text:
Compounds and methods are provided to treat inflammatory conditions with certain A2A adenosine receptor antagonists.
wherein (a) each R is individually hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl or phenyl(C1-C3)-alkyl;
(b) X is �CH2OH, �CO2R2, -OC(O)R2 or C(O)NR3R4;
(c) each of R2, R3 and R4 is individually H, C1-6-alkyl; C1-6-alkyl substituted with 1-3 C1-6-alkoxy, C3-C7 cycloalkyl, C1-6-alkylthio, halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino, or C6-10-aryl, wherein aryl may be substituted with 1-3 halogen, C1-6-alkyl, hydroxy, amino, mono(C1-6-alkyl)amino, or di(C1-6-alkyl)amino; C6-10-aryl; or C6-10-aryl substituted with 1-3 halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino or C1-6-alkyl;
(d) R1 is (X-(Z)-)n[(C3-C10)cycloalkyl]-(Z′)- wherein Z and Z′ are individually (C1-C6)alkyl, optionally interrupted by 1-3 S or noperoxide O, or is absent, and n is 1-3; or a pharmaceutically acceptable salt thereof.
2. A compound of
claim 1
3. A compound of
claim 2
4. A compound of
claim 1
5. A compound of
claim 1
6. A compound of
claim 5
7. A compound of
claim 1
8. A compound of
claim 7
9. A compound of
claim 7
10. A compound of
claim 7
11. A compound of
claim 1
12. A compound of
claim 1
13. Methyl-4-(3-{9-[(4S,5S,2R, 3R)-5-(N-ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-6-aminopurin-2-yl)}prop-2-ynyl)cyclohexane-carboxylate.
14. A therapeutic method to inhibit an inflammatory response comprising administering to a mammal in need of said therapy, an effective anti-inflammatory amount of a compound of formula (I):
wherein (a) each R is individually hydrogen, C1-C6 alkyl C3-C7 cycloalkyl, phenyl or phenyl(C1-C3)-alkyl;
(b) X is �CH2OH, �CO2R2, �OC(O)R2 or C(O)NR3R4;
(c) each of R2, R3 and R4 is individually H, C1-6-alkyl; C1-6-alkyl substituted with 1-3 C1-6-alkoxy, C3-C7-cycloalkyl, C1-6-alkylthio, halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino, or C6-10 -aryl, wherein aryl may be substituted with 1-3 halogen, C1-6-alkyl, hydroxy, amino, mono(C1-6-alkyl)amino, or di(C1-6-alkyl)amino; C6-10-aryl; or C6-10-aryl substituted with 1-3 halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino or C1-6-alkyl;
(d) each of R3 and R4 is individually hydrogen, C3-7-cycloalkyl, or any of the meanings of R2; and
(e) R1is (X-(Z)-)n[(C 3-Cl10)cycloalkyl]-(Z′)- wherein Z and Z′ are individually (C1-C6)alkyl, optionally interrupted by 1-3 S or noperoxide O, or is absent, and n is 1-3; or a pharmaceutically acceptable salt thereof.
15. The method of
claim 14
16. The method of
claim 15
17. The method of
claim 14
18. The method of
claim 14
19. The method of
claim 17
20. The method of
claim 14
21. The method of
claim 20
22. The method of
claim 20
23. The method of
claim 20
24. A method of
claim 14
25. A method of
claim 14
26. The method of
claim 14
27. The method of
claim 14
28. The method of
claim 14
29. The method of
claim 14
30. The method of
claim 14
31. The method of
claim 14
32. The method of
claim 14
33. The method of
claim 14
34. The method of
claim 14
35. The method of
claim 14
36. The method of
claim 14
37. The method of
claim 14
38. The method of
claim 14
39. The method of
claim 14
40. A therapeutic composition comprising a compound of
claim 1
41. The composition of
claim 37
42. The composition of
claim 38
43. The composition of
claim 38
44. The composition of
claim 43
[0001] This application is a continuation-in-part of Ser. No. 09/003,930, filed Jan. 8, 1998, pending. This application claims priority of U.S. provisional patent applications Ser. Nos. 60/118,029, filed Feb. 1, 1999, and 60/124,316 filed Mar. 12, 1999.
[0002] The present invention was made with the assistance of U.S. Government funding (NIH Grant ROL HL37942). The U.S. Government has certain rights in this invention.
[0003] The present invention relates to methods and compositions for preventing tissue injury, i.e., due to inflammatory activity.
[0004] The inflammatory response serves the purpose of eliminating harmful agents from the body. There is a wide range of pathogenic insults that can initiate an inflammatory response including infection, allergens, autoimmune stimuli, immune response to transplanted tissue, noxious chemicals, and toxins, ischemia/reperfusion, hypoxia, mechanical and thermal trauma. Inflammation normally is a very localized action which serves in expulsion, attenuation by dilution, and isolation of the damaging agent and injured tissue. The body's response becomes an agent of disease when it results in inappropriate injury to host tissues in the process of eliminating the targeted agent, or responding to a traumatic insult.
[0005] As examples, inflammation is a component of pathogenesis in several vascular diseases or injuries. Examples include: ischemia/reperfusion injury (N. G. Frangogiannis et al., in Myocardial Ischemia: Mechanisms, Reperfusion, Protection, M. Karmazyn, ed., Birkhuser Verlag (1996) at 236-284; H. S. Sharma et al., Med. of Inflamm, 6, 175 (1987)), atherosclerosis (R. Ross, Nature, 362, 801 (1993)), inflammatory aortic aneurysms (N. Girardi et al., Ann. Thor. Surg., 64, 251 (1997); D. I. Walker et al., Brit, J. Surg., 5, 609 (1972); R. L. Pennell et al., J. Vasc. Surg., 2, 859 (1985)), and restenosis following balloon angioplasty (see, R. Ross cited above). The cells involved with inflammation include leukocytes (i.e., the immune system cells - neutrophils, eosinophils, lymphocytes, monocytes, basophils, macrophages, dendritic cells, and mast cells), the vascular endothelium, vascular smooth muscle cells, fibroblasts, and myocytes.
[0006] The release of inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) by leukocytes is a means by which the immune system combats pathogenic invasions, including infections. TNFα stimulates the expression and activation of adherence factors on leukocytes and endothelial cells, primes neutrophils for an enhanced inflammatory response to secondary stimuli and enhances adherent neutrophil oxidative activity. See, Sharma et al., cited above. In addition, macrophages/dendritic cells act as accessory cells processing antigen for presentation to lymphocytes. The lymphocytes, in turn, become stimulated to act as pro-inflammatory cytotoxic cells.
[0007] Generally, cytokines stimulate neutrophils to enhance oxidative (e.g., superoxide and secondary products) and nonoxidative (e.g., myeloperoxidase and other enzymes) inflammatory activity. Inappropriate and over-release of cytokines can produce counterproductive exaggerated pathogenic effects through the release of tissue-darnaging oxidative and nonoxidative products (K. G. Tracey et al., J. Exp. Med., 117, 1211 (1988); and D. N. Mannel et al., Rev. Infect. Dis., 9 (suppl. 5), S602-S606 (1987)). For example, TNFα can induce neutrophils to adhere to the blood vessel wall and then to migrate through the vessel to the site of injury and release their oxidative and non-oxidative inflammatory products.
[0008] Although monocytes collect slowly at inflammatory foci, given favorable conditions, the monocytes develop into long-term resident accessory cells and macrophages. Upon stimulation with an inflammation trigger, monocytes/macrophages also produce and secrete an array of cytokines (including TNFα), complement, lipids, reactive oxygen species, proteases and growth factors that remodel tissue and regulate surrounding tissue functions.
[0009] For example, inflammatory cytokines have been shown to be pathogenic in: arthritis (C. A. Dinarello, Semin. Tmmunol., 4, 133 (1992)); ischemia (A. Seekamp et al., Agents-Actions-Supp., 41, 137 (1993)); septic shock (D. N. Mannel et al., Rev. Infect. Dis., 9 (suppl. 5), S602-S606 (1987)); asthma (N. M. Cembrzynska et al., Am. Rev. Respir. Dis., 147, 291 (1993)); organ transplant rejection (D. K. Imagawa et al., Transplantation, 51, 57 (1991); multiple sclerosis (H. P. Hartung, Ann. Neurol., 33, 591 (1993)); AIDS (T. Matsuyama et al., AIDS, 5, 1405 (1991)); and in alkali-burned eyes (F. Miyamoto et al., Opthalmic Res., 30, 168 (1997)). In addition, superoxide formation in leukocytes has been implicated in promoting replication of the human immunodeficiency virus (HIV) (S. Legrand-Poels et al., AIDS Res. Hum. Retroviruses, 6, 1389 (1990)).
[0010] It is well known that adenosine and some analogs of adenosine that nonselectively activate adenosine receptor subtypes decrease neutrophil production of inflammatory oxidative products (B. N. Cronstein et al., Ann. N.Y. Acad. Sci, 451, 291 (1985); P. A. Roberts et al., Biochem. J., 227, 669 (1985); D. J. Schrier et al., J. Immunol., 137, 3284 (1986); B. N. Cronstein et al., Clinical Immunol. and Immunopath., 42, 76 (1987); M. A. Iannone et al., in Topics and Perspective in Adenosine Research, E. Gerlach et al., eds., Springer-Verlag, Berlin, p. 286 (1987); S. T. McGarrity et al., J. Leukocyte Biol., 44, 411421 (1988); J. De La Harpe et al., J. Immunol., 143, 596 (1989); S. T. McGarrity et al., J. Immunol.,142, 1986 (1989); and C. P. Nielson et al., Br. J. Pharnacol., 97, 882 (1989)). For example, adenosine has been shown to inhibit superoxide release from neutrophils stimulated by chemoattractants such as the synthetic mimic of bacterial peptides, f-met-leu-phe (fMLP), and the complement component C5a (B. N. Cronstein et al., J. Immunol., 135, 1366 (1985)). Adenosine can decrease the greatly enhanced oxidative burst of PMN (neutrophil) first primed with TNF-α and then stimulated by a second stimulus such as f-met-leu-phe (G. W. Sullivan et al., Clin.Res., 41, 172A (1993)). Additionally, it has been reported that adenosine can decrease the rate of HIV replication in a T-cell line (S. Sipka et al., Acta. Biochim. Biopys. Hung., 23, 75 (1988)). However, there is no evidence that in vivo adenosine has anti-inflammatory activity (G. S. Firestein et al., Clin. Res., 41, 170A (1993); and B. N. Cronstein et al., Clin. Res., 41, 244A (1993)).
[0011] It has been suggested that there is more than one subtype of adenosine receptor on neutrophils that can have opposite effects on superoxide release (B. N. Cronstein et al., J. Clin. Invest., 85, 1150 (1990)). The existence of A2A receptor on neutrophils was originally demonstrated by Van Calker et al. (D. Van Calker et al., Eur. J. Pharmacology, 206, 285 (1991)).
[0012] There has been progressive development of compounds that are more and more potent and/or selective as agonists of A2A adenosine receptors (AR) based on radioligand binding assays and physiological responses. Initially, compounds with little or no selectivity for A2A receptors were developed, such as adenosine itself or 5′-carboxamides of adenosine, such as 5′-N-ethylcarboxamidoadenosine (NECA) (B. N. Cronstein et al., J. Immunol., 1,1366 (1985)). Later, it was shown that addition of 2-alkylamino substituents increased potency and selectivity, e.g., CV1808 and CGS21680 (M. F. Jarvis et al., J. Pharmacol. Exp. Ther., 251, 888 (1989)). 2-Alkoxy-substituted adenosine derivatives such as WRC-0090 are even more potent and selective as agonists at the coronary artery A2A receptor (M. Ueeda et al., J. Med. Chem., 34, 1334 (1991)). The 2-alklylhydrazino adenosine derivatives, e.g., SHA 211 (also called WRC-0474) have also been evaluated as agonists at the coronary artery A2A receptor (K. Niiya et al., J. Med. Chem., 35, 4557 (1992)).
[0013] There is one report of the combination of relatively nonspecific adenosine analogs, R-phenylisopropyladenosine (R-PIA) and 2-chloroadenosine (Cl-Ado) with a phosphodiesterase (PDE) inhibitor resulting in a lowering of neutrophil oxidative activity (M. A. lannone et al., Topics and Perspectives in Adenosine Research, E. Garlach et al., eds., Springer-Verlag, Berlin, pp. 286-298 (1987)). However, R-PIA and Cl-Ado analogs are actually more potent activators of A1 adenosine receptors than of A2A adenosine receptors and, thus, are likely to cause side effects due to activation of A1 receptors on cardiac muscle and other tissues causing effects such as �heart block.�
[0014] R. A. Olsson et al. (U.S. Pat. No. 5,278,150) disclose selective adenosine A2 receptor agonists of the formula:
[0015] wherein Rib is ribosyl, R1 can be H and R2 can be cycloalkyl. The compounds are disclosed to be useful for treating hypertension, atherosclerosis and as vasodilators.
[0016] Olsson et al. (U.S. Pat. No. 5,140,015) disclose certain adenosine A2 receptor agonists of formula:
[0017] wherein C(X)BR2 can be CH2OH and R1 can be alkyl- or alkoxyalkyl. The compounds are disclosed to be useful as vasodilators or an antihypertensives.
[0018] Linden et al. (U.S. Pat. No. 5,877,180) is based on the discovery that certain inflammatory diseases, such as arthritis and asthma, may be effectively treated by the administration of compounds which are selective agonists of A2A adenosine receptors, preferably in combination with a Type IV phosphodiesterase inhibitor. An embodiment of the Linden et al. invention provides a method for treating inflammatory diseases by administering an effective amount of an A2A adenosine receptor of the following formula:
[0019] wherein R and X are as described in the patent.
[0020] In a preferred embodiment, the Linden et al. invention involves the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the A2A adenosine receptor agonist. The Type IV phosphodiesterase (PDE) inhibitor includes racemic and optically active 4-(polyalkoxyphenyl)-2-pyrrolidones of the following formula:
[0021] wherein R′, R18, R19 and X are as disclosed and described in U.S. Pat. No. 4,193,926. Rolipram is an example of a suitable Type IV PDE inhibitor included within the above formula.
[0022] G. Cristalli (U.S. Pat. No. 5,593,975) discloses 2-arylethynyl, 2-cycloalkylethynyl or 2-hydroxyalkylethynyl derivatives, wherein the riboside residue is substituted by carboxy amino, or substituted carboxy amino (R3HNC(O)-). 2-Alkynylpurine derivatives have been disclosed in Miyasaka et al. (U.S. Pat. No. 4,956,345), wherein the 2-alkynyl group is substituted with (C3-C,16)alkyl. The ′975 compounds are disclosed to be vasodilators and to inhibit platelet aggregation, and thus to be useful as anti-ischemic, anti-atherosclerosis and anti-hypertensive agents.
[0023] However, a continuing need exists for selective A2 adenosine receptor agonists useful for therapeutic applications, that have reduced side effects.
[0024] The present invention comprises compounds and methods of their use for the treatment of inflammatory activity in mammalian tissue. The inflammatory tissue activity can be due to pathological agents or can be due to physical, chemical or thermal trauma, or the trauma of medical procedures, such as organ, tissue or cell transplantation, angioplasty (PCTA), inflammation following ischemia/reperfision, or grafting. The present compounds comprise a novel class of 2-alkynyladenosine derivatives, substituted at the ethyne position by substituted cycloalkyl moieties. Preferably, the riboside residue is substituted at the 5′-position (�X�) by an N-alkyl-(or cycloalkyl)carboxyamino (�aminocarbonyl�) moiety. Thus, the present invention provides a method for inhibiting the inflammatory response in a mammal, such as a human subject, and protecting the tissue subject to the response, by administering an effective amount of one or more compounds of the invention.
[0025] The compounds of the invention have the following general formula (I):
[0026] wherein (a) each R is individually hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl or phenyl(C1-C3)-alkyl;
[0027] (b) X is �CH2OH, �CO2R2, �OC(O)R2 or C(O)NR3R4;
[0028] (c) each of R2, R3 and R4 is individually H, C1-6 -alkyl; C1-6 -alkyl substituted with 1-3 C1-6-alkoxy, C3-C7 cycloalkyl, C1-6-alkylthio, halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino, or C6-10-aryl, wherein aryl may be substituted with 1-3 halogen, C1-6-alkyl, hydroxy, amino, mono(C1-6-alkyl)amino, or di(C1-6-alkyl)amino; C6-10-aryl; or C6≅10-aryl substituted with 1-3 halogen, hydroxy, amino, mono(C1-6-alkyl)amino, di(C1-6-alkyl)amino, or C1-6-alkyl;
[0029] (d) R′ is (X-(Z)-)n[(C3-C 10)cycloalkyl]-(Z′)- wherein Z and Z′are individually (C1-C6)alkyl, optionally interrupted by 1-3 S or nonperoxide O, or is absent, and n is 1-3; or a pharmaceutically acceptable salt thereof.
[0030] The invention provides a compound of formula I for use in medical therapy, preferably for use in treating or protecting tissue from inflammation such as an inflammatory response, as well as the use of a compound of formula I for the manufacture of a medicament for the treatment of an inflammatory response due to a pathological condition or symptom in a mammal, such as a human, which is associated with inflammation.
[0031] Although certain A2A adenosine receptor agonists have been reported to be vasodilators, and thus to be useful to directly treat hypertension, thrombus, atherosclerosis and the like, the tissue-protective activity of the compounds of formula (I) is not suggested by the prior art.
[0032] The invention also includes the use of a combination of these compounds with type IV phosphodiesterase inhibitors for synergistic decreases in the inflammatory response of immune cells.
[0033] The invention also provides a pharmaceutical composition comprising an effective amount of the compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier, and optionally, in combination with a Type W phosphodiesterase (PDE) inhibitor. Preferably, the composition is presented as a unit dosage form.
[0034] Additionally, the invention provides a therapeutic method for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of A2A adenosine receptors is implicated and agonism of said activity is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. It is believed that activation of A2A adenosine receptors inhibits inflammation by effecting neutrophils, mast cells, monocytes/macrophages, T-cells and/or eosinophils. Inhibition of these inflammatory cells results in tissue protection following tissue insults.
[0035] Among the inflammatory responses that can be treated (including treated prophylactically) with a compound of formula I, optionally with a Type IV PDE inhibitor, are inflammation due to (a) autoimmune stimulation (autoimmune diseases), such as lupus erythematosus, multiple sclerosis, infertility from endometriosis, type I diabetes mellitus including the destruction of pancreatic islets leading to diabetes and the inflammatory consequences of diabetes, including leg ulcers, Crohn's disease, ulcerative colitis, inflammatory bowel disease, osteoporosis and rheumatoid arthritis;
[0036] (b) allergic diseases such as asthma, hay fever, rhinitis, vernal conjunctivitis and other eosinophil-mediated conditions;
[0037] (c) skin diseases such as psoriasis, contact dermatitis, eczema, infectious skin ulcers, open wounds, cellulitis;
[0038] (d) infectious diseases including sepsis, septic shock, encephalitis, infectious arthritis, endotoxic shock, gram negative shock, Jarisch-Herxheimer reaction, shingles, toxic shock, cerebral malaria, bacterial meningitis, acute respiratory distress syndrome (ARDS), lyme disease, HIV infection, ( TNFα -enhanced HIV replication, TNFA inhibition of reverse transcriptase inhibitor activity);
[0039] (e) wasting diseases: cachexia secondary to cancer and HIV;
[0040] (f) organ, tissue or cell transplantation (e.g., bone marrow, cornea, kidney, lung, liver, heart, skin, pancreatic islets) including transplant rejection, and graft versus host disease;
[0041] (g) adverse effects from drug therapy, including adverse effects from amphotericin B treatment, adverse effects from immunosuppressive therapy, e.g., interleukin-2 treatment, adverse effects from OKT3 treatment, adverse effects from GM-CSF treatment, adverse effects of cyclosporine treatment, and adverse effects of aminoglycoside treatment, stomatitis and mucositis due to immunosuppression;
[0042] (h) cardiovascular conditions including circulatory diseases induced or exasperated by an inflammatory response, such as ischemia, atherosclerosis, peripheral vascular disease, restenosis following angioplasty, inflammatory aortic aneurysm, vasculitis, stroke, spinal cord injury, congestive heart failure, hemorrhagic shock, ischemia/reperfusion injury, vasospasm following subarachnoid hemorrhage, vasospasm following cerebrovascular accident, pleuritis, pericarditis, and the cardiovascular complications of diabetes;
[0043] (i) dialysis, including pericarditis, due to peritoneal dialysis;
[0044] (j) gout; and
[0045] (k) chemical or thermal trauma due to burns, acid, alkali and the like.
[0046] Of particular interest and efficacy is the use of the present compounds to treat inflammatory responses due to organ, tissue or cell transplantation, i.e., the transplantation of allogeneic or xenogeneic tissue into a mammalian recipient, autoimmune diseases and inflammatory conditions due to circulatory pathologies and the treatment thereof, including angioplasty, stent placement, shunt placement or grafting. Unexpectedly, it was found that administration of one or more compounds of formula (I) was effective after the onset of the inflammatory response, e.g., after the subject was afflicted with the pathology or trauma that initiates the inflammatory response.
[0047] The invention also includes a method for measuring the response, or binding a compound of formula I at or to designated A2A adenosine receptor sites comprising said receptors, in vivo or in vitro, with an amount of a compound of formula I effective to bind to said receptors. Tissue or cells comprising ligand bound receptor sites can be used to measure the selectively of test compounds for specific receptor subtypes, the amount of bioactive compound in blood or other physiological fluids, or can be used as a tool to identify potential therapeutic agents for the treatment of diseases or conditions associated with receptor site activation, by contacting said agents with said ligand-receptor complexes, and measuring the extent of displacement of the ligand and/or binding of the agent, or the cellular response to said agent (e.g., cAMP accumulation).
[0048] The following definitions are used, unless otherwise described. Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl, alkylaryl, etc. denote both straight and branched alkyl groups; but reference to an individual radical such as �propyl� embraces only the straight chain radical, a branched chain isomer such as �isopropyl� being specifically referred to. Aryl includes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H,O (C1-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
[0049] It will be appreciated by those skilled in the art that the compounds of formula (I) have more than one chiral center and may be isolated in optically active and racemic forms. Preferably, the riboside moiety of formula (I) is derived from D-ribose, i.e., the 3′, 4′-hydroxyl groups are alpha to the sugar ring and the 2′ and 5′ groups is beta (3R,4S,2R,5S). When the two groups on the cyclohexyl group are in the 4-position, they are preferably trans. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, or enzymatic techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine adenosine agonist activity using the tests described herein, or using other similar tests which are well known in the art.
[0050] Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
[0051] Specifically, (C1-C 6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl. As used herein, the term �cycloalkyl� encompasses bycycloalkyl (norbomyl, 2.2.2-bicyclooctyl, etc.) and tricycloalkyl (adamantyl, etc.), optionally comprising 1-2 N, O or S. Cycloalkyl also encompasses (cycloalkyl)alkyl. Thus, (C3-C 6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C 6)cycloalkyl(C1-C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl;, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl.
[0052] (C1-C 6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1-C6)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, b 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1 -hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1-6)alkoxycarbonyl (CO2R2) can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, puridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).
[0053] A specific value for R is amino, monomethylamino or cyclopropylamino.
[0054] A specific value for R′ is carboxy- or (C1-C4)alkoxycarbonyl-cyclohexyl(C1-C4)alkyl.
[0055] A specific value for R2 is H or (C1-C4)alkyl, i.e., methyl or ethyl.
[0056] A specific value for R3 is H, methyl or phenyl.
[0057] A specific value for R4 is H, methyl or phenyl.
[0058] A specific value for Z is �CH2� or �CH2�CH2�.
[0059] A specific value for X is CO2R2, (C2-C5)alkanoylmethyl or amido.
[0060] A specific value for n is 1.
[0061] Preferred compounds of formula (I) are those wherein each R is H, X is ethylaminocarbonyl and R′ is 4-carboxycyclohexylmethyl (DWH-146 a), R′ is 4-methoxycarbonylcyclohexylmethyl (DWH-146 e) or R1 is 4-acetoxymethyl-cyclohexylmethyl (JMR-193). They are depicted below (DWH-146 (acid) and methylester (e)) and JMR-193.
[0062] The synthesis of methyl 4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate (DWH-146 e) was accomplished by the cross coupling of an iodo-adenosine derivative (N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide) with methyl 4-(2-propynyl)-1 -cyclohexanecarboxylate by utilization of a Pd11 catalyst. The synthesis of the iodo-adenosine derivative was accomplished from guanosine. Guanosine is first treated with acetic anhydride, which acetalates the sugar hydroxyls, followed by the chlorination of position 6 with tetramethyl ammonium chloride and phosphorousoxychloride. Iodination of position 2 was accomplished via a modified Sandmeyer reaction, followed by displacement of the 6�Cl and sugar acetates with ammonia. The 2′ and 3′ hydroxyls were protected as the acetonide and the 5′ hydroxyl was iodized to the acid with potassium permanganate. Deptrotection of the 2′ and 3′ acetonide, Fisher esterification of the 5′ acid with ethanol and conversion of the resulting ethyl ester to the ethyl amide with ethylamine gave N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-p-D-ribofuranuoramide.
[0063] The acetylene (methyl 4-(2-propynyl)-1 -cyclohexanecarboxylate) was synthesized starting from trans-1,4-cyclohexanedimethanol. Initially the trans-diol was monotosylated followed by displacement of the tosylate with an acetylene anion. The hydroxyl of the resulting hydroxyl acetylene species was oxidized to the acid via Jones reagent followed by methylation with (trimethylsilyl)diazomethane to give methyl 4-(2-propynyl)-1-cyclohexanecarboxylate.
[0064] The cross-coupling reaction was performed under the following previously reported conditions. To a solution of N,N-dimethylfornamide (0.5 mL), acetonitrile (1 mL), triethylamine (0.25 mL), and N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-μ-D-ribofuranuroamide (25 mg, 0.06 mmol) was added bis(triphenylphosphine)palladium dichloride (1 mg, 2 mol%) and copper(I)iodide (0.06 mg, .5 mol %). To the resulting mixture was added methyl 4-(2-propynyl)-1-cyclohexanecarboxylate (54 mg, 0.3 mmol) and the reaction was stirred under N2 atmosphere for 16 hours. The solvent was removed under vacuum and the resulting residue was flash chromatographed in 20% methanol in chloroform (R17═0.45) to give 19 mg (off-white solid, mp 125� C. (decomposed)) of 4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3 ,4-dihydroxytetrahydro-Z-furanyl)-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate (DWH-146e).
[0065] DWH-146 e and JMR193 are substantially more potent as inhibitors in inflammatory model systems than the reference compound, CGS21680 (2-[p-(carboxyethyl)-phenyl-ethylamino]5′-N-ethylcarboxamidoadenosine). For example, DWH-146 e is about 80 times more potent at A2A receptors and 40 times more selective for A2A over A3 receptors than is CGS21680.
[0066] Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, malate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
[0067] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
[0068] The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
[0069] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
[0070] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
[0071] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0072] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0073] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
[0074] For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
[0075] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
[0076] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
[0077] Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0078] Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. Useful dosages of Type IV PDE inhibitors are known to the art. For example, see, U.S. Pat. No. 5,877,180, Col. 12.
[0079] Generally, the concentration of the compound(s) of formula (I) in a liquid composition, such as a lotion, will be from about 0.1-25% wt- %, preferably from about O.5-10 wt- %. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt- %, preferably about 0.5-2.5 wt- %.
[0080] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
[0081] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 μg/kg, e.g., from about 10 to about 75 μg/kg of body weight per day, such as 3 to about 50 μg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 μg/kg/day, most preferably in the range of 15 to 60 μg/kg/day.
[0082] The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 μg, conveniently 10 to 750 μg, most conveniently, 50 to 500 μg of active ingredient per unit dosage form.
[0083] Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.1 to about 10 nM, preferably, about 0.2 to 10 nM, most preferably, about 0.5 to about 5 nM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 μg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 μg/kg/hr or by intermittent infusions containing about 0.4-15 μg/kg of the active ingredient(s).
[0084] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. For example, it s desirable to administer the present compositions intravenously over an extended eriod of time following the insult that gives rise to inflammation.
[0085] The ability of a given compound of the invention to act as an A2A adenosine receptor agonist (or antagonist) may be determined using pharmacological models which are well known to the art, or using tests described below.
[0086] The invention will be firther described by reference to the following detailed examples, which are given for illustration of the invention, and are not intended to be limiting thereof.
[0087] Sodium hydride (1.68 g, 70 mmol) was added to a solution of 10 g (70 mmol) [4-(hydroxymethyl)cyclohexyl]methan-1-ol (5.1) in 700 mL of tetrahydrofuran and stirred for 1 hour p-toluenesulfonyl chloride (13.3 g, 70 mmol) was then added and the reaction mixture was refluxed for 5 hours. The reaction was then cooled to 0� C. and slowly quenched with water until there is no more reactive hydride. Once the hydride was quenched, the reaction mixture was diluted with ether (700 mL) and extracted 2 times with 10% aqueous potassium carbonate (700 mL). The organics were dried using sodium sulfate and the solvent was removed under reduced pressure. The product was purified by chromatography on silica gel column eluting with acetone-dichloromethane (5:95) to give 5.2 (35%). 1H NMR (300 MHz, CDCl3) δ7.75 (d, J═8.3 Hz, 2H), 7.32 (d, J═8.1 Hz, 2H), 3.79 (d, J═6.35 Hz, 2H), 3.39 (d, J═6.35 Hz, 2H), 2.42 (s, 3H), 1.75 (m, 4H), 1.59 (m, 1H), 1.37 (m, 1H), 0.9 (m, 4H). 13C NMR (300 MHz, CDCl3) δ145.3, 133.4, 130.3, 130.3, 128.3, 128.3, 75.8, 68.5, 40.6, 37.8, 28.9, 28.9, 28.9, 28.9, 22.1.
[0088] Lithium acetylide ethylenediamine complex (90%) (6.4 g, 70 mmol) was added very slowly to a solution of 5.2 (3 g, 10 mmol) in 40 mL of dimethylsulfoxide. The reaction mixture was allowed to stir for 5 days and then slowly quenched at 0� C. with water. This mixture was diluted with ether (300 mL) and extracted 3 times with saturated aqueous ammonium chloride (200 mL). The organics were dried with sodium sulfate. The solvent was removed under reduced pressure and the product was purified by chromatography on silica gel column eluting with ethyl acetate-hexanes (20:80) to give 5.3 (85%). 1H NMR (300 MHz, CDCl3) δ3.41 (d, J═6.5 Hz, 2H), 2.07 (dd, J═2.5, 6.5 Hz, 2H), 1.96-1.75 (m, 5H), 1.41 (m, 2H), 0.095 (m, 4). 13C NMR (300 MHz, CDCl3) δ83.8, 69.6, 68.9, 40.7, 37.7, 32.3, 32.3, 29.6, 29.6, 26.5.
[0089] A solution of chromium trioxide (1.1 g, 11 mmol) in 1.5 M sulfuric acid (40 mL, 27 mmol) was maintained at 0� C. while 5.3 (0.46 g, 3 mmol) in 80 mL of acetone was added over 2 hours. The reaction was then stirred for an additional 2 hours at room temperature. The reaction mixture was diluted with ether (200 mL) and extracted 2 times with water. The organics were dried with sodium sulfate. The solvent was removed under reduced pressure and the product was purified by chromatography on silica gel column eluting with acetone-dichloromethane (70:30) to give 5.4 (75%). 1H NMR (300 MHz, CDCl3) δ2.24 (dt, J═3.66, 12.1 Hz, 1H), 2.10 (dd, J═2.7, 6.5 Hz, 2H), 2.04-1.89 (m, 5H), 1.76 (d,J═2.3 Hz, 1H), 1.43 (dq,J═3.28, 13.1 Hz, 2H), 1.03 (dq, J═3.28, 13.1 Hz, 2H). 13C NMR (300 MHz, CDC13) δ183.2, 83.3, 69.9, 43.4, 36.7, 31.8, 28.9, 26.3.
[0090] (Trimethylsilyl)diazomethane (2.0 M) solution in hexanes (1 mL, 2 mmol) was added to a solution of 5.4 (0.34 g, 2 mmol) in 15 mL of methanol:dichloromethane (3:7). The solvents were removed under reduced pressure resulting in 100% conversion of starting material to product. 1H NMR (300 MHz, CDCl 3) δ2.24 (dt, J═3.66, 12.1 Hz, 1H), 2.10 (dd, J═2.7, 6.5 Hz, 2H), 2.06 (dd, J═1.54, 6.54 Hz, 1H), 2.00-1.89 (m, 3H), 1.76 (d, J═2.3 Hz, 1H), 1.43 (dq, J�3.28, 13.1 Hz, 2H), 1.03 (dq, J═3.28, 13.1 Hz, 2H). 13C NMR (300 MHz, CDC13) δ176.8, 83.3, 69.8, 51.9, 43.4, 36.7, 31.9, 29.2, 26.3.
[0091] A suspension of 113 g (0.4 mol) of dry guanosine (6.1), acetic anhydride (240 mL, 2.5 mol), dry pyridine (120 mL) and dry DMF (320 mL) was heated for 3.75 hours at 75� C. without allowing the temperature to exceed 80� C. The clear solution was then transferred to a 3L Erlenmyer flask and filled with 2-propanol. Upon cooling the solution to room temperature crystallization was initiated and allowed to proceed at 4� C. overnight. The white solid filtrate was filtered, washed with 2-propanol and recrystallized from 2-propanol to give 6.2 (96%). 1H NMR (300 Mhz, CDCl3) ε8.20 (s, 1H, H-8), 6.17 (d, J═5.41 Hz, 1 H, H-1′) 5.75 (t, J═5.39 Hz, 1H, H-2′), 5.56 (t, J═5.0, H-3′), 4.41 (m, 3H, H-4′,5′), 2.14 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.10 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) δ171.0, 170.3, 1702, 157.7, 154.8, 152.4, 136.7, 117.7, 85.5, 80.4, 73.0, 71.3, 64.0, 31.3, 21.2, 21.0.
[0092] To a 1000 mL flask was added 80 g (0.195 mol) [(2R,3R,4R,5R)-3-4-diacetyloxy-5-(2-amino-6-oxohyropurin-9-yl)oxolan-2-yl]methyl acetate (6.2), tetramethylammonium chloride (44 g, 0.4 mol), anhydrous acetonitrile (400 mL) and N,N-dimethlaniline (25 mL). The flask was placed in an ice salt bath and cooled to 2� C. To this solution was added dropwise POCl3 (107 mL 1.15 mol) at a rate that maintained the temperature below 5� C. (45 minutes). The flask was then removed from the ice bath, outfitted with a condenser, placed in an oil bath and allowed to reflux for 10 minutes whereas the solution changed to a red/brown color. The solvent was then removed under reduced pressure to yield an oily residue which was transferred to a beaker containing 1000 g of ice and 400 niL of CHCl3 and allowed to stir for 1.5 hours to decompose any remaining POCl3. The organic phase was then removed and the aqueous phase extracted with 3 �50 mL of CHCl3 and pooled with the organic phase. The pooled organic was then back extracted with 50 mL of water followed by stirring with 200 mL of saturated NaHCO3. The organic was further extracted with NaHCO3 until the aqueous extract was neutral (2X). The organic was finally extracted with brine and then dried over MgSO4 for 16 hours. To the solution was added 800 mL of 2-propanol after which the solution was concentrated under reduced pressure. To the oily solid was added 200 mL of 2-propanol and the solution was refrigerated overnight. The crystalline product was filtered, washed, and allowed to dry overnight to give 6.3 (77%). 1H NMR (300 MHz, CD3OD) δ8.31 (s, 1H, H-8), 7.00 (s,2H, NH2) 6.06 (d, J═5.8 Hz, 1H, H-1′), 5.83 (t, J═6.16 Hz, 1H, H-2′), 5.67 (m, 1H, H-3′), 4.29 (m, 3H, H-4′, 5′), 2.07 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) δ171.0, 170.4, 170.2, 160.8, 154.6, 150.8, 142.2, 124.5, 85.8, 80.6, 72.8, 71.2, 63.9, 21.4, 21.3, 21.1.
[0093] Isoamyl nitrite (5 mL, 37 mmol) was added to a mixture of 5.12 g (12 mmol) [(2R,3R,4R,5R)-3-,4-diacetyloxy-5-(2-amino-6-chloropurin-9-yl)oxolan-2-yl]methyl acetate (6.3), I2 (3.04 g, 12 mmol), CH2I2 (10 mL, 124 mmol), and CuI (2.4 g, 12.6 mmol) in THF (60 mL). The mixture was heated under reflux for 45 minutes and then allowed to cool to room temperature. To this solution was added 100 ml of sat. Na2S2O3 which removed the reddish color due to iodine. The aqueous was extracted 3X with chloroform, which was pooled, dried over MgSO4, and concentrated under reduced pressure. The product was then purified over a silica gel column using CHCl3�MeOH (98:2) to collect [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) (80% crystallized from EtOH). 1H NMR (300 MHz, CDCl3) δ8.20 (s, 1H H-8), 6.17 (d, J═5.41 Hz, 1H, H-1′), 5.75 (t, J═5.39 Hz, 1H, H-2′), 5.56 (t, J═5.40 Hz, 1H, H-3′), 4.38 (m, 3H, H-4′,5′), 2.14 (s, 1H, Ac), 2.11 (s, 1H, Ac), 2.10 (s, 1H, Ac).
[0094] To a flask containing 6.0 g (11.1 mmol) [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) was added 100 ml of liquid NH3 at �78� C. and the solution was allowed to stir for 6 hours. After which time it was allowed to come to r.t. overnight with concurrent evaporation of the NH3 to yield a brown oil. The product was crystallized from hot isopropanol to give 6.5 (80%), m.p. 143-145� C., r.f.═0.6 in 20% MeOH/CHCl3. 1H NMR (300 MHz, DMSO-d6) δ8.24 (s, 1H), 7.68 (s, 2H), 5.75 (d, J═6.16, 1H), 5.42 (d, J═5.40 Hz, 1H), 5.16 (d, J═4.62 Hz, 1H), 4.99 (t, J═5.39 Hz, 1H), 4.67 (d, J═4.81 Hz, 1H), 4.06 (d, J═3.37 Hz, 1H), 3.89 (m, 1H), 3.54 (m, 2H).
[0095] To a solution of 2.0 g (5.08 mmol) (4S,2R,3R,5R)-2-(6-amino-2-iodopurin-9-yl)-5(hydroxymethyl)oxolane-3,4-diol (6.6) in 100 mL acetone was added 9.6 g of p-toluenesulfonic acid and 5 ml of dimethoxypropane. The reaction was stirred at room temperature for 1 hour at which time 15 g of NaHCO3 and then stirred for an additional 3 hours. the residue was filtered and washed 2X with EtOAc. The filtrate was then concentrated under reduced pressure. The residue was chromatographed on a silica gel column with MeOH-CHCl3 (1:99) to give 6.6 (72%) as a solid, m.p. 185-187� C. 1H NMR (300 MHz, DMSO-d6) δ8.22 (s, 1H, H-8), 7.69 (s, 2H), NH2), 6.00 (d, J═2.70 Hz, 1H, H-1′), 5.21 (m, 1H, H-2′), 5.07 (bs, 1H, OH), 4.88 (m, 1H, H-3′), 4.13 (m, 1H, H-4′), 3.47 (m, 2H, H-5′), 1.49 and 1.28 (s, 3H, C(CH3)2).
[0096] To a stirred solution of 1.6 g (3.7 mmol) of [(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7-7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (6.6) in 200 mL of H2O was added 0.60 g of KOH and, dropwise, a solution of 1.70 g (10.8 mmi) of KMnO4 in 50 mL of H2O. The mixture was set aside in the dark at room temperature for 225 hours. The reaction mixture was then cooled to 5-10� C. and decolorized by a solution of 4 mL of 30% H2O2 in 16 mL of water, while the temperature was maintained under 10� C. using an ice-salt bath. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure to about 10 mL and then acidified to pH 4 with 2N HCl. The resulting precipitate was filtered off and washed with ether to yield 6.7 (70%) after drying as a white solid, m.p. 187-190� C. 1H NMR (300 MHz, DMSO-d6) δ8.11 (s, 1H, H-8), 7.62 (s, 2H, NH2), 7.46 (s, 1H, COOH), 6.22 (s, 1H, H-1 ′), 5.42 (d, J═5.71 Hz, 1H, H-2′), 5.34 (d, J═6.16 Hz, 1H, H-3′), 4.63 (s, 1H, H-4′), 1.46 and 1.30 (s, 3H, C(CH3)2).
[0097] A solution of 1.72 g (3.85 mmol) of (2S,1 R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]octane-2-carboxylic acid (6.7) in 80 mL of 50% HCOOH was stirred at 80� C. for 1.5 hours. The reaction mixture was evaporated under reduced pressure, dissolved in H2O, and the solution was evaporated again. This process was repeated until there was no odor of formic acid in the residue. Recrystallization from water yielded 1.33 g (85%) 6.8 as a white solid, m.p. 221-223� C. dec. 1H NMR (300 MHz, DMSO-d6) δ8.31 (s, 1H, H-8), 7.68 (s, 2H, NH2), 5.90 (d, J═6.55 Hz, 1H, H-1′), 4.42 (m, 1H, H-2′), 4.35 (d, J═2.31 Hz, 1H, H-4′), 4.22 (m, 1H, H-3′).
[0098] To a cooled (5� C.) and stirred solution of 1.29 g (3.17 mmol) of (2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolane-2-carboxylic acid (6.8) in 150 mL of absolute ethanol was added dropwise 1.15 mL of ice-cooled SOCl2. The mixture was stirred at room temperature overnight and then brought to pH 8 with saturated aqueous NaHCO3. The mixture was filtered, and then the filtrate was concentrated under reduced pressure to yield a white solid which was dried and then redissolved in 20 mL of dry ethylamine at �20� C. for 3 hours and then at room temperature overnight. The reaction mixture was diluted with absolute ethanol, and the precipitated product was filtered off and washed with dry ether to give 530 mg (72%) of 6.9 as a pure solid, m.p. 232-234� C. 1H NMR (300 MHz, DMSO-d6) δ8.34 (s, 1H, H-8), 8.12 (t, 1H, NH), 7.73 (s, 2H, NH2), 5.85, (d, J═6.93 Hz, 1H, H-1′), 4.54 (m, 1H, H-2′), 4.25 (d, J═1.92 Hz, 1H, H-4′), 4.13 (m, 1H, H-3′), 3.28 (m, 2H, CH2CH3), 1.00 (t, J═7.2 Hz, 3H, CH2CH3).
[0099] To a degassed solution of 25 mg (0.063 mmol) of [(2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-N-ethylcarboxamide (6.9), 16.9 mg (0.094 mmol) (5.5), and 0.75 mg CuI in 5 mL each of TEA and acetonitrile was added 15 mg of Pd(PPh3)4. The solution was stirred for 24 hours at 70� C. after which time the solution was filtered through celite and chromatographed on silica gel with MeOH-CHCl3 (5:95) to give DWH-146 e (24%).
[0100] Acetic anhydride (0.92 mL, 8.25 mmol) and pyridine (0.2 mL, 2.5 mmol) were added to a solution of 5.3 (250 mg, 1.65 mmol) in 25 mL ether. The reaction was allowed to stir at ambient temperature for 24 hours. Water was added to the reaction and the organic was further extracted with 10% NaHCO3. The organic layer was dried with MgSO4 and evaporated. The residue was chromatographed on silica gel with EtOAc-Hexanes (5:95) to yield 5.6 (47%).
[0101] To a degassed solution of 125 mg (0.29 mmol) of [(2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-N-ethylcarboxamide (6.9), 150 mg (0.77 mmol) (5.6), and 1.0 mg CuI in 1.3 mL of TEA and 4 mL DMF was added to 25 mg of Pd(PPh3)4. The solution was stirred for 72 hours at 60� C. after which time the solution was filtered through celite and chromatographed on silica gel with MeOH-CHCl3 (5:95) to give JMR193 (10%).
[0102] Binding to A2A receptors was evaluated with the radioligand 125-ZM241385. FIG. 2B depicts the competition by selective agonists for binding to recombinant human A2A adenosine receptors. DWH-146 e is highly selective for the recombinant human A2A (hA2A) subtype. Selectivity for the A3 receptor (not shown) is less impressive, but still about 50-fold. DWH-146 e is about 5 and 50 times more potent than WRC0470 and CGS21680, respectively (FIG. 1). An unexpected and interesting finding is that the ester, DWH-146 e also is about 50 times more potent than the acid, DWH-146 a (FIG. 1).
[0103] A. Materials.
[0104] f-met-leu-phe (fMLP), luminol, superoxide dismutase, cytochrome C, fibrinogen, adenosine deaminase, and trypan blue were obtained from Sigma Chemical. Ficoll-hypaque was purchased from ICN (Aurora, OH), and Cardinal Scientific (Santa Fe, NM) and Accurate Chemicals and Scientific (Westerbury, NY). endotoxin (lipopolysaccharide; E. coli K235) was from List Biologicals (Campbell, CA). Hanks balanced salt solution (HBSS), and limulus amebocyte lysate assay kit were from BioWittaker (Walkersville, MD). Human serum albumin (HSA) was from Cutter Biological (Elkhart, IN). Recombinant human tumor necrosis factor-alpha was supplied by Dianippon Pharmaceutical Co. Ltd. (Osaka, Japan). ZM241385 (4-(2-[7-amino-2-(2-furyl)-[ 1 ,2,4]-triazolo[2,3-a][1 ,3,5]triazin-5-yl amino]ethyl)phenol) was a gift from Simon Poucher, Zeneca Pharmaceuticals, Cheshire, UK. Stock solutions (1 mM and 10 mM in DMSO) were made and stored at �20� C.
[0105] B. Human neutrophil preparation
[0106] Purified neutrophils (�98% neutrophils and >95% viable by trypan blue exclusion) containing <1 platelet per 5 neutrophils and <50 pg/ml endotoxin (limulus amebocyte lysate assay) were obtained from normal heparinized (10 U/ml) venous blood by a one step Ficoll-hypaque separation procedure (A. Ferrante et al., J. Immunol. Meth., 36, 109 (1980)).
[0107] C. Release of inflammatory reactive oxygen species from primed and stimulated human neutrophils Chemiluminescence
[0108] Luminol-enhanced chemiluminescence, a measure of neutrophil oxidative activity, is dependent upon both superoxide production and mobilization of the lysosomal granule enzyme myeloperoxidase. The light is emitted from unstable high-energy oxygen species generated by activated neutrophils. Purified neutrophils (5-10 x 105/ml) were incubated in Hanks balanced salt solution containing 0.1% human serum albumin (1 ml) with or without DWH-146 a, DWH-146 e , CGS21680, or JMR193 with or without rolipram and with or without tumor necrosis factor-alpha (1 U/ml) for 30 minutes at 37� C. in a shaking water bath. Then luminol (1�10�4 M) enhanced f-met-leu-phe (1 mcM) stimulated chemiluminescence was read with a Chronology Photometer (Crono-log Corp., Havertown, PA) at 37� C. for 2-4 minutes. Chemiluminescence is reported as relative peak light emitted (═height of the curve) compared to samples with tumor necrosis factor-alpha and without DWH, JMR or rolipram.
[0109] D. Results
[0110] As shown in FIG. 2, JMR193 and DWH-146 e both decreased tumor necrosis factor-alpha-primed f-met-leu-phe-stimulated human neutrophil oxidative activity as measured by luminol-enhanced chemiluminescence more effectively than the adenosine A2A receptor agonist CGS21680. The horizonal axis gives the concentration of CGS21680, DWH-146 a, DWH-146 e or JMR193 (log nM). The vertical axis gives the resulting peak human neutrophil activity as relative amount of stimulated release of reactive oxygen species as measured with luminol-enhanced chemiluminescence compared to control samples which were not primed with tumor necrosis factor-alpha. Means SEM (n═4-5 separate experiments).
[0111] The data below the horizontal axis in FIG. 2 gives the EC50 for reducing the human neutrophil activity (based on the data in FIG. 2). Means SEM (n═4-5 separate experiments). *p<0.05 decreased IC50 compared to CGS21680.
[0112] JMR193 and DWH-146 e decreased the stimulated-neutrophil oxidative burst with EC50's less than 1 nM (0.8 and 0.3 nM, respectively). In contrast, the free acid A2A adenosine receptor agonists DWH-146 a and CGS21680 were not as effective in inhibiting the oxidative burst (53 and 9 nM, respectively; FIG. 2). DWH-146 e inhibition of the stimulated neutrophil oxidative burst was antagonized by the selective A2A AR antagonist ZM241385.
[0113] As shown in FIG. 3, JMR193 (1 nM) with rolipram (100 nM) synergistically decreased stimulated release of reactive oxygen species. Human neutrophils were primed with tumor necrosis factor-alpha (1 U/ml) and stimulated with f-met-leu-phe (1 μM). The vertical axis gives the percent inhibition of oxidative activity as measured by luminol-enhanced chemiluminescence. Means SEM (n═4 separate experiments. *p <0.05 synergy between JMR193 and rolipram compared to additive activity.
[0114] As shown in FIG. 4, the highly selective A2A adenosine receptor antagonist ZM241385 (100 nM) (ZM) counteracted human neutrophil oxidative activity inhibited by JMR193 (10 nM) as measured by luminol-enhanced chemiluminescence. Means SEM of 4 separate experiments. *p═0.0004 ZM241385 counteracted JMR193 inhibited oxidative activity.
[0115] E. Human neutrophil [cAMP]and neutrophil adherence to a biological surface
[0116] A 24 well tissue culture plate was coated with human fibrinogen (5 mg/ml in 1.5% sodium bicarbonate; 0.5 ml/well; Sigma Chemical) overnight at 37� C. in 5% CO2. Neutrophils (3-4�106/0.5 ml/sample) were incubated within a well of the coated plate for 45 minutes in 0.5 ml of HBSS containing 0.1% HSA and ADA (1 U/ml) in the presence and absence of recombinant human TNFα (10 U/ml), DWH-146 e (3-300 nM), rolipram (300 nM), and/or ZM241385 (100 nM). Following incubation, 0.5 ml HCl (0.2 N) was added to the wells and incubated for 45 minutes more at room temperature to extract the cAMP. The samples were then centrifuged in a microfuge for 2 minutes to remove cell debris. Half ml samples were frozen for cAMP analyses (B. Brooker et al., Science, 194, 270 (1976)). The wells were washed twice with normal saline and the remaining monolayer digested with 0.2 ml of 0.2 N NaOH containing SDS for 2 hours at room temperature. The protein samples were then frozen (−70� C.) for later protein analysis to determine relative PMN adherence (K. P. Stowell et al., Anal. Biochem., 85, 572 (1978)).
[0117] DWH-146 e (30-300 nM) alone and synergistically with rolipram (300 nM) increased human neutrophil cAMP content and with rolipram synergistically decreased neutrophil adherence to a fibrinogen-coated surface (FIG. 5). The effects of DWH-146 e (300 nM) +rolipram (300 nM) on neutrophil cAMP production and adherence were counteracted by the selective A2A adenosine receptor antagonist, ZM241385 (ZM; 100 nM). Mean SEM of 5 separate experiments. *p <0.05 increased neutrophil [cAMP] compared to without DWH-146 e ; **p <0.05 decreased neutrophil adherence compared to no DWH-146 e .
[0118] F. Adherent human neutrophil oxidative activity
[0119] Methods. Using methods modified from Section E, neutrophils (2�106/ml) from Ficoll-Hypaque separation were incubated 15 minutes 37� C. in 0.45 ml of Hanks balanced salt solution containing 0.1% human serum albumin and adenosine deaminase (1U/ml), rolipram (300 nM), and DWH-146 e (3-300 nM). Following incubation, cytochrome C (120 μM) and catalase (0.062 mg/ml) are added in the presence and absence of recombinant human tumor necrosis factor-alpha (1 U/ml) and 200 μl aliquots of cell suspension were immediately transferred to duplicate wells of a 96 well flat-bottomed tissue culture plate which had been coated overnight with human fibrinogen. The optical density of the samples were read at 550 nm against matched superoxide dismutase (200 U/ml) samples.
[0120] G. Results
[0121] As shown in FIG. 6, inhibition of tumor necrosis factor-alpha (TNF)-stimulated adherent human neutrophil superoxide release on a fibrinogen-coated surface was accomplished by rolipram (300 nM) and DWH-146 e . DWH-146 e decreased the oxidative burst of adhering neutrophils, and synergistically decreased the oxidative burst in the presence of rolipram, which by itself did not affect neutrophil oxidative activity. The horizontal axis gives the DWH-146 e concentration in nM and the vertical axis gives the amount of superoxide released by the neutrophils as measured by cytochrome c reduction. There was marked synergy with DWH-146 e and the type IV PDE inhibitor, rolipram, to decrease tumor necrosis factor-alpha-stimulated adherent human neutrophil oxidative activity. Means SEM of replicates from 4-5 separate experiments. *p <0.05 decreased superoxide release compared to without DWH-146 e; **p <0.05 decreased superoxide release compared to with rolipram and without DWH-146 e .
[0122] To determine whether or not DWH-146 e induced A2A adenosine receptor activation reduces plasma creatinine at 24 and 48 hours following I/R injury in rats, rat kidneys were subjected to 45 minutes ischemia and 24 or 48 hours of reperfusion. DWH-146 e (0.004 μg/kg/min) or vehicle was administered continuously via minipump beginning 5 hours prior to I/R. As shown in FIG. 7, DWH-146 e significantly decreased plasma creatinine in 7/7 rats (P <0.05) and in 6/6 rats treated with DWH-146 e (P <0.001), at 24 and 48 hours, respectively.
[0123] To determine whether or not the effect of DWH-146 e on reduction of plasma creatinine in rats subjected to I/R is A2A -receptor mediated, rat kidneys were subjected to 45 minutes ischemia followed by 48 hours reperfusion. DWH-146 e (0.004 μg/kg/min) was administered continuously via minipump beginning 5 hours prior ischemia. As shown in FIG. 8, the improvement in renal finction was reversed by the A2A antagonist ZM-241385 (0.003 μg/kg/min-equimolar delivery rate compared with DWH-146 e ) (*P <0.001 for Vehicle vs. DWH; **P <0.05 DWH vs. DWH/ZM. N═5 for Vehicle, DW; N═6 for DWH/ZM. ANOVA followed by Bonferroni correction).
[0124] DWH-146 e, at concentrations that have no hemodynamic effects, prevents renal edema, necrosis and red cell pooling in the inner medulla.
[0125] The protection against renal damage afforded by DWH-146 e (0.01 μg/kg/min s.c. for 48 hours) was correlated with a dramatic inhibition of neutrophil adherence to vascular endothelium. It is believed that inhibition by DWH-146 e of the interaction between neutrophils and vascular endothelium is responsible, at least in part, for the protection against renal damage.
[0126] To determine whether or not A2A -AR activation reduces neutrophils in the outer medulla of rats subjected to I/R, using Neurolucida�, the kidney was viewed under 100 �mag and the entire kidney was drawn. PMNs were counted by viewing kidney sections under 250 �mag. Kidney sections were overlaid with optical frames viewed under the microscope and all PMNs were counted within each frame. This system prevents counting of PMNs more than once. As shown in FIG. 9, the density of neutrophils was 15.65/mm2 for vehicle and 3.02/mm2 for DWH-146 e treatment.
[0127] A. Methods. An isolated, whole blood-perfused, ventilated rabbit lung model was used. Donor rabbits underwent lung harvest after pulmonary arterial PGE1, injection and Euro-Collins preservation solution flush, and lungs were preserved for 18 hours at 4� C. Group I lungs (n═9) served as control subjects. Group II lungs (n═9) were reperfused with whole blood that was first passed through a leukocyte-depleting filter. In group III (n═9), DWH-146 e was added to the blood reperfusate (25 μg/kg) immediately before reperfusion and was administered throughout the reperfusion period (1 μg/kg/min). All lungs were reperfused for 30 minutes, and pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), airway compliance (CPL) and arterial oxygenation were recorded. Mycloperoxidase activity (MPO) was recorded to quantify neutrophil sequestration, and wet/dry weight ratios were measured to demonstrate pulmonary edema.
[0128] B. Results. Arterial oxygentation in group II and group III was significantly higher than that of group I after 30 minutes of reperfusion (514.27 �35.80 and 461.12 �43.77 vs. 91.41 �20.58 mm Hg, p<0.001. As shown in FIG. 10, group III lungs displayed a progressive involvement in pO2 throughout reperfusion. Leukocyte depletion in group II lungs improved arterial oxygenation in early reperfusion. *p═0.004 (group II versus groups I and III); **p<0.001 (groups II and III versus group I).
[0129] As shown in FIG. 11, mean PVR in group II was significantly reduced when compared to controlled lungs (*p<0.001). PVR of group III lungs was significantly lower than even those lungs that underwent reperfusion with leukocyte-depleted blood (**p<0.001 versus groups I and II). Pulmonary vascular resistance was significantly reduced in group III (22,783 �357 dynes.s.cm�5) compared to both group II and group 1 (31,057�1743 and 36,911�2173 dynes.s.cm�5, p<0.001). Airway compliance was improved in groups II and III when compared to group I (1.68�0.08 and 1.68�0.05 vs. 1.36�0.13, p═0.03). Microvascular permeability in group III was reduced to 106.82�17.09 compared with 165.70�21.83 ng Evans-blue dye/gm tissue in group I (p═0.05). As shown in FIG. 12, myeloperoxidase activity in group III was significantly lower than in group I (*p═0.03). MPO═myeloperoxidase. Group III myeloperoxidase activity was 39.88 �4.87 compared with 88.70�18.69 ΔOD/gm/min in group I (p═0.03), and group II myeloperoxidase activity was 56.06�7.46.
[0130] C. Conclusions. DWH-146 e reduced lung neutrophil sequestration and dramatically improved pulmonary graft function. Neutrophils are important components of the inflammatory cascade of reperfusion injury and their source may include both the circulating blood and the lung graft itself. Selective adenosine-A2A activation interrupts the neutrophil-mediated inflammatory response and reduces lung reperfusion injury following transplantation.
[0131] Under light microscopy, control lungs in group I showed severe leukocyte infiltration and edema formation in the alveolar spaces after 18 hours of ischemic storage and 30 minutes of reperfusion. In group II, lungs that underwent reperfusion with leukocyte-depleted blood and in group III lungs (that received DWH-146 e during reperfusion, this infiltration was much less.
[0132] Leukocyte activation with release of inflammatory cytokines occurs after percutaneous coronary intervention and may play a role in restenosis. In the mouse, robust neointima formation in the presence of an intact endothelial lining occurs after ligation of the common carotid artery. Using this model, C57/BL6 mice were randomized at the time of carotid ligation to a 7 day infusion via osmotic pump of DWH-146 e , (n═7), or vehicle (n═8).
[0133] At 14 days after carotid ligation, histomorphometry demonstrated a significant reduction in neointimal area (0.005�0.004 mm2 vs. 0.021�0.014 mm2, p═0.02) and neointimal to medial area ratio (0.13�0.07 vs. 0.64�0.44, p═0.01) in the treated animals compared to controls. Medial area was similar in the two groups (0.034�0.007 mm2 vs. 0.036�0.009 mm2,p═0.81). This benefit in limiting neointimal growth persisted to 28 days. FIG. 13 summarizes the effect of DWH-146 e to inhibit neointimal growth in the mouse LCCA model. These experiments demonstrate that, in a mouse carotid artery ligation model, prolonged A2A stimulation (7 days) by DWH-146 e results in a significant reduction in neointimal formation for at least 21 days, possibly through its effect on leukocyte activation and function.
[0134] A. Materials.
[0135] Ficoll-hypaque was purchased from ICN (Aurora, OH) and Cardinal Scientific (Santa Fe, NM) and Accurate Chemicals and Scientific (Westbury, NY). Endotoxin (lipopolysaccharide; E. coli 011 1B4) was from List Biologicals (Campbell, CA). Hanks balanced salt solution (HBSS), and limulus amebocyte lysate assay kit were from BioWittaker (Walkersville, MD). Human serum albumin (HSA) was from Cutter Biological (Elkhart, IN). ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol) was a gift from Simon Poucher, Zeneca Pharmaceuticals, Cheshire, UK. Stock solutions (1 mM and 10 mM in DMSO) were made and stored at −20� C.
[0136] B. Production of TNFα by purified human adherent monocytes.
[0137] Methods: A monocyte rich monolayer (>65% monocytes) was prepared by incubating 1 ml of the mononuclear leukocyte fraction (5�105/ml) from a Ficoll-Hypaque separation (A. Ferrante et al., J. Immunol. Meth., 36, 109 (1980)) in wells of a 24 well tissue culture plate (1 hr; 37� C; 5% CO2). The non-adherent leukocytes were removed by washing and culture medium (1 ml Hanks balanced salt solution, containing 0.1% human serum albumin, adenosine deaminase [5 U/ml] and 1% heat-inactivated autologous serum) added to the wells containing the adherent mononuclear cells. As stated, the following were added: (1) endotoxin (100 ng/ml) and the A2A AR selective antagonist ZM241385 (100 nM) and, (2) A2A adenosine receptor selective agonists JMR193 (1-1000 nM), DWH146 e (1-1000 nM) and CGS21680 (10-1000 nM). The samples were then incubated for 4 hours (37� C; 5% CO2) and the supernatants harvested. Any suspended cells were removed by centrifugation and the cell-free samples frozen (−70� C). TNFα was assayed in the cell-free supernatants (n═6) by an ELISA kit (Coulter/Immunotech, Miami, FL).
[0138] C. Results.
[0139] As shown in FIGS. 10, 14, the A2A adenosine receptor agonists-decreased endotoxin-stimulated adherent monocyte production of TNFα. The A2A AR selective antagonist ZM241385 (100 nM) antagonized the effects of JMR193 on TNFα production (FIG. 15).
[0140] Thus, DWH146 e and JMR193 decrease LPS endotoxin-stimulated TNFα production by human monocytes by a mechanism that is dependent upon agonist binding to A2A adenosine receptors.
[0141] Preliminary experiments with experimental peritonitis have involved the injection of zymosan (Zym) as a potent stimulus of inflammation (Y. Zhang et al., Eur. J. Pharmacol., 313, 237 (1996)). As shown in FIG. 16, following injection of zymosan, the mean leukocyte concentration as determined in a neubauer hemocytometer was 7,325�1,893/mm3. Intraperitoneal injection of DWH-146 e at a dosage of 2.5 μg/kg one hour prior to zymosan inhibited the development of peritonitis with a mean � SEM leukocyte concentration of 2,012�374/mm3 6 hours later (p<0.05). Thus, these studies demonstrate that the A2A AR is instrumental in mediating PMN traversal into the peritoneum following zymosan challenge.
[0142] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
Referenced by
Legal Events
Original Image


text:
Squeezebox Hero is likely Las Vegas' most rockin' accordionist, playing lots of rock and pop not usually associated with the squeezebox. His real name is Botielus, but once this composer and multi-instrumentalist straps on his axe, he becomes a zany character!
For live concerts, he performs with custom backing tracks he arranged and recorded himself. But his favorite gig is brightening days (or nights) for folks, with "Squeezagrams" - modern singing telegrams, with accordion! Easy to order direct from his website to surprise anyone almost anywhere, they are unique and unforgettable!
Squeezebox Hero has appeared on "America's Got Talent" and other television and radio shows. He has performed solo and with a variety of bands and tribute shows around the world. He plays saxophones, synthesizers and keyboards too, but enjoys adding accordion whenever possible.
He even rents accordions! If you wanna try one out for a month, or need one for just a day, contact him for a special deal! The squeezebox hero also reluctantly gives lessons! He has given lessons before to beginners that already know something about reading music (just need to know how to have fun squeezing the accordion!).
In addition to his Squeezagrams, he loves performing full concerts. Either on stage or at parties/events. It's like having Weird Al at your event, but way cheaper! Plus, the Squeezebox Hero plays LOTS more accordion in his concerts than Al!
Great for almost any location or event, including but not limited to bars, schools, ski lodges, and even funerals (but only for folks that had/have a great sense of humor!).
More blather:
10/14 just finished rockin' the Ojai Oktoberfest (in Ojai CA). Super fun oktoberfest, workers, fans, lots of tips, good beer, SUPER emcee dude (sp?)
9/14 - Played the famous Whiskey a Go Go in Hollywood and was on live, national axs television! Played synths with a tribute band. Rave reviews!
9/14 - played for the super Oregon Cycle event (and separate concert as "Happy Happy Boy Boy" with the super Tony Dunne!)
7/2/12: Just performed at a great backyard birthday party! What a great and huge crowd. We even did the Tequila conga. The only bad part was that I was in day 2 of a cold and my voice was severely limited (at least one octave gone at the top). But I think that made me even funnier, so I guess good after all :-) I just finished a new video demo edit of 3 of my concerts using my backing tracks (so I sound like a band). Really excited about my upcoming concert at Freakin' Frog on 7/18/12
Hhenfrey 2/13/15
Oh my goodness I loooove it!!!!!!!!! So hilarious!!!! I just watched this and had my office mates cracking up. You are the beeeeesssst!!!! Hugs!!
Hhenfrey 2/13/15
Got my responses in a matter of minutes! He includes every point and even threw in a little extra. So funny! The most unique present you could ask for! Thumbs up all the way!
aaronkaufman added 5 days ago
HOLY F***N SH**, you are a GOD, this is absolutely hilarious. You are the man, thanks so much. You need to be on SNL.
nle1975 added 9 days ago
Absolutely wonderful!! I love how you really seem to enjoy making people laugh. I'll definitely be keeping you as my favorite fiverr artist. I'll make sure to tell all my friends to check out your services! Great job and thanks for helping me make my sister's birthday great! Thanks again, Nicole
seahostler SqueezeBoxHero may be the greatest man to have ever lived. This birthday video was northing short of an opus. Thank you SO MUCH!!!
tytanious I absolutley LOVE SqueezeboxHero, his work is just fantastic. His humor is spot on. I couldn't recommend him high enough.
badponymusic Absolutely brilliant. Appreciate the work you put into the clip mate. Thanks a bunch!
eddiehovas Buddy your a crazy fun nut and I love it! You did a great job! Thanks and I will for sure use you again and spread the wild word!
patrikedlund Just fanstastic
cainbrian You are hilarious!!! :)
cripflip ,my god- that is amazing! thank you soooo much!! i may be disowned but it will be worth it!
sdanilov Funny, zany, unique - a perfect birthday gift for a guy who has everything. Just perfecto in every way! :)
acm_207 Thank you so much for the perfect delivery. You're a true professional.
xnevans This guy was astounding ! SQ Box Hero is A definite buy…whip your $5 bucks and send it his way! And feel free to upgrade to the extras! He's funny, clever, personable and adds a ton of improv….way beyond other 5=ers stuff. Turned the BDay request around in a heartbeat.
jenergy Fantastic! Will hire again!
glebensw Mr Squeezeboxhero ROCKS!
Singing Telegrams and Parties!

subset: commoncrawl


text:
Flying is the only way to get close enough to a cloud to milk it or to visit your secret sky family. Break free from earthly bonds with this Groupon.
$125 for a Powered-Parachute Lesson ($289 Value)
During 30-minute lessons, a seasoned instructor relays the ins and outs of powered parachuting, a safe type of flight where a motorized two-person cart hangs from a 40-foot parachute. Clad in comfortable clothes and closed-toe shoes, passengers take the helm or just kick back and relax while an expert navigates the aircraft low or high over the picturesque Sonoran Desert at speeds agreed on by the passenger. After landing, riders receive a first-flight certificate and a pilot's log book to vouch for their introductory adventure.
Arizona Powerchutes
Randy Long entered the working world as a travel agent, a vocation that whet his appetite for globetrotting, adventure, and haggling with airlines. When he became a father and husband, he passed a passion for thrill seeking on to his family, and their recent escapades include scuba diving in Barbados and dog sledding in Alaska. It was this thirst for exploration and a love of aviation that drove Randy to become an FAA-certified powered-parachute instructor and found Arizona Powerchutes.
Powered parachutes are comprised of two-seater, wheeled carts that float 20 feet beneath 40-foot parachutes. At sunrise—or sunset during the cooler months—Randy and a passenger climb aboard the cart, and Randy hits the throttle, gathering speed for about 100 feet before the parachute fully inflates and hoists the cart into the air. Randy adjusts the altitude to his patron's comfort level and steers crafts over the exotic plants and mountain silhouettes of the Sonoran Desert, averaging a speed of 26 miles per hour. After journeys, powered parachutes float to land safely, as they are inspected by the pilot prior to each flight and by an FAA-approved facility after every 100 hours of operation.
97% of 71 customers recommend
"A fun time great experience. "
"GREAT experience! Very friendly, and professional, made the most of our 30 minutes in the air no time wasted. Knowledgeable, and informative, made the flight very..."
"GREAT experience! Very friendly, and professional, made the most of our 30 minutes in the air no time wasted. Knowledgeable, and informative, made the flight very personally rewarding."
"Owner of the business was extremely friendly and provided me with a great experience. I would definitely recommend his services to others."
Arizona Powerchutes
31414 N Cave Creek Rd.
Cave Creek, AZ 85351
+14805027640Get Directions


text:
Hi guys,
I'm in the process of designing something new and want to make sure it'll work for most keggle owners, so I have question:What's the diameter of the hole you cut in the top of your keggle?
KalP.S. I'm an equal opportunity employer and will take measurements in inches, centimeters, furlongs, Planck lengths or even parsecs. Measurements done in Canas or cubits, however, are highly discouraged.
The opening on mine is around 12 inches
I believe mine is about 9.877895e-18 Pc. I based it off the size of the spare lid that I had available.
11.5".. I found a stainless lid that fits it perfectly.
12.5" inches. Cut at 12 then ground it out a little to fit a lid.
15.5" I cut the tops off
12" =/-
Primary #1 - Midnight Ryeder (Midnight Wheat and Rye)
Primary #2 - Mango Habenero Berliner Weisse
Primary #3 - EMPTY!
Secondary #1 - Downtown Flanders Brown (brewed August 2012)
Keg #1 - Oktoberfest
Keg #2 - Chamomile Honey Wheat
Keg #3 - Pumpkin Ale
Bottled - NONE!
about 12.5"


text:
tree root causes sewer backflow into house.
We have had the outside drain repaired and the restoration company sent by the insurance company has cleaned and set up fans. There is damage to the kitchen counters and floor and into the dining room carpet. Also drained through the basement ceiling into the laundry room and ran into the adjacent family room carpet. Ack! Any tips on with insurance and restoration companies? Questions I should be asking? I was told anything on the inside would be covered, replaced.
You'll need to find out if the insurance company is paying you, the mortgage company, or the contractor.
In the case of my roof, the check was paid to me but had to signed my the mortgage company before I could cash it and then pay the contractor.
There will also be a deductable and proration of items unless your policy calls for actual replacement.
Normally I wouldnâÂÂt recommend using an insurance companyâÂÂs contractor/mitigation company. They have loyalty to the insurance company, not you. When insurance companies have a claim, their main priority is to minimize expenses, PERIOD!!!
Insurance companies do this (among other ways) by applying pressure on their vendor to do the repairs their way and with their pricing structure. ThatâÂÂs why insurance companies push contractors on their vendor programs so hard.
If itâÂÂs a franchise company doing the work, even more reason to be cautious. Franchises pay a major chunk of their profits to the franchisor. With these funds coming off the bottom line, they usually donâÂÂt have the funds to hire better quality staff which usually means lesser quality service or repairs.
I could tell you a million and one reasons to be cautious with insurance companies and franchisees but your insurance claim is fairly minor. Even the worst restoration companies in this industry should be able to handle this without much problem.
Unfortunately, the adjusters that handle these small claims are typically the most incompetent boneheads on the planet. You are going to need to stay on top of the adjuster to make sure he/she does things in a timely and equitable manner.
Good luck,
My next door neighbor had a massive Maple limb fall onto their place and crushed an exterior wall. The insurance company hired their people to do repairs and they in turn sub contracted it to a few non licensed fly by nighters. 3 years latter if she plugs in a toaster the breaker trips. The list goes on and on. These guys had to borrow my Hydro everyday and were constantly looking to borrow tools from me. Insist on approving the contractor.
They are very laid back people, certainly more than I. I think they're retired hippies, and live with it, I wouldn't. Peace man.


text:
The Sims 3 To Mesh With Social Networks 25
from the linked-sim-book-space dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
I miss the period of time when The Sims had just come out, and it was such a neat toy to play with. It was more of an architecture and people simulator with a tongue-in-cheek, 1950s nuclear family tone, especially the music. The game began to morph into something else, with people roleplaying and storytelling. Websites started charging for access to their downloads. EA went overboard with the number of expansions, adding crazy stuff like robots and wizards. Even the music shifted into hip techno tracks as the game got huge in the mainstream.
They advertise the game now using Sims that look like teenyboppers out of an MTV reality show, and the clever 1950s tone of the original is gone. It's no longer a life simulator as much as it is a mass-appeal sandbox for user-created clothes and stupid machinima sitcoms, which bores me. I don't need a videogame to write my own stories. Guess I'm not a part of the core audience anymore.
You think it will converge with nethack one day? That could be cool.
Having DLC on launch day is definitely a polite way of saying they've taken some files off the disk and put them online for purchase. It's just a way to increase the price of the game without people realising it.
While in some cases this is true here is something for you to consider:
Because they plan on having launch day DLC they can afford to expand the budget of the game and put more money into developing it. Without the DLC this would simply raise the price of the game but now you can sell the core game for a reasonable price and allow people to spend more to get a more expanded version if they so wish.
I'm not saying it's what's going on here, it's just a scenario where day one DLC is not so bad
Never underestimate the bandwidth of a station wagon full of tapes. -- Dr. Warren Jackson, Director, UTCS


text:
Roll over image to magnify & Click to Zoom
Why Set & Save?
Just click "Add to Set & Save" when placing items in your cart.
Retail price: $12.95
Vitacost price: $9.49
This advanced sulfate-free formula reinforces hair and prevents future breakage with Rice and Organic Quinoa Protein Complex, while Vitamin B5 penetrates leaving hair noticeably more radiant and resilient with each use.
Vitacost price: $9.99
Vitacost price: $7.46
Vitacost price: $7.46
|Enter Shipping Zip Code:
|Please enter a valid zip code


text:
Celebrating Memorial Day
The Sunday before the last Monday in May: an important occasion to memorialize loved ones who have died in war or in other ways. Themes can include: remembering people who have died; the power of memory; ritualizing our memories; gratitude for those who have gone before us; the cost of war.
Unitarian Universalist Perspectives
Words for Worship Services
- Chalice Lighting for a Service on the Separation of Church and State
- Memorial Day
- Memorial Day Prayer
- Prayer for Memorial Day
Prayer of the War Atheist
Tagged as: Hiroshima Day, Martin Luther King, Jr. Sunday, Memorial Day, Nonviolence, Peace, Remembrance
Spoken and Silent Meditation or Prayer
Tagged as: Connections, Gratitude, Interdependence, Memorial Day, Unitarian Universalism
Veterans Day Prayer
Tagged as: Connections, Generations, God, Memorial Day, Peace, Violence, War
For more information contact email@example.com.


text:
We come to Keswick very regularly with our daughter and teenage friends.Teenagers can be very surly so this is our secret weapon.None of those that we have taken there have had...
read more
We come to Keswick very regularly with our daughter and teenage friends.Teenagers can be very surly so this is our secret weapon.None of those that we have taken there have had anything but praise. The setting is perfect and activities can go on even in bad weather.The staff are entirely committed and I cannot recommend it too highly.
Fabulous day out with my family! Recommend to any budding rock climbers. Safe environment and fun too. Always a super place to visit when raining, if not on the pricey side.
Happy New Year and thank you for the great review. Glad that you had a good time with us and do come and see us again when you are next in Keswick We have a few new and very different activities planned for 2015, which should be open for Easter, weather and contractors permitting, so keep an eye on our...
My grandchildren tried this. One was unsure but the staff really supported him and made him feel comfortable to have a go. Good fun and they really enjoyed it.
Hello and a Happy New Year. Thank you for the lovely review and so pleased that your Grandchildren had such a good time with us, even the nervous one. Come and see us next time you are Grand child sitting and keep an eye out for the new activities we have planned for 2015: your Grand children will love them...
Not the biggest wall in the world but a good place to have a go at indoor climbing.
Has plenty to keep a regular climber occupied on a wet afternoon.
Nice staff who know their stuff and are helpful.
Hello and thank you for your lovely review. Yes, we are small but we do try to keep all standard of climbers entertained. We do change routes on a very regular basis, so there should be plenty of new challenges next time you come !
This was a great experience with one to one tuition for beginners at a great alternate place for enjoyment and exercise in the Keswick Area
Thank you for the review and the lovely comments and hope to see you next time you are in the area !
What a fantastic climbing wall. I took my daughter to this wall (she is a climbing wall fanatic!) and what a great time she had. The staff were very polite, helpful and professional. Well worth a visit.
Hello and thank you for the great review ! Please do come and climb with us again next time you are in Keswick: the staff are busy resetting the whole centre, so plenty of new routes to try.
As an experienced climber, I was pretty impressed by the quality of this centre. The staff were helpful and the facilities are great. There's a huge range of climbs ranging from super-beginner-easy to bloody difficult! I have now been three times and it never ceases to be a great place. I honestly couldn't comment on the instructors as I was...
Hello and many thanks for the great review. We do appreciate hearing from experienced climbers and their views on the wall and setting as we are not the tallest climbing wall but do we try to have a decent range of interesting challenges for all levels: glad there was enough to keep you interested and challenged. We are in the...
My self and partner had not done anything like this before and wanted to give it a try booked a instruction course midweek used the website for details booking really easy and we thought very good value as well for a 1 hour introduction our teacher was dillan he was very good freindly and showed us everything we needed to...
Hello and many thanks for taking the time to review Keswick Climbing Wall. I am pleased that you had such a good time with Dylan and I will pass on your comments to him. We want to make sure that after your lesson you have a chance to practice the skills you have just learned and so being able to...
Called in here while on circular walk. Needed a cuppa and loo and staff very helpful and friendly. We were able to use the coin operated drink & snack machines + loo. Reasonable prices. Watching the children enjoying the climbing walls and seeing facilities I can highly recommend it. Not many indoor places in the Lake district for the frequent...
Hello, and thank you for the lovely review. Do come and have a go next time you are in the area as we are not just for kids and we have plenty of interesting and challenging routes for all levels of climbers and ages. Hope to see you soon for a climbing lesson !
Climbing in the borrowdale valley followed by abseiling. A small friendly group led by Adam and Dappy who were great. Encouraging but no pressure. Lotsof fun
Hello, and thank you for your review. Really pleased that you enjoyed your time climbing outdoors with us. Despite being an indoor climbing wall, we always run outdoor sessions as well and run specific sessions to introduce you to outdoor climbing which is quite different to climbing walls: no brightly coloured holds for one !! I will pass your comments...
Register now for free — and start getting automatic notification of new reviews, responding to traveler feedback, adding new photos to your listing and much more.


text:
Executive PG Programme at IIM KozhikodeKOCHI: IIM Kozhikode (IIMK) campus in Kochi has announced admission to its Executive Post Graduate Programme (EPGP). The programme is a two-year non-residential programme custom designed in content so as to cater to the needs of the working ... New Indian Express, 2 weeks ago
Executive PG programme on IIM-K Kochi campus - The Hindu, 2 weeks ago
Goa Board Class 12th 2015 Exam Dates - 24Dunia, 2 months ago
The ethanol production grants program
This audit examined the effectiveness of the administration of the Ethanol Production Grants Program (EPGP). Objective, criteria and scope The objective of the audit was to examine the effectiveness of Industry's administration of the EPGP, ...Australian Policy Online, 1 month ago
How they came up trumps
Rachit Kothari, an IIT Kharagpur alumnus, bagged one of the two gold medals from the flagship Post Graduate Programme in Management (PGP) 2013-15 and is deciding his future with two companies: "I am still deciding between the offers of BCG (Boston Consulting ...Deccan Herald, 5 days ago
IIM Raipur admission 2015
IIM (Indian institute of management) Raipur has invited application for admission in executive fellow programme in management (EFPM). Last date for submission of application is February 27. Eligibility criteria The candidates applying for ...India Today, 1 month ago Editing details in CBSE CTET Feb 2015 online applications till Jan 23 24Dunia, 2 months ago IIM, Kozhikode Invites Applications for Executive PGP in Management 2015 24Dunia, 2 months ago IIM Raipur: RAT 2015 India Today, 2 months ago
IIMB slips in global MBA rankings this year
The Indian Institute of Management Bangalore (IIMB), was ranked 82nd in the Global MBA Rankings- 2015, released by the UK-based Financial Times. The institute fared much better last year at the 68th position. Officials from the institute said that the ...Deccan Herald, 1 month ago
UCSF names physician-scientist as executive vice chancellor, provost
Driven by science, humanism and service, Dan Lowenstein joins UCSF leadership team. Dan Lowenstein, UC San Francisco (Photoa by Marco Sanchez) By Lisa Cisneros, UC San Francisco A charismatic high school biology teacher named Brian ...UC Newswire, 2 months ago Driven by Science, Humanism and Service, Dan Lowenstein Joins UCSF Leadership Team Noodls, 2 months ago
JEE Main 2015: exam structure & number of attempts
will have two papers - Paper I for Bachelor of Engineering/Bachelor of Technology (BE/B Tech) and Paper II for Bachelor of Architecture/Bachelor of Planning (B Arch/B Planning). Candidates can appear for Paper I, Paper II or both, depending upon the ...24Dunia, 2 months ago
Maati Ke Rang 2015 ends in Dimapur
Maati Ke Rang, which started at the NEZCC Complex in Dimapur, Nagaland, on January 13, has ended with a mega cultural program, representing the diverse cultural flavours of India (through seven Zonal Cultural Centres).Giving a brief history of Maati ...24Dunia, 2 months ago
University of Delhi Invites Applications for Ph.D in Law Program 2015
full time Doctor of Philosophy (Ph.D) program in Law offered at Faculty of Law for the session 2015.Candidates who have secured 55% marks in LL.M or 60% marks in LL.B program are eligible to apply.The candidates would be required to appear for ...24Dunia, 2 months ago
Realtime News
on your WebpageAdd Widget >Get your members hooked!


text:
Send Ringtone to MobileGet this ringtone
We notice you have AdBlock installed. This prevents you from using all features on 4shared. Please consider whitelisting us in AdBlock or click the button below
Click to enable preview
Click to enable preview
Grupo Ta Na Mente - Fica (Pagodão) - download at 4shared. Grupo Ta Na Mente - Fica (Pagodão) is hosted at free file sharing service 4shared.
| Uploaded via: 4shared site
Share Embed Add to my account
Is owner
View only
Upload & Edit
Members may only view items in this folder.
Members may view, add, edit and delete items in this folder.
Checked by McAfee. No virus detected.
16 comments Add new comment View all
Wrong code entered. Please, try again.
Enter the code above to post message:
suellen R. 1 year ago Reply Spam SpamCancel
eu badoro e me amarroooo *-* amoooooo muito eles
kessyjhones35... 1 year ago Reply Spam SpamCancel
zika esse som
Gabriel A. 1 year ago Reply Spam SpamCancel
Boa de maais .. Viciante *-----*
Alexandra S. 1 year ago Reply Spam SpamCancel
Lindaaaaaaaaaaa!! Amooooooo o Ta Na Mente!
gabriel_omall... 1 year ago Reply Spam SpamCancel
muito boa
Remove Ads x
Download all files from this folder
Download folder as:
This file will be created in your 4shared folder
File name:
Zip as:
What is B1?


text:
Game Of The Week
The IHSA Football playoffs are about to give us some games to talk about this weekend for years to come. The winners from this weekend will face off for the championships of their classes next Thanksgiving weekend in Champaign, Urbana for the State Title trophy. Gately Stadium is the place to be this Saturday if you are looking for some great football action before the final cham ...Read more ›