensory irritative effect. It should be stressed that most
`of the phenyl substituted PGF2aIPha analogues, as pointed
`out earlier, have a poor IOP reducing effect in cats . Howev-
`er, the miotic effect in cats expressing a FP receptor function
`seems to correlate with the IOP reducing effect in primates
`and man . All analogues have been used as isopropyl esters
`and they have been administered topically on the eye .
`The miotic and irritative effects of a fixed dose (1 mcg) of
`phenyl substituted analogues of PGF2alpha-ie with an omega
`chain ranging from 3 to 12 carbon atoms (total number of
`carbons 15-24) (Scheme 7) are illustrated in Figure 4 . As
`can be seen, the position of the phenyl moiety in the omega
`chain is of fundamental importance for activity and selectiv-
`
`ity. The 17-phenyl substituted analogue was optimal be-
`cause this compound exhibited high biologic activity without
`irritating effect . Again differences in partition coefficient be-
`tween the compounds (Table II), and thus differences in
`bioavailability, cannot account for the striking differences in
`biologic activity. It can be seen that compounds 16, 17 and
`18 had higher log P values, but higher log P values reflect
`greater lipophilicity which usually enhances penetration into
`the eye . It should be observed that particularly the 16-phenyl
`13 but also the 18-,19-, 20-, and 20-methylene-phenyl ana-
`logues 14,15,16,17 exhibited irritation although significant-
`ly less than PGF2alpheie . Furthermore, it is noteworthy that
`the 16-phenyl analogue 13 of PGF2alPha-ie at a dose of 1 mcg
`exerted no miotic effect at all . Decreasing the chain length
`to the 15-phenyl-pentanor analogue 12 resulted in loss of
`biologic activity at the dose level tested as could be ex-
`pected and in general this compound is anticipated to have
`only weak activity. Elongation of the omega chain to the 20
`(4-phenyl-butyl)-PGF2a,pha-ie analogue 18 also resulted in
`loss of biologic activity in the eye, whereas the 20-methy-
`lene-phenyl analogue 17 exerted some biologic activity .
`These results indicate that the 17-phenyl-18,19,20-tri-
`nor- PGF2aIPha-ie is unique in that this compound exhibits a
`structural conformation with no affinity for PG receptors in-
`volved in the sensory irritative response (presumably PG .re-
`ceptors on sensory nerves), while retaining the affinity for
`FP receptors as demonstrated by the miotic response . In
`contrast, PGF2alpheie analogues with shorter or longer phe-
`nyl substituted omega chain did show some affinity for PG
`receptors mediating nociceptive impulses . However, this af-
`finity was much weaker than that of PGF2 SIpha-ie . It appears
`that the steric hindrance of the phenyl ring and the inter-
`atomic distances between functional groups in the mole-
`cules are important for drug-receptor interaction .
`
`10-
`
`6-
`
`8-
`
`7-
`
`6-
`
`5-
`
`4-
`
`3-
`
`2-
`
`0-
`
`TZ
`
`I
`
`-3.0
`
`-"2 .0
`
`i
`i
`
`i
`
`i
`
`i
`
`-1 .0
`
`i
`
`a
`
`a
`
`w
`
`EE
`
`w
`
`mE
`
`m
`
`aC
`
`L
`
`S C
`
`0
`
`cc
`
`PGF2a-ie C-15
`
`C-16
`
`C-17
`
`C-18
`
`C-19
`
`C-20
`
`C-21
`
`Compund :
`
`12
`
`13
`
`5
`
`14
`
`15
`
`16
`
`17
`
`C-24
`
`18
`
`Fig. 4. Miotic and irritative effects of phenyl substituted PGF2aIpha-ie analogues with different omega chain length . The compounds were
`administered topically on cat eyes and the dose was 1 mcg . The total number of carbon atoms in the analogues up to the phenyl ring is
`indicated below the abscissa. Empty columns indicate miosis and hatched columns ocular irritation . PGF2a1pt,a-ie included for comparison
`(n = 6 ; Mean +/- SEM) .
`
`Micro Labs Exhibit 1015-10
`
`
`
`
`
`
`
`
`
`
`
`

`

`Drugs Fut 1992, 17(8)
`
`701
`
`Table IV.• Miotic and irritative effects in the cat eye of 17-phenyl
`trinor prostaglandin analogues substituted in the phenyl ring.
`The compounds have been compared with PGF2alpha -ie and
`compound 5. Reduction in pupil diameter was determined 3 h
`after treatment. Dose 1 mcg (n = 6; Mean +/- SEM).
`
`Compound No.
`
`PGF2a,phaie
`
`5
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`Reduction in pupil
`diameter
`(mm)
`
`-8.24-0.7
`
`-9 .74-03
`
`-8 .3 +/- 0 .6
`
`-2 .54-0 .2
`
`-1 .24-0-2
`
`0 .0+/-0.0
`
`-0 .54-0 .2
`
`-8 .24-0 .2
`
`Irritation
`(0-3)
`
`2 .7 +/- 0.2
`
`0 .04-0 .0
`
`0 .04-0 .0
`
`0 .0+1-0 .0
`
`0 .3+/-0 .0
`
`0 .0+/-0 .0
`
`0 .0+/-0 .0
`
`0 .04-0 .0
`
`Effects of substituents on the phenyl ring
`
`The biological effects of different substituents on the ben-
`zene ring of 17-phenyl-18,19,20-trinor-PGF2a, pha ie have
`also been studied . The miotic and irritative effects in the cat
`eye were investigated as described above . The compounds
`investigated are shown in Scheme 8, and the synthesis of
`the compounds is outlined in Scheme 4 .
`
`the irritative response . However, introduction of the methyl
`group into position 4 of the phenyl ring 20 markedly de-
`creased the miotic effect (Table IV), probably reflecting a re-
`duction of biologic activity based on steric hindrance .
`Introduction of a methoxy group into the phenyl ring has
`been used to study the effect of an electron donating group
`in the vicinty of the benzene ring . Introduction of a methoxy
`group in position 3 of the phenyl ring 21 or in position 4 of the
`phenyl ring 22 resulted in markedly reduced miotic effect or
`complete loss of the miotic effect in cats (Table IV) . Even ten
`times higher doses of compound 22 had very little miotic ef-
`fect in the cat .
`Introduction of a trifluoromethyl group into position 4 in the
`phenyl ring 23, as can be expected, rendered the 17-phenyl-
`trinor- PGF2a,phaie analogue practically inactive (Table IV) .
`Even in a dose of ten times that in Table IV very little miotic
`effect could be elicited. Introduction of electron donating or
`electron receiving groups such as methoxy and trifluoro-
`methyl, respectively, conceivably gives this part of the mole-
`cule an ionic character that leads to decreased activity .
`Introduction of fluorine into position 4 on the phenyl ring
`24 did not appreciably change biologic activity as judged
`from the miotic response (Table IV) . There was no irritating
`effect of this compound either.
`Again, as evident from Table 11, the differences in log P val-
`ues between the compounds were not big enough to ac-
`count for the significant differences in biologic activity be-
`tween the compounds . These experiments thus show that
`it is
`possible
`to
`alter
`the
`biologic
`activity
`of
`17-phenyl-18,19,10-trinor-PGF 2a ,ph aie by introducing cer-
`tain substituents on the phenyl ring .
`
`Structure-activity relationships
`
`The effect of introduction of a methyl group, methoxy
`group, trifluoromethyl group or fluorine into the phenyl ring
`on the structure-activity relationship has been studied . As
`evident from Table IV introdid[ion of a methyl group into po-
`sition 2 on the benzene ring 19 did not change the miotic or
`
`Importance of ring structure on the omega chain
`
`From what is mentioned above, it is evident that by substi-
`tuting part of the omega chain