Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/16—Central respiratory analeptics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

• the present invention relates to certain N -subst ⁇ tuted-adenosme-5'- uronamide de ⁇ vatives and their use m the practice of medicine as compounds with activity as agonists of adenosine receptors, in particular, the adenosine A, and A 3 receptors
• A, receptors are coupled to the inhibition of adenylate cyclase through G t proteins and have also been shown to couple to other secondary messenger systems, including inhibition or stimulation of phosphoinositol turnover and activation of ion channels
• a 2 receptors are further divided into two subtypes, A 2A and A 2B , at which adenosine agonists activate adenylate cyclase with high and low affinity, respectively
• the A3 receptor sequence was first identified in a rat testes cDNA library, and this sequence, later cloned by homology to other G-protein coupled receptors from a rat bram cDNA library, was shown to correspond to a novel, functional adenosine receptor
• a 3 receptor mediates processes of inflammation, hypotension, and mast cell degranulation. This receptor apparently also has a role in the central nervous system.
• the A3 selective agonist IB-MECA induces behavioral depression and upon chronic administration protects against cerebral ischemia.
• A3 selective agonists at high concentrations were also found to induce apoptosis in HL-60 human leukemia cells.
• A3 antagonists for the A3 receptor are sought as potential antiinflammatory or possibly antiischemic agents in the brain. Recently, A3 antagonists have been under development as antiasthmatic, antidepressant, antiarrhythmic, renal protective, antiparkinson and cognitive enhancing drugs.
• N -(substituted phenylcarbamoyl)adenosine-5'- uronamides where the substituent is 2-chloro, 3-chloro or 4-methoxy, have been prepared (Baraldi et al., Advance ACS Abstracts. December 15, 1995) and demonstrated affinity at A3 receptors in the low nanomolar range (Ki values less than 10 nm).
• substituents such as 3-bromo, showed a ten-fold loss in activity and affinity for the A3 receptor (Baraldi et al., J. Med Chem.. 39:802-806 (1996).
• a number of A3 adenosine receptor agonists which have been previously synthesized are structurally related to adenosine itself, in which the ribose moiety is mainly intact. On the ribose, 5'-alkyluronamide groups are generally tolerated.
• Positions on the structure of adenosine providing flexibility of substitution, in general for adenosine agonists, have been the N and C 2 position. At the N position, most alkyl or aryl derivatives are A 1 selective. At the C 2 position, many C-, N-, or O-derivatives are A 2a selective. Benzyl derivatives at the N position have been shown to be A3 selective.
• R is hydrogen, alkyl, substituted alkyl, or aryl
• R 1 is heteroaryl-NR-C(X), heteroaryl-C(X)-, alkaryl-NR-C(X)-, substituted alkaryl-NR-C(X)-, aryl-NR-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-,
• R is hydrogen, alkyl, substituted alkyl, or aryl-NH-C(X)-, and
• X is O, S, or NR, with the proviso that, when R ⁇ is H, R is aryl-NH- C(X)- and X is O, the substituents on the aryl ring are not halo, methoxy or trifluoromethyl.
• R and R are, independently selected from the group consisting of halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitrile, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, amine, amide and other substituents routinely used in the field of nucleoside chemistry to modify these positions. Such modifications are expected to provide the compounds with activity as partial agonists.
• R 1 When R 1 is heteroaryl-NR-C(X), heteroaryl-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, the compounds tend to show affinity, and, in some cases, selectivity for the adenosine A j receptor. When R 1 is alkaryl-NR-C(X)-, substituted alkaryl-NR-C(X)-, or aryl-NR-C(X)-, the compounds tend to show affinity and selectivity for the A 3 receptor.
• Preferred substituents include p-sulfonamide, p-nitro, p-phenyl, 2,4-dichloro, p-methoxy, m-chloro, o-chloro and p-nitro.
• the compounds can be used in a method for fully or partially inhibiting adenylate cyclase (A j and A 3 ) in a mammal, including a human.
• the methods involve administering an effective amount of a compound of formula I sufficient to fully or partially inhibiting adenylate cyclase in the mammal.
• the compounds can be used in competitive binding assays to determine the activity of other compounds in their ability to bind the A j or A3 receptor.
• the compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients.
• Various chemical intermediates such as 2',3-isopropylidene-N-alkylcarboxamido adenosines, can be used to prepare the compounds.
• Figure 1 is a graph showing the effect of various adenosine agonists
• the present application discloses compounds useful as agonists or partial agonists of adenosine receptors, in particular, with activity as A j or A 3 agonists or partial agonists, and methods of preparation and use thereof.
• the compounds can be used in a method for treating a mammal, including a human, with excessive activity at adenosine receptors, in particular, A j or A3 receptors.
• the methods involve administering to the mammal an effective amount of a compound of formula I sufficient to modulate adenosine receptors in the mammal.
• the compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients.
• a compound is an agonist of an adenosine A, or A3 receptor if it is able to fully inhibit adenylate cyclase (A j and A3) and is able to displace [ IJ-AB-MECA in a competitive binding assay.
• a compound is a partial agonist of an adenosine A-, or A3 receptor if it is able to partially inhibit adenylate cyclase (A, and A 3 ) and is able to displace [ IJ-AB-MECA in a competitive binding assay.
• a compound is an antagonist of an adenosine A-. or A3 receptor if it is able to prevent the inhibition due to an agonist and is able to displace [ IJ-AB-MECA in a competitive binding assay.
• a compound is selective for the A j receptor if the ratio of A 2 /A j and A3/A, activity is greater than about 50, preferably between 50 and 100, and more preferably, greater than about 100.
• a compound is selective for the A3 receptor if the ratio of A j /A 3 and A 2 /A 3 activity is greater than about 50, preferably between 50 and 100, and more preferably, greater than about 100.
• alkyl refers to monovalent straight, branched or cyclic alkyl groups preferably having from 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms (“lower alkyl”) and most preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, «-propyl, zs ⁇ -propyl, n-butyl, iso-buty ⁇ , n-hexyl, and the like.
• alkylene and “lower alkylene” refer to divalent radicals of the corresponding alkane.
• moieties having names derived from alkanes such as alkoxyl, alkanoyl, alkenyl, cycloalkenyl, etc when modified by "lower,” have carbon chains of ten or less carbon atoms.
• alkenyl minimum of two carbons
• cycloalkyl minimum of three carbons
• substituted alkyl refers to an alkyl group, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, hydroxylamino, alkoxyamino, nitro, - SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-he
• alkoxy refers to the group “alkyl-O-", where alkyl is as defined above.
• Preferred alkoxy groups include, by way of example, methoxy, ethoxy, «-propoxy, w -propoxy, «-butoxy, tert-butoxy, sc -butoxy, «-pentoxy, «-hexoxy, 1 ,2-dimethylbutoxy, and the like.
• alkenyl refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation.
• alkynyl refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.
• acyl refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl- C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• acylamino refers to the group -C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
• such aryl groups can optionally be substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
• substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
• cycloalkyl refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings.
• Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
• halo or halogen refer to fluoro, chloro, bromo and iodo and preferably is either fluoro or chloro.
• heteroaryl refers to an aromatic carbocyclic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).
• heteroaryl groups can be optionally substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO
• Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
• Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).
• Heterocycle or “heterocyclic” refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to
• heterocyclic groups can be optionally substituted with 1 to
• heterocyclic groups can have a single ring or multiple condensed rings.
• any of the above groups that contain 1 or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
• “Pharmaceutically acceptable salts” refers to pharmaceutically acceptable salts of a compound of Formula I, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can be used as the pharmaceutically acceptable salt.
• protecting group refers to any group which when bound to one or more hydroxy 1, amino or carboxyl groups of the compounds (including intermediates thereof such as the aminolactams, aminolactones, etc.) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, amino or carboxyl group.
• Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), and the like which can be removed by conventional conditions compatible with the nature of the product.
• (R)-PIA (R)-N -(phenylisopropyl)adenosine
• DMSO dimethysulfoxide
• EDTA ethylenediamine tetraacetic acid
• I-AB-MECA N -(4-am ⁇ no-3- iodobenzyl)adenosine-5'-N-methyluronam ⁇ de
• IB-MECA N -(3- ⁇ odobenzyl)adenosme-5'-N-methyluronam ⁇ de
• Ki equilibrium inhibition constant
• NECA 5'-N-ethylcarboxamido adenosine
• THF tetrahydrofuran
• Tns tns(hydroxymethyl)am ⁇ nomethane.
• the compounds can be prepared as desc ⁇ bed below.
• an N-alkyl carboxamido adenosine such as 5'- (N-ethylcarboxam ⁇ do)adenosme (NECA)
• NECA N-ethylcarboxam ⁇ do
• a suitable protecting group is an isopropylidene ⁇ ng, although other protecting groups can be used and are intended to be within the scope of the invention.
• 2',3'-O- ⁇ sopropyhdene-N -(subst ⁇ tuted-carbonylam ⁇ no)-adenos ⁇ ne-5' alkyluronamides can be prepared from the isopropylidene protected N- alkylcarboxamido adenosines by reaction of the N -amine group with an approp ⁇ ate acid chlonde or other suitable activated carboxylic acid derivative, such as an anhyd ⁇ de, using routine amidation conditions (as shown in Figure 2a).
• 2',3'-O-isopropylidene-N -(substituted- carbamoylamino)-adenosine-5'-alkyluronamides can be prepared from the isopropylidene protected N-alkylcarboxamido adenosines by reaction of the N -amine group with an appropriate isocyanate using known chemistry (as shown in Figure 2b). The protecting groups are then removed to provide the desired compounds.
• N amine group with an acid halide or isocyanate N amine group with an acid halide or isocyanate.
• the compounds can be used for all indications for which agonists and antagonists of the A, or A3 receptor are effective.
• Compounds which effectively modulate the A, receptor can be used for:
• antinociception i.e., analgesics
• anticonvulsants i.e., sodium bigesics
• cardioprotection short term (e.g., prior to percutaneous angioplasty (PTDA), angioplasty, and cardiac surgeries) and long term (prevention of myocardial infarction, especially in high risk patients, reduction of infarct damage, especially in high risk patients);
• pain management generally, including different forms of neuropathic pain, e.g., diabetic neuropathy, post herpetic neuralgia;
• antilipid uses reduction of free fatty acids, triglycerides, glucose
• adjunct therapy in diabetes including insulin and non-insulin dependent diabetes mellitus: stimulation of insulin secretion from the pancreas, increase in tissue sensitivity to insulin;
• diagnostic uses, for example, to determine the presence of one or more of the above described medical conditions, or in a screening assay to determine the effectiveness of other compounds for binding to the A, Ado receptor (i.e., through competitive inhibition as determined by various binding assays), as described in Jacobson and Van Rhee, Purinergic approaches to experimental therapy, Jacobson and Jarvis, ed.,
• Compounds which effectively modulate the A 3 receptor can be used for: treating hypertension; mast cell degranulation; antitumor agents; treating cardiac hypoxia; and protection against cerebral ischemia; as described, for example, in Jacobson, TIPS May 1998, pp. 185-191, the contents of which are hereby incorporated by reference.
• the compounds can be administered via any medically acceptable means. Suitable means of administration include oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration, although oral or parenteral administration are preferred.
• the amount of the compound required to be effective as agonist or partial agonist of an adenosine receptor will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner.
• the factors to be considered include the condition being treated, the route of administration, the nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered.
• a suitable effective dose is in the range of about 0.1 ⁇ g/kg to about 10 mg/kg body weight per day, preferably in the range of about 1 mg/kg to about 3 mg/kg per day.
• the total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary. For example, for a 75 kg mammal, a dosage range would be about 75 mg to about 220 mg per day, and a typical dose would be about 150 mg per day. If discrete multiple doses are indicated, treatment might typically be 50 mg of a compound given 3 times per day.
• the compounds described above are preferably administered in a formulation including an active compound, i.e., a compound of formula I, together with an acceptable carrier for the mode of administration.
• an active compound i.e., a compound of formula I
• an acceptable carrier for the mode of administration.
• Suitable pharmaceutically acceptable carriers are known to those of skill in the art.
• compositions can optionally include other therapeutically active ingredients such as antivirals, antitumor agents, antibacterials, anti- inflammatories, analgesics, and immunosuppresants.
• the carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the formulations can include carriers suitable for oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration.
• Preferred carriers are those suitable for oral or parenteral administration.
• Formulations suitable for parenteral administration include sterile aqueous preparations of the active compound, which are preferably isotonic with the blood of the recipient. Such formulations may contain distilled water, 5% dextrose in distilled water or saline. Suitable formulations also include concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration.
• the compound can be incorporated into an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non- aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught.
• Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents.
• Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
• a syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients.
• a sugar e.g., sucrose
• accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
• the compounds can also be administered locally by topical application of a solution, ointment, cream, gel, lotion or polymeric material (for example, a Pluronic , BASF), which may be prepared by conventional methods known in the art of pharmacy.
• a solution, ointment, cream, gel, lotion or polymeric material for example, a Pluronic , BASF
• topical formulations may also contain preservatives, perfumes, and additional active pharmaceutical agents.
• Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.
• the compound may be administered in liposomes or microspheres (or microparticles).
• Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art.
• U.S. Patent No. 4,789,734 the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
• a review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes.” Drug Carriers in Biology and Medicine, pp.
• Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Patent Nos. 4,906,474, 4,925,673 and 3,625,214, the contents of which are hereby incorporated by reference.
• Preferred microparticles are those prepared from biodegradable polymers, such as polyglycohde, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.
• the formulations may further include one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
• optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
• Rat A Male Wistar rats (200-250 g) can be decapitated and the whole brain
• the membranes can be centrifuged and the pellets stored at -70 °C.
• Striatal tissues can be homogenized with a Polytron in 25 vol of 50 mM Tris HCl buffer containing 10 mM MgCL, pH 7.4. The homogenate can be centrifuged at 48,000 g for 10 min at 4°C and resuspended in Tris HCl buffer containing 2 IU/ml adenosine deaminase. After a 30 min incubation at 37 °C, the membranes can be centrifuged and the pellet stored at -70 °C.
• Radioligand binding assays Binding of [ 3 H]-DPCPX (l,3-dipropyl-8-cyclopentylxanthine) to rat brain membranes can be performed essentially according to the method previously described by Bruns et al, Proc. Natl. Acad. Sci. U.S.A., 77:5547- 5551 (1980), the contents of which are hereby incorporated by reference. In this method, displacement experiments are performed in 0.25 ml of buffer containing 1 nM [ 3 H]-DPCPX, 100 ⁇ of diluted membranes of rat brain
• Non specific binding is determined in the presence of 10 ⁇ M of CHA (N cyclohexyladenosine) and this is always ⁇ 10% of the total binding.
• the incubation time is typically around 120 min at 25 °C.
• Bound and free radioactivity can be separated by filtering the assay mixture through Whatman GF/B glass-fiber filters, using a Brandel cell harvester (Gaithersburg, MD, USA).
• the incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer.
• the filter bound radioactivity can be measured by liquid scintillation spectrometry.
• the protein concentration can be determined using known methodology, for example, using bovine albumin as a reference standard.
• Binding assays can be carried out according to the method described by Salvatore et al., Proc. Natl. Acad. Sci. U.S.A., 90:10365-10369 (1993), the contents of which are hereby incorporated by reference. In saturation studies using this method, an aliquot of membranes (8 mg protein ml) from HEK-293 cells transfected with the human recombinant A 3 adenosine receptor (Research Biochemical International, Natick, MA, USA) are incubated with from 10 to 12 different concentrations of [ JAB-MECA ranging from 0.1 to 5 nM.
• Competition experiments are carried out in duplicate in a final volume of 100 ⁇ in test tubes containing 0.3 nM [ 125 ]AB-MECA, 50 mM Tris HCl buffer, 10 mM MgCI 2 , pH 7.4 and 20 ⁇ of diluted membranes (12.4 mg protein/ml) and at least 6 to 8 different concentrations of examined ligands. Incubation time is typically around 60 min at 37 °C.
• Bound and free radioactivity are separated by filtering the assay mixture through Whatman GF/B glass-fiber filters using a Brandel cell harvester.
• Non-specific binding is defined as binding in the presence of 50 ⁇ M R-PIA and can be as high as about 30%.
• the incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer.
• the filter bound radioactivity is counted in a Beckman gamma 5500B ⁇ counter.
• the protein concentration can be determined according to known methodology, for example, using bovine albumin as reference standard.
• a weighted non linear least-squares curve fitting program for example, LIGAND can be used for computer analysis of saturation and inhibition experiments. Data are typically expressed as geometric mean, with 95% or 99% confidence limits in parentheses.
• Example 1 General procedure for the preparation of 2',3'-O- isopropylidene-N -(substituted- carbonylamino)adenosine-5'-N-ethyluronamide
• Example 12 Preparation of 2', 3'-O-isopropylidene-N -((S)- ⁇ - phenylethyl-carbamoylamino)adenosine-5'-N- ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 82% yield as a white solid, mp 109-111 °C.
• Example 13 Preparation of 2',3'-O-isopropyIidene-N -(5- methyl-isoxazoI-3-yl-carbamoylamino)adenosine- 5'-N-ethyluronamide
• Example 26 Preparation of N 6 -(4-acetyl-phenylcarbamoyl)- adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in an 83% yield as a white solid, mp 187°C.
• Nonspecific binding was determined m the presence of 200 ⁇ M NECA K j values were calculated according to Cheng-PrusofT assuming a K d for [ 125 I]-AB-MECA of 1 48 nM Binding of [ 3 H]R-PIA to A, receptors from rat cortical membranes and of [ 3 H]CGS 21680 to A 2A receptors for rat st ⁇ atal membranes was performed as desc ⁇ bed previously Adenosine deaminase (2 umts/mL) was present du ⁇ ng the preparation of the membranes Additional deaminase was not added du ⁇ ng incubation with the radiohgand
• Derivatives 3g, 3h and 3m showed a relatively high affinity (9.7-107 nM) at A 3 receptors with varying degrees of A 3 /A j selectivity.
• compound 3g was less active than IB-MECA (9.7 nM vs. 1.1 nM) but showed selectivity for A 3 vs. either A j or A 2A receptors which is comparable to IB-MECA.
• Compound 3g also showed a relatively high affinity, in the nanomolar range, at human A 3 adenosine receptors (56.1 ⁇ 9.1 nM), confirming the relatively high affinity of this compound versus this receptor subtype, independent of species.
• the lipophilicity of para substituents on a phenyl ring play a significant role in A 3 affinity. Substitution of the phenyl ring with a heterocycle (as in Compounds 3k-l) causes the compounds to lose affinity and selectivity for A 3 receptors, and increase in affinity for the A j receptor subtype.
• the R-isomer (3i) is more potent and selective that the S-isomer (3j) at the rat A 3 adenosine receptor subtype.
• substituents at N are of opposite configuration
• the R-isomer (3i) is more potent and selective that the S-isomer (3j) at the rat A 3 adenosine receptor subtype.
• a functional assay indicated that Compound 3g acted as a full agonist at rat A 3 receptor.
• the assay involved the agonist-induced inhibition of binding of guanine nucleotide to rat RBL-2H3 mast cell membranes, which contain a high density of A 3 receptors.
• Figure 1 shows that compound 3g was about as effective as the potent A 3 agonists I-AB-MECA and Cl-IB-MECA increased binding of [ 35 S]GTP- ⁇ -S in a dose dependent manner and with greater potency than NECA.
• Example 35 Pharmaceutical Formulations
• the silicone fluid and active compound are mixed together and the colloidal silicone dioxide is added to increase viscosity.
• the material is then dosed into a subsequent heat sealed polymeric laminate including the following: polyester release liner, skin contact adhesive composed of silicone or acrylic polymers, a control membrane which is a polyolefin, and an impermeable backing membrane made of a polyester multilaminate.
• the resulting laminated sheet is then cut into 10 sq. cm patches
• the active compound and the starch are granulated with water and dried. Magnesium stearate is added to the dried granules and the mixture is thoroughly blended. The blended mixture is compressed into tablets.
• the active compound and buffering agents are dissolved in the propylene glycol at about 50°C.
• the water for injection is then added with stirring and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving.
• the orphan receptor cDNA RDC7 encodes an A l adenosine receptor. F.MBO J. 1 91 , 10, 1677- 1682.
• S. RDC8 codes for an adenosine A 2 receptor with physiological constitutive activity. Biochem. Biophys. Res. Commun. 1990. 173, 1 169- 1 178.
• Adenosine Receptors pharmacology, structure-activity relationship and therapeutic potential. J. Med. Chem. 1992.35. 407-422.

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