A topical fluprostenol ophthalmic composition for the treatment of glaucoma and ocular hypertension

BACKGROUND OF THE INVENTION

0001The present invention relates to the treatment of glaucoma and ocular hypertension. In particular, the present invention relates to a topical ophthalmic composition as defined in claim 1 below.

0002Cloprostenol and fluprostanol, both known compounds, are synthetic analogues of PGF_2α, a naturally-occuring F-series prostaglandin (PG). Structures for PGF_2α (I), cloprostenol (II), and fluprostanol (III), are shown below: <chemistry id="chem0001" num="0001"><img file="EP1514548B1_D0001.tif" /></chemistry><chemistry id="chem0002" num="0002"><img file="EP1514548B1_D0002.tif" /></chemistry><chemistry id="chem0003" num="0003"><img file="EP1514548B1_D0003.tif" /></chemistry>

0003The chemical name for cloprostenol is 16-(3-chlorophenoxy)-17,18,19,20-tetranor PGF_2α. <nplcit id="ncit0001" npl-type="b"><text>Monograph No. 2397 (page 375) of The Merck Index, 11th Edition (1989</text></nplcit>) is incorporated herein by reference to the extent that it describes the preparation and known pharmacological profiles of cloprostenol. Fluprostenol has the chemical name 16-(3-trifluoromethylphenoxy)-17,18,19,20-tetranor PGF_2α. <nplcit id="ncit0002" npl-type="b"><text>Monograph No. 4121 (pages 656-657) of The Merck Index, 11th Edition (1989</text></nplcit>) is incorporated herein by reference to the extent that it describes the preparation and known pharmacological profiles of fluprostenol. Cloprostenol and iluprostenol are 16-aryloxy PGs and, in addition to the substituted aromatic ring, differ from the natural product, PGF_2α in that an oxygen atom is embedded within the lower (omega) chain. This oxygen interruption forms an ether functionality.

0004Naturally-occurring prostaglandins are known to lower intraocular pressure (IOP) after topical ocular instillation, but generally cause inflammation, as well as surface irritation characterized by conjunctival hyperemia and edema. Many synthetic prostaglandins have been observed to lower intraocular pressure, but such compounds also produce the aforementioned side effects. Various methods have been used in attempting to overcome the ocular side effects associated with prostaglandins. <patcit id="pcit0001" dnum="EP364417A1"><text>Stjemschantz et al. (EP 364 417 A1</text></patcit>) have synthesized derivatives or analogues of naturally-occurring prostaglandins in order to design out selectively the undesired side effects while maintaining the IOP-lowering effect. Others, including <patcit id="pcit0002" dnum="EP330511A2"><text>Ueno et al. (EP 330 511 A2</text></patcit>) and <patcit id="pcit0003" dnum="EP435682A2"><text>Wheeler (EP 435 682 A2</text></patcit>) have tried complexing prostaglandins with various cyclodextrins.

0005The Stjemschantz et al. publication is of particular interest, as it demonstrates that certain synthetically-modified PGF_2α analogues retain the potent IOP-lowering effect of the parent (PGF_2α isopropyl ester) while decreasing the degree of conjunctival hyperemia. In this publication, the only modification to the PG structure is to the omega chain: the chain length is 4-13 carbon atoms "optionally interrupted by preferably not more than two heteroatoms (O, S, or N)" and includes a phenyl ring (substituted or unsubstituted) on the terminus (see page 3, line 44 to page 4, line 7). Stjemschantz et al. exemplify two subclasses within this definition: <ol id="ol0001" compact="compact" ol-style=""><li>(1) carbon-only omega chains. i.e., <chemistry id="chem0004" num="0004"><img file="EP1514548B1_D0004.tif" /></chemistry> and (2) heteroatom-interrupted omega chains, i.e., <chemistry id="chem0005" num="0005"><img file="EP1514548B1_D0005.tif" /></chemistry></li></ol>

0006In particular, the 17-phenyl-18,19,20-trinor analogue of PGF_2α isopropyl ester (formula 1, n=2) displayed a superior separation of toward and untoward activities. Furthermore, the 13,14-dihydro analogue of 17-phenyl-18,19,20-trinor PGF_2α isopropyl ester displayed an even more favorable separation of activities. Both 17-phenyl PGF_2α and its 13,14-dihydro congener fall into the former (formula 1, carbon-only omega chain) subclass. Additional synthetic analogues employing the phenyl substituent on the end of the omega chain explored the effects of chain elongation, chain contraction, and substitution on the phenyl ring. However, such analogues showed no apparent therapeutic improvement over the preferred formulation, 13,14-dihydro-17-phenyl-18,19,20-trinor PGF_2α isopropyl ester.

0007Because they contain heteroatom (O) interruption of the omega chain, both cloprostenol and fluprostenol are generically included in the subclass defined in formula 2 by Stjemschantz et al. However, neither compound is specifically mentioned by Stjemschantz et al. and the disclosure is primarily related to carbon-only omega chains. The only example of a heteroatom-interrupted omega chain disclosed by Stjemschantz et al. is 16-phenoxy-17,18,19,20 tetranor PGF_2α isopropyl ester (see formula 2, n=1). The IOP data revealed by Stjemschantz et al. for 16-phenoxy-17-18,19,20-tetranor PGF_2α isopropyl ester (see Stjemschantz et al, page 17, Table V) indicate an initial <u>increase</u> in IOP (1-2 hours after administration) followed by a decrease. Moreover, this compound displays unacceptable hyperemia (see Stjemschantz et al., Table IV, line 40). In short, data from Stjemschantz et al. demonstrate that the oxygen-interrupted omega chain subgeneric class of compounds (see formula 2) displays an unacceptable therapeutic profile.

0008<patcit id="pcit0004" dnum="EP0603800A"><text>EP 0 603 800</text></patcit> is prior art pursuant to Article 54(3) EPC. It discloses a topical ophthalmic composition for the treatment of glaucoma and ocular hypertension, said composition comprising a combination of particular prostaglandins of the F and E series. Example E of <patcit id="pcit0005" dnum="EP0603800A"><text>EP 0 603 800</text></patcit> (which is the subject of the disclaimer in the claims) discloses compound (F) 0.0001 wt%, fluprostenol isopropyl ester 0.001 wt%, benzalkonium chloride 0.01 wt%, dextran 70 0.1 wt%,disodium edetate 0.05 wt%, potassium chloride 0.12 wt%, sodium chloride 0.77 wt%, hydroxypropyl methyl cellulose 0.3 wt%, HCl and/or NaOH to adjust pH, and purified water q.s. to 100%, wherein compound (F) has the following formula: <chemistry id="chem0006" num="0006"><img file="EP1514548B1_D0006.tif" /></chemistry>

SUMMARY OF THE INVENTION

0009It has now been unexpectedly found that fluprostenol isopropyl ester shows significantly greater IOP reduction than the compounds of Stjemschantz et al., while having a similar or lower side effect profile. In particular, it appears that the addition of a trifluoromethyl group to the <i>meta</i> position on the phenoxy ring at the end of the omega chain provides a compound having excellent IOP reduction without the significant side effects found with other, closely related compounds.

0010In addition, it has also been unexpectedly found that fluprostenol isopropyl ester is useful in treating glaucoma and ocular hypertension. In particular, topical application of ophthalmic compositions comprising fluprostenol isopropyl ester result in significant IOP reduction.

BRIEF DESCRIPTION OF THE DRAWING

0011<ul id="ul0001" list-style="none"><li><figref idref="f0001">Figure 1</figref> is a graph showing the relative hyperemia scores (cumulative) of five tested compounds (see Table 2, below), one of which is a compound of the present invention.</li><li><figref idref="f0002">Figure 2</figref> is a graph showing the relative IOP-lowering effects of five tested compounds (see Table 2, below), one of which is a compound of the present invention. The dose for each of the tested compounds was 0.3 µg.</li><li><figref idref="f0003">Figure 3</figref> is a graph similar to that of <figref idref="f0002">Figure 2</figref>, showing relative IOP-lowering effects of different concentrations of A (cloprostenol, isopropyl ester) and E (13,14-dihydro-17-phenyl-18,19,20-trinor PGF_2α, isopropyl ester).</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

0012The compound useful in the present invention has the following formula: <chemistry id="chem0007" num="0007"><img file="EP1514548B1_D0007.tif" /></chemistry>

0013The acid may be converted to the ester by conventional condensation with an alcohol (e.g., C₃ alkyl alcohol) or by reaction with an alkyl electrophile (e.g., C₃ alkyl iodide) in the presence of base, according to known procedures. Since such esterification reactions are well known, they are not further described here.

0014Compounds referred to herein include cloprostenol isopropyl ester (Table II, compound A), fluprostenol isopropyl ester (compound a), the 3-oxa form of doprostenol isopropyl ester (Table 1, compound 5), 13,14-dihydrofluprostanol isopropyl ester (compound 6), cloprostenol-1-ol (compound 7), and 13,14-dihydrodoprostenol-1-ol pivaloate (compound 8), of which only fluprostenol isopropyl ester (compound B) is of the present invention.

0015Fluprostenol isopropyl ester (compound B) is useful in lowering intraocular pressure and thus is useful in the treatment of glaucoma. The preferred route of administration is topical. The dosage range for topical administration is generally between about 0.001 and about 1000 micrograms per eye (µg/eye) and is preferably between about 0.01 and about 100 µg/eye and most preferably between about 0.05 and 10 µg/eye. The compound of the present invention can be administered as solutions, suspensions, or emulsions (dispersions) in a suitable ophthalmic vehicle.

0016In forming compositions for topical administration, the compound of the present invention is generally formulated as between about 0.00003 to about 3 percent by weight (wt%) solutions in water at a pH between 4.5 to 8.0. The compound is preferably formulated as between about 0.0003 to about 0.3 wt% and, most preferably, between about 0.003 and about 0.03 wt%. While the predse regimen is left to the discretion of the clinician, it is recommended that the resulting solution be topically applied by placing one drop in each eye one or two times a day.

0017Other ingredients which may be desirable to use in the ophthalmic preparations of the present invention include preservatives, co-solvents and viscosity building agents.

Antimicrobial Preservatives:

0018Ophthalmic products are typically packaged in multidose form, which generally require the addition of preservatives to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride; thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M®, or other agents known to those skilled in the art. Such preservatives are typically employed at a concentration between about 0.001% and about 1.0% by weight.

Co-Solvents:

0019Prostaglandins, and particularly ester derivatives, typically have limited solubility in water and therefore may require a surfactant or other appropriate cosolvent in the composition. Such co-solvents include: Polysorbate 20. 60 and 80; Pluronic® F-68, F-84 and P-103; Tyloxapol®; Cremophor® EL, sodium dodecyl sulfate; glycerol; PEG 400; propylene glycol; cydodextrins; or other agents known to those skilled in the art. Such co-solvents are typically employed at a concentration between about 0.01% and about 2% by weight.

Viscosity Agents:

0020Viscosity greater than that of simple aqueous solutions may be desirable to increase ocular absorption of the active compound, to decrease variability in dispensing the formulations, to decrease physical separation of components of a suspension or emulsion of formulation and/or otherwise to improve the ophthalmic formulation. Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose or other agents known to those skilled in the art. Such agents are typically employed at a concentration between about 0.01 % and about 2% by weight. <tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="14mm" /><colspec colnum="2" colname="col2" colwidth="62mm" /><colspec colnum="3" colname="col3" colwidth="70mm" /><thead><row><entry align="center" valign="top" /><entry align="center" valign="top"><b>COMPOUND NAME</b></entry><entry align="center" valign="top"><b>COMPOUND STRUCTURE</b></entry></row></thead><tbody><row><entry align="center"><b>5</b></entry><entry>3-oxacloprostenol isopropyl ester</entry><entry><chemistry id="chem0008" num="0008"><img file="EP1514548B1_D0008.tif" /></chemistry></entry></row><row><entry align="center"><b>6</b></entry><entry>13,14-dihydrofluprostenol isopropyl ester</entry><entry><chemistry id="chem0009" num="0009"><img file="EP1514548B1_D0009.tif" /></chemistry></entry></row><row><entry align="center"><b>7</b></entry><entry>cloprostenol-1-ol</entry><entry><chemistry id="chem0010" num="0010"><img file="EP1514548B1_D0010.tif" /></chemistry></entry></row><row><entry align="center"><b>8</b></entry><entry>13,14-dihydrocloprostenol-1-ol pivaloate</entry><entry><chemistry id="chem0011" num="0011"><img file="EP1514548B1_D0011.tif" /></chemistry></entry></row></tbody></tgroup></table></tables>

0021In the examples below, the following standard abbreviations are used: g = grams (mg = milligrams); mol = moles (mmol = millimoles); mol% = mole percent; mL = milliliters; mm Hg = millimeters of mercury; mp = melting point; bp = boiling point; h = hours; and min = minutes. In addition, "NMR" refers to nuclear magnetic resonance spectroscopy and "Cl MS" refers to chemical ionization mass spectrometry.

EXAMPLE 1 (Reference): Synthesis of 3-Oxacloprostenol (5)

0022<chemistry id="chem0012" num="0012"><img file="EP1514548B1_D0012.tif" /></chemistry>

A:_Ethyl (3-chlorophenoxy)acetate (<b>10</b>)

0023Acetone (320 ml), 75 g (450 mmol) of ethyl bromoacetate, and 40.0 g (310 mmol) of 3-chlorophenol were mixed together, then 69.8 g (505 mmol) of potassium carbonate was added. The mixture was mechanically stirred and heated to reflux for 4 h, and after cooling to room temperature, was poured Into 350 mL of ethyl acetate. To this was then cautiously added 400 mL of 1 <i>M</i> HCl, taking care to avoid excess foaming. The layers were separated and the aqueous layer was extracted with portions of ethyl acetate (3 X 200 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and the resulting solid was recrystallized from hexane to afford 58 g (87%) of <b>10</b> as a white solid, m.p. = 39-40°C. ¹H NMR δ 7.20-7.08 (m, 1 H), 6.95-6.82 (m, 2 H), 6.75-6.70 (m, 1 H), 4.53 (s, 2 H), 4.21 (q, J = 7.2 Hz, 2 H), 1.23 (t, J = 7.2 Hz, 3 H).

B: Dimethyl [3-(3-chlorophenoxy)-2-oxoprop-1-yl]phosphonate (<b>11</b>)

0024To 20.6 g (166 mmol, 238 mol%) of dimethyl methylphosphonate in 110 mL of THF at -78 °C was added dropwise 65 mL (162 mmol, 232 mol%) of a 2.5 <u>M</u> solution of <i>n</i>-BuLi in hexanes. After addition was complete, the mixture was stirred for an additional 1 h, after which 15.0 g (69.9 mmol) of aryloxyester <b>10</b> in 40 mL of THF was added dropwise. The reaction was stirred for 1 h and then quenched by the addition of 100 mL of saturated NH₄Cl. The mixture was poured into 200 mL of a 1/1 mixture of saturated NaCl/ethyl acetate, layers were separated, and the aqueous layer was further extracted with ethyl acetate (2 X 100 mL). Combined organic layers were dried over MgSO₄, filtered, and concentrated, to afford 20.5 g (100%) of <b>11</b> as a viscous oil. ¹H NMR δ 7.22 (t, J = 8.1 Hz, 1 H), 7.05-6.90 (m, 2 H), 6.85-6.78 (m, 1 H), 4.72 (s, 2 H), 3.84 (s, 3 H), 3.78 (s, 3 H), 3.27 (d, J = 22.8 Hz, 2 H).

C: (3a<i>R</i>, 4<i>R</i>, 5<i>R</i>, 6a<i>S</i>)-5-(Benzoyloxy)-4-[(<i>E</i>)-4-(3-chlorophenoxy)-3-oxo-1-butenyl]-hexahydro-2<i>H</i>-cyclopenta[b]furan-2-one (<b>13</b>)

0025Phosphonate <b>11</b> (20.5 g, 70.0 mmol), 2.6 g (62 mmol) of LiCl, and 200 mL of THF were mixed together at 0 °C and 6.10 g (60.4 mmol) of NEt₃ was added. Aldehyde <b>12</b> (14.0 g, 51.1 mmol) dissolved in 50 mL of CH₂Cl₂ was then added dropwise. After 1 h, the reaction was poured into 200 mL of a 1/1 mixture of saturated NH₄Cl/ethyl acetate, the layers were separated, and the aqueous layer was extracted with ethyl acetate (2 X 100 mL). Combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel eluting with ethyl acetate/hexanes, 3/2, to afford 16.2 g (72%) of <b>13</b> as a white crystalline solid, m.p. = 101.0-102.0 °C. ¹H NMR δ 8.0-7.9 (m, 2 H), 7.62-7.52 (m, 1 H), 7.50-7.38 (m, 2 H), 7.18 (t, J = 8.2 Hz, 1 H), 7.0-6.82 (m, 3 H), 6.75-6.70 (m, 1 H), 6.54 (d, J = 15.1 Hz, 1 H), 5.32 (q, J = 6.2 Hz, 1 H), 5.12-5.05 (m, 1 H), 4.66 (s, 2 H), 3.0-2.8 (m, 3 H), 2.7-2.2 (m, 3 H).

D:

(3a<i>R</i>,4<i>R,</i>5<i>R,</i>6a<i>S</i>)-5-(Benzoyloxy)-4-[(<i>E</i>)-(3<i>R</i>)-4-(3-chlorophenoxy)-3-hydroxy-1-butenyl]-hexahydro-2<i>H</i>-cyclopenta[b]furan-2-one(14)

0026To a solution of 9.70 g (22.0 mmol) of enone 13 in 60 mL of THF at -23 °C was added dropwise a solution of 11.1 g (34.6 mmol of (-)-<i>B</i>-chlorodiisopino-campheylborane in 30 mL of THF. After 4 h, the reaction was quenched by the dropwise addition of 5 mL of methanol and then warmed to room temperature. After pouring into 200 mL of a 2/1 mixture of ethyl acetate/saturated NH₄Cl, the layers were separated, and the aqueous phase was extracted with ethyl acetate (2 X 100 mL). Combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel eluting with ethyl acetate/hexanes, 3/2, to afford 4.7 g (48%) of 14 as a white solid, m. p. 101.0-102.5 °C. ¹H NMR δ 8.05-7.95 (m, 2 H), 7.62-7.40 (m, 3 H), 7.18 (t, J = 8.0 Hz, 1 H), 7.0-6.92 (m, 1 H), 6.85 (t, J = 2.1 Hz, 1 H), 6.77-6.70 (m, 1 H), 5.85 (d of d, J = 6.2, 15.5 Hz, 1 H), 5.72 (d of d, J = 4.5, 15.5 Hz, 1 H), 5.30 (q, J = 5.8 Hz, 1 H), 5.12-5.04 (m, 1 H), 4.58-4.48 (m, 1 H), 3.92 (d of d, J = 3.5, 9.3 Hz, 1 H), 3.80 (d of d, J = 7.3, 9.4 Hz, 1 H), 2.9-2.2 (m, 8 H).

E: (3a<i>R</i>, 4<i>R</i>, 5<i>R</i>, 6a<i>S</i>)-4-[(<i>E</i>)-(3<i>R</i>)-4-(3-Chlorophenoxy)-3-(tetrahydropyran-2-yloxy)-1-butenyl]-hexahydro-5-(tetrahydrocyran-2-yloxy)-2<i>H</i>-cyclopenta[b]furan-2-one (<b>16</b>)

0027To a mixture of 5.1 g (11.5 mmol) of <b>14</b> in 200 mL of methanol was added 1.7 g (12 mmol) of K₂CO₃. After 1 h, the mixture was poured into 100 mL of 0.5 M HCl and extracted with ethyl acetate (3 X 100 mL). The combined organic layers were washed successively with water (2 X 100 mL) and saturated NaCl (2 X 100 mL). The organic layer was dried over MgSO₄, filtered, and concentrated to afford 4.85 g of crude diol <b>15,</b> which was used in the next step without further purification.

0028To a mixture of 4.85 g of crude <b>15</b> and 2.4 g (28 mmol) of 3,4-dihydro-2<i>H-</i>pyran in 75 mL of CH₂Cl₂ at 0 °C was added 370 mg (1.9 mmol) of <i>p-</i>toluenesulfonic acid monohydrate. After stirring for 45 min, the reaction was poured into 40 mL of saturated NaHCO₃, layers were separated, and the aqueous layer was extracted with CH₂cl₂ (2 X 40 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated. The residue was chromatographed on silica gel eluting with 40% ethyl acetate In hexanes, to afford 6.0 g (100%) of <b>16</b> as an oil. ¹H NMR (COCl₃) δ (characteristic peaks only) 7.25-7.14 (m, 1 H), 6.95-6.87 (m, 2 H), 6.83-6.72 (m, 1 H), 5.8-5.4 (m, 4 H), 5.1-4.8 (m, 2 H).

F: (13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11-15-Bis(tetrahydrypyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4,5,6,17,18,19,20-nonanor-9-triethylsiloxy-13-prostenol triethylsilyl ether (<b>18</b>)

0029To a suspension of 400 mg (10.5 mmol) of lithium aluminum hydride in 20 mL of THF at 0 °C was added dropwise a solution of 4.5 g (8.8 mmol) of lactone 16 in 20 mL of THF. After 1 h at 0 °C the mixture was cautiously poured into 100 mL of a 1/1 mixture of ice-cold saturated NH₄Cl/ethyl acetate. The layers were separated, and the aqueous layer was extracted with ethyl acetate (2 X 50 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated to afford 4.5 g (100%) of diol <b>17</b> which was used in the next step without further purification.

0030Triethylsilyl chloride (3.0 g. 20 mmol) was added to a mixture of 4.5 g (8.8 mmol) of crude <b>17,</b> 40 mL of DMF, 1.85 g (27.0 mmol) of imidazole, and 310 mg (2.5 mmol) of 4-(dimethylamino)pyridine. After 2 h, the reaction was poured into 100 mL of a 1/1 mixture of ethyl acetate/saturated NH₄Cl, layers were separated, and the aqueous layer was extracted with ethyl acetate (2 X 25 mL). The combined organic layers were washed with water (3 X 25 mL), dried over MgSO₄, and concentrated. The residue was chromatographed on silica gel eluting with 20% ethyl acetate in hexane to afford 5.2 g (80%) of <b>18.</b>¹H NMR (CDCl₃) δ (characteristic peaks only) 7.22-7.12 (m, 1 H), 6.95-6.88 (m, 2 H), 6.83-6.71 (m, 1 H), 5.8-5.4 (m, 4 H), 5.1-4.8 (m, 2 H), 1.0-0.85 (m, 18 H), 0.7-0.5 (m, 12 H).

G: (13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4,5,6,17,18,19,20-nonanor-9-triethylsilyoxy-13-prostenal (<b>19</b>)

0031To a mixture of 1.6 g (12.6 mmol) of oxalyl chloride and 15 mL of CH₂Cl₂ at -78°C was added dropwise a solution of 1.54 g (19.7 mmol) of DMSO in 2 mL of CH₂Cl₂. After 10 min, 4.6 g (6.2 mmol) of bissilane <b>18</b> in 8 mL of CH₂Cl₂ was added dropwise. After 95 min, 3.0 g (30 mmol) of NEt₃ was added. The mixture was then warmed to room temperature and poured into 70 mL of saturated NH₄Cl-The solution was extracted with of CH₂Cl₂ (3 X 70 mL) and the combined organic layers were dried over MgSO₄, filtered, and concentrated. The residue was chromatographed on silica gel eluting with 20% ethyl acetate in hexane to afford 2.06 g (53%) of <b>19</b> as well as 1.5 g (26%) recovered <b>18.</b>¹H NMR (CDCl₃) δ (characteristic peaks only) 9.78 (t, J = 1.4 Hz, 1 H), 7.22-7.12 (m, 1 H), 6.95-6.88 (m, 2 H). 6.83-6.71 (m, 1 H), 5.8-5.4 (m, 4 H) 5.1-4.8 (m, 2 H), 1.0-0.85 (m, 18 H), 0.7-0.5 (m, 12 H).

H: (5<i>Z</i>, 13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2yloxy)-16-(3-chloro-phenoxy)-2,3,4,17,18,19,20-heptanor-9-triethylsilyloxy-5,13-prostadienoic acid methyl ester (<b>21</b>)

0032To a solution of 1.35 g (4.24 mmol) of phosphonate <b>20</b> and 2.60 g (9.84 mmol) of 18-crown-6 in 20 mL of THF at -78 °C was added dropwise 6.9 mL (3.45 mmol) of a 0.5 <i>M</i> solution in toluene of potassium hexamethyldisilazane. After stirring for 15 min, a solution of 1.65 g (2.64 mmol) of aldehyde <b>19</b> in 20 mL of THF was added dropwise. One hour later, the mixture was poured into 100 mL of saturated NH₄Cl/ethyl acetate, 1/1, layers were separated, and the aqueous layer was extracted with ethyl acetate (3 X 30 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated and the residue was chromatographed on silica gel eluting with 20% ethyl acetate in hexane to afford 1.135 g (63%) of <b>21.</b>¹H NMR (CDCl₃) δ (characteristic peaks only) 7.22-7.11 (m, 1 H), 6.97-6.86 (m, 2 H), 6.85-6.75 (m, 1 H), 6.4-6.2 (m, 1 H), 5.8-5.32 (m, 3 H), 3.66 (s, 3 H).

I: (5<i>Z</i>, 13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chloro-phenoxyl)-2,3,4,17,18,19,20-heptanor-9-triethylsilyloxy-5,13-prostadien-1-ol (<b>22</b>)

0033To a solution of 850 mg (1.25 mmol) of ester <b>21</b> in 10 mL of THF at 0 °C was added 2.4 mL (3.6 mmol) of a 1.5 <i>M</i> solution in toluene of dilsobutylaluminum hydride. After 1 h, the mixture was poured into 20 mL of saturated NH₄Cl and was extracted with ethyl acetate (3 X 20 mL). Combined organic layers were dried over MgSO₄, filtered, and concentrated down to 800 mg (98%) of <b>22</b> as an oil. ¹H NMR (CDCl₃) δ (characteristic peaks only) 7.25-7.15 (m, 1 H), 6.97-6.90 (m, 2 H), 6.86-6.75 (m, 1 H), 5.81-5.41 (m, 4 H).

J: (5<i>Z</i>, 13<i>E</i>-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chloro-phenoxy)-3-oxa-17,18,19,20-tetranor-9-triethylsilyloxy-5,13-prostadienoic acid isopropyl ester (<b>23</b>)

0034To a solution of 415 mg (6.37 mmol) of alcohol <b>22</b> In 4 mL of THF at -78 °C was added dropwise 0.35 mL (0.87 mol) of a 2.5 <i>M</i> solution in hexane of n-BuLi. After 15 min, this solution was transferred <i>via</i> syringe to a -78 °C solution of 195 mg (1.08 mmol) of isopropyl bromoacetate in 2 mL of THF. The mixture was kept at -78 °C for 40 min, warmed to room temperature overnight, and then poured into 20 mL of a 1/1 mixture of saturated NH₄Cl/ethyl acetate. Layers were separated, and the aqueous layer was extracted with ethyl acetate (2X10 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel (20% ethyl acetate In hexane) to afford 242 mg (53%) of <b>23</b> as an oil. ¹H NMR (CDCl₃) δ (characteristic peaks only) 7.24-7.15 (m, 1 H), 6.97-6.90 (m, 2 H), 6.86-6.75 (m, 1 H), 5.81-5.41 (m, 4 H), 1.57 (d. J = 5.7 Hz, 6 H).

K: (5<i>Z</i>, 13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-16-(3-Chlorophenoxy)-3-oxa-17,18,19,20-tetranor-9,11,15-trihydroxy-5,13-prostadienoic acid isopropyl ester (<b>5</b>)

0035To a solution of 230 mg (0.32 mmol) of silane <b>23</b> in 5 mL of THF at room temperature was added 0.33 mL (0.33 mmol) of a 1 <i>M</i> solution of Bu₄NF in THF. After 20 min, the reaction was poured into 4 mL of saturated NH₄Cl and was extracted with ethyl acetate (4X5 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel (ethyl acetate/hexane, 1/1), to afford 126 mg (65%) of desilytated compound <b>24.</b>

0036To 120 mg of <b>24</b> in 5 mL of methanol was added 0.4 mL of 2 M HCl. After 1 h, the mixture was added to 3 mL of saturated NaHCO₃, and the resulting mixture was extracted with ethyl acetate (3X8 mL). Combined organic layers were dried over MgSO₄, filtered, concentrated. The resulting residue was then chromatographed on silica gel eluting with ethyl acetate to afford 54 mg (56%) of <b>S.</b>¹³C NMR (CDCl₃) δ 169.92 (C), 159.26 (C), 135.13 (CH), 134.95 (CH), 134.81 (C), 124.93 (CH), 121.22 (CH), 115.06 (CH), 113.08 (CH), 77.75 (CH), 72.02 (CH), 71.94 (CH₂), 70.76 (CH₂), 68.77 (CH), 67.78 (CH₂). 66.50 (CH₂), 55.46 (CH), 49.93 (CH), 42.47 (CH₂), 25.85 (CH₂), 21.75 (CH₃). Cl MS, <i>m</i>/<i>z</i> calcd. for C₂₄H₃₄O₇Cl₁ (MH⁺), 469.1993, found 469.1993.

EXAMPLE 2 (Reference): Synthsesis of 13,14-Dihydrofluprostenol isopropyl Ester

0037<chemistry id="chem0013" num="0013"><img file="EP1514548B1_D0013.tif" /></chemistry>

A: (3a<i>R</i>, 4<i>R</i>, 5<i>R</i>, 6a<i>S</i>)-Hexahydro-5-hydroxy-4-[(3<i>R</i>)-4-(3-trifluromethyl)phenoxl-3-hydro-1-butyl] 2<i>H</i> cyclopenta[b]furan-2-one (<b>26</b>)

0038A mixture of 1.2 g (3.2 mmol) of diol <b>25</b> (for synthesis of diol <b>25,</b> see <patcit id="pcit0006" dnum="US4321275A"><text>U.S. Patent 4,321,275</text></patcit>) and 0.05 g of 10% (wt/wt) Pc/C in 20 mL of methanol was hydrogenated at 30 psi for 1.5 hours. After filtration through a short pad of Celite, concentration afforded 1.2 g of <b>26</b> as a colorless oil. ¹H NMR (CDCl₃) δ 7.44 (m, 2 H). 7.12 (m, 2 H), 4.95 (dt, 1 H), 4.15-3.80 (m, 4 H), 2.82 (dd, J = 10.8, 1 H), 2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 6 H).

B: (3a<i>R</i>, 4<i>R</i>, 5<i>R</i>, 6a<i>S</i>)-Hexahyrdro-5-(tetrahyrdropyran-2-yloxy)-4-[(3<i>R</i>)-4-(3-trifluoromethylphenoxy)-3-(tetrahydropyran-2-yloxy)-1-butyl]-2<i>H</i>-cyclopenta[b]furan-2-one (<b>27</b>

)

0039A mixture of 1.2 g (3.2 mmol) of diol <b>26</b> and 0.05 g of p-toluenesulfonic acid monohydrate in 100 mL of CH₂Cl₂ at 0 °C was treated with dihydropyran (1.1 ml, 12 mmol) and the solution was stirred for 2 h at 0 °C. After pouring into saturated NaHCO₃, phases were separated and the organic layer was dried over MgSO₄, filtered, concentrated, and purified by chromatography on silica gel (1/1, hexanes/ EtOAc) to afford 1.1 g of <b>27</b> as a clear, colorless oil. ¹H NMR (COCl₃) δ 8.04 (dd, J = 7.0. 1.8, 1 H), 7.44 (m, 2 H), 7.12 (m, 1 H), 4.95 (dt, 1 H), 4.8 (m. 1 H), 4.7 (m, 2 H), 4.15-3.80 (m, 4 H), 3.5 (m, 2 H), 2.82 (dd, J = 10.8, 1 H), 2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 6 H).

C: (5<i>Z</i>)-(9<i>S</i>, 11<i>R,</i> 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-9-hydroxy-17,18,19,20-tetranor-16-(3-trifluoromethylphenoxy)-5-prostenoic acid isopropyl ester (<b>31</b>)

0040To a solution of 2.1 g (3.9 mmol) of <b>27</b> in 100 mL of THF at -78 °C was added 3.9 mL (5.8 mmol) of a 1.5 M solution of diisobutyaluminum hydride in toluene. The solution was stirred for 2 h. then quenched by the sequentlal addition of 0.4 mL of isopropanol at -78 °C followed by 0.4 mL of water at 23 °C. Volatiles were removed under reduced pressure and the aqueous solution was extracted with Et₂/EtOAc (1/1). Organic extracts were dried over MgSO₄, filtered, and concentrated to furnish 1.9 g of lactol <b>28.</b>

0041To a 250 mL 3-necked round bottom flask equipped with a mechanical stirrer and a thermometer were added anhydrous DMSO (100 mL) and NaH (80% dispersion in mineral oil; 0.48 g, 16 mmol). The mixture was heated to 75 °C (internal) for 30 min, after which it was allowed to cool to room temperature for 1 h. Phosphonium bromide <b>29</b> (3.5 g, 8 mmol) was then added. After stirring for 30 minutes, 1.9 g (3.5 mmol) of lactol <b>28</b> in 50 mL of DMSO was added, and the resulting solution was heated to 50 °C for 2 h and then brought to room temperature for 16 h. The solution was then poured into 100 mL of water and approximately 2 mL of 50% NaOH added. The aqueous phase was extracted with ether (3 X 100 mL), then made acidic (pH = 5.5) by the addition of a 10% citric acid solution, and extracted with Et₂O:hexanes 2:1 (3 X 100 mL). The combined organic extracts were dried over MgSO₄. filtered, and concentrated to afford 1.9 g of 30 as a colorless oil.

0042To 1.9 g of carboxylic acid <b>30</b> dissolved in 10 mL acetone was added 0.95 g (6.0 mmol) of DBU and 1.0 g (6.1 mmol) of isopropyl iodide 1.0 g (6.1 mmol) at 23 °C. After 16 h. the solution was poured into 100 mL of water and extracted with 100 mL of EtOAc. The organic extract was dried over MgSO₄, filtered, concentrated, and purified by silica gel chromatography (3/2, hexanes/EtOAc) to afford 1.9 g of isopropyl ester <b>31</b> as a colorless oil. ¹H NMR (CDCl₃) δ 7.44 (t, 1 H), 7.12 (d, 1 H), 7.12 (dd, 2 H), 5.5-5.3 (m, 2 H), 4.99 (heptet, 1 H), 4.15-3.80 (m, 4 H), 2.82 (dd, J = 10.8, 1 H), 2.55 (m. 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 24 H), 1.23 (s, 3 H), 1.20 (s. 3 H).

D: (5<i>Z</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-17,18,19,20-Tetranor-16-(3-trifluoromethyl)-9,11,15-trihydroxy-5-prostenoic acid isopropyl ester (<b>6</b>)

0043Ester <b>31</b> (1.9 g, 2.8 mmol) was dissolved In 14 mL of a mixture of AcOH/THF/H₂O (4/2/1) and the solution was heated to 50 °C for 1 h. allowed to cool to 23 °C, poured Into a saturated solution of NaHCO₃, and extracted with Et₂O (2 X 100 mL) and EtOAc (100 mL). The combined organic extracts were dried over MgSO₄, filtered, concentrated, and purified by silica gel chromatography (1/1, hexanes/EtOAc) to furnish 0.5 g of triol <b>6</b> as a clear, colorless oil. ¹H NMR (CDCl₃) δ 7.44 (t, J = 7.8, 1 H). 7.12 (dd, J = 7.8, 2.0, 1 H), 7.12 (ddd, J = 15.6, 7.2, 2.0, 2 H), 5.5-5.3 (m, 2 H). 4.99 (heptet, J = 6.3, 1 H), 4.16-3.80 (m, 4 H), 3.2 (d, 1 H); 2.95 (s, 1 H). 2.82 (dd, J = 10.8, 1 H), 2.75 (d, J = 5.9, 1 H), 2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 24 H), 1.23 (s, 3 H), 1.20 (s, 3 H). CMR (CDCl₃) δ 173.5, 158.7, 132.1, 131.5, 130.0, 129.5, 129.2, 123.3, 120.8, 117.7, 117.6, 111.4, 111.4, 78.6, 74.4, 72.4, 69.9, 67.6, 52.6, 51.7, 42.5, 34.0, 31.5, 29.4, 26.8, 26.6, 24.9, 21.7.

EXAMPLE 3 (Reference): Synthesis of Cloprostenol-1-ol (7)

0044<chemistry id="chem0014" num="0014"><img file="EP1514548B1_D0014.tif" /></chemistry>

A: (5<i>Z</i>, 13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-16(3-chloprophenoxy)-9-hydroxy-17,18,19,20-tetranor-5,13-prostadienoic acid isopropyl ester (<b>34</b>)

0045A 1.5 <i>M</i> solution of diisobutylaluminum hydride In toluene (10 mL, 15 mmol) was added dropwise to a solution of 5.8 g (11.4 mmol) of lactone <b>16</b> in 55 mL of THF at -78 °C. After 1 h. 10 mL of methanol was added dropwise, and the mixture was stirred for 10 min at -78 °C before being warmed to room temperature. The mixture was then poured into 100 mL of a 1/1 solution of saturated aqueous potassium sodium tartrate/ethyl acetate and stirred. After separating layers, the aqueous phase was extracted with ethyl acetate (2 X 40 mL). Combined organic layers were dried over MgSO₄, filtered, concentrated, and purified by silica gel chromatography (3/2, ethyl acetate/hexane), to afford 4.4 g (76%) of lactol <b>33,</b> which was used immediately in the next step.

0046A 1 <i>M</i> solution of potassium t-butoxide in THF (50.0 ml) was added dropwise to 12.1 g (27.3 mmol) of phosphonium salt <b>29</b> in 100 mL of THF at 0 °C. After 30 min, a solution of 4.4 g (8.6 mmol) of lactol <b>33</b> in 20 mL of THF was added dropwise, and the mixture was stirred at room temperature overnight. The solution was then poured into 150 mL of a 1/1 mixture of ethyl acetate/saturated NH₄Cl. Layers were separated and the aqueous layer was extracted with ethyl acetate (2 X 100 mL). Combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was redissolved in 80 mL of acetone. To this was added 6.5 g (45 mmol) of DBU followed by 7.3 g (43 mmol) of isopropyl iodide. After stirring overnight, the reaction was poured into 100 mL of a 1/1 mixture of ethyl acetate/saturated NH₄Cl. Layers were then separated and the aqueous phase was further extracted with ethyl acetate (2 X 100 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and purified by silica gel chromatography (40% ethyl acetate in hexane) to afford 2.92 g (53% from lactone <b>16</b>) of ester <b>34.</b>

B: (5<i>Z</i>, 13<i>E</i>)-(9<i>S</i>, 11<i>R</i>, 15<i>R</i>)-16-(3-Chlorophenoxy)-17,18,19,20-tetranor-9,11,15-trihydroxy-5,13-prostadienol (<b>7</b>)

0047A solution of 500 mg (0.79 mmol) of <b>34</b> in 10 mL of THF was added dropwise to 61 mg (1.60 mmol) of lithium aluminum hydride in 20 mL of THF at 0 °C. After 40 min, the reaction was poured into 15 mL of saturated NH₄Cl, and the mixture was then extracted with ethyl acetate (3 X 40 mL). Combined organic layers were dried over MgSO₄ , filtered, and concentrated to afford 500 mg of crude <b>35.</b>

0048To a solution of 500 mg of <b>35</b> in 20 mL of methanol was added 0.5 mL of 2 <i>M</i> HCl. After 1 h, the reaction was quenched with 20 mL of saturated NaHCO₃ and the mixture was extracted with ethyl acetate (4 X 30 mL). The combined organic layers were dried over MgSO₄, filtered, and concentrated. Silica gel chromatography (EtOAc) provided 101 mg (31% from <b>34</b>) of <b>7.</b> "C NMR (CDCl₃ δ 159.27 (C), 135.44 (CH), 134.82 (C), 130.64 (CH), 130.26 (CH), 128.23 (CH), 121.25 (CH), 115.07 (CH), 113.08 (CH), 77.35 (CH), 72.35 (CH), 71.90 (CH₂), 70.89 (CH), 62.22 (CH₂), 55.40 (CH), 49.87 (CH). 42.79 (CH₂). 31.83 (CH₂), 26.77 (CH₂), 25.60 (CH₂), 25.33 (CH₂), Cl MS <i>m</i>/<i>z</i> calcd for C₂₂H₂₂O₅Cl, (MH⁺) 411.1938, found 411.1938.

EXAMPLE 4 (Reference): Synthesis of 13,14-Dihydrocloprostenol-ol Pivaloata (6)

0049<chemistry id="chem0015" num="0015"><img file="EP1514548B1_D0015.tif" /></chemistry>

A:

(3a<i>R,</i> 4<i>R,</i> 5<i>R</i>, 6a<i>S</i>)-4-[(3<i>R</i>)-4-(3-Chlorophenoxy)-3-hydroxybutyl]-hexahydro-5 hydroxy-2<i>H</i>-cyclopenta[<i>b</i>]furan-2-one (<b>37</b>):

0050A mixture of 2.4 g (5.4 mmol) of 14 and 250 mg of 10% (wt/wt) Pd/C in 35 mL of ethyl acetate was hydrogenated at 40 psi for 1 h. After filtration through a short pad of Celite, the filtrate was evaporated down to 2.3 g (100%) of hydrogenated product <b>36.</b>

0051The crude benzoate <b>36</b> was dissolved in 25 mL of methanol, and 610 mg (4.4 mmol) of K₂CO₃ was added. After 3.5 h, the mixture was poured into 100 mL of water/ethyl acetate (1/1). Layers were separated, and the aqueous phase was further extracted with ethyl acetate (2 X 50 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated. Silica gel chromatography (EtoAc) provided 1.50 g (82%) of 37 as a white solid, m.p. = 102.0-103.5 °C. ¹H NMR δ 7.22 (t, J = 8.2_. Hz, 1 H), 7.0-6.94 (m, 1 H), 6.91-6.88 (t, J = 2.1 Hz, 1 H), 6.83-6.77 (m, 1 H), 4.97 (dt, J = 3.0. 8.3 Hz. 1 H), 4.12-3.91 (m. 3 H), 3.82 (dd, J = 7.4, 9.0 Hz, 1 H), 2.85 (dd, J = 8.0, 16.5 Hz, 1 H), 2.6-1.4 (m, 11 H).

B: (3a<i>R</i>, 4<i>R</i>, 5<i>R,</i> 6a<i>S</i>)-4-[(3<i>R</i>)-4-(3-Chlorophenoxy)-3-(tetrahydropyran-2-yloxy)butyl]-hexahydro-5-(tetrahydropyran-2yloxy)-2<i>H</i>-cyclopenta[b]furan-2-one (<b>38</b>)

0052Diol <b>37</b> (3.4 g, 10 mmol) and 2.2 g (26 mmol) of 3,4-dihydro-2<i>H</i>-pyran were dissolved in 80 mL of CH₂Cl₂, and 240 mg (1.3 mmol) of <i>p</i>-toluenesulfonic acid monohydrate was added at 0 °C. After 1 h, the reaction was poured into 50 mL of saturated NaHCO₃ and the mixture was extracted with CH₂Cl₂ (3 X 40 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel (hexane/ethyl acetate, 1/1) to afford 4.5 g (87%) of bis-THP ether <b>38.</b>

C: (5<i>Z</i>)-(9<i>S</i>,11<i>R</i>, 15<i>R</i>)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-hydroxy-17,18,19,20-tetranor-5-prostenoic acid isopropyl ester (<b>41</b>)

0053A 1.5 <i>M</i> solution of diisobutylaluminum hydride in toluene (1.8 mL, 2.7 mmol) was added to the solution 1.05 g (2.06 mmol) of <b>38</b> In 10 mL of THF at -78 °C. After 1 h, 4 mL of methanol was added and the mixture was warmed to 25 °C, then poured into 40 mL of ethyl acetate/saturated aqueous potassium sodium tartrate (1/1). Layers were separated and the aqueous phase was further extracted with ethyl acetate (3 X 30 mL). The combined organic layers were then dried over MgSO₄, filtered, concentrated, and the residue was chromatographed on silica gel (ethyl acetate) to afford 740 mg (70%) of lactol <b>39.</b>

0054A 1.5 <i>M</i> solution of potassium t-butoxide in THF (8.6 mL, 8.6 mmol) was added dropwise to a mixture of 15 mL of THF and 1.92 g (4.33 mmol) of phosphonium salt <b>29</b> at 0 °C. After stirring 1 h, a solution of 740 mg (1.45 mmol) of lactol <b>39</b> in 5 mL of THF was added dropwise, and the reaction was allowed to warm to 25 °C overnight. The mixture was then poured into 100 mL of ethyl acetate/saturated NH₄Cl (1/1). Layers were separated, and the aqueous phase was further extracted with ethyl acetate (2 X 70 mL). Combined organic layers were dried over MgSO₄, filtered, and concentrated to afford 1.6 g of crude acid <b>40.</b>

0055Crude acid <b>40</b> (1.6 g) was dissolved in 11 mL of acetone and cooled to 0 °C, then 850 mg (5.6 mmol) of DBU was added dropwise to the sotution. The resulting mixture was stirred for 15 min at 0 °C and 30 min at 25 °C, after which 850 mg (5.0 mmol) of isopropyl iodide was added. The reaction was stirred overnight, poured into 100 mL of ethyl acetate/saturated NH₄Cl (1/1). Layers were separated, and the aqueous phase was further extracted with ethyl acetate (2 X 50 mL). Combined organic layers were dried over MgSO₄, filtered and concentrated. The resulting residue was purified by silica gel chromatography (ethyl acetate/hexanes, 3/2) to afford 560 mg (61% from lactol <b>39</b>) of isopropyl ester <b>41.</b>

D: (5<i>Z</i>)-(9<i>S</i>,11<i>R</i>,15<i>R</i>)-16-(3-Chlorophenoxy)-17.18.19.20-tetranor-9.11.15-trihydroxy-5-prostenyl) pivaloate (<b>8</b>)

0056A solution of 400 mg (0.63 mmol) of <b>41</b> in 5 mL of THF was added dropwise to a suspension of 35 mg (0.92 mmol) of lithium aluminum hydride in 5 mL of THF at 0 °C. After 2 h. the reaction was poured into 50 mL of a 1/1 mixture of ethyl acetate/saturated NaHCO₃. The layers were then separated, and the aqueous phase was extracted with ethyl acetate (2 X 2 mL). Combined organic layers were dried over MgSO₄, filtered, and concentrated. The resulting residue purified by silica gel chromatography (ethyl acetate) to afford 350 mg (95%) of diol <b>42</b>.

0057Pivaloyl chloride (90 mg, 0.75 mmol) was added to a mixture of 350 mg (0.60 mmol) of <b>42</b>, 60 mg (0.76 mmol) of pyridine, 22 mg (0.18 mmol) of 4-(dimethylamino)pyridine, and 7 mL of CH₂Cl₂. After 1.5 h the mixture was poured into 30 ml of saturated NH₄Cl/ethyl acetate (1/1). Layers were then separated and the aqueous phase was extracted with ethyl acetate (2 X 20 mL). The combined organic layers were dried over MgSO₄, filtered, concentrated, and purified by silica gel chromatography (ethyl acetate/hexane. 3/2) to afford 370 mg (93%) of pivaloate 43.

0058Water (10 drops) and concentrated HCl (3 drops) were added to a solution of 370 mg (0.56 mmol) of 43 in 5 mL of methanol. After stirring overnight, the reaction was quenched by the addition of 20 mL of saturated NaHCO₃, and the mixture was extracted with ethyl acetate (3 X 20 mL). The combined organic layers were dried over MugSO₄, filtered, and concentrated. The residue was chromatographed on silica gel (ethyl acetate/hexane, 3/2), to afford 165 mg (59%) of triol <b>8</b>. ¹³C NMR (CDCl₃) δ 178.77 (C), 159.27 (C), 134.80 (C), 130.20 (CH), 128.62 (CH), 121.19 (CH), 114.97 (CH), 112.97 (CH), 78.50 (CH), 74.46 (CH). 72.31 (CH₂), 69.86 (CH), 64.16 (CH₂), 52.53 (CH), 51.67 (CH), 42.50 (CH₂), 31.51 (CH₂), 29.40 (CH₂), 28.10 (CH₂), 27.12 (CH₃), 26.77 (CH₂), 26.65 (CH₂), 25.77 (CH₂),Cl MS, <i>m</i>/<i>z</i> calcd for C₂₇H₄₁O₆Cl₁ (MH⁺), 497.2670, found 497.2658

0059The studies detailed in the following Examples 5-9 compared the IOP lowering activity and side effects of five compounds: A) Cloprostenol, isopropyl ester; B) Fluprostenol, isopropyl ester; C) 16-Phenoxy-17,18,19,20-tetranor PGF_2α, isopropyl ester; D) 17-Phenyl-18,19,20-trinor PGF_2α, isopropyl ester; and E) 13,14-Dihydro-17-phenyl-18,19,20-trinor PGF_2α' isopropyl ester (latanoprost). The structures of these compounds are shown In the following Table 2. <tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="14mm" /><colspec colnum="2" colname="col2" colwidth="84mm" /><colspec colnum="3" colname="col3" colwidth="68mm" /><thead><row><entry align="center" valign="top" /><entry align="center" valign="top"><b>COMPOUND NAME</b></entry><entry align="center" valign="top"><b>COMPOUND STRUCTURE</b></entry></row></thead><tbody><row><entry align="center"><b>A</b></entry><entry>Cloprostenol, isopropyl ester</entry><entry><chemistry id="chem0016" num="0016"><img file="EP1514548B1_D0016.tif" /></chemistry></entry></row><row><entry align="center"><b>B</b></entry><entry>Fluprostenol, isopropyl ester</entry><entry><chemistry id="chem0017" num="0017"><img file="EP1514548B1_D0017.tif" /></chemistry></entry></row><row><entry align="center"><b>C</b></entry><entry>16-Phenoxy-17,18,19,20-tetranor PGF_2α' isopropyl ester</entry><entry><chemistry id="chem0018" num="0018"><img file="EP1514548B1_D0018.tif" /></chemistry></entry></row><row><entry align="center"><b>D</b></entry><entry>17-Phenyl-18,19,20-trinor PGF_2α' isopropyl ester</entry><entry><chemistry id="chem0019" num="0019"><img file="EP1514548B1_D0019.tif" /></chemistry></entry></row><row><entry align="center"><b>E</b></entry><entry>13,14-Dihydro-17-phenyl-18,19,20-trinor PGF_2α' isopropyl ester</entry><entry><chemistry id="chem0020" num="0020"><img file="EP1514548B1_D0020.tif" /></chemistry></entry></row></tbody></tgroup></table></tables>

0060As is apparent in Table 2, the five compounds differ only slightly in structure; however, as Examples 5 and 6 will show, such seemingly slight structural differences produce greatly different IOP-lowering effects and levels of hyperemia

EXAMPLE 5

0061Compounds A-E (Table 2, above) were tested for hyperemia in the guinea pig. The objective of the guinea pig conjunctival hyperemia model is to provide a primary screening indication of the potential of a prostaglandin for inducing conjunctival hyperemia in humans.

0062Guinea pigs were maintained in their cages during the study, and removed only for scoring and dosing. Eyes were evaluated using a magnifying lens with fluorescent illumination and scores for conjunctival hyperemia were recorded for upper bulbar conjunctiva according to the following criteria: <ul id="ul0002" list-style="none" compact="compact"><li><i>0 = Normal appearance of vessels at limbus and on superior rectus muscle</i></li><li><i>+1 = Enlargement of vessels normally visible at limbus and on superior rectus muscle</i></li><li><i>+2 Branch of vessels at limbus, new vessels visible</i></li><li><i>+3 = New vessels visible in open bulbar conjunctival areas</i></li><li><i>+4 = Diffuse redness in open bulbar conjunctival areas</i></li></ul> Scores of 0 or 1 indicated no hyperemia, and scores of 2-4 Indicated hyperemia (a score of 4 indicating the most hyperemia). Only integer scores were permitted in order to minimize subjectivity.

0063Baseline observations were made prior to unilateral dosing with a 10 µL aliquot of either the prostaglandin test formulation or the control formulation, followed by observations at 1, 2, 3 and 4 hours after dosing. Groups typically contained four animals, but ranged up to eight animals per group. The results of the study are presented in Table 3, below, as percent frequency of each score, and in <figref idref="f0001">Figure 1</figref> as percent incidence of hyperemia, defined as the percent of scores of +2 or +3 relative to the total number of observations for each dose. <tables id="tabl0003" num="0003"><img file="EP1514548B1_D0021.tif" /></tables>

Discussion:

0064Compound <b>C</b> (16-phenoxy-17,18,19,20-tetranor PGF_2α, isopropyl ester) produces significant hyperemia at low doses, and at 0.3 and 1.0 µg doses, all eyes received one or more scores of +3. Compound <b>D</b> (17-phenyl-18,19,20-trinor PGF_2α, isopropyl ester) produces less hyperemia than compound C, but significantly more than compound <b>E</b> (13,14-dihydro-17-phenyl-18,19,20-trinor PGF_2α, isopropyl ester), which produces only mild hyperemia. The hyperemia produced by compound <b>A</b> (cloprostenol, isopropyl ester) and compound <b>B</b> (fluprostenol, isopropyl ester) appear to be intermediate between that of compound <b>D</b> and compound <b>E</b>, but this degree of hyperemia is also mild, and cannot be distinguished from that produced by compound <b>E</b>.

EXAMPLE 6

0065In the study presented below, compounds <b>A-E</b> (Table 2, above) were tested for IOP-lowering effect in cynomolgus monkey eyes.

0066The right eyes of the cynomolgus monkeys used in this study were previously given laser trabeculoplasty to induce ocular hypertension in the lasered eye. Animals had been trained to sit in restraint chairs and conditioned to accept experimental procedures without chemical restraint. IOP was determined with a pneumatonometer after light corneal anesthesia with dilute proparacaine. The test protocol included a five-dose treatment regimen because of the typical delayed response to prostaglandins. The designated test formulations were administered to the lasered right eyes, and the normal left eyes remained untreated, although IOP measurements were taken. Baseline IOP values were determined prior to treatment with the test formulation, and then IOP was determined from 1 to 7 hours after the first dose, 16 hours after the fourth dose, and 1 to 4 hours after the fifth dose. Results are presented in Tables 4 and 5, below, and in <figref idref="f0002">Figures 2</figref> and <figref idref="f0003">3</figref>, as the mean percent reduction of IOP from baseline ± SEM. Prostaglandin doses are micrograms of compound contained in each treatment with 10 µL of the test formulation. In Table 4, the same amount (0.3 µg) of each of compounds <b>A-E</b> were compared for IOP reduction. In Table 5, various amounts of compound <b>A</b> (0.3 and 1.0 µg) were compared against various amounts of compound E (0.3, 1.0 and 3.0 µg) in order to determine the dose responses of the two different compounds. <tables id="tabl0004" num="0004"><table frame="all"><title><b>Table 4: Percent IOP Reduction in Lasered Cynomolgus Monkeys</b></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="50mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="20mm" colsep="1" /><thead><row><entry morerows="1" colsep="1" align="center" valign="middle">Compound (isopropyl ester)</entry><entry morerows="1" colsep="1" align="center" valign="middle">Baseline IOP (mm Hg)</entry><entry namest="col3" nameend="col6" align="center" valign="middle">Percent IOP Reduction (Hours after Last Dose/Dose#)</entry></row><row><entry colsep="1" align="center" valign="middle">16/4</entry><entry colsep="1" align="center" valign="middle">2/5</entry><entry colsep="1" align="center" valign="middle">4/5</entry><entry align="center" valign="middle">6/5</entry></row></thead><tbody><row><entry><b>A</b> (Cloprostanol)</entry><entry align="center">36.9</entry><entry align="center">23.6 ± 3.3</entry><entry align="center">30.2 ± 4.5</entry><entry align="center">31.2 ± 6.8</entry><entry align="center">24.4 ± 6.9</entry></row><row><entry><b>B</b> (Fluprostenol)</entry><entry align="center">41.6</entry><entry align="center">18.4 ± 5.9</entry><entry align="center">312 ± 3.7</entry><entry align="center">30.3 ± 3.8</entry><entry align="center">26.6 ± 3.6</entry></row><row><entry><b>C</b> (16-Phenoxy-17,18,19,20-tetranor PGF_2α)</entry><entry align="center">38.2</entry><entry align="center">30.2 ± 4.4</entry><entry align="center">25.3 ± 4.5</entry><entry align="center">23.6 ± 3.8</entry><entry align="center">28.9 ± 3.0</entry></row><row><entry><b>D</b> (17-Phenyt-18. 19,20-trinor PGF_2α')</entry><entry align="center">40.8</entry><entry align="center">25.6 ± 2.6</entry><entry align="center">36.0 ± 2.4</entry><entry align="center">39.8 ± 3.1</entry><entry align="center">30.3 ± 2.8</entry></row><row><entry><b>E</b> (13,14-Dihydro-17-phenyl-18,19, 20-trinor PGF_2α)</entry><entry align="center">39.7</entry><entry align="center">7.6 ± 2.9</entry><entry align="center">3.6 ± 2.7</entry><entry align="center">7.5 ± 2.7</entry><entry align="center">8.0 ± 3.4</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title><b>Table 5: Comparison of Percent IOP Redaction</b></title><tgroup cols="7" colsep="0"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="20mm" /><colspec colnum="7" colname="col7" colwidth="20mm" colsep="1" /><thead><row><entry morerows="1" colsep="1" align="center" valign="middle">Compound</entry><entry morerows="1" colsep="1" align="center" valign="middle">Dose (µg)</entry><entry morerows="1" colsep="1" align="center" valign="middle">Baseline IOP (mm Hg)</entry><entry namest="col4" nameend="col7" align="center" valign="middle">Percent IOP Reduction (Hours after Last Dose/Dose#)</entry></row><row><entry colsep="1" align="center" valign="middle">16/4</entry><entry colsep="1" align="center" valign="middle">2/5</entry><entry colsep="1" align="center" valign="middle">4/5</entry><entry align="center" valign="middle">6/5</entry></row></thead><tbody><row><entry valign="middle"><b>A*</b></entry><entry align="center" valign="middle">0.3</entry><entry align="center" valign="middle">36.9</entry><entry align="center" valign="middle">23.6 ± 3.3</entry><entry align="center" valign="middle">30.2 ± 4.S</entry><entry align="center" valign="middle">31.2 ± 6.8</entry><entry align="center" valign="middle">24.4 ± 6.9</entry></row><row><entry valign="middle"><b>A</b></entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">39.6</entry><entry align="center" valign="middle">34.8 ± 4.5</entry><entry align="center" valign="middle">36.7 ± 5.6</entry><entry align="center" valign="middle">38.7 ± 5.9</entry><entry align="center" valign="middle">35.8 ± 5.1</entry></row><row><entry valign="middle"><b>E</b></entry><entry align="center" valign="middle">0.3</entry><entry align="center" valign="middle">39.7</entry><entry align="center" valign="middle">7.6 ± 2.9</entry><entry align="center" valign="middle">3.6 ± 2.7</entry><entry align="center" valign="middle">7.5 ± 2.7</entry><entry align="center" valign="middle">8.0 ± 3.4</entry></row><row><entry valign="middle"><b>E**</b></entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">38.9</entry><entry align="center" valign="middle">23.2 ± 3.6</entry><entry align="center" valign="middle">22.0 ± 4.0</entry><entry align="center" valign="middle">18.8 ± 5.2</entry><entry align="center" valign="middle">20.2 ± 4.0</entry></row><row><entry valign="middle"><b>E</b></entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">30.1</entry><entry align="center" valign="middle">11.6 ± 6.5</entry><entry align="center" valign="middle">17.6 ± 5.8</entry><entry align="center" valign="middle">13.1 ± 5.0</entry><entry align="center" valign="middle">12.7 ± 5.0</entry></row></tbody></tgroup><tgroup cols="7" rowsep="0"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="20mm" /><colspec colnum="7" colname="col7" colwidth="20mm" /><tbody><row><entry namest="col1" nameend="col7" align="justify">*Cloprostenol, isopropyl ester **13,14-Dihydro-17-phenyl-18,19,20-trinor PGF_2α, Isopropyl ester</entry></row></tbody></tgroup></table></tables>

Discussion:

0067Table 4 shows that compounds <b>A, B, C,</b> and <b>D</b> produce similar degrees of IOP reduction with 0.3 µg doses; however, compound E is essentially inactive at this dose.

0068In Table 5, it is apparent that the IOP reduction with 1 µg of compound <b>A</b> is greater than that produced by 0.3 µg of compound <b>A</b>, and the response to either of these doses of compound <b>A</b> is greater than the maximum reduction produced by either dose of compound <b>E</b>. These observations indicate that compound <b>A</b> (cloprostenol, isopropyl ester) is both more potent and produces a greater maximum response for IOP reduction than compound <b>E</b> (13,14-dihydro-17-phenyl-18,19.20-trinor PGF_2α).

EXAMPLE 7 (Reference)

0069PGF_2α analogues are known to contract the iris sphincter of cats and this assay is a generally accepted reference for activity. For this reason, the pupil diameter of cats may be used to define the activity of PGF_2α analogues and, as demonstrated by <nplcit id="ncit0003" npl-type="s"><text>Stjemschantz and Resul (Drugs Future, 17:891-704 (1992</text></nplcit>)), predict the IOP-lowering potency.

0070Compounds of the present invention were therefore screened for pupillary constriction In the cat. Data for compounds 6, 7, and 8 are presented In Table 6, below. The response is quantitated as Area _1-5 values (area under the pupil diameter versus time curve from 1-5 hours), and the equivalent response dose (ED₅) is estimated from Its dose response relationship. <tables id="tabl0006" num="0006"><table frame="topbot"><title><b>Table 6: Cat Pupil Diameter Response</b></title><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Compound</b></entry><entry align="center" valign="top"><b>ED₅ (µg)</b></entry></row></thead><tbody><row rowsep="0"><entry>PGF_2α Isopropyl Ester</entry><entry align="char" char="." charoff="7">0.02</entry></row><row rowsep="0"><entry>Cloprostenol Isopropyl Ester</entry><entry align="char" char="." charoff="7">0.01</entry></row><row rowsep="0"><entry><b>6</b></entry><entry align="char" char="." charoff="7">0.2</entry></row><row rowsep="0"><entry><b>7</b></entry><entry align="char" char="." charoff="7">0.02</entry></row><row><entry><b>8</b></entry><entry align="char" char="." charoff="7">0.06</entry></row></tbody></tgroup></table></tables>

Discussion:

0071The two standard compounds, PGF_2α isopropyl ester and cloprostenol isopropyl ester, produced marked change in cat pupillary diameter, displaying ED₅ values of 0.02 and 0.01 µg, respectively. Compound 7 (cloprostenol-1-ol) and compound 8 (13,14-dihydrocloprostenol-1-ol pivaloate), displayed nearly equivalent potency. 13,14-Dihydrofluprostenol isopropyl ester (compound 6) was approximately one order of magnitude less potent, with an ED₅ of 0.2 µg.

EXAMPLE 8 (Reference)

0072In the study presented below, compound 6 (Table 1, above) was tested for IOP-lowering effect In cynomolgus monkey eyes.

0073The right eyes of the cynomolgus monkeys used In this study were previously given laser trabeculoplasty to induce ocular hypertension in the lasered eye. Animals had been trained to sit In restraint chairs and conditioned to accept experimental procedures without chemical restraint. IOP was determined with a pneumatonometer after light corneal anesthesia with dilute proparacaine. The test protocol Included a five-dose treatment regimen because of the typical delayed response to prostaglandins. The designated test formulations were administered to the lasered right eyes, and the normal left eyes remained untreated, although IOP measurements were taken. Baseline IOP values were determined prior to treatment with the test formulation, and then IOP was determined from 1 to 7 hours after the first dose, 16 hours after the fourth dose, and 1 to 4 hours after the fifth dose.

0074The equivalent response dose (ED₂₀) Is estimated from the dose response relationship to be the dose producing a 20% peak reduction In IOP. <tables id="tabl0007" num="0007"><table frame="bottom"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="36mm" /><colspec colnum="2" colname="col2" colwidth="22mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top"><b>Table 7: Monkey lop Response</b></entry></row><row><entry align="center" valign="top"><b>Compound</b></entry><entry align="center" valign="top"><b>ED₂₀ (µg)</b></entry></row></thead><tbody><row rowsep="0"><entry>PGF_2α Isopropyl Ester</entry><entry align="char" char="." charoff="9">0.4</entry></row><row><entry><b>6</b></entry><entry align="char" char="." charoff="9">0.3</entry></row></tbody></tgroup></table></tables>

Discussion

:

0075As can be seen in Table 7, above, compound 6, the 13,14-dihydro analogue of fluprostenol was quite potent in the monkey IOP model, producing a 20% reduction at 0.3 µg This was even more potent than the standard compound, PGF_2α isopropyl ester.

EXAMPLE 9

0076The following Formulations 1-8 are representative pharmaceutical compositions for topical use in lowering of intraocular pressure. Each of Formulations 1 through 8 may be formulated in accordance with procedures known to those skilled In the art.

FORMULATION 1 (Reference)

0077<tables id="tabl0008" num="0008"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="76mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Cloprostenol isopropyl ester (Table 2, Compound A)</entry><entry align="center">0.002</entry></row><row rowsep="0"><entry>Dextran 70</entry><entry align="center">0.1</entry></row><row rowsep="0"><entry>Hydroxypropyl methylcellulose</entry><entry align="center">0.3</entry></row><row rowsep="0"><entry>Sodium Chloride</entry><entry align="center">0.77</entry></row><row rowsep="0"><entry>Potassium chloride</entry><entry align="center">0.12</entry></row><row rowsep="0"><entry>Disodium EDTA (Edetate disodium)</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.2 - 7.5</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 2 (Reference)

0078<tables id="tabl0009" num="0009"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Cloprostenol, t-butyl ester</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA (Edetate disodium)</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.02</entry></row><row rowsep="0"><entry>Polysorbate 80</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 3 (Reference)

0079<tables id="tabl0010" num="0010"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Cloprostenol, methyl ester</entry><entry align="center">0.001</entry></row><row rowsep="0"><entry>Dextran 70</entry><entry align="center">0.1</entry></row><row rowsep="0"><entry>Hydroxypropyl methylcellulose</entry><entry align="center">0.5</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA (Edetate disodium)</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>NaOH and/or HCl</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 4 (Invention)

0080<tables id="tabl0011" num="0011"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="76mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Fluprostenol Isopropyl ester (Table 2, Compound <b>B</b>)</entry><entry align="center">0.003</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA (Edetate disodium)</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 5 (Reference)

0081<tables id="tabl0012" num="0012"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="48mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Compound 5 (Table 1)</entry><entry align="center">0.002</entry></row><row rowsep="0"><entry>Dextran 70</entry><entry align="center">0.1</entry></row><row rowsep="0"><entry>Hydroxypropyl methylcellulose</entry><entry align="center">0.3</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.77</entry></row><row rowsep="0"><entry>Potassium chloride</entry><entry align="center">0.12</entry></row><row rowsep="0"><entry>Disodium EDTA</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.2 - 7.5</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 6 (Reference)

0082<tables id="tabl0013" num="0013"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Compound 6 (Table 1)</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.02</entry></row><row rowsep="0"><entry>Polysorbate 80</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 7 (Reference)

0083<tables id="tabl0014" num="0014"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Compound 7 (Table 1)</entry><entry align="center">0.001</entry></row><row rowsep="0"><entry>Dextran 70</entry><entry align="center">0.1</entry></row><row rowsep="0"><entry>Hydroxypropyl methylcellulose</entry><entry align="center">0.5</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>NaOH and/or HCl</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>

FORMULATION 8 (Reference)

0084<tables id="tabl0015" num="0015"><table frame="topbot"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><thead><row><entry align="center" valign="top"><b>Ingredient</b></entry><entry align="center" valign="top"><b>Amount (wt%)</b></entry></row></thead><tbody><row rowsep="0"><entry>Compound 8 (Table 1)</entry><entry align="center">0.003</entry></row><row rowsep="0"><entry>Monobasic sodium phosphate</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Dibasic sodium phosphate (anhydrous)</entry><entry align="center">0.15</entry></row><row rowsep="0"><entry>Sodium chloride</entry><entry align="center">0.75</entry></row><row rowsep="0"><entry>Disodium EDTA</entry><entry align="center">0.05</entry></row><row rowsep="0"><entry>Benzalkonium chloride</entry><entry align="center">0.01</entry></row><row rowsep="0"><entry>HCl and/or NaOH</entry><entry align="center">pH 7.3 - 7.4</entry></row><row><entry>Purified water</entry><entry align="center">q.s. to 100%</entry></row></tbody></tgroup></table></tables>