WO2011077435A1 - Compositions and methods for reducing intraocular pressure - Google Patents

Compositions and methods for reducing intraocular pressure Download PDF

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Publication number
WO2011077435A1
WO2011077435A1 PCT/IL2010/001078 IL2010001078W WO2011077435A1 WO 2011077435 A1 WO2011077435 A1 WO 2011077435A1 IL 2010001078 W IL2010001078 W IL 2010001078W WO 2011077435 A1 WO2011077435 A1 WO 2011077435A1
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hydrocarbyl
independently
composition
compound
glaucoma
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PCT/IL2010/001078
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French (fr)
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Bilha Fischer
Shay Elyahu
Jesus Jeronimo Pintor
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Bar-Ilan University
Universidad Complutense De Madrid
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Publication of WO2011077435A1 publication Critical patent/WO2011077435A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65746—Esters of oxyacids of phosphorus the molecule containing more than one cyclic phosphorus atom

Definitions

  • the present invention relates to ophthalmic compositions and methods for lowering intraocular pressure and thereby treating ocular hypertension and/or glaucoma.
  • Extracellular nucleotides that activate G protein-coupled P2Y receptors are attractive pharmaceutical targets due to their ability to modulate various functions in many tissues and organs under normal and pathophysiological conditions (Hillmann et al, 2009; Burnstock and Verkhratsky, 2009).
  • Extracellular nucleotides and dinucleotides have been shown to play a role in ocular physiology and physiopathology (Crooke et al, 2008), and have been suggested as therapeutic agents for dry eye, retinal detachment and glaucoma (Guzman- Aranguez et al, 2007).
  • Ocular hypertension the most common cause of glaucoma, is a target for agents that reduce intraocular pressure (IOP) (Pintor, 2005).
  • IOP intraocular pressure
  • some nucleotides e.g., diadenosine triphosphate and diadenosine pentaphosphate, produce an increase in IOP while others such as ATP, adenosine tetraphosphate and diadenosine tetraphosphate decrease IOP (Peral et al, 2009; Pintor et al, 2003; Pintor et al, 2004).
  • Receptors for extracellular nucleotides including P2Y l 3 P2Y 2 and P2Y 4 have been identified in trabecular meshwork cells (TM), an area of tissue in the eye that is responsible for draining the aqueous humor (Soto et al, 2005).
  • TM trabecular meshwork cells
  • 2-MeS-ADP selective agonist 2-MeS-ADP reduces aqueous humor outflow in bovine ocular.
  • Other studies have reported the presence of P2Y[ and P2Y 2 receptors in bovine TM cells, and of P2Y b P2Y and P2Yii receptors in a human TM cell line (Crosson et al, 2004).
  • nucleotides for the treatment of glaucoma are limited, since they are degraded by extracellular enzymes, which reduce their potency, efficacy and duration of action.
  • nucleotides are chemically stable in a pH range of 4-1 1 (El-Tayeb et al, 2006), they are rapidly degraded at a more acidic or basic pH.
  • Nucleotides are hydrolyzed enzymatically by the ecto-nucleoside triphosphate diphosphohydrolase family of ectonucleotidases, i.e., e-NTPDase and alkaline phosphatases (Nahum et al, 2002), and ecto-nucleotide pyrophosphatases/phosphodiesterases, i.e., e-NPPs (Grobben et al, 2000; Zimmermann, 2001). Therefore, there is a need for the identification of enzymatically and chemically stable nucleotide scaffolds that can be used to develop selective and potent P2YR agonists.
  • ecto-nucleoside triphosphate diphosphohydrolase family of ectonucleotidases i.e., e-NTPDase and alkaline phosphatases (Nahum et al, 2002)
  • nucleotides A few attempts to improve the stability of nucleotides have been reported (Cusack et al, 1987; Misiura et al., 2005; owalska et al., 2007), including the use of phosphate bioisosteres of nucleotides such as phosphonate (Eliahu et al, 2009; Joseph et al., 2004), phosphoramide (Zhou et al, 2005) and boranophosphate (Nahum et al, 2002; Eliahu et al, 2009; Boyle et al, 2005; Barral et al, 2006) analogues.
  • phosphate bioisosteres of nucleotides such as phosphonate (Eliahu et al, 2009; Joseph et al., 2004), phosphoramide (Zhou et al, 2005) and boranophosphate (Nahum et al, 2002; Eliahu et al, 2009; Boy
  • US 7,084,128 discloses a method of reducing IOP by administration of certain mono- or di- nucleoside, preferably mono- or diadenosine, mono-, di-, tri-, terra-, penta- or hexaphosphate derivatives as defined therein, or a pharmaceutically-acceptable salt thereof, the particular compounds exemplified in this patent are 2'-(O)-,3'-(O)-(benzyl)methylenedioxy-adenosine-5'-triphosphate and 2'-(O)-,3'-(O)-(benzyl)methylenedioxy-2"-(O)-,3"-(O)-benzyl methylene dioxy-P',P 4 -di(adenosine 5'-)tetraphosphate, and as shown, these compounds, at a concentration of 0.25 mM, produced a time dependent reduction in IOP, which was maximal at 1-2 hours, with a reduction of 21-22%.
  • the two particular nucleoside triphosphate derivatives exhibiting the strongest hypotensive effect were 2-MeS-adenosine-5 '-O-triphosphate- 3,7-methylene and 2-MeS-adenosine-5 '-O- triphosphate-/3,7-dichloromethylene, with EC 50 values of 30.2 ⁇ and 95.5 nM, respectively, equally or more effective than anti-glaucoma drugs currently available.
  • the present invention provides an ophthalmic composition
  • an ophthalmic composition comprising an pharmaceutically acceptable carrier and a compound of the general formula I:
  • R] is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -N t Rs, heteroaryl, hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -NO 2 , -OR 4 , -SR 4 , -NR1R5 or heteroaryl, wherein R4 and R 5 each independently is H or hydrocarbyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl;
  • R 2 and R 3 each independently is H or hydrocarbyl
  • Y each independently is H, -OH, or -NH 2 ;
  • Zi, Z 2 and Z 3 each independently is -O " , -S " , or -BH 3 " ;
  • Wi and W 2 each independently is -O-, -NH-, or -C(XiX 2 )-, wherein Xi and X 2 each independently is H or halogen, provided that at least one of Wj, if present, and W 2 is not -O-;
  • n 0 or 1
  • n 3 or 4;
  • B + represents a pharmaceutically acceptable cation
  • the ophthalmic compositions of the invention are useful for reducing intraocular pressure and thereby treating ocular hypertension and/or glaucoma.
  • the present invention thus provides a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for use in reducing intraocular pressure.
  • the present invention relates to use of a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for the preparation of an ophthalmic composition.
  • the present invention relates to a method for reducing intraocular pressure in an individual in need thereof comprising administering to said individual a therapeutically effective amount of a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • Figs. 1A-1C show rates of hydrolysis of compounds 9 and 10B (7.5 mM) at pH 1.4 at 37°C, as monitored by HPLC.
  • Figs. 2A-2B show hydrolysis of ATP and compounds 1, 8, and 9 in human blood serum (180 ⁇ ) and RPMI-1640 (540 ⁇ ) over 24 h at 37°C, as monitored by HPLC.
  • Fig. 2A shows hydrolysis of 0.25 mM ATP and production of ADP and AMP; and Fig. 2B shows degradation of compounds 1, 8 and 9 in human blood serum.
  • Fig. 3A shows the effects of compounds 1, 8, 9 and 11-14 (100 ⁇ ) on rabbit intraocular pressure (IOP) measured over 8 hours vs. controls, i.e., an equal volume of saline administered to the contralateral eye and an equal volume of saline administered to other animals. Any treated eye has been measured twice before drug administration, and the values measured are almost identical to those obtained in the time course of the control animals.
  • IOP rabbit intraocular pressure
  • Fig 3B shows time course for the effects of compounds 1, 9, 13 and 14 (100 ⁇ ) on rabbit IOP over 8 h, using the same controls as in Fig. 3 A. Values are means ⁇ S.E.M. of results from ten independent experiments.
  • Fig. 3C shows time-course for the effects of compounds 11B, 12 A and 12B (100 ⁇ ) on rabbit IOP measured over 8 h. Values are means ⁇ S.E.M. of results from eight independent experiments.
  • Fig. 3D shows dose-response curves for the maximal effects on rabbit IOP of compounds 1, 9, 13 and 14 (100 ⁇ ). Values are means ⁇ S.E.M. of results from eight independent experiments.
  • Fig. 3E shows comparisons of the maximal effects obtained on rabbit IOP for compounds 1 and 9 (100 ⁇ , 10 ⁇ ), as compared to Xalatan (0.005 %), Trusopt (2%) and Timolol (0.5 %), each applied at a volume of 40 ⁇ , using the same controls as in Fig. 3A.
  • Fig. 3F shows the mean-time effect of compounds 1 and 9 (100 ⁇ 10 ⁇ ), calculated by measuring the time between 50% of IOP decrease after drug administration and 50% of IOP recovery (Morales et al, 2007), as compared to Xalatan (0.005 %), Trusopt (2%) and Timolol (0.5 %), each applied at a volume of 40 ⁇ , using the same controls as in Fig. 3A.
  • the present invention provides, in one aspect, an ophthalmic composition
  • an ophthalmic composition comprising a non-hydrolyzable nucleoside di- or triphosphate analogues of the general formula I as defined above, in which the ⁇ , ⁇ - or /3,7-bridging-oxygen, respectively, is replaced with a methylene or dihalomethylene group.
  • halogen includes fiuoro, chloro, bromo, and iodo, and is preferably chloro.
  • hydrocarbyl in any of the definitions of the different radicals Ri to R 5 refers to a radical containing only carbon and hydrogen atoms that may be saturated or unsaturated, linear or branched, cyclic or acyclic, or aromatic, and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and aryl.
  • alkyl typically means a straight or branched hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • Preferred are (C]-C 6 )alkyl groups, more preferably (CrC 4 )alkyl groups, most preferably methyl and ethyl.
  • alkenyl and alkynyl typically mean straight or branched hydrocarbon radicals having 2-8 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen- l-yl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, and the like.
  • Preferred are (C 2 - C )alkenyl and (C 2 -C 6 )alkynyl, more preferably (C 2 -C 4 )alkenyl and (C 2 -C4)alkynyl.
  • Each one of the alkyl, alkenyl and alkynyl may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, - NO 2 , -CN, -SCN, aryl, or heteroaryl, and/or interrupted by one or more heteroatoms selected from nitrogen, oxygen or sulfur.
  • halogen e.g., F, CI or Br
  • -OH, - NO 2 , -CN, -SCN aryl, or heteroaryl
  • heteroatoms selected from nitrogen, oxygen or sulfur.
  • cycloalkyl as used herein means a mono- or bicyclic saturated hydrocarbyl group having 3- 10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like, which may be substituted, e.g., with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO 2 , - CN, -SCN, (C r C 8 )alkyl, -O-(C r C 8 )alkyl, -S-(C r C 8 )alkyl, -NH 2 , -NH-(C,-C 8 )alkyl, or -N-((C)-C 8
  • cycloalkenyl as used herein means a mono- or bicyclic unsaturated hydrocarbyl group having 3- 10 carbon atoms and 1 double bond, and include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, hexahydropentalenyl, octahydronaphtalenyl, bicycle[4.2.0]oct-2-enyl, and the like.
  • aryl denotes an aromatic carbocyclic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl. Preferred are (C6-Cio)aryl, more preferably phenyl.
  • the aryl radical may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO 2 , -CN, -SCN, (C r C 8 )alkyl, -O-(C r C 8 )alkyl, -S-(C,-C 8 )alkyl, -NH 2 , -NH-(C,-C 8 )alkyl, or -N-((C r C 8 )alkyl) 2 .
  • halogen e.g., F, CI or Br
  • heteroaryl refers to a radical derived from a mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1 or 2, heteroatoms selected from N, O or S.
  • heteroaryl is a monocyclic ring, it is preferably a radical of a 5-6- membered ring such as, but not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl.
  • Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, pyrido[l,2-a]pyrimidinyl and 1,3-benzodioxinyl.
  • the heteroaryl may be substituted. It is to be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings.
  • R4 and R 5 each independently is H or hydrocarbyl as defined above or form together with the nitrogen atom to which they are attached a saturated or unsaturated heterocyclic ring optionally containing 1 or - 2 further heteroatoms selected from N, O or S.
  • heterocyclic ring denotes a mono- or poly-cyclic non-aromatic ring of 4-12 atoms containing at least one carbon atom and one to three, preferably 1-2 heteroatoms selected from N, O or S, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond.
  • the heterocyclic ring may optionally be substituted at any carbon atom as well as at a second nitrogen atom of the ring, if present, with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO 2 , -CN, -SCN, (C r C 8 )alkyl, -O-(C r C 8 )alkyl, -S-(C C 8 )alkyl, - NH 2 , -NH-(C r C 8 )alkyl, or -N-((C r C 8 )alkyl) 2 .
  • halogen e.g., F, CI or Br
  • Non-limiting examples of radicals - NR_(R5 include amino, dimethylamino, diethylamino, ethylmethylamino, phenylmethyl-amino, pyrrolidino, piperidino, tetrahydropyridino, piperazino, ethylpiperazino, hydroxyethyl piperazino, morpholino, thiomo ⁇ holino, thiazolino, and the like.
  • the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R[ is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR 4 R 5 , heteroaryl, or hydrocarbyl; R and R 5 each independently is H or hydrocarbyl, or R and R 5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein said hydrocarbyl each independently is (Ci-Cg)alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, or (C 6 -Ci 4 )aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S.
  • R[ is H, halogen,
  • said hydrocarbyl is selected from (Ci-C 6 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl; (C 2 - C 6 )alkenyl, preferably (C 2 -C 4 )alkenyl; (C 2 -C 6 )alkynyl, preferably (C 2 -C 4 )alkynyl; or (C 6 -Cio)aryl, preferably phenyl.
  • the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R 2 and R 3 each independently is H or hydrocarbyl selected from (C r C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, or (C 6 -Ci 4 )aryl.
  • said hydrocarbyl is selected from (C r C 6 )alkyl, preferably (C r C 4 )alkyl, more preferably methyl or ethyl; (C 2 -C 6 )alkenyl, preferably (C 2 - C 4 )alkenyl; (C 2 -C 6 )alkynyl, preferably (C 2 -C 4 )alkynyl; or (C 6 -Ci 0 )aryl, preferably phenyl.
  • the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein Y at positions 3 and 4 of the tetrahydrofuran moiety each independently is - OH.
  • the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R] is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R 5 each independently is H or hydrocarbyl, or R4 and R 5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; R 2 and R 3 each independently is H or hydrocarbyl; and Y each independently is -OH, wherein said hydrocarbyl each independently is (C C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, or (C 6 -Ci 4 )aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic
  • the compound comprised within the ophthalmic composition of the invention is a compound of the general formula I, wherein Rj is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R_j and R 5 each independently is H or hydrocarbyl, or R4 and R 5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; R 2 and R 3 each independently is H or hydrocarbyl; and Y each independently is -OH, wherein said hydrocarbyl is (Ci-C 6 )alkyl, preferably (C ⁇ - C 4 )alkyl, more preferably methyl or ethyl; (C 2 -C 6 )alkenyl, preferably (C 2 - C 4 )alkenyl; (C 2 -C
  • More particular compounds are those wherein R
  • Preferred compounds are those wherein Ri is H or -S-methyl; R 2 and R 3 are H; and Y each independently is -OH.
  • the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I as defined above wherein n is 0, i.e., a non-hydrolyzable nucleoside diphosphate derivative.
  • the compound used according to the method of the invention is a compound of the general formula I as defined above wherein n is 1, i.e., a non-hydrolyzable nucleoside triphosphate derivative.
  • W if present, is -O-; and W 2 is -C(X]X 2 )- or -NH-, preferably - C(X[X 2 )-, wherein X] and X 2 each independently is H or halogen selected from F, CI or Br, preferably CI.
  • More particular compounds are those wherein Z 2 , if present, and Z 3 each independently is -O " ; and Z is -O " or -BH 3 ⁇
  • nucleoside di- or triphosphate analogues of the general formula I including the nucleoside di- or triphosphate analogues of the general formula I, additional nucleoside di- or triphosphate analogues excluded from the general formula I by means of provisos, starting materials and intermediates, are herein identified by the Arabic numbers 1- 38 in bold. The full chemical structures of these compounds are depicted in Appendix A and/or in Schemes 1-3 hereinafter.
  • Compound 1 is also identified by the name 2MeS-adenosine-j8,7-CH 2 -5 '-triphosphate; compound 2 is also identified by the name adenosine-jS,7-CH 2 -5'-O-(l-boranotriphosphate); compound 3 is also identified by the name 2MeS-adenosine-i8,7-CH 2 -5'-O-(l-boranotriphosphate); compound 4 is also identified by the name 2MeS-adenosine-5'-O-(l- borano triphosphate); compound 5 is also identified by the name adenosine- triphosphate (ATP); compound 6 is also identified by the name adenosine- diphosphate (ADP); compound 7 is also identified by the name 2MeS-adenosine- diphosphate; compound 8 is also identified by the name 2MeS-adenosine-/3,7-CF 2 - 5 '-triphosphate; compound 9 is also identified by
  • the compound comprised within the ophthalmic composition of the invention is a nucleoside triphosphate analogue, i.e., a compound of the general formula I in which n is 1, wherein Y each independently is -OH; R 2 and R 3 are H; and (i) R, is -SCH 3 ; Z,, Z 2 and Z 3 are -O " ; W, is -O-; and W 2 is -CH 2 - (compound 1); (ii) Ri is -SCH 3 ; Z ] 5 Z 2 and Z 3 are O " ; W] is -O-; and W 2 is - CC1 2 - (compound 9); (iii) Rj is H; Zi is -BH 3 " ; Z 2 and Z 3 are -O " ; Wi is -O-; and W 2 is -CC1 2 -, characterized by being the isomer with a retention time (Rt) of 10.87 min when separated from a mixture of diastere
  • the compound comprised within the ophthalmic composition of the invention is a nucleoside diphosphate analogue, i.e., a compound of the general formula I in which n is 0, wherein Y each independently is -OH; Ri is -SCH 3 ; R 2 and R 3 are H; Z t and Z 3 are -O " ; and W 2 is -CF 2 - or -CC1 2 - (compounds 13 and 14, respectively).
  • the compounds of the general formula I may be synthesized according to any technology or procedure known in the art, e.g., as described in the Examples section hereinafter.
  • the compounds of the general formula I may have an asymmetric center, e.g., in the Pa, and may accordingly exist as pairs of diastereoisomers.
  • the separation and characterization of the different diastereoisomers may be accomplished using any technology known in the art, e.g., using a semi-preparative reverse-phase column and isocratic solution as described in the Examples section. According to the method of the invention, reducing of intraocular pressure could be carried out by administration of all such isomers and mixtures thereof.
  • the compounds of the general formula I are in the form of pharmaceutically acceptable salts.
  • the cation B is an inorganic cation of an alkali metal such as, but not limited to, Na + , + and Li + .
  • the cation B is ammonium (NH 4 + ) or is an organic cation derived from an amine of the formula R 4 N + , wherein each one of the Rs independently is selected from H, C r C 22 , preferably Ci-C 6 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine.
  • N, S and O such as pyrrolydine, piperidine and morpholine.
  • the cation B is a cationic lipid or a mixture of cationic lipids.
  • Cationic lipids are often mixed with neutral lipids prior to use as delivery agents.
  • Neutral lipids include, but are not limited to, lecithins; phosphatidyl-ethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; fatty acid esters; glycerol esters
  • Neutral lipids also include cholesterol and other 3/3 hydroxy-sterols.
  • Other neutral lipids contemplated herein include phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidyl glycerols.
  • cationic lipid compounds include, without being limited to, Lipofectin® (Life Technologies, Burlington, Ontario) (1 : 1 (w/ ) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); LipofectamineTM (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Miss
  • nucleotide analogues 8 and 9 exhibited high stability at pH 1.4 with ti /2 comparable to those of the analogues 1-3 (25 and 65 h, respectively).
  • Nucleoside diphosphate analogues 13 and 14 were significantly more stable at pH 1.4, as compared to their higher homologues 8 and 9, respectively, possibly due to the partial negative charge developing on the 5'-phosphonate (P a ) or 5 '-oxygen atom in analogues 8/9 or 13/14, respectively, upon hydrolysis, which may be better stabilized by a phosphate group, resulting in relatively more extensive hydrolysis of analogues 8/9 vs. 13/14, and hence shorter tj 2 values.
  • 2-MeS-a-borano-ATP (A-isomer), 4 is susceptible to hydrolysis by alkaline phosphatase resulting in oborano-2-MeS- AMP and 2-MeS-a-borano-ADP.
  • alkaline phosphatase hydrolyzed 60% of 2-MeS-a-borano-ATP (A-isomer) within 12 min at 37°C, whereas only traces of 2-MeS-a-borano-ATP remained after 100 min (Eliahu et al, 2009).
  • analogues 10 and 12 were substituted with a CF 2 or CC1 2 group in analogues 10 and 12, respectively, rendered these analogues completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37°C.
  • analogues 8- 14 were highly resistant to hydrolysis by NTPDasesl , 2, 3, and 8, and NPP1 and 3 (less than 5% hydrolysis of analogues), as compared to ATP.
  • analogue 12B was completely resistant to hydrolysis by NTPDasel , 2, and 3 and NPP3.
  • the extremely low hydrolysis of analogues is related primarily to the presence of a phosphonate moiety at ⁇ ?
  • analogues 1-3 While evaluating analogues 1-3 as agonists of the P2YiR, it has previously been found that the most effective P2Y]R agonist was analogue 1, with an EC 5 o of 80 nM (Eliahu et al, 2009); however, this analogue was still less potent agonist than the structurally related analogue 4A (EC 5 o of 2.6 nM) (Nahum et al, 2002).
  • the reduction in the activity of analogues 1-3 as compared to analogue 4A is possibly related to the elevated pK a of the terminal phosphonate, as compared to phosphate (pK a 8.4 vs.
  • the electronegativity of a dihalogenated methylene group such as CF 2 and CC1 2 lowers the p a of phosphonates from 8.4 to 6.7-7.0, making it closer to the p a of phosphate (Wang et al, 2004).
  • nucleotide analogues with a /3, ⁇ - dihalomethylene group were reported to be promising inhibitors of HIV-1 reverse transcriptase (Boyle et al, 2005; Wang et al, 2004), agonists of P2X 2/3 receptors (Spelta et al, 2003), and potent antagonists of the P2Y 12 receptor (Ingall et al, 1999; El-Tayeb et al, 2005).
  • analogues 8-14 are resistant to enzymatic hydrolysis, they are less potent P2Y[R agonists than the more rapidly hydrolyzed 2-MeS-ADP.
  • the activities of the corresponding 3,7-dihalomethylene analogues 8 and 9 are 300- to 3800-fold lower than that of 2-MeS-ADP, respectively.
  • the presence of a thiomethyl group at the adenine C-2 position was essential for P2Y]R activity, since analogues 11 A and 11B were inactive.
  • analogues 1, 9, 11B, 12A, 12B, 13 and 14 to reduce intraocular pressure (IOP) in male New- Zealand white rabbits, wherein the two compounds exhibiting the strongest hypotensive effect were analogues 1 and 9, with EC 5 o values of 30.2 ⁇ and 95.5 nM, respectively, equally or more effective than hypotensive drugs currently available.
  • IOP intraocular pressure
  • beta-blockers e.g., Timolol
  • cholinergic agents e.g., pilocarpine
  • prostaglandins e.g., Xalatan
  • eyelash growth iris pigmentation
  • muscle and joint pain Higginbotham et al., 2002.
  • the development of stable and potent nucleotide analogues should expand the limited repertoire of drugs currently available and used for treatment of glaucoma.
  • nucleotide analogues 8-14 were synthesized chemically and enzymatically stable nucleotide analogues 8-14, based on ADP and ATP scaffolds modified by ⁇ , ⁇ / ⁇ , ⁇ - dihalomethylene groups within the phosphate chain.
  • These analogues were agonists at the P2Y[R. Analogues 1 and 9, in particular, exhibited the ability to reduce IOP in normotense rabbits, with EC 5 o values of 30.2 ⁇ and 95.5 nM, respectively.
  • these analogues were more effective than several commonly prescribed glaucoma drugs, in particular, Xalatan and Trusopt, at reducing IOP.
  • compositions of the present invention can be provided in a variety of formulations and dosages. These compositions may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 Ed., 1995.
  • the compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the general formula I, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
  • compositions of the present invention may be formulated so as to have both pH and tonicity compatible with the eye.
  • This will normally require a buffer to maintain the pH of the composition at or near physiologic pH, i.e., in the range of 5-9, preferably 6 to 8, more preferably 6.8- 7.4; and may further require a tonicity agent to bring the osmolality of the composition to a level at or near 210-320 milliosmoles per kilogram (mOsm/kg).
  • the composition of the invention has an osmolality in the range of 50-700 mOsm/kg, preferably 100-600 mOsm/kg, more preferably 150-500 mOsm/kg, still more preferably 200-400 mOsm/kg, most preferably 200-350 mOsm/kg.
  • compositions of the invention may be administered to the eye of the subject by any suitable means.
  • the composition is in the form of a liquid, emulsion, gel or suspension of the compound of the general formula I, and it is administered as drops, spray, or gel.
  • the active agent i.e., the compound of the general formula I is applied to the eye via liposomes.
  • the active agent of the composition is contained within a continuous or selective-release device, e.g., membranes such as, but not limited to, those employed in the OcusertTM System (Alza Corp., Palo Alto, Calif.).
  • the active agent can be contained within, carried by, or attached to contact lenses, which are placed on the eye.
  • the active agent is contained within a swab or sponge, or within a liquid spray, which is applied to the ocular surface.
  • the active agent is directly injected into the ocular tissues, e.g., by subconjunctival, subscleral, or intravitreal injection, or onto the eye surface.
  • the ophthalmic composition of the present invention contains a physiologically compatible carrier or vehicle as those skilled in the ophthalmic art can select using conventional criteria.
  • vehicles may be selected from known ophthalmic vehicles that include, inter alia, saline solution, water, polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, cyclodextrins, in particular betahydroxypropyl cyclodextrin, petroleum derivatives, e.g., mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil, polysaccharides such as dextrans, an alginate such as sodium alginate optionally comprising guluronic acid and/or mannuronic acid, glycosaminoglycans such as sodium hyaluronate, and salts such as sodium chlor
  • compositions for the treatment of glaucoma may be administered daily, twice daily, or 3-4 times daily, and/or upon the occurrence of symptoms associated with the condition; and over a period of time consistent with treatment of the ocular hypertension and glaucoma, e.g., for a period of weeks, months, years, or decades.
  • the ophthalmic compositions of the present invention are useful for lowering intraocular pressure, thus can be used for prevention of treatment of ocular hypertension and/or glaucoma.
  • intraocular hypertension refers to an intraocular pressure in an eye of a patient that is above a normal level and is correlated as a risk factor for the development of visual field loss and glaucoma.
  • Glaucoma is a heterogeneous group of optic neuropathies that share certain clinical features, wherein the loss of vision is due to the selective death of retinal ganglion cells in the neural retina that is clinically diagnosed by characteristic changes in the visual field, nerve fiber layer defects, and a progressive cupping of the optic nerve head (ONH).
  • One of the main risk factors for the development of glaucoma is the presence of intraocular hypertension (elevated intraocular pressure, IOP). IOP also appears to be involved in the pathogenesis of normal tension glaucoma where patients have what is often considered to be normal IOP.
  • the elevated IOP associated with glaucoma is due to elevated aqueous humor outflow resistance in the trabecular meshwork (TM), a small- specialized tissue located in the iris-corneal angle of the ocular anterior chamber.
  • Glaucomatous changes to the TM include a loss in TM cells and the deposition and accumulation of extracellular debris including proteinaceous plaque-like material.
  • ONH glial cells In glaucomatous eyes, there are morphological and mobility changes in ONH glial cells.
  • IOP and/or transient ischemic insults there is a change in the composition of the ONH extracellular matrix and alterations in the glial cell and retinal ganglion cell axon morphologies.
  • Glaucoma is a disease of the eye characterized by increased pressure inside the eye with resultant optic nerve damage.
  • Glaucoma includes, but is not limited to, primary glaucomas, secondary glaucomas, juvenile glaucomas, congenital glaucomas, pseudoexfoliation glaucoma, acute angle closure glaucoma, absolute glaucoma, chronic glaucoma, narrow angle glaucoma, chronic open angle glaucoma, simplex glaucoma and familial glaucomas, including, without limitation, pigmentary glaucoma, high tension glaucoma, and low tension glaucoma and their related diseases.
  • the present invention provides a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for use in reducing intraocular pressure.
  • the present invention relates to use of a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for the preparation of an ophthalmic composition.
  • the present invention relates to a method for reducing intraocular pressure in an individual in need thereof comprising administering to said individual a therapeutically effective amount of a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • a compound of the general formula I as defined above or a diastereomer or mixture of diastereoisomers thereof.
  • compound 1 or 9, preferably 9, or a diastereomer or mixture of diastereoisomers thereof is administered.
  • Nucleotides were characterized also by 31 P NMR in D 2 O, using 85% H 3 PO 4 , and 19 F NMR using trifluorochloromethane as an external reference on Bruker AC-200 and DMX-600 spectrometers. High-resolution mass spectra were recorded on an AutoSpec-E FISION VG mass spectrometer by chemical ionization. Nucleotides were analyzed under electron spray ionization (ESI) conditions on a Q-TOF micro-instrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a column of Sephadex DEAE-A25, swollen in 1 M NaHCO 3 at 4°C for 1 day.
  • ESI electron spray ionization
  • the resin was washed with deionized water before use.
  • the LC separation was monitored by UV detection at 280 nm.
  • Final purification of the nucleotides and separation of diastereomers were achieved on a HPLC (Elite Lachrom, Merck-Hitachi) system using a semi-preparative reverse-phase column (Gemini 5u C-18 1 10A 250 x 10.00 mm; 5 micron; Phenomenex, Torrance, USA).
  • RPMI Roswell Park Memorial Institute 1640 buffer was obtained from Sigma- Aldrich. 2',3'-O- methoxymethylidene-adenosine and 2-MeS-adenosine were prepared, as previously described (Nahum et al., 2002; Griffin et al, 1967).
  • 2',3'-O-methoxymethylidene- 2-MeS-adenosine was purified with a MPLC system (Biotage, Kungsgatan, Uppsala, Sweden) using a silica gel (25+M) column and the following gradient scheme: 3 column volumes (CV) of 100:0 (A) CHC1 3 :(B) EtOH, 5 CV of a gradient from 100:0 to 90: 10 A:B and 4 CV of 90: 10 A:B at a flow rate of 12.5 ml/min. pH measurements were performed with an Orion micro combination pH electrode and a Hanna Instruments pH meter. Triethylammonium bicarbonate (TEAB) was prepared as previously described (Bachelet and Guibe, 1951).
  • TEAB Triethylammonium bicarbonate
  • Bis(tributyl ammonium)difluoromethylene diphosphonate salt was purchased from PJ Chemical Inc.
  • whole blood taken from healthy volunteers was obtained from a blood bank (Tel-Hashomer Hospital, Israel). Blood was stored for 12 h at 4°C, centrifuged in plastic tubes at 1500 x g for 15 min at RT. The serum was separated and stored at -80°C.
  • a H+ Dowex column was used for ion exchange chromatography. Thirty ml of Dowex was placed in a column with cotton wool at the bottom, and the column was washed with 10% NaOH (150 ml) until the pH of the effluent was basic, and then with distilled water until the pH of the effluent reached neutral. Then, the column was washed with 10% HC1 (300 ml) followed by distilled water until the effluent reached acidic and neutral pH, respectively. A flask containing Bu3N (2 eq) in EtOH was placed in an ice bath under the column and stirred.
  • the disodium form of dichloromethylene diphosphonate salt was dissolved in distilled water and poured onto the column, and the column was then washed with distilled water until the pH of the effluent was neutral. The effluent was dropped into the Bu 3 N/EtOH solution. The final solution of bis(tributylammonium) dichloromethylene diphosphonate salt was then freeze-dried.
  • the resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.5 M NH 4 HCO 3 ; total volume 2 1). The relevant fractions were collected and freeze-dried, and excess NH 4 HCO 3 was removed by repeated freeze-drying cycles with deionized water to obtain the product as a white powder.
  • the product was purified by LC yielding aompound 10 at a 21% yield (13 mg), aompound 11 at a 18% yield (14 mg) and aompound 12 at a 7.2% yield (18.6 mg).
  • the diastereomers of aompounds 10, 11 and 12 were separated on a HPLC column, under the conditions described below.
  • Purity data obtained on an analytical column retention time: 7.82 min (95.3% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 4.89 min (92.8% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 20 min at a flow rate of 1 ml/min.
  • Purity data obtained on an analytical column retention time: 6.88 min (98% purity) using Solvent System I with a gradient from 95:5 to 70:30 A:B over 10 min at a flow rate of 1 ml/min.
  • Diastereoisomers 12A and 12B were obtained at a 7.2% overall yield (13 mg) after
  • Deionized water (20 ml) was added and the reaction was treated with 18% HCl until the pH was 2.3, and then the mixture was stirred for 3 h at RT. Then, the mixture was treated with 24% NH 4 OH, and the pH was adjusted to 9. The solution was stirred for 45 min at RT and then freeze-dried. The resulting residue was applied to an activated Sephadex DEAE-A25 column (0-0.3 M NH 4 HCO 3 ; total volume of 2 1). The relevant fractions were collected and freeze-dried, and excess NH 4 HCO 3 was removed by repeated freeze-drying with deionized water to yield compound 13 as a white powder.
  • Purity data obtained on an analytical column retention time: 6.85 min (100.0% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 5.17 min (99.94% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 18 min at a flow rate of 1 ml/min.
  • the resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.3 M NH 4 HCO 3 ; total volume of 1.4 1). The relevant fractions were collected and freeze-dried, and excess NH 4 HCO 3 was removed by repeated freeze-drying with deionized water to yield compound 14 as a white powder.
  • the residue was separated using a HPLC system with a semi-preparative C-18 column and the following gradient scheme (A) 100 mM TEAA:(B) MeOH with gradients of 85: 15 to 75:25 over 10 min, 75:25 to 70:30 over 2 min, and 70:30 over 3 min at a flow rate of 5 mL/min. Retention time: 10.96 min.
  • Enzyme activity was determined by the release of p-nitrophenol from p- nitrophenyl phosphate measured by a UV-VIS spectrophotometer at 405 nm (Brandenberger and Hanson, 1953). Relative enzyme activity and resistance of compounds 5-14 to enzymatic hydrolysis were determined at 37°C using a solution of 0.2 mg of analogue in 77.5 ⁇ deionized water, 0.1 M Tris-HCl (pH 9.8) and 0.1 M MgCl 2 with calf intestine alkaline phosphatase (Fermentas Inc., Glen Burnie, MD; 10 unit/ ⁇ ; 1.25 ⁇ ; 12.5 u).
  • transfected cells were washed three times with Tris-saline buffer at 4°C, collected by scraping in the harvesting buffer (in 95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 45 mM Tris at pH 7.5), and washed twice by 300 g centriiugation for 10 min at 4°C. Cells were resuspended in the harvesting buffer containing 10 mg/ml aprotinin and sonicated.
  • Tris-saline buffer in 95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 45 mM Tris at pH 7.5
  • Nucleus and cellular debris were discarded by centrifugation at 300 g for 10 min at 4°C and the supernatant (crude protein extract) was aliquoted and stored at -80°C until used for activity assays. Protein concentration was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as a standard (Bradford, 1976). NTPDase protein extracts, 1/106 of final volume diluted accordingly to its specific activity, were added to the reaction mixture and pre-incubated at 37°C for 3 min.
  • BSA bovine serum albumin
  • the reaction was initiated by addition of ATP (Sigma-Aldrich, Oakville, ON, Canada) or compounds 8-14 at a final concentration of 100 ⁇ and the reaction was stopped after 20 min with 50 ⁇ of malachite green reagent (Sigma-Aldrich, Oakville, ON, Canada).
  • the released inorganic phosphate (Pi) was measured at 630 nm according to Baykov et al. (1988).
  • the activity obtained with protein extracts from untransfected cells was subtracted from the activity obtained with extracts from NTPDase-transfected cells. The activity of untransfected cell extracts never exceeded 5% of the activity of extracts from NTPDase-transfected cells.
  • the reaction was stopped after 20 min by transferring a 0.1 ml aliquot of the reaction mixture to 0.125 ml ice-cold 1 M perchloric acid (Fisher Scientific, Ottawa, ON, Canada). The samples were centrifuged for 5 min at 13,000xg. Supernatants were neutralized with 1 M KOH (Fisher Scientific, Ottawa, ON, Canada) at 4°C and centrifuged for 5 min at 13,000xg.
  • a nucleotide analogue (1.5 mg) was dissolved in 0.2 M HC1/KC1 buffer (0.8 ml) and the final pH was adjusted to 1.4 using 0.2 M HC1. Reactions continued at 37°C for 1 to 31 days with samples taken at 1-24 h intervals.
  • the stabilities of the compounds were evaluated by HPLC to monitor degradation products using a Gemini analytical column (5u C-18 110A; 150x4.60 mm) and the gradient elution system described for the hydrolysis of analogues in human blood serum at a flow rate of 1 ml/min (see above).
  • the hydrolysis rates of compounds 8-14 at pH 1.4 and 37°C were determined by measuring the change in the integration of the respective HPLC peaks with time.
  • Intraocular pressure was measured by means of a TonoVET rebound tonometer supplied by Tiolat Oy (Helsinki, Finland). The application of this tonometer to animals does not require the use of any anaesthetic.
  • TonoVET rebound tonometer supplied by Tiolat Oy (Helsinki, Finland).
  • the application of this tonometer to animals does not require the use of any anaesthetic.
  • different analogues were applied unilaterally to the cornea at a concentration of 100 ⁇ and a fixed volume of 10 ⁇ .
  • the contralateral eye received the same volume of saline solution (0.9% NaCl, vehicle).
  • Two IOP measurements were taken before any analogue was instilled. Experiments were performed following a blinded design where no visible indication was given to the experimenter as to the nature of the applied solution. IOP was followed up to 8 h to study the time course of the effect.
  • ATP analogues in which the 3,7-bridging oxygen is substituted by a methylene group are conventionally prepared via the activation of the 5 '-phosphate of nucleoside-5 '-monophosphate (NMP) to form a phosphoryl donor, followed by a reaction with methylene bisphosphonate salt (phosphoryl acceptor).
  • NMP nucleoside-5 '-monophosphate
  • Phosphoryl donors were prepared by activation of NMP with carbonyl diimidazole (CDI) (Padyukova et al, 1999), trifluoroacetic anhydride and N-methylimidazole (Mohamady and Jakeman, 2005) or dicyclohexylcarbodiimide (DCC) (Myers et al, 1963) followed by condensation with methylene bisphosphonic acid or its salt.
  • CDI carbonyl diimidazole
  • DCC dicyclohexylcarbodiimide
  • Compound 15 was first treated with POCl 3 in trimethylphosphate (TMP), in the presence of Proton Sponge ® at 0°C for 3 h, to obtain intermediate 16, which was then treated with bis(tributylammonium) dihalogen methylene-diphosphonate and tributylamine at 0°C for 1.5 h, providing the cyclic intermediates 17 and 18. Hydrolysis of intermediates 17 and 18 in 0.5 M TEAB and deprotection of the methoxymethylidene groups generated 5,y-CF 2 -2- MeS-ATP, 8, at a 46% overall yield, and /3,7-CCl 2 -2-MeS-ATP, 9, at a 10% overall yield, respectively.
  • TMP trimethylphosphate
  • each analogue was obtained as a pair of diastereoisomers in a 1 : 1 ratio.
  • P NMR spectra there was a slight difference between the chemical shifts for the two diastereoisomers of each analogue.
  • 2-MeS-ADP, 7, is a selective and highly potent P2YiR agonist. Yet, this agonist suffers from low chemical and enzymatic stability (Ravi et al, 2002). Therefore, we replaced the ⁇ ,/3-bridging oxygen with a dichloro- or difluoromethylene group in an attempt to generate potent P2Y]R agonists with increased chemical and metabolic stabilities, i.e., compounds 13 and 14.
  • the ⁇ - dihalomethylene-2-MeS-ADP compounds 13 and 14 were prepared, as previously reported (Davisson et al., 1987) and depicted in Scheme 3.
  • Alkaline phosphatase is a hydrolase that removes phosphate groups from nucleotides, thereby regulating extracellular nucleotide concentrations in vivo.
  • AP usually catalyzes the hydrolysis of phosphomonoesters yielding P s and the corresponding alcohol (Hull et al., 1976).
  • we evaluated the effect of cg8//3, ⁇ -dihalo-methylene groups in the compounds 8-14 on the resistance to hydrolysis by alkaline phosphatase, as compared to ATP, ADP and compounds 1-3. Results indicated that AP degraded 96% of ATP after 3 h (ti /2 1.4 h).
  • Nucleotides and their analogues undergo dephosphorylation by enzymes in physiological systems undergo dephosphorylation by enzymes in physiological systems (Schetinger et al, 2007; Terkeltaub, 2006). Blood serum contains such enzymes and, therefore, provides a good model system for assessing the metabolic stability of extracellular nucleotides.
  • NTPDasel, 2, 3 and 8 as well as NPP1 and 3 are the principal enzymes that metabolize extracellular nucleotides. As shown in Table 1 hereinbelow, in comparison to ATP, compounds 8-14 were barely hydrolyzed by NTPDases 1-3 and 8 ( ⁇ 5% over 24 h at 37°C) or NPP1 and 3 ( ⁇ 10% over 24 h at 37°C).
  • the ATP and ADP analogues 8-14 were all used as substrates of the ectonucleotidases identified on the left column at the concentration of 100 ⁇ .
  • the activity with 100 ⁇ ATP was set as 100% which were: 403 ⁇ 40; 1006 ⁇ 60; 533 ⁇ 42; 229 ⁇ 20 [nmol Pi min " '-mg protein " '] for NTPDasel, 2, 3 and 8, respectively.
  • Nucleotides are present in the aqueous humour (Pintor et ai, 2003), although their action has not been fully elucidated due to the multiple P2 receptor subtypes identified in intraocular tissues that are bathed by the aqueous humour (Pintor et al, 2004a).
  • IOP intraocular pressure
  • G protein- coupled P2Y] receptors in trabecular meshwork cells that control the evacuation of the aqueous humour (Soto et al, 2005), and ligand-gated ion channel P2X 2 receptors located on parasympathetic nerve terminals innervating the cilliary bodies (Markovskaya et al, 2008).
  • P2X receptor activation potentiates the release of acetylcholine, which induces contraction of cilliary muscle to open the trabecular meshwork to reduce IOP (Pintor and Peral, 2001).
  • activation of G protein-coupled P2Y 2 receptors increases IOP (Pintor and Peral, 2001).
  • compounds 8-14 we examined the effect of compounds 8-14, as compared to 2-MeS- /3,7-methylene-ATP, 1, on reduction of IOP in normal tense rats to identify novel and potent candidates for the treatment of ocular hypertension (Pintor and Peral, 2001).
  • the rank order of potency for reduction of IOP was 9 > 13 > 1 > 14 with EC 5 o values of 95.5 nM, and 7.9, 30.2 and 31.6 ⁇ , respectively (Fig. 3D).
  • compounds 1 and 9 were more effective than the prostglandin analogue Xalatan, the carbonic anhydrase inhibitor Trusopt, and equally effective as the beta-blocker Timolol (Fig. 3E).
  • the duration effect of the nucleotides as compared with the commercial compounds was sufficiently long.
  • the reduction of duration of IOP induced by compounds 1 and 9 was -3.5 and 4.5 h, comparable to Xalatan with an effective duration of 5.5 h (Fig. 3F).
  • Compound 8 a) trimethylphosphate, POCl 3 , Proton Sponge, 0°C, 3 h; b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu 3 N, 0°C, 1.5 h; c) 0.5 M TEAB, pH 7, RT, 1 h; and d) 1) 18% HCl, pH 2.3, RT, 3 h; and 2) 24% NH 4 OH, pH 9, RT, 45 min.
  • Compound 10 a) trimethylphosphate, PC1 3 , proton sponge, 0°C, 30 min; b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu 3 N, 0°C, 1 h; c) 2 M BH 3 -SMe in THF, 0°C, 5 min, and then RT, 60 min; d) 0.5 M TEAB, pH 7, RT, 1 h; and e) 1) 18% HC1, pH 2.3, RT, 3 h; and 2) 24% NH 4 OH, pH 9, RT, 45 min.
  • Compound 11 a) trimethylphosphate, PC1 3 , proton sponge, 0°C, 0.5 h; b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu 3 N, 0°C, 25 min; c) 2 M BHySMe in THF, 0°, 5 min then RT, 25 min; d) 0.5 M TEAB, pH 7, RT, 45 min; and e) 1) 18% HC1, pH 2.3, RT, 3 h; and 2) 24% NH 4 OH, pH 9, RT, 45 min.
  • Compound 15 a) CH 2 C1 2 , DMAP, TsCl, RT, 12 h; b) tetra-(n-butylammonium)difluoro methylenediphosphonate in dry DMF, RT, 72 h; c) 1) 18% HCl, pH 2.3, RT, 3 h; and 2) 24% NH 4 OH, pH 9, RT, 45 min.
  • Burnstock G. Verkhratsky A., Evolutionary origins of the purinergic signalling system, Acta Physiol., 2009, 195, 415-447 Bystrom C.E., Pettigrew D.W., Remington S.J., Branchaud B.P., ATP analogs with non-transferable groups in the gamma position as inhibitors of glycerol kinase, Bioorg. Med. Chem. Lett., 1997, 7, 2613-2616
  • Garrad R.C. Otero M.A., Erb L., Theiss P.M., Clarke L.L., Gonzalez F.A., Turner J.T., Weisman G.A., Stmctural basis of agonist-induced desensitization and sequestration of the P2Y 2 nucleotide receptor. Consequences of truncation of the C terminus, . Biol. Chem., 1998, 273, 29437-29444
  • Pintor J. Adenine nucleotides and dinucleotides as new substances for the treatment of ocular hypertension and glaucoma, Curr. Opin. Invest. Drugs, 2005, 6, 76-80

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Abstract

The present invention provides ophthalmic compositions comprising non-hydrolyzable nucleoside di-or triphosphate analogues in which the α,β- or β,γ-bridging-oxygen, respectively, is replaced with, e.g., a methylene or dihalomethylene group, such as 2MeS-adenosine-β,γ-CH2--5'-triphosphate and 2MeS-adenosine-β,γ-CCl25'-triphosphate. These compositions are useful for reducing intraocular pressure and thus for treatment of ocular hypertension and/or glaucoma.

Description

COMPOSITIONS AND METHODS FOR REDUCING INTRAOCULAR
PRESSURE
TECHNICAL FIELD
The present invention relates to ophthalmic compositions and methods for lowering intraocular pressure and thereby treating ocular hypertension and/or glaucoma.
BACKGROUND ART
Extracellular nucleotides that activate G protein-coupled P2Y receptors (P2YPvs) are attractive pharmaceutical targets due to their ability to modulate various functions in many tissues and organs under normal and pathophysiological conditions (Hillmann et al, 2009; Burnstock and Verkhratsky, 2009). Extracellular nucleotides and dinucleotides have been shown to play a role in ocular physiology and physiopathology (Crooke et al, 2008), and have been suggested as therapeutic agents for dry eye, retinal detachment and glaucoma (Guzman- Aranguez et al, 2007).
Ocular hypertension, the most common cause of glaucoma, is a target for agents that reduce intraocular pressure (IOP) (Pintor, 2005). When topically applied to New Zealand white rabbits, some nucleotides, e.g., diadenosine triphosphate and diadenosine pentaphosphate, produce an increase in IOP while others such as ATP, adenosine tetraphosphate and diadenosine tetraphosphate decrease IOP (Peral et al, 2009; Pintor et al, 2003; Pintor et al, 2004).
Receptors for extracellular nucleotides including P2Yl 3 P2Y2 and P2Y4 have been identified in trabecular meshwork cells (TM), an area of tissue in the eye that is responsible for draining the aqueous humor (Soto et al, 2005). Among these P2YR subtypes, activation of the P2Y,R by the selective agonist 2-MeS-ADP reduces aqueous humor outflow in bovine ocular. Other studies have reported the presence of P2Y[ and P2Y2 receptors in bovine TM cells, and of P2Yb P2Y and P2Yii receptors in a human TM cell line (Crosson et al, 2004). The therapeutic potential of endogenous nucleotides for the treatment of glaucoma is limited, since they are degraded by extracellular enzymes, which reduce their potency, efficacy and duration of action. In addition, although nucleotides are chemically stable in a pH range of 4-1 1 (El-Tayeb et al, 2006), they are rapidly degraded at a more acidic or basic pH. Nucleotides are hydrolyzed enzymatically by the ecto-nucleoside triphosphate diphosphohydrolase family of ectonucleotidases, i.e., e-NTPDase and alkaline phosphatases (Nahum et al, 2002), and ecto-nucleotide pyrophosphatases/phosphodiesterases, i.e., e-NPPs (Grobben et al, 2000; Zimmermann, 2001). Therefore, there is a need for the identification of enzymatically and chemically stable nucleotide scaffolds that can be used to develop selective and potent P2YR agonists.
A few attempts to improve the stability of nucleotides have been reported (Cusack et al, 1987; Misiura et al., 2005; owalska et al., 2007), including the use of phosphate bioisosteres of nucleotides such as phosphonate (Eliahu et al, 2009; Joseph et al., 2004), phosphoramide (Zhou et al, 2005) and boranophosphate (Nahum et al, 2002; Eliahu et al, 2009; Boyle et al, 2005; Barral et al, 2006) analogues.
US 7,084,128 discloses a method of reducing IOP by administration of certain mono- or di- nucleoside, preferably mono- or diadenosine, mono-, di-, tri-, terra-, penta- or hexaphosphate derivatives as defined therein, or a pharmaceutically-acceptable salt thereof, the particular compounds exemplified in this patent are 2'-(O)-,3'-(O)-(benzyl)methylenedioxy-adenosine-5'-triphosphate and 2'-(O)-,3'-(O)-(benzyl)methylenedioxy-2"-(O)-,3"-(O)-benzyl methylene dioxy-P',P4-di(adenosine 5'-)tetraphosphate, and as shown, these compounds, at a concentration of 0.25 mM, produced a time dependent reduction in IOP, which was maximal at 1-2 hours, with a reduction of 21-22%.
SUMMARY OF INVENTION
It has now been found, in accordance with the present invention, that certain non-hydrolyzable nucleoside di- or triphosphate derivatives in which the α,β- or ,γ-bridging-oxygen, respectively, is replaced with a methylene or dihalomethylene group are capable, upon administration to the cornea, to significantly reduce intraocular pressure (IOP) in male New-Zealand white rabbits, and are thus considered promising candidates for treatment of ocular hypertension and/or glaucoma through activation of certain P2Y receptors. The two particular nucleoside triphosphate derivatives exhibiting the strongest hypotensive effect were 2-MeS-adenosine-5 '-O-triphosphate- 3,7-methylene and 2-MeS-adenosine-5 '-O- triphosphate-/3,7-dichloromethylene, with EC50 values of 30.2 μΜ and 95.5 nM, respectively, equally or more effective than anti-glaucoma drugs currently available.
In one aspect, the present invention provides an ophthalmic composition comprising an pharmaceutically acceptable carrier and a compound of the general formula I:
Figure imgf000004_0001
or a diastereoisomer or mixture of diastereoisomers thereof,
wherein
R] is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -N tRs, heteroaryl, hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -NO2, -OR4, -SR4, -NR1R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl;
R2 and R3 each independently is H or hydrocarbyl;
Y each independently is H, -OH, or -NH2;
Zi, Z2 and Z3 each independently is -O", -S", or -BH3 "; Wi and W2 each independently is -O-, -NH-, or -C(XiX2)-, wherein Xi and X2 each independently is H or halogen, provided that at least one of Wj, if present, and W2 is not -O-;
n is 0 or 1;
m is 3 or 4; and
B+ represents a pharmaceutically acceptable cation.
The ophthalmic compositions of the invention are useful for reducing intraocular pressure and thereby treating ocular hypertension and/or glaucoma.
In another aspect, the present invention thus provides a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for use in reducing intraocular pressure.
In a further aspect, the present invention relates to use of a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for the preparation of an ophthalmic composition.
In still another aspect, the present invention relates to a method for reducing intraocular pressure in an individual in need thereof comprising administering to said individual a therapeutically effective amount of a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof. BRIEF DESCRIPTION OF DRAWINGS
Figs. 1A-1C show rates of hydrolysis of compounds 9 and 10B (7.5 mM) at pH 1.4 at 37°C, as monitored by HPLC. Fig. 1A shows HPLC chromatograms of compound 9 and hydrolysates at t=6, 52, and 216 h; Fig. IB shows kinetics of acidic hydrolysis of compound 9 (ti/2=65 h) and time-dependent formation of degradation products; and Fig. 1C shows kinetics of acidic hydrolysis of compound 10B (t1/2=8 h).
Figs. 2A-2B show hydrolysis of ATP and compounds 1, 8, and 9 in human blood serum (180 μ\) and RPMI-1640 (540 μΐ) over 24 h at 37°C, as monitored by HPLC. Fig. 2A shows hydrolysis of 0.25 mM ATP and production of ADP and AMP; and Fig. 2B shows degradation of compounds 1, 8 and 9 in human blood serum.
Fig. 3A shows the effects of compounds 1, 8, 9 and 11-14 (100 μΜ) on rabbit intraocular pressure (IOP) measured over 8 hours vs. controls, i.e., an equal volume of saline administered to the contralateral eye and an equal volume of saline administered to other animals. Any treated eye has been measured twice before drug administration, and the values measured are almost identical to those obtained in the time course of the control animals.
Fig 3B shows time course for the effects of compounds 1, 9, 13 and 14 (100 μΜ) on rabbit IOP over 8 h, using the same controls as in Fig. 3 A. Values are means±S.E.M. of results from ten independent experiments.
Fig. 3C shows time-course for the effects of compounds 11B, 12 A and 12B (100 μΜ) on rabbit IOP measured over 8 h. Values are means±S.E.M. of results from eight independent experiments.
Fig. 3D shows dose-response curves for the maximal effects on rabbit IOP of compounds 1, 9, 13 and 14 (100 μΜ). Values are means±S.E.M. of results from eight independent experiments.
Fig. 3E shows comparisons of the maximal effects obtained on rabbit IOP for compounds 1 and 9 (100 μΜ, 10 μΐ), as compared to Xalatan (0.005 %), Trusopt (2%) and Timolol (0.5 %), each applied at a volume of 40 μΐ, using the same controls as in Fig. 3A.
Fig. 3F shows the mean-time effect of compounds 1 and 9 (100 μΜ 10 μΐ), calculated by measuring the time between 50% of IOP decrease after drug administration and 50% of IOP recovery (Morales et al, 2007), as compared to Xalatan (0.005 %), Trusopt (2%) and Timolol (0.5 %), each applied at a volume of 40 μΐ, using the same controls as in Fig. 3A.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, in one aspect, an ophthalmic composition comprising a non-hydrolyzable nucleoside di- or triphosphate analogues of the general formula I as defined above, in which the α,β- or /3,7-bridging-oxygen, respectively, is replaced with a methylene or dihalomethylene group.
As used herein, the term "halogen" includes fiuoro, chloro, bromo, and iodo, and is preferably chloro.
The term "hydrocarbyl" in any of the definitions of the different radicals Ri to R5 refers to a radical containing only carbon and hydrogen atoms that may be saturated or unsaturated, linear or branched, cyclic or acyclic, or aromatic, and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and aryl.
The term "alkyl" as used herein typically means a straight or branched hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like. Preferred are (C]-C6)alkyl groups, more preferably (CrC4)alkyl groups, most preferably methyl and ethyl. The terms "alkenyl" and "alkynyl" typically mean straight or branched hydrocarbon radicals having 2-8 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen- l-yl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, and the like. Preferred are (C2- C )alkenyl and (C2-C6)alkynyl, more preferably (C2-C4)alkenyl and (C2-C4)alkynyl. Each one of the alkyl, alkenyl and alkynyl may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, - NO2, -CN, -SCN, aryl, or heteroaryl, and/or interrupted by one or more heteroatoms selected from nitrogen, oxygen or sulfur.
The term "cycloalkyl" as used herein means a mono- or bicyclic saturated hydrocarbyl group having 3- 10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like, which may be substituted, e.g., with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO2, - CN, -SCN, (CrC8)alkyl, -O-(CrC8)alkyl, -S-(CrC8)alkyl, -NH2, -NH-(C,-C8)alkyl, or -N-((C)-C8)alkyl)2. The term "cycloalkenyl" as used herein means a mono- or bicyclic unsaturated hydrocarbyl group having 3- 10 carbon atoms and 1 double bond, and include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, hexahydropentalenyl, octahydronaphtalenyl, bicycle[4.2.0]oct-2-enyl, and the like.
The term "aryl" as used herein denotes an aromatic carbocyclic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl. Preferred are (C6-Cio)aryl, more preferably phenyl. The aryl radical may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO2, -CN, -SCN, (CrC8)alkyl, -O-(Cr C8)alkyl, -S-(C,-C8)alkyl, -NH2, -NH-(C,-C8)alkyl, or -N-((CrC8)alkyl)2.
The term "heteroaryl" refers to a radical derived from a mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1 or 2, heteroatoms selected from N, O or S. When the heteroaryl is a monocyclic ring, it is preferably a radical of a 5-6- membered ring such as, but not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, pyrido[l,2-a]pyrimidinyl and 1,3-benzodioxinyl. The heteroaryl may be substituted. It is to be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings.
In the group -NR4R5, R4 and R5 each independently is H or hydrocarbyl as defined above or form together with the nitrogen atom to which they are attached a saturated or unsaturated heterocyclic ring optionally containing 1 or - 2 further heteroatoms selected from N, O or S. The term "heterocyclic ring" denotes a mono- or poly-cyclic non-aromatic ring of 4-12 atoms containing at least one carbon atom and one to three, preferably 1-2 heteroatoms selected from N, O or S, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Preferred are 5- or 6-membered heterocyclic rings. The heterocyclic ring may optionally be substituted at any carbon atom as well as at a second nitrogen atom of the ring, if present, with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -NO2, -CN, -SCN, (CrC8)alkyl, -O-(CrC8)alkyl, -S-(C C8)alkyl, - NH2, -NH-(CrC8)alkyl, or -N-((CrC8)alkyl)2. Non-limiting examples of radicals - NR_(R5 include amino, dimethylamino, diethylamino, ethylmethylamino, phenylmethyl-amino, pyrrolidino, piperidino, tetrahydropyridino, piperazino, ethylpiperazino, hydroxyethyl piperazino, morpholino, thiomoφholino, thiazolino, and the like.
In certain embodiments, the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R[ is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R and R5 each independently is H or hydrocarbyl, or R and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein said hydrocarbyl each independently is (Ci-Cg)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6-Ci4)aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S. In particular embodiments, said hydrocarbyl is selected from (Ci-C6)alkyl, preferably (Ci-C4)alkyl, more preferably methyl or ethyl; (C2- C6)alkenyl, preferably (C2-C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C4)alkynyl; or (C6-Cio)aryl, preferably phenyl.
In certain embodiments, the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R2 and R3 each independently is H or hydrocarbyl selected from (Cr C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6-Ci4)aryl. In particular embodiments, said hydrocarbyl is selected from (CrC6)alkyl, preferably (Cr C4)alkyl, more preferably methyl or ethyl; (C2-C6)alkenyl, preferably (C2- C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C4)alkynyl; or (C6-Ci0)aryl, preferably phenyl. In certain embodiments, the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein Y at positions 3 and 4 of the tetrahydrofuran moiety each independently is - OH.
In certain embodiments, the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I, wherein R] is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; R2 and R3 each independently is H or hydrocarbyl; and Y each independently is -OH, wherein said hydrocarbyl each independently is (C C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6-Ci4)aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S.
In particular embodiments, the compound comprised within the ophthalmic composition of the invention is a compound of the general formula I, wherein Rj is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R_j and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; R2 and R3 each independently is H or hydrocarbyl; and Y each independently is -OH, wherein said hydrocarbyl is (Ci-C6)alkyl, preferably (C\- C4)alkyl, more preferably methyl or ethyl; (C2-C6)alkenyl, preferably (C2- C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C4)alkynyl; or (C6-Ci0)aryl, preferably phenyl. More particular compounds are those wherein R| is H, -O-hydrocarbyl, or - S-hydrocarbyl; R2 and R3 each independently is H or hydrocarbyl; and Y each independently is -OH, wherein said hydrocarbyl each independently is methyl or ethyl. Preferred compounds are those wherein Ri is H or -S-methyl; R2 and R3 are H; and Y each independently is -OH. In certain embodiments, the compound comprised within the ophthalmic composition of the present invention is a compound of the general formula I as defined above wherein n is 0, i.e., a non-hydrolyzable nucleoside diphosphate derivative. In other embodiments, the compound used according to the method of the invention is a compound of the general formula I as defined above wherein n is 1, i.e., a non-hydrolyzable nucleoside triphosphate derivative. Particular compounds are those wherein W], if present, is -O-; and W2 is -C(X]X2)- or -NH-, preferably - C(X[X2)-, wherein X] and X2 each independently is H or halogen selected from F, CI or Br, preferably CI. More particular compounds are those wherein Z2, if present, and Z3 each independently is -O"; and Z is -O" or -BH3\
The compounds described in the specification, including the nucleoside di- or triphosphate analogues of the general formula I, additional nucleoside di- or triphosphate analogues excluded from the general formula I by means of provisos, starting materials and intermediates, are herein identified by the Arabic numbers 1- 38 in bold. The full chemical structures of these compounds are depicted in Appendix A and/or in Schemes 1-3 hereinafter. Compound 1 is also identified by the name 2MeS-adenosine-j8,7-CH2-5 '-triphosphate; compound 2 is also identified by the name adenosine-jS,7-CH2-5'-O-(l-boranotriphosphate); compound 3 is also identified by the name 2MeS-adenosine-i8,7-CH2-5'-O-(l-boranotriphosphate); compound 4 is also identified by the name 2MeS-adenosine-5'-O-(l- borano triphosphate); compound 5 is also identified by the name adenosine- triphosphate (ATP); compound 6 is also identified by the name adenosine- diphosphate (ADP); compound 7 is also identified by the name 2MeS-adenosine- diphosphate; compound 8 is also identified by the name 2MeS-adenosine-/3,7-CF2- 5 '-triphosphate; compound 9 is also identified by the name 2MeS-adenosine-/3,7- CC12- 5 '-triphosphate; compound 10 is also identified by the name 2MeS-adenosine- /3,7-CF2-5'-O-(l-boranotriphosphate); compound 11 is also identified by the name adenosine-jS,7-CCl2-5'-O-(l-boranotriphosphate); compound 12 is also identified by the name 2MeS-adenosine- 3,7-CCl2-5'-O-(l-boranotriphosphate); compound 13 is also identified by the name 2MeS-adenosine-o /3-CF2-5 '-diphosphate; and compound 14 is also identified by the name 2MeS-adenosine-a,/3-CCl2-5'- diphosphate.
In specific embodiments, the compound comprised within the ophthalmic composition of the invention is a nucleoside triphosphate analogue, i.e., a compound of the general formula I in which n is 1, wherein Y each independently is -OH; R2 and R3 are H; and (i) R, is -SCH3; Z,, Z2 and Z3 are -O"; W, is -O-; and W2 is -CH2- (compound 1); (ii) Ri is -SCH3; Z] 5 Z2 and Z3 are O"; W] is -O-; and W2 is - CC12- (compound 9); (iii) Rj is H; Zi is -BH3 "; Z2 and Z3 are -O"; Wi is -O-; and W2 is -CC12-, characterized by being the isomer with a retention time (Rt) of 10.87 min when separated from a mixture of diastereoisomers using a semi -preparative reverse-phase Gemini 5u column (C-18 1 10A, 250x 10 mm, 5 micron), and isocratic elution [100 mM triethylammonium acetate, pH 7: MeOH, 91 :9] with flow rate of 5 ml/min (compound 11 isomer B, i.e., compound 11B); or (vii) Rj is -SCH3; Z! is - BH3 ~; Z2 and Z3 are -O"; Wi is -O-; and W2 is -CC12- (compound 12, obtained as two diastereoisomers A and B), or a diastereoisomer or mixture of diastereoisomers thereof. Preferred compounds are those herein identified as compounds 1 and 9, more preferably compound 9.
In other specific embodiments, the compound comprised within the ophthalmic composition of the invention is a nucleoside diphosphate analogue, i.e., a compound of the general formula I in which n is 0, wherein Y each independently is -OH; Ri is -SCH3; R2 and R3 are H; Zt and Z3 are -O"; and W2 is -CF2- or -CC12- (compounds 13 and 14, respectively).
The compounds of the general formula I may be synthesized according to any technology or procedure known in the art, e.g., as described in the Examples section hereinafter.
The compounds of the general formula I may have an asymmetric center, e.g., in the Pa, and may accordingly exist as pairs of diastereoisomers. In cases a pair of diastereoisomers exists, the separation and characterization of the different diastereoisomers may be accomplished using any technology known in the art, e.g., using a semi-preparative reverse-phase column and isocratic solution as described in the Examples section. According to the method of the invention, reducing of intraocular pressure could be carried out by administration of all such isomers and mixtures thereof.
The compounds of the general formula I are in the form of pharmaceutically acceptable salts.
In certain embodiments, the cation B is an inorganic cation of an alkali metal such as, but not limited to, Na+, + and Li+.
In other embodiments, the cation B is ammonium (NH4 +) or is an organic cation derived from an amine of the formula R4N+, wherein each one of the Rs independently is selected from H, CrC22, preferably Ci-C6 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine.
In further embodiments, the cation B is a cationic lipid or a mixture of cationic lipids. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents. Neutral lipids include, but are not limited to, lecithins; phosphatidyl-ethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3/3 hydroxy-sterols. Other neutral lipids contemplated herein include phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidyl glycerols. Examples of cationic lipid compounds include, without being limited to, Lipofectin® (Life Technologies, Burlington, Ontario) (1 : 1 (w/ ) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine™ (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipid formulation), Metafectene (Biontex, Munich, Germany) (Polycationic lipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.) (ethanolic cationic lipids numbers 1 through 12: C52H,o6N6O44CF3CO2H, C88H178N8O4S24CF3CO2H, C40H84NO3P-CF3CO2H, C50H103N7O3-4CF3CO2H, C55H1 16N8O2 6CF3CO2H, C49H102N6O3-4CF3CO2H, C44H89N5O3-2CF3CO2H, C100H2o6N12O4S2 8CF3CO2H, C162H330N22O9 13CF3CO2H, C43H88N4O22CF3CO2H, C43H88N4O3 '2CF3CO2H, C41H78NO8P); Cytofectene (Bio- Rad, Hercules, Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER® (Gene Therapy Systems, San Diego, Calif.) (formulation of a neutral lipid (Dope) and a cationic lipid) and FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, Ind.) (Multi-component lipid based non-liposomal reagent).
As previously shown with respect to analogues 1-3, replacing the ,γ- bridging oxygen in ATP with a methylene group conferred a significant resistance to both enzymatic and chemical (ti/2 = 65, 19 and 14.5 h, respectively, at pH 1.4 and 37°C) hydrolysis; and replacing the Pa non-bridging oxygen by a borano isoster decreased enzymatic hydrolysis of the analogues by human blood serum, NTPDasei 2>3 8, NPP1 ,3 and alkaline phosphatase (Eliahu et al. , 2009). In the study described herein, we determined whether a /3,7-dihalomethylene group increases the chemical stability of these nucleotide analogues, as compared to the presence of a j3,7-methylene group. As shown in the Examples section hereinafter, nucleotide analogues 8 and 9 exhibited high stability at pH 1.4 with ti/2 comparable to those of the analogues 1-3 (25 and 65 h, respectively). Since hydrolytic cleavage of the Ρα-0-Ϋβ bond in both analogues 8 and 9 results in formation of 2-MeS-AMP and β,γ-dihalomethylene diphosphonate, we hypothesize that the lower chemical stability of analogue 9 as compared to 8 may be due to the higher electro-negativity of fluorine vs. chlorine, resulting in a better leaving group in 3,7-difluoromethylene diphosphonate. We further postulate that accessibility of a water molecule to nucleophilic attack on Pa or is more restricted for analogue 9 due to the steric hindrance of the larger chlorine atoms, as compared to analogue 8, resulting in longer t] 2. Analogues 10-12 were less stable at pH 1.4 (ti 2 = 8.0, 6.7 and 0.7 h, respectively) as compared to analogues 8 and 9, due to the low stability of the P-B bond under acidic conditions (Nahum and Fischer, 2004; Li et al., 1996). Nucleoside diphosphate analogues 13 and 14 were significantly more stable at pH 1.4, as compared to their higher homologues 8 and 9, respectively, possibly due to the partial negative charge developing on the 5'-phosphonate (Pa) or 5 '-oxygen atom in analogues 8/9 or 13/14, respectively, upon hydrolysis, which may be better stabilized by a phosphate group, resulting in relatively more extensive hydrolysis of analogues 8/9 vs. 13/14, and hence shorter tj 2 values.
As previously shown, analogue 1 is significantly more stable than ATP in human blood serum (t^ = 12.1 vs. 3.6 h, respectively) (Eliahu et al, 2009), whereas it has now been found that analogue 9 is even more stable exhibiting only 15% degradation after 24 h in human blood serum, as compared to ATP. It is postulated that the enhanced stability of analogue 9 is probably due to steric effects, more particularly, that the bulky chlorine atoms cause analogue 9 to be a weak substrate for dephosphorylating enzymes. Likewise, analogues 10-14 were non-substrates probably due to the replacement the non-bridging-oxygen by a borano group.
It has previously been shown that 2-MeS-a-borano-ATP (A-isomer), 4, is susceptible to hydrolysis by alkaline phosphatase resulting in oborano-2-MeS- AMP and 2-MeS-a-borano-ADP. As particularly shown, alkaline phosphatase hydrolyzed 60% of 2-MeS-a-borano-ATP (A-isomer) within 12 min at 37°C, whereas only traces of 2-MeS-a-borano-ATP remained after 100 min (Eliahu et al, 2009). The substitution of the P^P^bridging CH2 group with a CF2 or CC12 group in analogues 10 and 12, respectively, rendered these analogues completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37°C. Furthermore, analogues 8- 14 were highly resistant to hydrolysis by NTPDasesl , 2, 3, and 8, and NPP1 and 3 (less than 5% hydrolysis of analogues), as compared to ATP. In particular, analogue 12B was completely resistant to hydrolysis by NTPDasel , 2, and 3 and NPP3. The extremely low hydrolysis of analogues is related primarily to the presence of a phosphonate moiety at Ρ?,Ργ (8-12) or Pa,P/3 (13, 14). Bulky CI atoms further improve stability at the tested enzymes compared to F atoms, as shown for analogies 9 vs. 8. The further addition of a borano modification at Pa of 9, yielding analogue 12, produced the most stable analogue, indicating that all tested NTPDase and NPPs are not tolerant to steric hindrance at Pa.
While evaluating analogues 1-3 as agonists of the P2YiR, it has previously been found that the most effective P2Y]R agonist was analogue 1, with an EC5o of 80 nM (Eliahu et al, 2009); however, this analogue was still less potent agonist than the structurally related analogue 4A (EC5o of 2.6 nM) (Nahum et al, 2002). The reduction in the activity of analogues 1-3 as compared to analogue 4A, is possibly related to the elevated pKa of the terminal phosphonate, as compared to phosphate (pKa 8.4 vs. 6.5, respectively) (Bystrom et al, 1997; Myers et al, 1963). Under physiological conditions, i.e., pH 7.4, 91% of ATP is ionized, whereas the /3,7-methylene analogues of ATP are only 9% ionized in solution. Analogues 1-3 are 90% protonated at Ργ at pH 7.4, which likely prevents significant electrostatic interactions with positively charged amino residues in the P2Y]R (Major and Fischer, 2004; Major et al, 2004).
The electronegativity of a dihalogenated methylene group such as CF2 and CC12 lowers the p a of phosphonates from 8.4 to 6.7-7.0, making it closer to the p a of phosphate (Wang et al, 2004). Indeed, nucleotide analogues with a /3,γ- dihalomethylene group were reported to be promising inhibitors of HIV-1 reverse transcriptase (Boyle et al, 2005; Wang et al, 2004), agonists of P2X2/3 receptors (Spelta et al, 2003), and potent antagonists of the P2Y12 receptor (Ingall et al, 1999; El-Tayeb et al, 2005).
It has now been found that although analogues 8-14 are resistant to enzymatic hydrolysis, they are less potent P2Y[R agonists than the more rapidly hydrolyzed 2-MeS-ADP. In particular, whereas the activity of analogue 1 was 20- fold lower than that of 2-MeS-ADP (EC50 = 0.0025 μΜ) (Eliahu et al, 2009), the activities of the corresponding 3,7-dihalomethylene analogues 8 and 9 are 300- to 3800-fold lower than that of 2-MeS-ADP, respectively. Apparently, the presence of a thiomethyl group at the adenine C-2 position was essential for P2Y]R activity, since analogues 11 A and 11B were inactive. Yet, any halogen substitution at the phosphonate carbon was not tolerated by P2YjR. Considering the rank order of agonist potency obtained at the P2Y]R, analogue 1 > 8 > 9, it seems that the major parameter determining the affinity of this series of P2YjR agonists is the steric constraints of the ligand binding-site of P2Y]R rather than pKa values of the their phosphonate moieties (Blackburn et al, 1984; Bystrom et al, 1997). The smaller size of the /?,7-methylene group presumably allows tighter binding than the larger /3,7-dihalomethylene group, consistent with results for analogues 13 and 14.
The Examples hereinafter further demonstrate the ability of analogues 1, 9, 11B, 12A, 12B, 13 and 14 to reduce intraocular pressure (IOP) in male New- Zealand white rabbits, wherein the two compounds exhibiting the strongest hypotensive effect were analogues 1 and 9, with EC5o values of 30.2 μΜ and 95.5 nM, respectively, equally or more effective than hypotensive drugs currently available. This is a significant finding since nucleotide analogues activate different receptors than hypotensive drugs commonly used for treatment of glaucoma, and thus may represent alternatives for patients unable to use the currently approved drugs. Side effects are common with current glaucoma medications. For example, beta-blockers, e.g., Timolol, can cause bradycardia and hypertension (Higginbotham et al, 2002), whereas cholinergic agents, e.g., pilocarpine, produce fixed pupils, and induce myopia and cataracts (Hoyng and Van Beek, 2000; Alward, 1998). Prostaglandins, e.g., Xalatan, cause eyelash growth, iris pigmentation (Johnstone, 1997) and muscle and joint pain (Higginbotham et al., 2002). The development of stable and potent nucleotide analogues should expand the limited repertoire of drugs currently available and used for treatment of glaucoma.
We found no correlation between the potency of nucleotide analogues at the
P2Y]R and their ability to reduce IOP, since analogues 1 and 9 activated the P2YiR with EC50 values in the nanomolar and micromolar range, respectively, yet analogue 9 was more potent than analogue 1 at reducing IOP. These findings may imply that these analogues reduce IOP not via activation of P2Y)R as considered based on previous data (Markovskaya et al.y 2008), but via other P2Y]R-like receptors.
In summary, we synthesized chemically and enzymatically stable nucleotide analogues 8-14, based on ADP and ATP scaffolds modified by α,β/β,Ύ- dihalomethylene groups within the phosphate chain. These analogues were agonists at the P2Y[R. Analogues 1 and 9, in particular, exhibited the ability to reduce IOP in normotense rabbits, with EC5o values of 30.2 μΜ and 95.5 nM, respectively. Moreover, these analogues were more effective than several commonly prescribed glaucoma drugs, in particular, Xalatan and Trusopt, at reducing IOP. In addition, the duration of the hypotensive effect induced by analogue 9 was comparable to Xalatan, Trusopt and Timolol. In view of all the aforesaid, it is concluded that compound 9 is an attractive drug candidate for the treatment of glaucoma. Furthermore, although slightly less efficacious than Timolol, this compound represents a promising alternative to Timolol, as the latter is a /3-blocker and cannot be used for the treatment of patients suffering from cardiovascular problems, asthma, bronchitis or diabetes.
The ophthalmic compositions of the present invention can be provided in a variety of formulations and dosages. These compositions may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 Ed., 1995. The compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the general formula I, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
The compositions of the present invention, intended for direct application to the eye, may be formulated so as to have both pH and tonicity compatible with the eye. This will normally require a buffer to maintain the pH of the composition at or near physiologic pH, i.e., in the range of 5-9, preferably 6 to 8, more preferably 6.8- 7.4; and may further require a tonicity agent to bring the osmolality of the composition to a level at or near 210-320 milliosmoles per kilogram (mOsm/kg). In certain embodiments, the composition of the invention has an osmolality in the range of 50-700 mOsm/kg, preferably 100-600 mOsm/kg, more preferably 150-500 mOsm/kg, still more preferably 200-400 mOsm/kg, most preferably 200-350 mOsm/kg.
The compositions of the invention may be administered to the eye of the subject by any suitable means. In one embodiment, the composition is in the form of a liquid, emulsion, gel or suspension of the compound of the general formula I, and it is administered as drops, spray, or gel. In another embodiment, the active agent, i.e., the compound of the general formula I is applied to the eye via liposomes. In a further embodiment, the active agent of the composition is contained within a continuous or selective-release device, e.g., membranes such as, but not limited to, those employed in the OcusertTM System (Alza Corp., Palo Alto, Calif.).
In one embodiment, the active agent can be contained within, carried by, or attached to contact lenses, which are placed on the eye. In other embodiments, the active agent is contained within a swab or sponge, or within a liquid spray, which is applied to the ocular surface. In a further embodiment, the active agent is directly injected into the ocular tissues, e.g., by subconjunctival, subscleral, or intravitreal injection, or onto the eye surface.
In addition to the active agent, the ophthalmic composition of the present invention contains a physiologically compatible carrier or vehicle as those skilled in the ophthalmic art can select using conventional criteria. Such vehicles may be selected from known ophthalmic vehicles that include, inter alia, saline solution, water, polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, cyclodextrins, in particular betahydroxypropyl cyclodextrin, petroleum derivatives, e.g., mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil, polysaccharides such as dextrans, an alginate such as sodium alginate optionally comprising guluronic acid and/or mannuronic acid, glycosaminoglycans such as sodium hyaluronate, and salts such as sodium chloride and potassium chloride.
The optimal dosage for administration will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner. In particular, compositions for the treatment of glaucoma may be administered daily, twice daily, or 3-4 times daily, and/or upon the occurrence of symptoms associated with the condition; and over a period of time consistent with treatment of the ocular hypertension and glaucoma, e.g., for a period of weeks, months, years, or decades.
The ophthalmic compositions of the present invention are useful for lowering intraocular pressure, thus can be used for prevention of treatment of ocular hypertension and/or glaucoma.
The term "intraocular hypertension", "ocular hypertension", or "intraocular pressure", as used herein interchangeably, refers to an intraocular pressure in an eye of a patient that is above a normal level and is correlated as a risk factor for the development of visual field loss and glaucoma.
Glaucoma is a heterogeneous group of optic neuropathies that share certain clinical features, wherein the loss of vision is due to the selective death of retinal ganglion cells in the neural retina that is clinically diagnosed by characteristic changes in the visual field, nerve fiber layer defects, and a progressive cupping of the optic nerve head (ONH). One of the main risk factors for the development of glaucoma is the presence of intraocular hypertension (elevated intraocular pressure, IOP). IOP also appears to be involved in the pathogenesis of normal tension glaucoma where patients have what is often considered to be normal IOP. The elevated IOP associated with glaucoma is due to elevated aqueous humor outflow resistance in the trabecular meshwork (TM), a small- specialized tissue located in the iris-corneal angle of the ocular anterior chamber. Glaucomatous changes to the TM include a loss in TM cells and the deposition and accumulation of extracellular debris including proteinaceous plaque-like material. In addition, there are also changes that occur in the glaucomatous ONH. In glaucomatous eyes, there are morphological and mobility changes in ONH glial cells. In response to elevated IOP and/or transient ischemic insults, there is a change in the composition of the ONH extracellular matrix and alterations in the glial cell and retinal ganglion cell axon morphologies.
The term "glaucoma" as used herein is a disease of the eye characterized by increased pressure inside the eye with resultant optic nerve damage. Glaucoma includes, but is not limited to, primary glaucomas, secondary glaucomas, juvenile glaucomas, congenital glaucomas, pseudoexfoliation glaucoma, acute angle closure glaucoma, absolute glaucoma, chronic glaucoma, narrow angle glaucoma, chronic open angle glaucoma, simplex glaucoma and familial glaucomas, including, without limitation, pigmentary glaucoma, high tension glaucoma, and low tension glaucoma and their related diseases.
In another aspect, the present invention provides a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for use in reducing intraocular pressure.
In a further aspect, the present invention relates to use of a compound of the general formula I as defined above, or a diastereoisomer or mixture of diastereoisomers thereof, for the preparation of an ophthalmic composition.
In still another aspect, the present invention relates to a method for reducing intraocular pressure in an individual in need thereof comprising administering to said individual a therapeutically effective amount of a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof. In a particular embodiment of this method, compound 1 or 9, preferably 9, or a diastereomer or mixture of diastereoisomers thereof, is administered.
The invention will now be illustrated by the following non-limiting Examples. EXAMPLES Experimental General
All air- and moisture-sensitive reactions were carried out in flame-dried, argon-flushed, two-neck flasks sealed with rubber septa, and the reagents were introduced with a syringe. Progress of reactions was monitored by TLC on precoated Merck silica gel plates (60F-254). Visualization of reactants and products was accomplished by UV light. Compounds were characterized by nuclear magnetic resonance using Bruker AC-200, DPX-300 or DMX-600 spectrometers. 1H NMR spectra were measured at 200, 300 or 600 MHz. Nucleotides were characterized also by 31P NMR in D2O, using 85% H3PO4, and 19F NMR using trifluorochloromethane as an external reference on Bruker AC-200 and DMX-600 spectrometers. High-resolution mass spectra were recorded on an AutoSpec-E FISION VG mass spectrometer by chemical ionization. Nucleotides were analyzed under electron spray ionization (ESI) conditions on a Q-TOF micro-instrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a column of Sephadex DEAE-A25, swollen in 1 M NaHCO3 at 4°C for 1 day. The resin was washed with deionized water before use. The LC separation was monitored by UV detection at 280 nm. Final purification of the nucleotides and separation of diastereomers were achieved on a HPLC (Elite Lachrom, Merck-Hitachi) system using a semi-preparative reverse-phase column (Gemini 5u C-18 1 10A 250 x 10.00 mm; 5 micron; Phenomenex, Torrance, USA). The purity of the nucleotides was evaluated with an analytical reverse-phase column system (Gemini 5u C-18 1 1 OA, 150 x 4.60 mm; 5 micron; Phenomenex, Torrance, CA, USA) using two solvent systems: Solvent System I - (A) 100 mM triethylammonium acetate (TEA A), pH 7: (B) CH3CN; Solvent System II - (A) 0.01 M KH2PO4, pH 4.5: (B) CH3CN. The details of the solvent system gradients used for the separation of each product are given below. All commercial reagents were used without further purification, unless otherwise noted. All reactants in moisture- sensitive reactions were dried overnight in a vacuum oven. RPMI (Roswell Park Memorial Institute) 1640 buffer was obtained from Sigma- Aldrich. 2',3'-O- methoxymethylidene-adenosine and 2-MeS-adenosine were prepared, as previously described (Nahum et al., 2002; Griffin et al, 1967). 2',3'-O-methoxymethylidene- 2-MeS-adenosine was purified with a MPLC system (Biotage, Kungsgatan, Uppsala, Sweden) using a silica gel (25+M) column and the following gradient scheme: 3 column volumes (CV) of 100:0 (A) CHC13:(B) EtOH, 5 CV of a gradient from 100:0 to 90: 10 A:B and 4 CV of 90: 10 A:B at a flow rate of 12.5 ml/min. pH measurements were performed with an Orion micro combination pH electrode and a Hanna Instruments pH meter. Triethylammonium bicarbonate (TEAB) was prepared as previously described (Bachelet and Guibe, 1951). Bis(tributyl ammonium)difluoromethylene diphosphonate salt was purchased from PJ Chemical Inc. For preparation of human blood serum, whole blood taken from healthy volunteers was obtained from a blood bank (Tel-Hashomer Hospital, Israel). Blood was stored for 12 h at 4°C, centrifuged in plastic tubes at 1500 x g for 15 min at RT. The serum was separated and stored at -80°C.
Preparation of bis(tributylammonium)dichloromethylene diphosphonate salt
A H+ Dowex column was used for ion exchange chromatography. Thirty ml of Dowex was placed in a column with cotton wool at the bottom, and the column was washed with 10% NaOH (150 ml) until the pH of the effluent was basic, and then with distilled water until the pH of the effluent reached neutral. Then, the column was washed with 10% HC1 (300 ml) followed by distilled water until the effluent reached acidic and neutral pH, respectively. A flask containing Bu3N (2 eq) in EtOH was placed in an ice bath under the column and stirred.
The disodium form of dichloromethylene diphosphonate salt was dissolved in distilled water and poured onto the column, and the column was then washed with distilled water until the pH of the effluent was neutral. The effluent was dropped into the Bu3N/EtOH solution. The final solution of bis(tributylammonium) dichloromethylene diphosphonate salt was then freeze-dried.
Preparation of 2-MeS-adenosine-5 '-0-triphosphate^,y-methylene-dihalogen derivatives, 8 and 9
As depicted in Scheme 1 hereinafter, l,8-bis(dimethylamino)naphthalene (156 mg, 0.72 mmol, 2 eq) was added at 0°C to 2',3'-O-methoxymethylidene-2- MeS-adenosine, analogue 15, (130 mg, 0.36 mmol) in trimethylphosphate (2.5 ml) in a flame-dried two-neck flask under N2, and the reaction was stirred for 20 min until a clear solution was attained. POCl3 (66 μΐ, 0.72 mmol, 2 eq) was added at 0°C. The solution was stirred at 0°C for 1 h. A 0.5 M solution of bis(tributylammonium)dihalomethylene diphosphonate salt (660 mg, 1.13 mmol, 3 eq) in dry DMF (1.4 ml) and tributylamine (360 μΐ, 1.46 mmol, 4 eq) was added at 0°C and the reaction mixture was stirred for 90 min. A 0.5 M solution of TEAB (15 ml) was added at RT and the reaction mixture was stirred for 60 min, and then freeze-dried. The residue was dissolved in water and treated with 18% HC1 until the pH was 2.3, and the mixture was then stirred for 3 h at RT. Finally, the mixture was treated with 24% NH4OH solution, and the pH was adjusted to 9. The solution was stirred for 45 min at RT and then freeze-dried. The resulting residue was dissolved in deionized water (100 ml) and extracted with diethyl ether (2 x 50 ml) and then chloroform (50 ml). The aqueous phase was freeze-dried. The resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.5 M NH4HCO3; total volume 2 1). The relevant fractions were collected and freeze-dried, and excess NH4HCO3 was removed by repeated freeze-drying with deionized water to yield the product as a white powder. The product was purified by LC yielding analogue 8 at a 46% yield (1 10 mg) and analogue 9 at a 10% yield (19.6 mg). Finally, the nucleotide triethylammonium counter ions were exchanged for Na+ ions by passing analogues 8 and 9 through a Sephadex-CM C-25 Na+-form column. The spectral data for analogues 8 and 9 were consistent with the literature (Cusack et al, 1987). Preparation of adenosine-5 '-0-( GL-boranotriphosphate)- , -methylene-dihalogen derivatives, 10-12
As depicted in Scheme 2 hereinafter, 2',3'-O-methoxymethylidene adenosine derivatives (analogues 14 or 20; 50 mg, 0.14 mmol) were dissolved in trimethylphosphate (0.7 ml) in a flame-dried two-neck flask under N2. 1,8- bis(dimethylamino)naphthalene (60 mg, 0.28 mmol, 2 eq) was added at 0°C and the reaction was stirred for 20 min until a clear solution was attained. PC13 (23 μ\, 0.28 mmol, 2 eq) was added at 0°C and a white solid precipitated. The suspension was stirred at 0°C for 45 min. Then, a 0.5 M solution of bis(tributylammonium) dihalomethylene diphosphonate salt (245 mg, 0.42 mmol, 3 eq) in dry DMF (0.6 ml) and tributylamine (134 μΐ, 0.56 mmol, 4 eq) was added at 0° C and the reaction mixture was stirred for 50 min. A 2 M solution of BH3-SMe2 complex in THF (0.7 ml, 2.81 mmol, 10 eq) was added at 0°C and the reaction mixture turned clear. The solution was stirred for 5 min at 0°C and then for 60 min at RT. Finally, a 0.5 M TEAB solution (15 ml) was added at RT and the mixture was stirred for 60 min and then freeze-dried. The residue was dissolved in water, treated with 18% HC1 until the pH was 2.3, and then the mixture was stirred for 3 h at RT. Finally, the mixture was treated with 24% NH4OH, and the pH was adjusted to 9. The solution was stirred for 45 min at RT and then freeze-dried. The resulting residue was dissolved in deionized water (100 ml) and extracted with diethyl ether (2x30 ml) and chloroform (30 ml). The aqueous phase was freeze-dried. The resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.5 M NH4HCO3; total volume 2 1). The relevant fractions were collected and freeze-dried, and excess NH4HCO3 was removed by repeated freeze-drying cycles with deionized water to obtain the product as a white powder. The product was purified by LC yielding aompound 10 at a 21% yield (13 mg), aompound 11 at a 18% yield (14 mg) and aompound 12 at a 7.2% yield (18.6 mg). The diastereomers of aompounds 10, 11 and 12 were separated on a HPLC column, under the conditions described below. Finally, the purified diasteroisomers were passed through a Sephadex-CM C-25 Na+-form column to exchange triethylammonium ions for Na+. Separation of2-MeS-adenosine-5'-0-(a-boranotriphosphate)^,^-CF2 (10A, 10B) The separation of compound 10 diastereoisomers, 10A and 10B, was accomplished using a semi-preparative reverse-phase Gemini 5u column and isocratic elution with 80:20 (A) 100 raM triethylammonium acetate (TEAA), pH 7:(B) MeOH at a flow rate of 5 ml/min. Fractions containing purified isomers [Rt = 12.78 min (10A isomer); 14.75 min (10B isomer)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles with the solid residue dissolved each time in deionized water. Diastereoisomers 10A and 10B were obtained at 21% overall yield (18.6 mg) after LC separation. Characterization of2-MeS-adenosine-5'-0-(a-boranotriphosphate)- ,fCF2 (10A) Retention time on a semi-preparative column: 12.78 min. Ή NMR (D2O; 600 MHz): δ 8.43 (s; H-8; 1H), 6.13 (d; J= 5.40 Hz; H-l'; 1H), 4.80 (t; J= 5.40 Hz; H-2'; 1H), 4.65 (m; H-3'; 1H), 4.36 (m; H-4'; 1H), 4.31 (m; H-5'; 1H), 4.13 (m; H- 5"; 1H), 2.58 (s; CH3; 3H), and 0.45 (m; BH3; 3H) ppm. 31P NMR (D2O; 243 MHz): 5 83.50 (m; Pa-BH3), 4.71 (m; Ργ), and -2.25 (m; P^) ppm. 19F NMR (D2O 188 MHz): δ -115.38 (t, J = 79.27) ppm. MS-ES m/z: 584 (M"). TLC (NH4OH:H2O: isopropanol 2:8: 1 1), R = 0.12. Purity data obtained on an analytical column: retention time: 7.82 min (95.3% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 4.89 min (92.8% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 20 min at a flow rate of 1 ml/min.
Characterization of 2-MeS-adenosine-5'-0-(a-boranotriphosphate)^, -CF2 (10B) Retention time on a semi-preparative column: 14.75 min. Ή NMR (D2O; 600 MHz): 5 8.39 (s; H-8; 1H), 6.12 (d; J= 5.40 Hz; H-l'; 1H), 4.80 (t; J= 5.40 Hz; H-2'; 1H), 4.57 (m; H-3'; 1H), 4,37 (m; H-4'; 1H), 4.26 (m; H-5*; 1H), 4.19 (m; H- 5"; 1H), 2.58 (s; CH3; 3H), and 0.48 (m; BH3; 3H) ppm. 31P NMR (D2O; 243 MHz): δ 84.80 (m; Pa-BH3), 4.73 (m; Ργ), and -2.30 (m; P^) ppm. 19F NMR (D2O 188 MHz): δ -115.40 (t, J = 79.27) ppm. MS-ES m/z: 584 (M~). TLC (NH4OH:H2O: isopropanol 2:8: 1 1), Rf = 0.12. Purity data obtained on an analytical column: retention time: 8.17 min (87.0% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 3.08 min (94.95% purity) using Solvent System II with a gradient from 70:30 to 50:50 A:B over 10 min at a flow rate of 1 ml/min. Separation of adenosine-5'-0-(a-boranotriphosphate)^, -CCl2 (HA, 11B)
The separation of compound 11 diastereoisomers, 11A and 11B, was accomplished using a semi-preparative reverse-phase Gemini 5u column (C-18 1 10A; 250 10.00 mm; 5 micron) and isocratic elution with 91 :9 (A) 100 mM triethylammonium acetate (TEAA), pH 7:(B) MeOH at a flow rate of 5 ml/min. Fractions containing purified isomers [Rt = 8.37 min (11 A isomer); 10.87 min (11B isomer)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles with the solid residue dissolved each time in deionized water. Diastereoisomers 11 A and 11B were obtained at 18% overall yield (14 mg) after LC separation. Characterization of adenosine-5 '-O-fa-boranotriphosphate^^CC^ (HA)
Retention time on a semi-preparative column: 8.37 min. Ή NMR (D2O; 300 MHz): δ 8.51 (s; H-8; 1H), 8.14 (s; H-2; 1H), 6.04 (d; J = 5.7 Hz; H-l'; 1H), 4.78 (Η-2' and H-3' signals are hidden by the water signal), 4.30 (m; H-5'; 2H), 4.05 (m; H-4'; 1H), and 0.37 (m; BH3; 3H) ppm. 31P NMR (D2O; 243 MHz): δ 83.80 (m; Pa- BH3), 9.10 (d; J = 19.14 Hz; Ργ), and 2.02 (m; Ρ„) ppm. MS-ESI m/z: 570 (M ). HRMS-FAB (negative) m/z: calculated for CnH18BN5Oi ,Na2P3: 546.0104; found: 546.0104. TLC (NH4OH:H2O:isopropanol 2:8: 1 1), Rf= 0.3. Purity data obtained on an analytical column: retention time: 6.88 min (98% purity) using Solvent System I with a gradient from 95:5 to 70:30 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 1.55 min (98% purity) using Solvent System II with a gradient from 85: 15 to 70:30 of A:B over 10 min at a flow rate of 1 ml/min.
Characterization of adenosine-5'-0-(cL-boranotriphosphate)- ,y-CCl2 (HB)
Retention time on a semi-preparative column: 10.87 min. Ή NMR (D2O; 300 MHz): δ 8.49 (s; H-8; 1H), 8.14 (s; H-2; 1H), 6.04 (d; J = 5.7 Hz; Η-Γ; 1H), 4.78 (Η-2' signal is hidden by the water signal), 4.47 (m; H-3"; 1H), 4.23 (m; H-5*; H-5"; 2H), 4.15 (m; H-4'; 1H), and 0.37 (m; BH3; 3H) ppm. 31P NMR (D2O; 243 MHz): δ 84.58 (m; Pa-BH3), 9.14 (d; J = 18.46 Hz; Ργ), and 2.1 1 (m; P^) ppm. MS- ESI m/z: 570 (M"). TLC (NH4OH:H2O:isopropanol 2:8: 1 1), R = 0.3. Purity data obtained on an analytical column: retention time: 7.62 min (97% purity) using Solvent System I with a gradient from 95:5 to 70:30 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 1.32 min (98% purity) using Solvent System II with a gradient from 95:5 to 70:30 of A:B over 10 min at a flow rate of 1 ml/min.
Separation of2-MeS-adenosine-5'-0-(a-boranotriphosphate)^,y-CCl2 (12A, 12B) The separation of compound 12 diastereoisomers, 12A and 12B, was accomplished using a semi-preparative reverse-phase Gemini 5u column (C-18
110A; 250 x 10.00 mm; 5 micron) and isocratic elution with 82: 18 (A) 100 mM triethylammonium acetate (TEAA), pH 7:(B) MeOH at a flow rate of 5 mL/min.
Fractions containing purified isomers [Rt = 9.33 min (12A isomer); 10.61 min (12B isomer)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles with the solid residue dissolved each time in deionized water.
Diastereoisomers 12A and 12B were obtained at a 7.2% overall yield (13 mg) after
LC separation.
Characterization of 2-MeS-adenosine-5'-0-(a-boranotriphosphate)^,j-CCl2 (12A) Retention time on a semi-preparative column: 9.33 min. Ή NMR (D2O; 300
MHz): δ 8.34 (s; H-8; 1H), 6.04 (d; J = 5.10 Hz; H-l*; 1H), 4.78 (H-2' and H-3* signals are hidden by the water signal), 4.30 (m; H-4'; 1H), 4.05 (m; H-5'; H-5"; 2H), 2.49 (s; CH3; 3H), and 0.42 (m; BH3; 3H) ppm. 3lP NMR (D2O; 243 MHz): δ 83.40 (m; P«-BH3), 9.28 (d; J = 17.32 Hz; Ργ), and 2.65 (m; ?β) ppm. MS-ES m/z: 548 (M ). TLC (NH4OH:H20:isopropanol 2:8: 11), R = 0.15. Purity data obtained on an analytical column: retention time: 7.96 min (90.4% purity) using Solvent System I with a gradient from 95:5 to 65:35 A:B over 13 min at a flow rate of 1 ml/min. Retention time: 2.36 min (87.0% purity) using Solvent System II with a gradient from 95:5 to 60:40 A:B over 10 min at a flow rate of 1 ml/min. Characterization of 2-MeS-adenosine-5'-0-( .-boranotriphosphate)^,y-CCl2 (12B)
Retention time on a semi-preparative column: 10.61 min. Ή NMR (D2O; 300 MHz): δ 8.32 (s; H-8; 1H), 6.02 (d; J = 5.40 Hz; H-l'; 1H), 4.78 (Η-2' and H-3' signals are hidden by the water signal), 4.28 (m; H-4'; 1H), 4.18 (m; H-5'; H-5"; 2H), 2.48 (s; CH3; 3H), and 0.40 (m; BH3; 3H) ppm. 31P NMR (D2O; 243 MHz): 5 84.49 (m; Pa-BH3), 9.17 (d; J = 19.16 Hz; Ργ), and 2.35 (m; ?β) ppm. MS-ES m/z: 548 (M ). TLC (NH4OH:H2O:isopropanol 2:8:1 1), Ry = 0.15. Purity data obtained on an analytical column: retention time: 3.87 min (92.0% purity) using Solvent System I with a gradient from 85: 15 to 60:40 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 4.1 1 min (91.0% purity) using Solvent System II with a gradient from 90: 10 to 60:40 A:B over 10 min at a flow rate of 1 ml/min.
Preparation and characterization of2',3,-0-methoxymethylidene-5'-0-tosyl-2- MeS-adenosine, 34
As depicted in Scheme 3 hereinafter, a solution of 4-dimethylaminopyridine (132 mg, 1.07 mmol, 4 eq) in CH2C12 (1 ml) and a solution of TsCl (130 mg, 0.67 mmol, 2.5 eq) in CH2C12 (1 ml) were added to a suspension of 2',3'-O- methoxymethylidene 2-MeS-adenosine, 15, (95 mg; 0.27 mmol) in CH2C12 (3 ml) in a flame-dried two-neck flask under N2 at RT. The suspension turned clear and the reaction mixture was stirred for 2 h at RT. CH2C12 (30 ml) was added to the reaction mixture, which was then extracted with saturated NaHCO3 solution (3x30 ml). The organic phase was treated with Na2SO4 and filtered. The solvent was removed under reduced pressure and the residue was separated using aMPLC system with a C-18 (25+M) column and the following gradient scheme: 3 column volumes (CV) of 100:0 (A) CH2C12:(B) EtOH, 5 CV of a gradient from 100:0 to 90: 10 A:B and 4 CV of 90: 10 A:B at a flow rate of 25 ml/min. The relevant fractions were collected and the solvent was removed under reduced pressure yielding compound 34 at a 85% yield (1 15 mg) as a white solid. Ή NMR (CDC13; 200 MHz): δ 7.83 (s; H-8; 2H), 7.69, 7.23 (2m, 2H), 6.34, 6.29 (2 br s, NH2; 2H), 6.17, 6.05 (2d; J = 2.60 Hz; H-l'; 1H), 6.07, 6.01 (2s, CH-Ome; 1H), 5.56, 5.53 (2dd; J = 2.60 Hz; H-2'; 1H, J = 7.20 Hz; H-2'; 1H), 5.21, 5.06 (2dd, J= 7.20 Hz; H-3'; 1H, J = 3.20 Hz; H-3'; 1H), 4.66, 4.52 (2m, Η-4'; 1H), and 4.31 (m, H-5'; H-5"; 4H) ppm. MS-ES- z: 584 (M"). TLC (CHCl3:EtOH, 9: 1), R/= 0.64.
Preparation and characterization of2 3,-0-isopropylidene-5,-0-tosyl-2-MeS- adenosine, 35
As depicted in Scheme 3 hereinafter, a solution of 4-DMAP (134 mg, 1.09 mmol, 4 eq) in CH2C12 (2 ml) and a solution of TsCl (156 mg, 0.82 mmol, 3 eq) in CH2C12 (0.5 mL) were added to a solution of 2',3'-O-isopropylidene adenosine, compound 33, (97 mg; 0.27 mmol) in CH2C12 (1 ml) in a flame-dried two-neck flask under N2 at RT, and the reaction mixture was stirred for 2 h. CH2C12 (50 ml) was added to the reaction mixture which was then extracted with saturated NaHCO3 solution (3x30 ml). The organic phase was treated with Na2SO4 and filtered. The solvent was removed under reduced pressure and the residue was separated using a MPLC system with a silica gel (25+M) column and the following gradient scheme: 3 column volumes (CV) of 100:0 (A) CH2C12:(B) EtOH, 5 CV of a gradient from 100:0 to 90: 10 A:B and 4 CV of 90: 10 A:B at a flow rate of 25 ml/min. The relevant fractions were collected and the solvent was removed under reduced pressure yielding analogue 35 at a 51% yield (71 mg) as a white solid. Ή NMR (CDC13; 300 MHz): 5 7.90 (s; H-8; 1H), 7.67 (d, J = 8.40, 2H), 6.05 (s; Η- ; 1H), 5.35 (m; H-2'; 1H), 5.00 (m, H-3'; 1H), 4.50 (m, H-4'; 1 H), and 4.25 (m, H-5'; H-5"; 2H). MS-ES m/z: 508 (MH+). MS-ES+ m/z: 508 (MH+). TLC (CHCl3:EtOH 95:5), Rf= 0.79.
Preparation and characterization of2-MeS-adenosine-5'-0-diphosphate-^- difluoromethylene, 13
As depicted in Scheme 3 hereinafter, 2',3'-O-methoxymethylidene-5 '-O- tosyl-2-MeS-adenosine, analogue 34 (90 mg; 0.17 mmol), was dissolved in dry DMF (0.4 ml) in a flame-dried two-neck flask under N2. A solution of tris(tetra-n- butylammonium)difluoro methylenediphosphonate (Davisson et al., 1987) (0.28 mmol; 1.5 eq) in dry DMF (0.3 ml) was added at RT and the reaction was stirred for 72 h. Deionized water (20 ml) was added and the reaction was treated with 18% HCl until the pH was 2.3, and then the mixture was stirred for 3 h at RT. Then, the mixture was treated with 24% NH4OH, and the pH was adjusted to 9. The solution was stirred for 45 min at RT and then freeze-dried. The resulting residue was applied to an activated Sephadex DEAE-A25 column (0-0.3 M NH4HCO3; total volume of 2 1). The relevant fractions were collected and freeze-dried, and excess NH4HCO3 was removed by repeated freeze-drying with deionized water to yield compound 13 as a white powder. The residue was separated using a MPLC system with a C-18 (12+M) column and the following gradient scheme: 5 column volumes (CV) of 100:0 (A) TEAA:(B) MeOH, 7.5 CV of a gradient from 100:0 to 60:40 A:B and 3 CV of 60:40 A:B at a flow rate of 12 ml/min. Finally, triethylammonium ions were exchanged for Na+ by passing the pure aompound 13 through a Sephadex-CM C-25 Na+-form column. Compound 13 was obtained at a 34% yield (61 mg) after MPLC separation. Ή NMR (D2O; 300 MHz): δ 8.18 (s; H-8; 1H), 5.93 (d; J= 5.40 Hz; H-l'; 1H), 4.78 (Η-2' and H-3' signals are hidden by the water signal), 4.43 (m; H-4'; 1H), 4.19 (m; H-5'; 1H), 4.15 (m; H-5"; 1H), and 2.37 (s; CH3; 3H) ppm. 31P NMR (D2O; 81 MHz): δ 4.48 (m; Pa, ?β) ppm. , 9F NMR (D2O 188 MHz): δ 1 19.16 (dd, J, = 84.72 Hz, J2 = 80.95 Hz) ppm. MS-ES m/z: 506 (M"). HRMS-FAB (negative) m/z: calculated for ^Η,^Ν^ ^Ρ^: 573.9721; found: 573.966. TLC (NH4OH:H2O:isopropanol 2:8: 11), Rf = 0.5. Purity data obtained on an analytical column: retention time: 6.85 min (100% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 5.17 min (99.94% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 18 min at a flow rate of 1 ml/min.
Characterization of 2-MeS-adenosine, 5 ',5 '"-[P,P'-difluoro-metliylene
bisphosphonate], 38
Ή NMR (D2O; 300 MHz): δ 8.18 (s; H-8; 1H), 5.93 (d; J = 5.40 Hz; H-l'; 1H), 4.78 (H-2' and H-3' signals are hidden by the water signal), 4.43 (m; H-4'; 1H), 4.19 (m; H-5', 1H), 4.15 (m; H-5"; 1H), and 2.37 (s; CH3; 3H) ppm. 31P NMR (D2O; 81 MHz): δ 4.48 (m; Pa, P^) ppm. I9F NMR (D2O 188 MHz): δ 1 19.16 (dd, J, = 84.72 Hz, J2 = 80.95 Hz) ppm. MS-ES m/z: 506 (NT). HRMS-FAB (negative) m/z: calculated for C23H28F2N10O12Na3P2S2: 869.0460; found: 869.056. TLC (NH4OH:H2O:isopropanol 2:8: 1 1), Ry = 0.5. Purity data obtained on an analytical column: retention time: 6.85 min (100.0% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 15 min at a flow rate of 1 ml/min. Retention time: 5.17 min (99.94% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 18 min at a flow rate of 1 ml/min.
Preparation and characterization of 2-MeS-adenosine, 5',5"'-[P,P'-dichloro- methylene bisphosphonate], 14
As depicted in Scheme 3, 2',3'-O-isopropylidene-5'-O-tosyl-2-MeS- adenosine, compound 35 (42 mg, 0.08 mmol) was dissolved in dry DMF (0.2 ml) in a flame-dried two-neck flask under N2. A solution of tris(tetra-n- butylammonium)dichloro methylenediphosphonate (0.16 mmol; 2 eq) in dry DMF (0.3 ml) was added at RT and the reaction was stirred for 72 h. Neat TFA was added for 10 min under argon bubbling and then evaporated under reduced pressure to yield a yellow solid. The resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.3 M NH4HCO3; total volume of 1.4 1). The relevant fractions were collected and freeze-dried, and excess NH4HCO3 was removed by repeated freeze-drying with deionized water to yield compound 14 as a white powder. The residue was separated using a HPLC system with a semi-preparative C-18 column and the following gradient scheme (A) 100 mM TEAA:(B) MeOH with gradients of 85: 15 to 75:25 over 10 min, 75:25 to 70:30 over 2 min, and 70:30 over 3 min at a flow rate of 5 mL/min. Retention time: 10.96 min. The relevant fractions were collected and freeze-dried, and excess TEAA was removed by repeated freeze-drying with deionized water to yield compound 14 as a white powder. Finally, the triethylammonium ions were exchanged for Na+ by passing the pure compound 14 through a Sephadex-CM C-25 Na+-form column. Compound 14 was obtained at a 18% yield (9 mg) after HPLC separation. Retention time on a semi-preparative column: 14.75 min. Ή NMR (D2O; 600 MHz): δ 8.39 (s; H-8; 1H), 6.09 (d; J= 4.80 Hz; H-l'; 1H), 4.83 (m; H-2'; 1H), 4.78 (Η-3' signal is hidden by the water signal), 4.61 (m; H-4'; 1H), 4.38 (m; H-5'; H-5"; 2H), and 2.53 (s; CH3; 3H) ppm. 31P NMR (D2O; 243 MHz): δ 10.88 (d; J = 15.79 Hz) and 8.70 (d; J = 15.79 Hz) ppm. MS-ES m/z: 538 (Μ'). TLC (NH4OH:H2O:isopropanol 2:8: 1 1), R/= 0.26. Purity data obtained on an analytical column: retention time: 7.38 min (100% purity) using Solvent System I with a gradient from 85: 15 to 50:50 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 4.38 min (99.9% purity) using Solvent System II with a gradient from 85: 15 to 50:50 A:B over 10 min at a flow rate of 1 ml/min.
Evaluation of the stability of compounds 5-14 in human blood serum
The assay mixture containing 40 mM nucleotide derivative in deionized water (4.5 μΐ), human blood serum (180 μΐ) and RPMI-1640 medium (540 μΐ), was incubated at 37°C for 0-24 h and samples were removed at 0.5-12 h intervals. Each sample was then heated to 80°C for 30 min, treated with CM Sephadex (1-2 mg), stirred for 2 h, centrifuged for 6 min (13,000 rpm, 17,000 g) and then extracted with chloroform (2x500 μΐ). The aqueous layer was freeze-dried. Each resulting residue was applied to an activated starta™ X- AW weak anion exchange cartridge and then freeze-dried. The residue was purified using HPLC with a Gemini analytical column (5u, C-18, 1 10A; 150x4.60 mm) and gradient elution with Solvent System II at 100:0 A:B over 10 min for compound 5; 100:0 to 70:30 A:B over 3 min for compound 6; 90: 10 to 80:20 A:B over 10 min then 80:20 to 60:40 A:B over 5 min for compound 7; 85: 15 A:B over 10 min for compound 8; 90: 10 to 50:50 A:B over 18 min for compound 9; 80:20 to 30:70 A:B over 10 min for compound 14; and 80:20 to 30:70 A:B over 10 min for compound 13; or gradient elution with Solvent System I at 90: 10 to 60:40 A:B over 12 min for compounds 10A and 10B; 90: 10 A:B over 15 min for compounds 11 A and 11B; and 80:20 A:B over 15 min for compounds 12A and 12B at a flow rate of 1 ml/min. The hydrolysis rates for the nucleotide analogues in human blood serum were determined by measuring the change in the integration of the respective HPLC peaks with time. Evaluation of the stability of compounds 5-14 with alkaline phosphatase
Enzyme activity was determined by the release of p-nitrophenol from p- nitrophenyl phosphate measured by a UV-VIS spectrophotometer at 405 nm (Brandenberger and Hanson, 1953). Relative enzyme activity and resistance of compounds 5-14 to enzymatic hydrolysis were determined at 37°C using a solution of 0.2 mg of analogue in 77.5 μΐ deionized water, 0.1 M Tris-HCl (pH 9.8) and 0.1 M MgCl2 with calf intestine alkaline phosphatase (Fermentas Inc., Glen Burnie, MD; 10 unit/μΐ; 1.25 μΐ; 12.5 u). After 3 h, the reaction was stopped by incubation of the sample at 80°C for 15 min. Each sample was applied to an activated starta™ X-AW weak anion exchange cartridge and then freeze-dried. The residue was purified by HPLC with a Gemini analytical column (5u C-18 1 10A; 150x4.60 mm) using the gradient elution system described for the hydrolysis of the compounds in human blood serum at a flow rate of 1 ml/min. The hydrolysis rates for compounds 5-14 with alkaline phosphatase were determined by measuring the change in the integration of the respective HPLC peaks with time.
NTPDasel, 2, 3 and 8 (EC 3.6.1.5) assays
Activity was measured as previously described ( ukulski et al, 2005) at 37°C in 0.2 ml of Tris-Ringers buffer: (in mM) 120 NaCl, 5 C1, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 5 glucose, 80 Tris, pH 7.4; (Sigma-Aldrich, Oakville, ON, Canada). Ectonucleotidases were produced by transiently transfecting COS-7 cells in 10 cm plates by use of Lipofectamine (Invitrogen), as previously described (Kukulski et al, 2005). For the preparation of protein extracts, transfected cells were washed three times with Tris-saline buffer at 4°C, collected by scraping in the harvesting buffer (in 95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 45 mM Tris at pH 7.5), and washed twice by 300 g centriiugation for 10 min at 4°C. Cells were resuspended in the harvesting buffer containing 10 mg/ml aprotinin and sonicated. Nucleus and cellular debris were discarded by centrifugation at 300 g for 10 min at 4°C and the supernatant (crude protein extract) was aliquoted and stored at -80°C until used for activity assays. Protein concentration was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as a standard (Bradford, 1976). NTPDase protein extracts, 1/106 of final volume diluted accordingly to its specific activity, were added to the reaction mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by addition of ATP (Sigma-Aldrich, Oakville, ON, Canada) or compounds 8-14 at a final concentration of 100 μΜ and the reaction was stopped after 20 min with 50 μΐ of malachite green reagent (Sigma-Aldrich, Oakville, ON, Canada). The released inorganic phosphate (Pi) was measured at 630 nm according to Baykov et al. (1988). The activity obtained with protein extracts from untransfected cells was subtracted from the activity obtained with extracts from NTPDase-transfected cells. The activity of untransfected cell extracts never exceeded 5% of the activity of extracts from NTPDase-transfected cells.
NPP1 and 3 (EC 3.1.4.1; EC 3.6.1.9) assays
Evaluation of the activity of human NPP1 and NPP3 with ATP (Sigma- Aldrich, Oakville, ON, Canada) and compounds 8-14 was performed, as previously described (Levesque et al, 2007) with some modifications. Reactions were carried out at 37°C in 0.2 ml of the following mixture: (in mM) 1 CaCl2, 140 NaCl, 5 C1, and 50 Tris, pH 8.5; (Sigma-Aldrich, Oakville, ON, Canada). Human NPP1 or NPP3 extract was added to the reaction mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by addition of ATP or compounds 8-4 at a final concentration of 100 μΜ. The reaction was stopped after 20 min by transferring a 0.1 ml aliquot of the reaction mixture to 0.125 ml ice-cold 1 M perchloric acid (Fisher Scientific, Ottawa, ON, Canada). The samples were centrifuged for 5 min at 13,000xg. Supernatants were neutralized with 1 M KOH (Fisher Scientific, Ottawa, ON, Canada) at 4°C and centrifuged for 5 min at 13,000xg. An aliquot of 20 ml was separated by reverse-phase HPLC to evaluate the degradation of ATP and compounds 8-14 levels using a SUPELCOSIL™ LC-18-T column (15 cm x 4.6 mm; 3 mm Supelco; Bellefonte, Pennsylvania, USA) with a mobile phase composed of 25 mM TBA, 5 mM EDTA, 100 mM KH2PO4/K2HPO4, pH 7.0 and 2% methanol at a flow rate of 1 ml/min. Evaluation of the chemical stability of compounds 8-14
A nucleotide analogue (1.5 mg) was dissolved in 0.2 M HC1/KC1 buffer (0.8 ml) and the final pH was adjusted to 1.4 using 0.2 M HC1. Reactions continued at 37°C for 1 to 31 days with samples taken at 1-24 h intervals. The stabilities of the compounds were evaluated by HPLC to monitor degradation products using a Gemini analytical column (5u C-18 110A; 150x4.60 mm) and the gradient elution system described for the hydrolysis of analogues in human blood serum at a flow rate of 1 ml/min (see above). The hydrolysis rates of compounds 8-14 at pH 1.4 and 37°C were determined by measuring the change in the integration of the respective HPLC peaks with time.
Intracellular calcium measurements
Human 1321N1 astrocytoma cells stably expressing the turkey P2Yi receptor were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 500 μg/ml Geneticin (G-418; Life Technologies, Inc). Changes in the intracellular free calcium concentration, [Ca2+] were detected by dual-excitation spectrofluorometric analysis of suspensions from cells loaded with fura-2, as previously described (Grynkiewicz et al, 1985; Garrad et al, 1998). Cells were treated with the indicated nucleotide analogue at 37°C in 10 mM Hepes-buffered saline (pH 7.4) containing 1 mM CaCl2 and 1 mM MgCl2 and the maximal increase in [Ca ] was determined at different analogue concentrations to calculate the EC50. Concentration-response data were analyzed with the Prism curve-fitting program (GraphPAD Software, San Diego, CA). Data were obtained from three experiments performed in triplicate.
Animals
Twenty-four male New Zealand white rabbits (2.5±3.0 kg) were kept in individual cages with free access to food and water on controlled 12 h/12 h light/dark cycles. All the protocols used adhere to the The Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmology and Vision Research and also are in accordance with the European Communities Council Directive (86/609/EEC). Intraocular pressure measurements
Intraocular pressure (IOP) was measured by means of a TonoVET rebound tonometer supplied by Tiolat Oy (Helsinki, Finland). The application of this tonometer to animals does not require the use of any anaesthetic. For single dose experiments, different analogues were applied unilaterally to the cornea at a concentration of 100 μΜ and a fixed volume of 10 μΐ. The contralateral eye received the same volume of saline solution (0.9% NaCl, vehicle). Two IOP measurements were taken before any analogue was instilled. Experiments were performed following a blinded design where no visible indication was given to the experimenter as to the nature of the applied solution. IOP was followed up to 8 h to study the time course of the effect. Some of the analogues were assayed over a range of doses from 1 nM to 100 μΜ to generate dose-response curves. For these experiments, IOP was measured as the maximal response obtained with each dose of the analogue. In all experiments, on any given day, only a single dose was tested on a single animal, which was washed out at least 2 days between doses. The commercial hypotensive agents Xalatan® (latanoprost; 0.005%), Trusopt® (dorzolamide chlorhydrate; 2%) and Timolol® (timolol maleate; 0.5%) were assayed by applying a volume of 40 μ\.
Statistical analysis
All data are presented as the means ± s.e.m. Significant differences were determined by two-tailed Student's t-tests. The plotting and fitting of dose-response curves was carried out with Microcal Origin v.7.0 software (Microcal Software, U.S.A.).
Example 1. Synthesis of compounds 8-14
ATP analogues in which the 3,7-bridging oxygen is substituted by a methylene group are conventionally prepared via the activation of the 5 '-phosphate of nucleoside-5 '-monophosphate (NMP) to form a phosphoryl donor, followed by a reaction with methylene bisphosphonate salt (phosphoryl acceptor). Phosphoryl donors were prepared by activation of NMP with carbonyl diimidazole (CDI) (Padyukova et al, 1999), trifluoroacetic anhydride and N-methylimidazole (Mohamady and Jakeman, 2005) or dicyclohexylcarbodiimide (DCC) (Myers et al, 1963) followed by condensation with methylene bisphosphonic acid or its salt.
Recently, we reported a facile 3-step one pot synthesis of /3,7-methylene-2- MeS-ATP, 1, at a 35% overall yield (Eliahu et al, 2009). Here, we used this synthetic procedure to prepare the j3,Y-CF2/CCl2-2-MeS-ATP, 8 and 9. As depicted in Scheme 1 hereinafter, in order to ensure the selective phosphorylation of 2-MeS- adenosine at the 5'-OH, we used 2',3'-methoxy-methylidene-2-MeS-adenosine, 15, as the starting material. Compound 15 was first treated with POCl3 in trimethylphosphate (TMP), in the presence of Proton Sponge® at 0°C for 3 h, to obtain intermediate 16, which was then treated with bis(tributylammonium) dihalogen methylene-diphosphonate and tributylamine at 0°C for 1.5 h, providing the cyclic intermediates 17 and 18. Hydrolysis of intermediates 17 and 18 in 0.5 M TEAB and deprotection of the methoxymethylidene groups generated 5,y-CF2-2- MeS-ATP, 8, at a 46% overall yield, and /3,7-CCl2-2-MeS-ATP, 9, at a 10% overall yield, respectively. The formation of compounds 8 and 9 was confirmed by the presence of three typical signals in 31P NMR, as previously described (Cusack et al, 1987): 3.24 (m, P7), -4.89 (m, P^), and -10.61 (d, Pa) ppm for compound 8, and 8.15 (d, Ργ), 1.04 (dd, P0), and -10.22 (d, Pa) ppm for compound 9.
As depicted in Scheme 2, the preparation of a-borano-/3,7-CF2-2-MeS-ATP,
10, c-borano-]S,7-CCl2-ATP, 11, and a-borano- 3,7-CCl2-2-MeS-ATP, 12, required the protection of the 2' and 3' hydroxyl groups in the starting materials 15 and 21 by methoxy-methylidene groups, which remained throughout the entire synthesis until removal in the last step. In the first synthetic step, we used PC13, which was rapidly and completely consumed in less than 30 min to form intermediates 22 and 23. Next, dihalogen-bisphosphonate salt was added and the reactions progressed for 50 min at 0°C, followed by addition of BH3-SMe2 at 0°C and stirring for 1 h at RT to obtain the cyclic intermediates 27-29. Finally, hydrolysis of the cyclic intermediates in 0.5 M TEAB produced compounds 30-32, which were then deprotected to generate compounds 10, 11 and 12 at 21%, 18% and 7% overall yields, respectively, after LC. The identity and purity of compounds 10-12 were established by Ή and 31P NMR, ESI or MALDI negative mass spectrometry, and HPLC using two solvent systems. 31P NMR spectra of compounds 10-12 showed a typical Pa signal as a multiplet at about 80 ppm. Ή NMR spectra showed borane hydrogen atoms as a very broad signal at about 0.4 ppm. Due to the chiral center at Pa, each analogue was obtained as a pair of diastereoisomers in a 1 : 1 ratio. In both the Ή and 3 ,P NMR spectra, there was a slight difference between the chemical shifts for the two diastereoisomers of each analogue. These isomers were well separated by reverse-phase HPLC with about a 1-2 min difference in their retention times with the A isomer eluting before the B isomer.
2-MeS-ADP, 7, is a selective and highly potent P2YiR agonist. Yet, this agonist suffers from low chemical and enzymatic stability (Ravi et al, 2002). Therefore, we replaced the α,/3-bridging oxygen with a dichloro- or difluoromethylene group in an attempt to generate potent P2Y]R agonists with increased chemical and metabolic stabilities, i.e., compounds 13 and 14. The αβ- dihalomethylene-2-MeS-ADP compounds 13 and 14 were prepared, as previously reported (Davisson et al., 1987) and depicted in Scheme 3. Specifically, the 5'-OH of the 2 ',3 '-protected 2-MeS-adenosine analogues 15 and 33 were activated with tosyl chloride to obtain intermediates 34 and 35, which were then coupled with tris(tetra-n-butylammonium) dihalogen diphosphonate salt to provide intermediates 36 and 37. Finally, removal of the protecting group provided the β- dihalomethylene-2-MeS-ADP derivatives, 13 and 14, at 34% and 18% overall yields, respectively, after LC/MPLC. The relatively high yield of compound 13 may be due to the higher nucleophility of the difluoro diphosphonate salt, as compared to compound 14. In addition to compound 13, a small amount (4%) of the dinucleotide, analogue 38, was isolated, due to coupling of analogue 36 with analogue 34. Interestingly, the corresponding dichloro analogue was not detected under the identical reaction conditions. Example 2. Hydrolytic stability of compounds 8-14
In order to explore the potential of compounds 8-14 as drug candidates, we first evaluated their hydrolytic stability. Previously, we investigated the effect of a β,γ-methylene group on the hydrolytic stability of compounds 1-3 at acidic pH, which mimics the acidity of gastric juice, i.e., pH 1.4 at 37°C (Eliahu et al, 2009). 2-MeS-J3,' ¾-ATP, 1, a-B,fty-CH2-ATP, 2 and 2-MeS-a-B,ftT-CH2-ATP, 3, were found to be highly stable at pH 1.4 with t]/2 values of 65, 19, and 14.5 h, respectively. Although we intend to administer these analogues topically, a higher stability of compounds 8-14 at acidic pH, as compared to compounds 1-3, may suggest a wider range of therapeutic applications.
The hydrolysis of 2-MeS- 3,7-CF2/CCl2-ATP, 9, at pH 1.4 and 37°C was monitored by HPLC over 216 h (Fig. 1A) and the hydrolysis rate was determined by integration of the HPLC peaks with time to fit a pseudo first-order exponential decay rate equation (Fig. IB), yielding a half-life of -65 h. Compound 9 was degraded to 2-MeS-AMP and 2-MeS-adenosine.
The hydrolysis of 2-MeS-/3,7-CF2-ATP, 10 (B isomer) was similarly determined, yielding a half-life of ~8 h (Fig. 1C). The half-lives of compounds 8, 11 (isomer A) and 12 (isomer B) were 25, 6.7 and 0.7 h, respectively (data not shown). 2-MeS-a, 3-dichloromeyhylene-ADP, 14, was highly stable at pH 1.4, with only 15% hydrolysis of the starting material after 6 days, and 2-MeS-a,]8-difluoro methylene-ADP, 13, was highly stable with 61% of the starting material remaining after 30 days. The rest of the nucleotide was degraded to 2-MeS-adenosine.
Example 3. Resistance of compounds 8-14 to degradation by alkaline
phosphatase
Alkaline phosphatase (AP) is a hydrolase that removes phosphate groups from nucleotides, thereby regulating extracellular nucleotide concentrations in vivo. AP usually catalyzes the hydrolysis of phosphomonoesters yielding Ps and the corresponding alcohol (Hull et al., 1976). In this study, we evaluated the effect of cg8//3, γ-dihalo-methylene groups in the compounds 8-14 on the resistance to hydrolysis by alkaline phosphatase, as compared to ATP, ADP and compounds 1-3. Results indicated that AP degraded 96% of ATP after 3 h (ti/2=1.4 h). Likewise, ADP was completely degraded by AP after 3 h, whereas 2-MeS-ADP was 87% degraded after 3 h. Yet, compounds 8-14 were completely resistant to hydrolysis by AP under the same conditions, i.e., 0.2 mg analogue/77.5 μΐ in the presence of 12.5 units of AP (data not shown).
Example 4. Resistance of compounds 8-14 to hydrolysis in human blood serum
Nucleotides and their analogues undergo dephosphorylation by enzymes in physiological systems (Schetinger et al, 2007; Terkeltaub, 2006). Blood serum contains such enzymes and, therefore, provides a good model system for assessing the metabolic stability of extracellular nucleotides. Previous studies have used human blood serum (Arzumanov et al, 1996) to demonstrate the metabolic stability of phosphonate modified nucleotide analogues (Boyle et al, 2005; Wang et al, 2004). Similarly, we have shown with human blood serum that the half-lives of /3,γ- CH2-ATP and a-B, 3,Y-CH2-ATP, 1-3, were increased by 3.5-20 fold, as compared to ATP (Eliahu et al, 2009).
In this study, we investigated the effect of incorporation of a β,γ- dihalomethylene group in compounds 8-14 on the metabolic stability of these compounds in human blood serum at 37°C.
As compared to ATP, which is hydrolyzed to ADP and AMP with a half-life of 4.9 h (Fig. 2A), compound 8 was hydrolyzed to the corresponding nucleoside 5'- monophosphate with a half-life of 12.4 h, whereas compound 9 was only 15% degraded over 24 h (Fig. 2B). Furthermore, replacement of the Pa non-bridging oxygen in compounds 10-12 with a BH3 group endowed complete resistance to hydrolysis in human blood serum over 24 h (data not shown). Similarly, replacement of the Pa bridging oxygen in 2-MeS-ADP with CF2/CC12 produced compounds 13 and 14 that were completely resistant to hydrolysis in human blood serum over 24 h, as compared to ADP (t1 2=1.4 h) or 2-MeS-ADP (t, 2=23.5 h) (data not shown). Example 5. Compounds 8-14 are substrates for ecto-5'-nucIeotidases
NTPDasel, 2, 3 and 8 as well as NPP1 and 3 are the principal enzymes that metabolize extracellular nucleotides. As shown in Table 1 hereinbelow, in comparison to ATP, compounds 8-14 were barely hydrolyzed by NTPDases 1-3 and 8 (<5% over 24 h at 37°C) or NPP1 and 3 (<10% over 24 h at 37°C).
Table 1: Relative hydrolysis [%] of compounds 8-14 by human ecto-nucleotidases*
Figure imgf000042_0001
* The ATP and ADP analogues 8-14 were all used as substrates of the ectonucleotidases identified on the left column at the concentration of 100 μΜ. The activity with 100 μΜ ATP was set as 100% which were: 403±40; 1006±60; 533±42; 229±20 [nmol Pi min" '-mg protein"'] for NTPDasel, 2, 3 and 8, respectively. The 100% of the activity with ATP as substrate for NPP1 and 3 was 619 and 245 [nmol nucL min^ mg protein"1], respectively. ND = not detectable.
Example 6. Activities of compounds 8-14 at the P2Yi receptor
Earlier, we reported that 2-MeS-/3,7-methylene-ATP, 1, is a potent and selective P2Yj receptor (P2Y]R) agonist (Eliahu et al, 2009). In this study, we examined whether a /5,7-dihalomethylene substitution in compounds 8-14 would enhance agonist potency at the P2YiR due to the reduced pKa of the terminal phosphonate (~7), as compared to the pKa of 8.4 (Blackburn et al, 1984) for the corresponding methylene derivatives. Since compounds 8-14 should be 90% ionized under physiological conditions (vs. 9% ionization in 1), we expected that negatively charged phosphonates in compounds 8-14 would increase binding to the P2Y]R. Accordingly, the activities of compounds 8-14 were determined at the P2YiR heterologously expressed in human 1321N1 astrocytoma cells that are devoid of endogenous P2Y receptors (Parr et al, 1994). P2YiR activities were evaluated by monitoring increases in [Ca ], induced by the analogues, as compared to 2-MeS-ADP, 7, (EC50=0.0025 μΜ at the P2Y,R).
Surprisingly, and as shown in Table 2, compounds 8-14 were found to be weaker agonists of the P2Y,R than 2-MeS-ADP, 7, with EC50 values of 0.57 to 9.54 μΜ. Replacement of the non-bridging oxygen with BH3 in 2-MeS-a-borano-/3,7- dichloromethylene-ATP, 12A (A isomer), produced the most potent P2Y]R agonist in this series of analogues (EC50=0.57 μΜ), whereas the B isomer, 12B, was about 2-fold less potent (EC50=1.20 μΜ). Compounds 11A and 11B that lack a 2-MeS group were inactive at the P2YiR.
Replacement of the non-bridging oxygen in 2-MeS-j3,7-difluoromethylene-
ATP, 8, with BH3 to generate compound 10B resulted in a 3-fold reduction in P2YiR activity (EC5o=0.76 μΜ for compound 8 vs. EC50=2.13 μΜ for compound 10B), whereas the A isomer (compound 10A) had no activity. Although the α,β- dihalomethylene substitution in compounds 13 and 14 decreased their rate of degradation by ecto-nucleotidases as shown in Table 1, as compared to 2-MeS- ADP, compounds 13 and 14 were weak P2Y[R agonists with EC50 values of 0.98 and 3.1 μΜ, respectively. None of the compounds tested antagonized the effect of equimolar concentrations of 2-MeS-ADP on P2Y,R activation in 1321N1 cell transfectants (data not shown). Table 2: Activity of compounds 7-14 at the P2Y,R, i.e., EC50 (μΜ) values for
94- analogue-induced increases in [Ca ]
Figure imgf000043_0001
Example 7. Effect of nucleotide analogues on intraocular pressure
Nucleotides are present in the aqueous humour (Pintor et ai, 2003), although their action has not been fully elucidated due to the multiple P2 receptor subtypes identified in intraocular tissues that are bathed by the aqueous humour (Pintor et al, 2004a). Studies suggest that intraocular pressure (IOP) is regulated by G protein- coupled P2Y] receptors in trabecular meshwork cells that control the evacuation of the aqueous humour (Soto et al, 2005), and ligand-gated ion channel P2X2 receptors located on parasympathetic nerve terminals innervating the cilliary bodies (Markovskaya et al, 2008). P2X receptor activation potentiates the release of acetylcholine, which induces contraction of cilliary muscle to open the trabecular meshwork to reduce IOP (Pintor and Peral, 2001). In contrast, activation of G protein-coupled P2Y2 receptors increases IOP (Pintor and Peral, 2001). In the current study, we examined the effect of compounds 8-14, as compared to 2-MeS- /3,7-methylene-ATP, 1, on reduction of IOP in normal tense rats to identify novel and potent candidates for the treatment of ocular hypertension (Pintor and Peral, 2001).
As shown in Figs. 3A-3F, compounds 8 and 11A increased IOP (Fig. 3 A), whereas compounds 1, 9, 11B, 12A/B, 13 and 14 reduced IOP (Figs. 3A-C). Compounds 1 and 9 reduced IOP by -32%, and were more effective than Ap4A and Ap4 (Pintor et al, 2003; Pintor et al, 2004). Based on concentration response curves, compounds 1, 9, 13 and 14 exhibited the strongest hypotensive effects with pD2 values of 7.02±0.70, 4.52±0.71 , 5.1+0.3 and 4.5±0.4, respectively. The rank order of potency for reduction of IOP was 9 > 13 > 1 > 14 with EC5o values of 95.5 nM, and 7.9, 30.2 and 31.6 μΜ, respectively (Fig. 3D). In comparison to the other agents shown to reduce IOP, compounds 1 and 9 were more effective than the prostglandin analogue Xalatan, the carbonic anhydrase inhibitor Trusopt, and equally effective as the beta-blocker Timolol (Fig. 3E). The duration effect of the nucleotides as compared with the commercial compounds was sufficiently long. The reduction of duration of IOP induced by compounds 1 and 9 was -3.5 and 4.5 h, comparable to Xalatan with an effective duration of 5.5 h (Fig. 3F). APPENDIX
Figure imgf000045_0001
Figure imgf000045_0002
Scheme 1: S nthesis of compounds 8 and 9
Figure imgf000046_0001
9 X=C1
Reaction conditions
Compound 8: a) trimethylphosphate, POCl3, Proton Sponge, 0°C, 3 h; b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu3N, 0°C, 1.5 h; c) 0.5 M TEAB, pH 7, RT, 1 h; and d) 1) 18% HCl, pH 2.3, RT, 3 h; and 2) 24% NH4OH, pH 9, RT, 45 min.
Compound 9: a) trimethylphosphate, POCl3, proton sponge, 0°C, 1 h; b) 1 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu3N, 0°C, 40 min, and then RT, 5 min; c) 0.5 M TEAB, pH 7, RT, 1 h. and d) as described for compound 8. Scheme 2: Synthesis of compounds 10-12
Figure imgf000047_0001
24 R = SMe; X = Cl 27 R = SMe; X - CI
25 R = H; X = CI 28 R = H; X = CI
26 R = SMe; X = F 29 R = SMe; X = F
Figure imgf000047_0002
30 R = SMe; X = CI 10 R = SMe; X = F
31 R - H; X = C1 11 R = H; X = C1
32 R = SMe; X = F 12 R = SMe; X = CI
Reaction conditions
Compound 10: a) trimethylphosphate, PC13, proton sponge, 0°C, 30 min; b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu3N, 0°C, 1 h; c) 2 M BH3-SMe in THF, 0°C, 5 min, and then RT, 60 min; d) 0.5 M TEAB, pH 7, RT, 1 h; and e) 1) 18% HC1, pH 2.3, RT, 3 h; and 2) 24% NH4OH, pH 9, RT, 45 min.
Compound 11 : a) trimethylphosphate, PC13, proton sponge, 0°C, 0.5 h; b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu3N, 0°C, 25 min; c) 2 M BHySMe in THF, 0°, 5 min then RT, 25 min; d) 0.5 M TEAB, pH 7, RT, 45 min; and e) 1) 18% HC1, pH 2.3, RT, 3 h; and 2) 24% NH4OH, pH 9, RT, 45 min.
Compound 12: a) trimethylphosphate, PC13, proton sponge, 0°C, 45 min; b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu3N, 0°C, 50 min; c) 2 M BHySMe in THF, 0°C, 5 min then RT, 60 min; d) and e) as described for 10. Scheme 3: Synthesis of compounds 13-14
Figure imgf000048_0001
15 R-R = CH(OMe) 34 R-R = CH(OMe)
33 R-R = C(CH3)2 35 R-R = C(CH3)2
Figure imgf000048_0002
36 R-R = CH(OMe); X = F 13 X = F 37 R-R = C(CH3)2; X = CI 14 X = Cl
Figure imgf000048_0003
Reaction conditions
Compound 15: a) CH2C12, DMAP, TsCl, RT, 12 h; b) tetra-(n-butylammonium)difluoro methylenediphosphonate in dry DMF, RT, 72 h; c) 1) 18% HCl, pH 2.3, RT, 3 h; and 2) 24% NH4OH, pH 9, RT, 45 min.
Compound 33: a) CH2C12) DMAP, TsCl, RT, 12 h; b) tetra-(n-butylammonium)dichloro methylenediphosphonate in dry DMF, RT, 72 h; and c) TFA, Argon bubbling, RT, 10 min. REFERENCES
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Claims

An ophthalmic composition comprising a pharmaceutically acceptable and a compound of the general formula I:
Figure imgf000056_0001
or a diastereoisomer or mixture of diastereoisomers thereof,
wherein
Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -NO2, -OR4, -SR4, -NR4R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O, or S, wherein the additional nitrogen is optionally substituted by alkyl;
R2 and R3 each independently is H or hydrocarbyl;
Y each independently is H, -OH, or -NH2;
Z\, Z2 and Z3 each independently is -O", -S", or -BH3 ";
Wi and W2 each independently is -O-, -NH-, or -C(XiX2)-, wherein X\ and X2 each independently is H or halogen, provided that at least one of W] 5 if present, and W2 is not -O-;
n is 0 or 1 ;
m is 3 or 4; and
B+ represents a pharmaceutically acceptable cation.
2. The composition of claim 1 , wherein R] is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S,
wherein said hydrocarbyl each independently is (Ci-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6-Ci4)aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S.
3. The composition of claim 2, wherein said hydrocarbyl is (C]-C6)alkyl, preferably (C C4)alkyl, more preferably methyl or ethyl; (C2-C6)alkenyl, preferably (C2-C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C4)alkynyl; or (C6-Ci0)aryl, preferably phenyl.
4. The composition of claim 1, wherein R2 and R3 each independently is H or hydrocarbyl selected from (CrC8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6- CH)aryl.
5. The composition of claim 4, wherein said hydrocarbyl each independently is (Ci-C6)alkyl, preferably (Ci-C4)alkyl, more preferably methyl or ethyl; (C2- C6)alkenyl, preferably (C2-C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C )alkynyl; or (C6-C10)aryl, preferably phenyl.
6. The composition of claim 1, wherein Y each independently is -OH.
7. The composition of claim 1, wherein R\ is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; R2 and R3 each independently is H or hydrocarbyl; and Y each independently is -OH,
wherein said hydrocarbyl each independently is (C]-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or (C6-Ci4)aryl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S.
8. The composition of claim 7, wherein said hydrocarbyl is (Ci-C6)alkyl, preferably (C]-C4)alkyl, more preferably methyl or ethyl; (C2-C6)alkenyl, preferably (C2-C4)alkenyl; (C2-C6)alkynyl, preferably (C2-C4)alkynyl; or (C6-C10)aryl, preferably phenyl.
9. The composition of claim 8, wherein R) is H, -O-hydrocarbyl, or -S- hydrocarbyl; R2 and R3 each independently is H or hydrocarbyl; Y each independently is -OH; and said hydrocarbyl each independently is methyl or ethyl.
10. The composition of claim 9, wherein Ri is H or -S-methyl; R2 and R3 are H; and Y each independently is -OH.
11. The composition of any one of claims 1 to 10, wherein Wb if present, is -O-; W2 is -C(X,X2)- or -NH-, preferably -C(XiX2)-; and Xi and X2 each independently is H or a halogen selected from F, CI or Br, preferably CI.
12. The composition of claim 1 1, wherein Z2, if present, and Z3 each independently is -O"; and Zi is -O" or -BH3 ".
13. The composition of claim 12, wherein n is 1 ; Y each independently is -OH; R2 and R3 are H; and
(i) Ri is -SCH3; Z,, Z2 and Z3 are -O"; W, is -O-; and W2 is -CH2- (herein identified compound 1);
(ii) R, is -SCH3; Z,, Z2 and Z3 are O"; W[ is -O-; and W2 is -CC12- (herein identified compound 9);
(iii) R, is H; Z, is -BH3 "; Z2 and Z3 are -O"; W, is -O-; and W2 is -CC12-, characterized by being the isomer with a retention time (Rt) of 10.87 min when separated from a mixture of diastereoisomers using a semi- preparative reverse-phase Gemini 5u column (C-18 11 OA, 250 10 mm, 5 micron), and isocratic elution [100 mM triethylammonium acetate, pH 7: MeOH, 91 :9] with flow rate of 5 ml/min (herein identified compound 11B); or (iv) Ri is -SCH3; Z, is -BH3 "; Z2 and Z3 are -O"; Wi is -O-; and W2 is -CC12- (herein identified compound 12);
14. The composition of claim 12, wherein n is 0; Y each independently is OH; Ri is -SCH3; R2 and R3 are H; Zx and Z3 are -O"; and W2 is -CF2- or -CC12- (herein identified compounds 13 and 14, respectively).
15. The composition of claim 13, comprising compound 1 or 9, preferably 9.
16. The composition of any one of claims 1 to 15, formulated as ophthalmic drops, emulsion, suspension, gel, ointment, or a membranous ocular eye patch.
17. The composition of claim 16, for reducing intraocular pressure.
18. The composition of claim 17, for prevention or treatment of intraocular hypertension and/or glaucoma.
19. The composition of claim 18, wherein the glaucoma is primary open angle glaucoma, normal pressure glaucoma, acute angle closure glaucoma, absolute glaucoma chronic glaucoma, congenital glaucoma, juvenile glaucoma, narrow angle glaucoma, chronic open angle glaucoma, or simplex glaucoma.
20. A compound of the general formula I, or a diastereoisomer or mixture of diastereoisomers thereof, for use in reducing intraocular pressure.
21. Use of a compound of the general formula I, or a diastereoisomer or mixture of diastereoisomers thereof, for the preparation of an ophthalmic composition.
22. A method for reducing intraocular pressure in an individual in need thereof comprising administering to said individual a therapeutically effective amount of a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof.
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