WO2009048558A1 - Bloqueurs de canaux maxi-k et procédés d'utilisation - Google Patents

Bloqueurs de canaux maxi-k et procédés d'utilisation Download PDF

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WO2009048558A1
WO2009048558A1 PCT/US2008/011556 US2008011556W WO2009048558A1 WO 2009048558 A1 WO2009048558 A1 WO 2009048558A1 US 2008011556 W US2008011556 W US 2008011556W WO 2009048558 A1 WO2009048558 A1 WO 2009048558A1
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alkyl
aryl
alkenyl
heterocyclyl
ene
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PCT/US2008/011556
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English (en)
Inventor
Ed Brnardic
James B. Doherty
Charles Ellwood
Martin Fillmore
Mike Malaska
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Merck & Co., Inc.
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Publication of WO2009048558A1 publication Critical patent/WO2009048558A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered

Definitions

  • Glaucoma is a degenerative disease of the eye wherein the intraocular pressure is too high to permit normal eye function. As a result, damage may occur to the optic nerve head and result in irreversible loss of visual function. If untreated, glaucoma may eventually lead to blindness. Ocular hypertension, i.e., the condition of elevated intraocular pressure without optic nerve head damage or characteristic glaucomatous visual field defects, is now believed by the majority of ophthalmologists to represent merely the earliest phase in the onset of glaucoma.
  • the present invention relates to novel indole diterpene alkaloids which are useful as potassium channel antagonists, particularly as Maxi-K channel antagonists. These alkaloids are useful in treating glaucoma and other associated conditions, and macular degeneration. More particularly, the present invention includes compounds of formula I and pharmaceutically acceptable salts thereof:
  • R is H, OPO3, COR2, OCOR2, -NHR2, OR2, CH(R2)2, OCNHR2, (CHR2) n NR2C(O)O(CH 2 )nR2, (C(Ri) 2 ) p NRiR 2 ; -NCOR 2 , (CH 2 ) n ORl, Ci- 6 alkyl, (CH2)nC6-10 aryl, (CH 2 ) n heterocyclyl, C2-6 alkenyl, (CH2) n C3-6 cycloalkyl, THF, - (CH2) n NR2(CH2)n J , -(CH2) n O) s R2-, or OCOC(R2)2N(R2)2, saicl alkyl, alkenyl, cfcloalkyi; aryl and heterocyclyl optionally substituted with 1 to 3 groups of R a ;
  • Ri and Ri a independently represent H, (CH2) n OCONH(CH2) n R, (CH2) n C00R, (CH2) n OR, COR, COO-C6-10aryl, COOalkylR, OCOC(R)2N(R)2, C0NR(CH2) n R, (CH2) n OCOalkylR, halogen, COalkyl, Ci.6alkyl, C2-6alkenyl, (CH2) n C3-6cycloalkyl, (CH2) n C(S)N(R)2, (CH 2 ) n C5-10 heterocyclyl, (CH 2 ) n C6-10 aryl, (-(CH 2 ) n O) s , (CH2)nC(O)(CH 2 ) n C(O)OR, (CH2) n OP(O)(OR)2, or -(CHR) n N(R2)2, said alkyl, alkenyl, aryl, and heterocycl
  • R2 is H, (CH2) n C00R, (CH2) n 0R, COR, NRCOR, C0NR(CH2) n R, halogen, (CH2) n COalkyl, Ci_6alkyl, C2-6alkenyl, (CH2) n C3-6cycloalkyl, (CH2) n C5-10 heterocyclyl, (CH2) n C6-10 aryl, (-(CH2)nO)s, or -CHRN(R)2, said alkyl, alkenyl, aryl, and heterocyclyl optionally substituted with 1 to 3 groups of R a ;
  • R3 is H, Ci-6 alkyl, -Si((R)2)R, (CH2) n C5-10 heterocyclyl, -P(O)(OR)2,or -PO3, said alkyl, and heterocyclyl optionally substituted with 1 to 3 groups of R a ;
  • R.5 is CO2R1, CH2OR, or halo;
  • Rl 3 is H, or CONR
  • R21 and R23 independently represent hydrogen, CN, COOR, CONR 1 R 2 , CONHR 1 , halogen, SO 2 R, C5-10 heterocyclyl, NO 2 , H, OR, COR, Ci-6 alkyl, C2-6 alkenyl, (CH2) n C5-l0 aryl, said alkyl, alkenyl, aryl, and heterocyclyl optionally substituted with 1 to 3 groups of R a ;
  • Ra is OH, Ci-6 alkyl, C3-6 cycloalkyl, (CH2) n C6-10 aryl, -O(CH2) n C6-10 aryl, CF3, halo, -SR, -O-, NO2, (CH 2 )n0Rl, C 2 -6 alkenyl, CN, N(Ri )2, COOR3, SO2R, -0P(0)(0R)2, -OPO3, or - OSi(R 2 )Ri;
  • the claimed compounds lack the tremorgenic liability of other indole diterpenes, yet retain excellent potency against the Maxi-K channel.
  • the present invention is directed to novel indole diterpenes of formula I described above.
  • the compounds of this invention block the Maxi-K channel and do not have a tremorgenic effect.
  • This invention is also directed to a method for treating ocular hypertension or glaucoma which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound having structural formula I.
  • An embodiment of this invention is realized when Ri is hydrogen, C 1-6 alkyl, or halogen, and all other variables are as originally described.
  • Another embodiment of this invention is realized when Ri is hydrogen and all other variables are as originally described.
  • Another embodiment of this invention is realized when Ri 3 is hydrogen and all other variables are as originally described.
  • Ri 4 is hydrogen, Cl -6 alkyl, or (C(O))pC3-6 cycloalkyl and all other variables are as originally described.
  • Still another embodiment of this invention is realized when R21 and R23 are independently hydrogen and halogen.
  • R21 is CN, halogen, COOR, C2-6 alkenyl, OR, or NO2.
  • Another embodiment of this invention is realized by the compound of Formula II:
  • Rl 0 a and Ri Q ⁇ together 0 or -N(Ri)COR2, and all other variables are as previously described.
  • Rl ⁇ a and RlO ⁇ together -O and Ri a is hydrogen.
  • Ri a , Ri 4, RlOa 5 and RlO ⁇ are as described herein.
  • Ri a , Rl 4, RlOa 5 and Ri ob are as described herein.
  • the compounds of the present invention may have asymmetric centers, chiral axes and chiral planes, and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. (See E.L. Eliel and S.H. Wilen Stereochemistry of Carbon Compounds (John Wiley and Sons, New York 1994), in particular pages 11 19-1190).
  • any variable e.g. aryl, heterocycle, Rl, R ⁇ etc.
  • its definition on each occurrence is independent at every other occurrence.
  • combinations of substituents/or variables are permissible only if such combinations result in stable compounds.
  • alkyl refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 10 carbon atoms unless otherwise defined. It may be straight, branched or cyclic. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopropyl, cyclopentyl, and cyclohexyl. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with "branched alkyl group”.
  • Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, unless otherwise defined, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings, which are fused. Examples of such cycloalkyl elements include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • Alkenyl is C2-C6 alkenyl.
  • Alkoxy refers to an alkyl group of indicated number of carbon atoms attached through an oxygen bridge, with the alkyl group optionally substituted as described herein.
  • Said groups are those groups of the designated length in either a straight or branched configuration and if two or more carbon atoms in length, they may include a double or a triple bond.
  • Exemplary of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy, allyloxy, propargyloxy, and the like.
  • Halogen (halo) refers to chlorine, fluorine, iodine or bromine.
  • Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl and the like.
  • An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms.
  • aryl groups are phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl and phenanthrenyl, preferably phenyl, naphthyl or phenanthrenyl.
  • Aryl groups may likewise be substituted as defined.
  • Preferred substituted aryls include phenyl and naphthyl.
  • heterocyclyl or heterocyclic represents a stable 3- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
  • a fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring.
  • heterocycle or heterocyclic includes heteroaryl moieties.
  • heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydropyrrolyl, 1,3-dioxolanyl, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholin
  • heterocycle is selected from 2-azepinonyl, benzimidazolyl, 2-diazapinonyl, dihydroimidazolyl, dihydropyrrolyl, imidazolyl, 2-imidazolidinonyl, indolyl, isoquinolinyl, morpholinyl, piperidyl, piperazinyl, pyridyl, pyrrolidinyl, 2-piperidinonyl, 2-pyrimidinonyl, 2-pyrollidinonyl, quinolinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, and thienyl.
  • heteroatom means O, S or N, selected on an independent basis.
  • heteroaryl refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two additional carbon atoms is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Examples of such heterocyclic, elementsinclude, but are.not.
  • This invention is also concerned with compositions and methods of treating ocular hypertension or glaucoma by administering to a patient in need thereof one of the compounds of formula I alone or in combination with one or more of the following active ingredients, in combination with a ⁇ -adrenergic blocking agent such as timolol, betaxolol, levobetaxolol, carteolol, and levobunolol; a parasympathomimetic agent such as epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine, carbonic anhydrase inhibitor such as dorzolamide, acetazolamide, metazolamide or brinzolamide, an EP4 agonist (such as those disclosed in WO 02/24647, WO 02/42268, EP 1114816, WO 01/46140, PCT Appln.
  • a ⁇ -adrenergic blocking agent such as timolol, betaxo
  • 4,690,931 particularly eliprodil and i?-eliprodil as set forth in WO 94/13275, including memantine; an agonist of 5-HT2 receptors as set forth in PCT/USOO/31247, particularly l-(2-aminopropyl)-3-methyl-l//-imdazol- 6-ol fumarate and 2-(3-chloro-6-methoxy-indazol-l-yl)-l-methyl-ethylamine or a mixture thereof.
  • hypotensive lipid (the carboxylic acid group on the ⁇ -chain link of the basic prostaglandin structure is replaced with electrochemically neutral substituents) is that in which the carboxylic acid group is replaced with a C ⁇ . ⁇ alkoxy group such as OCH3 (PGF2a 1- OCH3), or a hydroxy group (PGF2 a 1 -OH).
  • a C ⁇ . ⁇ alkoxy group such as OCH3 (PGF2a 1- OCH3), or a hydroxy group (PGF2 a 1 -OH).
  • Preferred potassium channel blockers are calcium activated potassium channel blockers. More preferred potassium channel blockers are high conductance, calcium activated potassium (Maxi-K) channel blockers. Maxi-K channels are a family of ion channels that are prevalent in neuronal, smooth muscle and epithelial tissues and which are gated by membrane potential and intracellular Ca2+.
  • the present invention is based upon the finding that Maxi-K channels, if blocked, inhibit aqueous humor production by inhibiting net solute and H2O efflux and therefore lower
  • this finding suggests ⁇ hatMaxi-K channel -blockers are.useful for treating other. ophthalmological dysfunctions such as macular edema and macular degeneration. It is known that lowering IOP promotes blood flow to the retina and optic nerve. Accordingly, the compounds of this invention are useful for treating macular edema and/or macular degeneration. It is believed that Maxi-K channel blockers which lower IOP are useful for providing a neuroprotective effect. They are also believed to be effective for increasing retinal and optic nerve head blood velocity and increasing retinal and optic nerve oxygen by lowering IOP, which when coupled together benefits optic nerve health. As a result, this invention further relates to a method for increasing retinal and optic nerve head blood velocity, increasing retinal and optic nerve oxygen tension as well as providing a neuroprotective effect or a combination thereof.
  • This invention is also concerned with the use of a compound of Formula I in the manufacture of a medicament for the treatment of ocular diseases such as ocular hypertension and/or glaucoma.
  • a number of marketed drugs function as potassium channel antagonists. The most important of these include the compounds Glyburide, Glipizide and Tolbutamide. These potassium channel antagonists are useful as antidiabetic agents.
  • the compounds of this invention may be combined with one or more of these compounds to treat diabetes. Potassium channel antagonists are also utilized as Class 3 antiarrhythmic agents and to treat acute infarctions in humans.
  • a number of naturally occuring toxins are known to block potassium channels including Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin, Kaliotoxin, Dendrotoxin(s), mast cell degranuating (MCD) peptide, and ⁇ -Bungarotoxin ( ⁇ - BTX).
  • the compounds of this invention may be combined with one or more of these compounds to treat arrhythmias.
  • Depression is related to a decrease in neurotransmitter release.
  • Current treatments of depression include blockers of neurotransmitter uptake, and inhibitors of enzymes involved in neurotransmitter degradation which act to prolong the lifetime of neurotransmitters.
  • Alzheimer's disease is also characterized by a diminished neurotransmitter release.
  • Alzheimer's disease cholinergic potentiators such as the anticholinesterase drugs (e.g., physostigmine (eserine), and Tacrine (tetrahydroaminocridine)); nootropics that affect neuron metabolism with little effect elsewhere (e.g., Piracetam, Oxiracetam; and those drugs that affect brain vasculature such as a mixture of ergoloid mesylates amd calcium channel blocking drugs including Nimodipine. Selegiline, a monoamine oxidase B inhibitor which increases brain dopamine and norepinephrine has reportedly caused mild improvement in some Alzheimer's patients.
  • the anticholinesterase drugs e.g., physostigmine (eserine), and Tacrine (tetrahydroaminocridine)
  • nootropics that affect neuron metabolism with little effect elsewhere
  • Piracetam e.g., Piracetam, Oxiracetam
  • Aluminum chelating agents have been of interest to .those who believcAlzheimer.'s disease is due to aluminum toxicity. Drugs that affect behavior, including neuroleptics, and anxiolytics have been employed. Anxiolytics, which are mild tranquilizers, are less effective than neuroleptics.
  • the present invention is related to novel compounds which are useful as potassium channel antagonists.
  • the compounds of this invention may be combined with anticholinesterase drugs such as physostigmine (eserine) and Tacrine (tetrahydroaminocridine), nootropics such as Piracetam, Oxiracetam, ergoloid mesylates, selective calcium channel blockers such as Nimodipine, or monoamine oxidase B inhibitors such as Selegiline, in the treatment of
  • anticholinesterase drugs such as physostigmine (eserine) and Tacrine (tetrahydroaminocridine)
  • nootropics such as Piracetam, Oxiracetam, ergoloid mesylates
  • selective calcium channel blockers such as Nimodipine
  • monoamine oxidase B inhibitors such as Selegiline
  • the compounds of this invention may also be combined with Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin, Kaliotoxin, Dendrotoxin(s), mast cell degranuating (MCD) peptide, ⁇ -Bungarotoxin ( ⁇ -BTX) or a combination thereof in treating arrhythmias.
  • the compounds of this invention may further be combined with Glyburide, Glipizide, Tolbutamide or a combination thereof to treat diabetes.
  • each of the claimed compounds are potassium channel antagonists and are thus useful in the described neurological disorders in which it is desirable to maintain the cell in a depolarized state to achieve maximal neurotransmitter release.
  • the compounds produced in the present invention are readily combined with suitable and known pharmaceutically acceptable excipients to produce compositions which may be administered to mammals, including humans, to achieve effective potassium channel blockage.
  • salts of the compounds of formula I will be pharmaceutically acceptable salts.
  • Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.
  • suitable “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, ⁇ iV'-dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, iV-ethylmorpholine, 7V-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine,- tromethamine.and.the like.-
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid and the like.
  • Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
  • composition is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
  • the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.
  • the Maxi-K channel blockers used can be administered in a therapeutically effective amount intravenously, subcutaneously, topically, transdermally, parenterally or any other method known to those skilled in the art.
  • Ophthalmic pharmaceutical compositions are preferably adapted for topical administration to the eye in the form of solutions, suspensions, ointments, creams or as a solid insert.
  • Ophthalmic formulations of this compound may contain from 0.01 ppm to 5% and especially 0.1 ppm to 1% of medicament. Higher dosages as, for example, about 10% or lower dosages can be employed provided the dose is effective in reducing intraocular pressure, treating glaucoma, increasing blood flow velocity or oxygen tension.
  • For a single dose from between 1 ng to 5000 ⁇ g, preferably 10 ng to 500 ⁇ g, and especially 100 ng to 200 ⁇ g of the compound can be applied to the human eye.
  • the pharmaceutical preparation which contains the compound may be conveniently admixed with a non-toxic pharmaceutical organic carrier, or with a non-toxic pharmaceutical inorganic carrier.
  • a non-toxic pharmaceutical organic carrier or with a non-toxic pharmaceutical inorganic carrier.
  • pharmaceutically acceptable carriers are, for example, water, mixtures of water and water-miscible solvents such as lower alkanols or aralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly, ethyl cellulose, ethyl oleate,_carboxyrnethykcellulose, polyvinylpyrrolidone, isopropyl myristate. and .other. conventionally employed acceptable carriers.
  • the pharmaceutical preparation may also contain non-toxic auxiliary substances such as emulsifying, preserving, wetting agents, bodying agents and the like, as for example, polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500, 4,000, 6,000 and 10,000, antibacterial components such as quaternary ammonium compounds, phenylmercuric salts known to have cold sterilizing properties and which are non- injurious in use, thimerosal, methyl and propyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredients such as sodium borate, sodium acetates, gluconate buffers, and other conventional ingredients such as sorbitan monolaurate, triethanolamine, oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodium sulfosuccinate, monothioglycerol, thiosorbitol, ethylenediamine tetracetic acid, and the like.
  • auxiliary substances such as e
  • suitable ophthalmic vehicles can be used as carrier media for the present purpose including conventional phosphate buffer vehicle systems, isotonic boric acid vehicles, isotonic sodium chloride vehicles, isotonic sodium borate vehicles and the like.
  • the pharmaceutical preparation may also be in the form of a microparticle formulation.
  • the pharmaceutical preparation may also be in the form of a solid insert. For example, one may use a solid water soluble polymer as the carrier for the medicament.
  • the polymer used to form the insert may be any water soluble non-toxic polymer, for example, cellulose derivatives such as methylcellulose, sodium carboxymethyl cellulose, (hydroxyloweralkyl cellulose), hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose; acrylates such as polyacrylic acid salts, ethylacrylates, polyactylamides; natural products such as gelatin, alginates, pectins, tragacanth, karaya, chondrus, agar, acacia; the starch derivatives such as starch acetate, hydroxymethyl starch ethers, hydroxypropyl starch, as well as other synthetic derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralized carbopol and xanthan gum, gellan gum, and mixtures of said polymer.
  • cellulose derivatives such as methylcellulose, sodium carboxymethyl
  • Suitable subjects for the administration of the formulation of the present invention include primates, man and other animals, particularly man and domesticated animals such as cats and dogs.
  • the pharmaceutical preparation may contain non- toxic auxiliary substances such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate buffers; and other conventional ingredients such as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraacetic acid, and the like.
  • the ophthalmic solution or suspension may be administered as often as necessary to maintain an acceptable IOP level in the eye. It is contemplated that, administration to the- . mammalian eye will be about once or twice daily.
  • the novel formulations of this invention may take the form of solutions, gels, ointments, suspensions or solid inserts, formulated so that a unit dosage comprises a therapeutically effective amount of the active component or some multiple thereof in the case of a combination therapy.
  • HPLC purification was performed by redissolving the residue in a small volume of DMSO and filtering through a 0.45 micron (nylon disc) syringe filter. The solution was then purified via reverse-phase preparatory HPLC purification system using a 50 mm Varian Dynamax HPLC 21.4 mm Microsorb Guard-8 C 8 column. The initial gradient of 40-80% MeOHiH 2 O was selected as appropriate for the target compound. This initial gradient was maintained for 0.5 minutes then ramped up to 100% MeOH:0% H 2 O over 5 minutes. 100% MeOH was maintained for 2 more minutes before it was re-equilibrated back to the initial starting gradient. Total run time was 8 minutes. The resulting fractions were analyzed, combined as appropriate, and then evaporated to provide purified material.
  • Proton magnetic resonance ( 1 H NMR) spectra were recorded on either a Varian INOVA 400 MHz ( 1 H) NMR spectrometer, Varian INOVA 500 MHz ( 1 H) NMR spectrometer, Bruker ARX 300 MHz ( 1 H) NMR spectrometer, Bruker DPX 400 MHz ( 1 H) NMR spectrometer, or a Bruker DRX 500 MHz ( 1 H) NMR spectrometer. All spectra were determined in the solvents indicated. Although chemical shifts are reported in parts per million (ppm) downfield of tetramethylsilane, they are referenced to the residual proton peak of the respective solvent peak for 1 H NMR.
  • ppm parts per million
  • LCMS spectra were obtained using a ThermoFinnigan AQA MS ESI instrument.
  • the samples were sent through a Phenomenex Aqua 5 micron Cj 8 125A 50 x 4.60 mm column.
  • the initial gradient was 55% MeOH: 1% CH 3 CN in H 2 O which was ramped up to 100% MeOH over 3 minutes. 100% MeOH was maintained for 2 minutes before it was re-equilibrated to the initial starting gradient.
  • the spray setting for the MS probe was at 350 uL/min with a cone voltage at 25 mV and a probe temperature at 45O 0 C.
  • reaction mixture was allowed to cool and was transferred to a 125-mL separatory funnel, taken up in 30-40 mL of ethyl acetate, and washed x 2 with a 1 :1 brine/water mixture and once with a saturated aqueous KF solution.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo. Purification was performed by HPLC. LCMS m/e 530.4 (M+Na). (II) isolated.
  • the reaction was filtered through a 0.45 micron (nylon disc) syringe filter.
  • the sample was then washed one time with a 10% HCl solution, one time with a saturated sodium bicarbonate solution and concentrated under nitrogen.
  • the sample was then taken up in DMSO and purified via HPLC using a 50 mm Varian Dynamax HPLC 21.4 mm Microsorb Guard-8 Cg column.
  • the purification method starts with 40:60 MeOHiH 2 O maintained for 0.5 minute and is ramped up to 100:0 MeOH:H 2 O over approximately 5 minutes (i.e.; total time to 100% MeOH is 5.2 minutes). 100% MeOH is maintained for 2 more minutes before it is re-equilibrated back to 40:60 MeOH:H 2 O.
  • Example 10 10(R), 14(5)-Dihydroxypaxillin-6-ene (CCCLXTV)
  • Compound CCCLXVIII was prepared in the same manner as compound CCCLXVII.
  • Compound CCCLXIX was prepared in the same manner as compound CCCLXVII.
  • the purification method starts with 40:60 MeOH:H 2 O maintained for 0.5 minute and is ramped up to 100:0 MeOH:H 2 O over approximately 5 minutes (i.e.; total time to 100% MeOH is 5.2 minutes). 100% MeOH is maintained for 2 more minutes before it is re-equilibrated back to 40:60 MeOH:H 2 O. Total run time is 8 minutes.
  • the activity of the compounds can also be quantified by the following assay.
  • the identification of inhibitors of the Maxi-K channel is based on the ability of expressed Maxi-K channels to set cellular resting potential after transfection of both alpha and betal subunits of the channel in HEK-293 cells and after being incubated with potassium channel blockers that selectively eliminate the endogenous potassium conductances of HEK-293 cells.
  • the transfected HEK-293 cells display a hyperpolarized membrane potential, negative inside, close to E K (-80 mV) which is a consequence of the activity ofthe Maxi-K channel.
  • Blockade of the Maxi-K channel by- incubation with Maxi-K channel blockers will cause cell depolarization. Changes in membrane potential can be determined with voltage-sensitive fluorescence resonance energy transfer (FRET) dye pairs that use two components, a donor coumarin (CC 2 DMPE) and an acceptor oxanol (DiSBAC 2 (3)).
  • FRET voltage-sensitive fluorescence resonance energy transfer
  • Oxanol is a lipophilic anion and distributes across the membrane according to membrane potential. Under normal conditions, when the inside ofthe cell is negative with respect to the outside, oxanol is accumulated at the outer leaflet ofthe membrane and excitation of coumarin will cause FRET to occur. Conditions that lead to membrane depolarization will cause the oxanol to redistribute to the inside ofthe cell, and, as a consequence, to a decrease in FRET. Thus, the ratio change (donor/acceptor) increases after membrane depolarization, which determines if a test compound actively blocks the Maxi-K channel.
  • the HEK-293 cells were obtained from the American Type Culture Collection ,
  • HEK-293 cells were plated in 100 mm tissue culture treated dishes at a density of 3x10 6 cells per dish, and a total of five dishes were prepared. Cells were grown in a medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine serum, IX L-Glutamine, and IX Penicillin/Streptomycin, at 37 0 C, 10% CO 2 .
  • DMEM Dulbecco's Modified Eagle Medium
  • cells were put under selection media which consisted of DMEM supplemented with both 600 ⁇ g/mL G418 and 0.75 ⁇ g/mL puromycin. Cells were grown until separate colonies were formed. Five colonies were collected and transferred to a 6 well tissue culture treated dish. A total of 75 colonies were collected. Cells were allowed to grow until a confluent monolayer was obtained. Cells were then tested for the presence of Maxi-K channel alpha and betal subunits using an assay that monitors binding of 125 I-iberiotoxin-D 19Y/Y36F to the channel.
  • the transfected cells (2E+06 Cells/mL) are then plated on 96-well poly-D-lysine plates at a density of about 100,000 cells/well and incubated for about 16 to about 24 hours.
  • the medium is aspirated of the cells and the cells washed one time with 100 ⁇ l of Dulbecco's phosphate buffered saline (D-PBS).
  • D-PBS Dulbecco's phosphate buffered saline
  • One hundred microliters of about 9 ⁇ M coumarin (CC 2 DMPE)-0.02% pluronic-127 in D-PBS per well is added and the wells are incubated in the dark for about 30 minutes.
  • the cells are washed two times with 100 ⁇ l of Dulbecco's phosphate-buffered saline and 100 ⁇ l of about 4.5 ⁇ M of oxanol (DiSBAC 2 (3)) in (mM) 140 NaCl, 0.1 KCl, 2 CaCl 2 , 1 MgCl 2 , 20 Hepes-NaOH, pH 7.4, 10 glucose is added.
  • mM 140 NaCl, 0.1 KCl, 2 CaCl 2 , 1 MgCl 2 , 20 Hepes-NaOH, pH 7.4, 10 glucose is added.
  • Three micromolar of an inhibitor of endogenous potassium conductance of HEK-293 cells is added.
  • a Maxi-K channel blocker is added (about 0.01 micromolar to about 10 micromolar) and the cells are incubated at room temperature in the dark for about 30 minutes.
  • the plates are loaded into a voltage/ion probe reader (VIPR) instrument, and the fluorescence emission of both CC 2 DMPE and DiSBAC 2 (3) are recorded for 10 sec.
  • VPR voltage/ion probe reader
  • 100 ⁇ l of high-potassium solution (mM): 140 KCl, 2 CaCl 2 , 1 MgCl 2 , 20 Hepes-KOH, pH 7.4, 10 glucose are added and the fluorescence emission of both dyes recorded for an additional 10 sec.
  • the ratio CC 2 DMPE/DiSBAC 2 (3), before addition of high-potassium solution equals 1.
  • the ratio after addition of high-potassium solution varies between 1.65-2.0. When the Maxi-K channel has been completely inhibited by either a known standard or test compound, this ratio remains at 1. It is possible, therefore, to titrate the activity of a Maxi-K channel inhibitor by monitoring the concentration-dependent change in the fluorescence ratio.
  • the compounds of this invention were found to cause concentration-dependent inhibition of the fluorescence ratio with IC 50 in the range of about InM to about 20 ⁇ M, more preferably from about 10 nM to about 500 nM.
  • the activity for blocking Maxi-K channels by compounds of the present invention is 1 ⁇ M or less.
  • the following compounds of -formulas CCCLXIIr CeeLX/and eexrV have-IC50 values of 445 nM; 16OnM;- and ' 115OTnMr ⁇ respectively.
  • Pipettes were typically filled with solutions containing (mM): 150 KCl, 10 Hepes (4-(2-hydroxyethyl)-l-piperazine methanesulfonic acid), 1 Mg, 0.01 Ca, and adjusted to pH 7.20 with KOH. After forming a high resistance (>l ⁇ 9 ohms) seal between the plasma membrane and the pipette, the pipette was withdrawn from the cell, forming an excised inside-out membrane patch.
  • solutions containing (mM): 150 KCl, 10 Hepes (4-(2-hydroxyethyl)-l-piperazine methanesulfonic acid), 1 Mg, 0.01 Ca and adjusted to pH 7.20 with KOH. After forming a high resistance (>l ⁇ 9 ohms) seal between the plasma membrane and the pipette, the pipette was withdrawn from the cell, forming an excised inside-out membrane patch.
  • the patch was excised into a bath solution containing (mM): 150 KCl, 10 Hepes, 5 EGTA (ethylene glycol bis( ⁇ - aminoethyl ether)-N,N,N',N'-tetraacetic acid), sufficient Ca to yield a free Ca concentration of 1 - 5 ⁇ M, and the pH was adjusted to 7.2 with KOH. For example, 4.193 mM Ca was added to give a free concentration of 1 ⁇ M at 22 0 C.
  • An EPC9 amplifier (HEKA Elektronic, Lambrect, Germany) was used to control the voltage and to measure the currents flowing across the membrane patch.
  • the input to the headstage was connected to the pipette solution with a Ag/ AgCl wire, and the amplifier ground was connected to the bath solution with a Ag/ AgCl wire covered with a tube filled with agar dissolved in 0.2 M KCl.
  • the identity of Maxi-K currents was confirmed by the sensitivity of channel open probability to membrane potential and intracellular calcium concentration.
  • K values for channel block were calculated by fitting the fractional block obtained at each compound concentration with a Hill equation.
  • the K, values for channel block by the compounds described in the present invention range from 0.001 nM to greater than 10 ⁇ M.
  • the activity for blocking Maxi-K channel currents by the compounds of the present invention is 100 nM or less.

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  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation de puissants bloqueurs de canaux potassiques ou une formulation de ceux-ci dans le traitement d'un glaucome et d'autres pathologies qui conduisent à une pression intraoculaire élevée dans l'œil d'un patient. La présente invention concerne également l'utilisation de tels composés pour produire un effet neuroprotecteur sur l'œil d'une espèce mammalienne, en particulier l'être humain.
PCT/US2008/011556 2007-10-11 2008-10-07 Bloqueurs de canaux maxi-k et procédés d'utilisation WO2009048558A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872338A (zh) * 2018-09-04 2020-03-10 中国海洋大学 一种吲哚二萜类化合物及其制备方法和用途
WO2020151566A1 (fr) * 2019-01-22 2020-07-30 凯复制药有限公司 Composé inhibant la transduction du signal pge2/ep4, son procédé de préparation et ses applications thérapeutiques

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Publication number Priority date Publication date Assignee Title
US6924306B2 (en) * 2000-01-18 2005-08-02 Merck & Co. Inc. Method for treating ocular hypertension
US20050239863A1 (en) * 2002-06-17 2005-10-27 Garcia Maria L Novel maxi-k channel blockers, methods of use and process for making the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924306B2 (en) * 2000-01-18 2005-08-02 Merck & Co. Inc. Method for treating ocular hypertension
US20050239863A1 (en) * 2002-06-17 2005-10-27 Garcia Maria L Novel maxi-k channel blockers, methods of use and process for making the same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872338A (zh) * 2018-09-04 2020-03-10 中国海洋大学 一种吲哚二萜类化合物及其制备方法和用途
WO2020151566A1 (fr) * 2019-01-22 2020-07-30 凯复制药有限公司 Composé inhibant la transduction du signal pge2/ep4, son procédé de préparation et ses applications thérapeutiques
CN111989311A (zh) * 2019-01-22 2020-11-24 凯复(苏州)生物医药有限公司 抑制pge2/ep4信号传导的化合物、其制备方法及其在医药上的应用
JP2022522993A (ja) * 2019-01-22 2022-04-21 キーセラ・（スーチョウ）・ファーマシューティカルズ・カンパニー・リミテッド Ｐｇｅ２／ｅｐ４シグナル伝達を阻害する化合物、その製造方法及びその医薬における応用
JP7488269B2 (ja) 2019-01-22 2024-05-21 キーセラ・（スーチョウ）・ファーマシューティカルズ・カンパニー・リミテッド Ｐｇｅ２／ｅｐ４シグナル伝達を阻害する化合物、その製造方法及びその医薬における応用

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