An important goal in animal husbandry is to develop biologically active agents which can increase the quantity and improve the quality of meat obtained from livestock animals.
xe2x80x9cIncreasing the quantityxe2x80x9d of food obtained from livestock animals refers to, inter alia, promoting the growth of livestock animals, increasing the efficiency of feed utilized in raising livestock animals and/or enhancing the production of lean body mass in livestock animals. Biologically active agents with these attributes are commonly referred to as xe2x80x9canabolic agentsxe2x80x9d.
xe2x80x9cImproving the qualityxe2x80x9d of food obtained from livestock animals refers to, inter alia, reducing the quantity of subcutaneous fat in meat and, in poultry, the size of the abdominal xe2x80x9cfat padxe2x80x9d. Subcutaneous fat can cause elevated cholesterol and triglyceride levels in individuals who consume large quantities of meat, has minimal nutritional value and decreases the overall yield of meat. Therefore, the reduction or elimination of this type of fat from meat is desirable. On the other hand, intramuscular fat, commonly referred to as xe2x80x9cmarblingxe2x80x9d, contributes positively to the flavor of meat and maintains a high Quality Grade. Marbling is therefore considered a desirable quality. Biologically active agents which are lipolytic can reduce subcutaneous fat while retaining the intramuscular fat.
Certain publications have appeared generally disclosing arylpropanolamines such as U.S. Pat. No. 5,013,761 and WO 97/10825. There continues to be a need for the development of agents which are anabolic and lipolytic to improve the economics of meat production by increasing the yield and improving the quality of meat obtained from livestock animals. Further increases in profitability could be achieved by the development of long-lasting anabolic/lipolytic agents which, because they require less frequent dosing, are more convenient and economical to use.
It has now been found that indazolyloxy propanolamimines such as those represented by Structural Formula (I) and (III) below are both anabolic and lipolytic when administered to livestock (Example 7 and Table 1). It has also been found that the anabolic and lipolytic effects of these compounds are longer lasting than other aryloxy propanolamines. Specifically, the percent decrease in serum urea nitrogen level (SUN) after forty-eight hours and the increase in non-esterified fatty acid levels (NEFA) after twenty-four hours in cattle treated with the indazolyloxy propanolamines of the present invention have been found to be significantly greater than for other aryloxy propanolamines (see Example 8 and Table 2). The percent decrease in SUN is indicative of anabolic activity and the increase in NEFA is indicative of lipolytic effects. Based on these results, novel compounds and novel methods of improving meat production from livestock animals are disclosed herein.
One embodiment of the present invention is a compound represented by Structural Formula (I): 
and physiologically acceptable salts thereof; where R1 and R2 are independently xe2x80x94H or a C1-C4 straight chained or branched alkyl group;
Ring A, Ring B and Ring C are independently substituted or unsubstituted, provided, however, that Ring C is not substituted in the para position with a group represented by Structural Formula (II): 
R3 and R4 are independently xe2x80x94H, a straight or branched C1-C4 alkyl or, taken together with the nitrogen atom to which they are bonded, form a non-aromatic heterocyclic ring, and Ring D is substituted with zero, one, two or three additional substituents.
Another embodiment of the present invention is a compound represented by Structural Formula (III): 
and physiologically acceptable salts thereof; where
R1 and R2 are independently xe2x80x94H, a C1-C4 straight chained or a branched alkyl group;
Ring A, Ring B, Ring C and Ring E are independently substituted or unsubstituted, provided, however, that Ring E is not substituted in the position meta to xe2x80x94Nxe2x80x94 and ortho to the carbon bonded to oxygen with xe2x80x94CONR3R4;
R3 and R4 are independently xe2x80x94H or a straight or branched chain C1-C4 alkyl group or, taken together with the nitrogen atom to which they are bonded, form a non-aromatic heterocyclic ring.
Another embodiment of the present invention is a method of increasing the quantity and improving the quality of meat obtained from a livestock animal. The method comprises administering to the animal an effective amount of one or more compounds represented by Structural Formula (I). Another embodiment of the present invention is a method of increasing the quantity and improving quality of meat obtained from a livestock animal. The method comprises administering to the animal an effective amount of one or more compounds represented by Structural Formula (III).
The indazolyloxy propanolamines of the present invention have both anabolic and lipolytic activity and can therefore be administered to livestock to increase the output of meat and to decrease its fat content. Moreover, the duration of the anabolic and lipolytic activity of these indazolyloxy propanolamines is longer in vivo than other structurally related aryloxy propanolamines. Consequently, indazolyloxy propanolamines are more convenient and economical to use than other aryloxy propanolamines because indazolyloxy propanolamines need to be administered less frequently and/or at lower doses. The compounds of the present invention are intended for the treatment of healthy animals.
The present invention is directed to a compound represented by Structural Formula (I) and (III). Also included is a method of improving livestock production by administering one or more compounds of the present invention to the livestock.
In a preferred embodiment, the compound is represented by Structural Formula (IV): 
Ring A, Ring B, Ring C, R1 and R2 are as described above for Structural Formula (I). Ring E is substituted or unsubstituted.
X is xe2x80x94CHxe2x80x94 or xe2x80x94Nxe2x80x94.
In Structural Formula (IV), R1 and R2 are preferably methyl and Rings A-C have no further substitution.
In a preferred embodiment, the compound of the present invention is represented by Structural Formula (V): 
X is xe2x80x94Nxe2x80x94 or xe2x80x94CHxe2x80x94.
Ring E is substituted or unsubstituted. Examples of suitable substituents for Ring E include halo, xe2x80x94CN, xe2x80x94OR5, C1-C4 alkyl, C1-C4 haloalkyl, xe2x80x94CO2R5, xe2x80x94CONR6R7, xe2x80x94CONH(C1-C4 alkyl), xe2x80x94SR5, xe2x80x94CSNR6R7, xe2x80x94CSNR6R7, xe2x80x94SO2R5, xe2x80x94SO2NR6R7, xe2x80x94SOR5, xe2x80x94NR6R7. Preferred substituents are xe2x80x94CN, xe2x80x94CONH2, xe2x80x94SO2CH3, xe2x80x94SO2NH2 or are represented by Structural Formulas (VI) or (VII): 
xe2x80x94SR2CH3 is a more preferred substituent.
R5 is xe2x80x94H, C1-C4 alkyl or aryl.
R6 and R7 are independently xe2x80x94H, C1-C4 alkyl, aryl, xe2x80x94(CH2)naryl, or combine with the nitrogen atom to which each is bound to form morpholinyl, piperidinyl, pyrrolidinyl, or piperazinyl.
n is 0, 1, 2, or 3.
In another preferred embodiment, the compound of the present invention is represented by Structural Formula (IV) or (V), provided, however, that Ring E is not substituted in the position meta to xe2x80x94Xxe2x80x94 and ortho to the carbon bonded to oxygen with xe2x80x94CONR3R4. R3 and R4 are independently xe2x80x94H or a straight or branched chain C1-C4 alkyl group or, taken together with the nitrogen atom to which they are bonded, form a non-aromatic heterocyclic ring.
Also included in the present invention are Compounds 1-5, shown in Tables 1 and 2.
Physiologically acceptable salts of the compounds disclosed herein, including the compounds represented by Structural Formulas (I),(III), (IV), (V) and Compounds 1-5, shown in Tables 1 and 2, are included. Salts can be formed from those compounds which comprise acidic functional groups by reacting with a suitable base. Such salts include those derived from inorganic bases such as ammonium and alkali and alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, as well as salts derived from basic organic amines such as aliphatic and aromatic amines, aliphatic diamines, hydroxy alkamines, and the like. Such bases useful in preparing the salts of this invention thus include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylenediamine, cyclohexylamine, ethanolamine and the like.
Because of the amine moiety, salts of the compounds disclosed herein can also be prepared by reacting with a suitable acid. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic, acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, mono-hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, 2-butyne-1,4 dioate, 3-hexyne-2, 5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, xcex2-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like salts.
When substituted, Rings A-E can have one, two or three substituents in addition to those shown in Structural Formulas (I)-(V). Suitable substituents are those which do not significantly decrease the anabolic and lipolytic properties of the compound. Examples of suitable substituents include halogens, hydroxy, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x80x94S(O)Rxe2x80x2, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94COORxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94CN, xe2x80x94NO2, xe2x80x94OCONRxe2x80x2Rxe2x80x3, xe2x80x94OCONHRxe2x80x2, xe2x80x94NHCOORxe2x80x2, xe2x80x94NRxe2x80x3COORxe2x80x2, xe2x80x94NHRxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94CN, C1-C4 alkyl, C1-C4 haloalkyl, xe2x80x94CONRxe2x80x2Rxe2x80x3, xe2x80x94CONHRxe2x80x2, xe2x80x94CSNRxe2x80x2Rxe2x80x3, xe2x80x94CSNRxe2x80x2Rxe2x80x3, xe2x80x94SO2NRxe2x80x2Rxe2x80x3. Rxe2x80x2 and Rxe2x80x3 are independently xe2x80x94H, C1-C4 alkyl or aryl. In addition, when Rxe2x80x2 and Rxe2x80x3 are bonded to the same nitrogen atom (e.g, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94CONRxe2x80x2Rxe2x80x3 or xe2x80x94OCONRxe2x80x2Rxe2x80x3), then R and Rxe2x80x2, taken together with the nitrogen atom, can form a non-aromatic heterocyclic ring.
Aryl groups include carbocyclic aromatic groups such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl, and heteroaryl groups such as N-imidazolyl, 2-imidazolyl, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.
Aryl groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include 1-benzimidazolinyl, 2-benzimidazolonyl, 1-benzimidthioazolinyl, 2-benzimidthioazolonyl, 1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazolyl, 2-benzooxazolyl, 2-benzimidazolyl, 2-quinolinyl, 3-quinolinyl, 1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl. Also included within the scope of the term aryl group, as it is used herein, is a group in which one or more carbocyclic aromatic rings and/or heteroaromatic rings are fused to a cycloalkyl or non-aromatic heterocyclic ring.
Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings which include one, two or three heteroatoms selected from nitrogen, oxygen and sulfur in the ring that will afford a stable structure. The ring can be five, six, seven or eight-membered. Examples include 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahyrothiophenyl, 3-tetrahyrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl and 4-thiazolidinyl.
As used herein, aliphatic groups include straight chained, branched or cyclic C1-C20 hydrocarbons which are completely saturated or which contain one, two or three units of unsaturation.
Suitable substituents on an aliphatic group, aryl group (carbocyclic and heteroaryl), non-aromatic heterocyclic ring or benzyl group are those which do not significantly reduce the anabolic effects or alter the lipolytic effects of the compound. Examples include xe2x80x94OH, halogen (xe2x80x94Br, xe2x80x94Cl, xe2x80x94I and xe2x80x94F), xe2x80x94OR, xe2x80x94Oxe2x80x94COR, xe2x80x94CN, xe2x80x94NO2, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94NHR, xe2x80x94NR2, xe2x80x94COOR, xe2x80x94COR, xe2x80x94CHO, xe2x80x94CONH2, xe2x80x94CONHR, xe2x80x94CONR2, xe2x80x94SH, xe2x80x94SR and xe2x80x94NHxe2x80x94C(xe2x95x90NH)xe2x80x94NH2. R is C1-C6 alkyl, benzyl, or phenyl.
A substituted non-aromatic heterocyclic ring can also have xe2x95x90O, xe2x95x90S, xe2x95x90NH or xe2x95x90NR where R is as defined above as a substituent. A substituted aliphatic, substituted aromatic, substituted non-aromatic heterocyclic ring or substituted benzyl group can have one, two or three substituents.
In the structural formulas depicted herein, the bond by which a chemical group or moiety is connected to the remainder of the molecule or compound is indicated by the following symbol:

For example, the corresponding symbol in Structural Formula (II) indicates the bond by which the oxygen of the pyridyloxy group is connected to Ring C of Structural Formula (I).
The present invention includes solvates of the compounds of Structural Formula I and the physiologically acceptable salts thereof. A particular compound of the present invention or a physiologically acceptable salt thereof may form solvates with water or common organic solvents. Such solvates are included within the scope of compounds of the present invention.
In addition, it will be appreciated that diastereomers exist for the compounds of Structural Formula I and, depending on the substituents, further diastereomers may exist. The compounds of the present invention include mixtures of two or more diastereomers as well as each individual stereoisomer.
It will also be appreciated that some of the heterocycles may exist in tautomeric forms. All such forms are included within the scope of the present invention.
As used herein, xe2x80x9ccrystallizingxe2x80x9d refers to providing a solvent or solvent mixture in which the indazolyloxy propanolamine compound is highly soluble and in which an ammonium salt(s) thereof is insoluble or only slightly soluble. The compound is dissolved in the solvent or solvent mixture and then converted to the ammonium salt by the addition of at least one equivalent of the appropriate acid, after which the ammonium salt precipitates. To minimize contamination of the precipitated product, between about 1.0 and about 1.1 equivalents of acid are preferably used. Impurities present in the indazolyloxy propanolamine are preferably highly soluble in the solvent or solvent mixture, resulting in a precipitated salt which is purified relative to the free base prior to precipitation. More preferably, the precipitated compound is crystalline.
Purification by acidic (or basic) extraction refers to dissolving a compound with a basic functional group such as an amine (or a compound with an acidic functional group, such as a carboxylic acid) in aqueous or alcoholic acid (or aqueous or alcoholic base, in the case of a compound with an acidic functional group). The aqueous solution can then be washed with organic solvents that are not miscible with water to remove organic impurities. The pH of the solution is then adjusted to the isoelectric point of the compound, thereby precipitating the compound or allowing its extraction into an organic solvent.
Purification by precipitation at the isoelectric point refers to adjusting the pH of an aqueous solution of a compound with both an acidic and basic functional group (e.g., an amino acid) to its isoelectric point, thereby causing the compound to precipitate from solution. xe2x80x9cIsoelectric pHxe2x80x9d is the pH at which the compound is electrically neutral and therefore least soluble in aqueous solution. Preferably, impurities present in the compound are highly soluble at the isoelectric point. As a result, the precipitated compound is purified relative to the compound prior to precipitation. More preferably, the precipitated compound is crystalline. Optionally, a compound with both an acidic and basic functional group can be purified by both by acidic (or basic) extraction and by precipitation at its isoelectric point.
Livestock animals are animals raised for food production. Ruminants or xe2x80x9ccud-chewingxe2x80x9d animals such as cows, bulls, heifers, steers, sheep, buffalo, bison, goats and antelopes are examples of livestock. Other examples of livestock include pigs and avians (poultry) such as chickens, ducks, turkeys and geese. Yet other examples of livestock include fish, shellfish and crustaceans raised in aquaculture. Also included are exotic animals used in food production such as alligators, water buffalo and ratites (e.g., emu, rheas or ostriches). The method of the present invention is preferably used with avians.
An xe2x80x9ceffective amountxe2x80x9d of a compound of the present invention is the quantity which, when administered to a livestock animal, increases the quantity of meat and/or quality of meat obtained from the animal.
Increasing the quantity of meat obtained refers to promoting a greater amount of growth in the animal with a treatment compared with the absence of the treatment. Alternatively, increasing the quantity of meat obtained refers to promoting formation of lean body mass. The formation of lean body mass is promoted, for example, when there is a higher ratio of muscle to fat as a result of a treatment than in the absence of the treatment. Alternatively, increasing the quantity of meat obtained refers to improving the efficiency of utilization of food. Food utilization is more efficient when there is a greater body weight gain per a given amount of feed consumed by an animal as a result of a treatment than in its absence. Thus, increasing the quantity of meat obtained from a livestock animal generally results in an improvement in the economics, e.g., in increase in the profitability of producing meat.
Increasing the quality of meat refers to an improvement in carcass quality of the animal. Improved carcass quality refers, for example, to the formation of less fatty tissue (subcutaneous fat), to a decreased size of the fat pad in poultry and/or to greater leanness (improved yield). Thus, improved carcass quality generally results in meat that is more healthy to consume, e.g., is less likely to cause elevated cholesterol and/or triglyceride levels. Improving the quality of meat can also improve the economics and increase the profitability of producing meat because high quality grade meat can command higher selling prices at market.
The effective amount to be administered will vary somewhat depending upon the particular animal species being treated and the particular active ingredient employed, but generally will be from about 0.5 to about 1000 parts per million (ppm: milligrams compound per kilogram food) of total daily feed intake. Such amount will provide a dosage of about 0.02 to about 50 mg/kg. A preferred embodiment employs about 0.5 to about 200 ppm, and more preferably from about 1 to about 40 ppm. For example, when practicing the method in animals such as poultry, the compound will be added to the daily feed ration at about 2 to 100 parts per million of the daily feed ration.
The method of the present invention is preferably practiced by orally administering an effective amount of a compound of the present invention to a livestock animal. Other routes of administration can be employed, for instance intranasal (e.g., by intranasal misting device), in ovo or subcutaneous, intramuscular or intravenous injection; however, such routes are less practical.
For oral administration, a compound of the present invention is preferably admixed with suitable carriers or diluents commonly employed in animal husbandry. Animal feedstuffs comprising a compound of the present invention are provided as a further embodiment of this invention. Typical carriers and diluents commonly employed in such feedstuffs include corn meal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybean mill run, cottonseed oil meal, bone meal, ground corn, corncob meal, wheat middlings, limestone, dicalcium phosphate, sodium chloride, urea, distillers dried grain, vitamin and/or mineral mixes, cane molasses or other liquid carriers and the like. Such carriers promote a uniform distribution of the active ingredient, and more typically about 20 to about 98 percent by weight.
While the preferred method for orally administering the compounds of the present invention is via the daily feed rations, the compounds can be incorporated into salt blocks and mineral licks, as well as being added directly to link tank formulations or drinking water for convenient oral consumption. The compounds can additionally be formulated with polymorphous materials, waxes and the like for long-term controlled release, and administered to animals as a bolus or tablet only as needed to maintain the desired daily payout of active ingredient. Compounds can also be administered orally by gavage treatment and/or applied transdermally.
For parenteral administration, the compounds of the present invention can be admixed with conventional carriers such as water, propylene glycol, polyethylene glycols, n-methyl pyrrolidone, glycerol formal, corn oil, sesame oil, calcium stearate, polymeric materials and the like. Such formulations can be molded into pellets and administered as an injection or as a slow-release subcutaneous implant, sustained rumen delivery device or intranasal device. Such administrations can be made as often as needed to ensure the proper dosing of active ingredient to obtain the desired rate of growth promotion and improvement in leanness and feed efficiency.
The compounds of the present invention can be prepared by procedures disclosed in WO 97/10825 to Bell et al., WO 98/09625 to Crowell et al., U.S. Pat. Nos. 5,808,080 and 6,046,227. The entire teachings of these references are incorporated herein by reference. A reaction scheme for preparing these compounds is shown below: 
X and Ring D in the Scheme are as described above.
The amination of the epoxides in the Scheme is carried out under conditions known in the art for this type of reaction. For example, the epoxide may be combined with the amine in an alcohol, preferably ethanol at room temperature, to the reflux temperature of the reaction mixture. For example, the reaction is carried out under conditions generally described in Atkins et al., Tetrahedron Letters 27:2451 (1986) the entire teachings of which are incorporated herein by reference. An example of specific conditions for reacting an epoxide with an amine is provided in Example 6.
Substituents which interfere with the reaction shown in the Scheme can be present, provided that they are first converted to a protected form. Suitable protecting groups are known to those skilled in the art and are disclosed in Green and Wuts, xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, 1991, the teachings of which are incorporated herein by reference.
An alternative method of preparing the compounds of the present invention involves deprotecting a cyclic sulfate-containing compound to reveal hydroxy substituents, and comprises combining the cyclic sulfate-containing compound with a trialkylsilyl halide in a solvent for a time sufficient to deprotect the hydroxyl group.
Specifically, the process comprises reacting a compound of the formula (VIII): 
with thionyl halide in a solvent for a time sufficient to yield a sulfite compound of the formula (IX): 
where Ring A and Ring B are as described above.
Suitable thionyl halides include thionyl bromide and thionyl chloride. Thionyl chloride is preferred. Typically, 2 equivalents of thionyl halide are used per mole of compound (VIII).
Any solvent can be used in this step, so long as it does not interfere with the reaction. THF is preferably used. The reaction is typically conducted until product is obtained, generally for about 120 to 240, preferably 180, minutes. The reaction can be conducted at any temperature, but generally is conducted at a temperature less than about 0xc2x0 C., preferably from about xe2x88x9210 to 0xc2x0 C., more preferably from about xe2x88x9210 to xe2x88x925xc2x0 C., most preferably from about xe2x88x929 to xe2x88x928xc2x0 C.
In a preferred embodiment, the NH group in the 1H-indazole ring is first N-protected prior to conversion of the compound to the corresponding sulfate. Selection of the protecting group is preferably made such that it can be removed under the same conditions as for removing the sulfate group to reveal a hydroxy group.
Conditions for N-protecting the nitrogen atoms depend on the particular protecting group chosen. Suitable reaction conditions are described in xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d Peter G. M. Wuts (Editor), Theodora W. Greene, 3rd ed. (April 1999), Vch Pub.
Thereafter, compound (IX) can be converted into corresponding sulfate compound of the formula (X): 
where Ring A and Ring B are as described above.
Suitably, the conversion comprises combining sulfite compound (IX) with a catalytic amount of a ruthenium compound and an oxidizing agent in solvent for a time sufficient to oxidize said sulfite compound (IX) to a sulfate compound (X).
Suitable ruthenium compounds include ruthenium chloride or ruthenium oxide. Generally, about 0.001 to 0.25 equivalents of ruthenium compound are used per mole of sulfite compound (IX).
Suitable oxidizing agents include sodium periodate, sodium hypochlorite, sodium bromate, calcium hypochloride, sodium chlorate or ozone. Generally, about greater than about 2.0, preferably 2.5, equivalents of oxidizing agent are used per mole of sulfite compound (IX).
The conversion is typically conducted in any solvent that would not interfere with the reaction, such as CCl4, CHCl3, CH3CN, or water or mixtures thereof. The conversion is typically conducted for about 30 to 120, preferably 60, minutes. Preferably, the conversion is conducted in a mixture of CHCl3, CH3CN and water. The conversion is typically conducted at a temperature of from about xe2x88x9210 to 25xc2x0 C.
Thereafter, the sulfate compound (X) can be reacted with a primary amine of the formula (XI): 
where X, Ring C and Ring E are as described above, in solvent for a time sufficient to yield a compound (XII): 
Typically, about 0.9 to 2 equivalents of amine are used per mole of sulfate compound (X). Preferably, about 0.9 to 1.1 eq. are used. Most preferably, about 1.0 eq. (a stoichiometric amount) of amine is used.
Typically, any solvent can be used in this step, as long as it does not interfere with the reaction. Preferably, the reaction is conducted in CH3CN. The reaction is typically conducted for about 60-180, preferably 120, minutes at a temperature of from about 78 to 85xc2x0 C.
Finally, the compound (XII) can be combined with trialkylsilyl halide in solvent for a time sufficient to yield compound (IV): 
where R1, R2, X, Ring A, Ring B, Ring C, and Ring E are as described above.
Suitable trialkylsilyl halides include trialkylsilyl iodide, trialkylsilyl bromide or trialkylsilyl chloride. Preferably, the trialkylsilyl halide is trimethylsilyl iodide.
The reaction may be carried out in any solvent, so long as the solvent does not interfere with the reaction. Preferably, the solvent is an aprotic solvent such as carbon tetrachloride, acetonitrile, dimethyl sulfoxide, dimethylformamide, sulfolane, tetrahydrofuran, diethyl ether, methyl-t-butyl ether, 1,2-dimethoxy-ethane, dioxane, chloroform, methylene chloride, toluene or acetone or mixtures thereof.
The reaction is typically conducted for 5-25, preferably 15, minutes at a temperature of less than 0xc2x0 C., preferably xe2x88x9210 to 0xc2x0 C., most preferably xe2x88x929 to xe2x88x928xc2x0 C.
A method of preparing the compounds of the present invention involves formation of salts of novel indazolyloxy propanolamines in which the amine group is substituted with an alkyl 2-[4-(2-yl-2-methylpropyl)phenoxy]pyridine-carboxylate group (hereinafter xe2x80x9cindazolyloxy propanolamine estersxe2x80x9d). These indazolyloxy propanolamine esters can be readily crystallized in high yield and in high purity, substantially free (typically less than 1 ppm) of epoxide precursors. In addition, the corresponding carboxylic acids of indazolyloxy propanolamine esters (hereinafter xe2x80x9cindazolyloxy propanolamine carboxylic acidsxe2x80x9d) can be purified by precipitation at their isoelectric point in high yield and high purity. Both indazolyloxy propanolamine esters and carboxylic acids can be prepared in high yield from readily accessible starting materials.
This method can be used to prepare an indazolyloxy propanolamine ester represented by Structural Formula (XIII): 
and ammonium salts thereof.
R8 is a C1 to C6 straight or branched chain alkyl group or a C7 to C9 substituted or unsubstituted aralkyl group. Preferably, R8 is methyl or ethyl.
The method comprises the step of reacting an epoxide starting material with an amine starting material. The epoxide starting material is represented by Structural Formula (XIV): 
and the amine starting material is represented by Structural Formula (XV):. 
This method of can also be used to prepare an indazolyloxy propanolamine carboxylic acid or a carboxylate salt thereof from an indazolyloxy propanolamine ester represented by Structural Formula (XIII). The indazolyloxy propanolamine carboxylic acid is represented by Structural Formula (XVI): 
The method comprises the step of hydrolyzing the xe2x80x94COOR8 group of the indazolyloxy propanolamine ester.
Because of the amine moiety present in the indazolyloxy propanolamines esters disclosed herein, ammonium salts of these compounds can be prepared by reacting with a suitable acid. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic, acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, mono-hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 2-butyne-1,4 dioate, 3-hexyne-2, 5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, xcex2-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate salts.
Carboxylate salts can be formed from the indazolyloxy propanolamines acids disclosed herein which have a carboxylic acid functional group by reacting with a suitable base. Such salts include those derived from inorganic bases such as ammonium and alkali and alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, as well as salts derived from basic organic amines such as aliphatic and aromatic amines, aliphatic diamines, hydroxy alkylamines, and the like. Such bases useful in preparing the salts of this invention thus include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, sodium hydroxide, lithium hydroxide, potassium hydroxide, methylamine, diethylamine, ethylenediamine, cyclohexylamine, ethanolamine and the like.
Preferred carboxylate salts of the compound represented by Structural Formula (XVI) include alkali metal carboxylate salts such as the sodium carboxylate salt, the potassium carboxylate salt and the lithium carboxylate salt.
The preparation of the indazolyloxy propanolamine ester represented by Structural Formula (XIII) from the epoxide and amine represented by Structural Formulas (XIV) and (XV), respectively, is generally carried out in a solvent at room temperature to the reflux temperature of the reaction mixture. Temperatures of 40-140xc2x0 C. are generally preferred. The solvents that may be used in the reaction include: alcoholic solvents, such as methanol, ethanol or isopropanol, (with the preferred solvent corresponding to the particular ester being used in the reaction to prevent transesterification); aromatic solvents, such as benzene, toluene, xylenes, chlorobenzene, dichlorobenzene, other haloaromatics, nitrobenzene, benzonitrile, or trifluoromethylbenzene; or dipolar aprotic solvents, such as dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMAC) or N,N-dimethylformamide; or other solvents where the reagents are soluble and the temperature can be elevated to the range previously described. Equimolar amounts of epoxide and amine can be used. Alternatively, up to about a five fold excess of one reagent is used. Preferably, however, between about 1.1 to about 2.0 equivalents of amine relative to epoxide is preferred.
Indazolyloxy propanolamine esters, prepared as described above, can be purified by any suitable means, including by converting to a suitable ammonium salt and crystallizing, as described above.
Amine starting materials can be prepared as generally described in Examples 3 and 4. A phenoxide salt of the alkylamino phenol represented by Structural Formula (XVII) is reacted with a 2-halo pyridine ester represented by Structural Formula (XVIII): 
R8 in Structural Formula (XVIII) is as described for Structural Formula (XIII). X is a halo group. The coupling reaction and the preparation of the compound represented by Structural Formula (XVII) can be carried out according to procedures described in the aforementioned WO 97/10825 to Bell et al., WO 98/09625 to Crowell et al., U.S. Pat. Nos. 5,808,080 and 6,046,227.
Generally, the reaction may be carried out by mixing the alkylamino phenol with a base in the presence of the 2-halopyridine ester. Equimolar amounts of the starting materials are preferably used. However, molar excesses up to about five or ten fold of one starting material relative to the other can be used. The coupling is performed by mixing the amino phenol with a base in a suitable solvent or solvent system. The reaction can be carried out at temperatures as low as room temperature, but is preferably carried out by heating the mixture at reflux while azeotropically removing water formed during the deprotonation step. The 2-halopyridine ester is then added and the reaction continued until the reaction is complete.
Suitable solvents include dipolar aprotic, ethereal, and aromatic solvents, as well as combinations thereof. Dipolar aprotic solvents include solvents such as DMSO, N,N-dimethylacetamide, N-methylpyrrolidinone, 1,3-dimethyl-2-imidazolindinone (DMI), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) with a lower boiling solvent to azeotropically remove water such as benzene, toluene, isooctane, xylenes or other solvents capable of forming binary azeotropes with water but which are inert under the reaction conditions. Other suitable compounds for azeotropic removal of water may be found in Advances In Chemistry Series 116xe2x80x94Azeotropic Data III, American Chemical Society: Washington D.C., 1973. Ethereal solvents include tetrahydrofuran, dioxane and 1,2-dimethoxyethane. Aromatic solvents include benzene, toluene, chlorobenzene, anisole and 1,2-dichlorobenzene. Preferred is the use of an aromatic solvent such as chlorobenzene containing 0.1-10 equivalents of a dipolar aprotic solvent such as N,N-dimethylacetamide. Suitable bases include alkali metal alkoxides, such as alkali metal methoxides, ethoxides and tert-butoxides (preferably corresponding to the alkyl group of the ester to prevent transesterification), alkali metal hydroxides, such as lithium hydroxide, sodium hydroxide and potassium hydroxide, or alkali metal carbonates, such as Na2CO3 or K2CO3. Although excesses of base up to at least about ten fold can be used, preferred is the use of stoichiometric quantities of hydroxide or alkoxide bases or 1.5-5 equivalents of K2CO3.
The preparation of the epoxide starting material represented by Structural Formula (XIV) can be carried out by reacting 4-hydroxyindazole with (2S)-(+)-glycidyl 3-nitrobenzenesulfonate in an inert solvent (e.g., acetone, methyl ethyl ketone, methyl isobutylketone, dimethyl sulfoxide, N,N-dimethylacetamide (DMAC) or DMF) and in the presence of a base. Although about equimolar amounts of the starting materials are preferred, molar excess up to about five to about ten-fold of one starting material relative to the other can also be used. Suitable bases include non-nucleophilic bases such as potassium carbonate, sodium carbonate and alkali metal alkoxides; potassium carbonate is preferred. Although an excess of base can be used, between about 1.05 and about 1.50 equivalents of K2CO3 are preferred. The reaction is carried out at temperatures ranging from about ambient temperature to about 40xc2x0 C., preferably about 30xc2x0 C. Other sulfonate esters can also be used (e.g., tosylate, nosylate or mesylate) as well as halides such as epibromohydrin or epichlorohydrin. The nosylate is preferred. Specific reaction conditions are described in Examples 1 and 2.
The hydrolysis of the indazolyloxy propanolamine ester represented by Structural Formula (XIII) to form the carboxylic acid represented by Structural Formula (XVI) or the carboxylate salt thereof can be carried out by any suitable means, including by procedures disclosed in Larock, R. C. Comprehensive Organic Transformations; VCH: New York, 1989, pp. 981-985; March, xe2x80x9cAdvanced Organic Chemistryxe2x80x9d, third(3rd) edition, John Wiley Sons (1985), pages 375-76 and references cited therein, the entire relevant teachings of which are incorporated herein by reference. This reaction is referred to herein as the xe2x80x9chydrolysis reactionxe2x80x9d.
Preferably, the hydrolysis reaction is carried out in alcoholic base such as lithium hydroxide, sodium hydroxide or potassium hydroxide in methanol or ethanol. Methanolic sodium hydroxide is preferred. The hydrolysis can be carried out with one equivalent of base relative to indazolyloxy propanolamine ester. Alternatively, an excess of base, for example, an excess up to about ten to about twenty-fold can be used to form the carboxylate salt. Preferably, the excess of base is between about zero and about 30 percent. Generally, the reaction temperature varies between room temperature and the reflux temperature of the solvent, and is typically 40-70xc2x0 C. The carboxylic acid product (or carboxylate salt thereof) is isolated by conventional means, for example, by removal of the solvent in vacuo.
The indazolyloxy propanolamine carboxylic acid or carboxylate salt thereof can be purified by basic (or acidic) extraction, e.g., by dissolving in aqueous base (e.g., aqueous sodium hydroxide, potassium hydroxide or lithium hydroxide), and/or by precipitation at its isoelectric point. Preferably, the aqueous solution is extracted with one or more organic solvents which are not miscible with water to remove impurities. The pH of the aqueous solution can then adjusted to its isoelectric point with aqueous acid (e.g., aqueous HCl, H2SO4, acetic acid or a sulfonic acid), thereby causing the indazolyloxy propanolamine carboxylic acid to precipitate.
The indazolyloxy propanolamine carboxylic acids or carboxylate salts thereof can also be purified by using either cationic ion-exchange resins, such as AMB 15 (H form), 50WX2-400 (H form), or IR50S (H form) and eluting with an alcoholic solution of an alkali metal acetate, such as 6% (w/v) sodium acetate in methanol; or by using anionic ion-exchange resins, such as IRA900 (chloride form) or A21 (acetate form) and eluting with a basic solution of water and either alcohol or acetonitrile.
The individual optically active isomers of the compounds prepared by the present invention may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley Sons, 1981.
The compounds employed as initial starting materials in the synthesis of the compounds of this invention are well known and, to the extent not commercially available, are readily synthesized by standard procedures commonly employed by those of ordinary skill in the art.
The invention is illustrated by the following examples, which are not meant to be limiting in any way.