WO2011077435A1 - Compositions et procédés pour la réduction de la pression intraoculaire - Google Patents

Compositions et procédés pour la réduction de la pression intraoculaire Download PDF

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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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English (en)
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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/fr

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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 System
    • 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 System
    • 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

Abstract

La présente invention porte sur des compositions ophtalmiques comprenant des analogues de nucléoside diphosphate ou triphosphate non hydrolysables dans lesquels l'oxygène de pontage α,β ou β,γ, respectivement, est remplacé, par exemple, par un groupe méthylène ou dihalogénométhylène, tels que le 2MeS-adénosine-β,γ-CH2-5'-triphosphate et le 2MeS-adénosine-β,γ-CCl2-5'-triphosphate. Ces compositions sont utiles pour la réduction de la pression intraoculaire et donc pour le traitement de l'hypertension oculaire et/ou du glaucome.
PCT/IL2010/001078 2009-12-22 2010-12-22 Compositions et procédés pour la réduction de la pression intraoculaire WO2011077435A1 (fr)

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CN103788140A (zh) * 2014-01-26 2014-05-14 江西科技师范大学 核苷5′-β,γ亚甲基及双卤代亚甲基三磷酸合成方法
US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
US9370530B2 (en) 2010-01-11 2016-06-21 Inotek Pharmaceuticals Corporation Combination, kit and method of reducing intraocular pressure
US9522160B2 (en) 2013-03-15 2016-12-20 Inotek Pharmaceuticals Corporation Ophthalmic formulations
US9718853B2 (en) 2012-01-26 2017-08-01 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-YL)-3,4-dihydroxytetrahydrofuran-2-YL)] methyl nitrate and processes of preparation thereof
US9789131B1 (en) 2016-04-21 2017-10-17 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
WO2019092546A1 (fr) * 2017-11-10 2019-05-16 Olon S.P.A. Procédé efficace de préparation de cangrelor
US10765693B2 (en) 2018-09-26 2020-09-08 Astrocyte Pharmaceuticals, Inc. Polymorphic compounds and uses thereof
US11839615B2 (en) 2018-02-09 2023-12-12 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating addiction and related disorders

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US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
US9718853B2 (en) 2012-01-26 2017-08-01 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-YL)-3,4-dihydroxytetrahydrofuran-2-YL)] methyl nitrate and processes of preparation thereof
WO2013132489A1 (fr) * 2012-03-05 2013-09-12 Bar-Ilan University Analogues de nucléoside 5'-phosphorothioate et leurs utilisations
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