Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological 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
  • A61P37/08—Antiallergic agents
• • 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
  • C07D213/62—Oxygen or sulfur atoms
  • C07D213/63—One oxygen atom
  • C07D213/65—One oxygen atom attached in position 3 or 5

Definitions

• This invention relates to certain compounds containing a substituted pyridyl group linked to a substituted phenyl group by an alkyl or heteroatom-containing tether and their use for treating diseases arising from or related to leukotrienes, particularly leukotriene B4. As such their utility lies in antagonizing the effects of leukotrienes.
• the family of bioactive lipids known as the leukotrienes exe ⁇ pharmacological effects on respiratory, cardiovascular, and gastrointestinal systems.
• the leukotrienes are generally divided into two sub-classes, the peptidoleukotrienes (leukotrienes C4, D4 and E4) and the dihydroxyleukotrienes (leukotriene B4).
• This invention is primarily concerned with the hydroxyleukotrienes (LTB) but is not limited to this specific group of leukotrienes.
• Leukotrienes are critically involved in mediating many types of cardiovascular, pulmonary, dermatological, renal, allergic, and inflammatory diseases including asthma, adult respiratory distress syndrome, cystic fibrosis, psoriasis, and inflammatory bowel disease.
• LTB4 has been established as an inflammatory mediator in vivo. It has also been associated with airway hyper-responsiveness in the dog as well as being found in increased levels in lung lavages from humans with severe pulmonary dysfunction.
• the compounds and pharmaceutical compositions of the present invention are valuable in the treatment of diseases in subjects, including human or animals, in which leukotrienes are a factor.
• R is Ci to C20-aliphatic, unsubstituted or substituted phenyl Cj to C ⁇ o-aliphatic where substituted phenyl has one or more radicals selected from the group consisting of lower alkoxy, lower alkyl, trihalomethyl, and halo, or R is Cj to C20-aliphatic-O-, or R is unsubstituted or substituted phenyl C to Cjo-aliphatic-O- where substituted phenyl has one or more radicals selected from the group consisting of lower alkoxy, lower alkyl, trihalomethyl, and halo
• Rl is -(Ci to C5 aliphatic)R 4 , -(Ci to C 5 aliphatic)CHO f -(Ci to C 5 aliphatic)CH20R8, -R4, -CH 2 OH, or CHO;
• R2 is H, halo, lower alkyl, lower alkoxy, -CN, -(CH ⁇ nR ⁇ -CH(NH2)(R4), or -(CH2) n R9 where n is 0 - 5 and where R9 is -N(R7)2 where each R7 is independently H, or an aliphatic group of 1 to 10 carbon atoms, or acyl of 1-6 carbon atoms, or a cycloalkyl-(CH 2 )n- group of 4 to 10 carbons where n is 0-3, or both R7 groups form a ring having 4 to 6 carbons; or
• R3 is hydrogen, lower alkyl, lower alkoxy, halo, -CN, R4, NHCONH2, or OH; each R 4 group is independently -COR5 where R5 is -OH, a pharmaceutically acceptable ester-forming group -OR ⁇ , or -OX where X is a pharmaceutically acceptable cation, or R5 is -N(R7)2 where each R7 is independently H, or an aliphatic group of 1 to 10 carbon atoms, or a cycloalkyl-(CH2)n- group of 4 to 10 carbons where n is 0-3, or both R7 groups form a ring having 4 to 6 carbons, or R 4 is a sulfonamide, or an amide, or tetrazol-5-yl; and
• R8 is hydrogen, Ci to C ⁇ alkyl, or Ci to C ⁇ -acyl, exlcuding those compounds where R2 and R3 are other than hydrogen and are substituted in the 2 and 6 positions.
• this invention covers pharmaceutical compositions containing the instant compounds and a pharmaceutically acceptable excipient
• Treatment of diseases related to or caused by leukotrienes, particularly LTB4, or related pharmacologically active mediators at the end organ are within the scope of this invention.
• This treatment can be effected by administering one or more of the compounds of formula I alone or in combination with a pharmaceutically acceptable excipient.
• Processes for making these compounds are also included in the scope of this invention, which processes comprise: a) forming a salt, or b) hydrolyzing an ester to give a salt or acid; c) forming an ester, d) forming an amide; e) oxidizing a thio ether, f) forming a compound of formula I by treating a 6- halomethylpyridyl compound with tha appropriate mercaptobenzoate or hydroxybenzoate.
• Aliphatic is intended to include saturated and unsaturated radicals. This includes normal and branched chains, saturated or mono or poly unsaturated chains where both double and triple bonds may be present in any combination.
• the phrase "lower alkyl” means an alkyl group of 1 to 6 carbon atoms in any isomeric form, but particularly the normal or linear form.
• Lower alkoxy means the group lower alkyl-O-.
• Halo means fluoro, chloro, bromo or iodo.
• Acyl means the radical having a terminal carbonyl carbon.
• substituted phenyl ring When reference is made to a substituted phenyl ring, it is meant that the ring can be substituted with one or more of the named substituents as may be compatible with chemical synthesis. Multiple substituents may be the same or different, such as where there are three chloro groups, or a combination of chloro and alkyl groups and further where this latter combination may have different alkyl radicals in the chloro/alkyl substituent pattern.
• a pharmaceutically acceptable ester-forming group in R2 and R3 covers all esters which can be made from the acid function(s) which may be present in these compounds.
• the resultant esters will be ones which are acceptable in its application to a pharmaceutical use. By that it is meant that the mono or diesters will retain the biological activity of the parent compound and will not have an untoward or deleterious effect in their application and use in treating diseases.
• esters are, for example, those formed with one of the following radicals: Ci to C6 alkyl, phenyl Ci-C6alkyl, cycloalkyl, aryl, arylalkyl, alkylaryl, alkylarylalkyl, aminoalkyl, indanyl, pivaloyloxymethyl, acetoxymethyl, propionyloxymethyl, glycyloxymethyl, phenylglycyloxymethyl, or thienylglycyloxymethyl.
• ester-forming radicals are those where R3 is alkyl, particularly alkyl of 1 to 10 carbons, (ie CH3- (CH2) n - where n is 0-9), or phenyl-(CH2) n - where n is 0-4.
• R2 When R2 is referred to as being an amine, that includes the radical -NH2 and mono- or dialkylate derivatives of this -NH2 radical.
• Preferred alkylated amines are the mono- or disubstituted amines having 1 to 6 carbons.
• R2 When R2 is referred to as being an amide, that includes all acylate derivatives of the NH2 radical.
• the preferred amides are those having 1 to 6 carbons. Where there is an acid group, amides may be formed.
• the most preferred amides are those where -R ⁇ is hydrogen or alkyl of 1 to 6 carbon atoms. Particularly preferred is the diethylamide or dimethylamide.
• salts of the instant compounds are intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.
• compositions are prepared in a standard manner, in a suitable solvent.
• the parent compound in a suitable solvent is reacted with an excess of an organic or inorganic acid, in the case of acid addition salts, or an excess of organic or inorganic base in the case where R4 is OH.
• N-oxides may also be prepared by means of selected oxidizing agents. These oxides are useful as intermediates in preparing the compounds of fo ⁇ nula I and have useful pharmaceutical activity in and of themselves. Hence one can administer the N- oxides of formula I to a subject who is susceptible to or is suffering from a disease related to or caused by LTB4 or similar leukotrienes.
• a chiral center is created or another form of an isomeric center is created in a compound of this invention, all forms of such isomer(s) are intended to be covered herein.
• These compounds may be used as a racemic mixture or the racemates may be separated and the individual enantiomer used alone.
• Olefins may have the cis or trans configuration (E or Z); either are useful in the practice of this invention.
• these compounds can be used in treating a variety of disease assoicated with or attributing their origin or affect to leukotrienes, particularly
• these compounds can be used to treat allergic diseases such of a pulmonary and non-pulmonary nature.
• these compounds will be useful in antigen-induced anaphylaxis; for treating asthma and allergic rhinitis; psoriasis, or irritable bowel disease; ocular diseases such as uveitis, and allergic conjunctivitis.
• the preferred compounds are those where Z is O or S(O) ⁇ ; m is 0-3; n is 0-2; R is alkoxy of 8 to 15 carbon atoms or unsubstituted or substituted pheny-Cj to C Q- aliphatic-O-; and Ri is -(Cj to C5 a!iphatic)R4 or -(Ci to C 5 -aIiphatic)CH2ORs.
• Another set of preferred compounds are the anilines, those where R2 is N(R7>2, particularly where R7 is hydrogen.
• a third set of preferred compounds are those where both R2 and R3 are hydrogen.
• the most preferred compounds are: l-fluoro-3-[2-thia-3-[2-(E-2-carboxyethenyl)-3-[4-(4-methoxyphenyl)butyloxy]- 6-pyridyl]propyl]benzene, lithium salt;
• Synthesis There are several methods for preparing these compounds.
• One generic process comprises preparing a 6-(halomethyl)pyridyl adduct and then condensing this fragment with the appropriate mercaptan or alcohol to make compounds where Z is sulfur or oxygen.
• functional groups such as acid groups will be protected; any acid group may be derivatized in some manner to render it unreactive.
• protecting groups may be removed to provide the parent functionality, e.g. an acid. Further modification of these reactive groups can then be carried out, such as forming a salt, an amide, an ester or the like.
• Sulfonamides are prepared from the corresponding amines by literature methods.
• Tetrazoles are prepared from the corresponding acid halide, e.g., the acid chloride, by literature methods.
• Scheme I outlines a means for making a substituted phenylalkyl tail which is R.
• the starting alcohol represented here as the 3-octyn-l-ol, is commercially available (Lancaster Synthesis). To migrate the triple bond to the w-carbon, KH and 1,3-
• Protecting the alcohol is accomplished by forming a silyl ether illustrated here as the r-butyldiphenylsilyl ether. Other silyl ethers could be used.
• the alcohol is dissolved in a polar solvent, for example dimethylformamide, and imidazole is added followed by the desired silane. All this is carried out under an inert atmosphere such as argon. Ambient temperature is acceptable for effecting the reaction.
• Adding the phenyl group is done in a dry environment using an amine for a solvent and an inert atmosphere.
• a solvent such as triethylamine under argon is added the silylether followed by a halophenyl compound, eg. iodoanisole, a palladium catalyst (P 3P)2PdCl and Cul, both of the latter in catalytic amounts.
• Heat is used to effect the reaction, usually a temperature of up to about 50°C will be sufficient. 25 Two or more hours, up to six but often about four at the elevated temperature will usually cause the reaction to go to completion.
• the triple bond is then saturated, preferably by catalytic hydrogenation.
• the silyl ether can be dissolved in a saturated solvent such as an alcohol, a heavy metal catalyst added (Pd-C) and the mixture put under H2 for a time sufficient to reduce the triple bond. Stirring for 2 to 6 hours will usually effect the reaction.
• Recovering the alcohol is done by treating the silyl ether with a fluoride source such as tetrabutylammonium fluoride. Reactants are combined at a mildly reduced temperature, eg.0°C, then the reaction is allowed to run its course at ambient temperature or there about Several hours may be needed for the reaction to go to completion. Product was recovered by extraction means.
• a fluoride source such as tetrabutylammonium fluoride
• Converting the alcohol to the iodo compound is accomplished using a phosphine, imidazole and I2.
• this transformation is accomplished by adding to a solution of alcohol under argon, a molar excess of triphenylphosphine, for example, and a three-fold excess of imidazole followed by iodine. Materials are combined at room temperature, but then the reaction pot may be heated to between 50 - 70°C for a brief period, 10 minutes to an hour to complete the reaction. Standard procedures are then used to recover and purify the product.
• Scheme II illustrates an alternative process for making R groups.
• an alkylsilazide is added to an inert solvent under an inert atmosphere.
• the phosphonium salt is added. This addition can be done at room temperature or thereabouts. After a brief period of mixing, this mixture is usually a suspension, the benzaldehyde is added slowly at about room temperature. A slight molar excess of the phosphonium salt is employed After an additional brief period of stirring at about room temperature, the reaction is quenched with water.
• the solution is acidified and the acid extracted with a suitable organic solvent Further separatory and purification procedures may be employed as desired.
• the alcohol is made by reducing the acid using a reducing agent. Lithium aluminum hydride or similar reducing agents may be employed, and conditions may be varied as needed to effect the reduction.
• the tosylate is prepared in an inert solvent employing a base such as pyridine. Suitable conditions include carrying out the reaction at room temperature or thereabouts for a period of 1 to 5 hours. Other leaving groups similar in function to the tosylate may be prepared and will be useful as a means for forming the R moiety.
• R alkyl
• haloalkylbenzoates are commercially available or can be made by methods known in the art.
• Thiourea is added to a solution of haloalkylbenzoate at ambient temperature or thereabouts. Any appropriate solvent may be used, acetone for example.
• a precipitate of the thiouronium salts should form under these conditions.
• the precipitate is collected and dissolved in water and the pH adjusted to about 10.5 with a base, for example a solution of NaOH. Refluxing is then commenced for between 1 and 4 hours.
• Product, as the free acid is then recovered by some other separatory and purification means. Esterification is then carried out by mixing the acid with an alcohol, bubbling HC1 through the solution, and letting sit the resulting solution for a time not more than several days; two days usually is sufficient to effect the reaction.
• the starting material is available from Aldrich. It is treated with a mild oxidizing agent such as MnC-2 to oxidize the 2-hydroxyethyl group to the corresponding -aldehyde.
• the R group is then formed.
• an ether is prepared under basic conditions using an a-halo intermediate.
• a tosylate made as per Scheme HI, can also be used in this step.
• Introducing the acid function at position 2 is accomplished by means of a triphenylphosphoranylidene reagent. The acetate form is illustrated here but other similar reagents could be used.
• the N-oxide is then formed by means of a peroxy acid. Trifluoroacetic anhydride is used to oxidize the 6-position methyl group.
• This hydroxymethyl group is then converted to the corresponding halide, (in the hydrohal ⁇ de form) in this case the chloride, by means of thionyl chloride.
• An alkyl hydroxybenzoate is then reacted with the 6-chloromethyl compound in the presence of tetrabutylammonium iodide and a weak base.
• the resulting diester can be hydrolyzed to the salt or, further, acidified to give the free acid.
• An oxidant can be used to regenerate the N-oxide which can then be treated with base to hydrolyze the esters.
• Esters can be converted to salts, the free acids and other derivatives.
• Catalytic hydrogenation can be used to reduce the double bond in the Ri group described here.
• Scheme V illustrates a method for making compounds where Z is a S and m is 0.
• the starting hydrochloride is described in Scheme IV. Instead of treating the hydrochloride with an alcohol, in this instance the mercapto analog of the hydroxybenzoate described above is used.
• the resulting thioether can be hydrolyzed to give the salt or treated further to give the free acid from which other derivatives of the carboxyl function can be prepared, including alcohols and aldehydes.
• the double bond in the Ri group can be reduced by catalytic means using a heavy metal catalyst and hydrogen.
• the sulfone and sulfoxide can be prepared by treating the thioether with an oxidizing agent A peroxy acid or other oxidizing agent can be used.
• 2-hydroxypicolinic acid is converted to the alkyl ester using the corresponding alcohol and an acid to catalyze the reaction.
• the hydroxyl group is then converted to the triflu ⁇ romethy sulfonate by means of trifluoromethanesulfonic anhydride - and a base, e.g. pyridine.
• the lipid tail is attached using the appropriate alkyl catechol boronateunderpalladium coupling conditions. For example, 1-iododecene and catechol borane are reacted to form the alkyl catechol boronate.
• alkylation reaction is effected using Pd(OAc)2-
• the ester is reduced to the corresponding aldehyde with a hydride such as d ⁇ sobutylaluminum hydride (DIBAL).
• DIBAL d ⁇ sobutylaluminum hydride
• a Wittig olef ⁇ nation is then carried out using, for example, methyl(triphenylphosphoranyUdene)acetate.
• the resulting pyridyl methyl acrylate is then oxidized to the N-oxide with an oxidizing agent such as 3-chloroperoxybenzoic acid. This oxide is then rearranged to the 2-pyridone with trifluoroacetic anhydride.
• a trifluoromethylsulfonate is then formed using trifluoromethanesulfonic anhydride and pyridine. Carbomethylation is then effected by means of Pd(OAc)2, a simple alcohol, and carbon monoxide. Selectively reducing the pyridyl-ester (using a hydride such as NaBH4 in a low molecular weight alcohol) yields the 2-(hydroxymethyl)-pyridine. This compound is treated with thionyl chloride to form the 6-chloromethyl compound. This intermediate is transformed to the ethers or thioether of formula I in the same manner as is illustrated in Schemes IV - VI.
• compositions of the present invention comprise a pharmaceutical carrier or diluent and some amount of a compound of the formula (I).
• the compound may be present in an amount to effect a physiological response, or it may be present in a lesser amount such that the user will need to take two or more units of the compositon to effect the treatment intended.
• These compositions may be made up as a solid, liquid or in a gaseous form. Or one of these three forms may be transformed to another at the time of being administered such as when a solid is delivered by aerosol means, or when a liquid is delivered as a spray or aerosol.
• compositions and the pharmaceutical carrier or diluent will, of course, depend upon the intended route of administration, for example parenterally, topically, orally or by inhalation.
• the pharmaceutical composition will be in the form of a sterile injectable liquid such as an ampule or an aqueous or non-aqueous liquid suspension.
• the pharmaceutical composition will be in the form of a cream, ointment, liniment, lotion, pastes, and drops suitable for administration to the eye, ear, or nose.
• the pharmaceutical composition will be in the form of a tablet, capsule, powder, pellet, atroche, lozenge, syrup, liquid, or emulsion.
• examples of appropriate pharmaceutical carriers or diluents include: for aqueous systems, water; for non-aqueous systems, ethanol, glycerin, propylene glycol, com oil, cottonseed oil, peanut oil, sesame oil, liquid parafins and mixtures thereof with water; for solid systems, lactose, kaolin and mannitol; and for aerosol systems, dichlorodifluoromethane, chlorotrifluoroethane and compressed carbon dioxide.
• the instant compositions may include other ingredients such as stabilizers, antioxidants, preservatives, lubricants, suspending agents, viscosity modifiers and the like, provided that the additional ingredients do not have a detrimental effect on the therapeutic action of the instant compositions.
• the pharmaceutical preparations thus described are made following the conventional techniques of the pharmaceutical chemist as appropriate to the desired end product.
• Formulations for treating psoriasis can take the form of oral or topical preparations. Topically applied formulations are preferred. Ointments, creams, liniments, lotions, pastes and similar preparations are examples of preferred topical formulations. Aerosols may also be used. These dosage forms will contain between 0.01 and 5 percent by weight of the active ingredient
• a compound of formula I is administered, that is applied, to a subject in a composition comprising a nontoxic amount sufficient to produce an inhibition of the symptoms of a disease state.
• the dosage of the composition is selected from the range of from 50 mg to 1000 mg of active ingredient for each administration.
• equal doses will be administered 1 to 5 times daily with the daily dosage regimen being selected from about 50 mg to about 5000 mg.
• the amount applied will depend on the size of the affected area and the severity and progress of the disease, ie. psoriasis.
• a disease mediated by LTB4 which comprises administering to a subject a therapeutically effective amount of a compound of formula I, preferably in the form of a pharmaceutical composition.
• a therapeutically effective amount of a compound of formula I preferably in the form of a pharmaceutical composition.
• inhibiting the inflammatory response resulting from psoriasis by administration of an effective amount of a compound of formula I is included within the scope of this disclosure.
• the administration may be carried out in dosage units at suitable intervals or in single doses as needed. Usually this method will be practiced when relief of symptoms is specifically required.
• the method is also usefully carried out as continuous or prophylactic treatment It is within the skill of the art to determine by routine experimentation the effective dosage to be administered from the dose range set forth above, taking into consideration such factors as the degree of severity of the condition or disease being treated, and so forth.
• compositions and their method of use also include the combination of a compound of formula I with Hi Mockers where the combination contains sufficient amounts of both compounds to treat antigen-induced respiratory anaphylaxis or similar allergic reaction.
• Hi blockers useful here include: cromolyn sodium, compounds from the ethanolamines class (diphenhydramine), ethylenediamines (pyrilamine), the alkylamine class (chlorpheniramine), the piperazine class (chlorcyclizine), and the phenothiazine class (promethazine).
• Hi blockers such as 2-[4-(5-bromo-3-methylpyrid-2-yl)butylamino]-5-[(6-methylpyrid-3-yl)methyl]-4- pyrimidone are particularly useful in this invention.
• the specificity of the antagonist activity of a number of the compounds of this invention is demonstrated by relatively low levels of antagonism toward agonists such as potassium chloride, carbachol, histamine and PGF2.
• the receptor binding affinity of the compounds used in the method of this invention is measured by the ability of the compounds to bind to [3_H]-LTB4 binding sites on human U937 cell membranes.
• the LTB4 antagonist activity of the compounds used in the method of this invention is measured by their ability to antagonize in a dose dependent manner the LTB4 elicited calcium transient measured with fura-2, the fluorescent calcium probe. The methods employed were as follows: U-937 Cell Culture Conditions
• U-937 cells were obtained from Dr. John Bomalaski (Medical College of PA) and Dr. John Lee (SmithKline Beecham Corp., Dept of Immunology) and grown in RPMI- 1640 medium supplemented with 10% (v/v) heat inactivated fetal calf serum, in a humidified environment of 5% CO2, 95% air at 37°C. Cells were grown both in T-flasks and in Spinner culture. For differentiation of the U937 cells with DMSO to macrophage- like cells, the cells were seeded at a concentration of 1 x l(fi cells/ml in the above medium with 1.3% DMSO and the incubation continued for 4 days. The cells were generally at a density of 0.75-1.25 x 10 ⁇ cells/ml and were harvested by centrifugation at 800 x g for 10 min.
• [ 3 H]-LTB4 binding assays were performed at 25°C, in 50 mM Tris-HCl (pH 7.5) buffer containing 10 mM CaCl2, 10 mM MgCl2, [ 3 H]-LTB4, U937 cell membrane protein (standard conditions) in the presence or absence of varying concentrations of LTB4, or test compounds. Each experimental point represents the means of triplicate determinations. Total and non-specific binding of [ 3 H]-LTB4 were determined in the absence or presence of 2 mM of unlabeled LTB4, respectively. Specific binding was calculated as the difference between total and nonspecific binding.
• the radioligand competition experiments were performed, under standard conditions, using approximately 0.2 nM [ 3 H]-LTB4, 20-40 mg of U937 cell membrane protein, increasing concentrations of LTB4 (0.1 mM to 10 mM) or other competing ligands (0.1 mM to 30 mM) in a reaction volume of 0.2 ml and incubated for 30 minutes at 25°C.
• the unbound radioligand and competing drugs were separated from the membrane bound ligand by a vacuum filtration technique.
• the membrane bound radioactivity on the filters was determined by liquid scintillation spectrometry.
• the cells were centrifuged at 800 x g for 10 minutes and resuspended in cold fresh buffer B at 5 x i cells/ml. Cells were maintained on ice in the dark until used for fluorescent measurements. Fluorescent Measurements - Calcium Mobilization
• the fluorescence of fura-2-containing U937 cells was measured with a fluorometer designed by the Johnson Foundation Biomedical Instrumentation Group.
• a fluorometer was equipped with temperature control and a magnetic stirrer under the cuvette holder.
• the wave lengths are set at 339 nm for excitation and 499 nm for emission. All experiments were performed at 37°C with constant mixing.
• U-937 cells were diluted with fresh buffer (B) to a concentration of 1 x 10 ⁇ cells/ml and maintained in the dark on ice. Aliquots (2 ml) of the cell suspension were put into 4 ml cuvettes and the temperature brought up to 37°C, (maintained in 37°C, water bath for 10 min). Cuvettes were transferred to the fluorometer and fluorescence measured for about one minute before addition of stimulants or antagonists and followed for about 2 minutes post stimulus. Agonists and antagonists were added as 2 ml aliquots. Antagonists were added first to the cells in the fluorometer in order to detect potential agonist activity. Then after about one minute 10 nM LTB4 (a near maximal effective concentration) was added and the maximal Ca ⁇ + mobilization [Ca- ⁇ +j was calculated using the following formula:
• F was the maximum relative fluorescence measurement of the sample. Fmax was determined by lysing the cells with 10 ml of 10% Triton X-100 (final Concentration 0.02%). After Fmax was determined 67 ml of 100 mM EDTA solution (pH 10) was added to totally chelate the Ca ⁇ + and quench the fura-2 signal and obtain the Fmin.
• the [Ca2 + j " i level for 10 nM LTB4 in the absence of an antagonist was 100% and basal [Ca2 + ] j was 0%.
• the IC50 concentration is the concentration of antagonist which blocks 50% of the lOnM LTB4 induced [Ca 2+ ]j mobilization.
• the EC50 for LTB4 induced increase in [Ca 2 "* " ] j mobilization was the concentration for half maximal increase.
• the Ki for calcium mobilization was determined using the formula:
• the LTB4 concentration was 10 nM and the EC50 was 2 nM.
• 6-(4-Methoxyphenyl)hexan-l-f-butyldiphenylsilyl ether (2.0g, 4.6mmol) in tetrahydrofuran (20mL) was cooled to 0 ⁇ C and treated with tetrabutylammonium fluoride (14mL, 14mmol, 1M in tetrahydrofuran). The cooling bath was removed and the reaction was stirred at room temperature for 24 hours. The reaction was diluted with ethyl acetate and was washed with H2O and brine and dried (Na2S ⁇ 4).
• 3-Hydroxy-6-methyl-2-pyridine carboxaldehye obtained above was dissolved in dry dimethylfo ⁇ namide (lOmL) and treated with 1-iodododecane (2. lmL, 8.62mmol) and anhydrous K2CO3 (3.0g, 21.7mmol) under an argon atmosphere.
• the reaction was heated at 90° C for lh with vigorous stirring.
• the reaction mixture was poured into ethyl acetate (lOOmL); the ethyl acetate solution was washed with H2O (3X20mL) and brine and dried (MgS ⁇ 4).
• reaction solution was slowly added to a cooled (0 C) saturated aqueous Na2C ⁇ 3 solution (lOOmL).
• the aqueous solution was extracted with ethyl acetate (2X50mL) and the combined ethyl acetate extracts were washed with H2O (2X20mL) and brine and dried (MgSO4); the solvent was removed in vacuo.
• the product mixture was dissolved in methanol (20mL), treated with anhydrous K2CO3 (500mg), and vigorously stirred for 20 minutes.
• the reaction was diluted with ethyl acetate (75mL) and washed with H2O (30mL).
• Methyl 3-[l-oxa-2-[2-(E-2-carboxymethylethenyl)-3- dodecyloxy-6-pyridyl]ethyl]benzoate 130mg, 0.254mmol was dissolved in dry CH2CI2 (1.5mL), cooled to 0 «C, and treated with 85% m-chloroperoxybenzoic acid
• 3-Aminophenol f ⁇ utylcarbamate 3-Aminophenol (2.0g, 18.3mmol; Aldrich) was dissolved in CH2CI2 (18mL) and DMF (6mL) and treated with d butyl dicarbonate (5.0mL, 21.7mmol). The reaction was stirred under an argon atmosphere for 18 hours. The reaction solution was diluted with EtOAc and washed with H 2 O and brine and dried (MgSO 4 ).
• Example 17 Preparation of 3-rN-r2-r2-(E-2-CarboxyethenvI)-3-(8-(4-methoxyphenyl)octyloxy)-6- pyridvHmethvHlaminobenzoic acid, dilithium salt
• the captioned compound was prepared according to the method set out in Scheme 5 above by reacting the appropriate r-BOC-protected aminobenzoic acid with 2-Q ⁇ -2- carboxymethyIethenyl)-3-dodecyloxy-6-(chloromethyl)-pyridine hydrochloride or a similar intermediate, the captioned compound was prepared.
• Methyl 4-r2-oxythia-3-r2-(E-2-carboxymethylethenyl)-3-r8-(4- • mefl ⁇ oxyphenyl)octyloxy1-6-pyridyllpropynbenzoate Methyl 4-[2-thia-3-[2-(E-2- carix)xymethylethenyl)-3-[8-(4-methoxyphenyl)octyloxy]-6-pyridyl]propyl]benzoate (HOmg, 0.186mmol) was dissolved in dry CH2CI2 (4mL) under an argon atmosphere and cooled to -20°C.
• the thiouronium salt was dissolved in H2O (65mL) and the pH was adjusted to 10.5 by the addition of 10% NaOH. The mixture was refluxed for 2 hours. After cooling to room temperature the solution was extracted with EtOAc and the organic layer was discarded. The aqueous solution was acidified to pH 1.5 and extracted three times with EtOAc. The organic extracts were dried (MgSO4), filtered and the solvent evaporated. The crude acid was then dissolved in anhydrous MeOH (125mL), cooled to 0°C, and dry HCl gas was 5 bubbled through the solution for 30 minutes. The reaction was then left for two days at room temperature.
• reaction was heated at 60°C under an atmosphere of argon for 1.5 hours. Upon cooling to room temperature the reaction was diluted with EtOAc and washed with H20, 10-% NaOH, H2O, and brine and dried (MgSO4).
• This reaction can also be used to make other sulfoxides and sulfones of this inventions including 3-[2-dioxythia-[2-(E-2-carboxyethenyl)-3- [4-(4- methoxyphenyl)butyloxy]-6-pyridyl]propyl]benzoic acid, dilithium salt, and 3-[2- oxythia-[2-(E-2-carboxyethenyl)-3-[4-(4-methoxyphenyl)butyloxy]-6- pyridyl]propyl]benzoic acid, dilithium salt.
• Example 22 3-r2-Thia-3-r2-(2-carboxyethanyl)-3-r8-(4-methoxyDhenyl)octvIoxy1-6- pyridynpropyn benzoic acid, dilithium salt 22(a).
• Example 24 4-r2-Oxythia-3-r2-(E-2-carboxyethenyl)-3-r8-(4-metho ⁇ yphenyl)octyloxyl-6- pyridvnpropyllphenylacetic acid, dilithium salt 24(a). Methyl 4-r2-oxythia-3-r2-(E-2-carboxymethylethenyl)-3-r8-(4- methoxyphenyl)octyloxy1-6-pyridvnpropynphenylacetate.
• the resulting acid chloride was dissolved in dry CH2CI2 (5mL), cooled to 0 °C, and treated with triethylamine (52mL, 0.37mmol). Diethylamine was then introduced into the reaction via a cooling finger; reaction was stirred for 15 minutes.
• This tetrazole is prepared via the acid chloride described above according to Duncia, Pierce, and Santella, /. Org. Chem., 1991, 56, 2395-2400.
• Example 27 Formulations for pharmaceutical use incorporating compounds of the present invention can be prepared in various forms and with numerous excipients. Means for making various formulations can be found in standard texts such as Remington's Pharmaceutical Sciences, and similar publications and compendia. Specific examples of formulations are given below.
• Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitable mixer/blender.
• Step 2 Add sufficient water portionwise to the blend from Step 1 with careful mixing after each addition. Such additions of water and mixing until the mass is of a consistency to permit its conversion to wet granules.
• Step 3 The wet mass is converted to granules by passing it through an oscillating granulator using a No. 8 mesh (2.38 mm) screen.
• Step 4 The wet granules are then dried in an oven at 410°F (60°C) until dry. Step 5.
• the dry granules are lubricated with ingredient No. 5.
• Step 6 The lubricated granules are compressed on a suitable tablet press.
• Step 1 Melt ingredient No. 2 and No. 3 together and stir until uniform.
• Step 2. Dissolve ingredient No. 1 in the molten mass from Step 1 and stir until uniform.
• Step 3. Pour the molten mass from Step 2 into supository moulds and chill.
• Step 4. Remove the suppositories from moulds and wrap.
• Example 28 Inhalation Formulation A compound of formula 1, 1 to 10 mg ml, is dissolved in isotonic saUne and aerosoUzed from a nebuUzer operating at an air flow adjusted to deliver the desired amount of drug per use.
• Formulations for pharmaceutical use incorporating compounds of the present invention can be prepared in various forms and with numerous excipients. Means for making various formulations can be found in standard texts such as Remington's Pharmaceutical Sciences, and similar publications and compendia. Specific examples of formulations are given below.
• the stearyl alcohol, white wax and white petrolatum are melted together (steam bath for example) and cholesterol and the active ingredient are added. Stirring is commenced and continued until the solids disappear. The source of heat is removed and the mix allowed to congeal and packaged in metal or plastic tubes.
• the stearyl alcohol and white petrolatum are combined over heat. Other ingredients are dissolved in water, then this solution is added to the warm (ca 50 to 100°C) alcohol/petrolatum mixture and stirred until the mixture congeals. It can then be packed in tubes or another appropriate package form.

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• 1992
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