Patent Application
20060211694
1. Field of the Invention
The instant invention relates to substituted pyridinones that are useful for treating diseases and conditions caused or exacerbated by unregulated p38 MAP kinase activity. Pharmaceutical compositions containing the pyridinone compounds, methods of preparing the compounds and methods of treatment using the compounds are also disclosed.
2. Description of the Related Art
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. MAP kinases are a family of proline-directed serine/threonine kinases that activate their substrates by dual phosphorylation. Four distinct subgroups of MAP kinases, p38 alpha, p38 beta, p38 gamma, and p38 delta have been identified and each of these consists of a specific module of kinases that function downstream of an activating stimulus by phosphorylating and activating transcription factors (e.g. ATF2, CHOP and MEF2C) as well as other kinases (e.g. MAPKAP-2 and MAPKAP-3). One subgroup of the MAP kinases is the p38 MAP kinase cascade, which is activated by a variety of signals including proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) as well as bacterial lipopolysaccharides, and environmental stress (e.g., osmotic shock and ultraviolet radiation). Upon activation, the p38 cascade leads to the induction of gene expression of several factors involved in inflammation and immunity including TNF, interleukin-6, granulocyte-macrophage colony stimulating factor (GM-CSF), and HIV long terminal repeat (Paul et al., Cell Signal., 1997, 9, 403-410). The products of the p38 phosphorylation inhibit or modulate the production of inflammatory cytokines, including TNF and IL-1, and cyclooxygenase-2, and also potentially block the effects of these cytokines on their target cells, which therefore inhibit or modulate inflammation.
p38 MAP kinases have also been shown to help prevent apoptosis during ischemia in cardiac myocytes, which suggests that p38 MAP kinase inhibitors can be used for treating ischemic heart disease, p38 MAP kinase is also required for T-cell HIV-1 replication and may be a useful target for AIDS therapy. p38 Pathway inhibitors have also been used to increase cancer cell sensitivity to cancer therapy.
TNF is a cytokine and a potent proinflammatory mediator implicated in inflammatory conditions such as arthritis, asthma, septic shock, non-insulin dependent diabetes mellitus, multiple sclerosis, asthma, and inflammatory bowel disease. TNF has also been implicated in viral infections, such as HIV, influenza virus, and herpes virus including herpes simplex virus type-1 (HSV-1 ), herpes simplex virus type-2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), human herpesvirus-8 (HHV-8), pseudorabies and rhinotracheitis, among others.
Excessive or unregulated TNF production has also been shown to produce elevated levels of IL-1 . Inhibition of TNF, therefore, should reduce levels of IL-1 and ameliorate disease states caused by unregulated IL-1 synthesis. Such disease states include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, bone resorption diseases, reperfusion injury, graft versus host reaction, alallograft rejections, fever and myalgias due to infection, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS related complex (ARC), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, and pyresis.
IL-1 has also been shown to mediate a variety of biological activities such as the activation of T-helper cells, induction of fever, stimulation of prostaglandin or collagenase production, neutrophil chemotaxis, and the suppression of plasma iron levels (Rev. Infect. Disease, 6, 51 (1984) ). Elevated levels of IL-1 have also been implicated in mediating or exacerbating a number of disease states including rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, ulcerative colitis, anaphylaxis, muscle degeneration, cachexia, Reiter's syndrome, type I and type II diabetes, bone resorption diseases, ischemia reperfusion injury, arteriosclerosis, brain trauma, multiple sclerosis, sepsis, septic shock, and toxic shock syndrome. Viruses sensitive to TNF inhibition, such as HIV-1, HIV-2, HIV-3, are also affected by IL-1 production. In rheumatoid arthritis, both IL-1 and TNF induce collagenase synthesis and ultimately lead to tissue destruction within arthritic joints (Lymphokine Cytokine Res. (11): 253-256, (1992) and Clin. Exp. Immunol. 989:244-250, (1992)).
IL-6 is another pro-inflammatory cytokine, which is associated with many conditions including inflammation.
Consequently, TNF, IL-1 and IL-6 affect a wide variety of cells and tissues and are important inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines by inhibition or modulation of p38 kinase is of benefit in controlling, reducing and alleviating many of these disease states and conditions. Therefore, the present invention concerns finding small molecule inhibitors or modulators of p38 kinase and the p38 kinase pathway.
In a broad aspect, the invention provides compounds of Formula I:
and pharmaceutically acceptable salts thereof, wherein
The invention also includes the intermediates that are useful in making the compounds of the invention.
These compounds bind and/or interact with p38 kinase and/or TNF. Preferably, they inhibit the activity of p38 kinase and/or TNF. They are therefore used in treating p38 map kinase or TNF mediated disorders. Preferably they are used in treating p38 alpha or TNF mediated disorders.
The instant invention also includes pharmaceutical compositions comprising at least one compound of formula I and at least one pharmaceutically acceptable carrier, solvent, adjuvant or excipient.
The instant invention also includes methods of treating a TNF mediated disorder, a p38 kinase mediated disorder, inflammation and/or arthritis in a subject, the method comprising treating a subject having or susceptible to such disorder or condition with a therapeutically-effective amount of a compound of Formula I.
A preferred class of compounds of formula I are those wherein,
Another preferred class of compounds of formula I are those wherein:
A more preferred class of compound are those wherein
Another more preferred class of compound are those wherein
Yet another more preferred class of compound are those wherein
Yet another more preferred class of compound are those wherein
Still yet another more preferred class of compound are those wherein
Still yet another more preferred class of compound are those wherein
An even more preferred class of compounds is those compounds wherein
Still yet, an even more preferred class of compounds is those compounds wherein
Another embodiment of the invention is those compounds of formula III wherein
More preferred compounds of formula III are those wherein
Other more preferred compounds of formula III are those wherein
Still other more preferred compounds of formula III are those wherein
Still other more preferred compounds of formula III are those wherein
Still other more preferred compounds of formula III are those wherein
Still other more preferred compounds of formula III are those wherein
Another embodiment of the invention is those compounds of formula IV, wherein
More preferred compounds of formula IV are those wherein
Other preferred compounds of formula IV are those wherein
Other preferred compounds of formula IV are those wherein
Even more preferred compound of formula IV are those wherein
Still more preferred are those compounds wherein
The invention also includes a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, solvent, adjuvant or excipient and a compound according to Formula I.
Another preferred class of compounds of formula I are those of formula X:
or a pharmaceutically acceptable salt thereof, wherein
More preferred compounds of formula X are those wherein
Other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Yet even more preferred compounds or salts of formula X are those wherein
Still yet even more preferred compounds or salts of formula X are those wherein
Other still yet even more preferred compounds of formula X are those wherein
Other still yet even more preferred compounds of formula X are those wherein
Even more preferred compounds of formula X are those wherein R2 is benzyloxy, or phenethyloxy, each of the above is unsubstituted or substituted with 1, 2, or 3, groups that are independently fluoro, chloro, bromo, CF3, or (C1-C4)alkyl.
Other preferred compounds of formula X are those wherein
More preferred compounds of formula X are those wherein
Other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Still other more preferred compounds of formula X are those wherein
Still yet more preferred compounds of formula X are those wherein
Still yet more preferred compounds of formula X are those wherein
Still yet more preferred compounds of formula X are those wherein
Still yet more preferred compounds of formula X are those wherein
Still yet even more preferred compounds of formula X are those wherein
A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, solvent, adjuvant or excipient and a compound according to formula X.
The present invention comprises a pharmaceutical composition for the treatment of a TNF mediated disorder, a p38 kinase mediated disorder, inflammation, and/or arthritis, comprising a therapeutically-effective amount of a compound of Formula I, or a therapeutically-acceptable salt or tautomer thereof, in association with at least one pharmaceutically-acceptable carrier, adjuvant, solvent, excipient, or diluent.
The present invention also comprises a therapeutic method of treating a TNF mediated disorder, a p38 kinase mediated disorder, inflammation and/or arthritis in a subject, the method comprising treating a subject having or susceptible to such disorder or condition with a therapeutically-effective amount of a compound of Formula I and/or Formula X.
Specific diseases or conditions that may be treated using compounds of Formula I, Formula III, Formula IV, and/or Formula X include:
inflammation;
arthritis, including but not limited to, rheumatoid arthritis, spondylarthropathies, gouty arthritis, gouty arthritis, osteoarthritis, systemic lupus erthematosus and juvenile arthritis, osteoarthritis, gouty arthritis and other arthritic conditions;
neuroinflammation;
pain (i.e., use as an analgesic) including but not limited to neuropathic pain;
fever (i.e., use as an antipyretic);
pulmonary disorders or lung inflammation, including adult respiratory distress syndrome, pulmonary sarcoisosis, asthma, silicosis, and chronic pulmonary inflammatory disease;
cardiovascular diseases including arteriosclerosis, myocardial infarction, thrombosis, congestive heart failure, and cardiac reperfusion injury;
cardiomyopathy;
reperfusion injury;
renal reperfusion injury;
ischemia including stroke and brain ischemia;
brain trauma;
brain edema;
liver disease and nephritis;
ulceratiuve diseases such as gastric ulcer;
ulcerative diseases such as gastric ulcer;
ophthalmic diseases such as retinitis, retinopathies, uveitis, ocular photophobia, and of acute injury to the eye tissue;
ophthalmological conditions such as corneal graft rejection, ocular neovascularization, retinal neovascularization including neovascularization following injury or infection, diabetic retinopathy, retrolental fibroplasias and neovascular glaucoma;
diabetes;
diabetic nephropathy;
skin-related conditions such as psoriasis, eczema, burns, dermatitis, keloid formation, scar tissue formation, and angiogenic disorders;
viral and bacterial infections, including sepsis, septic shock, gram negative sepsis, malaria, meningitis, opportunistic infections, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), pneumonia, and herpes virus;
myalgias due to infection;
influenza;
endotoxic shock;
toxic shock syndrome;
autoimmune disease including graft vs. host reaction and allograft rejections;
treatment of bone resorption diseases, such as osteoporosis;
multiple sclerosis;
disorders of the female reproductive system such as endometriosis;
pathological, but non-malignant, conditions such as hemaginomas, including infantile hemagionmas, angiofibroma of the nasopharynz and avascular necrosis of bone;
benign and malignant tumors/neoplasia including cancer, such as colorectal cancer, brain cancer, bone cancer, epithelial call-derived neoplasia (epithelial carcinoma) such as basal cell carcinoma, adenocarcinoma, gastrointestinal cancer such as lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamus cell and basal cell cancers, prostate cancer, renal cell carcimoma, and other known cancers that affect epithelial cells throughout the body;
leukemia;
lymphoma;
systemic lupus erthrematosis (SLE);
angiogenesis including neoplasia;
metastasis; and
central nervous system disorders (including, but not limited to, central nervous system disorders having an inflammatory or apoptotic component), such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, and peripheral neuropathy.
The invention also covers a method of treating a p38 kinase or TNF-alpha mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of any of the preceding compounds and at least one pharmaceutically acceptable carrier, adjuvant, solvent or excipient.
The following compounds of the invention are meant to give the reader an understanding of the compounds falling within the invention:
As used herein, the term “alkenyl” refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl, and 2-methyl-3-heptene.
The term “alkoxy” represents an alkyl attached to the parent molecular moiety through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.
The term “thioalkoxy” represents an alkyl attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxy groups include, for example, thiomethoxy, thioethoxy, thiopropoxy and thioisopropoxy.
As used herein, the term “alkyl” includes those alkyl groups of a designed number of carbon atoms. Alkyl groups may be straight or branched. Examples of “alkyl” include methyl, ethyl, propyl, isopropyl, butyl, iso-, sec- and tert-butyl, pentyl, hexyl, heptyl, 3-ethylbutyl, and the like. “Cx-Cy alkyl” represents an alkyl group of the specified number of carbons. For example, C1-C4 alkyl includes all alkyl groups that include at least one and no more than four carbon atoms. It also contains subgroups, such as, for example, C2-C3 alkyl or C1-C3 alkyl.
The term “aryl” refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring may optionally be fused or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene, indanyl, and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl. The most preferred aryl group is phenyl.
The term “arylalkyl” refers to an aryl group, as defined above, attached to the parent molecular moiety through an alkyl group, as defined above. Preferred arylalkyl groups include, benzyl, phenethyl, phenpropyl, and phenbutyl. More preferred arylalkyl groups include benzyl and phenethyl. The most preferred arylalkyl group is benzyl.
The term “arylalkoxy” refers to an aryl group, as defined above, attached to the parent molecular moiety through an alkoxy group, as defined above. Preferred arylaloxy groups include, benzyloxy, phenethyloxy, phenpropyloxy, and phenbutyloxy. The most preferred arylalkoxy group is benzyloxy.
The term “cycloalkyl” refers to a C3-C8-cyclic hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. More preferred cycloalkyl groups include cyclopropyl.
The term “cycloalkylalkyl,” as used herein, refers to a C3-C8 cycloalkyl group attached to the parent molecular moiety through an alkyl group, as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
The terms “halogen” or “halo” indicate fluorine, chlorine, bromine, or iodine.
The term “heterocycloalkyl,” refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur, wherein the non-aromatic heterocycle is attached to the core. The heterocycloalkyl ring may be optionally fused to or otherwise attached to other heterocycloalkyl rings, aromatic heterocycles, aromatic hydrocarbons and/or non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups have from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, 1,2,3,4-tetrahydroisoquinoline, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.
The term “heteroaryl” refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heteroaryl ring may be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or heterocycloalkyl rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl. Preferred heteroaryl groups include pyridyl.
As used herein, the term “p38 mediated disorder” refers to any and all disorders and disease states in which p38 plays a role, either by control of p38 itself, or by p38 causing another factor to be released, such as but not limited to IL-1, IL-6 or IL-8. A disease state in which, for instance, IL-1 is a major component, and whose production or action, is exacerbated or secreted in response to p38, would therefore be considered a disorder mediated by p38.
As TNF-beta has close structural homology with TNF-alpha (also known as cachectin), and since each induces similar biologic responses and binds to the same cellular receptor, the synthesis of both TNF-alpha and TNF-beta are inhibited by the compounds of the present invention and thus are herein referred to collectively as “TNF” unless specifically delineated otherwise.
Non-toxic pharmaceutically acceptable salts include, but are not limited to salts of inorganic acids such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, and nitric or salts of organic acids such as formic, citric, malic, maleic, fumaric, tartaric, succinic, acetic, lactic, methanesulfonic, p-toluenesulfonic, 2-hydroxyethylsulfonic, salicylic and stearic. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts. The present invention also encompasses prodrugs of the compounds of Formula I.
The present invention also encompasses the acylated prodrugs of the compounds of Formula I. Those skilled in the art will recognize various synthetic methodologies, which may be employed to prepare non-toxic pharmaceutically acceptable addition salts and acylated prodrugs of the compounds encompassed by Formula I.
The compounds of this invention may contain one or more asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates, chiral non-racemic or diastereomers. In these situations, the single enantiomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent; chromatography, using, for example a chiral HPLC column; or derivatizing the racemic mixture with a resolving reagent to generate diastereomers, separating the diastereomers via chromatography or selective crystallization, and removing the resolving agent to generate the original compound in enantiomerically enriched form. Any of the above procedures can be repeated to increase the enantiomeric purity of a compound.
When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include the cis, trans, Z- and E-configurations. Likewise, all tautomeric forms are also intended to be included.
The present invention also encompasses the prodrugs of the compounds of Formula I. Those skilled in the art will recognize various synthetic methodologies that may be employed to prepare non-toxic pharmaceutically acceptable prodrugs of the compounds encompassed by Formula I. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable solvates, such as water, ethanol, mineral oil, vegetable oil, and dimethylsulfoxide.
The compounds of general Formula I may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a compound of general Formula I and a pharmaceutically acceptable carrier. One or more compounds of general Formula I may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing compounds of general Formula I may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques. In some cases such coatings may be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Formulations for oral use may also be presented as lozenges.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin, or cetyl alcohol. Sweetening agents and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring, and coloring agents, may also be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds of general Formula I may also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
Compounds of general Formula I may be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives, and buffering agents can be dissolved in the vehicle.
For disorders of the eye or other external tissues, e.g., mouth and skin, the formulations are preferably applied as a topical gel, spray, ointment or cream, or as a suppository, containing the active ingredients in a total amount of, for example, 0.075 to 30% w/w, preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w. When formulated in an ointment, the active ingredients may be employed with either paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example at least 30% w/w of a polyhydric alcohol such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, polyethylene glycol and mixtures thereof. The topical formulation may desirably include a compound, which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs. The compounds of this invention can also be administered by a transdermal device. Preferably topical administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. In either case, the active agent is delivered continuously from the reservoir or microcapsules through a membrane into the active agent permeable adhesive, which is in contact with the skin or mucosa of the recipient. If the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent may also function as the membrane. The transdermal patch may include the compound in a suitable solvent system with an adhesive system, such as an acrylic emulsion, and a polyester patch. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier, it may comprise a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make-up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base, which forms the oily, dispersed phase of the cream formulations. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate, among others. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus, the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredients are dissolved or suspended in suitable carrier, especially an aqueous solvent for the active ingredients. The anti-inflammatory active ingredients are preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% and particularly about 1.5% w/w. For therapeutic purposes, the active compounds of this combination invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient. The daily dose can be administered in one to four doses per day. In the case of skin conditions, it may be preferable to apply a topical preparation of compounds of this invention to the affected area two to four times a day.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
For administration to non-human animals, the composition may also be added to the animal feed or drinking water. It may be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to the feed or drinking water.
The disclosures in this application of all articles and references, including patents, are incorporated herein by reference.
The invention is illustrated further by the following examples, which are not to be construed as limiting the invention in scope or spirit to the specific procedures described in them.
The starting materials and various intermediates may be obtained from commercial sources, prepared from commercially available compounds, or prepared using well-known synthetic methods.
The compound names in this application were created using ACD Name Pro program, or the ChemDraw ultra program. In all cases, the given name will enable one skilled in the art to determine the structure of the named compound.
General Synthetic Procedures
The compounds of the instant invention may be prepared according to the method described in Scheme 1, using methods well known in the art.
R5 is as defined above.
Alternatively, the compounds of the instant invention may be prepared according to the method described in Scheme 2, using methods well known in the art.
Q at each occurrence is independently alkyl, halogen, alkoxy, arylalkoxy, thioalkoxy, alkoxycarbonyl, arylalkoxycarbonyl, CO2H, CN, amidinooxime, NR6R7, NR6R7alkyl, —C(O)NR6R7, amidino, haloalkyl, or haloalkoxy; and n is 0, 1, 2, 3, 4, or 5.
The invention is illustrated further by the following examples, which are not to be construed as limiting the invention in scope or spirit to the specific procedures described in them. Those having skill in the art will recognize that the starting materials may be varied and additional steps employed to produce compounds encompassed by the invention, as demonstrated by the following examples. Those skilled in the art will also recognize that it may be necessary to utilize different solvents or reagents to achieve some of the above transformations. In some cases, protection of reactive functionalities may be necessary to achieve the above transformations. In general, such need for protecting groups, as well as the conditions necessary to attach and remove such groups, will be apparent to those skilled in the art of organic synthesis. When a protecting group is employed, deprotection step may be required. Suitable protecting groups and methodology for protection and deprotection such as those described in Protecting Groups in Organic Synthesis by Greene and Wuts are well known and appreciated in the art.
Unless otherwise specified, all reagents and solvents are of standard commercial grade and are used without further purification. The appropriate atmosphere to run the reaction under, for example, air, nitrogen, hydrogen, argon and the like, will be apparent to those skilled in the art.
4-Benzyloxy-2(1H)-pyridone (3.0 9, 0.015 mol), 4-methylbenzyl bromide (3.15 g, 0.17 mol), and potassium carbonate (3.0 g, 0.022 mol) were heated at 80° C. for 2 hours. Contents were allowed to cool, diluted with water and a solid (5.52 g) was filtered. The solid was recrystallized from EtOAc to give colorless flakes, 2.46 g (54% yield), mp 142.8-143.3° C. FABHRMS m/z 306.1494 (M+H, C20H20NO2 requires 306.1494). 1H NMR (CDCl3/300 MHz): 7.50-7.40 (m, 5H); 7.20-7.05 (m, 5H); 6.07-6.00 (m, 1H); 5.95-5.90 (m, 1H); 5.05 (s, 2H); 5.00 (s, 2H); 2.32 (s, 3H).
Anal. Calcd for C20H19NO2: C, 78.66; H, 6.27; N, 4.59. Found: C, 78.54; H, 6.38; N, 4.58.
The material prepared in Example 1 (2.1 g, 0.007 mol) and sodium acetate (738 mg, 0.009 mol) in glacial acetic acid (15 mL) were cooled to 15° C. Bromine (0.412 mL, 0.008) in glacial acetic acid (5 mL) was added dropwise. Contents were stirred 2 hours, coming to room temperature. Water (200 mL) was added and a light yellow solid was filtered. The solid was recrystallized from EtOAc to give colorless needles, 646 mg (24 % yield). Mp 150.4-151.2° C. FABHRMS m/z 384.0599 (M+H, C20H19BrNO2 requires 384.0601). 1H NMR (CDCl3/300 MHz) δ: 7.42-7.30 (m, 5H); 7.22-7.08 (m, 5H); 6.02 (d, 1H); 5.20 (s, 2H); 5.12 (s, 2H); 2.32 (s, 3H).
Anal. Calcd for C20H18BrNO2: C, 62.51; H, 4.72; N, 3.65. Found: C, 62.11; H, 4.48; N, 3.54.
The material of Example 3 was prepared according to the procedure of Example 1. FABHRMS m/z 370.0443 (M+H, C19H17BrNO2 requires 370.0428). 1H NMR (CDCl3/300 MHz) δ: 7.43 (d, 2H); 7.40-7.33 (m, 5H); 7.20-7.07 (m, 3H); 6.04-6.01 (m, 1H); 6.00-5.92 (m, 1H); 5.03 (s, 2H); 4.98 (s, 2H)
Anal. Calcd for C19H16BrNO2: C, 61.64; H, 4.36; N, 3.78. Found: C, 61.58; H, 4.38; N, 3.74.
The material of Example 4 was prepared according to the procedure of Example 2. FABHRMS m/z 447.9548 (M+H, C19H16Br2NO2 requires 447.9522). 1H NMR (CDCl3/300 MHz) δ: 7.50-7.15 (m, 10H); 6.06 (d, 1H); 5.20 (s, 2H), 5.10 (s, 2H).
Anal. Calcd for C19H15Br2NO2: C, 50.81; H, 3.37; N, 3.12. Found: C, 50.58; H, 3.04; N, 3.15.
The material of Example 5 was prepared according to the procedure of Example 1. FABHRMS m/z 326.0893 (M+H, C19H17ClNO2 requires 326.0948). 1H NMR (CDCl3/300 MHz) δ: 7.40-7.32 (m, 5H); 7.24 (AB quartet, 4H); 7.10 (d, 1H); 6.03-6.00 (m, 1H); 5.98-5.92 (m, 1H); 5.03 (s, 2H); 4.99 (s, 2H).
Anal. Calcd for C19H16ClNO2: C, 70.05; H, 4.95; N, 4.30. Found: C, 69.78; H, 4.72; N, 4.29.
The material of Example 6 was prepared according to the procedure of Example 2. FABHRMS m/z 404.0035 (M+H, C19H16BrClNO2 requires 404.0053). 1H NMR (CDCl3/300 MHz): 7.43-7.20 (m, 10H); 6.08 (d, 1H); 5.20 (s, 2H); 5.10 (s, 2H).
Anal. Calcd for C19H15BrClNO2: C, 56.39; H, 3.74; N, 3.46. Found: C, 56.15; H, 3.61; N, 3.35.
The material of Example 7 was prepared according to the procedure of Example 1. FABHRMS m/z 310.1226 (M+H, C19H17FNO2 requires 310.1243). 1H NMR (CDCl3/300 MHz) δ: 7.45-7.25 (m, 5H); 7.12 (d, 1H); 7.07-6.93 (m, 4H); 6.04-6.02 (m, 1H); 6.00-5.94 (m, 1H); 5.08 (s, 2H); 5.00 (s, 2H).
Anal. Calcd for C19H16FNO2: C, 73.77; H, 5.21; N, 4.53. Found: C, 73.75; H, 5.22; N, 4.58.
The material of Example 8 was prepared according to the procedure of Example 2. 1H NMR (CDCl3/300 MHz) δ: 7.43-7.25 (m, 6H); 7.21 (d, 1H); 7.10-6.93 (m, 3H); 6.08 (d, 1H); 5.22 (s, 2H); 5.12 (s, 2H).
Anal. Calcd for C19H15BrFNO2: C, 58.78; H, 3.89; N, 3.61. Found: C, 58.79; H, 3.83; N, 3.61.
The material of Example 9 was prepared according to the procedure of Example 1. FABHRMS m/z 310.1243 (M+H, C19H17FNO2 requires 310.1231). 1H NMR (CDCl3/300 MHz) δ: 7.43-7.00 (m, 10H); 6.01-5.92 (m, 2H); 5.10 (s, 2H); 4.99 (s, 2H).
Anal. Calcd for C19H16FNO2: C, 73.77; H, 5.21; N, 4.53. Found: C, 73.66; H, 5.45; N, 4.46.
The material of Example 10 was prepared according to the procedure of Example 2. FABHRMS m/z 388.0373 (M+H, C19H16FNO2 requires 388.0348). 1H NMR (CDCl3/300 MHz): 7.52 (d of t, 1H); 7.44-7.26 (m, 7H); 7.15-7.00 (m, 2H); 6.03 (d, 1H); 5.20 (s, 2H); 5.15 (s, 2H).
Anal. Calcd for C19H15BrFNO2: C, 58.78; H, 3.89; N, 4.61. Found: C, 58.59; H, 4.00; N, 3.57.
The material of Example 11 was prepared according to the procedure of Example 2. 1H NMR (CDCl3/300 MHz) δ: 7.50-7.30 (m, 6H); 6.20 (d, 1H); 5.24 (s, 2H).
Anal. Calcd for C12H10BrNO2 (0.3H2O): C, 50.48; H, 3.74; N, 4.91. Found: C, 50.79; H, 3.41; N, 4.82.
To the material of Example 11 (1.0 g, 0.0036 mol) in DMF (5 mL) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 4 mL). Contents were stirred one hour before adding dropwise a solution of 4-benzyloxybenzyl chloride (931 mg, 0.004 mol) in DMF (5 mL). Contents were heated at 75° C. for four hours. Contents were allowed to cool and poured into water (75 mL). A solid (1.9 g) was filtered and recrystallized from EtOAc to give white flakes, 986 mg (57% yield). mp 137.6-144.3° C. FABHRMS m/z 476.0854 (M+H, C26H23BrNO3 requires 476.0861). 1H NMR (CDCl3/300 MHz): 7.45-7.15 (m, 13H); 6.92 (d, 2H); 6.01 (d, 1H); 5.20 (s, 2H); 5.08 (s, 2H); 5.03 (s, 2H)
Anal. Calcd for C26H22BrNO3: C, 65.55; H, 4.66; N, 2.94. Found: C, 65.54; H, 4.56; N, 2.94.
The material of Example 13 was prepared according to the procedure of Example 1. FABHRMS m/z 350.1391 (M+H, C21H20NO4 requires 350.1392). 1H NMR (CDCl3/300 MHz): 8.00 (d, 2H); 7.40-7.25 (m, 7H); 7.10 (d, 1H); 6.03-6.01 (m, 1H); 6.00-5.93 (m, 1H); 5.12, (s, 2H); 5.00 (s, 2H); 3.95 (s, 3H).
Anal. Calcd for C21H19NO4: C, 72.19; H, 5.48; N, 4.01. Found: C, 72.20; H, 5.28; N, 3.97.
The material of Example 14 was prepared according to the procedure of Example 2. FABHRMS m/z 428.0480 (M+H, C21H19BrNO4 requires 428.0497). 1H NMR (CDCl3/300 MHz): 8.00 (d, 2H); 7.42-7.31 (m, 7H); 7.23 (d, 1H); 6.08 (d, 1H); 5.22 (d, 2H); 5.20 (s, 2H); 3.95 (s, 3H).
Anal. Calcd for C21H18BrNO4 (0.5 HBr, 0.5 H2O): C, 52.80; H, 4.11; N, 2.93. Found: C, 52.77; H, 4.26; N, 2.85.
The material of Example 14 (1.0 g, 0.0023 mol) and 2.5N aqueous sodium hydroxide (1.9 mL) were refluxed in methanol (10 mL) for 5 hours. Contents were allowed to cool and made acidic with 3N HCl to pH 2.5. A white solid was filtered and triturated with warm ethanol to give the desired as a white solid (1.0 g). mp 250.8-255.7° C. FABHRMS m/z 414.0360 (M+H, C20H17BrNO4 requires 414.0341). 1H NMR (DMSO-d6/300 MHz): 8.00-7.80 (m, 3H); 7.53-7.27 (m, 7H); 6.50 (d, 1H); 5.32 (s, 2H); 5.20 (s, 2H).
Anal. Calcd for C20H16BrNO4: C, 57.99; H, 3.89; N, 3.38. Found: C, 57.55; H, 3.70; N, 3.36.
The material of Example 16 was prepared according to the procedure of Example 1. FABHRMS m/z 317.1270 (M+H, C20H17N2O2 requires 317.1290). 1H NMR (CDCl3/300 MHz) δ: 7.60 (d, 2H); 7.42-7.30 (m, 7H); 7.13 (d, 1H); 6.05-5.98 (m, 2H); 5.11 (s, 2H); 5.00 (s, 2H).
Anal. Calcd for C20H16N2O2: C, 75.93; H, 5.10; N, 8.86. Found: C, 75.61; H, 5.07; N, 8.77.
The material of Example 17 was prepared according to the procedure of Example 2. FABHRMS m/z 395.0376 (M+H, C20H16BrN2O2 requires 395.0395). 1H NMR (CDCl3/300 MHz) δ: 7.61 (d, 2H); 7.48-7.30 (m, 6H); 7.23 (d, 2H); 6.12 (d, 1H); 5.22 (s, 2H); 5.20 (s, 2H)
Anal. Calcd for C20H15BrN2O2: C, 60.78; H, 3.83; N, 7.09. Found: C, 60.49; H, 3.72; N, 7.02.
The material of Example 18 was prepared according to the procedure of Example 1. FABHRMS m/z 348.1949. (M+H, C23H26NO2 requires 348.1964). 1H NMR (CDCl3/300 MHz): 7.40-7.28 (m, 7H); 7.20 (d, 2H); 7.10 (d, 1H); 6.02 (d, 1H); 5.97-5.90 (m, 1H); 5.02 (d, 2H); 4.98 (d, 2H)
Anal. Calcd for C23H25NO2: C, 79.51; H, 7.25; N, 4.03. Found: C, 79.51; H, 7.21; N, 4.08.
The material of Example 19 was prepared according to the procedure of Example 2. FABHRMS m/z 426.1023 (M+H, C23H25BrNO2 requires 426.1069). 1H NMR (CDCl3/300 MHz) δ: 7.43-7.20 (m, 10H); 6.02 (d, 1H); 5.20 (s, 2H); 5.10 (s, 2H); 1.30 (s, 9H).
Anal. Calcd for C23H24BrNO2: C, 64.79; H, 5.67; N, 3.29. Found: C, 64.49; H 5.56; N, 3.19.
To 4-benzyloxy-2(1H)-pyridone (1.0 g, 0.005 mol) and potassium carbonate (1.0 g, 0.007 mol) in DMF (10 mL) was added bromoethane (0.82 mL, 0.011 mol). Contents were heated at 75° C. overnight. Contents were allowed to cool and partitioned between EtOAc and water. The EtOAc layer was dried over MgSO4, filtered, and concentrated in vacuo leaving a waxy solid, which was recrystallized from EtOAc/hexanes to give a white solid (720 mg). To the white solid (700 mg, 0.003 mol) in glacial acetic acid (10 mL), bromine (0.17 mL, 0.00325 mol) in glacial acetic acid (5 mL) was added dropwise at 15° C. Contents were stirred one hour at room temperature and a yellow solid (1.1 g) was filtered. The solid was partitioned between EtOAc and 2.5N sodium hydroxide. The EtOAc layer was dried over MgSO4, filtered, and concentrated in vacuo leaving a colorless oil (710 mg), which solidified and was recrystallized from EtOAc/hexanes to give the desired as a white solid (600 mg). mp 61.3-64.8° C. FABHRMS m/z 310.0267 (M+H, C14H15BrNO2 requires 310.0263). 1H NMR (CDCl3/300 MHz) δ: 7.45-7.30 (m, 6H); 7.22 (d, 1H); 6.07 (d, 1H); 5.20 (s, 2H); 4.00 (q, 2H); 1.32 (t, 3H).
Anal. Calcd for C14H14BrNO2: C, 54.56; H, 4.58; N, 4.55. Found: C, 54.21; H, 4.38; N, 4.43.
The material of Example 12 (120 mg, 0.25 mmol) and 10% palladium/carbon (30 mg) in glacial acetic acid (2 mL) were shaken at 55 lbs of hydrogen for 4 hours. Contents were filtered and the filtrate was concentrated in vacuo leaving an oil, which was crystallized from EtOAc/hexanes to give the desired as a white solid, 59 mg (80% yield). Mp 102.0-108° C. FABHRMS m/z 295.9952 (M+H, C12H10BrNO3 requires 295.9922). 1H NMR (DMSO-d6/300 MHz) δ: 11.40 (br s, 1H); 9.40 (br s, 1H); 7.60 (d, 1H); 7.10 (d, 2H); 6.70 (d, 2H); 6.02 (d, 1H); 4.93 (s, 2H).
Anal. Calcd for C12H10BrNO3 (1.4 H2O): C, 44.85; H, 4.02; N, 4.36. Found: C, 45.07; H, 4.10; N, 4.35.
To 4-benzyloxy-2(1H)-pyridone (1.0 g, 0.005 mol) and potassium carbonate (760 mg, 0.0055 mol) in DMF (10 mL) was added methyl iodide (0.342 mL, 0.0055 mol). Contents were stirred overnight. Contents were partitioned between EtOAc and water. The EtOAc layer was dried over MgSO4, filtered, and concentrated in vacuo leaving a white solid (960 mg).
To the white solid (332 mg, 0.0015 mol) in glacial acetic acid (10 mL), bromine (256 mg, 0.0016 mol) in glacial acetic acid (5 mL) was added dropwise at 15° C. Contents were stirred one hour at room temperature and the desired was filtered as a white solid, 262 mg (59% yield). mp 105.3-105.6° C. FABHRMS m/z 296.0097 (M+H, C13H13BrNO2 requires 296.0110). 1H NMR (CDCl3/300 MHz) δ: 7.45-7.30 (m, 6H); 7.22 (d, 1H); 6.07 (d, 1H); 5.20 (s, 2H); 4.00 (q, 2H); 1.32 (t, 3H)
Anal. Calcd for C13H12BrNO2 (HBr, 0.3H2O): C, 41.04; H, 3.60; N, 3.68. Found: C, 41.00; H, 3.87; N, 3.52.
The material of Example 22 was partitioned between EtOAc and 2.5N sodium hydroxide. The EtOAc layer was dried over MgSO4, filtered, and concentrated in vacuo leaving a red oil, which solidified and was recrystallized from EtOAc/hexanes to give the desired as colorless crystals (103 mg). mp 61.3-64.8° C. FABHRMS m/z 294.0112 (M+H, C13H13BrNO2 requires 294.0130). 1H NMR (CDCl3/300 MHz): 7.45-7.30 (m, 6H); 7.22 (d, 1H); 6.07 (d, 1H) 5.20 (s, 2H); 4.00 (q, 2H); 1.32 (t, 3H)
Anal. Calcd for C13H12BrNO2: C, 53.08; H, 4.11; N, 4.76. Found: C, 53.06; H, 4.20; N, 4.74.
The material of Example 17 (500 mg, 0.00127 mol), hydroxylamine hydrochloride (90 mg, 0.0013 mol) and sodium bicarbonate (109 mg) were refluxed in ethanol (15 mL) overnight. Contents were allowed to cool and a solid was filtered and washed with water to give the desired as a white solid, 447 mg, (82% yield). mp 210.2-212.2° C. FABHRMS m/z 428.0634 (M+H, C20H19BrN3O3 requires 428.0610). 1H NMR (DMSO-d6/300 MHz): 9.66 (s, 1H); 7.98 (d, 1H); 7.65 (d, 2H); 7.55-7.35 (m, 5H); 7.30 (d, 2H); 6.54 (d, 1H); 5.82 (s, 2H); 5.35 (s, 2H); 5.17 (s, 2H).
Anal. Calcd for C20H18BrN3O3: C, 56.09; H, 4.24; N, 9.81. Found: C, 55.92; H, 4.01; N, 9.52.
To the material of Example 11 (924 mg, 0.0033 mol) in DMF (5 mL) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 3.6 mL). Contents were stirred one hour before adding dropwise a solution of 4-methanesulfonyloxymethyl-1-piperidine-1-carboxylic acid tert-butyl ester (J. Labelled Compd, Radiopharm, 38(7), 1996, 595-606) (1.0 g, 0.0036 mol) in DMF (5 mL). Contents were heated at 75° C. overnight. Contents were allowed to cool and poured into water (100 mL). A solid was filtered and recrystallized from EtOAc to give white crystals (546 mg). The white crystals were refluxed in 4 N HCl/dioxane (10 mL) for 3 hours, allowed to cool and filtered to give the desired as a white solid, 415 mg (30% yield). mp 207.9° C. FABHRMS m/z 377.0852 (M+H, C18H23BrClN2O2 requires 377.0865). 1H NMR (DMSO-d6/300 MHz) δ: 8.90 (br, 1H); 8.64 (br, 1H); 7.80 (d, 1H); 7.50-7.30 (m, 5H); 6.48 (d, 1H); 5.30 (s, 2H); 3.83 (d, 2H); 3.20 (d, 2H); 2.88-2.64 (m, 2H); 2.10-1.90 (m, 1H); 1.60 (d, 2H); 1.50-1.40 (m, 2H).
Anal. Calcd for C18H22BrClN2O2 (0.3 H2O): C, 51.58; H, 5.43; N, 6.68. Found: C, 51.59; H, 5.42; N, 6.81.
The material of Example 26 was prepared according to the procedure of Example 1. FABHRMS m/z 360.1213 (M+H, C20H17F3NO2 requires 360.1211). 1H NMR (CDCl3/300 MHz) δ: 7.60 (d, 2H) 7.41-7.30 (m, 7H); 7.13 (d, 1H); 6.05-6.01 (m, 1H); 6.00-5.95 (m, 1H); 5.13 (s, 2H); 5.00 (s, 2H)
Anal. Calcd for C20H16F3NO2: C, 66.85; H, 4.49; N, 3.90. Found: C, 66.64; H, 4.26; N, 3.93.
The material of Example 27 was prepared according to the procedure of Example 2. FABHRMS m/z 438.0308 (M+H, C20H16BrF3NO2 requires 438.0316). 1H NMR (CDCl3/300 MHz) δ: 7.65-7.20 (m, 10H); 6.13-6.03 (m, 1H); 5.30-5.13 (m, 4H).
Anal. Calcd for C20H15BrF3NO2: C, 54.81; H, 3.45; N, 3.20. Found: C, 54.69; H, 3.34; N, 3.19.
To the material of Example 11 (3.1 g, 0.011 mol) in DMF (20 mL) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 12 mL). Contents were stirred one hour before adding dropwise a solution of 3-methanesulfonyloxymethyl-1-piperidine-1-carboxylic acid tert-butyl ester (Bioorg. Med. Chem. Lett, 8(13), 1998, 1595-1600) (4.2 g, 0.015 mol) in DMF (5 mL). Contents were heated at 75° C. overnight. Contents were allowed to cool, poured into water (100 mL) and a solid was filtered. The solid was stirred in 4 N HCl/dioxane (15 mL) for 3 hours and filtered to give the desired as a white solid, 752 mg (18% yield). mp 138.1-139.2° C. FABHRMS m/z 377.0859 (M+H, C18H22BrN2O2 requires 377.0865). 1H NMR (DMSO-d6 /300 MHz): 9.50-9.10 (br, 2H); 8.00 (d, 1H); 7.50-7.30 (m, 5H); 6.93 (d, 1H); 5.30 (s, 2H); 4.30-3.90 (m, 3H); 3.40-3.10 (m, 3H); 2.80-2.50 (m, 3H); 2.40-2.00 (m, 1H); 1.90-1.60 (m, 4H); 1.40-1.10 (m, 1H).
Anal. Calcd for C18H21BrN2O2 (2HCl, 0.25 H2O): C, 47.55; H, 5.21; N, 6.16. Found: C, 47.48; H, 5.46; N, 6.27.
To the material of Example 11 (500 mg, 0.0018 mol) in DMF (5 ML) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 2 mL). Contents were stirred one hour before adding dropwise a solution of methanesulfonic acid 2-thiophen-3-yl-ethyl ester (J.A.C.S, 109(6), 1987, 1858-1859) (412 mg, 0.002 mol) in DMF (5 mL). Contents were heated at 75° C. overnight. Contents were allowed to cool, poured into water (100 mL), and extracted into EtOAc, dried over MgSO4, filtered, and concentrated in vacuo leaving a light yellow oil. The oil was purified by silica gel chromatography eluting with 50% EtOAc/hexanes to give the desired as a white solid, 199 mg (28% yield). mp 134.0-134.3° C.
FABHRMS m/z 390.0144 (M+H, C18H17BrNO2S requires 390.0163). 1H NMR (CDCl3/300 MHz): 7.43-7.20 (m, 6H); 6.92-6.80 (m, 3H); 5.90 (d, 1H); 5.20 (s, 2H); 4.13 (t, 2H); 3.10 (t, 2H).
Anal. Calcd for C18H16BrNO2S: C, 55.39; H, 4.13; N, 3.59. Found: C, 55.21; H, 3.87; N, 3.52.
To the material of Example 11 (1.0 g, 0.0036 mol) in DMF (10 mL) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 4 mL). Contents were stirred one hour before adding dropwise a solution of methanesulfonic acid 2-thiophen-2-yl-ethyl ester (J. Chem. Res. Miniprint, 6, 2000, 701-715) (824 mg, 0.004 mol) in DMF (5 mL). Contents were heated at 75° C. overnight. Contents were allowed to cool, poured into water (100 mL), and filtered a solid. The solid was recrystallized from EtOAc to give the desired as a white solid, 411 mg (29% yield). mp 128.0-129.5° C. FABHRMS m/z 390.0160 (M+H, C18H17BrNO2S requires 390.0163). 1H NMR (CDCl3/300 MHz) δ: 7.48-7.30 (m, 5H); 7.12 (d, 1H); 6.95-6.80 (m, 2H); 6.75-6.68 (m 1H); 5.95 (d, 1H); 5.20 (s, 2H); 4.16 (t, 2H); 3.30 (t, 2H).
Anal. Calcd for C18H16BrNO2S: C, 55.39; H, 4.13; N, 3.59. Found: C, 55.06; H, 4.01; N, 3.56.
To the material of Example 11 (500 mg, 0.0018 mol) in DMF (5 mL) was added dropwise sodium bis(trimethylsilyl)amide (1M in THF, 2 mL). Contents were stirred one hour before adding dropwise a solution of 3-trifluoromethylbenzyl bromide (478 mg, 0.002 mol) in DMF (5 mL). Contents were heated at 75° C. for 2 hours. Contents were allowed to cool, poured into water (100 mL), and extracted with EtOAc, which was dried over MgSO4, filtered, and concentrated in vacuo leaving a white solid. The solid was recrystallized from EtOAc to give the desired as a white solid, 341 mg (43% yield). mp 181.5-181.6° C. FABHRMS m/z 438.0301 (M+H, C20H16BrF3NO2 requires 438.0316). 1H NMR (CDCl3/300 MHz): 7.60-7.20 (m, 10H); 6.10 (d, 1H); 5.14 (s, 2H); 5.20 (s, 2H).
Anal. Calcd for C20H15BrF3NO2: C, 54.81; H, 3.45; N, 3.20. Found: C, 54.81; H, 3.36; N, 3.13.
The material of Example 32 was prepared according to the procedure of Example 31.
FABHRMS m/z 438.0280 (M+H, C20H16BrF3NO2 requires 438.0316). 1H NMR (CDCl3/300 MHz) δ: 7.68 (d, 1H); 7.55-7.20 (m, 8H); 7.15 (d, 1H); 6.10 (d, 1H); 5.40 (s, 2H); 5.13 (s, 2H).
Anal. Calcd for C20H15BrF3NO2: C, 54.81; H, 3.45; N, 3.20. Found: C, 54.48; H, 3.36; N, 3.17.
The material of Example 33 was prepared according to the procedure of Example 1.
FABHRMS m/z 376.1158 (M+H, C20H17F3NO3 requires 376.1161). 1H NMR (CDCl3/300 MHz) δ: 7.40-7.05 (m, 10H); 6.05-5.95 (m, 2H); 5.06 (s, 2H); 4.98 (s, 2H)
Anal. Calcd for C20H16F3NO3: C, 64.00; H, 4.30; N, 3.73. Found: C, 63.97; H, 4.26; N, 3.57.
The material of Example 34 was prepared according to the procedure of Example 2.
FABHRMS m/z 454.0240 (M+H, C20H16BrF3NO3 requires 454.0266). 1H NMR (CDCl3/300 MHz) δ: 7.45-7.10 (m, 10H); 6.08 (d, 1H); 5.20 (s, 2H); 5.12 (s, 2H).
Anal. Calcd for C20H15BrF3NO3: C, 52.88; H, 3.33; N, 3.08. Found: C, 52.53; H, 3.09; N, 2.92.
4-hydroxy-6-methyl-2-pyrone (0.2 mol, 25.2 g) and benzylamine (0.2 mol, 21.4 g) were added to water (800 mL) and heated to reflux with stirring for 2 hours. After cooling to room temperature, a light brown solid was collected by filtration. (33.4 g, 77%): 1H NMR (DMSO-d6/300 MHz) δ: 10.5 (s, 1H), 7.4-7.1 (m, 5 H), 5.8-5.6 (m, 2H), 5.2 (s,2H), 5.1 (s, 2H), 2.2 (s, 3H). ESHRMS m/z 216.100 (M+H, C12H13NO2 requires 216.102).
1-benzyl-4-hydroxy-6-methylpyridin-2(1H)-one (10 mmol, 2.15 g), dichloromethane (100 mL), benzylbromide (11 mmol, 1.88 g), sodium hydroxide (2.5 N, 20 mmol, 8 mL), and benzyltriethylammonium chloride (0.5 g) were vigorously stirred at room temperature for 16h. Hydrochloric acid (1 N) was added until the mixture produced an acidic reaction to pH paper. The mixture was then extracted with ethyl acetate (3×50 mL). The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered, and concentrated. The product was obtained by flash chromatography eluting with ethyl acetate: hexanes (1:2). The appropriate fractions were concentrated to a clear oil. (1.3 g, 43%): 1H NMR (DMSO-d6/300 MHz) δ: 7.4-7.1 (m, 10 H), 6.0-5.9 (m, 2H), 5.2 (s,2H), 5.1 (s, 2H), 2.2 (s, 3H). ESHRMS m/z 306.147 (M+H, C20H19NO2 requires 306.149).
The product from example 35, 1-benzyl-4-(benzyloxy)-6-methylpyridin-2(1H)-one (4.2 mmol, 1.3 g), acetic acid (50 ML), and sodium acetate (5.0 mmol, 0.41 g) were stirred at room temperature. Bromine (4.2 mmol, 0.67 g) was added drop wise with stirring. After M hour, water (100 mL) was added and the mixture was extracted with ethyl acetate (3×50 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate solution and brine. After drying over magnesium sulfate and concentrating, the mixture was purified by flash column chromatography eluting with ethyl acetate:hexanes (1:2). The appropriate fractions were concentrated to yield a light oil. (1.0 9, 62%): 1H NMR (DMSO-d6/300 MHz) 7.4-7.0 (m, 10 H), 6.5 (s, 1H), 5.29 (s,2H), 5.27 (s, 2H), 2.2 (s, 3H). ESHRMS m/z 384.057 (M+H, C20H18NO2Br requires 384.060).
The product from example 35, 1-benzyl-4-(benzyloxy)-6-methylpyridin-2(1H)-one (4.2 mmol, 1.3 g), acetic acid (50 mL), and sodium acetate (5.0 mmol, 0.41 g) were stirred at room temperature. Bromine (4.2 mmol, 0.67 g) was added drop wise with stirring. After ½ hour, water (100 mL) was added and the mixture was extracted with ethyl acetate (3×50 mL) . The combined organics were washed with saturated aqueous sodium bicarbonate solution and brine. After drying over magnesium sulfate and concentrating, the mixture was purified by flash column chromatography eluting with ethyl acetate:hexanes (1:2). The appropriate fractions were concentrated to yield a white solid. (0.3 g, 15%): 1H NMR (DMSO-d6/300 MHz) 7.5-7.0 (m, 10 H), 5.42 (s,2H), 5.07 (s, 2H), 2.45 (s, 3H). ESHRMS m/z 463.966 (M+H, C20H17NO2Br2 requires 463.968).
1-benzyl-4-hydroxy-6-methylpyridin-2(1H)-one (from example 35) (10 mmol, 2.15 g), N,N′-dimethylformamide (30 mL), potassium carbonate (20 mmol, 2.76 g), and 4-bromobenzenesulfonyl chloride (10 mmol, 2.55 g) were stirred at room temperature for 16 hours. Hydrochloric acid (1N) was added until the mixture was acidic to pH paper. Brine (50 mL) was added and the mixture extracted with ethyl acetate (3×50 mL). The combined organic extracts were washed with brine and dried over magnesium sulfate, and filtered. After concentrating, the material was purified by flash column chromatography eluting with ethyl acetate:hexanes (1:2). The appropriate fractions were concentrated to a clear oil, which solidified upon standing several days to a white solid. (3.3 g, 76%): 1H NMR (DMSO-d6/400 MHz) 7.9 (m, 4H), 7.32-7.00 (m, 5H), 7.3 (m, 1H), 6.12 (d, J=2.4 Hz, 1H), 6.02 (d, J=2.8 Hz, 1H), 5.20 (s, 2H), 2.2 (s, 3H). ESHRMS m/z 436.002 (M+H, C19H16NO4SBr requires 436.004).
1-benzyl-6-methyl-2-oxo-1,2-dihydropyridin-4-yl 4-bromobenzenesulfonate (3.0 mmol, 1.3 g), N,N′-dimethylformamide (30 mL), 3-chlorobenzyl alcohol (3.0 mmol, 0.43 g), and sodium hydroxide (60%, 3.3 mmol, 0.13 g) were stirred at room temperature under nitrogen for 4 hours. Hydrochloric acid (1 N, 10 mL) was added and the mixture extracted with ethyl acetate (3×25 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate solution and brine. After drying over magnesium sulfate and concentrating, the mixture was purified by flash column chromatography eluting with ethyl acetate:hexanes (1:1) to obtain a light yellow oil. (14.3 g, 64%): 1H NMR (DMSO-d6/300 MHz) δ: 7.4-7.0 (m, 10 H), 6.0-5.8 (m, 2H) , 5.2 (s,2H) , 5.0 (s, 2H) , 2.1 (s, 3H). ESHRMS m/z 340.110 (M+H, C20H18NO2Cl requires 340.110).
The product of example 38 (SC-83316), 1-benzyl-4-[(3-chlorobenzyl)oxy]-6-methylpyridin-2(1H)-one (0.91 mmol, 310 Mg), acetic acid (20 mL), and sodium acetate (0.91 mmol, 80 Mg) were stirred at room temperature when bromine (0.91 mmol, 145 Mg) was added. After stirring for one hour, the mixture was concentrated, dissolved in ethyl acetate, and washed successively with saturated aqueous sodium bicarbonate solution, brine, and water. After drying over magnesium sulfate and concentrating, the product was recrystallized from tetrahydrofuran/hexanes to yield a white solid. (240 Mg, 63%): 1H NMR (DMSO-d6/300 MHz) 7.6-7.0 (m, 10 H), 6.5 (s, 1H), 5.33 (s,2H), 5.33 (s, 2H), 2.3 (s, 3H). ESHRMS m/z 420.019 (M+H, C20H17NO2BrCl requires 420.019).
2,6-Dichlorobenzyl alcohol (12.57 g, 71 mmol) was dissolved in DMF, (100 mL). Sodium hydride (2.84 g, 71 mmol, of a 60% dispersion in mineral oil) was added in portions. Reaction mixture stirred for one hour. 4-Nitropyridine-1-oxide (10.0 g, 71 mmol) dissolved in DMF (150 mL) was added via cannula. Reaction stirred at room temperature for 16 hours. The solvent was evaporated, and the residue was purified by flash column chromatography (dichloromethane-methanol, 10:1) to yield an orange oil. Addition of ethyl acetate caused the formation of a yellow precipitate 13.5 g (70%).
4-[(2,6-Dichlororbenzyl)oxy]pyridine-1-oxide (Step 1) (4.0 g, 14.8 mmol) was dissolved in acetic anhydride (30 mL). The reaction mixture was heated at reflux for 6 hours. Stirred at room temperature for 16 hours. The solvent was evaporated, and the residue was dissolved in dichloromethane (50 mL). Extracted with 2.5 N NaOH (2×25 mL), and H2O (25 mL). The organic phase was dried over MgSO4, filtered, and evaporated to yield a brown solid. Recrystallized from hot ethyl acetate. Obtained a tan solid (0.984 g, 25%).
4-[(2,6-Dichlorobenzyl)oxy]pyridine 1-oxide (0.500 g, 1.85 mmol) was dissolved in DMF (20 mL). Potassium carbonate (0.511 g, 3.70 mmol) was added, followed by benzyl bromide (0.260 mL, 0.400 g, 2.20 mmol). The reaction mixture was stirred at room temperature for 16 hours. Solvent was evaporated, and the residue dissolved in ethyl acetate (50 mL). Extracted with H2O (2×25 mL), and brine (25 mL). The organic phase was dried over MgSO4, filtered, and evaporated to yield a tan solid. Recrystallized from ethyl acetate/hexane. Filtered a light tan solid (0.502 g, 75%). mp 151.6-152.0° C. 1H NMR (CDCl3/300 MHz) δ: 7.31 (m, 8H), 7.12 (d, 1H, J=7.45 Hz), 6.13 (d, 1H, J=2.42 Hz), 5.90 (dd, 1H, J=2.62 Hz), 5.22 (s, 2H) , 5.10 (s, 2H) ESHRMS m/z 360.0551 (M+H C19H15Cl2NO2 requires 360.0558).
1-Benzyl-4-[2,6-(dichlorobenzyl)oxy]pyridin-2(1H)-one (0.400 g, 1.11 mmol) was dissolved in acetic acid (10 mL). Sodium acetate (0.091 g, 1.11 mmol was added, and the mixture was cooled to 15° C. Bromine (0.195 g, 1.22 mmol) was added via syringe. The reaction stirred at room temperature for 2 hours. Water (15 mL) was added, and the mixture transferred to a separatory funnel. Ethyl acetate (50 mL) was added and the layers were separated. The organic phase was washed with aqueous NaHCO3 (2×25 mL), dried over MgSO4, filtered, and evaporated to yield a white solid. Recrystallization from ethyl acetate/hexanes afforded a white solid (0.380 g, 62%). mp 156.3-157.9° C. 1HNMR (CDCl3/300 MHz) δ: 7.34 (m, 9H), 6.24 (d, 1H, J=7.65 Hz), 5.37 (s, 2H) , 5.18 (s, 2H). ESHRMS m/z 439.9646 (M+H C19H14BrCl2NO2 requires 439.9641).
The title compound was prepared by a procedure similar to the one described in Example 1. mp 124.6-125.0° C. 1HNMR (CDCl3/300 MHz) δ: 7.36 (m, 9H), 7.14 (d, 1H, J=7.65 Hz), 6.04 (d, 1H, J=2.62 Hz), 5.98 (d, 1H, J=2.82 Hz), 5.10 (s, 2H), 5.09 (s, 2H). ESHRMS m/z 326.0950 (M+H C19H16ClNO2 requires 326.0948).
Anal. Calc'd. for C19H16ClNO2: C, 70.05; H, 4.95; N, 4.30; Cl, 10.88. Found: C, 69.87; H, 4.74; N, 4.42, Cl, 11.08.
The title compound was prepared by a procedure similar to the one described in Example 2. mp 143.3-145.5° C. 1HNMR (CDCl3/300 MHz) δ: 7.63 (d, 2H, J=1.81 Hz), 7.44 (m, 9H), 6.06 (d, 1H, J=7.65 Hz), 5.29 (s, 2H), 5.17 (s, 2H). ESHRMS m/z 406.0036 (M+H C19H15BrClNO2 requires 406.0032). Anal. Calc'd. for C19H15Cl BrNO2: C, 56.39; H, 3.74; N, 3.46; Cl, 8.76. Found: C, 56.01; H, 3.38; N, 3.36, Cl, 9.01.
The title compound was prepared by a procedure similar to the one described in Example 2. mp 149.0-149.7° C. 1HNMR (CDCl3/300 MHz) δ: 7.25 (m, 10H), 6.04 (d, 1H, J=7.65 Hz), 5.17 (s, 2H), 5.15 (s, 2H), 2.34 (s, 3H). ESHRMS m/z 386.0583 (M+H C20H18BrNO2 requires 386.0581).
The title compound was prepared by a procedure similar to the one described in Example 1. mp 95.5-95.7° C. 1HNMR (CDCl3/300 MHz) δ: 7.34 (m, 9H), 7.13 (d, 1H, J=7.45 Hz), 5.96 (m, 1H), 5.95 (d, 1H, J=7.45 Hz), 5.09 (s, 2H), 4.96 (s, 2H). ESHRMS m/z 326.0977 (M+H C19H16ClNO2 requires 326.0948).
The title compound was prepared by a procedure similar to the one described in Example 2. mp 180.6-182.1° C. 1HNMR (CDCl3/300 MHz) δ: 7.33 (m, 10H), 7.14 (d, 1H, J=7.45 Hz), 6.08 (d, 1H, J=7.45 Hz), 5.13 (s, 2H), 4.15 (s, 2H). ESHRMS m/z 386.0211 (M+H C19H16BrNOS requires 386.0214).
The title compound was prepared by a procedure similar to the one described in Example 2. mp 133.2-133.5° C. 1HNMR (CDCl3/300 MHz) δ: 7.81 (d, 1H, J=7.65 Hz), 7.68 (d, 1H, J=7.65 Hz), 7.61 (t, 1H, J=7.65 Hz), 7.38 (m, 7H), 6.01 (d, 1H, J=7.85 Hz), 5.39 (s, 2H), 5.16 (s, 2H). ESHRMS m/z 438.0313 (M+H C20H15BrF3NO2 requires 403.0316).
A mixture of N,O-dibenzyl-2-pyridone (2.0 g, 6.87 mmol), N-iodosuccinimide (1.7 g), dichloroacetic acid (0.15 mL) in acetonitrile (40.0 mL) was heated at 65° C. under argon atmosphere for 3.5 h, with constant stirring. The reaction mixture was concentrated to dryness, and the residue was purified by silica gel flash chromatography using EtOAc/hexanes 1:1 v/v to give the title compound 2.3 g (80%) as a flaky white solid: 1H-NMR (CDCl3) δ: 7.4-7.2 (m, 10 H), 7.19 (1H, d, J=7.6 Hz), 5.95 (d, 1H, J=7.6 Hz), 5.2 (s, 1H), 5.15 (s, 2H); ER-MS m/z=418 (MH+); HR-MS m/z calcd C19H17NO2 418.0304, found 418.0277.
A solution of 1-benzyl-4-(benzyloxy)-3-iodopyridin-2(1H)-one (1.9 g, 4.56 mmol) and vinyl-tri-butyltin (2.5 mL) in acetonitrile (20 0 mL) containing DMF (2.0 mL) was degassed using house vacuum and purged with argon. Then added PdCl2(PPh3)2 (0.3 g) and the mixture was heated at 65° C. under argon atmosphere for 4 h, with stirring. The solvents were distilled in vacuo, and the residue was triturated with EtOAc and filtered through a pad of celite. The filtrate was concentrated and the residue was purified by silica gel flash chromatography using 25% EtOAc in hexanes to give the title compound (0.75 g. 50%) as an orange colored solid.
1H-NMR (CDCl3) δ: 7.4-7.2 (m, 10 H), 7.14 (d, 1H, J=7.6 Hz), 7.05 (dd, 1H, J=12.0 Hz), 6.47 (dd, 1H, J=2.8 Hz), 6.07 (d, 1H, J=7.6 Hz), 5.4 (dd, 1H, J=2.8 Hz), 5.13 (s, 4H); ER-MS m/z=418 (MH+); ER-MS m/z=318 (MH+); HR-MS m/z calcd C21H20NO2 318.1494, found 318.1480.
To a solution of 1-benzyl-4-(benzyloxy)-3-vinylpyridin-2(1H)-one (0.5 g, 1.6 mmol) in EtOH (10.0 mL) and EtOAc (10.0 mL) was added Pd/C (10%, 0.25 g) and stirred in an atmosphere of hydrogen gas at 30 psi for 16 h. The catalyst was removed by filtration, the filtrate was concentrated to dryness and the resulting residue was purified by silica gel flash chromatography using EtOAc/hexanes (1:1, v/v) to afford the title compound (0.32 g, 64%) as a pale yellow powder: 1H-NMR (CD3OD) δ: 7.52 (d, 1H, J=7.6 Hz), 7.39-7.2 (m, 10 H), 6.41 (d, 1h, J=7.6 Hz), 5.18 (s, 2H), 5.15 (s, 2H), 2.58 (q, 2H, J=7.2 Hz), 1.03 (t, 3H, J=7.2 Hz), ER-MS m/z=320 (MH+); HR-MS m/z calcd C21H22NO2320.1651, found 320.1648.
Step A
A mixture of 2-chlorophenylisocyanate (3.0 g, 19.53 mmol), and diketene (3.3 g, 39.28 mmol) in toluene (10.0 mL) containing triethylamine (0.05 mL) was heated to reflux for 6 h, under an atmosphere of argon. Toluene was distilled in vacuo and the resulting residue was purified by silica gel flash chromatography using 25% EtOAc in hexanes as the eluent to afford the title compound (0.85 g, see ref: Heterocycles 27 (9), 2063, 1988.) as a pale yellow solid: 1H-NMR (CD3OD) δ: 7.63 (m, 1H), 7.52 (m, 2H), 7.4 (m, 1H), 6.14 (s, 1H), 2.58 (s, 3H), and 1.95 (s, 3H); ES-MS m/z=278 (MH+).
Step B
To a solution of 3-acetyl-1-(2-chlorophenyl)-4-hydroxy-6-methylpyridin-2(1H)-one ( 0.56 g, 2.02 mmol) in DMF (5.0 mL), benzyl bromide (0.3 mL) and potassium carbonate (0.3 g, 2.16 mmol) were added. The mixture was stirred at room temperature for 3 h, and at 65° C. for 1 h under argon atmosphere. The reaction mixture was concentrated in vacuo and the residue was partitioned between 5% citric acid (25 mL) and EtOAc (50.0 mL). The organic phase was washed with brine, dried (Na2SO4), filtered, and concentrated to dryness. The resulting residue was purified by silica gel flash chromatography using 50% EtOAc in hexanes to afford the title compound (0.58 g, 75%) as a pale yellow amorphous substance: 1H-NMR (CD3OD) δ: 7.65-7.3 (m, 9H), 6.5 (s, 1H), 5.31 (s, 2H), 2.42 (s, 3H), and 2.01 (s, 3H); ER-MS m/z=368 (MH +); HR-MS m/z calcd C21H19NO3Cl, 368.1060, found 368.1053.
Step A
A suspension of N-benzyl-4-hydroxy-2-pyridone ((0.75 g, 3.7 mmol), NBS (0.7 g, 1.05 mmol) in dichloromethane was stirred at room temperature for 1.5 h under argon atmosphere. It was diluted with dichloromethane (25 mL), cooled and filtered. The solids were washed with dichloromethane and dried in vacuo. The filtrate and the washings were combined and washed with water, dried (Na2SO4), filtered, and concentrated to dryness. The resulting residue was washed with EtOAc, and dried in vacuo to give a combined mass of 0.65 g of the title compound as a white powder: 1H NMR (CD3OD) δ: 7.54 (d, 1H, J=7.6 Hz), 7.27 (m, 5H), 6.12 (d, 1H, J=7.6 Hz), 5.15 (s, 2H); ES-MS: m/z=280 (MH+).
Step B
To a cold (−30° C.) suspension of 1-benzyl-3-bromo-4-hydroxypyridin-2(1H)-one (0.78 g, 2.8 mmol) in dichloromethane (10.0 mL), was added triethylamine (0.6 mL, 4.28 mmol ), followed by the addition of triflic anhydride (0.7 mL, 4.17 mmol). The resulting mixture was stirred at −30° C. under argon atmosphere for 1 h. The reaction mixture was then poured into ice/water mixture (50 mL) and the products were extracted with dichloromethane (2×25 mL). The combined organic extracts were washed with water (2×20 mL), dried (Na2SO4), filtered, and concentrated to dryness. The residue was dried in vacuo to afford the desired trifluorosulfonate (1.0 g) as a pale yellow solid which used as such in the next step: 1H-NMR (CDCl3) δ: 7.35 (m, 6H), 6.26 (d, 1H, J=8.0 Hz); 19F-NMR (CDCl3) δ: −73.73 ppm; ES-MS: m/z=412 (MH+)
Step C
To a solution of 1-benzyl-3-bromo-2-oxo-1,2-dihydropyridin-4-yl trifluoromethanesulfonate (1.0 g) in DMF (5.0 mL) was added phenylacetylene (0.4 mL) and degassed using house vacuum. The reaction flask was then purged with argon, added diisopropylethylamine (0.53 mL), and PdCl2(PPh3)2 (0.35 g) were added. The resulting mixture was stirred at room temperature for 15 min and heated at 65° C. under an argon atmosphere for 3h. The dark colored reaction mixture was concentrated in vacuo, and the residue was partitioned between EtOAc (50 mL) and 5% aqueous citric acid (25 mL). The organic extracts were washed with water, dried (Na2SO4), filtered, and concentrated to dryness. The resulting material was purified by silica gel flash chromatography using 25% EtOAc in hexanes as the eluent. The appropriate fractions were combined, concentrated under reduced pressure, and the residue was crystallized from EtOAc/hexanes to afford the title compound (0.35 g) as shiny flakes: 1H NMR (CDCl3) δ: 7.57 (m, 2H), 7.38 (m, 8H), 7.21 (d, 1H, J=6.8 Hz), 6.25 (d, 1H, J=6.8 Hz), and 5.16 (d, 2H), ES-MS: m/z=364 (MH+);
HR-MS m/z (MH+) calcd C20H15NOBr 364.0337, found 364.0337.
Step D
A mixture of 1-benzyl-3-bromo-4-(phenylethynyl)pyridin-2(1H)-one (0.3 g), and platinum oxide (0.05 g) in a solvent mixture of EtOAc (10.0 mL) and EtOH ( 10.0 mL) was stirred in an atmosphere of hydrogen at 15 psi in a Fischer porter bottle for 45 min. The catalyst was removed by filtration, and filtrate was concentrated. The resulting residue was purified by silica gel flash chromatography using 25% EtOAc in hexanes as the eluent. The appropriate fractions (visualized under an UV lamp) were combined and concentrated under reduced pressure. The resulting residue was crystallized from EtOAc/hexanes to give the title compound (0.16 g) as shiny flakes: 1H-NMR (CD3OD) δ: 7.56 (d, 1H, J=6.8 Hz), 7.31-7.17 (m, 10 H), 6.24 (d, 1H, J=6.8 Hz), 5.19 (s, 2H), 2.96 (m, 2H), and 2.91 (m, 2H); ES-MS m/z=368 (MH+); HR-MS m/z (MH+) calcd C20H19NOBr 368.0650, found 368.0630.
Step A
A mixture of 4-hydroxy-6-methyl-2-pyrone (2.5 g, 0.02 mol) and 3-fluorobenzylamine (2.5 g, 0.02 mol) in n-butanol (15.0 mL) was heated to reflux for 16 h under argon atmosphere. Butanol wad distilled in vacuo, the residue was triturated with EtOAc, cooled and filtered the precipitate. It was washed with cold EtOAc, and dried to give 0.86 g of the title compound as a pale yellow powder: 1H-NMR δ: (CD3OD) δ: 7.31 (m, 1H), 7.0-6.85 (m, 2H), 6.83 (d, 1H, J=9.6 Hz), 5.96 (d, 1H, j=2.0 Hz), 5.80 (d, 1H, J=2.0 Hz), 5.30 (s, 2H), and 2,24 (s, 3H); ES-MS m/z=234 (MH+)
Step B
A mixture of 1-(3-fluorobenzyl)-4-hydroxy-6-methylpyridin-2(1H)-one ( 0.8 g, 0.0034 mol), NBS (0.64 g, 0.0036 mol) in dichloromethane (15.0 mL) was stirred at room temperature, under argon atmosphere. After 1.5 h, the reaction mixture was diluted with dichloromethane (15.0 mL), cooled and filtered the solids. The residue was washed with dichloromethane and dried in vacuo to give 0.93 g of the title compound as a white powder: 1H-NMR δ: (CD3OD) δ: 7.33 (m, 1H), 7.2-6.8 (m, 3H), 6.07 (s, 1H), 5.34 (s, 2H), 2.26 (s, 3H); ES-MS m/z=312 (MH+); HR-MS m/z calcd C13H12NO2BrF 312.0035, found 312.0016.
Step C
To a suspension of 3-bromo-1-(3-fluorobenzyl)-4-hydroxy-6-methylpyridin-2(1H)-one (0.86 g, 0.0028 mol) in dichloromethane (15.0 mL) cooled to −30° C., triethylamine (0.5 mL, 0.004 mol) and triflic anhydride (0.7 mL, 0.0042 mol) were added and stirred for 1 h. The resulting orange solution was poured into ice cold water (25 mL) and extracted with dichloromethane (2×25 mL) The combined organic extracts were washed with water, dried (Na2SO4), filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash chromatography using 1:1 EtOAc/hexanes v/v to afford (85%) the title compound as a light brown solid: 1H-NMR (CDCl3) δ:g.32 (m, 1H), 7.0-6.85 (m, 3H), 6.18 (s, 1H), 5.32 (s, 2H), and 2.34 (s, 3H); ES-MS m/z=444 (MH+); HR-MS m/z calcd C14H11NO4BrF4S 443.9528, found 443.9492.
Step D
A solution of 3-bromo-1-(3-fluorobenzyl)-6-methyl-2-oxo-1,2-dihydropyridin-4-yl trifluoromethanesulfonate (1.0 g, 0.0022 mol) and phenylacetylene (0.3 mL, 0.0029 mol) in DMF (5.0 mL) was degassed using house vacuum, and purged with argon (3 cycles). Diisopropylethylamine, (0.5 mL) was then added, followed by PdCl2(PPh3)2 (0.36 g). The reaction mixture was heated at 65° C. for 1.5 h under argon atmosphere. The solvents were distilled in vacuo, and the residue was purified by silica gel flash chromatography using EtOAc/hexanes (2:3 v/v) to afford 0.65 g (70%) of the title compound as a brown colored amorphous solid:
1H-NMR (CD3OD) δ: 7.59 (m, 2H), 7.45-7.3 (m, 4H), 7.05-6.85 (m, 3H), 6.44 (s, 1H), 5.41 (s, 2H), and 2.31 (s, 3H);
19F-NMR (CD3OD) δ: −116.33 (m); ES-MS, m/z=396 (MH+); HR-MS m/z cald C21H16NOBrF 396.0399, found 396.0373.
Step E
To a solution of 3-bromo-1-(3-fluorobenzyl)-6-methyl-4-(phenylethynyl)pyridin-2(1H)-one (0.55 g, 0.0014 mol) in EtOAc (10.0 mL) and EtOH (10.0 mL) was added PtO2 (0.05 g) and stirred in an atmosphere of hydrogen gas at 15 psi for 30 min. The catalyst was removed by filtration, the filtrate was concentrated and the residue was purified by silica gel flash chromatography using 25% EtOAc in hexanes as the eluent. The appropriate fractions were combined (visualized under UV) and concentrated to dryness. The residue was crystallized from EtOAc/hexanes to provide 0.21 g of the title compound as a light brown crystalline solid: 1H-NMR δ: (CD3OD) δ: 7.35 (m, 1H), 7.31-7.16 (m, 5H), 6.99(m, 1H), 6.91 (m, 1H), 6.81 (m, 1H), 6.20 (s, 1H), 5.41 (s, 2H), 2.94 (m, 4H), and 2.24 (s, 3H); 19F-NMR (CD3OD) δ: −115.01 (m); ES-MS, m/z=400 (MH+); HR-MS m/z calcd C21H20NOBrF 400.0712, found 400.0695.
Step A
A mixture of 2,6 dichlorophenylisocyanate (4.8 g, 0.025 mol), and diketene (4.3 g, 0.05 mol) in toluene (15.0 mL) was heated to reflux for 4 h under an atmosphere of argon. After removal of the solvent in vacuo, the residue was purified by silica gel flash chromatography using EtOAc/hexanes (1:3 v/v). The appropriate fractions, as monitored by ES mass spectrometry (MH+ m/z=312) were combined and concentrated under reduced pressure. The resulting yellow solid (2.3 g) was further purified by reverse-phase HPLC using 10-90% acetonitrile/water gradient (45 min) at a flow rate of 100 mL/min. The appropriate fractions, as monitored by ES mass spectrometry (MH+ m/z=312) were combined and concentrated to half the volume. The solid that separated was extracted with EtOAc (2×25 mL). The combined extracts were washed with water, dried (Na2SO4), filtered, and concentrated to dryness to give the title compound (0.77 g) as a pale yellow powder: 1H-NMR (CD3OD) δ: 7.62 (m, 2H), 7.52 (m, 1H), 6.19 (s, 1H), 2.59 (s, 3H), and 1.96 (s, 3H); ES-MS m/z=312 (MH+); HR-MS, m/z calc C14H12NO3Cl2 312.0189, found 312.0214.
Step B.
A mixture of 3-acetyl-1-(2,6-dichlorophenyl)-4-hydroxy-6-methylpyridin-2(1H)-one 0.7 g (0.002mol) in n-butanol (3.0 mL) containing sulfuric acid (1.5 mL) was heated at 120° C. for 4 h. The dark reaction mixture was cooled, added ice/water (25 mL), and extracted with EtOAc (2×25 ml). The combined organic extracts were washed with water, dried (Na2SO4), filtered, concentrated under reduced pressure and the resulting material was purified by silica gel flash chromatography using 25% EtOAc in hexanes as the eluent to afford the title compound (0.14 g) as a pale yellow powder: 1H-NMR (CD3OD) δ: 7.6 (m, 2H), 7.48 (m, 1H), 6.10 (dd, 1H), 5.78 (d, 1H, J=2.4 Hz), 1.91 (s, 3H); ES-MS m/z=270 (MH+); HR-MS, m/z calc C12H10NO2Cl2 270.0083, found 270.0103.
Step C
A mixture of 1-(2,6-dichlorophenyl)-4-hydroxy-6-methylpyridin-2(1H)-one (0.125 g, 0.46 mmol) and benzylbromide (0.1 mL) in DMF (2.5 mL) was stirred at room temperature for 16 h. The reaction mixture was diluted with water (10.0 mL) and extracted with EtOAc (2×20 mL). The combined organic extracts were washed with water, dried (Na2SO4), filtered, concentrated under reduced pressure and the resulting material was purified by silica gel flash chromatography using 25% EtOAc in hexanes to afford the title compound (0.11 g) as a pale yellow syrup: 1H-NMR (CD3OD) δ: 7.61 (m, 2H), 7.55-7.3 (m, 6H), 6.23 (d, 1H, J=2.0 Hz), 6.01 (d, 1H, J=2.0 Hz), 5.12 (s, 2H), and 1.93 (s, 3H); ES-MS m/z=360 (MH+); HR-MS, m/z calc C19H16NO2Cl2, 360.0553, found 360.0569.
Step D
A mixture of 4-(benzyloxy)-1-(2,6-dichlorophenyl)-6-methylpyridin-2(1H)-one (0.1 g, 0.278 mmol) and N-bromosuccinimide (0.055 g, 0.3 mmol) in dichloroethane (3.0 mL) was stirred at room temperature for 1 h, and heated at 60° C. under argon for 30 min. The reaction mixture was then diluted with dichloroethane (15 mL), washed with water, dried (Na2SO4), filtered, concentrated under reduced pressure and the resulting material was crystallized from EtOAc/hexanes to furnish the title compound (0.075 g) as pale yellow needles. 1H NMR (CD3OD) δ: 7.64 (m, 2H), 7.55 (m, 3H), 7.38 (m, 3H), 6.65 (s, 1H), 5.34 (s, 2H), and 2.00(s, 3H); ES-MS m/z=439 (MH+); HR-MS, m/z calc C19H16NO2Cl2Br, 439.9635, found 439.9669.
Step A
A mixture of 1-(3-fluorobenzyl)-4-hydroxypyridin-2(1H)-one 1.0 g, 0.004 mo) and N-bromosuccinimide (0.85 g, 0.0048 mol) in dichloromethane ( 20.0 mL) was stirred at room temperature for 30 min. The resulting white precipitate was filtered, washed with dichloromethane, and dried in vacuo to afford the title compound (1.1 g) as a white powder: 1H NMR (CD3OD) δ: 7.57 (d, 1H, J=7.6 Hz), 7.38 (m, 1H), 7.15 (m, 3H), 6.14 (d, 1H, J=7.6 Hz), and 5.15 (s, 2H); ES-MS m/z=298 (MH+).
Step B
To a cold suspension (−30° C.) of 3-bromo-1-(3-fluorobenzyl)-4-hydroxypyridin-2(1H)-one (1.0 g, 0.0034 mol) in dichloromethane (10.0 mL), was added triethylamine (0.65 mL. 0.0046 mol), followed by the addition of a solution of trifluoromethanesulfonic anhydride (0.73 mL, 0.0043 mol) in dichloromethane (3.0 mL). The resulting mixture was stirred at −30° C. under an atmosphere of argon for 1 h, and then poured into a mixture of ice/water (50 mL) and dichloromethane (25 mL). The organic phase was washed with water, dried (Na2SO4), filtered, concentrated under reduced pressure and the resulting material was purified by flash chromatography using 25% EtOAc in hexanes to give the title compound (1.25 g, 84%) as a white solid: 1H-NMR (CDCl3) δ: 7.35 (m, 2H), 7.03 (m, 3H), 6.29 (d, 1H, J=2.4 Hz), and 5.14 (s, 2H); ES-MS m/z=430 (MH+); HR-MS, m/z calc C13H9NO4F4Br, 429.9366, found 429.9364.
Step C
To a degassed solution of 3-bromo-1-(3-fluorobenzyl)-2-oxo-1,2-dihydropyridin-4-yl trifluoromethanesulfonate (0.6 g, 0.0014 mol) in DMF (5.00 mL), diisopropylethylamine amine (0.4 ml, 0.0029 mol), phenylacetylene (0.3 mL. 0.0027 mol) and PdCl2(PPh3)2 (0.2 g) were added and the mixture was heated at 65° C. for 2 h under an atmosphere of argon. The solvents were then distilled in vacuo and the residue was purified by silica gel flash chromatography using 25% EtOAc in hexanes. Appropriate fractions, as monitored by ES-mass spectrometry (MH+ m/z=382) were combined and concentrated under reduced pressure. The resulting residue was crystallized from EtOAc/hexanes to afford the title compound (0.19 g) as shiny yellow flakes: 1H NMR (CD3OD) δ: 7.73 (d, 1H, J=7.2 Hz), 7,59 (m, 2H), 7.44 (m, 4H), 7.1( m, 3H), 6.49 (d, 1H, J=7.2 Hz), 5.22 (s, 2H); ES-MS m/z=382 (MH+); HR-MS, m/z calc C20H14NOFBr, 382.0237, found 382.0226.
Step D
A suspension of 3-bromo-1-(3-fluorobenzyl)-4-(phenylethynyl)pyridin-2(1H)-one ( 0.15 g, 0.39 mmol) in EtOAc (5.0 mL), and EtOH (5.0 mL) containing PtO2 (0.02 g) was stirred in an atmosphere of hydrogen (10 psi) in a Fischer-Porter bottle for 30 min. It was filtered, and the filtrate was concentrated under reduced pressure. The resulting material was purified by silica gel chromatography using 25% EtOAc in hexane. The appropriate fractions were combined, concentrated under reduced pressure, and the residue was crystallized from EtOAc/hexanes to afford the title compound (0.08 g) as a white crystalline solid:
1H-NMR (CD3OD) δ: 7.58 (d, 1H, J=6.8 Hz), 7.4-7.0 (m, 9H), 6.26 (d, 1H. J=6.8 Hz), 5.19 (s, 2H), 2.97 (m, 2H), and 2.90 (m, 2H); ES-MS m/z=386 (MH+); HR-MS, m/z calc C20H18NOFBr, 386.0550, found 386.0585.
Step A
To a chilled solution of 1-benzyl-4-hydroxypyridin-2(1H)-one (0.375 g, 1.86 mmol) in anhydrous acetonitrile (10 mL) was added triethylamine (0.206 g, 2.04 mmol) followed by N-methyl-N-phenylcarbamoyl chloride (0.379 g, 2.24 mmol). The reaction mixture was stirred under nitrogen atmosphere at 0° C. for 30 min then at room temperature for 1 h. The reaction was monitored by TLC (5% methanol in dichloromethane). The solvent was removed under reduced pressure and the residue was washed with 10% citric acid and extracted with EtOAc. The organic extracts were combined, washed with water dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure to afford a yellow syrup. The residue was purified by flash chromatography (silica gel) using 5% MeOH in CH2Cl2 to give the desired product (0.382 g, 61%) as a white semisolid.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (d6-DMSO, 400 MHz) δ: 7.8 (d, 1H), 7.39 (m, 10H), 6.19 (s, 2H), 5.03 (s, 2H), 3.29 (s, 3H); HR-MS (ES) m/z calcd for C20H18N2O3 (MH+)=335.1396, observed 335.1418.
Step B
To a solution of 1-benzyl-2-oxo-1,2-dihydropyridin-4-yl methyl(phenyl)carbamate (0.38 g, 1.13 mmol) in anhydrous CH2Cl2 (7 mL) was added N-Bromosuccinimide (NBS, 0.24 g, 1.34 mmol). The reaction was stirred overnight at room temperature under nitrogen atmosphere. The reaction mixture was purified by flash chromatography (silica gel) using EtOAc/hexanes (1:1 v/v). The appropriate fractions were collected according to ES MS (M+H 413) and concentrated. The dried product showed about 14% of di-brominated product by analytical HPLC. The compounds were separated by reverse phase HPLC using a 10-90% acetonitrile in water, 30 min gradient at a 100 mL/min flow rate, to afford (after lyophilization) the salt of the desired compound. The salt was diluted in EtOAc and washed with NaHCO3. The organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated to afford the desired compound (0.271 g, 58%) as a beige solid.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (d6-DMSO, 400 Hz) δ: 7.83 (d, 1H), 7.39 (m, 10H), 6.48 (s, 1H), 5.12 (s, 2H), 3.33 (s, 3H); HR-MS (ES) m/z calcd for C20H17O3Br (MH+)=413.0495, observed 413.0496.
Step A
Heated a reaction mixture of 4-(benzyloxy)-1-(3-fluorobenzyl)pyridin-2(1H)-one (4.83 g, 15.6 mmol) in anhydrous acetonitrile (55 mL) and N-iodosuccinimide (NIS, 3.86 g, 17.1 mmol) under nitrogen atmosphere at 65° C. for 4 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by flash chromatography (silica gel) using EtOAc/hexanes (1:1 v:v). The appropriate fractions were collected according to ES MS (M+H 436) and washed with Na2SO3 to remove the color impurities. The fractions were concentrated under reduced pressure and dried in vacuo to afford the desired product (6.15 g, 90%) as a light yellow solid.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.73 (d, 1H), 7.47 (d, 2H), 7.39 (m, 4H), 7.08 (m, 3H), 6.39 (d, 1H), 5.29 (s, 2H), 5.19 (s, 2H); HR-MS (ES) m/z calcd for C19H15NO2FI (MH+)=436.0210, observed 436.0196.
Step B
Degassed a solution of 4-(benzyloxy)-1-(3-fluorobenzyl)-3-iodopyridin-2(1H)-one (2.01 g, 4.62 mmol) in anhydrous acetonitrile (25 mL) under argon atmosphere. Triethylamine (1.11 g, 11 mmol) was added and quickly degassed. The reaction mixture was chilled in an ice bath for 15 minutes before adding bistriphenylphosphine-palladium chloride (0.34 g, 0.48 mmol) and cuprous iodide (0.2 g). The reaction was stirred at room temperature for 30 min before heating at 60° C. under an atmosphere of argon for 2 h. The reaction mixture was filtered through a bed of celite and the filtrate was concentrated under reduced pressure. The dark brown residue was diluted with CH2Cl2 (100 mL) and washed with water. The organic extracts were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The dark brown residue was purified by flash chromatography (silica gel) using 30% EtOAc in hexane. The appropriate fractions were combined and concentrated under reduced pressure to afford the desired product (1.34 .g, 72%) as a light yellow solid.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.74 (d, 1H), 7.47 (d, 2H), 7.35 (m, 4H), 7.09 (m, 3H), 6.46 (d, 1H), 5.26 (s, 2H), 5.13 (s, 2H), 0.18 (s, 9H); HR-MS (ES) m/z calcd for C24H24NO2FSi (MH+)=406.1638, observed 406.1610.
Step C
To a solution of 4-(benzyloxy)-1-(3-fluorobenzyl)-3-[(trimethylsilyl)ethynyl]pyridin-2(1H)-one (1.31 g, 3.2 mmol) in anhydrous acetonitrile (25 ML) at 0° C. was added tetrabutylammonium fluoride (0.611 g, 1.93 mmol). The reaction was stirred at 0° C. for 15 min then for 1 h at room temperature. The reaction was concentrated under reduced pressure and the residue was diluted with EtOAc and washed with water. The organic extracts were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) using EtOAc in hexanes (1:1 v/v). The appropriate fractions were combined and concentrated under reduced pressure to afford the desired product (0.779 g, 72%) as a gold solid.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.73 (d, 1H), 7.43 (d, 2H), 7.35 (m,4H), 7.09 (m,3H), 6.45 (d, 1H), 5.27 (s, 2H), 5.13 (s,2H), 3.78 (s, 1H); HR-MS (ES) m/z calcd for C21H16NO2F (MH+)=334.1243, observed 334.1234.
Step A
In a Fischer-Porter bottle, added a solution of 4-(benzyloxy)-1-(3-fluorobenzyl)pyridin-2(1H)-one (4.5 g, 14.56 mmol) in absolute ethanol (20 mL). Flushed the solution with nitrogen then added palladium catalyst (1.05 g). Sealed bottle and evacuated system. The system was purged with hydrogen gas (2×15 psi) to check for leaks. The reaction was charged with hydrogen (35 psi) and stirred at room temperature for 45 min. The system was evacuated and flushed with nitrogen. The reaction was filtered and the catalyst was carefully washed with fresh ethanol. The filtrate was concentrated under reduced pressure and the residue was recrystallized in hot EtOAc to afford the desired product (1.94 g, 61%) as a beige solid after drying in vacuo.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.54 (d, 1H), 7.32 (m, 1H), 7.06 (m, 3H), 6.05 (dd, 1H), 5.83 (s, 1H), 5.09 (s, 2H); HR-MS (ES) m/z calcd for C12H10NO2F (MH+)=220.0774, observed 220.0787.
Step B
Heated a reaction mixture of 1-(3-fluorobenzyl)-4-hydroxypyridin-2(1H)-one (1.005 g, 4.5 mmol) in benzylamine (15 mL) at reflux (185° C.) under nitrogen atmosphere for 24 h. The reaction was monitored by ES-MS (MH+309). The solvent was removed by vacuum distillation to give a yellow residue. The residue was recrystallized in hot EtOAc. The white solid was allowed to cool and then filtered to afford the desired product (0.862, 61%) after drying in vacuo.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.31 (m, 7H), 7.03 (m, 3H), 5.98 (dd, 1H), 5.45 (s, 1H), 5.00 (s, 2H), 4.30 (s, 2H); HR-MS (ES) m/z calcd for C19H17N2OF (MH+)=309.1403, observed 309.1375.
Step C
To a solution of 4-(benzylamino)-1-(3-fluorobenzyl)pyridin-2(1H)-one (0.50 g, 1.62 mmol) in anhydrous CH2Cl2 (10 mL) was added N-bromosuccinimide (NBS, 0.30 g, 1.7 mmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 3 h. The reaction mixture was purified by flash chromatography (silica gel) using EtOAc in hexanes (1:1 v/v). The appropriate fractions were combined and concentrated. The jelly like residue was recrystallized in hot EtOAc/hexanes (1:1 v/v) to afford the desired product (0.216 g, 35%) as a white solid after drying in vacuo.
MS and 1H-NMR were consistent with the desired structure. 1H-NMR (CD3OD, 400 Hz) δ: 7.41 (d, 1H), 7.31 (m, 6H), 7.04 (m, 3H), 5.99 (d, 1H), 5.08 (s, 2H), 4.53 (s, 2H); HR-MS (ES) m/z calcd for C19H16N2OFBr (MH+)=387.0508, observed 387.0504.
Biological Evaluation
p38 Kinase Assay
Cloning of human p38a:
The coding region of the human p38a cDNA was obtained by PCR-amplification from RNA isolated from the human monocyte cell line THP.1. First strand cDNA was synthesized from total RNA as follows: 2 μg of RNA was annealed to 100 ng of random hexamer primers in a 10 μl reaction by heating to 70° C. for 10 minutes followed by 2 minutes on ice. cDNA was then synthesized by adding 1 μl of RNAs in (Promega, Madison Wis.), 2 μl of 50 mM dNTP's, 4 μl of 5× buffer, 2 μl of 100 mM DTT and 1 μl (200 U) of Superscript II™ AMV reverse transcriptase. Random primer, dNTP's and Superscript II™ reagents were all purchased from Life-Technologies, Gaithersburg, Mass. The reaction was incubated at 42° C. for 1 hour. Amplification of p38 cDNA was performed by aliquoting 5 μl of the reverse transcriptase reaction into a 100 μl PCR reaction containing the following: 80 μl dH.sub.2 0, 2. μl 50 mM dNTP's, 1 μl each of forward and reverse primers (50 μmol/μl), 10 μl of 10× buffer and 1 μl Expand™ polymerase (Boehringer Mannheim). The PCR primers incorporated Bam HI sites onto the 5′ and 3′ end of the amplified fragment, and were purchased from Genosys. The sequences of the forward and reverse primers were 5′-GATCGAGGATTCATGTCTCAGGAGAGGCCCA-3′ and 5′GATCGAGGATTCTCAGGACTCCATCTCTTC-3′ respectively. The PCR amplification was carried out in a DNA Thermal Cycler (Perkin Elmer) by repeating 30 cycles of 94° C. for 1 minute, 60° C. for 1 minute and 68° C. for 2 minutes. After amplification, excess primers and unincorporated dNTP's were removed from the amplified fragment with a Wizard™ PCR prep (Promega) and digested with Bam HI (New England Biolabs). The Bam HI digested fragment was ligated into BamHI digested pGEX 2T plasmid DNA (PharmaciaBiotech) using T-4 DNA ligase (New England Biolabs) as described by T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989). The ligation reaction was transformed into chemically competent E. coli DH10B cells purchased from Life-Technologies following the manufacturer's instructions. Plasmid DNA was isolated from the resulting bacterial colonies using a Promega Wizard™ miniprep kit. Plasmids containing the appropriate Bam HI fragment were sequenced in a DNA Thermal Cycler (Perkin Elmer) with Prism™ (Applied Biosystems Inc.) cDNA clones were identified that coded for both human p38a isoforms (Lee et al. Nature 372, 739). One of the clones that contained the cDNA for p38a-2 (CSB-2) inserted in the cloning site of PGEX 2T, 3′ of the GST coding region was designated pMON 35802. The sequence obtained for this clone is an exact match of the cDNA clone reported by Lee et al. This expression plasmid allows for the production of a GST-p38a fusion protein.
Expression of Human p38a
GST/p38a fusion protein w as expressed from the plasmid pMON 35802 in E. coli, stain DH10B (Life Technologies, Gibco-BRL). Overnight cultures were grown in Luria Broth (LB) containing 100 mg/ml ampicillin. The next day, 500 ml of fresh LB was inoculated with 10 ml of overnight culture, and grown in a 2 liter flask at 37° C. with constant shaking until the culture reached an absorbance of 0.8 at 600 nm. Expression of the fusion protein was induced by addition of isopropyl b-D-thiogalactosidase (IPTG) to a final concentration of 0.05 mM. The cultures were shaken for three hours at room temperature, and the cells were harvested by centrifugation. The cell pellets were stored frozen until protein purification.
Purification of P38 Kinase-alpha
All chemicals were from Sigma Chemical Co. unless noted. Twenty grams of E. coli cell pellet collected from five 1 L shake flask fermentations was resuspended in a volume of PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2 HPO.sub.4, 1.8 mM KH.sub.2 PO.sub.4, pH 7.3) up to 200 ml. The cell suspension was adjusted to 5 mM DTT with 2 M DTT and then split equally into five 50 ml Falcon conical tubes. The cells were sonnicated (Ultrasonics model W375) with a 1 cm probe for 3.times.1 minutes (pulsed) on ice. Lysed cell material was removed by centrifugation (12,000×g, 15 minutes) and the clarified supernatant applied to glutathione-sepharose resin (Pharmacia).
Glutathione-Sepharose Affinity Chromatography
Twelve ml of a 50% glutathione sepharose-PBS suspension was added to 200 ml clarified supernatant and incubated batchwise for 30 minutes at room temperature. The resin was collected by centrifugation (600.times.g, 5 min) and washed with 2.times.150 ml PBS/1% Triton X-100, followed by 4.times.40 ml PBS. To cleave the p38 kinase from the GST-p38 fusion protein, the glutathione-sepharose resin was resuspended in 6 ml PBS containing 250 units thrombin protease (Pharmacia, specific activity>7500 units/mg) and mixed gently for 4 hours at room temperature. The glutathione-sepharose resin was removed by centrifugation (600.times.g, 5 min) and washed 2.times.6 ml with PBS. The PBS wash fractions and digest supernatant containing p38 kinase protein were pooled and adjusted to 0.3 mM PMSF.
Mono Q Anion Exchange Chromatography
The thrombin-cleaved p38 kinase was further purified by FPLC-anion exchange chromatography. Thrombin-cleaved sample was diluted 2-fold with Buffer A (25 mM HEPES, pH 7.5, 25 mM beta-glycerophosphate, 2 mM DTT, 5% glycerol) and injected onto a Mono Q HR 10/10 (Pharmacia) anion exchange column equilibrated with Buffer A. The column was eluted with a 160 ml 0.1 M-0.6 M NaCl/Buffer A gradient (2 ml/minute flowrate). The p38 kinase peak eluting at 200 mM NaCl was collected and concentrated to 3-4 ml with a Filtron 10 concentrator (Filtron Corp.).
Sephacryl S100 Gel Filtration Chromatography
The concentrated Mono Q-p38 kinase purified sample was purified by gel filtration chromatography (Pharmacia HiPrep 26/60 Sephacryl S100 column equilibrated with Buffer B (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM DTT, 5% glycerol)). Protein was eluted from the column with Buffer B at a 0.5 ml/minute flowrate and protein was detected by absorbance at 280 nm. Fractions containing p38 kinase (detected by SDS-polyacrylamide gel electrophoresis) were pooled and frozen at −80° C. Typical purified protein yields from 5 L E. coli shake flasks fermentations were 35 mg p38 kinase.
In Vitro Assay
The ability of compounds to inhibit human p38 kinase alpha was evaluated using two in vitro assay methods. In the first method, activated human p38 kinase alpha phosphorylates a biotinylated substrate, PHAS-I (phosphorylated heat and acid stable protein-insulin inducible), in the presence of gamma 32P-ATP (32P-ATP). PHAS-I was biotinylated prior to the assay and provides a means of capturing the substrate, which is phosphorylated during the assay. p38 Kinase was activated by MKK6. Compounds were tested in 10 fold serial dilutions over the range of 100 μM to 0.001 μM using 1% DMSO. Each concentration of inhibitor was tested in triplicate.
All reactions were carried out in 96 well polypropylene plates. Each reaction well contained 25 mM HEPES pH 7.5, 10 mM magnesium acetate and 50 μM unlabeled ATP. Activation of p38 was required to achieve sufficient signal in the assay. Biotinylated PHAS-I was used at 1-2 μg per 50 μl reaction volume, with a final concentration of 1.5 μM. Activated human p38 kinase alpha was used at 1 μg per 50 μl reaction volume representing a final concentration of 0.3 μM. Gamma 32P-ATP was used to follow the phosphorylation of PHAS-I. 32P-ATP has a specific activity of 3000 Ci/mmol and was used at 1.2 μCi per 50 μl reaction volume. The reaction proceeded either for one hour or overnight at 30° C.
Following incubation, 20 μl of reaction mixture was transferred to a high capacity streptavidin coated filter plate (SAM-streptavidin-matrix, Promega) prewetted with phosphate buffered saline. The transferred reaction mix was allowed to contact the streptavidin membrane of the Promega plate for 1-2 minutes. Following capture of biotinylated PHAS-I with 32P incorporated, each well was washed to remove unincorporated 32P-ATP three times with 2M NaCl, three washes of 2M NaCl with 1% phosphoric, three washes of distilled water and finally a single wash of 95% ethanol. Filter plates were air-dried and 20 μl of scintillant was added. The plates were sealed and counted.
A second assay format was also employed that is based on p38 kinase alpha induced phosphorylation of EGFRP (epidermal growth factor receptor peptide, a 21 mer) in the presence 33P-ATP. Compounds were tested in 10 fold serial dilutions over the range of 100 μM to 0.001 μM in 1% DMSO. Each concentration of inhibitor was tested in triplicate. Compounds were evaluated in 50 μl reaction volumes in the presence of 25 mM Hepes pH 7.5, 10 mM magnesium acetate, 4% glycerol, 0.4% bovine serum albumin, 0.4 mM DTT, 50 μM unlabeled ATP, 25 μg EGFRP (200 μM), and 0.05 μCi 33P-ATP. Reactions were initiated by addition of 0.09 μg of activated, purified human GST-p38 kinase alpha. Activation was carried out using GST-MKK6 (5:1, p38:MKK6) for one hour at 30° C. in the presence of 50 μM ATP. Following incubation for 60 minutes at room temperature, the reaction was stopped by addition of 150 μl of AG 1.times.8 resin in 900 mM sodium formate buffer, pH 3.0 (1 volume resin to 2 volumes buffer). The mixture was mixed three times with pipetting and the resin was allowed to settle. A total of 50 μl of clarified solution head volume was transferred from the reaction wells to Microlite-2 μlates. 150 μl of Microscint 40 was then added to each well of the Microlite plate, and the plate was sealed, mixed, and counted.
TNF Cell Assays
Method of Isolation of Human Peripheral Blood Mononuclear Cells:
Human whole blood was collected in Vacutainer tubes containing EDTA as an anticoagulant. A blood sample (7 ml) was carefully layered over 5 ml PMN Cell Isolation Medium (Robbins Scientific) in a 15 ml round bottom centrifuge tube. The sample was centrifuged at 450-500.times.g for 30-35 minutes in a swing out rotor at room temperature. After centrifugation, the top band of cells were removed and washed 3 times with PBS w/o calcium or magnesium. The cells were centrifuged at 400 .times.g for 10 minutes at room temperature. The cells were resuspended in Macrophage Serum Free Medium (Gibco BRL) at a concentration of 2 million cells/mi.
LPS Stimulation of Human PBMs
PBM cells (0.1 ml, 2 million/ ml) were co-incubated with 0.1 ml compound (10-0.41 μM, final concentration) for 1 hour in flat bottom 96 well microtiter plates. Compounds were dissolved in DMSO initially and diluted in TCM for a final concentration of 0.1% DMSO. LPS (Calbiochem, 20 ng/ml, final concentration) was then added at a volume of 0.010 ml. Cultures were incubated overnight at 37° C. Supernatants were then removed and tested by ELISA for TNF-a and IL1-b. Viability was analyzed using MTS. After 0.1 ml supernatant was collected, 0.020 ml MTS was added to remaining 0.1 ml cells. The cells were incubated at 37° C. for 2-4 hours, then the O.D. was measured at 490-650 nM.
Maintenance and Differentiation of the U937 Human Histiocytic Lymphoma Cell Line
U937 cells (ATCC) were propagated in RPMI 1640 containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine (Gibco). Fifty million cells in 100 ml media were induced to terminal monocytic differentiation by 24 hour incubation with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma). The cells were washed by centrifugation (200.times.g for 5 min) and resuspended in 100 ml fresh medium. After 24-48 hours, the cells were harvested, centrifuged, and resuspended in culture medium at 2 million cells/ml.
LPS Stimulation of TNF production by U937 Cells
U937 cells (0.1 ml, 2 million/ml) were incubated with 0.1 ml compound (0.004-50 μM, final concentration) for 1 hour in 96 well microtiter plates. Compounds were prepared as 10 mM stock solutions in DMSO and diluted in culture medium to yield a final DMSO concentration of 0.1% in the cell assay. LPS (E coli, 100 ng/ml final concentration) was then added at a volume of 0.02 ml. After 4 hour incubation at 37° C., the amount of TNF-.alpha. released in the culture medium was quantitated by ELISA. Inhibitory potency is expressed as IC50 (μM).
Rat Assay
The efficacy of the novel compounds in blocking the production of TNF also was evaluated using a model based on rats challenged with LPS. Male Harlen Lewis rats [Sprague Dawley Co.] were used in this model. Each rat weighed approximately 300 g and was fasted overnight prior to testing. Compound administration was typically by oral gavage (although intraperitoneal, subcutaneous and intravenous administration were also used in a few instances) 1 to 24 hours prior to the LPS challenge. Rats were administered 30 μg/kg LPS [salmonella typhosa, Sigma Co.] intravenously via the tail vein. Blood was collected via heart puncture 1 hour after the LPS challenge. Serum samples were stored at −20° C. until quantitative analysis of TNF-.alpha. by Enzyme Linked-Immuno-Sorbent Assay (“ELISA”) [Biosource]. Additional details of the assay are set forth in Perretti, M., et al., Br. J. Pharmacol. (1993), 110, 868-874, which is incorporated by reference in this application.
Mouse Assay
Mouse Model of LPS-Induced TNF Alpha Production
TNF alpha was induced in 10-12 week old BALB/c female mice by tail vein injection with 100 ng lipopolysaccharide (from S. Typhosa) in 0.2 ml saline. One hour later mice were bled from the retroorbital sinus and TNF concentrations in serum from clotted blood were quantified by ELISA. Typically, peak levels of serum TNF ranged from 2-6 ng/ml one hour after LPS injection.
The compounds tested were administered to fasted mice by oral gavage as a suspension in 0.2 ml of 0.5% methylcellulose and 0.025% Tween 20 in water at 1 hour or 6 hours prior to LPS injection. The 1 hour protocol allowed evaluation of compound potency at Cmax plasma levels whereas the 6 hour protocol allowed estimation of compound duration of action. Efficacy was determined at each time point as percent inhibition of serum TNF levels relative to LPS injected mice that received vehicle only.
Induction and Assessment of Collagen-Induced Arthritis in Mice
Arthritis was induced in mice according to the procedure set forth in J. M. Stuart, Collagen Autoimmune Arthritis, Annual Rev. Immunol. 2:199 (1984), which is incorporated herein by reference. Specifically, arthritis was induced in 8-12 week old DBA/1 male mice by injection of 50 μg of chick type II collagen (CII) (provided by Dr. Marie Griffiths, Univ. of Utah, Salt Lake City, Utah) in complete Freund's adjuvant (Sigma) on day 0 at the base of the tail. Injection volume was 100 A1. Animals were boosted on day 21 with 50 μg of CII in incomplete Freund's adjuvant (100 μl volume). Animals were evaluated several times each week for signs of arthritis. Any animal with paw redness or swelling was counted as arthritic. Scoring of arthritic paws was conducted in accordance with the procedure set forth in Wooley et al., Genetic Control of Type II Collagen Induced Arthritis in Mice: Factors Influencing Disease Suspectibility and Evidence for Multiple MHC Associated Gene Control., Trans. Proc., 15:180 (1983). Scoring of severity was carried out using a score of 1-3 for each paw (maximal score of 12/mouse). Animals displaying any redness or swelling of digits or the paw were scored as 1. Gross swelling of the whole paw or deformity was scored as 2. Ankylosis of joints was scored as 3. Animals were evaluated for 8 weeks. 8-10 animals per group were used.
Results obtained using the above-described assays are set forth in Tables I, II, and III below. p38 Assay results are expressed as IC50 (μm).
In the preceding table, the “n” value in the parenthesis is the number of times that the particular compound was assayed. If an “n” value is not reported, then n=1. If n is not 1, then the average from all trials is reported.
In the preceding tables, the “n” value relates to the number of times that the assay of the particular compound was performed. If an “n” value is not reported, then the “n” value is 1.
For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, hard or soft capsule, lozenges, dispensable -powders, suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are tablets or capsules.
The active ingredient may also be administered by injection (IV, IM, subcutaneous or jet) as a composition wherein, for example, saline, dextrose, or water may be used as a suitable carrier. The pH of the composition may be adjusted, if necessary, with suitable acid, base, or buffer. Suitable bulking, dispersing, wetting or suspending -agents, including mannitol and PEG 400, may also be included in the composition. A suitable parenteral composition can also include a compound formulated as a sterile solid substance, including lyophilized powder, in injection vials. Aqueous solution can be added to dissolve the compound prior to injection.
The amount of therapeutically active compounds that are administered and the dosage regimen for treating a disease condition with the compounds and/or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the severity of the inflammation or inflammation related disorder, the route and frequency of administration, and the particular compound employed, and thus may vary widely. The pharmaceutical compositions may contain active ingredients in the range of about 0.1 to 1000 mg, preferably in the range of about 7.0 to 350 mg. A daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight and most preferably between about 0.5 to 30 mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day. In the case of skin conditions, it may be preferable to apply a topical preparation of compounds of this invention to the affected area two to four times a day.
For disorders of the eye or other external tissues, e.g., mouth and skin, the formulations are preferably applied as a topical gel, spray, ointment or cream, or as a suppository, containing the active ingredients in a total amount of, for example, 0.075 to 30% w/w, preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w. When formulated in an ointment, the active ingredients may be employed with either paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example at least 30% w/w of a polyhydric alcohol such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, polyethylene glycol and mixtures thereof. The topical formulation may desirably include a compound, which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs. The compounds of this invention can also be administered by a transdermal device. Preferably topical administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. In either case, the active agent is delivered continuously from the reservoir or microcapsules through a membrane into the active agent permeable adhesive, which is in contact with the skin or mucosa of the recipient. If the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent may also function as the membrane. The transdermal patch may include the compound in a suitable solvent system with an adhesive system, such as an acrylic emulsion, and a polyester patch. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier, it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make-up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base, which forms the oily, dispersed phase of the cream formulations. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate, among others. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus, the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredients are dissolved or suspended in suitable carrier, especially an aqueous solvent for the active ingredients. The antiinflammatory active ingredients are preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% and particularly about 1.5% w/w. For therapeutic purposes, the active compounds of this combination invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.
The invention and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.
| Number | Date | Country | |
|---|---|---|---|
| 60357029 | Feb 2002 | US | |
| 60436915 | Dec 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10367987 | Feb 2003 | US |
| Child | 11226556 | Sep 2005 | US |
