Dinucleoside polyphosphate compositions and their therapeutic use as purinergic receptor agonists

TECHNICAL FIELD

[0002] The present invention relates to novel compositions of dinucleoside polyphosphates with purinergic receptor agonist activity, which facilitate secretory mechanisms, such as increasing the hydration of mucus secretions, stimulating the production of mucins and increasing ciliary beat frequency to facilitate clearance of retained secretions.

BACKGROUND OF THE INVENTION

[0003] Chronic obstructive pulmonary disease (COPD) affects 15 million patients in the U.S. and is the sixth leading cause of death. It is characterized by the retention of mucus secretions in the lungs. Many patients diagnosed with COPD have a disorder called chronic bronchitis (CB), and 600,000 patients are hospitalized each year due to an acute exacerbation of CB. Cystic fibrosis and Primary Ciliary Dyskinesia (PCD) are other examples of lung disorders which assume a clinical profile similar to COPD. Ciliary dyskinesia, whether primary or secondary, results in retained secretions that can only be cleared by coughing.

[0004] Another disease state characterized by the accumulation of retained mucous secretions is sinusitis, an inflammation of the paranasal sinuses typically associated with an upper respiratory infection. Sinusitis is this country's most common health-care complaint, affecting an estimated 31 million people. (A. Moss and V. Parsons, National Center for Health Statistics, 1986: 66-7, DHHS Publication No. (PHS)86-1588 (1985)).

[0005] Otitis media (OM) is a viral or bacterial infection of the middle ear, which primarily afflicts children under the age of three years. It is usually precipitated by an upper respiratory infection which spreads into the middle ear via the nasopharynx and eustachian tube. Approximately 25-50 million office visits are made each year for diagnosis and treatment of OM. By age three, about 75% of children will have had at least one episode of acute OM (J. Klein, Clin. Infect. Dis. 19, 823-33 (1994)). Following appropriate treatment with antibiotics, accumulated fluid in the middle ear remains, causing hearing impairment and potential language and cognitive development delays. Enhanced ability to clear secretions in the middle ear would reduce or eliminate significant sequelae of otitis media.

[0006] An additional disorder resulting from retained secretions is pneumonia. Patients who are immobilized for a variety of reasons are at high risk for developing pneumonia. Despite extra vigilance and numerous interventions, pneumonia develops in over 400,000 patients per year, with significant morbidity and mortality.

[0007] Mucous secretions are normally removed via the mucociliary clearance (MCC) system. MCC relies on the integrated action of three components: 1) mucus secretion by goblet cells and submucosal glands; 2) the movement of cilia on epithelial cells which propels the mucus across the luminal surface; and 3) ion transport into and out of luminal epithelial cells which concomitantly controls the flow of water into the mucus.

[0008] Similarly, disorders of secretion may lead to improper hydration at various body sites, thereby resulting in pathological conditions. These include dry eye disease, nasolacrimal duct obstruction, retinal detachment, glaucoma or ocular hypertension, retinal degeneration, vaginal dryness, gastroesophageal reflux, dry mouth, and constipation.

[0009] Enhancement of mucociliary clearance or balanced tissue hydration would thus be useful in the prevention, management and treatment of such disorders. Furthermore, enhancement of mucociliary clearance would also have diagnostic applications such as sputum induction and detection of lung cancer.

[0010] It is now known that P2Y receptor agonists modulate all components of the MCC system by: (1) increasing both the rate and total amount of mucin secretion by goblet cells in vitro (Lethem, et al., Am. J. Respir. Cell. Mol. Biol. 9, 315-22 (1993)); (2) increasing cilia beat frequency in human airway epithelial cells in vitro (Drutz, et al., Drug Dev. Res. 37(3), 185 (1996)); (3) increasing Cl− secretion, hence, water secretion from airway epithelial cells in vitro (Mason, et al., Br. J. Pharmacol. 103, 1649-1656 (1991); and (4) releasing surfactant from Type II alveolar cells (Gobran, Am. J. Physiol. 267, L625-L633 (1994)). In addition to such actions, P2Y agonists have also been shown to increase intracellular Ca++ due to stimulation of phospholipase C by the P2Y2 receptor (Brown, et al., Mol. Pharmacol. 40, 648-655 (1991); Yerxa and Johnson, Drugs of the Future 24(7): 759-769 (1999)). U.S. Pat. Nos. 5,789,391; 5,763,447; 5,635,160; 5,935,555; 5,656,256; 5,628,984; 5,902,567; 5,292,498; 5,837,861; 5,900,407; 5,972,904; 5,981,506; 5,958,897; 5,968,913; 6,022,527; 6,133,247; and 6,143,279, and PCT International Patents WO97/29756, WO97/35591, WO96/40059, WO97/05195, WO94/08593, WO98/19685, WO98/15835, WO98/03182, WO98/03177, WO98/34942, WO98/34593, WO99/09998, WO99/32085, WO99/61012, WO00/30629, WO00/50024, and WO 96/40059 disclose beneficial therapeutic effects of dinucleotides and related compounds in sinusitis, otitis media, ciliary dyskinesia, pneumonia associated with immobilization, lung disease, cystic fibrosis, dry eye disease, vaginal dryness, bronchitis, edematous retinal disorders, retinal degeneration and detachment, and gastrointestinal disease. These and all other U.S. patents cited and herein are specifically incorporated herein by reference in their entirety.

[0011] Dinucleotides in the prior art are disclosed in the following references (International Patent Nos. WO 96/40059, WO 96/02554A1, WO-A-9815563, and WO 98/55494; Theoclitou, et al., J. Chem. Soc. Perkin Trans. I, 2009-2019 (1996); Guranowski, et al., Nucleosides and Nucleotides 14, 731-734 (1995); Visscher, et al., Nucleic Acids Research 20, 5749-5752 (1992); Holler, et al., Biochemistry 22, 4924-4933 (1983); Orr, et al., Biochem. Pharmacol. 673-677 (1988); Plateau, et al., Biochemistry 24, 914-922 (1985); Hagmeier, et al., J. Chromatography 237, 174-177 (1982); Scheffzek, et al., Biochemistry 35, 9716-9727 (1996); Stridh, et al, Antiviral Res., 97-105 (1981); Tarasova, et al., Chem. Abs. 110, 154770 (1988); Hata, et al., Chem Lett., 987-990 (1976); Huhn, et al., 28, 1959-1970 (1993); Tumanov, et al., Chem. Abs. 109-6867d (1987); Pintor, et al., Molecular Pharmacology 51, 277-284 (1997); and U.S. Pat. Nos. 4,855,304; 5,635,160; 5,495,550; and 5,681,823).

[0012] Applicants have discovered new dinucleoside compounds which are effective in clearing retained mucous secretions and/or balancing tissue hydration.

SUMMARY OF THE INVENTION

[0013] The invention provides novel pharmaceutical compositions comprising compounds of general Formula I, which feature: (1) a dinucleotide with a sugar moiety selected from the group consisting of: 3′-deoxyribofuranosyl, 2′,3′-dideoxyribofuranosyl, arabinofuranosyl, 3′-deoxyarabinofuranosyl, xylofiranosyl, 2′-deoxyxylofuranosyl, and lyxofuranosyl (Formula I); (2) a dinucleotide with an azapurine base (Formula II); and (3) a dinucleotide with a 6-substituted purine (Formula III). The invention also provides methods for using such compounds in the clearance of retained mucous secretion, the enhancement of ciliary beat frequency and the restoration of proper tissue hydration. Accordingly, a broad embodiment of the invention is directed to compounds of general Formulae I, II and III, or the pharmaceutically acceptable non-toxic salts thereof.

[0014] The compounds of the present invention are selective agonists of the P2Y purinergic receptor family; thus, they may be useful in the treatment of respiratory system disorders including chronic obstructive pulmonary diseases (chronic bronchitis, PCD, and cystic fibrosis), and may also be useful in the treatment of immobilized patients who are at risk for developing pneumonia. Furthermore, because of their general ability to clear retained mucus secretions and stimulate ciliary beat frequency, the compounds of the present invention may also be useful in the treatment of sinusitis and otitis media. Additionally, it is postulated that the compounds of the present invention could facilitate secretory mechanisms at other body sites, such as the eye, for the treatment of dry eye, retinal degeneration and detachment, and edematous retinal disorders; the gastrointestinal tract, for treatment of gastrointestinal disease and dry mouth; and vagina, for vaginal dryness.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The invention provides novel compositions of dinucleoside polyphosphate compounds, which may be used therapeutically as purinergic receptor agonists. The invention also provides methods for using such compositions to facilitate clearance of retained mucous secretions and enhancement of ciliary beat frequency. The method comprises administering to a mammal a pharmaceutical composition comprising a therapeutically effective amount of P2Y receptor ligands. The “therapeutic effective amount” used herein means an amount effective to treat a disease or disorder, which is an amount effective to reverse, halt, or delay the progression of the disease state, or to confer protection from subsequent damage and degeneration.

[0016] This invention provides that P2Y receptors are localized to multiple cell types. Applicants believe that the activation of P2Y receptors by the novel compounds of the present invention triggers specific cellular mechanisms, which result in observed P2Y receptor-mediated therapeutic effects.

[0017] A. New Sugar Moieties

[0018] P2Y agonists include dinucleoside polyphosphates as depicted by general Formula I, wherein one of the novel features is the presence of a sugar moiety selected from the group consisting of: 3′-deoxyribofuranosyl, 2′,3′-dideoxyribofuranosyl, arabinofuranosyl, 3′-deoxyarabinofuranosyl, xylofuranosyl, 2′-deoxyxylofuranosyl, and lyxofuranosyl:

[0019] wherein:

[0020] X is oxygen, methylene, dihalomethylene (with difluoromethylene and dichloromethylene preferred), or imido;

[0021] n=0, 1 or 2;

[0022] m=0, 1 or 2;

[0023] n+m=0,1, 2, 3, or 4;

[0024] Z=OH or H;

[0025] Z′=OH or H;

[0026] Y=OH or H;

[0027] Y′=OH or H; and

[0028] B and B′ are each independently a purine residue or a pyrimidine residue, as defined in Formula Ia or Ib, linked through the 9- or 1-position, respectively;

[0029] R1 is hydrogen, chlorine, amino, monosubstituted amino, disubstituted amino, alkylthio, arylthio, or aralkylthio, wherein the substituent on sulfur contains up to a maximum of 20 carbon atoms, with or without unsaturation;

[0030] R2 is hydroxy, alkenyl, oxo, amino, mercapto, thione, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy, monosubstituted alkylamino, heterocyclic, monosubstituted cycloalkylamino, monosubstituted aralkylamino, monosubstituted arylamino, diaralkylamino, diarylamino, dialkylamino, acylamino, or diacylamino,

[0031] Rx is O, H or is absent;

[0032] R2 and Rx are optionally taken together to form a 5-membered fused imidazole ring of 1,N6-etheno adenine derivatives, optionally substituted on the 4- or 5-positions of the etheno moiety with alkyl, aryl or aralkyl moieties as defined below;

[0033] R3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio, or aralkylthio as defined below; or T(C1-6alkyl)OCONH(C1-6alkyl)W, wherein T and W are independently amino, mercapto, hydroxy or carboxyl; or pharmaceutically acceptable esters, amides or salts thereof; or absent;

[0034] Thus, the substituted derivatives of adenine include adenine 1-oxide; 1,N6-(4-or 5-substituted etheno) adenine; N6-substituted adenine; or N-substituted 8-aminoadenine, where R′ of the 6- or 8-HNR′ groups are chosen from among: arylalkyl (C1-6) groups with the aryl moiety optionally functionalized as described below; alkyl; and alkyl groups with functional groups therein, such as: ([6-aminohexyl]carbamoylmethyl)-, and ω-acylated- amino(hydroxy, thiol and carboxy)alkyl(C2-10)- and their ω-acylated-amino (hydroxy, thiol and carboxy) derivatives where the acyl group is chosen from among, but not limited to, acetyl, trifluoroacetyl, benzoyl, substituted-benzoyl, etc., or the carboxylic moiety is present as its ester or amide derivative, for example, the ethyl or methyl ester or its methyl, ethyl or benzamido derivative. The ω-amino(hydroxy, thiol) moiety may be alkylated with a C1-4alkyl group;

[0035] J is carbon or nitrogen, with the provision that when J is nitrogen, R3 is not present;

[0036] wherein the alkyls are straight-chain, branched or cyclic;

[0037] wherein the aryl groups are optionally substituted with lower alkyl, aryl, amino, mono- or dialkylamino, NO2, N3, cyano, carboxylic, amido, sulfonamido, sulphonic acid, phosphate, or halo group;

[0038] wherein:

[0039] R4 is hydroxy, oxo, mercapto, thione, amino, cyano, C7-12arylalkoxy, C1-6alkylthio, C1-6alkoxy, C1-6alkylamino or diC1-4alkylamino, wherein the alkyl groups are optionally linked to form a heterocycle;

[0040] R5 is hydrogen, acetyl, benzoyl, C1-6alkyl, C1-5alkanoyl, aroyl, or absent;

[0041] R6 is hydroxy, oxo, mercapto, thione, C1-4alkoxy, C7-12arylalkoxy, C1-6alkylthio, S-phenyl, arylthio, arylalkylthio, triazolyl, amino, C1-6alkylamino, C1-5disubstituted amino, or di-C1-4alkylamino, wherein said dialkyl groups are optionally linked to form a heterocycle or linked to form a substituted ring such as morpholino, pyrrolo, etc.; or

[0042] R3 and R6 taken together form a 5-membered fused imidazole ring between positions 3 and 4 of the pyrimidine ring and form a 3,N4-ethenocytosine derivative, wherein said etheno moiety is optionally substituted on the 4- or 5-positions with C1-4alkyl, phenyl or phenyloxy; wherein at least one hydrogen of said C1-4alkyl, phenyl or phenyloxy is optionally substituted with a moiety selected from the group consisting of halogen, hydroxy, C1-4alkoxy, C1-4alkyl, C6-10aryl, C7-12arylalkyl, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino, C1-4alkylamino, and di-C1-4alkylamino, wherein said dialkyl groups are optionally linked to form a heterocycle;

[0043] R7 is selected from the group consisting of hydrogen, hydroxy, cyano, nitro, C1-6alkyl or phenyl; substituted C2-8alkynyl, halogen, substituted C1-4alkyl, CF3, C2-3alkenyl, C2-3alkynyl, allylamino, bromovinyl, ethyl propenoate, or propenoic acid and C2-8alkenyl;; or

[0044] R6 and R7 together form a 5 or 6-membered saturated or unsaturated ring bonded through N or O or S at R6, such ring optionally contains substituents that themselves contain functionalities; and

[0045] R8 is selected from the group consisting of hydrogen, amino, di-C1-4alkylamino, C1-4alkoxy, C7-12arylalkoxy, C1-4alkylthio, C7-12arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, and phenylthio; provided that when R8 is amino or substituted amino, R7 is hydrogen.

[0046] The furanosyl moieties are as depicted in the D-configuration, but may be L-, or D- and L-. The D-configuration is preferred. The nucleoside residue can be in the alpha- or beta- and D- or L-configurations, but most preferably the beta-D-configuration.

[0047] B. Azapurine

[0048] Novel P2Y agonists also include dinucleoside polyphosphates as depicted by said general Formula II with the novel feature of an azapurine base, as described below. The nucleoside residue may include the sugar moieties: ribofuranosyl, 2′-deoxyribofuranosyl, 3′-deoxyribofuranosyl, 2′,3′-dideoxyribofuranosyl, arabinofuranosyl, 3′-deoxyarabinofuranosyl, xylofuranosyl, 2′-deoxyxylofuranosyl, and lyxofuranosyl. The furanosyl moieties can be in the alpha- or beta- and D- or L-configurations, but most preferably the beta-D-configuration.

[0049] wherein:

[0050] X, n, m, n+m, Z, Z′, Y, and Y′ are the same as described in Section A above. B and B′ are each independently a purine residue or a pyrimidine residue, as defined in Formula IIa or Ib linked through the 9- or 1-position, respectively, provided that at least one of B and B′ is a purine;

[0051] R1 is hydrogen, chlorine, amino, monosubstituted amino, disubstituted amino, alkylthio, arylthio, or aralkylthio, wherein the substituent on sulfur contains up to a maximum of 20 carbon atoms, with or without unsaturation;

[0052] R2 is hydroxy, oxo, alkenyl, amino, mercapto, thione, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy, monosubstituted alkylamino, heterocyclic, monosubstituted cycloalkylamino, monosubstituted aralkylamino, monosubstituted arylamino, diaralkylamino, diarylamino, dialkylamino, acylamino, or diacylamino;

[0053] Rx is O, H or is absent;

[0054] R2 and Rx are optionally taken together to form a 5-membered fused imidazole ring of 1,N6-etheno adenine derivatives, optionally substituted on the 4- or 5-positions of the etheno moiety with alkyl, aryl or aralkyl moieties as defined below;

[0055] The substituted derivatives of adenine are as described above;

[0056] J is nitrogen and R3 is not present;

[0057] wherein the alkyls are straight-chain, branched or cyclic;

[0058] wherein the aryl groups are optionally substituted with lower alkyl, aryl, amino, mono- or dialkylamino, NO2, N3, cyano, carboxylic, amido, sulfonamido, sulphonic acid, phosphate, or halo groups.

[0059] C. 6-Substituted Purine

[0060] Novel P2Y agonists also include dinucleoside polyphosphates as depicted by said general Formula III with the novel feature of a 6-substituted purine base, as described below. The nucleoside residue may include a variety of sugar moieties, such as ribofuranosyl, 2′-deoxyribofuranosyl, 3′-deoxyribofuranosyl, 2′,3′-dideoxyribofuranosyl, arabinofuranosyl, 3′-deoxyarabinofuranosyl, xylofiranosyl, 2′-deoxyxylofuranosyl, and lyxofuranosyl. The furanosyl moieties can be in the alpha- or beta- and D- or L-configurations, but most preferably the beta-D-configuration.

[0061] wherein:

[0062] X, n, m, n+m, Z, Z′, Y, and Y′ are the same as described in Section A and Section B above; and

[0063] B and B′ are each independently a purine residue or a pyrimidine residue, as defined in Formula IIIa or Ib, linked through the 9- or 1-position, respectively, provided that at least one of B and B′ is a purine;

[0064] R1 is hydrogen, chlorine, amino, monosubstituted amino, disubstituted amino, alkylthio, arylthio, or aralkylthio, wherein the substituent on sulfur contains up to a maximum of 20 carbon atoms, with or without unsaturation;

[0065] R2 is mercapto, thione, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, acyloxy, or di-substituted amino;

[0066] Rx is O (adenine 1-oxide derivatives), H or is absent (adenine derivatives);

[0067] R3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio, or aralkylthio as defined below; or T(C1-6alkyl)OCONH(C1-6alkyl)W wherein T and W are independently amino, mercapto, hydroxy or carboxyl, or pharmaceutically acceptable esters, amides or salts thereof;

[0068] J is carbon or nitrogen, with the provision that when J is nitrogen, R3 is not present;

[0069] wherein the alkyls are straight-chain, branched or cyclic;

[0070] wherein the aryl groups are optionally substituted with lower alkyl, amino, monoalkylamino, dialkylamino, NO2, N3, cyano, carboxylic, amido, sulfonamido, sulphonic acid, phosphate, or halo groups;

[0071] B and B′ can also be a pyrimidine with the general formula of Formula Ib, as described above.

[0072] In the general structure of Formulae Ia, IIa and IIIa, the dotted lines are intended to indicate the presence of single or double bonds in these positions; the relative positions of the double or single bonds being determined by whether the R2 and Rx substituents are capable of keto-enol tautomerism.

[0073] In the general structure of Formulae Ib, IIb and IIIb, the dotted lines in the 2- to 6-positions are intended to indicate the presence of single or double bonds in these positions; the relative positions of the double or single bonds being determined by whether the R4, R5, R6, R7, and R8 substituents are capable of keto-enol tautomerism.

[0074] In the general structures of Formula Ia, Ib, IIa, IIb, IIIa, and IIIb above, the acyl groups comprise alkanoyl or aroyl groups. The alkyl groups contain 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms optionally substituted by one or more appropriate substituents, as described below. The aryl groups including the aryl moieties of such groups as aryloxy are preferably phenyl groups optionally substituted by one or more appropriate substituents, as described below. The above-mentioned alkenyl and alkynyl groups contain 2 to 8 carbon atoms, particularly 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionally substituted by one or more appropriate substituents as described below.

[0075] Appropriate substituents on the above-mentioned alkyl, alkenyl, alkynyl, and aryl groups are selected from halogen, hydroxy, C1-4alkoxy, C1-4alkyl, C6-12aryl, C6-12arylalkoxy, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic, amino and substituted amino wherein the amino is singly or doubly substituted by a C1-4alkyl, and when doubly substituted, the alkyl groups optionally being linked to form a heterocycle.

[0076] Compounds of Formulae I, II and III can be made in accordance with known procedures described by Zamecnik, et al., Proc. Natl. Acad. Sci. USA 89, 838-42 (1981); and Ng and Orgel, Nucleic Acids Res. 15:3572-80 (1987), Pendergast, et al., U.S. Pat. No. 5,837,861, or variations thereof.

[0077] Compounds encompassed by the present invention can be prepared by condensation of a nucleoside mono-, di-, or triphosphate, activated with a condensing agent such as, but not limited to, carbonyldiimidazole or dicyclohexylcarbodiimide, with a second molecule of the same or a different mono-, di-, or triphosphate to form the desired dinucleotide polyphosphate. Another method of preparation is the sequential condensation of a nucleoside phosphate, activated as above, with a non-nucleoside mono-, di- or polyphosphate moiety, such as, but not limited, to a monophosphate or pyrophosphate anion to yield the desired dinucleotide polyphosphate, the non-isolated intermediate in such a case being a mononucleotide polyphosphate. Yet another preparative approach is the sequential condensation of a mono-, di- or polyphosphate moiety, activated as mentioned above, or in the form of an acid halide or other derivative reactive toward nucleophilic displacement, with a nucleoside phosphate or polyphosphate to yield the desired dinucleotide polyphosphate. The desired dinucleotide polyphosphate may be formed by modification of a pre-formed dinucleotide polyphosphate by substitution or derivatization of a moiety or moieties on the purine, pyrimidine or carbohydrate ring. Nucleoside phosphates used as starting materials may be commercially available, or may be made from the corresponding nucleosides by methods well known to those skilled in the art. Likewise, where nucleosides are not commercially available, they may be made by modification of other readily available nucleosides, or by synthesis from heterocyclic and carbohydrate precursors by methods well known to those skilled in the art. The compounds araATP and araCTP are available from Sigma (St. Louis, Mo.). Tetra-acetyl xylofuranose obtained from Pfanstiehl (Waukegan, Ill.) can be converetd to xylouridine by the method of Gosselin, et al. (J. Med. Chem. 29(2): 203-213 (1986)). Various lyxo and arabinofuranosyl pyrimidines can be synthesized by the method of Iwai, et al. (pp. 388-394, in Synthetic Procedures in Nucleic Acid Chemistry, Eds. W. Werner Zorbach and R. Stuart Tipson, Interscience Publishers, New York, (1968).

[0078] Those having skill in the art will recognize that the starting materials may be varied and additional steps employed to produce compounds encompassed by the present invention, as demonstrated by the following examples. In some cases protection of certain reactive functionalities may be necessary to achieve some of the above transformations. In general the need for such protecting groups will be apparent to those skilled in the art of organic synthesis as well as the conditions necessary to attach and remove such groups.

[0079] The compounds of the present invention also encompass their non-toxic pharmaceutically acceptable salts, such as, but not limited to, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as manganese, magnesium or calcium; or an ammonium or tetraalkyl ammonium salt, i.e., NX4+(wherein X is C1-4). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. The present invention also encompasses the acylated prodrugs of the compounds disclosed herein. Those skilled in the art will recognize various synthetic methodologies which may be employed to prepare non-toxic pharmaceutically acceptable salts and acylated prodrugs of the compounds.

[0080] This invention further provides a method of using a pharmaceutical composition comprising P2Y receptor agonists for the treatment of a variety of disorders related to tissue fluid secretions, mucociliary clearance, and retinal degeneration; such disorders include, for example, chronic obstructive pulmonary disease, pneumonia, cystic fibrosis, bronchitis, sinusitis, otitis media, nasolacrimal duct obstruction, dry eye disease, edematous retinal disorders, retinal degeneration, vaginal dryness, dry mouth and gastrointestinal disease.

[0081] U.S. Pat. Nos. 5,292,498 and 5,635,160 disclose a method of hydrating mucous secretions in the lungs by administering UTP, related uridine phosphate compounds, and a dinucleotide, A2P4, for the treatment of cystic fibrosis. U.S. Pat. No. 5,763,477 teaches a method of treating pneumonia in immobilized patients by promoting drainage of mucous secretions in congested airways of such patients comprising the administration of uridine phosphates, including UTP and U2P4. U.S. Pat. No. 5,789,391 teaches a method of treating sinusitis by promoting drainage of congested mucous secretions in the sinuses of said patient comprising administering uridine phosphates, including UTP and U2P4. A method for treating dry eye disease by stimulating tear secretion comprising the administration of mono- and dinucleotides is disclosed in U.S. Pat. No. 5,900,407. U.S. Pat. No. 6,159,952 discloses a method for treating bronchitis, by promoting clearance of retained mucous secretions in the bronchi, bronchioles and small terminal airways comprising the administration of uridine phosphates including UTP and U2P4. WO 00/30629 discloses a method of treating vaginal dryness by stimulating cervical and vaginal secretions comprising the administration of ATP, CTP, UTP, and U2P4. WO97/29756 teaches a method of treating otitis media by administering UTP and related compounds in an amount effective to promote fluid drainage from the middle ear by hydrating mucous secretions in the middle ear and by increasing ciliary beat frequency in the middle ear and Eustachian tube. WO97/35591 discloses a method of treating ciliary dyskinesia by stimulating ciliary beat frequency to promote mucociliary or cough clearance of retained secretions in the lungs, sinuses, or ears of a patient by administering mono- and dinucleotide polyphosphates. WO98/15835 discloses a method of inducing sputum production or for diagnostic purposes or expectoration by administering uridine 5′-triphosphate (UTP), P1, P4−(uridine 5′−) tetraphosphate (U2P4) or related compounds. U.S. patent application Ser. No. 09/747,777 discloses a method of treating gastrointestinal diseases or disorders in which the mucosal barrier of the gastrointestinal tract is impaired by administering a mono- or dinucleotide polyphosphate in an amount effective to regulate mucous or mucin secretions or to correct abnormal fluid transport in the gastrointestinal system. U.S. patent application Ser. No. 09/570,231 discloses a method of treating retinal degeneration by administering mono- and dinucleotide polyphosphates in an amount effective to mount a neuroprotective effect on the retinal glial and neuronal cells. U.S. patent application Ser. No. 09/774,752 teaches a method of treating retinal detachment and retinal edematous disorders by stimulating the removal of extraneous intra-retinal or subretinal pathological fluid. U.S. patent application Ser. No. 09/512,867 discloses a method of stimulating mucosal hydration, treating dry eye, treating rhinosinusitis, treating dry mouth, treating corneal injury, and treating vaginal dryness by administering uridine diphosphate derivatives and certain dinucleotide triphosphate derivatives in an amount effective to increase hydration. U.S. patent application Ser. No. 60/171,710 discloses a method of treating gastrointestinal diseases or disorders in which the mucosal barrier of the gastrointestinal system is impaired, or in which the fluid transport across the lumenal tract is abnormal, by administering mono- and dinucleotide polyphosphates in an amount effective to regulate mucus or mucin secretions or correct abnormal fluid transport in the gastrointestical system. All the references cited above are specifically incorporated herein by reference in their entirety. This invention applies Applicants' novel compounds in the treatment of a variety of disorders as described in the above references.

[0082] The compounds of the present invention are selective agonists of the P2Y receptors. An embodiment of the present invention is the use of these novel compounds in the treatment of mammals including humans suffering from chronic obstructive pulmonary diseases such as chronic bronchitis, PCD, and cystic fibrosis, as well as the treatment or prevention of pneumonia, including pneumonia due to immobility. A further embodiment is the use of such novel compounds in the treatment of sinusitis, otitis media and nasolacrimal duct obstruction in mammals, including humans, because of their general ability to clear retained mucus secretions and stimulate ciliary beat frequency. Yet a further embodiment of the compounds of the present invention is their use for treating mammals including humans with dry eye, retinal degeneration and edematous retinal disorders, including retinal detachment. Another embodiment of the present invention is the use of these compounds in the treatment of vaginal dryness. Yet another embodiment of the present invention is their use in treating gastrointestinal disorders. A further embodiment of the present invention is their use in the treatment of glaucoma. To summarize, these purinergic receptor agonists can have beneficial therapeutic effects in sinusitis, otitis media, ciliary dyskinesia, pneumonia associated with immobilization, lung disease, cystic fibrosis, dry eye disease, vaginal dryness, bronchitis, edematous retinal disorders, retinal degeneration, detachment and edema, dry mouth and gastrointestinal disease.

[0083] Though the compounds of the present invention are primarily concerned with the treatment of human subjects, they may also be employed for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes.

[0084] The compounds of general Formula I, II and III may be administered orally, topically, parenterally, by inhalation or spray, intra-operatively, rectally, or vaginally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term topically as used herein includes patches, gels, creams, aerosols, ointments, or nose, ear or eye drops. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intra-articular, intrathecal, and intravitreal injection or infusion techniques. In addition, there is provided a pharmaceutical formulation comprising a compound of general Formulae I, II and III, and a pharmaceutically acceptable carrier. One or more compounds of general Formulae I, II and III may be present in association with one or more non-toxic pharmaceutically acceptable carriers or diluents or adjuvants and, if desired, other active ingredients. One such carrier would be sugars, where the compounds may be intimately incorporated in the matrix through glassification or simply admixed with the carrier (e.g., lactose, sucrose, trehalose, mannitol) or other acceptable excipients for lung or airway delivery.

[0085] One or more compounds of general Formula I, II and III can be administered separately or together, or separately or together with mucolytics such as DNAse or acetylcysteine; agents used to treat and manage glaucoma, including anticholinesterase inhibitors, carbonic anhydrase inhibitors, osmotic agents, parasympathomimetic agents, sympathomimetic agents, prostaglandins, hypotensive lipids and beta-adrenergic agents; agents used to treat edematous retinal disorders, including corticosteroids, carbonic anhydrase inhibitors, anti-inflammatory agents (such as COX-2 inhibitors and non-steroidal anti-inflammatory agents), and pharmaceuticals that promote digestion of collagen and fibrous tissues that connect vitreous and retina; agents used to treat dry eye disease; agents used to manage corneal injury; agents used to treat dry mouth; agents used to treat sinusitis, otitis media, and nasolacrimal duct obstruction; agents used to treat pneumonia, cystic fibrosis, bronchitis, and other chronic obstructive pulmonary disease; agents used to treat vaginal dryness; anti-infective agents; and agents used for neuronal protection.

[0086] The pharmaceutical compositions containing compounds of general Formula I, II and III can be in a form suitable for oral use, for example, as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions; and such compositions may contain one or more agents selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

[0087] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

[0088] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example: sodium carboxymethylcellulose, methylcellulose and sodium alginate. Dispersing or wetting agents may be a naturally-occurring phosphatide or condensation products of an allylene oxide with fatty acids, or condensation products of ethylene oxide with long chain aliphatic alcohols, or condensation products of ethylene oxide with partial esters from fatty acids and a hexitol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anydrides. Those skilled in the art will recognize the many specific excipients and wetting agents encompassed by the general description above. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0089] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring, and coloring agents, may also be present.

[0090] Compounds of general Formulae I, II and III can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are sterile water, saline solution, or Ringer's solution.

[0091] The compounds of general Formulae I, II, and III can also be administered in the form of suppositories for ear, rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient, which is solid at ordinary temperatures but liquid at body temperature and will, therefore, melt to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0092] Another means of administration of the active compounds is intra-operative instillation of a gel, cream, powder, foam, crystals, liposomes, spray or liquid suspension form of said compound, such that a therapeutically effective amount of said compound contacts the target site of said patient via systemic absorption and circulation.

[0093] Dosage levels of the order of from about 10−7 M to about 10−1 M, preferably in the range 10−5 to 10−1 M, are useful in the treatment of the above-indicated conditions. The effective dose ranges between about 0.1 to about 1000 mg, preferably between about 0.1 to about 100 mg, and most preferably between about 0.5 to about 50 mg for single doses. The amount of active ingredients that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0094] A further embodiment of the invention is the use of specific dinucleotide polyphosphates as specific agonists for different subtypes of purinergic receptors. For example, dinucleoside tetraphosphates may have specificity and affinity for P2Y2 and/or P2Y4 receptors, whereas dinucleotide triphosphates have specificity for P2Y6 receptors.

[0095] The invention is illustrated further by the following examples, which are not to be construed as limiting the invention in scope or spirit to the specific procedures described in them.

EXAMPLE 1

P1-(8-Azaadenosine-5′-) P4-(uridine-5′-) tetraphosphate

[0096] 8-Azaadenosine monophosphate sodium salt (prepared from 8-azaadenosine by the method of Yoshikawa, et al.; Tetrahedron Lett., 5065-5068 (1967)) is dissolved in water, applied to a column of Biorad AG-MP50 strong cation exchange resin in its tributylamine form, the column eluted with water, and the eluate lyophilized. The resulting tributylamine salt of 8-azaadenosine MP is condensed in DMF at 50° C. for 24h with uridine 5′-cyclic trimetaphosphate, which is prepared by activating UTP with dicyclohexylcarbodiimide (see example 1). The reaction mixture is evaporated under high vacuum overnight. The residue is dissolved in water, filtered to remove a little residual dicyclohexylurea, and purified by semipreparative ion-exchange chromatography (Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min). The appropriate product fractions are collected, then evaporated and lyophilized to give the title compound. The isolated ammonium salt of the title compound is converted to the tetrasodium salt by dissolving in water and applying this solution to a column of Biorad AG-MP50 strong cation exchange resin in its hydrogen form. Elution with water is followed by adjusting the eluent pH to 8 with sodium hydroxide, then removal of solvent in vacuo.

EXAMPLE 2

P1-(6-Mercaptopurine riboside 5′-) P4-(uridine 5′-) tetraphosphate

[0097] 6-Mercaptopurine riboside monophosphate sodium salt (prepared from 6-mercaptopurine riboside by the method of Yoshikawa, et al. (1967) (Tetrahedron Lett., 5065-5068) is dissolved in water, applied to a column of Biorad AG-MP50 strong cation exchange resin in its tributylamine form, the column eluted with water, and the eluate lyophilized. The resulting tributylamine salt of 6-mercaptopurine riboside MP is condensed as above in DMF with uridine 5′-cyclic trimetaphosphate, which is prepared by activating UTP with dicyclohexylcarbodiimide (see example 1). After evaporating the DMF from the reaction mixture in vacuo, the residue is dissolved in water and purified by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min and appropriate product fractions collected and lyophilized.

EXAMPLE 3

P1-(6-Mercaptopurine riboside 5′-) P4-(2′-deoxyuridine 5′-) tetraphosphate

[0098] 6-Mercaptopurine riboside monophosphate sodium salt (prepared from 6-mercaptopurine riboside by the method of Yoshikawa, et al. (1967) (Tetrahedron Lett., 5065-5068) is dissolved in water, applied to a column of Biorad AG-MP50 strong cation exchange resin in its tributylamine form, the column eluted with water, and the eluate lyophilized. The resulting tributylamine salt of 6-mercaptopurine riboside MP is condensed as above in DMF with deoxyuridine 5′-cyclic trimetaphosphate, which is prepared by activating dUTP (Sigma, St. Louis, Mo.) with dicyclohexylcarbodiimide (prepared analogously to UcTP, see example 1). After evaporating the DMF from the reaction mixture in vacuo, the residue is dissolved in water and purified by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min and appropriate product fractions collected and lyophilized.

EXAMPLE 4

P1-(4-Thiouridine 5′-) P4-(arabinocytidine 5′-) tetraphosphate

[0099] 4-Thiouridine monophosphate sodium salt (Sigma, St. Louis, Mo.) is dissolved in water and applied to a column of Biorad AG-MP50 strong cation exchange resin in its tributylamine form. The column is eluted with water and the eluate lyophilized. The resulting tributylamine salt of 4-thio-UMP is condensed in DMF as above with cytidine arabinoside 5′-cyclic trimetaphosphate, prepared by activation of araCTP (Sigma, St. Louis, Mo.) with dicyclohexylcarbodiimide (prepared analogously to UcTP, see example 1). After evaporating the DMF from the reaction mixture in vacuo, the residue is dissolved in water and separated by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min. The appropriate product fractions are collected and lyophilized to give the title compound.

EXAMPLE 5

P1-(2′-Deoxyadenosine 5′-) P4-(6-thiohexylpurine riboside 5′-) tetraphosphate

[0100] A solution of 2′-deoxyadenosine 5′-triphosphate (dATP, Sigma, St. Louis, Mo.) trisodium salt in water is passed through a column of BioRad AG-MP 50 strong cation exchange resin in its tributylamine form and eluted with distilled water. To this solution is added tributylamine and the suspension is shaken until the pH of the aqueous fraction has risen to 8. The layers are separated and the aqueous solution evaporated to small volume, then lyophilized. The residue is dissolved in dry DMF and the solvent is evaporated at 0.1 mmHg. Dicyclohexylcarbodiimide (DCC) is added to an aliquot of the foregoing dATP solution and the solution is stirred at room temperature for 30 min. The deposited dicyclohexylurea is removed by filtration, the reaction mixture is extracted with ether, and the residue is dissolved in dry DMF. Condensing this 2′-deoxyadenosine 5′-cyclic trimetaphosphate (dAcTP) and 6-thiohexylpurine riboside 5′-monophosphate (prepared from 6-thiohexylpurine riboside by the method of Yoshikawa, et al.; (1967) (Tetrahedron Lett., 5065-5068) is as described above. After evaporating the DMF from the reaction mixture in vacuo, the residue is dissolved in water, filtered, and separated by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min, 30 min. The appropriate product fractions are collected and lyophilized.

EXAMPLE 6

P1-(6-Eicosanyloxypurine riboside 5′-) P4-(uridine 5′-) tetraphosphate

[0101] 6-Chloropurine riboside monophosphate sodium salt (prepared from 6-chloropurine riboside by the method of Yoshikawa, et al. (1967) (Tetrahedron Lett., 5065-5068) is dissolved in water, applied to a column of Biorad AG-MP50 strong cation exchange resin in its tributylamine form, the column eluted with water, and the eluate lyophilized. The resulting tributylamine salt of 6-chloropurine riboside MP is condensed as above in DMF with uridine 5′-cyclic trimetaphosphate, which is prepared by activating UTP with dicyclohexylcarbodiimide (see example 1). After evaporating the DMF from the reaction mixture in vacuo, the residue is dissolved in water and purified by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min followed by collecting and lyophilizing the product fractions. This tetraphosphate dinucleotide is dissolved in DMF and treated with the sodium salt of 1-eicosanol (Aldrich, Milwaukee, Wis.) at 40° C. for 48 h. All solvents are evaporated in vacuo and the residue purified by ion exchange chromatography on a Hamilton PRP X-100 column, eluting with isocratic 1.0 M ammonium bicarbonate, 5 mL/min. The appropriate product fractions are collected and lyophilized.

[0102] The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes particular embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

	Number	Date	Country
Parent	09774752	Jan 2001	US
Child	09817017	Mar 2001	US

Dinucleoside polyphosphate compositions and their therapeutic use as purinergic receptor agonists

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