Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
  • C07D253/02—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 not condensed with other rings
  • C07D253/06—1,2,4-Triazines
  • C07D253/065—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members
  • C07D253/07—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members with hetero atoms, or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/79—Acids; Esters
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
  • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
  • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

• the present invention relates to derivatives and/or analogues of sphingosine and pharmaceutical compositions, including such derivatives and/or analogues, which are useful as drugs for the treatment of fungal infections, allergic diseases, immune disorders, etc.
• Sphingosine is a compound having the chemical structure shown in the general formula described below, in which Y 1 is hydrogen. It is known that various sphingolipids, having sphingosine as a constituent, are widely distributed in the living body including on the surface of cell membranes of cells in the nervous system.
• a sphingo lipid is one of the lipids having important roles in the living body.
• a disease called lipidosis is caused by accumulation of a specified sphingo lipid in the body.
• Sphingolipids present on cell membranes function to regulate cell growth; participate in the development and differentiation of cells; function in nerves; are involved in the infection and malignancy of cells; etc. Many of the physiological roles of sphingo lipids remain to be solved.
• ceramide a derivative of sphingosine, has an important role in the mechanism of cell signal transduction has been indicated, and studies about its effect on apoptosis and cell cycle have been reported.
• Sphingosine- 1 -phosphate is an important cellular metabolite, derived from ceramide that is synthesized de novo or as part of the sphingomeyeline cycle (in animals cells). It has also been found in insects, yeasts and plants.
• the enzyme, ceramidase acts upon ceramides to release sphingosine, which is phosphorylated by spingosine kinase, a ubiquitous enzyme in the cytosol and endoplasmic reticulum, to form sphingosine- 1 -phosphate.
• the reverse reaction can occur also by the action of sphingosine phosphatases, and the enzymes act in concert to control the cellular concentrations of the metabolite, which concentrations are always low. In plasma, such concentration can reach 0.2 to 0.9 ⁇ M, and the metabolite is found in association with the lipoproteins, especially the HDL.
• sphingosine- 1 -phosphate formation is an essential step in the catabolism of sphingoid bases.
• sphingosine- 1 -phosphate is a potent messenger molecule that perhaps uniquely operates both intra- and inter-cellularly, but with very different functions from ceramides and sphingosine.
• the balance between these various sphingolipid metabolites may be important for health.
• sphingosine- 1 -phosphate promotes cellular division (mitosis) as opposed to cell death (apoptosis), which it inhibits. Intracellularly, it also functions to regulate calcium mobilization and cell growth in response to a variety of extracellular stimuli.
• sphingosine - 1 -phosphate In common with the lysophospholipids, especially lysophosphatidic acid, with which it has some structural similarities, sphingosine-1 -phosphate exerts many of its extra-cellular effects through interaction with five specific G protein-coupled receptors on cell surfaces. These are important for the growth of new blood vessels, vascular maturation, cardiac development and immunity, and for directed cell movement.
• Sphingosine-1 phosphate is stored in relatively high concentrations in human platelets, which lack the enzymes responsible for its catabolism, and it is released into the blood stream upon activation of physiological stimuli, such as growth factors, cytokines, and receptor agonists and antigens. It may also have a critical role in platelet aggregation and thrombosis and could aggravate cardiovascular disease.
• physiological stimuli such as growth factors, cytokines, and receptor agonists and antigens. It may also have a critical role in platelet aggregation and thrombosis and could aggravate cardiovascular disease.
• the relatively high concentration of the metabolite in high-density lipoproteins (HDL) may have beneficial implications for atherogenesis.
• sphingosine-1 - phosphate together with other lyso lipids such as sphingosylphosphorylcholine and lysosulfatide, are responsible for the beneficial clinical effects of HDL by stimulating the production of the potent antiatherogenic signaling molecule nitric oxide by the vascular endothelium.
• lysophosphatidic acid it is a marker for certain types of cancer, and there is evidence that its role in cell division or proliferation may have an influence on the development of cancers.
• Fungi and plants have sphingolipids and the major sphingosine contained in these organisms has the formula described below. It is known that these lipids have important roles in the cell growth of fungi and plants, but details of the roles remain to be solved.
• derivatives of sphingo lipids and their related compounds exhibit a variety of biological activities through inhibition or stimulation of the metabolism pathways. These compounds include inhibitors of protein kinase C, inducers of apoptosis, immuno-suppressive compounds, antifungal compounds, and the like. Substances having these biological activities are expected to be useful compounds for various diseases.
• the present invention provides a derivative or analogue of sphingosine that is able to regulate the functions of sphingo lipid, and pharmaceutical compositions comprising said derivative or analogue.
• X is selected from the group consisting of CR 3 and N;
• Y is selected from the group consisting of CR 3 and N;
• Z is selected from the group consisting of CR 3 and N;
• W is NR 3 or O
• V is oxo or represents two H atoms
• R 1 is an aryl group and is selected, preferably, from the group consisting of phenyl and substituted derivatives thereof;
• R 2 is an aryl group and is selected, preferably from the group consisting of phenyl, furanyl, thienyl, pyridyl, pyranyl and substituted derivatives thereof;
• R 3 is a hydrocarbyl or substituted hydrocarbyl radical which is selected, preferably, from the group consisting of H and alkyl and more preferably, R 3 is selected from the group consisting of H and lower alkyl, e. g. Ci to C 6 alkyl ;
• R 4 is a hydrocarbyl or substituted hydrocarbyl radical which is selected, preferably, from the group consisting of H and alkyl and, more preferably, R 4 is selected from the group consisting of H and lower alkyl, e. g. Ci to C 6 alkyl;
• R 3 or R 4 may together form a cyclic alkyl ring having from 3 to 6 carbon atoms;
• a is 0 or an integer of from 1 to 6, e. g. 0 or an integer of from 1 to 3;
• b is 0 or 1 ;
• c is 0 or an integer of from 1 to 6, e. g. 0 or an integer of from 1 to 5;
• d is 0 or 1 ;
• e is 0 or 1 ;
• x is l
• y is 0 or an integer of from 1 to 2;
• z is 0 or an integer of from 1 to 2;
• a diphenylethyl-l,2-dione e.g. benzil
• methyl oxalamidrazonate in ethanol
• a methyl 5,6-diphenyl-l,3,4-triazine-2- carboxylate (as a mixture of geometric isomers if the dione is asymmetrical).
• These triazines can undergo Diels-Aler reactions with a pyrrolidine enamine compound to give a methyl 5,6-diphenylpyridine-2-carboxylate derivative.
• These compounds can be reduced with diisobutylaluminum hydride to the corresponding aldehyde derivatives, which then can be converted into a number of homo logs and derivatives.
• the aldehyde can be converted into a secondary amine by reacting it with a primary amine in the presence of a reducing agent, such as sodium cyanoborohydride.
• a reducing agent such as sodium cyanoborohydride.
• the aldehyde may be reduced to an alcohol and treated with an alkyl halide in the presence of a mild base to produce alkyl ethers.
• these compounds may be used to prepare many other homo logs, many of which are described in the Specific Examples section below.
• .salt also refers to an -v.-'- salt which rnav •. form in vivo as a result of admitti.stratioji oi an ⁇ cidjjnj ⁇ theniaUjj ⁇ a ⁇ ⁇ rod ⁇ ! ⁇
• cxaninic, iautomcr ⁇ may be related by transfer of a nroton, hydrouen atom, or hvd ⁇ dcJML lJji] ⁇ 4er ⁇ ch ⁇ istrvi ⁇ j ⁇ E]ici ⁇ ⁇ voi ⁇ os ⁇ lbic_ ⁇ ]2X ⁇ isornin ⁇
• R 3 and R 4 may be independently selected from the group consisting of hydrogen, straight or branched chain alkyl having 1 to 12 carbons, cycloalkyl having 3 to 6 carbons, alkenyl having 2 to 6 carbons and 1 or 2 double bonds, alkynyl having 2 to 6 carbons and 1 or 2 triple bonds, aryl, preferably a carbocyclic aryl group having from 6 to 14 carbon atoms or a heterocyclic aryl group having from 2 to 14 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, halo, e.g.
• Ci to Ci 2 haloalkyl e.g. trifluoromethyl, hydroxyl, Ci to C 12 alkoxy, Ci to C12 alkylcarbonyl, formyl, oxycarbonyl, carboxy, Ci to Ci 2 alkyl carboxylate, Ci to Ci 2 alkyl amide, aminocarbonyl, amino, cyano, diazo, nitro, thio, sulfoxyl, or sulfonyl groups.
• R 1 and R 2 are aryl groups which may be any carbocyclic aryl or heterocyclic aryl group including but not limited to benzene, pyridine, pyrazine, pyridazine, pyrimidine, triazine, thiophene, furan, thiazole, thiadiazole, isothiazole, oxazole,oxadiazole, isooxazole, naphthalene, quinoline, tetralin, chroman, thiochroman, tetrahydroquinoline, dihydronaphthalene, tetrahydronaphthalen, chromene, thiochromene, dihydroquinoline, indan, dihydrobenzofuran, dihydrobenzothiophene, indene, benzofuran, benzothiophene, coumarin and coumarinone.
• Such aryl groups can be bonded to the above moiety at any position.
• Such aryl group may itself be substituted with any common organic functional group including but not limited to alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, hydroxyl, alkoxyl, alkylcarbonyl, formyl, oxycarbonyl, carboxyl, alkyl carboxylate, alkyl amide, aminocarbonyl, amino, cyano, diazo, nitro, thio, sulfoxyl, or sulfonyl groups.
• the carbocyclic aryl group will comprise from 6 to 14 carbon atoms, e.g. from 6 to 10 carbon atoms.
• the heterocyclic aryl group will comprise from 2 to 14 carbon atoms and one or more, e.g. from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
• the compounds have a side chain which terminates in a phosphonic acid or a phosphonic acid ester group.
• R 1 is selected from the group consisting of phenyl and substituted derivatives thereo f .
• R 2 is selected from the group consisting of phenyl, furanyl, thienyl, pyridyl, pyranyl and substituted derivatives thereof.
• R 3 and R 4 are H or lower alkyl.
• a is 0 or an integer of from 1 to 3 and c is 0 or an integer of from 1 to 5.
• R 1 is represented, preferably, by the general formula
• R 5 is selected from the group consisting of H, alkyl, trifluoromethyl, trifluoromethyloxy, halo, e. g.chloro, and loweralkylthio.
• R 2 is selected from the group consisting of furanyl, thienyl, pyridyl and pyranyl or R 2 is represented by the general formula
• R 5 is selected from the group consisting of H, alkyl, trifluoromethyl, trifluoromethyloxy, halo, e. g. chloro, and loweralkylthio .
• R is H
• c is 1, 2 or 3 and a is 1.
• Z is N
• X and Y are CR 3
• W is NR 3
• R 2 is phenyl and R 5 is selected from the group consisting of H and methyl or R 2 is pyridyl and R 5 is ethyl
• d is 0 and therefore the compounds have a side chain which terminates in a carbon-oxygen radical such as a carboxylic acid, an ester thereof, an ether, an alcohol, or an alkyl carboxy group.
• a carbon-oxygen radical such as a carboxylic acid, an ester thereof, an ether, an alcohol, or an alkyl carboxy group.
• R 1 may be represented by the general formula
• R is selected from the group consisting of H, alkyl, trifluoromethyl, trifluoromethyloxy, halo, e. g. chloro, and loweralkylthio
• R 2 may also be represented by the general formula
• R 5 is selected from the group consisting of H, lower alkyl, trifluoromethyl, trifluoromethyloxy, halo, e. g. chloro, and loweralkylthio or R 2 is selected from the group consisting of furanyl, thienyl, pyridyl and pyranyl.
• R 3 is H and more preferably, a is 1.
• x is 1
• z is 0
• R 4 is selected from the group consisting of H, methyl and ethyl.
• carbon-oxygen compounds of this invention preferably is Z is N, X and Y are CR 3 , R 2 is pyridyl, R 4 is selected from the group consisting of methyl and ethyl and R 5 is selected from the group consisting of H, methyl, ethyl, propyl and trifluoromethyl, or
• R 4 is selected from the group consisting of methyl and ethyl and
• R 5 is selected from the group consisting of H, methyl, ethyl, propyl and trifluoromethyl, or
• X and Z are N and Y is CR
• Me refers to methyl.
• Et refers to ethyl.
• tBu refers to t-butyl
• iPr refers to i-propyl
• Ph refers to phenyl
• “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid and the like.
• Alkyl refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon.
• the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, most preferably 1 to 4 carbons.
• Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
• Alkenyl refers to a straight-chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon—carbon double bond.
• the alkenyl group has 2 to 12 carbons. More preferably it is a lower alkenyl of from 2 to 7 carbons, most preferably 2 to 4 carbons.
• the alkenyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, O, S, NO 2 , halogen, dimethyl amino, and SH.
• Alkynyl refers to a straight-chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon—carbon triple bond.
• the alkynyl group has 2 to 12 carbons. More preferably it is a lower alkynyl of from 2 to 7 carbons, most preferably 1 to 4 carbons.
• the alkynyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, O, S, NO 2 , halogen, dimethyl amino, and SH.
• Alkoxyl refers to an “O-alkyl” group.
• Aryl refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups.
• the aryl group may be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO 2 , amine, thioether, cyano, alkoxy, alkyl, and amino.
• Alkaryl refers to an alkyl that is covalently joined to an aryl group.
• the alkyl is a lower alkyl.
• Carbocyclic aryl refers to an aryl group wherein the ring atoms are carbon.
• Heterocyclic aryl refers to an aryl group having from 1 to 3 heteroatoms as ring atoms, the remainder of the ring atoms being carbon. Heteroatoms include oxygen, sulfur, and nitrogen.
• Hydrocarbyl refers to a hydrocarbon radical having only carbon and hydrogen atoms.
• the hydrocarbyl radical has from 1 to 20 carbon atoms, more preferably from 1 to 12 carbon atoms and most preferably from 1 to 7 carbon atoms.
• substituted hydrocarbyl refers to a hydrocarbyl radical wherein one or more, but not all, of the hydrogen and/or the carbon atoms are replaced by a halogen, nitrogen, oxygen, sulfur or phosphorus atom or a radical including a halogen, nitrogen, oxygen, sulfur or phosphorus atom, e.g. fluoro, chloro, cyano, nitro, hydroxyl, phosphate, thiol, etc.
• Amide refers to -C(O)-NH-R', wherein R' is alkyl, aryl, alkylaryl or hydrogen.
• Thioamide refers to -C(S)-NH-R, wherein R is alkyl, aryl, alkylaryl or hydrogen.
• “Amine” refers to a — N(R")R" group, wherein R" and R'" are independently selected from the group consisting of alkyl, aryl, and alkylaryl.
• Thioether refers to — S— R", wherein R" is alkyl, aryl, or alkylaryl.
• Compounds were also assessed for their ability to activate or block activation of the human S1P3 receptor in T24 cells stably expressing the human S1P3 receptor.
• Ten thousand cells/well were plated into 384-well poly-D-lysine coated plates one day prior to use.
• the growth media for the S1P3 receptor expressing cell line was McCoy's 5 A medium supplemented with 10% charcoal-treated fetal bovine serum (FBS), 1% antibiotic-antimycotic and 400 ⁇ g/ml geneticin.
• FBS charcoal-treated fetal bovine serum
• the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/Hepes buffer).
• the cells were then dye loaded with 2 uM Fluo-4 diluted in the HBSS/Hepes buffer with 1.25 mM Probenecid and incubated at 37 0 C for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR
• Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates.
• the positive control, Sphingosine-1 -Phosphate (SlP) was diluted in HBSS/Hepes buffer with 4 mg/ml fatty acid free bovine serum albumin.
• the FLIPR transferred 12.5 ⁇ l from the ligand microplate to the cell plate and took fluorescent measurements for 75 seconds, taking readings every second, and then for 2.5 minutes, taking readings every 10 seconds. Drugs were tested over the concentration range of 0.61 nM to 10,000 nM. Data for Ca +2 responses were obtained in arbitrary fluorescence units and not translated into Ca +2 concentrations.
• IC50 values were determined through a linear regression analysis using the Levenburg Marquardt algorithm.
• NA is defined as “Not Active”
• ND is defined as “Not Determined”
• % efficacy is defined as “percent of receptor activity induced by a test compound at the highest dose test (10 ⁇ M) relative to the receptor activity induced by 5 nM sphingosine-1 - phosphate.”
• % inhibition is defined as "percent of receptor activity induced by 5 nM sphingosine-1 -phosphate that is inhibited by a test compound at the highest dose tested (10 ⁇ M).”
• mice Male C57/Blk6 mice (Charles River, Wilmington, MA at 8 weeks of age and weighing ⁇ 30 grams) received a single (IP) injection of vehicle or Compound 65 (3 mg/kg). Four hours post-injection animals were anesthetized with iso/02 mix, blood was collected by retro-orbital bleeding into a BD Biosciences Microtainer tube containing the anticoagulant dipotassium-EDTA (-300-500 ul of blood collected).
• Advia 120 analyzes K-EDTA anticoagulated whole blood using cytochemical reactions and flow-cytometry measurements to enumerate and differentiate leukocytes (white blood cells), enumerate and characterize erythrocytes (red blood cells), thrombocytes (platelets), and reticulocytes (immature red blood cells).
• Leukocytes are enumerated and differentiated using a combination of two methods, a Peroxidase method and a Basophil Lobularity method, which generate relative and absolute counts for neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
• a Peroxidase method a cell suspension passes through the flowcell where the absorption (correlating to cytoplasmic peroxidase staining) and forward light-scattering (correlating to cell size) are measured.
• Basophil Lobularity method a suspension cell nuclei are passed though a flowcell where the low-angle light scatter and high-angle light scatter are measured correlating to nuclear size and complexity.
• leukocyte differentials When necessary leukocyte differentials may be performed manually from Romanowski stained blood smears.
• an adaptation of the classic cyan-methemoglobin spectrophotometric methodology was used to measure total hemoglobin concentration.
• erythrocytic and thrombocytic indices were derived by the Advia 120 software. The results are summarized in Table 2.
• An art-accepted model or assay for measuring an analgesic effect of a compound in chronic pain is the model known as Kim and Chung 1992, Pain 150, pp 355-363 (Chung model).
• This model involves the surgical ligation of the L5 (and optionally the L6) spinal nerves on one side in experimental animals. Rats recovering from the surgery gain weight and display a level of general activity similar to that of normal rats. However, these rats develop abnormalities of the foot, wherein the hindpaw is moderately everted and the toes are held together. More importantly, the hindpaw on the side affected by the surgery appears to become sensitive to low-threshold mechanical stimuli and will perceive pain instead of the faint sensation of touch.
• tactile allodynia This sensitivity to normally non-painful touch, called “tactile allodynia", develops within the first week after surgery and lasts for at least two months.
• the allodynia response includes lifting the affected hindpaw to escape from the stimulus, licking the paw and holding it in the air for many seconds. None of these responses is normally seen in the control group.
• rats are anesthetized before surgery. The surgical site is shaved and prepared either with betadine or Novacaine. Incision is made from the thoracic vertebra XlIl down toward the sacrum. Muscle tissue is separated from the spinal vertebra (left side) at the L4 - S2 levels.
• the L6 vertebra is located and the transverse process is carefully removed with a small rongeur to expose the L4 - L6 spinal nerves.
• the L5 and L6 spinal nerves are isolated and tightly ligated with 6-0 silk thread. The same procedure is done on the right side as a control, except no ligation of the spinal nerves is performed.
• the wounds are sutured.
• a small amount of antibiotic ointment is applied to the incised area, and the rat is transferred to the recovery plastic cage under a regulated heat-temperature lamp.
• the test drugs are administered by intraperitoneal (i.p.) injection or oral gavage (p.o.).
• i.p. administration the compounds are formulated in H 2 O and given in a volume of 1 ml/kg body weight by injecting into the intraperitoneal cavity.
• p.o. administration the compounds are formulated in H 2 O and given in a volume of 1 ml/kg body weight using an 18-gauge, 3 inch gavage needle that is slowly inserted through the esophagus into the stomach.
• Tactile allodynia is assessed via von Frey hairs, which are a series of fine hairs with incremental differences in stiffness. Rats are placed in a plastic cage with a wire mesh bottom and allowed to acclimate for approximately 30 minutes. To establish the pre-drug baseline, the von Frey hairs are applied perpendicularly through the mesh to the mid-plantar region of the rats' hindpaw with sufficient force to cause slight buckling and held for 6-8 seconds. The applied force has been calculated to range from 0.41 to 15.1 grams. If the paw is sharply withdrawn, it is considered a positive response. A normal animal will not respond to stimuli in this range, but a surgically ligated paw will be withdrawn in response to a 1-2 gram hair.
• the 50% paw withdrawal threshold is determined using the method of Dixon, W.J., Ann. Rev. Pharmacol. Toxicol. 20:441-462 (1980) hereby incorporated by reference. Tactile allodynia is measured prior to and 15, 30, and 60 minutes after drug administration. The post-drug threshold is compared to the pre-drug threshold and the percent reversal of tactile sensitivity is calculated based on a normal threshold of 15.1 grams.
• Table 3 below indicates the degree of pain reversal obtained in the Chung model with exemplary compounds of the invention.
• the intraperitonial (i.p.) and/or intravenous (iv) administration of the compounds was dosed (as indicated) and the peak percentage of reversal of allodynia was measured at 15, 30 or 60 minutes after administration, as is indicated in the table. Data are expressed as the highest % allodynia reversal (out of 3 time points: 15 min, 30 min, or 60 min. post- drug) with a minimum of a 20% allodynia reversal in the rat Chung model. Comparisons between groups (drug treated vs. saline treated) were made using a two-tailed, 2-sample, unpaired t-test.
• DIBAL-H diisobutylaluminum hydride
• DME 1 ,2-dimethoxyethane
• MeI iodomethane i-PrOH: isopropanol
• MgSO 4 magnesium sulfate
• KOH potassium hydroxide
• K 2 COs potassium carbonate
• NaOEt sodium ethoxide
• NaOH sodium hydroxide
• Example 1 l-(2-p-Tolylethynyl)benzene (Compound 1).
• General Procedure A To a solution of lithium phenylacetylide (15.2 ml, 15.2 mmol) in DME (20 ml) under Argon at -78 0 C was added triisopropoxylborane (3.5 ml, 15.2 mmol). The mixture was stirred at -78 0 C for 1.5 hours.
• Example 2 l-(2-(4-Ethylphenyl)ethynyl)benzene (Compound 2) Following General Procedure A, lithium phenylacetylide (14.0 ml, 14.1 mmol), triisopropoxylborane (3.2 ml, 14.1 mmol), l-bromo-4-ethylbenzene (2 g, 10.8 mmol) and Pd(PPh 3 ) 4 (375 mg, 0.32 mmol) in DME (30ml) and THF (10 ml) were reacted to obtain the title compound as a yellow oil.
• Example 3 l-(2-(4-n-Proylphenyl)ethynyl)benzene (Compound 3) Following General Procedure A, lithium phenylacetylide (13.0 ml, 13.1 mmol), triisopropoxylborane (3.0 ml, 13.1 mmol), l-bromo-4-n-propylbenzene (2 g, 10.1 mmol) and Pd(PPh 3 ) 4 (348 mg, 0.30 mmol) in DME (30 ml) and THF (10 ml) were reacted to obtain the title compound as a yellow oil.
• Example 4 l-(2-(4-Trifluoromethylphenyl)ethynyl)benzene (Compound 4) Following General Procedure A, lithium phenylacetylide (17.3 ml, 17.3 mmol), triisopropoxylborane (4.0 ml, 17.3 mmol), l-bromo-4-trifluoromethyl-benzene (3 g, 13.3 mmol) and Pd(PPh 3 ) 4 (462 mg, 0.40 mmol) in DME (40 ml) and THF (15 ml) were reacted to obtain the title compound as a yellow solid.
• Example 5 l-(2-(4-n-Nonanylphenyl)ethynyl)benzene (Compound 5) Following General Procedure A, lithium phenylacetylide (12.4 ml, 12.4 mmol), triisopropoxylborane (2.8 ml, 12.4 mmol), l-bromo-4-n-nonanylbenzene (2.7 g, 9.5 mmol) and Pd(PPh 3 ) 4 (331 mg, 0.40 mmol) in DME (30 ml) and THF (10 ml) were reacted to obtain the title compound as a yellow solid.
• Example 6 l-Phenyl-2-p-tolylethane-l,2-dione (Compound 6).
• General Procedure B To a suspension of iodosobenzene (2.5 g, 11.3 mmol) in CH 2 Cl 2 (30 ml) was added RuCl 2 (PPh 3 ) 4 (45 mg, 0.04 mmol). A solution of 1 -(2-p-tolylethynyl)benzene (Compound 1, 835 mg, 4.3 mmol) in CH 2 Cl 2 (10 ml) was cannulated into the suspension. The resulting mixture was stirred at room temperature overnight resulting in a homogeneous solution. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (10% ethyl acetate in hexane) to produce the title compound as a yellow oil.
• Example 8 l-(4-n-Propyl-phenyl)-2-phenyl-ethane-l,2-dione (Compound 8).
• iodosobenzene 2.2 g, 10.0 mmol
• RuCl 2 (PPh 3 ) 4 38 mg, 0.04 mmol
• l-(2-(4-/?-propylphenyl)ethynyl)benzene Compound 3, 860 mg, 3.9 mmol
• CH 2 Cl 2 50 ml
• Ethyl 5,6-Diphenyl-l,2,4-triazine-3-carboxylate (Compound 11).
• General Procedure C A solution of ethyl oxalamidrazonate (Compound 37, 236 mg, 1.8 mmol) in ethanol (20 ml) was cannulated slowly into a stirring solution of benzil (500 mg, 2.4 mmol) in ethanol (20 ml) under argon at room temperature. After the addition was completed, the reaction was stirred at room temperature overnight
• Example 12 and Example 17 Ethyl 5-Phenyl-6-p-tolyl-[l,2,4]triazine-3-carboxylate (Compound 12), and Ethyl 6-Phenyl-5-p-tolyl-[l,2,4]triazine-3-carboxylate (Compound 17).
• Example 13 and Example 18 Ethyl 6-(4-Ethylphenyl)-5-phenyl -[l,2,4]triazine-3-carboxylate (Compound 13), and Ethyl 5-(4-Ethylphenyl)-6-phenyl-[l,2,4]triazine-3-carboxylate (Compound 18).
• Ethyl 5-Phenyl-6-(4-propylphenyl)-[l, 2,4] triazine-3-carboxylate (Compound 14).
• ethyl oxalamidrazonate (Compound 37, 460 mg, 1.5 mmol)
• l-(4-n-propylphenyl)-2-phenyl-ethane-l,2-dione (Compound 8, 588 mg, 2.3 mmol) in ethanol (40 ml) were reacted and the product was recrystalized from 5% ethyl acetate in hexane to produce the title compound as yellow solid.
• Example 15 and Example 19 Ethyl 6-(4-Trifluoromethyl -phenyl)-5-phenyl -[l,2,4]-triazine-3-carboxylate (Compound 15), and Ethyl 5-(4-Trifluoromethylphenyl)-6-phenyl- [1,2,4] - triazine-3-carboxylatate (Compound 19).
• Ethyl 5,6-diphenylpyridine-2-carboxylate (Compound 21).
• General Procedure D Ethyl 5,6-diphenyl-[l,2,4]-triazine-3-carboxylate (Compound 11, 200 mg, 0.66 mmol) and crude 1-vinylpyrrolidine (Compound 38, 2 g) in CHCl 3 (20 ml) was heated at 75 0 C overnight under nitrogen. The solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (20 % ethyl acetate in hexane) to yield the title compound as a light yellow solid.
• 6-Phenyl-5-p-tolyl-pyridine-2-carboxylic acid ethyl ester (Compound 22). Following General Procedure D, ethyl 5-phenyl-6-/?-tolyl-[l,2,4]-triazine-3- carboxylate (Compound 12, 177 mg, 0.56 mmol) and crude 1-vinylpyrrolidine (Compound 38, 730 mg) in CHCl 3 (10 ml) were reacted to produce the title compound as a yellow solid.
• Ethyl 6-Phenyl-5-(4-trifluoromethylphenyl)-pyridine-2-carboxylate (Compound 25). Following General Procedure D, ethyl 6-(4- tirfluoromethylphenyl)-5 -phenyl -[l,2,4]triazine-3-carboxylate (Compound 15), (378 mg, 1.01 mmol) and crude 1-vinylpyrrolidine (Compound 38, 780 mg) in CHCI3 (10 ml) were reacted to produce the title compound as a yellow oil.
• Ethyl S-Phenyl- ⁇ -p-tolyl-pyridine ⁇ -carboxylate (Compound 27). Following General Procedure D, ethyl 6-phenyl-5-/?-tolyl-[l,2,4]triazine-3-carboxylate (Compound 17, 361 mg, 1.13 mmol) and crude 1-vinylpyrrolidine (Compound 38, 806 mg) in CHCl 3 (10 ml) were reacted to produce the title compound as a yellow oil.
• Ethyl 5-(4-Ethylphenyl)-6-phenyl-pyridine-2-carboxylic acid ethyl ester (Compound 28). Following General Procedure D, ethyl 5-(4-ethylphenyl)-6- phenyl-[l,2,4]triazine-3-carboxylate (Compound 18, 245 mg, 0.74 mmol) and crude 1-vinylpyrrolidine (Compound 38, 572 mg) in CHCl 3 (10 ml) were reacted to produce the title compound as a yellow oil.
• Ethyl 5-Phenyl-6-(4-trifluoromethyl-phenyl)-pyridine-2-carboxylate (Compound 29). Following General Procedure D, ethyl 5-(4- trifluoromethylphenyl)-6-phenyl-[l,2,4]-triazine-3-carboxylate (Compound 19, 1 g, 2.68 mmol) and crude 1-vinylpyrrolidine (Compound 38, 1.4 g) in CHCI3 (20 ml) were reacted to produce the title compound as a yellow oil.
• Methyl 5,6-diphenylpyridine-2-carboxylate (Compound 30).
• General Procedure E A solution of ethyl 5,6-diphenylpyridine-2-carboxylate (Compound 21, 30 mg, 0.1 mmol) and cone. H 2 SO 4 (3 drops) in MeOH (5 ml) was heated at 50 0 C overnight. The mixture was diluted with water, and the products were extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over Na 2 SO 4 . The filtered solvent was concentrated in vacuo and the residue was purified by column chromatography (20 % ethyl acetate in hexane) to obtain the title compound as a yellow solid.
• Methyl 5-Phenyl-6-(4-trifluoromethylphenyl)-pyridine-2-carboxylate (Compound 35). Following General Procedure E, ethyl 5-phenyl-6-(4- trifluoromethylphenyl)-pyridine-2-carboxylate (Compound 29, 103 mg, 0.28 mmol) and cone. H 2 SO 4 (5 drops) in MeOH (5 ml) were reacted to produce the title compound as a white solid.
• Example 37 Ethyl oxalamidrazonate (Compound 37).
• a solution of anhydrous hydrazine (0.5 ml, 15.0 mmol) in ethanol (5 ml) was added dropwise to a stirred solution of ethyl thiooxamate (2 g, 15.0 mmol) in ethanol (45 ml) under argon at room temperature.
• the mixture was stirred at room temperature for 1 hour, and the solvent was removed in vacuo and dried under high vacuum to get a white solid which was maintained in argon atmosphere after drying. The white solid was used in the next step without further purification.
• Example 39 1-Propenylpyrrolidine (Compound 39). Following General Procedure F, K2CO3 (3.8 g, 28.1 mmol), pyrrolidine (1 g, 14.0 mmol) and propionaldehyde (1.6 g, 28.1 mmol) in toluene (10 ml) were reacted to produce the title compound as a brown oil.
• the resulting white precipitate was filtered off, rinsed with THF (10 ml), and the combined filtrates were added to a solution OfNaBH 4 (945 mg, 24.9 mmol) in H 2 O (20 ml) slowly in order to maintain an internal temperature of 10 0 C to 15 0 C. After the addition was completed, the reaction was stirred at room temperature for 4 hours, and then it was made acidic with HCl (20 %). The layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with NaHCO 3 (aq), and water, and brine, and dried over Na 2 SO 4 . The filtered solution was concentrated in vacuo, and the residue was purified by column chromatography (20 % ethyl acetate in hexane) to yield a white solid.
• Ethyl (2Z,4i ⁇ )-2-azido-5-(4-ethylphenyl)penta-2,4-dienoate (Compound 44).
• General Procedure I A solution of NaOEt in ethanol was prepared in situ by dissolving Na (948 mg, 41.3 mmol) in 30 ml of ethanol. To this solution was added a solution of (£)-3-(4-ethylphenyl)acrylaldehyde (Compound 42, 1.Ig , 6.9 mmol) and ethyl azidoacetate (13 ml, 41.3 mmol) in EtOH (20 ml) dropwise at -10 0 C.
• Example 46 3-Ethoxycarbonyl-l,l,l-triphenyl-6-(4-ethylphenyl)-2-aza-l ⁇ , 5 -phosphahexa-l, 3,5-triene (Compound 46).
• General Procedure J A solution of triphenylphosphine (1.2 g, 4.54 mmol) in diethyl ether (10 ml) was added dropwise to a solution of ethyl (2Z,4ii)-2-azido-5-(4-ethylphenyl)penta-2,4-dienoate (Compound 44, 1.2 g, 4.54 mmol) in diethyl ether (20 ml) at 0 0 C.
• Example 55 and Example 60 ⁇ -Phenyl-S-p-tolylpyridine-l-carbaldehyde (Compound 55) and (6-phenyl-5-p- tolylpyridin-2-yl)methanol (Compound 60).
• ethyl ⁇ -phenyl-S-p-tolylpyridine ⁇ -carboxylate (Compound 22, 1.1 g, 3.47 mmol) and DIBAL-H (5.2 ml, 5.21 mmol, 1.0M in cyclohexane) in CH 2 Cl 2 (30 ml) were reacted to produce Compound 55 and Compound 60 after separation by column chromatography (silica gel, 15% ethyl acetate in hexane).
• Example 56 and Example 61 5-(4-Ethylphenyl)-6-phenylpyridine-2-carbaldehyde (Compound 56) and [5-(4- Ethylphenyl)-6-phenylpyridin-2-yl]-methanol (Compound 61).
• Example 63 ⁇ 3-[(5,6-Diphenylpyridin-2-ylmethyl)-amino]-propyl ⁇ -phosphonic Acid (Compound 63).
• General Procedure M To a solution of 5,6-diphenylpyridine-2- carbaldehyde (Compound 54, 95 mg, 0.37 mmol) and (3-amino-propyl)- phosphonic acid (51 mg, 0.37 mmol) in MeOH (3 ml) was added Bu 4 NOH (0.4 ml, 0.37 mmol, IM in MeOH) under argon. The mixture was stirred at 50 0 C for 30 min. before adding NaCNBH 3 (23 mg, 0.37 mmol) to the mixture.
• Example 86 and Example 87 5-(4-Isopropyl-phenyl)-6-phenyl-pyridine-2-carbaldehyde (Compound 86) and (5-(4-Isopropylphenyl)-6-phenylpyridin-2-yl)methanol (Compound 87).
• Example 76 4- ⁇ [5-(4-Ethyl-phenyl)-6-phenyl-pyridin-2-ylmethyl]-amino ⁇ -butyric Acid (Compound 76).
• 5-(4-Ethyl-phenyl)-6-phenyl-pyridine-2-carbaldehyde (Compound 56, 20 mg, 0.07 mmol), 5-amino-pentanoic acid (17 mg, 0.14 mmol), NaCNBH 3 (4 mg, 0.07 mmol) and HOAc (ldrop) in MeOH (2 ml) were reacted overnight. The solvent was removed and the resulting crude solid was purified by MPLC column chromatography (silica gel, 0 - 100 % ethyl acetate in hexane) to obtain the title compound as a white solid.
• Example 88 [4-(Tert-butyl-dimethyl-silanyloxy)-butyl] -triphenyl- ⁇ s -phosphane Iodide Salt (Compound 88).
• Tert-butyl(4-iodobutoxy)dimethylsilane (2 g, 6.4 mmol) was treated with triphenylphosphine (2.2 g, 8.3 mmol) in THF (30 ml).
• Example 92 3-(4-Ethylphenyl)-6-(5-iodopentyl)-2-phenylpyridine (Compound 92).
• Iodine 31 mg, 0.12 mmol
• triphenylphosphine 32 mg, 0.12 mmol
• To the resulting yellow slurry was added dropwise a solution of 5-(5-(4-ethylphenyl)-6- phenylpyridin-2-yl)pentan-l-ol (Compound 91, 28 mg, 0.08 mmol) and imidazol in CH 2 Cl 2 .
• Methyl 5,6-Diphenyl-pyrazine-2-carboxylate (93).
• General Procedure O To a solution of benzyl (500 mg, 2.38 mmol) and 2,3 -diaminopropionic acid monohydro chloride (334 mg, 2.38 mmol) in MeOH (10 ml) was added NaOH (380 mg, 9.51 mmol) at room temperature. After the mixture was refluxed for 6 hours, it was cooled down in an ice-bath, and cone. H 2 SO 4 (1 ml) was added dropwise, and the reaction mixture was stirred under reflux for 3 hours. MeOH was removed under vacuum, and the residue was dissolved in water, and extracted with ethyl acetate.
• Methyl ⁇ -Phenyl-S-p-tolylpyrazine-l-carboxylate (Compound 94) and Methyl 5-Phenyl-6-p-tolylpyrazine-2-carboxylate (Compound 95).
• l-phenyl-2-/?-tolylethane-l,2-dione (287 mg, 1.3 mmol) and 2,3- diaminopropionic acid monohydro chloride (180 mg, 1.3 mmol) NaOH (205 mg, 5.2 mmol) in MeOH (10 ml) was refluxed for 48 hours. Then it was cooled down in an ice-bath, and cone.
• the present invention contemplates and includes a compound comprising a 6-membered heteroaromatic ring including one, two or three enchained nitrogen atoms at the 1 , or 1 and 3 or 1 , 3 and 4 positions, respectively, and the remaining ring atoms being carbon, an aryl radical directly bonded to said 6-membered heteroaromatic ring at both of the 5 and 6 positions and a side chain at the 2 position of said 6-membered heteroaromatic ring, wherein said side chain terminates with an end group selected from the group consisting of a phosphonic acid, a lower alkyl ester thereof, a carboxylic acid, a lower alkyl ester thereof, a lower alkyl ether and a lower alkylcarboxy and a compound comprising a 6- membered heteroaro

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