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1. WO2009073147 - PROCÉDÉ DE PRÉPARATION DE BIS(THIOHYDRAZIDE AMIDES)

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PROCESS FOR PREPARING BIS(THIOHYDRAZIDE AMIDES)

RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
61/004,476, filed on November 28, 2007, and U.S. Provisional Application No. 61/004,740, filed on November 29, 2007. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION
It has been reported in U.S. Patent No. 6,800,660, U.S. Patent No. 6,762,204, U.S. Patent No. 7,037,940, U.S. Patent No. 7,001,923, and U.S. Patent No.
6,924,312 that certain bis(thio-hydrazide amide) compounds significantly enhance the anti-cancer activity of taxol and taxol analogs. In addition, methods of preparing bis(thio-hydrazide amide) compounds have been reported in U.S. Patent No.
6,825,235. The entire teachings of these patents are incorporated by reference herein. Moreover, the bis(thio-hydrazide amide), N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide), in combination with Taxol has been shown to increase the time to progress of patients suffering from stage IV metastatic melanoma in relation to patients treated with Taxol alone. However, the synthesis of bis(thio-hydrazide amide) compounds has been complicated by the formation of regioisomers, and the effective administration of N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) has been challenging due to low solubility of the compound. Therefore, a need exists for a method of preparing bis(thio-hydrazide amide) compounds that minimizes the formation of unwanted regioisomers and results in a bis(thio-hydrazide amide) compound in a form that has improved dissolution rate.

SUMMARY OF THE INVENTION
In one embodiment, the invention relates to a method of regioselectively preparing a hydrazide represented by formula (I):

(D
wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group; and
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; comprising the steps of:
a) dissolving a thioglycolic acid represented by formula (II):



(II)
in an aqueous solution, wherein the aqueous solution comprises at least about 1 equivalent of Mn+(0H")n in relation to the thioglycolic acid; and wherein n is 1 or 2; and when n is 1, Mn+ is selected from Li+, Na+, K+, Rb+ or Cs+; and when n is 2, Mπ+ is selected from Be2+, Mg2+, Sr2+ or Ba2+.
b) dissolving in water at least about 1 equivalent of a hydrazine in relation to the thioglycolic acid used in step a), wherein the hydrazine is represented by formula (III):

HN NH2
R3
(III) ; and
c) adding the thioglycolic acid solution to the hydrazine solution, thereby regioselectively preparing a hydrazide represented by formula (I).

In another embodiment, the invention relates to a method of preparing a bis(thio-hydrazide amide) represented by formula (IV):



(IV)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the steps of:
a) dissolving a hydrazide represented by formula (I):



(I)
in a solution comprising an organic solvent and optionally at least about 1 equivalent in relation to the hydrazide of a non-nucleophilic base;
b) a bis(acid chloride) represented by formula (V):



(V)
c) dissolving between about 0.4 and 0.65 equivalents of the bis(acid chloride) in an organic solvent; and - A - d) adding the bis(acid chloride) solution to the hydrazide solution, thereby preparing a bis(thio-hydrazide amide) represented by formula (IV).

In another embodiment, the invention relates to a method of preparing a bis(thio-hydrazide amide) represented by formula (IV):



(IV)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the step of combining l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a hydrazide represented by formula (I):



(0
and a bis(carboxylic acid) represented by formula (VI):



(VI)
in an organic solvent selected from the group consisting of DMF and methylene chloride, thereby forming a compound represented by formula (IV).

In another embodiment, the invention is related to a method of purifying a compound represented by Structural Formula (VII):



(VII)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
Y' is a covalent bond or an optionally substituted straight chained hydrocarbyl group, or, Y', taken together with both >C=Z groups to which it is bonded, is an optionally substituted aromatic group;
Ri', R2', R3', and R4' are independently -H, an optionally substituted aliphatic group, an optionally substituted aryl group, or Rj' and R3' taken together with the carbon and nitrogen atoms to which they are bonded, and/or R2' and R4' taken together with the carbon and nitrogen atoms to which they are bonded, form a non-aromatic ring optionally fused to an aromatic ring;
R7' and R8' are independently -H, an optionally substituted aliphatic group, or an optionally substituted aryl group; and
Z is O or S;
comprising dissolving the compound in a solvent consisting of THF-acetone or THF-CH3CN, and precipitating the compound with water.

In another embodiment, the invention relates to a method of preparing a crystalline bis(thio-hydrazide amide) represented by formula (IV):



(IV) or a tautomer thereof, wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the steps of:
a) dissolving the bis(thio-hydrazide amide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of bis(thio-hydrazide amide) to water; and
c) collecting the precipitated bis(thio-hydrazide amide).

In another embodiment, the present invention relates to a crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) represented by the following structural formula:



or a tautomer thereof, wherein the crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) is prepared by a method comprising the steps of:
a) dissolving the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) to water; and
c) collecting the precipitated N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide).

In another embodiment, the invention relates to a pharmaceutical
composition comprising the crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) and a pharmaceutically acceptable diluent or a pharmaceutically acceptable carrier, wherein the crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) is prepared by a method comprising the steps of: a) dissolving the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) to water; and
c) collecting the precipitated N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) .

In another embodiment, the invention relates to N-malonyl-bis(N'-methyl- N'-thiobenzoylhydrazide) represented by the following structural formula:



or a tautomer thereof, wherein at least about 90 % of the N-malonyl-bis(N' -methyl -N'-thiobenzoylhydrazide) has a particle size of less than about 150 μm.

In another embodiment, the invention relates to a method of preparing an N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm, comprising the steps of:
a) milling N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) through a 500 μm screen, thereby forming N-malonyl-bis(N'-methyl-N'- thiobenzoylhydrazide) having a particle size of less than about 500 μm; and b) milling the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of 500 μm through a 150 μm screen, thereby forming N- malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm.

In another embodiment, the invention relates to a pharmaceutical composition comprising an N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm and a pharmaceutically acceptable diluent or a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of preparing bis(thio-hydrazide amide) and methods of purifying and improving the dissolution rate of bis(thio-hydrazide amide). In one embodiment, the invention relates to a method of regioselectively preparing a hydrazide represented by formula (I):



(I)
wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group; and
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; comprising the steps of:
a) dissolving a thioglycolic acid represented by formula (II):



(H)
in an aqueous solution, wherein the aqueous solution comprises at least about I equivalent of Mn+(0H')n in relation to the thioglycolic acid; and wherein n is 1 or 2; and when n is 1, Mn+ is selected from Li+, Na+, K+, Rb+ or Cs+; and when n is 2, Mn+ is selected from Be2+, Mg2+, Sr2+ or Ba2+.
b) dissolving in water at least about 1 equivalent of a hydrazine in relation to the thioglycolic acid used in step a), wherein the hydrazine is represented by formula (III):

HN NH2

(III) ; and
c) adding the thioglycolic acid solution to the hydrazine solution, thereby regioselectively preparing a hydrazide represented by formula (I).

In one embodiment of the regioselective preparation, the thioglycolic acid solution comprises between about 0.9 and about 1.1 equivalents of Mn+(0H')n in relation the thioglycolic acid. Typically, about 1.01 equivalents of Mn+(0H')n in relation to the thioglycolic acid is present in the thioglycolic acid solution. In one embodiment, Mn+(0H")n is NaOH.
In another embodiment, the thioglycolic acid solution prepared in step a) of the regioselective method comprises between about 0.9 L and about 1.15 L of water per mole of the thioglycolic acid.
In another embodiment, the hydrazine solution comprise between about 1 equivalent and about 1.75 equivalents of a hydrazine in relation to the thioglycolic acid. Typically, the hydrazine solution comprises about 1.3 equivalents of the hydrazine in relation to the thioglycolic acid. In another embodiment, the hydrazine solution prepared in step b) of the regioselective method comprises between about 0.9 L and about 1.15 L of water per mole of the thioglycolic acid. Typically, the hydrazine solution is maintained at between about -10 0C and about 15 0C during addition of the thioglycolic acid solution.
In another embodiment, R1 is an optionally substituted phenyl. In another embodiment, Ri is an unsubstituted phenyl. In another embodiment, R3 is a lower alkyl group, such as methyl or ethyl.
In another embodiment, the regioselective method further comprising the step of extracting the hydrazide represented by formula (I) into a water immiscible organic solvent (e.g., ethyl acetate, isopropyl acetate, butyl acetate, methylene chloride, chloroform, diethyl ether, petroleum ether, toluene, or combinations thereof) and separating the organic layer from the water layer. Typically, between about 0.75 L and about 1.0 L of organic solvent is used for the extraction per mole of thioglycolic acid. In one embodiment, the organic solvent used for extracting the hydrazide is methylene chloride. In another embodiment, the organic layer is washed with an aqueous basic solution, such as a LiOH5 NaOH or KOH solution, one or more times. Typically, the aqueous basic solution is a NaOH solution having a normality of between about 0.5 N and about 0.05 N. More typically, the aqueous basic solution is a NaOH solution that has a normality of about 0.1 N. Between about 0.75 L and about 1.0 L of basic solution is used for the wash per mole of thiolglycolic acid. In another embodiment, the organic layer is washed with water one or more times following the last basic wash, wherein the water from the last water wash has a pH of about 8 or less than 8. Typically, between about 0.75 L and about 1.0 L of water per mole of thioglycolic acid is used in each wash.
In another embodiment, the regioselective method, further comprising the steps of:
concentrating the organic layer to between about 0.2 L and about 0.5 L per mole of thioglycolic acid;
adding between about 0.75 L and about 1.25 L of hexane(s) or heptane(s) to the concentrated organic layer to form a slurry;
stirring the slurry for about 1 hour to about 24 hours; and
collecting the solid hydrazide.
Typically, the yield of the hydrazide represented by formula (I) is about 80%, about 85%, about 90%, about 95%, about 98%, or greater, and the purity is about 95%, about 98 %, about 99%, or greater.

In another embodiment, the invention relates to a method of preparing a bis(thio-hydrazide amide) represented by formula (IV):



(IV)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:

Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the steps of:
a) dissolving a hydrazide represented by formula (I):



(I)
in a solution comprising an organic solvent and optionally at least about 1 equivalent in relation to the hydrazide of a non-nucleophilic base;
b) a bis(acid chloride) represented by formula (V):



(V)
c) dissolving between about 0.4 and 0.65 equivalents of the bis(acid chloride) in an organic solvent; and
d) adding the bis(acid chloride) solution to the hydrazide solution, thereby preparing a bis(thio-hydrazide amide) represented by formula (IV).

In one embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), the hydrazide solution is cooled to between about -10 0C and about 15 0C before addition of the bis(acid chloride) and the temperature is maintained in this range during addition of the bis(acid chloride) and formation of the bis(thio-hydrazide amide).
In another embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), the non-nucleophilic base is triethylamine.

Typically, between about 1 equivalent and about 1.1 equivalents of the non-nucleophilic base is used in relation to the hydrazide.
In another embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), between about 0.5 equivalents and about 0.6 equivalents of the bis(acid chloride) is used in relation to the hydrazide. In one embodiment, the bis(acid chloride) is malonyl dichloride. In one embodiment, the bis(acid chloride) may be freshly distilled.
In another embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), the method further comprising the step of adding water to the reaction mixture prepared in step d) to quench the reaction. Typically, the water is added to the reaction mixture after the reaction mixture has been allowed to react for between about 5 minutes and about 2 hours. In one embodiment, between about 0.75 L and about 1.25 L of water per gram of hydrazide is used to quench the reaction.
In another embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), the organic solvent used in steps a) and c) is a water immiscible organic solvent, such as ethyl acetate, methylene chloride, chloroform, diethyl ether, petroleum ether, toluene, or combinations thereof. In one embodiment, the organic solvent used in steps a) and c) is ethyl acetate. In this embodiment, after the reaction is complete, the reaction mixture is allowed to separate into an aqueous layer and an organic layer. In one embodiment, the organic layer is separated from the water layer; and the volume of the organic layer is reduced until the bis(thio-hydrazide amide) precipitates out. Typically, the volume of the organic layer is reduced by about one-third.
In another embodiment of the method of preparing the bis(thio-hydrazide amide) represented by formula (IV), Ri is an unsubstituted phenyl. In another embodiment, Ri is a substituted phenyl. In another embodiment, R3 is an alkyl. In another embodiment, R3 is methyl.
In another embodiment, the invention relates to a method of preparing a bis(thio-hydrazide amide) represented by formula (IV):

(IV)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the step of combining l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a hydrazide represented by formula (I):



(I)
and a bis(carboxylic acid) represented by formula (VI):



(VI)
in an organic solvent selected from the group consisting of DMF and methylene chloride, thereby forming a compound represented by formula (IV).

In one embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), between about 0.5 and about 0.6 equivalents of bis(carboxylic acid) in relation to the hydrazide is used. In one embodiment, the bis(carboxylic aid) is malonic acid.

In another embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), between about 1 and about 1.5 equivalents of EDC in relation to the hydrazide is used. In one embodiment, the EDC is added portionwise such that the reaction temperature is maintained at less than about 3O0C. In another embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), the organic solvent is DMF. Typically, between about 4.5 L and about 5.5 L of DMF per kilogram of hydrazide is used.
In another embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), the reaction is diluted by addition of an alcohol after the reaction is substantially complete. The reaction is substantially complete when about 10 %, 5%, 2%, 1%, or less of the hydrazide represented by formula (I) remains in the reaction mixture. The amount of hydrazide left in the reaction mixture can be deterimined by any method known in the art such as thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC). Typically, the reaction is complete in between about 0.5 hours and about 4 hours. In one embodiment, the alcohol used to dilute the reaction mixture is ethanol. Typically, between about 3.5 L and about 4.5 L of ethanol per kilogram of hydrazide is used to dilute the reaction mixture.
In another embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), between about 18 L and about 22 L of water is added to the mixture to precipitate out the compound represented by formula (IV). Typically, the water is added after the addition of an alcohol (e.g., ethanol) to the reaction mixture.
In another embodiment of the method of preparing a bis(thio-hydrazide amide) represented by formula (IV), Ri is an unsubstituted phenyl. In another embodiment, Ri is a substituted phenyl. In another embodiment, R3 is an alkyl, such as methyl or ethyl.
In another embodiment, the invention relates to a method of preparing a crystalline bis(thio-hydrazide amide) represented by represented by formula (IV):

(IV)
or a tautomer thereof, wherein:
Ri is H, an optionally substituted aliphatic group, or an optionally substituted aryl group;
R3 is an optionally substituted aliphatic group or an optionally substituted aryl group; and
Y is a covalent bond or a methylene which is optionally substituted with one or two independently selected methyl or ethyl group, comprising the steps of:
a) dissolving the bis(thio-hydrazide amide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of bis(thio-hydrazide amide) to water; and
c) collecting the precipitated bis(thio-hydrazide amide).

In one embodiment of the preceding method, between about 25 kg and about

30 kg of acetone is used for each kilogram of bis(thio-hydrazide amide). In another embodiment, the acetone is maintained at between about 15 0C and about 25 0C during dissolution of the bis(thio-hydrazide amide). In another embodiment, between about 60 kg and about 70 kg of water is used per kilogram of bis(thio-hydrazide amide). In another embodiment, the mixture of bis(thio-hydrazide amide) in acetone and water is cooled to between about -5 0C and 15 0C before collecting the precipitated bis(thio-hydrazide amide). In another embodiment, the method further comprises the step of washing the precipitated bis(thio-hydrazide amide) with a mixture of acetone and water. In another embodiment, the bis(thio-hydrazide amide) is N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) represented by the following structural formula:
or a tautomer thereof.

In another embodiment, the present invention relates to a crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) represented by the following structural formula:



or a tautomer thereof, wherein the crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) is prepared by a method comprising the steps of:
a) dissolving the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) to water; and
c) collecting the precipitated N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide).
Alternatively, a bis(thio-hydrazide amide) represented by formula (IV) can be crystallized from aqueous ethanol or aqueous THF solution.

In another embodiment, the invention relates to a pharmaceutical composition comprising the crystalline N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) and a pharmaceutically acceptable diluent or a
pharmaceutically acceptable carrier, wherein the crystalline N-malonyl-bis(N'-methyl-N' -thiobenzoylhydrazide) is prepared by a method comprising the steps of: a) dissolving the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) in acetone, wherein the acetone optionally comprises up to about 15 % water;
b) adding the solution of N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) to water; and
c) collecting the precipitated N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide).
Alternatively, the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) can be crystallized from aqueous ethanol or aqueous THF solution.

In another embodiment, the invention relates to N-malonyl-bis(N '-methyl - N'-thiobenzoylhydrazide) represented by the following structural formula:



or a tautomer thereof, wherein at least about 90 % of the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) has a particle size of less than about 150 μm. In one embodiment, the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) is crystalline. In another embodiment, the invention relates to a method of preparing an N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm, comprising the steps of:
a) milling N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) through a 500 μm screen, thereby forming N-malonyl-bis(N'-methyl-N'- thiobenzoylhydrazide) having a particle size of less than about 500 μm; and b) milling the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of 500 μm through a 150 μm screen, thereby forming N- malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm.
Alternatively, the N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a desired particle size can be prepared, for example, by micronization, jet milling, fitz milling or cone milling. The desired particle size, for example, can be less than 500 μm , less than 220 μm , less than 150 μm , less than 100 μm, less than 50 μm or less than 5 μm .
In another embodiment, the invention relates to a pharmaceutical composition comprising an N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) having a particle size of less than about 150 μm and a pharmaceutically acceptable diluent or a pharmaceutically acceptable carrier.
In another embodiment, the present invention relates to methods of purifying bis(thio-hydrazide amides) represented by Structural Formula I and
pharmaceutically acceptable salts and solvates of the compounds represented by Structural Formula (IV) or (VII).
In another embodiment, the invention is related to a method of purifying a compound represented by Structural Formula (VII):



(VII)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein:
Y' is a covalent bond or an optionally substituted straight chained hydrocarbyl group, or, Y', taken together with both >C=Z groups to which it is bonded, is an optionally substituted aromatic group;
Ri', R2', R3', and R4' are independently -H, an optionally substituted aliphatic group, an optionally substituted aryl group, or Ri' and R3' taken together with the carbon and nitrogen atoms to which they are bonded, and/or R2' and R4' taken together with the carbon and nitrogen atoms to which they are bonded, form a non-aromatic ring optionally fused to an aromatic ring;
R7' and R8' are independently -H, an optionally substituted aliphatic group, or an optionally substituted aryl group; and
Z is O or S;

comprising dissolving the compound in a solvent consisting of THF-acetone or THF-CH3CN, and precipitating the compound with water.

In one embodiment, the compound is dissolved in a solvent consisting of THF-acetone or THF-CH3CN, and precipitated with water.
Another embodiment, further comprises dissolving the precipitate in a second solvent consisting of THF-acetone or THF-CH3CN, and precipitating the compound with water.
Another embodiment, further comprises washing the precipitate with THF-water.
In another embodiment, the THF -water is in a ratio of 1:3 (v/v).
In another embodiment, the compound, solvent, and water are stirred for 3 to 4 hours.
In another embodiment, the compound, second solvent, and water are stirred for 3 to 4 hours.
In another embodiment, the temperature is 0-4 °C.
In one embodiment, the solvent is THF-acetone.
In another embodiment, the solvent is THF-CH3CN.
In one embodiment, the second solvent is THF-acetone.
In one embodiment, the second solvent is THF-CH3CN.
In one embodiment, the solvent is THF-acetone and the second solvent is THF-CH3CN.
In one embodiment, the solvent is THF-CH3CN and the second solvent is THF-acetone.
In one embodiment, Y in Structural Formula (VII) is a covalent bond,

-C(R55R6')-, -(CH2CH2)-, trans-(CH=CH)-, cis-(CH=CH)- or -(C≡C)- group, preferably -C(R55R6')-. Ri '-R4' are as described above for Structural Formula (VII). R55 and R6' are each independently -H, an aliphatic or substituted aliphatic group, or R55 is -H and R65 is an optionally substituted aryl group, or, R55 and R65, taken together, are an optionally substituted C2-C6 alkylene group.

In specific embodiments, Y taken together with both >C=Z groups to which it is bonded, is an optionally substituted aromatic group. In this instance, certain bis(thio-hydrazide amides) are represented by Structural Formula (VIII):



(VIII)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein Ring A is substituted or unsubstituted and V is -CH- or -N-. The other variables in Structural Formula (VIII) are as described herein for Structural Formula (VII).
In particular embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (IX):



(IX)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein
Ri '-R8' are as described above for Structural Formula (VII).
In Structural Formulas (VII), (VIII) and (IX), Ri' and R2' are the same or different and/or R3' and R4- are the same or different; preferably, Ri' and R2' are the same and R3' and R4' are the same. In Structural Formulas (VII) and (IX), Z is preferably O. Typically in Structural Formulas (VII) and (IX), Z is O; Ri' and R2' are the same; and R3' and R4' are the same. More preferably, Z is O; Ri' and R2' are the same; R3' and R4' are the same, and R7' and R8' are the same.
In other embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (IX): Ri' and R2' are each an optionally substituted aryl group, preferably an optionally substituted phenyl group; R3' and R4' are each an optionally substituted aliphatic group, preferably an alkyl group optionally substituted with -OH, halogen, phenyl, benzyl, pyridyl, or C1-C8 alkoxy and R6' is -H or methyl, more preferably, methyl or ethyl group optionally substituted with -OH, halogen, phenyl, benzyl, pyridyl, or C1-C8 alkoxy and R6' is -H or methyl optionally substituted with -OH, halogen or C1-C4 alkoxy; and R5' and R6' are as described above, but R5' is preferably -H and R6' is preferably -H, an aliphatic or substituted aliphatic group.
Alternatively, Ri' and R2' are each an optionally substituted aryl group; R3' and R4' are each an optionally substituted aliphatic group; R5' is -H; and R6' is -H, an aliphatic or substituted aliphatic group. Preferably, Ri' and R2' are each an optionally substituted aryl group; R3' and R4' are each an alkyl group optionally substituted with -OH, halogen, phenyl, benzyl, pyridyl, or C1-C8 alkoxy and R6' is -H or methyl; and R5' is -H and R6' is -H or methyl. Even more preferably, Ri' and R2' are each an optionally substituted phenyl group, preferably optionally substituted with -OH, halogen, C 1-4 alkyl or C1-C4 alkoxy; R3' and R4' are each methyl or ethyl optionally substituted with -OH, halogen or C1-C4 alkoxy; and R5' is -H and R6' is -H or methyl. Suitable substituents for an aryl group represented by Ri' and R2' and an aliphatic group represented by R3', R4' and R6' are as described below for aryl and aliphatic groups.
In another embodiment, the bis(thio-hydrazide amides) are represented by

Structural Formula (IX): Ri' and R2' are each an optionally substituted aliphatic group, preferably a C3-C8 cycloalkyl group optionally substituted with at least one alkyl group, more preferably cyclopropyl or 1-methylcyclopropyl; R3' and R4' are as described above for Structural Formula (VII), preferably both an optionally substituted alkyl group; and R5' and R6' are as described above, but R5' is preferably -H and R6' is preferably -H, an aliphatic or substituted aliphatic group, more preferably — H or methyl.
Alternatively, the bis(thio-hydrazide amides) are represented by Structural Formula (IX): Ri' and R2' are each an optionally substituted aliphatic group; R3' and R4' are as described above for Structural Formula (VII), preferably both an optionally substituted alkyl group; and R5' is -H and R6' is -H or an optionally substituted aliphatic group. Preferably, Ri' and R2' are both a C3-C8 cycloalkyl group optionally substituted with at least one alkyl group; R3' and R4' are both as described above for Structural Formula (VII), preferably an alkyl group; and R5' is -H and R6' is -H or an aliphatic or substituted aliphatic group. More preferably, Rf and R2' are both a C3-C8 cycloalkyl group optionally substituted with at least one alkyl group; R3' and R4' are both an alkyl group group optionally substituted with -OH, halogen, phenyl, benzyl, pyridyl, or C1-C8 alkoxy and R6' is -H or methyl; and R5' is -H and R6' is -H or methyl. Even more preferably, Ri' and R2' are both cyclopropyl or 1-methylcyclo-propyl; R3' and R4' are both an alkyl group, preferably methyl or ethyl optionally substituted with -OH, halogen or C1-C4 alkoxy; and R5' is -H and R6' is -H or methyl.
In particular embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (X):



(X)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein
Ri', R2', R3', R4', R7', Rg', and Z are as defined above for Structural Formula (IX).
In specific embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (IX):



(XI)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein: Ri' and R2' are both phenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Ri' and R2' are both phenyl, R3' and R4' are both ethyl, and R5' and R6' are both -H; Rf and R2' are both 4-cyanophenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Ri' and R2' are both 4-methoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both phenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Rf and R2' are both phenyl, R3' and R4' are both ethyl, R5' is methyl, and R6' is -H; Rf and R2' are both
4-cyanophenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,5-dimethoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,5-dimethoxyphenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Rf and R2' are both 3-cyanophenyl, R3' and R4- are both methyl, and R5- and R6- are both -H; Rf and R2' are both 3 -fluorophenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both
4-chlorophenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Rf and R2' are both 2-dimethoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 3-methoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,3-dimethoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both
2,3-dimethoxyphenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; R]' and R2' are both 2,5-difluorophenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,5-difluorophenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; R1 ' and R2' are both 2,5-dichlorophenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both
2,5-dimethylphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,5-dimethoxyphenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both phenyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 2,5-dimethoxyphenyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Rf and R2' are both cyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both cyclopropyl, R3' and R4' are both ethyl, and R5' and R6' are both -H; Rf and R2' are both
cyclopropyl, R3' and R4' are both methyl, R5' is methyl, and R6' is -H; Rf and R2' are both 1-methylcyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both 1-methylcyclopropyl, R3' and R4' are both methyl, R5' is methyl and R6' is -H; Rf and R2' are both 1-methylcyclopropyl, R3' and R4' are both methyl, R5' is ethyl, and R6' is -H; R1' and R2' are both 1 -methyl cyclopropyl, R3' and R4' are both methyl, R5' is ^-propyl, and R6' is -H; R1' and R2' are both 1-methylcyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both methyl; R]' and R2' are both 1-methylcyclopropyl, R3' and R4' are both ethyl, and R5' and R6' are both -H; Ri' and R2' are both 1-methylcyclopropyl, R3' is methyl, R4' is ethyl, and R5' and R6' are both -H; Ri' and R2' are both 2-methylcyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Ri' and R2' are both
2-phenylcyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Ri' and R2' are both 1-phenylcyclopropyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both cyclobutyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rj' and R2' are both cyclopentyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rj' and R2' are both cyclohexyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Ri' and R2' are both cyclohexyl, R3' and R4' are both phenyl, and R5' and R6' are both -H; Ri' and R2' are both methyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Rf and R2' are both methyl, R3' and R4' are both t-butyl, and R5' and R6' are both -H; Rj' and R2' are both methyl, R3' and R4' are both phenyl, and R5' and R6' are both -H; Ri' and R2' are both t-butyl, R3' and R4' are both methyl, and R5' and R6' are both -H; Ri' and R2' are ethyl, R3' and R4' are both methyl, and R5' and R6' are both -H; or Rj' and R2' are both ^-propyl, R3' and R4' are both methyl, and R5' and R6' are both -H.
In particular embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (XI):



(XI)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein Ri', R2', R3', and R4' are as defined above for Structural Formula IVa.
In specific embodiments, the bis(thio-hydrazide amides) are represented by Structural Formula (XII):

(XII)
or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof, wherein: Rf and R2' are both phenyl, and R3' and R4' are both
ø-CH3-phenyl; R, ' and R2' are both o-CH3C(O)O-phenyl, and R3' and R4' are phenyl; Ri' and R2' are both phenyl, and R3' and R4' are both methyl; Ri' and R2' are both phenyl, and R3' and R4' are both ethyl; Ri' and R2' are both phenyl, and R3' and R4' are both ^-propyl; Ri' and R2' are both/?-cyanophenyl, and R3' and R4' are both methyl; R1' and R2' are both/7-nitro phenyl, and R3' and R4' are both methyl; Ri' and R2' are both 2,5-dimethoxyphenyl, and R3' and R4' are both methyl; Ri' and R2' are both phenyl, and R3' and R4' are both n-butyl; Ri' and R2' are both
/?-chlorophenyl, and R3' and R4' are both methyl; Ri' and R2' are both 3-nitrophenyl, and R3' and R4' are both methyl; Rf and R2' are both 3-cyanophenyl, and R3' and R4' are both methyl; Ri' and R2' are both 3 -fluorophenyl, and R3' and R4' are both methyl; Ri' and R2' are both 2-furanyl, and R3' and R4' are both phenyl; Ri' and R2' are both 2-methoxyphenyl, and R3' and R4' are both methyl; R1' and R2' are both 3-methoxyphenyl, and R3' and R4' are both methyl; R1' and R2' are both
2,3-dimethoxyphenyl, and R3' and R4' are both methyl; Ri' and R2' are both
2-methoxy-5-chlorophenyl, and R3' and R4' are both ethyl; Ri' and R2' are both 2,5-difluorophenyl, and R3' and R4' are both methyl; Ri 'and R2' are both
2,5-dichlorophenyl, and R3' and R4' are both methyl; R1' and R2' are both
2,5-dimethylphenyl, and R3' and R4' are both methyl; R]' and R2' are both
2-methoxy-5-chlorophenyl, and R3' and R4' are both methyl; R1' and R2' are both 3,6-dimethoxyphenyl, and R3' and R4' are both methyl; Rj' and R2' are both phenyl, and R3' and R4' are both 2-ethylphenyl; Ri' and R2' are both 2-methyl-5-pyridyl, and R3' and R4' are both methyl; or Rj' is phenyl; R2' is 2,5-dimethoxyphenyl, and R3' and R4' are both methyl; Ri' and R2' are both methyl, and R3' and R4' are both /?-CF3-phenyl; Rf and R2' are both methyl, and R3' and R4' are both o-CH3-phenyl; Ri' and R2' are both -(CH2)3COOH; and R3' and R4' are both phenyl; R1' and R2' are both represented by the following structural

formula:
,and R3' and R4' are both phenyl; R1' and R2' are both rc-butyl, and R3' and R4' are both phenyl; Rj' and R2' are both H-pentyl, R3' and R4' are both phenyl; Rj' and R2' are both methyl, and R3' and R4' are both 2-pyridyl; Rj' and R2' are both cyclohexyl, and R3' and R4' are both phenyl; R)' and R2' are both methyl, and R3' and R4' are both 2-ethylphenyl; Rf and R2' are both methyl, and R3' and R4' are both 2,6-dichlorophenyl; Rf-R4' are all methyl; Rf and R2' are both methyl, and R3' and R4' are both f-butyl; Rf and R2' are both ethyl, and R3' and R4' are both methyl; Rf and R2' are both t-butyl, and R3' and R4' are both methyl; Rf and R2' are both cyclopropyl, and R3' and R4' are both methyl; Rf and R2' are both cyclopropyl, and R3' and R4' are both ethyl; Rf and R2' are both 1-methylcyclopropyl, and R3' and R4' are both methyl; Rf and R2' are both
2-methylcyclopropyl, and R3' and R4' are both methyl; Rf and R2' are both
1-phenylcyclopropyl, and R3' and R4' are both methyl; Rf and R2' are both
2-phenylcyclopropyl, and R3' and R4' are both methyl; Rf and R2' are both cyclobutyl, and R3' and RV are both methyl; Rf and R2' are both cyclopentyl, and R3' and R4' are both methyl; Rf is cyclopropyl, R2' is phenyl, and R3' and R4' are both methyl.
Preferred examples of bis(thio-hydrazide amides) include Compounds (I)-(18) and pharmaceutically acceptable salts and solvates thereof:



Compound (1)



Compound (5)
Compound (6)

Compound (7)



Compound (8)



Compound (9)



Compound (10)
Compound (11)
Compound (12)


Compound (13)



Compound (16) ncj



As used herein, the term "bis(thio-hydrazide amide)" and references to the Structural Formulas of this invention also include pharmaceutically acceptable salts and solvates of these compounds and Structural Formulas. Examples of acceptable salts and solvates are described in US Publication No.: 20060135595 and US Patent Application Serial No.: 11/432,307 filed 1 l-May-2006, titled Synthesis Of Bis(Thio-Hydrazide Amide) Salts, the entire contents of each of which are incorporated herein by reference.
It is to be understood when one tautomeric form of a disclosed compound is depicted structurally, other tautomeric forms are also encompassed.
Certain compounds of the invention may be obtained as different
stereoisomers (e.g., diastereomers and enantiomers). The invention includes all isomeric forms and racemic mixtures of the disclosed compounds and methods of treating a subject with both pure isomers and mixtures thereof, including racemic mixtures. Stereoisomers can be separated and isolated using any suitable method, such as chromatography.
An "alkyl group" is saturated straight or branched chain linear or cyclic hydrocarbon group. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10, and a cyclic alkyl group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. An alkyl group is preferably a straight chained or branched alkyl group, e.g, methyl, ethyl, H-propyl, /sσ-propyl, H-butyl, sec-butyl, tert-buty\, pentyl, hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbon atoms. A C1-C8 straight chained or branched alkyl group or a C3-C8 cyclic alkyl group is also referred to as a "lower alkyl" group. Suitable substitutents for an alkyl group are those which do not substantially interfere with the anti-cancer activity of the disclosed compounds. Suitable substituents are as described below for aliphatic groups. Preferred substituents on alkyl groups include, -OH, -NH2> -NO2, -CN, -COOH, halogen, aryl, C1-C8 alkoxy, C1-C8 haloalkoxy and -CO(C 1-C8 alkyl). More preferred substituents on alkyl groups include -OH, halogen, phenyl, benzyl, pyridyl, and Cl-C8 alkoxy. More preferred substituents on alkyl groups include -OH, halogen, and C 1-C4 alkoxy.
A "straight chained hydrocarbyl group" is an alkylene group, i.e., -(CH2)y-, with one or more (preferably one) internal methylene groups optionally replaced with a linkage group, y is a positive integer (e.g., between 1 and 10), preferably between 1 and 6 and more preferably 1 or 2. A "linkage group" refers to a functional group which replaces a methylene in a straight chained hydrocarbyl. Examples of suitable linkage groups include a ketone (-C(O)-), alkene, alkyne, phenylene, ether (-0-), thioether (-S-), or amine (-N(Ra)-), wherein Ra is defined below. A preferred linkage group is -C(R55R6')-, wherein R5' and R6' are defined above. Suitable substitutents for an alkylene group and a hydrocarbyl group are those which do not substantially interfere with the anti -cancer activity of the disclosed compounds. R5' and R6' are preferred substituents for an alkylene or hydrocarbyl group represented by Y'.
An aliphatic group is a straight chained, branched or cyclic non-aromatic hydrocarbon which is completely saturated or which contains one or more units of unsaturation. Typically, a straight chained or branched aliphatic group has from 1 to about 20 carbon atoms, preferably from 1 to about 10, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. An aliphatic group is preferably a straight chained or branched alkyl group, e.g, methyl, ethyl, n-propyl, wø-propyl, rø-butyl, sec-butyl, ter/-butyl, pentyl, hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbon atoms. A C1-C8 straight chained or branched alkyl group or a C3-C8 cyclic alkyl group is also referred to as a "lower alkyl" group.
The term "aromatic group" may be used interchangeably with "aryl," "aryl ring," "aromatic ring," "aryl group" and "aromatic group." Aromatic groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furanyl, pyridyl, pyrimidy, pyranyl, pyrazolyl, pyrroyl, pyrazinyl, thiazole, oxazolyl, and tetrazole. The term "heteroaryl group" may be used interchangeably with "heteroaryl," "heteroaryl ring," "heteroaromatic ring" and "heteroaromatic group." Heteroaryl groups are aromatic groups that comprise one or more heteroatom, such as sulfur, oxygen and nitrogen, in the ring structure. Preferably, heteroaryl groups comprise from one to four heteroatoms.
Aromatic 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 benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.
Non-aromatic heterocyclic rings are non-aromatic rings which include one or more heteroatoms such as nitrogen, oxygen or sulfur in the ring. The ring can be five, six, seven or eight-membered. Preferably, heterocyclic groups comprise from one to about four heteroatoms. Examples include tetrahydrofuranyl,
tetrahyrothiophenyl, morpholino, thiomorpholino, pyrrolidinyl, piperazinyl, piperidinyl, and thiazolidinyl.
Suitable substituents on an aliphatic group (including an alkylene group), non-aromatic heterocyclic group, benzylic or aryl group (carbocyclic and heteroaryl) are those which do not substantially interfere with the anti-cancer activity of the disclosed compounds. A substituent substantially interferes with anti-cancer activity when the anti-cancer activity is reduced by more than about 50% in a compound with the substituent compared with a compound without the substituent. Examples of suitable substituents include -Ra, -OH, -Br, -Cl, -I, -F, -ORa, -O-COR3, -COR\ -CN, -NO2, -COOH, -SO3H, -NH2, -NHRa, -N(RaRb), -COORa, -CHO, -CONH2, -CONHR8, -CON(RaRb), -NHCORa, -NRcCORa, -NHCONH2, -NHCONR3H, -NHCON(R3R"), -NR0CONH2, -NR0CONR3H, -NRcCON(R3Rb), -C(=NH)-NH2, -C(=NH)-NHRa, -C(=NH)-N(RaRb), -C(=NRC)-NH2, -C(=NRc)-NHRa, -C(=NRC)-N(RaRb), -NH-C(=NH)-NH2, -NH-C(=NH)-NHRa, -NH-C(=NH)-N(RaRb), -NH-C(=NRC)-NH2, -NH-C(=NRC)-NHR3, -NH-C(=NRc)-N(R3Rb), -NRdH-C(=NH)-NH2, -NRd-C(=NH)-NHRa, -NRd-C(=NH)-N(RaRb), -NRd-C(=NRc)-NH2, -NRd-C(=NRc)-NHRa, -NRd-C(=NRc)-N(RaRb), -NHNH2, -NHNHR3, -NHRaRb, -SO2NH2, - SO2NHR3, -SO2NR3R", -CH=CHR3, -CH=CRaRb, -CRc=CRaRb,-CRc=CHRa, -CRc=CRaRb, -CCR3, -SH, -SR3, -S(O)R3, -S(O)2R3.
Ra-Rd are each independently an alkyl group, aromatic group, non-aromatic heterocyclic group or -N(RaRb), taken together, form a non-aromatic heterocyclic group. The alkyl, aromatic and non-aromatic heterocyclic group represented by Ra-Rd and the non-aromatic heterocyclic group represented by -N(R3Rb) are each optionally and independently substituted with one or more groups represented by R . Preferably Ra-Rd are unsubstituted.
R# is R+, -OR+, -O(haloalkyl), -SR+, -NO2, -CN, -NCS, -N(R+)2, -NHCO2R+, -NHC(O)R+, -NHNHC(O)R+, -NHC(0)N(R+)2, -NHNHC(0)N(R+)2, -NHNHCO2R+,
-C(O)C(O)R+, -C(O)CH2C(O)R+, -CO2R+, -C(O)R+, -C(0)N(R+)2, -OC(O)R+, -OC(O)N(R+)2, -S(O)2R+, -SO2N(R+)2, -S(O)R+, -NHSO2N(R+)2, -NHSO2R+, -C(=S)N(R+)2, or -C(=NH)-N(R+)2.
R+ is -H, a C1-C4 alkyl group, a monocyclic heteroaryl group, a non-aromatic heterocyclic group or a phenyl group optionally substituted with alkyl, haloalkyl, alkoxy, haloalkoxy, halo, -CN, -NO2, amine, alkylamine or dialkylamine. Preferably R+ is unsubstituted. Optionally, the group -N(R+)2 is a non-aromatic heterocyclic group, provided that non-aromatic heterocyclic groups represented by R+ and -N(R+)2 that comprise a secondary ring amine are optionally acylated or alkylated.
Preferred substituents for a phenyl group, including phenyl groups represented by Ri-R4, include C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, phenyl, benzyl, pyridyl, -OH, -NH2, -F, -Cl, -Br, -I, -NO2 or -CN. More preferred for a phenyl group, including phenyl groups represented by Ri-R4, include Ri and R2 are optionally substituted with -OH, -CN, halogen, C 1-4 alkyl or C1-C4 alkoxy
Preferred substituents for a cycloalkyl group, including cycloalkyl groups represented by Ri and R2, are alkyl groups, such as a methyl or ethyl group.
The bis(thio-hydrazide amide) disclosed herein can be prepared by the methods described in U.S. Publication Nos. 20060135595, 2003/0045518 and

2003/0119914, U.S. Application Serial No.: 11/432,307, filed 1 l-May-2006, titled Synthesis Of Bis(Thio-Hydrazide Amide) Salts, U.S. Provisional Patent No.: 60/708,977 filed 16-Aug-2005, titled Bis(Thio-Hydrazide Amide) Formulation and also according to methods described in U.S. Publication No. 2004/0225016 Al,
entitled TREATMENT FOR CANCERS. The entire teachings of these applications are incorporated herein by reference.
The present invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1: Synthesis of Compound 1 (Method 1)
Step A: Synthesis of Thiobenzoic Acid N-Methyl Hydrazide



Thiobenzoic acid Λ'-methyl-hydrazide





\ Q regioisomer

Sodium hydroxide (0.19 kg per kg of (thiobenzoyl)thioglycolic acid, 1.01
eq.) was dissolved in water (5 L per kg of (thiobenzoyl)thioglycolic acid) then
cooled to between O0C and 1O0C. (Thiobenzoyl)thioglycolic acid was added and the

15 mixture was stirred until all solids were dissolved. The temperature was maintained between O0C and 1O0C.
In a separate reactor, methyl hydrazine (0.28 kg per kg of
(thiobenzoyl)thioglycolic acid, 1.3 eq.) was dissolved in cold water (50C, 5 L per kg of (thiobenzoyl)thioglycolic acid). The solution was cooled so that the internal
0 temperature was between O0C and 50C then the (thiobenzoyl)thioglycolic acid
solution was added slowly to it. The internal temperature of the reaction mixture
was maintained between O0C and 1O0C. The vessel that contained the
(thiobenzoyl)thioglycolic acid was rinsed with 0.40 L of water per kg of
(thiobenzoyl)thioglycolic acid and the rinse was added to the reaction mixture.

After 30 minutes, the reaction was monitored by HPLC to determine completeness. Upon completion, methylene chloride (3.1 L per kg of
(thiobenzoyl)thioglycolic acid) was added to the reaction mixture, and the mixture was stirred for about 5 minutes until all the solids were dissolved. The organic layer was separated from the aqueous layer, then washed with 0.1 N aqueous NaOH solution (4.0L per kg of (thiobenzoyl)thioglycolic acid). The organic layer was analyzed by HPLC to determine the presence of regioisomer. If the level of regioisomer exceeded 0.5%, then the NaOH wash was repeated. The organic layer was washed with water (4.0 L per kg of (thiobenzoyl)thioglycolic acid) two or more times until the pH in the final aqueous wash was less than 8.
The organic layer was concentrated to about 1.54 L per kg of
(thiobenzoyl)thioglycolic acid then heptane (5 L per kg of (thiobenzoyl)thioglycolic acid) was added. The slurry was stirred overnight at ambient temperature then the solid thiobenzoic acid N-methyl hydrazide was filtered out. The filter cake was washed three times with heptane (0.5 L per kg (thiobenzoyl)thioglycolic acid then dried at ambient temperature under vacuum until a constant weight was achieved.

Step B: Synthesis of Compound 1


Compound 1

Malonyl dichloride was distilled under vacuum at approximately 250C to 350C. The temperature of the bath used to heat the molonyl dichloride did not exceed 5O0C. Malonyl dichloride was considered freshly distilled if it had been distilled within 7 days of use and had been stored under an inert atmosphere at a temperature of O0C to 50C.
Thiobenzoic acid N-methyl hydrazide was dissolved in ethyl acetate (13 L per kg of the hydrazide) and triethyl amine (0.64 kg per kg of the hydrazide, 1.05 eq.). The solution was cooled to an internal temperature of between 30C and 70C. The freshly distilled malonyl dichloride (0.477 kg per kg of hydrazide, 0.56 eq.) was dissolved in anhydrous ethyl acetate (2 L per kg of hydrazide) and added to the solution containing the hydrazide at a rate that maintained the temperature between 30C and 1O0C. The reaction was stirred for about 20 minutes then quenched by addition of cold (O0C to 1O0C) water (5 L per kg of hydrazide). The mixture was stirred an additional 15 minutes then the organic layer was separated and clarified by filtration.
The organic layer was concentrated under vacuum to about two-thirds of its original volume (approximately 5 L per kg of hydrazide was removed) then stirred at ambient temperature overnight to allow crystallization of Compound 1. Compound 1 was collected by filtration and rinsed three times with ethyl acetate (1 L per kg of hydrazide). Compound 1 was then dried under vacuum at 400C until a constant weight was achieved.

EXAMPLE 2: Synthesis of Compound 1 (Method 2)



coupling agent
solvent



Thiobenzoic acid N-methyl hydrazide (100 g, 1.0 eq) and malonic acid (35.1 g, 0.56 eq.) were dissolved in MN-dimethylformamide (DMF, 500 mL, 5 L/kg). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 150 g, 1.5 kg/kg, 1.3 eq.) was added in portion maintaining the internal temperature of the reaction mixture less than 22 0C using water-ice bath. After 2h at room temperature (17 ~ 25 0C) , the reaction was diluted with ethyl alcohol (SDA 3 A 200 proof, 200 mL, 5 L/kg) followed by polish filtration and subsequent washing with ethyl alcohol (SDA 3A 200 proof, 200 mL, 5 L/kg) to give clear yellow solution. Water (deionized, 2000 mL, 20 L/kg; 1950 mL - 2100 mL) was added to the reaction solution, while the internal temperature of the reaction solution went up to 37 0C. The reaction solution was slowly cooled down to room temperature (17 - 25 0C) and stirred at ambient temperature (17 ~ 25 0C) overnight to complete crystallization. The following day (18h ~ 24h), the solids were isolated by filtration and rinsed on the filter with 30% aq. acetone (3 x 800 mL, 3 x 8 L/kg) followed by heptane (1 x 500 mL, 1 x 5 L/kg).

The solids were dried at 55 0C under vacuum, producing yellow solid (82~83% yield, 99.3 ~ 99.7% pure (HPLC a/a%)).

EXAMPLE 3: Purification of Compound 1
A. Synthesis of Compound 1
Compound 1 used in the following purification procedures was prepared by the following procedure. 274.3 kg of RO/DI water was added to a reactor (10 °C). 5.475 g of NaOH pellets were added until a clear solution was obtained. 28.35 kg of S-(thiobenzoyl)thioglycolic acid was added to the reactor (0-5 °C). 7.965 kg of methylhydrazine was added to the reactor while stirring (0-10 °C). The reactor temperature was increased to 20.0 0C over 150 minutes. The precipitate of thiobenzoyl-N'-methylhydrazide (19.05 kg) was collected.
18.95 kg of thiobenzoyl-N'-methylhydrazide was added to a reactor and 221.6 kg of anhydrous ethyl acetate was added with stirring. After the material was dissolved, the reactor was cooled to 4.0 0C. 12.13 kg of TEA was added to the reactor, followed by a solution of malonyl chloride (9.30 kg) in anyhydrous ethyl acetate (34.0kg) (-5 to 10 0C). The reactor temperature was increased to 11.4 °C over 95 min. 96.15 kg of RO/DI water was added (2.5 °C +/- 2.5 °C) and stirred for 20 min. NaCl solution (11.430 kg in 19.07 kg RO/DI water) was added in two portions (draining aqueous layer after each addition) and stirred (27 min, 19 min). The organic layer was washed with ACS ethyl acetate. Ethyl acetate was removed by distillation at 100 +/- 50 mm Hg (23-28 °C). The cake was washed with ethyl acetate. The product was dried under vacuum and N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide) (15.233 kg) was collected.

B. Method 1
Purification procedure (THF-CH 3CN-H2O): To a solution of Compound 1 (2g, 95%) in THF (20ml) was added CH3CN (6ml), cooled to 0-4 0C. To the mixture was added cold water (0-4 0C, 60ml) with stirring. The whole solution was stirred for 3 h followed by filtration. The precipitate was washed with cold THF-H2O (1 :3, v/v), and dried to give Compound 1 (1.8 g, 98%).

C. Method 2
Purification procedure (THF-Acetone-H20): To a solution of Compound 1 (2g, 95%) in THF (20ml) was added acetone (6ml), cooled to 0-4 0C. To the mixture was added cold water (0-4 0C, 60ml) with stirring. The whole solution was stirred for 3 hr, and filtered. The precipitate was washed with cold THF-H2O (1 :3), and dried to give Compound 1 in a yield and purity similar to the method described above.
99+% pure Compound 1 was obtained by combination of the above two procedures sequentially.
The data from purification procedures by re-slurrying Compound 1 in various solvent systems is provided in Tables 1 and 2.

Table 1


Table 2

-P- o

* with less water

EXAMPLE 4: Purification of Compound 1
In another embodiment, 1 kg of Compound 1 was dissolved in 22.15 kg of acetone while maintaining the temperature at 150C to 250C. Once all solids were dissolved, the solution was clarified by filtering it through a 1.2 μm filter using nitrogen pressure directly into a crystallization vessel containing 61.4 kg of water. The flasked that contained the solution of Compound 1 in acetone was rinsed with with 2.14 kg of acetone and the rinse was passed through the 1.2 μm filter. The water/acetone mixture was cooled to 50C to 150C for 6 to 12 hours with continuous mixing to allow Compound 1 to crystalize. The mixture was then warmed to 150C to 250C and allowed to stir at ambient temperature for an additional 8 to 16 hours. The solid product was then collected by filtration under nitrogen pressure. 3 kg of acetone was added to the crystallization vessel followed by 3.80 kg of water and the wash mixture was cooled to O0C to 1O0C then used to wash the filtration cake in two approximately equal portions. The fitration cake was then washed with 2.60 kg of n-heptane under nitrogen pressure. The solid product was then collected and dried at ambient temperature under vacuum for at least 16 hours.
Alternatively, Compound 1 was suspended in acetone (14 L/kg), and water (2 L/kg) was added into suspension with stirring until all solid was dissolved at ambient temperature. Water (30.4 L/kg) was added into a crystallization vessel and cooled to 0 °C. The acetone/water solution of compound 1 was added to the water in crystallization vessel over 20-40 minutes while maintaining the temperature below 5 0C. The resulting suspension was agitated for 20-24 hours at ambient temperature. The resulting solid was filtered washed with cold 50% aqueous acetone followed by rc-heptane, and dried under vacuum at 50 0C.

Compound 1 formed by this method underwent particle size reduction by milling through a 500 μm screen followed by milling through a 150 μm screen to form a product having a particle size of less 150 μm. Compound 1 having particle size less than 150 μm dissolved in a solution of 50% Cremephor 50% ethanol in less than 20 minutes with continuous shaking.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.