Manufacture of trans-[tetrachlorobis(1h-indazole)ruthenate (III)] and compositions thereof

Abstract

To provide the manufacture of trans-[tetrachlorobis(1H-indazole)ruthenate (III)] and compositions thereof.SOLUTION: The present invention relates to the preparation of compositions comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]. Synthesis and formulation preparation are detailed. Impurity profiles are also discussed. Compositions herein are useful for anti-cancer applications. According to an embodiment, the invention provides a composition that comprises sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)], and cesium. In some embodiments, the compositions can further comprise RuIIICl3(Hind)2(H2O), RuIIICl3(Hind)2(CH3CN), and RuIIICl3(Hind) (HN=C(Me) ind).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 501,984, filed May 5, 2017, the entirety of which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates generally to chemical synthesis and, in particular, to a method for making alkali metal salts of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]. [Background technology]

[0003] trans-[tetrachlorobis(1H-indazole)ruthenate(III)] sodium salt (also known as KP1339, NKP-1339, IT-139, and Na[Ru III Several methods exist in the literature for the preparation of Cl(Hind)[. For example, W. Peti et al, Eur. J. Inorg. Chem. 1999, 1551-1555, disclose the following synthetic scheme: [ka]

[0004] This method results in the requirement of large amounts of solvent due to the limited solubility of tetramethylammonium chloride salt. Furthermore, toxicity concerns exist regarding the use of tetramethylammonium salt. U.S. Pat. No. 8,362,266 describes an additional process for making the compound M-trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (wherein M is an alkali metal cation), comprising: (1) reacting trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium with an inorganic salt of the alkali metal cation M in an aqueous solution or a mixture of water and a first, water-soluble organic solvent to form the compound M-trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and an inorganic salt of indazole; and (2) extracting the indazole from M-trans-[tetrachlorobis(1H-indazole)ruthenate(III)] using a second, substantially water-insoluble organic solvent. This method is summarized in the following scheme: [ka]

[0005] While the above methods are effective, the need for an extraction step and associated hold time can limit useful batch sizes. Additionally, the purity of the compound is directly related to the length of time the compound is in the basic aqueous environment. Overall yields for this method range from 20-35%. Therefore, methods that do not utilize extraction processes, avoid the aqueous basic environment, and produce compounds with high yields and high purity levels are highly desirable. Furthermore, methodologies that avoid extraction and large amounts of organic solvents are also desirable. It is believed that methodologies that primarily focus on precipitation, followed by filtration, would meet this need. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 8,362,266 [Non-patent literature]

[0007] [Non-Patent Document 1] W.Peti et al,Eur.J.Inorg.Chem.1999,1551-1555 [Brief explanation of the drawings]

[0008] [Figure 1] Purity of IT-139 drug substance in bulk solution prepared and stored at refrigerated (2-8°C) and room temperature (18-22°C). [Figure 2] HPLC chromatogram of IT-139 stored in a refrigerator (2-8°C) for 18 hours. [Figure 3] HPLC chromatogram of IT-139 stored at room temperature (18-22°C) for 18 hours. [Figure 4] HPLC chromatogram using HPLC method #3 for Formula Ib prepared using the previous synthetic methodology disclosed in US Pat. No. 8,362,266. [Figure 5] HPLC chromatogram of Formula Ib prepared using previous synthetic methodology using HPLC method #2. [Figure 6] HPLC chromatogram of Formula Ib prepared using the synthetic methodology of the present invention using HPLC method #3. [Figure 7] HPLC chromatogram of Formula Ib prepared using the synthetic methodology of the present invention using HPLC method #2. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of Certain Embodiments of the Invention 1. Overview As described herein, the present invention provides a method for producing trans-[tetrachlorobis(1H-yne The present invention provides a method for preparing alkali metal salts of ruthenate (III) (ruthenate (III)). Such compounds include compounds of formula I: [ka] wherein M is an alkali metal cation.

[0010] The present invention provides synthetic intermediates useful in the preparation of such compounds.

[0011] The present invention also provides a method for the preparation of cesium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] as shown in formula Ia below. [ka]

[0012] The present invention also provides a process for the preparation of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] as shown in formula Ib below. [ka]

[0013] 2.Definition trans-[tetrachlorobis(1H-indazole)ruthenate(III)]nat Ru, KP1339, NKP-1339, IT-139, and Na[Ru III Cl4(Hind)2] all correspond to the same compound (Formula Ib), and it should be understood that these terms may be used interchangeably.

[0014] As used herein, the term amorphous refers to a non-crystalline solid that lacks long-range order.

[0015] Compounds of the present invention include those generally described above, and are further described by the embodiments, subembodiments, and species disclosed herein. As used herein, the following definitions apply unless otherwise indicated. For purposes of this invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th In addition, general principles of organic chemistry are identified in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.

[0016] As used herein, the terms "aliphatic" or "aliphatic group" refer to a hydrocarbon moiety that may be straight-chained (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclics) and may be fully saturated or contain one or more unsaturated (but non-aromatic) units. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. In some embodiments, aliphatic groups contain 1-10 carbon atoms. In other embodiments, aliphatic groups contain 1-8 carbon atoms. In still other embodiments, aliphatic groups contain 1-6 carbon atoms, and in still other embodiments, aliphatic groups contain 1-4 carbon atoms. Aliphatic groups include, but are not limited to, straight-chain or branched alkyl, alkenyl, and alkynyl groups, as well as hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0017] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. The heteroatom includes any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocyclic ring (=N-, as in 3,4-dihydro-2H-pyrrolyl, -NH-, as in pyrrolidinyl, or =N(R) as in N-substituted pyrrolidinyl). † )-includes).

[0018] As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0019] As used herein, "a divalent, saturated or unsaturated, straight or branched C 1-12 The term "hydrocarbon chain" refers to divalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

[0020] The term "aryl," used alone or as part of a larger moiety, such as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring."

[0021] As described herein, compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens in the designated moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents can be the same or different at every position. Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is not substantially altered when subjected to conditions that allow for its production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein.

[0022] The monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0-4 R ○ ;-(CH2) 0-4 OR ○ ;-O-(CH2) 0-4 C(O)OR ○ ;-(CH2) 0-4 CH(OR ○ )2;-(CH2) 0-4 SR ○ ;-(CH2) 0-4 Ph(R ○ may be substituted with);-(CH2) 0-4 O(CH2) 0-1 Ph(R ○ -CH=CHPh(R ○ -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R ○ )2;-(CH2) 0-4 N(R ○ )C(O)R ○ ;-N(R ○)C(S)R ○ ;-(CH2) 0-4 N(R ○ )C(O)NR ○ 2;-N(R ○ )C(S)NR ○ 2;-(CH2) 0-4 N(R ○ )C(O)OR ○ ;-N(R ○ )N(R ○ )C(O)R ○ ;-N(R ○ )N(R ○ )C(O)NR ○ 2;-N(R ○ )N(R ○ )C(O)OR ○ ;-(CH2) 0-4 C(O)R ○ ;-C(S)R ○ ;-(CH2) 0-4 C(O)OR ○ ;-(CH2) 0-4 C(O)SR ○ ;-(CH2) 0-4 C(O)OSiR ○ 3;-(CH2) 0-4 OC(O)R ○ ;-OC(O)(CH2) 0-4 SR-、SC(S)SR ○ ;-(CH2) 0-4 SC(O)R ○ ;-(CH2) 0-4 C(O)NR ○ 2;-C(S)NR ○ 2;-C(S)SR ○ ;-SC(S)SR°、-(CH2) 0-4 OC(O)NR ○ 2;-C(O)N(OR ○ )R ○ ;-C(O)C(O)R ○ ;-C(O)CH2C(O)R ○ ;-C(NOR ○ )R ○ ;-(CH2) 0-4 SSR ○ ;-(CH2) 0-4 S(O)2R ○ ;-(CH2) 0-4 S(O)2OR○ ;-(CH2) 0-4 OS(O)2R ○ ;-S(O)2NR ○ 2;-(CH2) 0-4 S(O)R ○ ;-N(R ○ )S(O)NR ○ 2;-N(R ○ )S(O)2R ○ ;-N(OR ○ )R ○ ;-C(NH)NR ○ 2;-P(O)2R ○ ;-P(O)R ○ 2;-OP(O)R ○ 2;-OP(O)(OR ○ )2;SiR ○ 3;-(C 1-4 Linear or branched alkylene)ON(R ○ )2; or -(C 1-4 Linear or branched alkylene)C(O)ON(R ○ )2, where each R ○ are optionally substituted as defined below and independently represent hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definitions, R ○ two independent occurrences of together with the intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0023] R ○ (or two independent R ○ The monovalent substituents on the ring formed by joining together the occurrences of -(CH) with the intervening atoms are independently selected from halogen, -(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2)0 -2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● 、 -(C 1-4 Straight or branched alkylene)C(O)OR ● , or -SSR ● At this time, each R ● is unsubstituted or, if preceded by "halo", substituted with only one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. ○ Divalent substituents on a saturated carbon atom of include =0 and =S.

[0024] Divalent substituents on a saturated carbon atom of an "optionally substituted" group include ═O, ═S, ═NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2))2-3 O-, or -S(C(R * 2)) 2-3 S- is mentioned, and then R * Each independent occurrence of hydrogen, C 1-6 A divalent substituent attached to a substitutable carbon adjacent to an "optionally substituted" group is selected from an aliphatic (optionally substituted as defined below) or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. * 2) 2-3 O-, where R * Each independent occurrence of hydrogen, C 1-6 aliphatic (optionally substituted as defined below) or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. The tetravalent substituent attached to the adjacent substitutable methylene carbon of an "optionally substituted" group is a dicobalt hexacarbonyl cluster, which, when depicted with the methylene bearing it, is: [ka] is expressed by

[0025] R * Suitable substituents on the aliphatic group include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", substituted with only one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0026] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † In this case, each R † are independently hydrogen, C 1-6 aliphatic (optionally substituted as defined below), unsubstituted -OPh, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, R † two independent occurrences of together with the intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0027] R † Suitable substituents on the aliphatic group are independently halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ●is unsubstituted or, if preceded by "halo", substituted with only one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0028] Protecting hydroxyl groups is well known in the art and is described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3 rdedition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Examples of suitable protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, aryl alkyl ethers, and alkoxy alkyl ethers. Examples of suitable esters include formates, acetates, propionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formate, benzoylformate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxycrotonate, benzoate, p-benylbenzoate, and 2,4,6-trimethylbenzoate. Examples of carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropyl ether, and other trialkylsilyl ethers. Examples of alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ethers, or derivatives thereof. Examples of alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzoyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Examples of arylalkyl include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

[0029] Protected amines are well known in the art and include those described in detail in Greene (1999). Mono-protected amines further include, but are not limited to, aralkyl amines, carbamates, allyl amines, amides, and the like. Examples of mono-protected amino moieties include t-butyloxycarbonylamino (-NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxocarbonylamino (-NHCBZ), allylamino, benzylamino (-NHBn), fluorenylmethylcarbonyl (-NHFmoc), formamide, acetamide, chloroacetamide, dichloroacetamide, trichloroacetamide, phenylacetamide, trifluoroacetamide, benzamide, t-butyldiphenylsilyl, and the like. Di-protected amines include amines substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides such as phthalimide, maleimide, succinimide, etc. Di-protected amines also include 2,2,5,5-tetramethyl-[1.2.5]azadisilolidine, such as pyrrole. and azides.

[0030] Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal, 1,3-dioxane, 1,3-dioxolane, semicarbazones, and derivatives thereof.

[0031] Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Protected carboxylic acids further include, but are not limited to, optionally substituted C 1-6 Included are aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, etc. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl esters, each group optionally substituted. Additional protected carboxylic acids include oxazolines and orthoesters.

[0032] Protected thiols are well known in the art and include those described in detail in Greene (1999). Protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioesters, to name a few.

[0033] Unless otherwise specified, structures depicted herein are intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations at each asymmetric center, Z and E double bond isomers, and Z and E stereoisomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise specified, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise specified, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which a hydrogen is replaced by deuterium or tritium, or a carbon is replaced by a methyl group, are also intended to include compounds in which a hydrogen is replaced by a methyl group, or a methyl group, are replaced by a methyl group, are also intended to include compounds in which a hydrogen is replaced by a methyl group, or a methyl group, are replaced by a methyl group, are also intended to include compounds in which a hydrogen is replaced by a methyl group, are also intended to include compounds in which a hydrogen is replaced by a methyl group, or a methyl group, are also intended to include compounds in which a carbon is replaced by a methyl group, are also intended to include compounds in which a hydrogen is replaced by a methyl group, are also intended to include compounds in which a carbon ... 13 C or 14 Compounds having the present structures except for the C-enriched carbons are within the scope of this invention. Such compounds are useful as analytical tools or probes in biological assays, such as in neutron scattering experiments.

[0034] As used herein, the phrase "unit dosage form" refers to a physically discrete unit of the formulation of the present invention appropriate for the subject to be treated. However, it should be understood that the total daily usage of the composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific active agent used, the specific composition used, the age, weight, general health, sex, and diet of the subject, the time of administration, the excretion rate of the specific active agent used, the duration of treatment, drugs and / or additional therapies used in combination with or simultaneously with the specific compound(s) used, and similar factors well known in the medical arts.

[0035] The term "about," when referring to a measurable value, such as an amount, time period, or the like, refers to a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value, since such variations are appropriate in practicing the methods of the present disclosure.

[0036] 3. Description of Exemplary Embodiments 3.1 Drug Substances In certain embodiments, compounds of the present invention are generally prepared according to Scheme I, shown below. Scheme I [ka]

[0037] In one aspect, the present invention provides a method for preparing a compound of Formula I according to the steps shown in Scheme I above. In step S-1, ruthenium chloride (III) reacts with indazole to form the indazolium salt of trans-[tetrachlorobis(1H-indazole)ruthenic acid (III)]. This step (S-1) is well known in the art. See Keppler et al., Inorganic Chemistry, 26, 1987. In step S-2, the indazolium salt is converted to the cesium salt of trans-[tetrachlorobis(1H-indazole)ruthenic acid (III)] of Formula Ia by treatment with cesium chloride. Those skilled in the art will recognize this as a salt exchange from the indazolium salt to the cesium salt. In step S-3, the cesium salt of Formula Ia is converted to the sodium salt of trans-[tetrachlorobis(1H-indazole)ruthenic acid (III)] of Formula Ib by treatment with sodium aluminum sulfate. Those skilled in the art will recognize this as a salt exchange from the cesium salt to the sodium salt.

[0038] In certain embodiments, the above-described synthetic steps may be performed sequentially, with isolation of each intermediate after each step. Alternatively, steps S-1, S-2, and S-3 shown in Scheme I above may be performed in a manner that does not involve isolation of the intermediate.

[0039] Those skilled in the art will recognize that steps S-1, S-2, and S-3 involve the preparation of first the indazolium salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)], then the cesium salt, and then the sodium salt. Furthermore, U.S. Pat. No. 8,362,266 describes the preparation of Formula Ib directly from an indazolium salt. One aspect of the present invention involves the preparation of Formula Ia as an intermediate in the synthesis of Formula Ib. The cesium salt intermediate has been found to be preferable to existing methods because the purity of the product and the overall yield can be significantly increased over existing methods. While not wishing to be bound by any particular theory, the inventors believe that this increase in yield and purity is due to the difficulty of isolating the indazolium salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]. The inventors have not previously described this material. However, we found that isolation as a pure, solvent-free material was extremely difficult due to the presence of residual water and hydrochloric acid in the filtered material. One proposed decomposition pathway for this material is shown in Scheme II below. Scheme II [ka]

[0040] Scheme II shows the compound A (mer, trans-[Ru IIIThe preparation of ruthenium ion complex (Cl3(Hind)2(H2O)] is shown. The impurity, Compound A, is also known in the literature as the water complex. The formation of Compound A can be limited by the exclusion of water or by maintaining a significantly high concentration of chloride ions. For example, Formula Ib is much more stable in a solution of sodium chloride or hydrochloric acid than in pure water. Those skilled in the art will recognize that maintaining a chloride ion concentration reduces the possibility of water replacing the chloride on the ruthenium complex. Furthermore, it has been found that the rate of aquation (or preparation of Compound A) is greatly increased in basic solutions.

[0041] Since the primary decomposition products are aquation reactions, particularly those that are promoted in basic aqueous solutions, it may be preferable to avoid reaction steps that involve dissolving the compound of formula I in water.

[0042] One embodiment of the present invention provides a method for preparing Formula Ib by preparing trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium, isolating the material by filtration, and drying to a theoretical yield of 200% to 500% by weight for use in S-2. In another embodiment, the present invention provides a method for preparing Formula Ib by preparing trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium, isolating the material by filtration, and drying to a theoretical yield of 245% to 425% by weight for use in S-2.

[0043] Another aspect of the present invention is the introduction of step S-2 in the preparation of formula Ib. Step S-2 involves the preparation of the cesium intermediate, formula Ia. Surprisingly, it has been discovered that the cesium intermediate is a key step in the present invention because it can be isolated by precipitation and filtration, can be dried without inducing decomposition (as observed with indazolium salts), and the dried powder is stable at ambient conditions. As noted above, trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium is isolated by filtration as what can best be described as a muddy substance. The stability of this compound is improved by the presence of hydrochloric acid (chloride ions). Washing the filtrate with a polar solvent (e.g., methanol) also leads to decomposition. Therefore, the best case scenario is Trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium is prepared, isolated by filtration, and used directly in S-2 without delay. S-2 consists of mixing trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium with cesium chloride in a suitable solvent. Suitable solvents can be alcohols having 1 to 5 carbon atoms, diols having 2 to 4 carbon atoms, water, ketones having 1 to 6 carbon atoms, cyclic ethers having 4 to 7 carbon atoms, amides having 1 to 4 carbon atoms, DMSO, sulfolane, esters having 4 to 6 carbon atoms, chlorinated hydrocarbons having 1 or 2 carbon atoms, liquid aromatic hydrocarbons, nitriles having 2 to 6 carbon atoms, or mixtures thereof.

[0044] In one aspect of the present invention, step S-2 comprises mixing trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium with cesium chloride in ethanol and methyl ethyl ketone to obtain Formula Ia. The cesium salt intermediate was collected by filtering the reaction mixture and washing with ethanol. In some embodiments, step S-2 utilizes 1 to 10 equivalents of cesium chloride in the reaction mixture. In other embodiments, step S-2 utilizes 2 to 4 equivalents of cesium chloride in the reaction mixture. In a preferred embodiment, the present invention provides a method for preparing Formula Ib, in which 2.8 equivalents of cesium chloride are used in step S-2. The preferred solvent for step S-2 is an ethanol-containing mixture, most preferably a methanol-methyl ethyl ketone (MEK) mixture. This is because MEK mixtures can form crystalline MEK solvates of the cesium salt, which aids in purification. The resulting MEK solvates are then readily converted to the more stable hydrate form of the cesium salt by treatment with aqueous ethanol.

[0045] Another aspect of the present invention is step S-3, which converts the cesium salt intermediate (Formula Ia) to the desired sodium salt, Formula Ib. Previous methodologies for providing Formula Ib described herein involve treating an aqueous solution of trans-[tetrachlorobis(1H-indazole)ruthenic acid (III)] with a sodium salt under basic conditions. As discussed above, aqueous basic conditions lead to decomposition to Compound A. To address this issue, the inventors developed step S-3, which converts Formula Ia to Formula Ib by mixing sodium aluminum sulfate (NaAl(SO4)2). This salt exchange was carried out by mixing sodium aluminum sulfate and Formula Ia in water. The reaction was carried out at a high concentration such that the reaction mixture was heterogeneous. The driving force in the reaction is the differential solubility of sodium aluminum sulfate and cesium aluminum sulfate. Sodium aluminum sulfate is soluble in water and provides a source of sodium ions. Cesium aluminum sulfate is insoluble in water and precipitates from the reaction mixture. Therefore, the cesium counterion is constantly removed from the reaction solution, resulting in the formation of Formula Ib. The insoluble aluminum cesium sulfate and Formula Ib are isolated by filtration. Formula Ib is dissolved in a suitable solvent, and the aluminum cesium sulfate is removed by filtration. Suitable solvents include low molecular weight alcohols (having 1-5 carbon atoms), ketones having 3-6 carbon atoms, nitriles having 2-5 carbon atoms, esters having 3-6 carbon atoms, amides having 1-4 carbon atoms, diols having 1-4 carbon atoms, DMSO, sulfolane, water, or combinations thereof. The most preferred solvent for solid extraction is acetonitrile. Formula Ib is then precipitated using a suitable anti-solvent and recovered by filtration. Suitable anti-solvents include ethers having 3-8 carbon atoms, cyclic, acyclic, or aromatic hydrocarbons having 5-8 carbon atoms, chlorinated hydrocarbons having 1-4 carbon atoms, benzotrifluoride, chlorobenzene, and methyl carbonate. The most preferred anti-solvent is methyl tert-butyl ether (MTBE).

[0046] In some embodiments, the present invention provides a method for preparing formula Ib, wherein the concentration of sodium aluminum sulfate in step S-3 is 0.5M to 1.65M. In a preferred embodiment, the present invention provides a method for preparing Formula Ib, wherein the concentration of sodium aluminum sulfate in step S-3 is 1.1 M.

[0047] In some embodiments, the present invention provides a method for preparing Formula Ib, wherein the reaction temperature in step S-3 is −5° C. to 50° C. In a preferred embodiment, the present invention provides a method for preparing Formula Ib, wherein the reaction temperature in step S-3 is 20° C. to 25° C.

[0048] In some embodiments, the present invention provides a method for preparing Formula Ib, wherein the reaction time in step S-3 is 12 hours to 168 hours. In a preferred embodiment, the present invention provides a method for preparing Formula Ib, wherein the reaction time in step S-3 is 30 hours.

[0049] Another aspect of the present invention is a purification step in which residual cesium is removed from Formula Ib. This process involves stirring Formula Ib with methanol in the presence of 4 Å molecular sieves, followed by precipitation with MTBE. While not wishing to be bound by any particular theory, it is believed that the cesium atom has an affinity for the 4 Å pores present in the molecular sieves. Furthermore, it has been found that traces of solvent impurities can be removed from the desired product by stirring and washing with a water-saturated MTBE solution. This final purification step provides the purest Formula Ib.

[0050] Characterization of the ruthenium containing target compound required multiple techniques. Nuclear magnetic resonance spectroscopy of ruthenium compounds is challenging due to the 5 / 2 nuclear spin state, so alternative characterization methods were used, including HPLC and x-ray diffraction (crystallography). To fully characterize the purity of IT-139, the inventors intentionally prepared several compounds believed to be impurities in the final composition of IT-139, namely, compounds A, C, and D. The identities of impurities A, C, and D were confirmed by x-ray diffraction. Compound B is an unstable complex believed to be an intermediate in the formation of compound C. The structure of the impurity is as follows: [ka]

[0051] Once the impurity compounds were prepared and identified, their retention times were analyzed by HPLC so that the identity and percentage of the impurities could be rapidly quantified by HPLC analysis. During this process, the inventors observed that the water complex, Compound A, resulted in multiple peaks on HPLC, and the chromatographic profile changed as a function of time. The inventors observed that the water complex reacted with acetonitrile in the mobile phase to form an acetonitrile adduct, Compound B, and that this adduct subsequently reacted with acetonitrile to form a tetrahydrofuran. It was found that the covalent bond between the hydroxyl group and the hydroxyl group forms compound C (see Inorganic Chemistry, 2008, v47, pp. 6513-6523). This reaction is shown in Scheme III below. [ka]

[0052] The relative retention times for each compound are listed in the table below (Table 1). [Table 1]

[0053] In some embodiments, the relative retention times (RRT) listed in Table 1 may be defined by ranges. For example, the RRT for compound A may be 1.09 + / - 0.02, the RRT for compound B may be 1.28 + / - 0.02, the RRT for compound C may be 1.06 + / - 0.03, and the RRT for compound D may be 1.59 + / - 0.03.

[0054] Because the water complex (compound A) rapidly forms compounds B and C in the mobile phase in HPLC analysis, the amount of compound A in the sample submitted to HPLC analysis is determined to be the sum of the peak areas corresponding to compounds A, B, and C. One advantage of the synthetic methodology of the present invention over other synthetic methodologies is the high purity level that can be achieved by the present invention. Previous methodologies described above result in a final product (drug substance) containing 4-8% compound A as an impurity. In contrast, less than 2% compound A is easily achievable with the present invention. One embodiment of the present invention One embodiment provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and compound A, wherein the composition contains 2.0% by weight or less of compound A. One embodiment provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and compound A, wherein the composition contains 1.0% by weight or less of compound A. One embodiment provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and compound A, wherein the composition contains 1.5% by weight or less of compound A. One embodiment provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and compound A, wherein the composition contains 0.5% by weight or less of compound A. One embodiment provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and compound A, wherein the composition contains 3.0% by weight or less of compound A.

[0055] Another advantage of the present invention over previous synthetic methodologies is the reduced amount of impurities purified. Previous synthetic methodologies described above (see, e.g., U.S. Pat. No. 8,362,266) were often analyzed by HPLC methods (e.g., HPLC Method #3, described below) that did not resolve the impurities (Compound A, Compound B, and Compound C) from the drug substance (Formula Ib). Figure 4 shows the drug substance prepared using the other synthetic methodology analyzed by HPLC Method #3, while Figure 5 shows the same drug substance analyzed by an analytical method (HPLC Method #2, described below) that resolves Formula Ib from the impurities Compound A, Compound B, and Compound C. As a result, a purity of approximately 99.5% was reported for the drug substance synthesized using the other methodology (analysis by HPLC Method #3). However, the same material analyzed by HPLC Method #2 showed that the purity was actually approximately about 76.4% Formula Ib, contaminated with about 7.3% Compound A, about 11.0% Compound B, and about 0.36% Compound C. The present invention provides a composition comprising Formula Ib in about 99.9% purity as analyzed using HPLC Method #3. The present invention provides a composition comprising about 96.3% Formula Ib, about 1.1% Compound A, about 1.7% Compound B, and about 0.2% Compound C. The HPLC data are reproduced in the table below (Table 2). [Table 2]

[0056] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Cl3(Hind) (HN=C(Me)ind).

[0057] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and RuIII A composition is provided that includes Cl3(Hind) (HN=C(Me)ind) and cesium.

[0058] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is greater than or equal to about 95.5 weight percent of the composition; The Ru III Cl3(Hind)2(H2O) is less than or equal to about 1.0 weight percent of the composition; The Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 2.5 weight percent of the composition; The Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 2.0 weight percent of the composition; Cesium is less than or equal to about 0.5 weight percent of the composition; A composition is provided.

[0059] One embodiment of the present invention is a catalyst containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], cesium, and optionally Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind), the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; The Ru IIICl3(Hind)2(H2O) is from about 0 to about 1.0 weight percent of the composition; The Ru III Cl3(Hind)2(CH3CN) is from about 0 to about 2.5 weight percent of the composition; The Ru III Cl(Hind) (HN=C(Me)ind) is from about 0 to about 2.0 weight percent of the composition; Cesium is about 0 to about 0.5 weight percent of the composition; A composition is provided.

[0060] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; The Ru III Cl3(Hind)2(H2O) is from about 0.001 to about 1.0 weight percent of the composition; The Ru III Cl3(Hind)2(CH3CN) is from about 0.001 to about 2.5 weight percent of the composition; The Ru III Cl(Hind) (HN=C(Me)ind) is from about 0.001 to about 2.0 weight percent of the composition; Cesium is about 0.0001 to about 0.5 weight percent of the composition; A composition is provided.

[0061] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: IIICl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; The Ru III Cl3(Hind)2(H2O) is from about 0.001 to about 0.75 weight percent of the composition; The Ru III Cl3(Hind)2(CH3CN) is from about 0.001 to about 1.5 weight percent of the composition; The Ru III Cl(Hind) (HN=C(Me)ind) is from about 0.001 to about 1.25 weight percent of the composition; Cesium is about 0.0001 to about 0.25 weight percent of the composition; A composition is provided.

[0062] One embodiment of the present invention is a method for producing a tetrachlorobis(1H-indazole)ruthenate (III)-containing ruthenate, comprising: III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; The Ru III Cl3(Hind)2(H2O) is from about 0.001 to about 0.5 weight percent of the composition; The Ru III Cl3(Hind)2(CH3CN) is from about 0.001 to about 0.5 weight percent of the composition; The Ru IIICl(Hind) (HN=C(Me)ind) is about 0.001 to about 0.5 weight percent of the composition; Cesium is about 0.0001 to about 0.01 weight percent of the composition; A composition is provided.

[0063] 3.2 Medicines An additional embodiment of the present invention provides a method for preparing a drug product containing the sodium salt of trans-[tetrachlorobis(1H-indazole)ruthenic acid (III)] (ie, IT-139).

[0064] One aspect of the present invention provides a method for preparing a sterile, lyophilized drug product containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]. This formulation is considered suitable for administration to patients. The formulation consists of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], a pH buffer, and a cryoprotectant. A general method for providing the above formulation includes the steps of preparing an aqueous buffer solution, preparing an aqueous cryoprotectant solution, dissolving sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in the buffer solution, adding the cryoprotectant solution, sterile filtering (e.g., aseptic filtering), filling vials under sterile conditions, and lyophilizing under sterile conditions. Suitable buffers include, but are not limited to, citric acid, TRIS, acetic acid, EDTA, HEPES, tricine, and imidazole. Phosphate buffers can be used, but are not preferred. A preferred aspect of the present invention is the use of a citric acid / sodium citrate buffer. Suitable cryoprotectants include, but are not limited to, sugars, monosaccharides, disaccharides, polysaccharides, polyalcohols, mannitol, sorbitol, and the like. Examples of cryoprotectants include mannitol, sucrose, trehalose, dextran, and dextrose. A preferred embodiment of the present invention is the use of mannitol as a cryoprotectant.

[0065] As described hereinabove, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] can decompose to Compound A in water (Scheme II). Those skilled in the art will recognize that limiting this decomposition reaction would be advantageous in obtaining the highest purity product. It has been discovered that cooling sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] during the formulation process significantly reduces the amount of Compound A present in the lyophilized product. In one embodiment of the present invention, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] solution is cooled to 4° C. during the formulation process. In another embodiment of the present invention, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] solution is cooled to 2-8° C. during the formulation process. In another embodiment of the present invention, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] solution is cooled to 2-15° C. during the formulation process.

[0066] One embodiment of the present invention provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], a suitable buffer, and mannitol. In some embodiments, the suitable buffer comprises a citrate buffer. For example, in some embodiments, the citrate buffer comprises sodium citrate and citric acid.

[0067] One embodiment of the present invention provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, and mannitol.

[0068] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)].

[0069] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt.

[0070] One embodiment of the present invention provides a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, and mannitol, wherein the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is amorphous.

[0071] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)], wherein the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is amorphous.

[0072] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and cesium salts wherein the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is amorphous.

[0073] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.4 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; A composition is provided.

[0074] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.4 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; A composition is provided.

[0075] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, mer, trans-[Ru III Cl(Hind)(H2O)] is from about 0.01 to about 0.2 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; A composition is provided.

[0076] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, mer, trans-[RuIII Cl(Hind)(H2O)] is from about 0.01 to about 0.40 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; A composition is provided.

[0077] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, the composition is a lyophilized powder; mer, trans-[Ru III Cl(Hind)(H2O)] is from about 0.01 to about 0.40 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; A composition is provided.

[0078] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and cesium salts A composition comprising: the composition is a lyophilized powder; mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.3 weight percent of the composition; Cesium is about 0.00001 to about 0.1 weight percent of the composition; A composition is provided.

[0079] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru IIICl3(Hind)2(H2O)] and a cesium salt, mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.3 weight percent of the composition; Cesium is about 0.00001 to about 0.1 weight percent of the composition; A composition is provided.

[0080] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, the composition is a lyophilized powder; sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 11.5 to about 14.0 weight percent of the composition; citric acid is about 43.9 to about 53.7 weight percent of the composition; sodium citrate is about 25.7 to about 23.1 weight percent of the composition; mannitol is about 11.5 to about 14.0 weight percent of the composition; mer, trans-[Ru III Cl(Hind)(H0)] is between about 0.01 and about 0.3 weight percent of the composition; Cesium is about 0.00001 to about 0.1 weight percent of the composition; A composition is provided.

[0081] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, the composition is a lyophilized powder; sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 10.2 to about 15.3 weight percent of the composition; citric acid is about 39.0 to about 58.5 weight percent of the composition; sodium citrate is about 20.5 to about 30.8 weight percent of the composition; mannitol is about 10.2 to about 15.3 weight percent of the composition; mer, trans-[Ru III Cl(Hind)(H0)] is between about 0.01 and about 0.3 weight percent of the composition; Cesium is about 0.00001 to about 0.1 weight percent of the composition; A composition is provided.

[0082] One embodiment of the present invention is a mixture of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, and mer, trans-[Ru III Cl3(Hind)2(H2O)] and a cesium salt, the composition is a lyophilized powder; sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 10.2 to about 15.3 weight percent of the composition; mer, trans-[Ru III Cl(Hind)(H0)] is about 0.01 and about 0.3 weight percent composition; Cesium is about 0.00001 to about 0.1 weight percent of the composition; A composition is provided.

[0083] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 49.86 weight percent of the composition; mannitol is about 49.86 weight percent of the composition; citric acid is about 0.187 weight percent of the composition; sodium citrate is about 0.093 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0084] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is about 0.001 to about 0.25 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0085] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 30 to about 70 weight percent of the composition; mannitol is about 30 to about 70 weight percent of the composition; citric acid is about 0.001 to about 1 weight percent of the composition; sodium citrate is about 0.0001 to about 1 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0086] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O), sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 49.86 weight percent of the composition; mannitol is about 49.86 weight percent of the composition; citric acid is about 0.187 weight percent of the composition; sodium citrate is about 0.093 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to 0.5 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0087] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O), sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is about 0.001 to about 0.25 weight percent of the composition; RuIII Cl3(Hind)2(H2O) is about 0 to about 0.5 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0088] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III A composition comprising Cl3(Hind)2(H2O) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 30 to about 70 weight percent of the composition; mannitol is about 30 to about 70 weight percent of the composition; citric acid is about 0.001 to about 1 weight percent of the composition; sodium citrate is from about 0.0001 to about 1 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to 0.5 weight percent of the composition; Cesium is 0.25 weight percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0089] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, sodium citrate, and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 49.61 weight percent of the composition; mannitol is about 49.86 weight percent of the composition; citric acid is about 0.187 weight percent of the composition; sodium citrate is about 0.093 weight percent of the composition; the cesium is about 0.25 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0090] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, sodium citrate, and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is about 0.001 to about 0.25 weight percent of the composition; Cesium is about 0.1 to about 0.5 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0091] One embodiment of the present invention is a composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, sodium citrate, and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 30 to about 70 weight percent of the composition; mannitol is about 30 to about 70 weight percent of the composition; citric acid is about 0.001 to about 1 weight percent of the composition; sodium citrate is from about 0.0001 to about 1 weight percent of the composition; Cesium is about 0.01 to about 1 weight percent of the composition; In some such embodiments, the composition is a lyophilized powder.

[0092] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 46.61 weight percent of the composition; mannitol is about 49.86 weight percent of the composition; citric acid is about 0.187 weight percent of the composition; sodium citrate is about 0.093 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is 1.0 weight percent or less of the composition; Cesium is 0.25 weight percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0093] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O) and Ru IIICl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 46.61 weight percent of the composition; mannitol is about 49.86 weight percent of the composition; citric acid is about 0.187 weight percent of the composition; sodium citrate is about 0.093 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is 1.0 weight percent or less of the composition; Cesium is 0.25 weight percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0094] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is about 0.001 to about 0.25 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition; Cesium is 0.25 percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0095] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 30 to about 70 weight percent of the composition; mannitol is about 30 to about 70 weight percent of the composition; citric acid is about 0.001 to about 1 weight percent of the composition; sodium citrate is from about 0.0001 to about 1 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru IIICl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is about 1.0% of the composition weight percentage or less, Cesium is 0.25 percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0096] One embodiment of the present invention is a method for producing a ruthenate comprising the steps of: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]; mannitol; citric acid; sodium citrate; and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III A composition comprising Cl3(Hind) (HN=C(Me)ind) and cesium, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 20 to about 80 weight percent of the composition; mannitol is about 20 to about 80 weight percent of the composition; citric acid is from about 0.0001 to about 5 weight percent of the composition; sodium citrate is from about 0.00001 to about 5 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition; Cesium is 0.25 percent or less of the composition; In some such embodiments, the composition is a lyophilized powder.

[0097] 3.3 Unit Dosage Form In some embodiments, the present invention provides a unit dosage form comprising the formulation or composition described herein. As used herein, the term "unit dosage form" refers to a physically discrete unit of the provided formulation appropriate for the subject to be treated. However, it should be understood that the total daily usage of the provided formulation will be determined by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or organism will depend on various factors, including the disorder being treated and the severity of the disorder, the activity of the specific active agent used, the specific formulation used, the age, weight, general health, sex, and diet of the subject, the time of administration, the excretion rate of the specific active agent used, the duration of treatment, drugs and / or additional therapies used in combination with or simultaneously with the specific compound(s) used, and similar factors well known in the medical field.

[0098] The compositions of the present invention can be provided as a unit dosage form. In some embodiments, a vial containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate is the unit dosage form.

[0099] In some embodiments, the vial containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] of the present invention, mannitol, citric acid, sodium citrate, and cesium is a unit dosage form.

[0100] In some embodiments, the present invention provides a method for preparing a ruthenate (III) solution containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, sodium citrate, and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(H d) 2(CH3CN) and Ru IIIThe vial containing Cl3(Hind) (HN=C(Me)ind) and cesium is a unit dosage form.

[0101] Still further encompassed by the present invention are pharmaceutical packs and / or kits containing the compositions described herein, or unit dosage forms comprising a provided composition and a container (e.g., a foil or plastic package, or other suitable container). Optionally, such kits are additionally provided with instructions for use.

[0102] In some embodiments, the present invention may be provided as a unit dosage form. Indeed, a vial containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate is the unit dosage form shown in Table 3. [Table 3]

[0103] In some embodiments, the pharmaceutical components described in Table 3 further comprise cesium; At this time, The cesium is not more than 0.25 weight percent of the composition.

[0104] In some embodiments, the pharmaceutical components listed in Table 3 contain cesium and Ru. III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Cl3(Hind) (HN=C(Me)ind), At this time, the cesium is less than or equal to about 0.25 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition.

[0105] In some embodiments, the pharmaceutical composition is selected from those in Table 4. [Table 4]

[0106] In some embodiments, the pharmaceutical components described in Table 4 further comprise cesium; At this time, The cesium is not more than 0.25 weight percent of the composition.

[0107] In some embodiments, the pharmaceutical components listed in Table 4 contain cesium and Ru. III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Cl3(Hind) (HN=C(Me)ind), At this time, the cesium is less than or equal to about 0.25 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition.

[0108] In some embodiments, the present invention may be provided as a unit dosage form. Indeed, a vial containing sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], mannitol, citric acid, and sodium citrate is the unit dosage form shown in Table 5. [Table 5]

[0109] In some embodiments, the pharmaceutical components described in Table 5 further comprise cesium; At this time, The cesium is not more than 0.25 weight percent of the composition.

[0110] In some embodiments, the pharmaceutical components listed in Table 5 contain cesium and Ru. III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Cl3(Hind) (HN=C(Me)ind), At this time, the cesium is less than or equal to about 0.25 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition.

[0111] In some embodiments, the pharmaceutical composition is selected from those in Table 6. [Table 6]

[0112] In some embodiments, the pharmaceutical components described in Table 6 further comprise cesium; At this time, The cesium is not more than 0.25 weight percent of the composition.

[0113] In some embodiments, the pharmaceutical components listed in Table 6 contain cesium and Ru. III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Cl3(Hind) (HN=C(Me)ind), At this time, the cesium is less than or equal to about 0.25 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is about 1.0% of the composition. It is less than a weight percentage.

[0114] In some embodiments, the pharmaceutical component is as set forth in any of Tables 3-6 and further comprises cesium. In some embodiments, the cesium is present in an amount of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, Present in an amount of 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 weight percent.

[0115] 3.4 Treatment method In some embodiments, the present invention provides methods for treating cancer in a subject in need thereof, comprising administering to the subject a provided IT-139 composition as described above and herein. In some such embodiments, the subject is a human patient.

[0116] In some embodiments, the present invention provides methods for treating cancer in a subject in need thereof, comprising administering a provided composition of IT-139 as described above and herein in combination with a chemotherapeutic agent.

[0117] In some embodiments, the present invention provides methods for treating cancer in a subject in need thereof, comprising administering a provided composition of IT-139 described above and herein in combination with an immuno-cancer agent.

[0118] According to another embodiment, the present invention relates to a method of treating cancer selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver and biliary tract, kidney cancer, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cell, buccal cavity and pharynx (oral cavity), lip, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, and leukemia, comprising administering IT-139 or a pharmaceutically acceptable composition thereof.

[0119] According to another embodiment, the present invention relates to a method of treating cancer selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver and biliary tract, kidney cancer, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cell, buccal cavity and pharynx (oral cavity), lip, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, and leukemia, comprising administering IT-139 or a pharmaceutically acceptable composition thereof in combination with a chemotherapeutic agent.

[0120] According to another embodiment, the present invention provides a method for treating cancer selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract, kidney cancer, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cell, buccal cavity and pharynx (oral cavity), lip, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, and leukemia. The present invention relates to a method for treating cancer, comprising administering IT-139 or a pharmaceutically acceptable composition thereof in combination with an immuno-cancer agent.

[0121] Another embodiment provides a method for treating cancer by reducing the amount of GRP78 in cancer cells following administration of IT-139.

[0122] According to another embodiment, the present invention provides a method for treating cancer by reducing the amount of GRP78 in cancer cells following administration of IT-139 in combination with a chemotherapeutic agent, wherein administration of IT-139, or a pharmaceutically acceptable composition thereof, results in a reduction in the amount of GRP78 compared to administration of the chemotherapeutic agent.

[0123] According to another embodiment, the present invention provides a method for treating cancer by reducing the amount of GRP78 in cancer cells following administration of IT-139 in combination with an immuno-oncology agent, wherein administration of IT-139, or a pharmaceutically acceptable composition thereof, results in a reduction in the amount of GRP78 compared to administration of the immuno-oncology agent alone.

[0124] The order in which therapeutic agents are administered should be carefully considered. While not wishing to be bound by any particular theory, the mechanism of action and downregulation of GRP78 suggests that for maximum therapeutic efficacy, any chemotherapeutic agent should be administered first, followed by IT-139. As mentioned above, treatment with a series of chemotherapeutic agents results in ER stress, which induces the production of GRP78. This process is a cell survival mechanism. Administration of IT-139 reduces stress-induced GRP78 levels, eliminating cell survival pathways. The net result is increased cancer cell death and enhanced antitumor efficacy.

[0125] According to one embodiment of the present invention, there is provided a method for treating cancer in a patient in need thereof, comprising: 1) administering a chemotherapeutic agent to a patient; 2) subsequently administering IT-139 or a pharmaceutically acceptable composition thereof to the patient; 3) optionally repeating steps 1 and 2; The present invention provides a method comprising:

[0126] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered to a patient one day after the chemotherapy. In other embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered to a patient one day after the chemotherapy. In yet other embodiments, IT-139 is administered to a patient one to seven days after the chemotherapy.

[0127] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered simultaneously with a chemotherapeutic agent, ie, IT-139 or a pharmaceutically acceptable composition thereof and a chemotherapeutic agent are administered within about 20 to 28 hours of each other, or within about 22 to 26 hours of each other, or within about 24 hours of each other.

[0128] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered prior to the chemotherapeutic agent, ie, at least about 8 to 16 hours, at least about 10 to 14 hours, or at least about 12 hours prior to the chemotherapeutic agent.

[0129] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 20-28 hours before the chemotherapy agent or at least about 22-28 hours after the chemotherapy agent. It is administered 6 hours before, or at least about 24 hours before, the chemotherapy agent.

[0130] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 44-52 hours before the chemotherapeutic agent, or at least about 46-50 hours before the chemotherapeutic agent, or at least about 48 hours before the chemotherapeutic agent.

[0131] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 64 to 80 hours before the chemotherapeutic agent, or at least about 70 to 74 hours before the chemotherapeutic agent, or at least about 72 hours before the chemotherapeutic agent.

[0132] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered before the chemotherapeutic agent, or at least about 8 to 16 hours after the chemotherapeutic agent, or at least about 10 to 14 hours after the chemotherapeutic agent, or at least about 12 hours after the chemotherapeutic agent.

[0133] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 20-28 hours after the chemotherapy agent, or at least about 22-26 hours after the chemotherapy agent, or at least about 24 hours after the chemotherapy agent.

[0134] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 44-52 hours after the chemotherapy agent, or at least about 46-50 hours after the chemotherapy agent, or at least about 48 hours after the chemotherapy agent.

[0135] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 64 to 80 hours after the chemotherapy agent, or at least about 70 to 74 hours after the chemotherapy agent, or at least about 72 hours after the chemotherapy agent.

[0136] In certain embodiments, the chemotherapeutic agent is selected from the group consisting of gemcitabine, nanoparticle albumin paclitaxel, paclitaxel, docetaxel, cabazitaxel, oxaliplatin, cisplatin, carboplatin, doxorubicin, daunorubicin, sorafenib, everolimus, and vemurafenib. In certain embodiments, the chemotherapeutic agent is gemcitabine.

[0137] According to one embodiment of the present invention, there is provided a method for treating pancreatic cancer in a patient in need thereof, comprising: 1) administering gemcitabine and albumin nanoparticle paclitaxel; 2) subsequently administering IT-139 or a pharmaceutically acceptable composition thereof; and 3) optionally repeating steps 1 and 2. The present invention provides a method comprising:

[0138] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered simultaneously with gemcitabine. In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof and gemcitabine are administered within about 20 to 28 hours of each other, or within about 22 to 26 hours of each other, or within about 24 hours of each other.

[0139] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered before gemcitabine, ie, at least about 8 to 16 hours before gemcitabine, or at least about 10 to 14 hours before gemcitabine, or at least about 12 hours after gemcitabine. It is administered before

[0140] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 20-28 hours before gemcitabine, or at least about 22-26 hours before gemcitabine, or at least about 24 hours before gemcitabine.

[0141] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 44-52 hours before gemcitabine, or at least about 46-50 hours before gemcitabine, or at least about 48 hours before gemcitabine.

[0142] According to one embodiment of the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering IT-139 or a pharmaceutically acceptable composition thereof in combination with an immuno-oncology agent. In certain embodiments, the immuno-oncology agent is administered to the patient prior to administration of IT-139 or a pharmaceutically acceptable composition thereof.

[0143] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered simultaneously with the immuno-oncology agent. In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof and the immuno-oncology agent are administered within about 20 to 28 hours of each other, or within about 22 to 26 hours of each other, or within about 24 hours of each other.

[0144] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered prior to the immuno-cancer agent, ie, at least about 8-16 hours, at least about 10-14 hours, or at least about 12 hours prior to the immuno-cancer agent.

[0145] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 20-28 hours before the immuno-cancer agent, or at least about 22-26 hours before the immuno-cancer agent, or at least about 24 hours before the immuno-cancer agent.

[0146] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 44-52 hours before the immuno-cancer agent, or at least about 46-50 hours before the immuno-cancer agent, or at least about 48 hours before the immuno-cancer agent.

[0147] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 64 to 80 hours prior to the immuno-oncology agent, or at least about 70 to 74 hours prior to the immuno-oncology agent, or at least about 72 hours prior to the immuno-oncology agent.

[0148] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered after the immuno-cancer agent, ie, at least about 8 to 16 hours after the immuno-cancer agent, at least about 10 to 14 hours after the immuno-cancer agent, or at least about 12 hours after the immuno-cancer agent.

[0149] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 20-28 hours after the immuno-oncology agent, or at least about 22-26 hours after the immuno-oncology agent, or at least about 24 hours after the immuno-oncology agent.

[0150] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is The immuno-oncology agent is administered at least about 44 to 52 hours after the immuno-oncology agent, or at least about 46 to 50 hours after the immuno-oncology agent, or at least about 48 hours after the immuno-oncology agent.

[0151] In certain embodiments, IT-139 or a pharmaceutically acceptable composition thereof is administered at least about 64 to 80 hours after the immuno-oncology agent, or at least about 70 to 74 hours after the immuno-oncology agent, or at least about 72 hours after the immuno-oncology agent.

[0152] In certain embodiments, the immuno-oncology agent is selected from the group consisting of a cytokine, a checkpoint inhibitor, and an antibody other than a PD-1 antibody, hi certain embodiments, the immuno-oncology agent is selected from the group consisting of an interferon, an interleukin, a PD-L1 antibody, alemtuzumab, ipilimumab, ofatumumab, atezolizumab, and rituximab.

[0153] According to one embodiment of the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering IT-139 or a pharmaceutically acceptable composition thereof in combination with a PD-1 antibody. In certain embodiments, the PD-1 antibody is administered prior to administration of IT-139 or a pharmaceutically acceptable formulation thereof.

[0154] According to one embodiment of the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering IT-139, or a pharmaceutically acceptable composition thereof, in combination with a PD-L1 antibody. In certain embodiments, the PD-L1 antibody is administered prior to administration of IT-139, or a pharmaceutically acceptable formulation thereof.

[0155] According to one embodiment of the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering IT-139 or a pharmaceutically acceptable composition thereof in combination with an immuno-oncology agent other than a PD-1 antibody. In certain embodiments, the immuno-oncology agent other than a PD-1 antibody is administered prior to administration of IT-139 or a pharmaceutically acceptable formulation thereof. [Example]

[0156] In order that the invention described herein may be more fully understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the invention in any way.

[0157] Analysis method The following analytical methods were utilized to characterize the compounds of the present invention.

[0158] HPLC Method 1: Quantitation and identity of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] sodium were determined by high-pressure liquid chromatography with UV detection at 292 nm. The IT-139 drug was dissolved in water at a concentration of 1 mg / mL, and 10 μL was injected onto an Agilent Zorbax SB-C18 (3 μm, 4.6 × 150 mm) HPLC column. Mobile phase A consisted of 0.1% trifluoroacetic acid in water, and mobile phase B consisted of 0.1% trifluoroacetic acid in acetonitrile. Separation was achieved by a gradient flow of 1.0 mL / min, with mobile phase A going from 90% to 10% from time zero to 12 min, and mobile phase B going from 10% to 90% over 12 min. The gradient was then reversed from 10% mobile phase A and 90% mobile phase B to 90% A and 10% B from 12 to 13 min. This continued until the end of the 15 minute run. The analyte retention time was 7.7 minutes. The sample temperature was maintained at 5°C and the column temperature was maintained at 25°C.

[0159] HPLC Method 2: Two HPLC methods were used because two different impurities co-elute using HPLC Method 1. Related substances in the IT-139 drug were determined by high-pressure liquid chromatography with UV detection at 292 nm. The IT-139 drug was dissolved in water at a concentration of 1 mg / mL, and 10 μL was injected onto an Agilent Zorbax SB-C18 (3 μm, 4.6 × 150 mm) HPLC column. Mobile phase A consisted of 0.1% trifluoroacetic acid in water, and mobile phase B consisted of 0.1% trifluoroacetic acid in acetonitrile. Separation was achieved by a gradient flow of 1.0 mL / min, with mobile phase A going from 90% to 10% from time zero to 15 min, and mobile phase B going from 10% to 90% over 15 min. The gradient was held at 10% A and 90% B from 15 min to 19.9 min. The gradient was then reversed to 90% A and 10% B from 19.9 to 20 minutes and held until the end of the run at 26 minutes. The sample temperature was maintained at 5°C and the column temperature was maintained at 25°C.

[0160] HPLC Method 3: Analysis of Formula Ib from Compound A, Compound B, and Compound C without degradation uses high-pressure liquid chromatography with UV detection at 297 nm. IT-139 drug substance was dissolved in water at a concentration of 0.5 mg / mL, and 10 μL was injected onto a Phenomenex Luna, Phenyl-Hexyl (150 × 3 mm × 3 μm) HPLC column. The mobile phase consisted of 25% (vol / vol) methanol in 20 mM ammonium acetate buffer and 4 mM acetic acid. Separation was achieved by maintaining the column temperature at 25°C and using an isocratic flow of 1.0 mL / min for a total run time of 27 minutes.

[0161] Elemental Analysis: Galbraith Laboratories (Knoxville, TN) performed all elemental analysis measurements. Carbon, hydrogen, and nitrogen analyses were performed using standard operating procedure ME-14, which requires weighing 1-5 mg into a tin capsule and then combusting at 920-980°C in a PerkinElmer 2400 Series II CHNS / O analyzer. Sodium and ruthenium analyses were performed by inductively coupled plasma atomic emission spectroscopy using standard operating procedure ME-70 and an ICP-OES Optima 5300 instrument. Cesium analyses were performed by inductively coupled plasma atomic emission spectroscopy using standard operating procedure ME-30.

[0162] X-ray diffraction: X-ray data were collected on a Bruker D8 Venture single crystal diffractometer equipped with a PHOTON 100 CMOS detector, an IμS copper MX source, and an Oxford Cryostream Plus cryogenic device, or on a Bruker Smart Apex2 single crystal diffractometer using copper radiation with room temperature data collection.

[0163] Example 1 Purification of Ruthenium Chloride RuCl3.xH2O (100.0 g) was dissolved in 600 mL of concentrated HCl and 99% ethanol. The resulting concentrated ruthenium chloride solution remaining in the distillation flask was cooled to ambient temperature and filtered through a medium-porosity glass Buchner funnel, the Buchner funnel and flask were rinsed with concentrated HCl, and the combined filtrate was diluted with additional concentrated HCl to a total volume of approximately 500 mL.

[0164] Example 2 Preparation of indazolium salts [ka] 300.0 g (2.54 mol; 6.64 equiv.) of 1H-indazole was combined with 800 mL of water. 4 L of concentrated HCl was added, and the mixture was stirred until dissolved (20 min). This indazole solution was charged into a 15 L jacketed stirred glass and Teflon reactor equipped with a large, efficient paddle stirrer, an internal thermoprobe, an air-cooled reflux condenser with a gas outlet at the top (for HCl gas release), and a 0.5 L addition funnel with a stem extended with polyethylene tubing. An additional 4.0 L of concentrated HCl was combined with the indazole solution in the reactor, and the mixture was stirred and heated until the internal temperature reached 90°C. The stirring speed was then increased to 250 rpm, and the temperature was maintained at 90°C for at least 30 minutes. The RuCl3 solution from Example 1 was then carefully added dropwise over a period of approximately 5 hours from a funnel with a stem extended with polyethylene tubing, while maintaining high-speed stirring at 250 rpm. After complete addition, the addition funnel was rinsed with a small amount of concentrated HCl (2 x 50 mL), and the rinse was also added to the reactor. The combined volume of the reaction mixture was 9.5 L, and the product precipitated in the form of tan microcrystalline flakes. After complete addition, the reaction was stirred at 250 rpm at 90°C for an additional 10 hours. The reaction mixture was cooled to 25°C with stirring and transferred to a polyethylene plastic bucket through the bottom drain valve. The precipitated product was collected by filtration through a medium-porosity 3 L glass filter funnel. The reactor and stirring paddle were filled with 2M The HCl wash was added to the material on the filter funnel. The resulting solid was thoroughly rinsed with approximately 2 L of additional 2 M HCl and then partially dried overnight under suction. This gave 598 g of the crude indazolium salt, wet with the remaining 2 M HCl, as a brown, sticky solid. HPLC analysis: Method 1: 97.7%, Method 2: 98.0%.

[0165] Example 3 Preparation of cesium salts [ka] A 10 L wide-mouth flask was charged with the wet indazolium salt from Example 2, and 180.0 g (1.07 mol; 2.8 equiv.) of solid powdered CsCl was added. Pure, non-denatured ethanol 99% (1.8 L) was combined with MEK (2.0 L) and then combined with the mixture of indazolium salt and CsCl in the 10 L flask. The mixture was mechanically stirred at 22 °C using a Teflon paddle. High-speed stirring was performed, first at 200 rpm for 5 minutes and then at 700 rpm for 2 hours. The resulting orange slurry was collected by filtration using a medium-pore glass Buchner funnel (3 L). The solid was thoroughly washed with 99% ethanol, and the filter cake material was partially dried under suction for approximately 1 hour. The resulting material, containing the cesium salt in the form of an orange MEK solvate mixed with the remaining CsCl, was transferred to a large 4 L beaker. One liter of a 2:1 (v / v) mixture of ethanol and water was added to the crude Cs salt solid in a beaker. The slurry was mechanically stirred in an open beaker at 350 rpm for 15 minutes. During this time, the bright orange color of the MEK solvate slurry changed to the cinnamon-red color of the hydrate. The solid was collected by filtration using the same Buchner funnel previously used to filter the Cs salt. The resulting solid was thoroughly washed with approximately 1 liter of 99% ethanol. The material was dried by suction and then in vacuo for 14 hours (overnight). The result was 226.90 g (0.350 mol) of a cinnamon-red solid. HPLC analysis: no free indazole detected; Method 1: 98.6% purity; Method 2: 99.0% purity. Found elemental analysis results: Cs: 21.6%, Ru: 16.6%, Cl: 21.96%. Theoretical values ​​for dihydrate: Cs: 20.5%, Ru: 15.6%, Cl: 21.88%. Theoretical values ​​for monohydrate: Cs: 21.1%, Ru: 16.0%, Cl: 22.51%. X-ray diffraction analysis of a single crystal from a vacuum-dried sample showed an occupancy density of approximately 50% for two hydration water molecules in the crystal structure.

[0166] Example 4 Preparation of the sodium salt [ka] 1000 g of solid Al2(SO4)3.18H2O (3.0 mol Al) was slowly added to stirred DI water (2.0 L), followed by 13.0 g of solid Na2SO42 (3.0 mol Na). The mixture was stirred to complete dissolution (approximately 30 min), and the total volume of the solution was adjusted to 2.7 L by adding DI water. Prior to use, the resulting 1.1 M solution was filtered through a fine 0.45 micron SteriCup Durapore membrane to yield 2.7 L of a 1.1 M solution of NaAl(SO4)2. This 1.1 M NaAl(SO4)2 solution was combined with 226.9 g of Cs salt (0.350 mol) from Example 5 in a 4 L beaker. 6.0 g of solid powdered CsCl was added to the mixture to cause the formation of Cs alum. The mixture was magnetically stirred using a large Teflon-coated rod stirrer at ambient temperature for 30 hours. During this time, the reddish-brown slurry of cesium salts changed to a coffee-brown slurry of IT-139 mixed with fine, white, salt-like crystals of cesium aluminum salts. The solid was collected by filtration using a medium-bore Buchner funnel (3 L). The reaction flask and solid were thoroughly washed with a total of approximately 1.5 L of saturated (=1.5 M) aqueous Na2SO4 (in three portions (3 x 0.5 L) until the filtrate was colorless). The solid was dried by suction on the Buchner funnel and then dried in vacuo for at least 1 day. The thoroughly dried solid was transferred to a 2 L wide-mouth Erlenmeyer flask along with 700 mL of acetonitrile. The mixture was mechanically stirred for 15 minutes. The resulting orange slurry was filtered on a medium-bore Buchner funnel to remove insoluble sulfate salts from the mixture. The salt cake was rinsed with an additional 300 mL of acetonitrile (3 × 100 mL, until colorless) and then discarded. The combined orange filtrate in the 5 L round-bottom flask was diluted with 4 L of MTBE (four 1 L portions added with gentle stirring), and the flask was allowed to stand for 30 min to complete the precipitation. The precipitated crude Na salt was collected by filtration, rinsed thoroughly with MTBE (2 × 0.5 L), and then dried under suction and vacuum. The result was 190.4 g (100% of theory, calculated as the dihydrate) of crude product as a fluffy brown solid, retaining MTBE in the form of a solvate, with an HPLC purity of 98.4% by Method 1.Elemental analysis indicates that the product contains 0.1-0.8 wt% cesium. The structure of the product was confirmed by x-ray diffraction.

[0167] Example 5 Removal of residual cesium 190.4 g of material from Example 4 was transferred to a dry 10 L flask. An equal weight of activated 4A molecular sieve powder (191 g) was added. [Aldrich 688363-1KG, sodium aluminosilicate, "SYLOSIV A4" (manufactured by Grace Davidson)]. Methyl ethyl ketone (4.2 L) was added to the flask and the mixture was mechanically stirred. Methanol (600 mL) was added to the stirred slurry over a 5 minute period. The mixture was slowly added over a period of time. Stirring (800 rpm) was continued for 30 minutes, at which time nearly all of the dark brown clumps of material had dissolved. The resulting orange slurry was filtered through a Whatman glass fiber GF-B filter disk placed on top of a fine-porosity Buchner funnel. The spent molecular sieves were rinsed with an additional 0.4 L (2 x 200 mL) of MEK and discarded. The combined filtrate was precipitated by the slow addition of 10 L of MTBE with mechanical stirring. Stirring was stopped, and the mixture was allowed to precipitate for 30 minutes. The precipitated product was collected by filtration (3 L Buchner funnel), rinsed thoroughly with 1 L of MTBE (2 x 0.5 L), and dried by suction for approximately 2 hours, until the Buchner funnel was no longer cold. This yielded 184 g of purified sodium salt. The purified material was treated with wet MTBE to remove traces of solvent. 184 g of the purified sodium salt was combined with 3.3 L of wet MTBE (water-saturated MTBE) in a 5 L wide-mouth Erlenmeyer and mechanically stirred (200 rpm) for 40 minutes. The resulting brown solid was collected by filtration. The solid was rinsed with wet MTBE, dried by suction, and then thoroughly dried overnight (15 hours) in vacuo. The result was 176.11 g of a coffee-brown, granular, heavy solid, 98.7% pure by HPLC (Method 1). This corresponds to 85% of the overall yield from RuCl3.xH2O (starting from Example 1). Elemental analysis determined that 35-750 ppm cesium remained.

[0168] Example 6 Solution Stability Test The compound from Example 5 was prepared at room temperature (20°C) using the room temperature solution and refrigerated (2-8°C) using the cold (2-8°C) solution. A total volume of 500 mL was obtained for each. A citric acid solution was prepared by dissolving 19.2 grams of citric acid in 1 L of water. A sodium citrate solution was prepared by dissolving 29.4 grams of sodium citrate in 1 L of water. The sodium citrate solution was added to the citric acid solution until the pH increased from 2.0 to 3.4. A mannitol solution was prepared by dissolving 13.3 g of mannitol in 200 mL of water. A solution of Formula Ib was prepared by adding 16.6 mL of citrate buffer to 400 mL of water. 3.33 g of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] was added to the solution and stirred for 10 minutes. 50 mL of mannitol solution was added, followed by 33.4 mL of water, to a final volume of 500 mL of IT-139 bulk solution. Samples were stirred at room temperature (18-22 °C) using a magnetic stir plate on a laboratory bench, and refrigerated samples were stirred using a magnetic stir plate in a refrigerator (2-8 °C). 100 μL aliquots were taken from each sample immediately after dissolution (T = 0) and at 0.5, 1, 2, 3, 4, 5, 6, 18, 24, 32, and 48 h. Each sample was added to 1.9 mL of methanol in an HPLC vial and mixed by vortexing. The purity of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in the bulk solution stored at room temperature (18-22 °C) decreased from 96.8% to 12.1% after 18 h. The purity of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in bulk solution stored refrigerated (2-8°C) decreased from 97.05% to 95.4% after 18 hours and to 89.8% after 48 hours. Figure 1 shows the percentage of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] after 18 hours at room temperature (18-22°C) and 48 hours refrigerated (2-8°C).Table 7 shows the percentage of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] sodium (RT 7.7 min) and impurities (sum of RRTs 0.7, 1.9, and unspecified RRTs) in each sample based on HPLC peak area. HPLC chromatograms for IT-139 samples stored at refrigerated or room temperature at 18 hours are shown in Figures 2 and 3, respectively. [Table 7]

[0169] Example 7 Preparation of Compound A [ka] Indazole (400 mg, 3.40 mmol, 1 equiv.) was dissolved in 2 mM HCl (1.6 L) in a large beaker at 80 °C. The solution was cooled to room temperature and then diluted with Na[Ru] as an aqueous solution (400 mL HO). III Cl4(Hind)2 (1.8 g, 3.40 mmol, 1 equiv.) was added. The resulting brownish-red solution was stirred for 5 min and then allowed to stand without stirring. After 1 day, crystals began to form at the bottom of the beaker. After a total of 3 days, a significant amount of crystals had formed, which were collected by vacuum filtration, washed with HO (2 x 350 mL), and dried under reduced pressure overnight to give Ru. III Cl3(Hind)2(H2O) (930 mg, 59.2%) was obtained as dark red crystals. The product was suitable for x-ray crystallography, which was used to confirm the structure.

[0170] Example 8 Preparation of Compound C [ka] In a 100 mL round-bottom flask equipped with a reflux condenser, add Ru IIICl3(Hind)2(HO) (100 mg, 0.217 mmol) and CH3CN (6 mL) were added. The resulting dark red suspension was heated to 50 °C. After several hours, the material was solubilized and stirred at 50 °C. After 4 days, a noticeable precipitate formed in the reaction flask. The crude reaction mixture was centrifuged to give a dark brown solid, which was washed with cold Et2O (three times, isolated each time via centrifugation). The resulting light brown powder was >95% pure (HPLC analysis). A small amount (approximately 40 mg) of the product was dissolved in a minimal amount of CH3CN (approximately 20 mL), sonicated to dissolve, and then sealed in a vial. After 1–2 days, diffraction-quality red crystals formed, and the structure was confirmed by X-ray crystallography.

[0171] Example 9 Preparation of Compound D [ka] In a 50 mL round-bottom flask equipped with a reflux condenser, add Hind[Ru III The mixture was charged with Cl4(Hind)2] (198 mg, 0.331 mmol) and THF (10 mL). The resulting brownish-red suspension was briefly sonicated to break up large chunks of material. The reaction mixture was heated to reflux, and after 20 minutes, the material was completely solubilized, yielding a dark red solution. After an additional 20 minutes of refluxing, the reaction mixture was cooled to room temperature, and various aliquots (0.1–1.0 mL) were diluted with various amounts of EtO or MTBE (1–15 mL). After 1–2 days, several crystallization trials produced dark red crystals. The most successful attempts involved a dilution conversion factor of 1:2–3 (i.e., 1 volume of reaction mixture was diluted with 2–3 volumes of either EtO or MTBE). The crystals were washed with either cold EtO or cold MTBE, depending on the antisolvent used. This yielded red crystals, which were used to confirm the structure via X-ray crystallography.

[0172] Example 10 IT-139 Formulation Process IT-139 was prepared in the cold using a cold (2-8°C) solution to a total volume of 1 L. Citrate buffer was prepared by adding sodium citrate solution (29.4 g / L in water) to citric acid solution (19.2 g / L in water) until the pH increased from 2.0 to 3.4. A working citrate buffer solution was then prepared by adding 33 mL of citrate buffer to 767 mL of water. 6.66 grams of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] was added to 800 mL of cold (2-8°C) working citrate buffer solution and stirred using a magnetic stir plate and stir bar for 20 minutes while the solution cooled to 2-8°C. A working mannitol solution was prepared by dissolving 13.3 g of mannitol in 200 mL of water. 100 mL of cold (2-8 °C) mannitol solution was added to 800 mL of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] dissolved in working citrate buffer and stirred for 5 min while cooling the solution to 2-8 °C. 100 mL of cold (2-8 °C) water was added to the drug solution to a final volume of 1 L. The IT-139 drug bulk solution was filtered through a 0.22 μm pass-through filter and aseptically filled into lyophilization vials, with a final fill volume of 30 mL in 50 mL clear glass vials. The vials were partially stoppered and loaded into a lyophilizer with shelves pre-cooled to -5 °C. The lyophilization cycle consisted of 3 hours of freezing at -40°C, primary drying for 15 hours at 0.1 mbar and -10°C, then 10 hours at 0.1 mbar and -5°C, and secondary drying for 2 hours at 0.05 mbar and 5°C, then 2 hours at 0.05 mbar and 10°C, then 2 hours at 0.05 mbar and 15°C, then 2 hours at 0.05 mbar and 20°C, for a total drying time of 36 hours. Vials were fully stoppered, sealed and stored at -20°C.

[0173] Example 11 IT-139 Formulation Process 2 IT-139 was prepared in the cold using a cold (2-8°C) solution to a total volume of 2.8 L of the drug. Citrate buffer was prepared by adding sodium citrate solution (29.4 g / L in water) to citric acid solution (19.2 g / L in water) until the pH increased from 2.0 to 3.4. A working citrate buffer solution was then prepared by adding 9.3 g of citrate buffer to 1960 g of water. 36.3 g of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] was added to 1969.3 grams of the cold (2-8°C) working citrate buffer solution and stirred using a magnetic stir plate and stir bar for 20 minutes while the solution cooled to 2-8°C. A working mannitol solution was prepared by dissolving 35 g of mannitol in 525 mL of water. 525 mL of cold (2-8°C) mannitol solution was added to the solution of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] sodium in working citrate buffer and stirred for 5 minutes while the solution was cooled to 2-8°C. 300 mL of cold (2-8°C) water was added to the drug solution to a final volume of 2.8 L. The IT-139 drug bulk solution was filtered through a 0.22 μm pass-through filter and aseptically filled into lyophilization vials, with a final fill weight of 25.1 grams in 50 mL clear glass vials. The vials were partially stoppered and loaded into a lyophilizer with the shelf pre-cooled to -5°C. The lyophilization cycle The vials consisted of freezing at -40°C for 6 hours, primary drying at 0.2 mbar and -10°C for 50 hours, and secondary drying at 0.2 mbar and 30°C for 33 hours. The vials were backfilled with nitrogen, fully stoppered, sealed, and stored at 4°C.

[0174] Example 12 Batch Analysis of IT-139 Drug Substance trans-[tetrachlorobis(1H-indazole)ruthenate(III)]sodium and Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru IIIBatch analytical data for drug substance containing Cl3(Hind) (HN=C(Me)ind) and cesium are reproduced in Table 8. [Table 8-1] [Table 8-2] The present invention provides, for example, the following items. (Item 1) A composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] and cesium. (Item 2) Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III Item 1. The composition of claim 1, further comprising Cl3(Hind) (HN=C(Me)ind). (Item 3) the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is greater than or equal to about 95.5 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is less than or equal to about 1.0 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 2.5 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 2.0 weight percent of the composition; Cesium is less than or equal to about 0.5 weight percent of the composition; Item 2. The composition according to item 2. (Item 4) the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; cesium is about 0.0001 to about 0.5 weight percent of the composition; The composition optionally comprises Ru III Cl3(Hind)2(H2O), Ru III Cl3(Hind)2(CH3CN), and Ru III Cl3(Hind) (HN=C(Me)ind), wherein: Ru III Cl3(Hind)2(H2O) is from about 0 to about 1.0 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is from about 0 to about 2.5 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is about 0 to about 2.0 weight percent of the composition; Item 1. The composition according to item 1. (Item 5) the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is about 0.001 to about 1 0.0 weight percentage, Ru III Cl3(Hind)2(CH3CN) is from about 0.001 to about 2.5 weight percent of the composition; Ru III Cl(Hind) (HN=C(Me)ind) is from about 0.001 to about 2.0 weight percent of the composition; Cesium is about 0.0001 to about 0.5 weight percent of the composition; Item 3. The composition according to item 3. (Item 6) the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; Ru IIICl(Hind)(H0) is from about 0.001 to about 0.75 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is from about 0.001 to about 1.5 weight percent of the composition; Ru III Cl(Hind) (HN=C(Me)ind) is from about 0.001 to about 1.25 weight percent of the composition; Cesium is about 0.0001 to about 0.25 weight percent of the composition; Item 6. The composition according to item 5. (Item 7) the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 95.5 to about 99.9 weight percent of the composition; Ru III Cl3(Hind)2(H2O) is about 0.001 to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is about 0.001 to about 0.5 weight percent of the composition; Ru III Cl(Hind) (HN=C(Me)ind) is about 0.001 to about 0.5 weight percent of the composition; Cesium is about 0.0001 to about 0.01 weight percent of the composition. Item 7. The composition according to item 6. (Item 8) Formula Ia: [ka] Compound. (Item 9) A composition comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, and mannitol. (Item 10) mer, trans-[Ru III10. The composition of claim 9, further comprising Cl3(Hind)2(H2O)]. (Item 11) 11. The composition of claim 10, further comprising a cesium salt. (Item 12) Item 12. The composition of claim 11, wherein the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is amorphous. (Item 13) mer, trans-[Ru III Cl(Hind)(H2O)] is from about 0.01 to about 0.4 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; Item 13. The composition according to item 12. (Item 14) mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.3 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; Item 14. The composition according to item 13. (Item 15) mer, trans-[Ru III Cl(Hind)(H0)] is from about 0.01 to about 0.2 weight percent of the composition; Cesium is about 0.00001 to about 0.01 weight percent of the composition; Item 15. The composition according to item 14. (Item 16) 16. The composition according to any one of items 9 to 15, wherein the composition is a lyophilized powder. (Item 17) sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is about 0.001 to about 0.25 weight percent of the composition; The composition according to any one of items 9 to 16. (Item 18) Ru III Cl3(Hind)2(H2O) and Ru III Cl3(Hind)2(CH3CN) and Ru III 18. The composition according to any one of items 9 to 17, further comprising Cl3(Hind) (HN=C(Me)ind). (Item 19) Ru III Cl3(Hind)2(H2O) is less than or equal to about 0.5 weight percent of the composition; Ru III Cl3(Hind)2(CH3CN) is less than or equal to about 1.25 weight percent of the composition; Ru III Cl3(Hind) (HN=C(Me)ind) is less than or equal to about 1.0 weight percent of the composition; Item 19. The composition according to item 18. (Item 20) Compounds of formula Ia: [ka] 1. A method for preparing The method comprises reacting trans-[tetrachlorobis(1H-indazole)ruthenate(III)]indazolium with cesium chloride in a suitable solvent. (Item 21) Compounds of formula Ib: [ka] 1. A method for preparing Compounds of formula Ia: [ka] under conditions suitable to effect salt exchange. (Item 22) 21. A method for treating cancer in a patient in need thereof, the method comprising administering IT-139, or a pharmaceutically acceptable salt thereof, of the composition described in any of items 1 to 20.

Claims

1. A composition for treating cancer in a patient in need thereof, comprising: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], sodium citrate, citric acid, mannitol, mer, trans-[Ru III Cl 3 (Hind) 2 (H 2 O)], Cesium salts Including, The composition, wherein the sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is amorphous.

2. The composition of claim 1, wherein mer,trans-[Ru III Cl 3 (Hind) 2 (H 2 O)] is about 0.01 to about 0.4 weight percent of the composition; cesium is about 0.00001 to about 0.01 weight percent of the composition; The composition of claim 1.

3. The composition of claim 2, wherein mer,trans-[Ru III Cl 3 (Hind) 2 (H 2 O)] is about 0.01 to about 0.3 weight percent of the composition; cesium is about 0.00001 to about 0.01 weight percent of the composition; The composition of claim 2.

4. The composition of claim 1, wherein mer,trans-[Ru III Cl 3 (Hind) 2 (H 2 O)] is about 0.01 to about 0.2 weight percent of the composition; cesium is about 0.00001 to about 0.01 weight percent of the composition; The composition of claim 3.

5. A composition described in any of claims 1 to 4, wherein the composition is a freeze-dried powder.

6. The composition of claim 1, wherein sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is about 40 to about 60 weight percent of the composition; mannitol is about 40 to about 60 weight percent of the composition; citric acid is about 0.01 to about 0.5 weight percent of the composition; sodium citrate is from about 0.001 to about 0.25 weight percent of the composition; The composition according to any one of claims 1 to 5.

7. The composition of claim 1, further comprising Ru III Cl 3 (Hind) 2 (H 2 O), Ru III Cl 3 (Hind) 2 (CH 3 CN), and Ru III Cl 3 (Hind) (HN═C(Me)ind).

8. The composition of claim 7, wherein the Ru III Cl 3 (Hind) 2 (H 2 O) is about 0.5 weight percent or less of the composition; the Ru III Cl 3 (Hind) 2 (CH 3 CN) is less than or equal to about 1.25 weight percent of the composition; the Ru III Cl 3 (Hind) (HN═C(Me)ind) is less than or equal to about 1.0 weight percent of the composition; The composition of claim 7.

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