Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
  • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
  • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
  • C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D237/14—Oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
  • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
  • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
  • C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D237/14—Oxygen atoms
  • C07D237/16—Two oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/13—Crystalline forms, e.g. polymorphs
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• Thyroid hormones are critical for normal growth and development and for maintaining metabolic homeostasis (Paul M. Yen, Physiological reviews, Vol. 81(3): pp. 1097-1126 (2001)). Circulating levels of thyroid hormones are tightly regulated by feedback mechanisms in the hypothalamus/pituitary/thyroid (HPT) axis. Thyroid dysfunction leading to hypothyroidism or hyperthyroidism clearly demonstrates that thyroid hormones exert profound effects on cardiac function, body weight, metabolism, metabolic rate, body temperature, cholesterol, bone, muscle and behavior.
• HPT hypothalamus/pituitary/thyroid
• TRs thyroid hormone receptors
• THRs thyroid hormone receptors
• TRs belong to the superfamily known as nuclear receptors. TRs form heterodimers with the retinoid receptor that act as ligand-inducible transcription factors. TRs have a ligand binding domain, a DNA binding domain, and an amino terminal domain, and regulate gene expression through interactions with DNA response elements and with various nuclear co-activators and compressors.
• the thyroid hormone receptors are derived from two separate genes, a and ⁇ . These distinct gene products produce multiple forms of their respective receptors through differential RNA processing.
• Thyroid hormone receptors al, ⁇ and ⁇ 2 bind thyroid hormone. It has been shown that the thyroid hormone receptor subtypes can differ in their contribution to particular biological responses. Recent studies suggest that TR i plays an important role in regulating TRH (thyrotropin releasing hormone) and on regulating thyroid hormone actions in the liver. TR 2 plays an important role in the regulation of TSH (thyroid stimulating hormone) (Abel et. al., J. Clin. Invest., Vol 104: pp. 291-300 (1999)). TR i plays an important role in regulating heart rate (B. Gloss et. al. Endocrinology, Vol. 142: pp. 544-550 (2001); C.
• thyroid hormone mimetics may yield desirable reductions in body weight, lipids, cholesterol, and lipoproteins, with reduced impact on cardiovascular function or normal function of the hypothalamus/pituitary/thyroid axis (see, e.g., Joharapurkar et al., J. Med. Chem., 2012, 55 (12), pp 5649-5675).
• thyroid hormone analogs which avoid the undesirable effects of hyperthyroidism and hypothyroidism while maintaining the beneficial effects of thyroid hormones would open new avenues of treatment for patients with metabolic disease such as obesity, hyperlipidemia, hypercholesterolemia, diabetes and other disorders and diseases such as liver steatosis and NASH, atherosclerosis, cardiovascular diseases, hypothyroidism, thyroid cancer, thyroid diseases, resistance to thyroid hormone and related disorders and diseases.
• the present invention provides methods for synthesizing thyroid hormone analogs such as pyridazinone compounds and prodrugs thereof.
• An ideal method of synthesizing the thyroid hormone analogs and their prodrugs would, for example, provide product compounds in high purity and high yield.
• the present invention is directed at providing one or more of these desirable features.
• the present disclosure describes a synthetic process, which may be used to prepare 6- (4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one ("Int. 7”), a compound that is useful as an intermediate for making pyridazinone compounds as thyroid hormone analogs, as follows:
• R 1 is isopropyl or isopropenyl
• X is halo and R 2 is H or an amine protecting group
• the solvent can be an aprotic organic solvent, such as THF, diethyl ether, toluene, or dioxane
• the reaction temperature can be 0-60 °C, 20-50 °C, 30-45 °C, or 35-45 °C
• the reaction time can be 10 min to 10 hours, 1-8 hours, or 3-5 hours
• the amount of the Grignard reagent (R'MgX) can be 3-10 equivalents or 3-6 equivalents of the compound of Formula (I).
• the base is used to isomerize the compound of Formula (II). It can be an organic base or an inorganic base. Examples of bases include, but are not limited to, triethylamine, pyridine, KOH, NaOH, and carbonates. The isomerization can also be achieved under other conditions, e.g., treatment with an acid or heating in an aprotic solvent.
• the oxidizing agent is not particularly limited.
• amine protecting groups include, but are not limited to, substituted alkyl, acyl (e.g., benzoyl or acetyl) and silyl. Hydroxy and amine protecting groups have been discussed in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.
• step (a) is performed by contacting R'MgX with the compound of Formula (I), in which R 1 is isopropenyl and X is Br.
• the solvent used in this reaction can be THF with a volume to weight ratio of THF to the compound of Formula (I) ranging between 7 and 30 (or between 7 and 15). This step may be performed in the presence of a
• Lewis acid e.g., a lithium halide
• step (a) is performed by contacting R'MgX with the compound of Formula (I), in which R 1 is isopropyl and X is CI.
• the solvent used in this reaction can be THF with a volume to weight ratio of THF to the compound of Formula (I) ranging between 7 and 30 (or between 7 and 15).
• This step may be performed in the presence of a Lewis acid (e.g., a lithium halide).
• the base in step (b) is a metal hydroxide (e.g., potassium hydroxide).
• a metal hydroxide e.g., potassium hydroxide
• the oxidizing agent in step (b) is bromine and step (b) is performed in the presence of an acid.
• the R 2 group in Formula (I) and Formula (II) is acetyl or benzoyl.
• R 2 is benzoyl
• the process further comprises providing the compound of
• a polar aprotic solvent e.g., dimethylacetamide (DMAC)
• a base e.g., CS2CO 3
• step (a) the compound of
• Formula (I) is purified in an acidic solution at a temperature between 80 and 100 °C.
• the process further comprises step (c) when present, removing the amine protecting group R 2 of the compound of Formula (III) to form 6-(4-amino-2,6- dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one.
• the compound, e.g., Int. 7, made by the method described herein has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the compound, i.e., 6-(4-amino-2,6-dichlorophenoxy)-4- isopropylpyridazin-3(2H)-one, made by the method described herein has less than 1.5% of 6- (4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one, e.g., less than 1.0 % of 6-
• the compound made by the above-described process is free 6-(4-amino-2,6-dichlorophenoxy)-5-isopropylpyridazin-3(2H)-one.
• the synthetic process of this invention may further comprise the following step to synthesize pyridazinone compounds as thyroid hormone analogs and their prodrugs:
• R 3 is H or CH 2 R a , in which R a is hydroxyl, O-linked amino acid, -OP(0)(OH) 2 or
• Rb being lower alkyl, alkoxy, alkyl acid, cycloalkyl, aryl, heteroaryl, or -(CH 2 ) n -heteroaryl and n being 0 or 1 ;
• the compound of Formula (IV) is 2-(3,5-dichloro-4-((5- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile ("Compound A”) and the above step is performed by contacting 6-(4- amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one with ethyl (2- cyanoacetyl)carbamate and a metal nitrite followed by treatment with potassium acetate in DMAC.
• the process further comprises forming a morphic form of 2-(3,5- dichloro-4-((5 -isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 - tetrahydro- l,2,4-triazine-6-carbonitrile ("Compound A”) (Form I) characterized by an X-ray powder diffraction pattern including peaks at about 10.5, 18.7, 22.9, 23.6, and 24.7 degrees 2 ⁇ .
• Formula (IV) is of Formula (V)
• step (d) is performed by contacting 6-(4-amino-2,6-dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one with ethyl (2-cyanoacetyl)carbamate followed by treatment with potassium acetate in DMAC to form 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-l,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5- dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile ("Compound A”) and converting Compound A to the compound of Formula (V) in a suitable manner, e.g., using one of the techniques described in U.S. Patent 8,076,334.
• the compound of Formula (IV) e.g., 2-(3,5-dichloro-4-((5- isopropyl-6-oxo-l,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4- triazine-6-carbonitrile
• Compound A made by the method described herein has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the compound of Formula (IV) made by the method described herein is Compound A in Form I, and has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the compound of Formula (IV) made by the method described herein is Compound A in Form I, and Form I has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the composition comprising a compound of Formula (IV), such as Compound A, made by the method described herein has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of the corresponding -isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4-isopropyl-6-oxo-l,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5- dioxo-2,3,4,5-tetrahydro- 1 ,2,4-triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• a compound of Formula (IV) such as Compound A, made by the method described herein
• the composition comprising a compound of Formula (IV), such as Compound A, made by the method described herein is free of the corresponding ⁇ - isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4-isopropyl-6-oxo-l,6- dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• a compound of Formula (IV) such as Compound A
• the composition comprising a compound of Formula (IV), such as Compound A, made by the method described herein has less than 1.5% (e.g., less than 0.1 %) of heavy metal, e.g. , silver.
• the composition comprising a compound of Formula (IV), such as Compound A, made by the method described herein is free of heavy metal, e.g., silver, gold, or platinum.
• the synthetic methods described herein include advantages compared to the previous methods, such as those disclosed in U.S. Patent 7,452,882.
• the overall yield of 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-l,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo- 2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile (“Compound A") is greatly increased (e.g., > 40% versus -9% when made according to the method disclosed in U.S. Patent 7,452,882). Also, regioselectivity of the synthesis is far superior.
• the invention features a composition comprising greater than
• isopropylpyridazin-3(2H)-one regioisomer i.e., and/or has less than 1.5% of heavy metal.
• the compound of Formula (IV), e.g., Compound A has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the compound of Formula (IV) is Compound A in Form I, and has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the compound of Formula (IV) is Compound A in Form I, and Form I has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the content of impurities is less than 15%, less than 14%, less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.5%, or less than 0.2%.
• the compound of Formula (IV), such as Compound A has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of the corresponding ⁇ - isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4-isopropyl-6-oxo-l,6- dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• the compound of Formula (IV) has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of the corresponding ⁇ - isopropylpyridazin-3(2H)-one regioi
• the compound of Formula (IV), such as Compound A is free of the corresponding -isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• the -isopropylpyridazin-3(2H)-one regioisomer e.g., 2-(3,5-dichloro-4-((4- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phen
• the compound of Formula (IV), such as Compound A has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of heavy metal, e.g., silver, gold, or platinum.
• the compound of Formula (IV), such as Compound A, made by the method described herein is free of heavy metal, e.g., silver.
• the invention features a morphic form of 2-(3,5-dichloro-4-((5-isopropyl-6- oxo-l,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6- carbonitrile ("Compound A”) (Form I) characterized by an X-ray powder diffraction ("XRPD”) pattern including peaks at about 10.5, 18.7, 22.9, 23.6, and 24.7 degrees 2 ⁇ .
• XRPD X-ray powder diffraction
• Form I is characterized by an X-ray powder diffraction pattern further including peaks at about 8.2, 11.2, 15.7 16.4, 17.7, 30.0, and 32.2 degrees 2 ⁇ .
• Form I is characterized by an X-ray powder diffraction pattern including peaks at about 8.2, 10.5, 18.7, 22.9, 23.6, and 24.7 degrees 2 ⁇ .
• Form I is characterized by an X-ray powder diffraction pattern including peaks at about 8.2, 10.5, 11.2, 15.7 16.4, 17.7, 18.7, 22.9, 23.6, and 24.7 degrees 2 ⁇ . In one embodiment, Form I is characterized by an X-ray powder diffraction pattern including peaks at about 8.2, 10.5, 11.2, 15.7 16.4, 17.7, 18.7, 22.9, 23.6, 24.7, 30.0, and 32.2 degrees 2 ⁇ .
• Form I is characterized by an X-ray powder diffraction pattern substantially similar to that set forth in FIG. 1.
• the present disclosure describes a process of preparing Form I.
• the process comprises mixing a sample containing Compound A (e.g., either crude or purified preparation of Compound A) with an organic solvent, such as alcohol (e.g., ethanol), ketone (e.g., methyl isobutyl ketone, i.e., MIBK), or an aqueous solution including alcohol or ketone.
• an organic solvent such as alcohol (e.g., ethanol), ketone (e.g., methyl isobutyl ketone, i.e., MIBK), or an aqueous solution including alcohol or ketone.
• the resulting mixture e.g., a slurry or suspension
• the organic solvent is ethanol.
• the starting Compound A which goes into the form conversion can be a solvate, such as a hydrate (e.g., a monohydrate or dihydrate), or a solvate of an organic solvent (for example dimethyl acetamide, ethanol or MIBK).
• a solvate such as a hydrate (e.g., a monohydrate or dihydrate), or a solvate of an organic solvent (for example dimethyl acetamide, ethanol or MIBK).
• the starting Compound A can be an ansolvate (e.g., an anhydrate).
• the process is performed by heating the Compound A with the organic solvent to an elevated temperature (e.g., about 60-110 °C or about 80 °C) to form a slurry or suspension, followed by cooling (e.g., to a temperature about 0-60 °C, about 40-60 °C, about 45-55 °C, or at about room temperature) to give Compound A Form I.
• the organic solvent is ethanol and slurry containing Compound A can be cooled to a temperature greater than about 40 °C to obtain Form I.
• the organic solvent is MIBK, and slurry containing Compound A can be cooled to room temperature to obtain Form I.
• an ethanol suspension of Compound A is heated to an elevated temperature (e.g., about 80 °C) and then cooled to a temperature not lower than about 40 °C (e.g., about 45-55 °C), filtered (e.g., about 45-55 °C), washed with warmed (e.g., 45-55°C) ethanol and dried at e.g., 45-55°C to obtain Form I of Compound A that is substantially free of any solvate of Compound A such as ethanol solvate.
• Form I of Compound A as prepared has ethanol solvate content of ⁇ 5% (e.g., ⁇ 2%, ⁇ 1%, ⁇ 0.5%, or ⁇ 0.1%).
• the process further comprises, after cooling the mixture, filtering the mixture.
• the filtration step can be performed at a temperature between about 0 °C and about
• 60 °C (e.g., about 40-60 °C, about 45-55 °C, or at about room temperature) to obtain a filter cake.
• the process further comprises, after filtering the mixture, rinsing the filter cake.
• the rinsing step can be performed at a temperature between about 0 °C and about
• an organic solvent e.g. , an alcohol such as ethanol
• the process further comprises, after rinsing the filter cake, drying the rinsed filter cake.
• the drying step can be performed at a temperature between about 0 °C and about 60 °C (e.g., about 40-60 °C, about 45-55 °C, or at about room temperature) to obtain Form I of Compound A.
• Form I has a purity of greater than 91%, e.g., greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, or greater than 97.5%.
• Form I has a purity of greater than 98%, e.g., greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the disclosure provides compounds such as
• the disclosure also provides a method for treating a resistance to thyroid hormone (RTH) in a subject in need thereof.
• the method comprises administering to a subject having at least one TR mutation a therapeutically effective amount of a compound of Formula (IV):
• R 3 is H or CH 2 R a , in which R a is hydroxyl, O-linked amino acid, -OP(0)(OH) 2 or
• R b being lower alkyl, alkoxy, alkyl acid, cycloalkyl, aryl, heteroaryl, or
• RTH thyroid hormone
• the compound used in the above method is 2-(3,5-dichloro-4-((5- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile ("Compound A”), e.g., Compound A in Form I.
• the subject to be treated by the above method has obesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axis alteration, atherosclerosis, a cardiovascular disorder, tachycardia, hyperkinetic behavior, hypothyroidism, goiter, attention deficit hyperactivity disorder, learning disabilities, mental retardation, hearing loss, delayed bone age, neurologic or psychiatric disease or thyroid cancer.
• the THR mutation is selected from the group consisting of a substitution of threonine (T) for the wild type residue alanine (A) at amino acid position 234 of SEQ ID NO: 1 (A234T); a substitution of glutamine (Q) for the wild type residue arginine (R) at amino acid position 243 of SEQ ID NO: 1 (R243Q); a substitution of histidine (H) for the wild type residue arginine (R) at amino acid position 316 of SEQ ID NO: 1 (R316H); and a substitution of threonine (T) for the wild type residue alanine (A) at amino acid position 317 of SEQ ID NO: 1 (A317T).
• the compound used in the method restores activity of mutant THRp.
• the purity of compound of Formula (IV), such as Compound A is obtained from reslurrying a crude compound from a suitable solvent described herein.
• the compound is not a solvate (e.g., a hydrate).
• the compound of Formula (IV), e.g., Compound A has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the compound of Formula (IV) is Compound A in Form I, and has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the compound of Formula (IV) is Compound A in Form I, and Form I has a purity of greater than 85%, e.g., greater than 86%, greater than 90%, greater than 92.5%, greater than 95%, greater than 96%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, or greater than 99.8%.
• the compound of Formula (IV), such as Compound A has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of the corresponding ⁇ - isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4-isopropyl-6-oxo-l,6- dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• the compound of Formula (IV) has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of the corresponding ⁇ - isopropylpyridazin-3(2H)-one regioi
• the compound of Formula (IV), such as Compound A is free of the corresponding -isopropylpyridazin-3(2H)-one regioisomer (e.g., 2-(3,5-dichloro-4-((4- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile, the -isopropylpyridazin-3(2H)-one regioisomer of Compound A).
• the -isopropylpyridazin-3(2H)-one regioisomer e.g., 2-(3,5-dichloro-4-((4- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phen
• the compound of Formula (IV), such as Compound A has less than 1.5% (e.g., less than 1.0%, e.g., less than 0.5%) of heavy metal, e.g., silver, gold, or platinum.
• the subject is a mammal. In another embodiment, the subject is a human.
• the disclosure further provides a method for determining a responsiveness of a subject to the compound of Formula (IV) or a pharmaceutically acceptable salt thereof, the method comprising:
• TR thyroid hormone receptor
• the compound of Formula (IV) is 2-(3,5-dichloro-4-((5- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile ("Compound A").
• the TR is TR .
• the subject treated by the method of this invention has obesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axis alteration, atherosclerosis, a cardiovascular disorder, tachycardia, hyperkinetic behavior, hypothyroidism, goiter, attention deficit hyperactivity disorder, learning disabilities, mental retardation, hearing loss, delayed bone age, neurologic or psychiatric disease or thyroid cancer.
• a method for determining a responsiveness to the compound of Formula (IV) can be used together with the method for treating a resistance to thyroid hormone. That is, before the treatment, a subject is tested to determine the responsiveness to the compound.
• Figure 1 is an X-ray powder diffractogram (XRPD) of 2-(3,5-dichloro-4-((5- isopropyl-6-oxo- 1 , 6-dihydropyridazin-3 -yl)oxy)phenyl)-3 ,5 -dioxo-2, 3 ,4,5 -tetrahydro- 1 ,2,4- triazine-6-carbonitrile ("Compound A”) Form I.
• XRPD X-ray powder diffractogram
• FIG. 1 is a differential scanning calorimetry (DSC) diagram of Compound A Form
• Figures 3A and 3B are MacPymol modeling images to show T3 and Compound A in THR , respectively.
• Figure 4 is a MacPymol modeling image to show superimposed T3 and Compound A in THR .
• Figure 5A is a MacPymol modeling image to show polar interactions between T3 and wild type ⁇ , where T3 interacts with Arg320 very specifically.
• Figure 5B is a MacPymol modeling image to show polar interactions between
• Figure 6 is a MacPymol modeling image to show that mutations lead to many changes in the polar region of the ligand binding domain ("LBD").
• Figure 7A is a MacPymol modeling image to show interactions between T3 and
• THR mutants Ala234Thr, Arg243Gln, Arg316His, Ala317Thr.
• Figure 7B is a MacPymol modeling image to show interactions between Compound A and THR mutants: Ala234Thr, Arg243Gln, Arg316His, Ala317Thr; indicating that, compared to T3, the negatively charged heterocycle in Compound A accommodates mutations better.
• FIGS 8A and 8B are MacPymol modeling images of T3 and Compound A in Arg316His mutant, respectively.
• T3-Arg320 interaction is likely weaker due to rotation of Arg320 away from ligand in the mutant, while Compound A maintains favorable interaction with Arg320 and is well positioned for the CN group to form a pi-cation interaction with the mutated His316.
• Figures 9A and 9B are MacPymol modeling images of Compound A in the WT
• the phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure, and are not meant to be limiting in any fashion.
• the terms “may,” “optional,” “optionally,” or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
• the phrase “optionally present” means that an object may or may not be present, and, thus, the description includes instances wherein the object is present and instances wherein the object is not present.
• TR thyroid hormone receptor
• TR nucleic acids and polypeptides from various species have previously been described. See, e.g. , R. L. Wagner et al. (2001), Molecular Endocrinology
• the residues at the 234, 243, 316, and 317 positions of human TR are underlined in SEQ ID NO: 1.
• the portion of the human TR nucleotide sequence that encodes the above amino acid sequence is SEQ ID NO: 2.
• the nucleotide sequence of human TR is provided, e.g. , by Genbank Accession No. NM_000461.4, incorporated herein by reference.
• alkyl refers to a branched or unbranched saturated hydrocarbon group typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, i-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
• alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms.
• lower alkyl intends an alkyl group of 1 to 6 carbon atoms, for example, 1, 2, 3, 4, 5, or 6 carbon atoms.
• substituted alkyl refers to alkyl substituted with one or more substituent groups, and the terms
• heteroatom-containing alkyl and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra.
• alkenyl refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n- propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like.
• alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms.
• lower alkenyl intends an alkenyl group of 2 to 6 carbon atoms.
• substituted alkenyl refers to alkenyl substituted with one or more substituent groups
• heteroatom-containing alkenyl and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom, e.g., N, P, O, or S.
• alkynyl refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms.
• substituted alkynyl refers to alkynyl substituted with one or more substituent groups
• heteroatom-containing alkynyl and heteroalkynyl refer to alkynyl in which at least one carbon atom is replaced with a heteroatom
• alkoxy intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy” group may be represented as -O-alkyl where alkyl is as defined above.
• a "lower alkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t- butyloxy, etc.
• alkyl acid refers to an acid substituent that is on an alkyl group, such as -(CH 2 ) 0 COOH, in which o is an integer between 1 and 6.
• the alkyl group can either be linear or branched.
• aryl refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety).
• Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms.
• aryl groups may contain one aromatic ring or two fused or linked aromatic rings, e.g.
• substituted aryl refers to an aryl moiety substituted with one or more substituent groups
• heteroatom-containing aryl and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. If not otherwise indicated, the term “aryl” includes rings that are unsubstituted, substituted, and/or have heteroatom-containing aromatic substituents.
• aralkyl refers to an alkyl group with an aryl substituent
• alkaryl refers to an aryl group with an alkyl substituent, wherein “alkyl” and “aryl” are as defined above.
• aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms.
• Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms.
• amino is used herein to refer to the group -NZ'Z 2 wherein Z 1 and Z 2 are hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
• halo and halogen are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.
• heteroatom-containing refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g. , nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur.
• heteroalkyl refers to an alkyl substituent that is heteroatom-containing
• heterocycle or “heterocyclic” refers to a cyclic moiety that is heteroatom-containing
• heteroaryl and heteroaryomatic respectively refer to “aryl” and “aromatic” substituents that are heteroatom- containing, and the like.
• heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like.
• heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc.
• heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc.
• Hydrocarbyl refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms, and further including about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like.
• Substituted hydrocarbyl refers to hydrocarbyl substituted with one or more substituent groups
• heteroatom-containing hydrocarbyl refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom.
• O-linked amino acid means any amino acid, naturally occurring or synthetic, linked to a molecule via an oxygen of a carboxyl group of the amino acid, preferably via the carboxyl group of the carboxy terminus of the amino acid.
• protecting group means that a particular functional moiety, e.g. , O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound.
• a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group must be selectively removed in good yield by readily available, preferably nontoxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction.
• oxygen, sulfur, nitrogen and carbon protecting groups may be utilized.
• certain exemplary oxygen protecting groups may be utilized. These oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g. , MOM
• Nitrogen protecting groups are utilized. Nitrogen protecting groups, as well as protection and deprotection methods are known in the art. Nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g.
• sulfur protecting groups include, but are not limited to those oxygen protecting group describe above as well as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinyl sulfonyl, and optionally substituted maleic acid. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention.
• substituted as in “substituted hydrocarbyl,” “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents.
• substituents include, without limitation, functional groups and the hydrocarbyl moieties Ci- C 2 4 alkyl (including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-C 6 alkyl), C2-C24 alkenyl (including C2-C1 8 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C1 8 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C 30 aryl (including C5-C2 0 aryl, and further including C5-C12 aryl), and C6-C 30 aralkyl (including C6-C2 0 aralkyl, and further including C 6 -C 12 aralkyl).
• Ci- C 2 4 alkyl including Ci-Cis alkyl, further including C1-C12 alkyl, and
• Examples of functional groups include, without limitation: halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C2 0 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C2 0 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C2 0 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (- CO)-X where X is halo), C 2 -C 2 4 alkylcarbonato (-O-(CO)-O-alkyl), C 6 -C 2 o arylcarbonato (- O-(CO)-O-aryl), carboxy (-COOH), carboxyla
• the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.
• the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.
• telescoping a process refers to collapsing a multistep process into a smaller number of steps or unit operations.
• a unit operation includes transformations, but also encompasses handling and isolation steps. Centrifugation, filtration, distillation, decantation, precipitation/crystallization, and packaging are examples of unit operations.
• telescoping and other process improvements in the literature (see, e.g., /. Org. Chem., 2007, 72, 9757-9760).
• the disclosure provides methods of synthesizing a compound, e.g., one that is useful as an intermediate for synthesizing the pyridazinone compounds as thyroid hormone analogs.
• a compound e.g., one that is useful as an intermediate for synthesizing the pyridazinone compounds as thyroid hormone analogs.
• Pyridazinone compounds as thyroid hormone analogs, as well as their prodrugs, have been disclosed in e.g., U.S. Patents 7,452,882, 7,807,674, and 8,076,334.
• the invention features a method of making 6-(4-amino-2,6- dichlorophenoxy)-4-isopropylpyridazin-3(2H)-one ("Int. 7”) or a salt thereof, the method comprising:
• R 1 is isopropyl or isopropenyl
• X is halo and R 2 is H or an amine protecting group
• the present disclosure also describes a method for synthesizing the pyridazinone compounds as thyroid hormone analogs, as well as their prodrugs.
• Such compounds include those disclosed in U.S. Patents 7,452,882, 7,807,674, and 8,076,334.
• the disclosure describes a method of making a compound of Formula (IV) or a pharmaceutically acceptable salt thereof:
• R 3 is H or CH 2 R a , in which R a is hydroxyl, O-linked amino acid, -OP(0)(OH) 2 or
• R b being lower alkyl, alkoxy, alkyl acid, cycloalkyl, aryl, heteroaryl, or
• the method comprises: (a) contacting R'MgX or R J Li with a compound of Formula (I):
• R 1 is isopropyl or isopropenyl
• X is halo
• R 2 is H or an amine protecting
• compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components.
• methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps.
• steps or order for performing certain actions is immaterial so long as the invention remains operable.
• two or more steps or actions can be conducted simultaneously.
• the synthetic processes of the invention can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used.
• the processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, ester or prodrug thereof.
• Stage 1 Synthesis of 3,5-dichloro-4-((6-chloropyridazin-3-yl)oxy)aniline (Compound 2) and N-(3,5-dichloro-4-((6-oxo- l,6-dihydropyridazin-3-yl)oxy)phenyl)benzamide or N- (3,5-dichloro-4-((6-oxo- l ,6-dihydropyridazin-3-yl)oxy)phenyl)acetamide (Compound 4)
• Compound 2 is prepared by contacting 3,6-dichloropyridazine with 2,6-dichloro-4- aminophenol in the presence of a small amount of a suitable base such as a metal carbonate (e.g. , cesium or potassium carbonate) or a metal alkoxide (e.g., potassium t-butoxide) in a suitable organic solvent (e.g. , DMSO or DMAC) at a suitable reaction temperature (e.g. , 60 to 120 °C) until completion of reaction, typically about 3 to 30 hours, for example about 3 to 15 hours.
• a suitable base such as a metal carbonate (e.g. , cesium or potassium carbonate) or a metal alkoxide (e.g., potassium t-butoxide) in a suitable organic solvent (e.g. , DMSO or DMAC)
• a suitable reaction temperature e.g. , 60 to 120 °C
• Compound 4 is prepared by protecting 2 with a suitable amine protecting reagent
• a suitable organic solvent such as acetic acid
• a suitable reaction temperature e.g. , 100 to 120 °C
• the crude product is purified with a suitable solvent (e.g. , a mixture of water and acetic acid) at a suitable temperature (e.g., 88- 100 °C).
• a suitable solvent e.g. , a mixture of water and acetic acid
• Compound 6 is prepared by contacting Compound 4 with an isopropyl Grignard in a suitable organic solvent (such as tetrahydrofuran or dioxane) followed by an oxidation step.
• a suitable organic solvent such as tetrahydrofuran or dioxane
• the oxidation step can be performed in the presence of an oxidizing reagent such as bromine in a suitable organic solvent such as acetic acid at a suitable reaction temperature (e.g. , 60 to 90 °C) until completion of reaction, typically about 2 to 10 hours, for example about 2 to 5 hours.
• Int. 7 is obtained by deprotecting Compound 6 (where R 2 is Bz) with a base such as metal hydroxide (e.g., KOH or NaOH) or metal carbonate (e.g., sodium carbonate).
• a base such as metal hydroxide (e.g., KOH or NaOH) or metal carbonate (e.g., sodium carbonate).
• Int. 7 is obtained by deprotecting Compound 6 (where R 2 is Ac) with an acid such as trifluoroacetic acid.
• Compound 7 is prepared by contacting Compound 4 with an isopropenyl Grignard in a suitable organic solvent (such as tetrahydrofuran or 2-methyl THF) followed by isomerization (e.g., from 5A to 6) and deprotection under the treatment of a base such as metal hydroxide (e.g., KOH).
• a suitable reaction temperature e.g. , 60 to 90 °C
• completion of reaction typically about 10 to 60 hours, for example about 16 hours at 90 °C.
• the Grignard reaction can be performed in the presence of a Lewis acid such as LiCl or LiBr at a suitable reaction temperature (e.g. , room temperature to 40 °C) until completion of reaction, typically about 2 to 10 hours, for example about 2 to 5 hours.
• a Lewis acid such as LiCl or LiBr
• the synthesis of compound 5 or 5A results in improved yield of Int. 7 relative to other methods known in the art.
• the synthesis of 5 or 5A results in a yield of greater than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or greater than 90%.
• the Grignard reaction improves regioselectivity, resulting
• Int. 8 is prepared by contacting 6-(4-amino-2,6-dichlorophenoxy)-4- isopropylpyridazin-3(2H)-one with ethyl (2-cyanoacetyl)carbamate and a metal nitrite such as sodium nitrite in the presence of an acid (such as HCl) in a suitable solvent (e.g. , a mixture of acetic acid and water) at a suitable reaction temperature (e.g. , below 10 °C) until the reaction is complete.
• an acid such as HCl
• a suitable solvent e.g. , a mixture of acetic acid and water
• Compound A is prepared by contacting Int. 8 and a base such as sodium acetate or potassium acetate in a suitable solvent (e.g. , DMAC) at a suitable reaction temperature (e.g. , at about 120 °C) until the reaction is complete.
• a suitable solvent e.g. , DMAC
• the conversion from 6-(4-amino-2,6-dichlorophenoxy)-4- isopropylpyridazin-3(2H)-one (“Int. 7") to a compound of Formula (IV) other than MGL- 3916 (such as prodrugs thereof) is performed under conditions described in, e.g., U.S. Patents 7,452,882, 7,807,674, and 8,076,334, which are hereby incorporated by reference in their entireties.
• the synthetic methods described herein result in superior regioselectivity, with the Grignard installation of the isopropenyl or isopropyl group versus the biaryl ether formation in the synthetic route previously disclosed in, e.g., U.S. Patent 7,452,882, which gave poor regioselectivity. Further, by telescoping the biaryl ether formation into the benzamide protection, the methods disclosed herein avoid the isolation of the biaryl ether product, which was nearly practically impossible because of filtration times of greater than 1 week per batch when synthesizing this product in kilogram quantities.
• the present invention provides, compounds with high purity and/or in specific morphic form (e.g., Form I), compositions described herein and methods for the treatment or prevention of obesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axis alteration, atherosclerosis, a cardiovascular disorder, tachycardia, hyperkinetic behavior, hypothyroidism, goiter, attention deficit hyperactivity disorder, learning disabilities, mental retardation, hearing loss, delayed bone age, neurologic or psychiatric disease or thyroid cancer.
• the methods disclosed herein are suitable for both large- scale and small-scale preparations of the desired compounds.
• the thyroid hormone analogs may be prepared on a large scale, for example on an industrial production scale rather than on an experimental/laboratory scale.
• a batch-type process according to the methods of the disclosure allows the preparation of batches of at least 1 g, or at least 5 g, or at least 10 g, or at least 100 g, or at least 1 kg, or at least 100 kg of thyroid hormone analogs.
• the methods allow the preparation of a thyroid hormone analog having a purity of at least 98%, or at least 98.5% as measured by HPLC.
• the present invention also provides pharmaceutical compositions comprising a compound of Formula IV in combination with at least one pharmaceutically acceptable excipient or carrier.
• a "pharmaceutical composition” is a formulation containing a compound of the present invention in a form suitable for administration to a subject.
• the pharmaceutical composition is in bulk or in unit dosage form.
• the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
• the quantity of active ingredient (e.g. , a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
• the dosage will also depend on the route of administration.
• routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.
• Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that are required.
• the phrase "pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• “Pharmaceutically acceptable excipient or carrier” means an excipient or carrier that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
• a "pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g. , inhalation), transdermal (topical), and transmucosal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• therapeutically effective amount refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect.
• the effect can be detected by any assay method known in the art.
• the precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
• Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
• the disease or condition to be treated is a metabolic disorder.
• an effective amount of any one of the compounds of this invention or a combination of any of the compounds of this invention or a pharmaceutically acceptable salt or ester thereof is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
• the compounds or compositions can thus be administered orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories or washings), transdermally (e.g., skin electroporation) or by inhalation (e.g., by aerosol), and in the form or solid, liquid or gaseous dosages, including tablets and suspensions.
• buccal cavity e.g., buccal cavity
• parenterally e.g., intramuscularly, intravenously, or subcutaneously
• rectally e.g., by suppositories or washings
• transdermally e.g., skin electroporation
• the administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum.
• the therapeutic composition can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.
• Useful pharmaceutical carriers for the preparation of the compositions hereof can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like.
• the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.
• formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile.
• Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
• the compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like.
• Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.
• the pharmaceutical preparations can also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain other therapeutically valuable substances, including additional active ingredients other than those of formula I.
• the compounds of the present invention are useful as medicaments for the treatment of a resistance to thyroid hormone (RTH) in a subject who has at least one TR mutation.
• the subject may have a disease, such as obesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axis alteration, atherosclerosis, a cardiovascular disorder, tachycardia, hyperkinetic behavior,
• hypothyroidism goiter, attention deficit hyperactivity disorder, learning disabilities, mental retardation, hearing loss, delayed bone age, neurologic or psychiatric disease or thyroid cancer.
• the therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art.
• the drug can be dosed according to body weight. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated.
• the drug can be administered by fixed does, e.g., dose not adjusted according to body weight.
• a daily dosage of from about 0.5 mg to about 1000 mg should be appropriate, although the upper limit may be exceeded when indicated.
• the dosage is preferably from about 5 mg to about 400 mg per day.
• a preferred dosage may be from about 20 mg to about 100 mg per day.
• the daily dosage can be administered as a single dose or in divided doses, or for parenteral administration it may be given as continuous infusion.
• an effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer.
• dosage effective manner refers to amount of an active compound to produce the desired biological effect in a subject or cell.
• compositions can be included in a container, pack, or dispenser together with instructions for administration.
• pharmaceutically acceptable salts refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof.
• pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.
• the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxy naphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
• salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like.
• the present invention also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, lysine, arginine, morpholine, 2- hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine, tetramethylammonium hydroxide and the like.
• a metal ion e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion
• an organic base such as ethanolamine, diethanolamine, triethanolamine, trometh
• the compounds of the present invention can also be prepared as esters, for example, pharmaceutically acceptable esters.
• a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g. , a methyl, ethyl or other ester.
• an alcohol group in a compound can be converted to its corresponding ester, e.g. , an acetate, propionate or other ester.
• the compounds of the present invention can also be prepared as prodrugs, for example, pharmaceutically acceptable prodrugs.
• prodrug and “prodrug” are used interchangeably herein and refer to any compound which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of
• the compounds of the present invention can be delivered in prodrug form.
• the present invention is intended to cover prodrugs of the presently claimed compounds, methods of delivering the same and compositions containing the same.
• Prodrugs are intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when such prodrug is administered to a subject.
• Prodrugs in the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
• Prodrugs include compounds of the present invention wherein a hydroxy, amino, sulfhydryl, carboxy or carbonyl group is bonded to any group that may be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl group, respectively.
• Examples of prodrugs include, but are not limited to, esters (e.g. , acetate,
• dialkylaminoacetates formates, phosphates, sulfates and benzoate derivatives
• carbamates e.g. , ⁇ , ⁇ -dimethylaminocarbonyl
• esters e.g. , ethyl esters, morpholinoethanol esters
• N-acyl derivatives e.g.
• the compounds, or pharmaceutically acceptable salts, esters or prodrugs thereof are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally.
• the compound is administered orally.
• One skilled in the art will recognize the advantages of certain routes of administration.
• the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
• An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
• the compounds described herein, and the pharmaceutically acceptable salts thereof are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent.
• suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions.
• the compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
• the invention features a method for treating or alleviating a symptom of resistance to thyroid hormone in a subject by administering to a subject expressing a mutant ⁇
• the disclosure also provides a method of determining a responsiveness of a subject having resistance to thyroid hormone (RTH) to a compound of Formula (IV) disclosed herein by providing a sample from the subject; and detecting at least one TR mutation (e.g., a gene mutation or a mutation in the ligand-binding domain of TR polypeptide, e.g. , a polypeptide as defined in SEQ ID NO: 1); and the presence of said mutation indicates the subject is responsive to a compound of Formula (IV), such as Compound A, e.g., Form I thereof.
• the method can further include treating the subject who has the mutation by administering with a therapeutically effective amount of a compound of Formula (IV), such as Compound A, e.g., Form I thereof.
• the subject that shows or will show responsiveness to a compound of Formula (IV) such as Compound A has obesity, hyperlipidemia,
• hypercholesterolemia diabetes, non-alcoholic steatohepatitis, fatty liver, bone disease, thyroid axis alteration, atherosclerosis, a cardiovascular disorder, tachycardia, hyperkinetic behavior, hypothyroidism, goiter, attention deficit hyperactivity disorder, learning disabilities, mental retardation, hearing loss, delayed bone age, neurologic or psychiatric disease or thyroid cancer.
• the disclosure also provides a method which includes determining the presence of a TR gene mutation in a sample from a subject; and selecting, based on the presence of an TR gene mutation, a therapy that includes the administration of a
• a compound of Formula (IV) such as Compound A, e.g., Form I thereof.
• the disclosure also provides a method which includes amplifying a nucleic acid in a sample from a subject with a primer that is complementary to a mutant TR nucleic acid sequence comprising a TR gene mutation in a nucleic acid sequence as defined in SEQ ID NO: 2; determining the presence of the amplified nucleic acid, and selecting, based on the presence of the amplified nucleic acid, a therapy that includes the administration of a therapeutically effective amount of a compound of Formula (IV), or treating the subject by administering a therapeutically effective amount of a compound of Formula (IV) based on the presence of the amplified nucleic acid.
• the mutant TR described herein is a mutant TR polypeptide or a nucleic acid sequence encoding a mutant TR polypeptide.
• the mutant TR comprises one or more mutations at amino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1. More preferably, mutation is selected from the group consisting of a substitution of threonine (T) for the wild type residue alanine (A) at amino acid position 234 of SEQ ID NO: 1 (A234T); a substitution of glutamine (Q) for the wild type residue arginine (R) at amino acid position 243 of SEQ ID NO: 1 (R243Q); a substitution of histidine (H) for the wild type residue arginine (R) at amino acid position 316 of SEQ ID NO: 1 (R316H); and a substitution of threonine (T) for the wild type residue alanine (A) at amino acid position 317 of SEQ ID NO: 1 (A317T).
• the mutant TR comprises a nucleic acid sequence encoding a mutant TR polypeptide having one or more mutations at amino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1.
• a nucleic acid sequence encoding a mutant TR polypeptide or a peptide fragment that is characteristic of the mutant TR polypeptide can be detected using any suitable method.
• a nucleic acid sequence encoding a mutant TR polypeptide can be detected using whole-genome resequencing or target region resequencing (the latter also known as targeted resequencing) using suitably selected sources of DNA and polymerase chain reaction (PCR) primers in accordance with methods well known in the art.
• PCR polymerase chain reaction
• the method typically and generally entails the steps of genomic DNA purification, PCR amplification to amplify the region of interest, cycle sequencing, sequencing reaction cleanup, capillary electrophoresis, and data analysis.
• High quality PCR primers to cover region of interest are designed using in silico primer design tools.
• Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products.
• the products are typically terminated with a fluorescent tag that identifies the terminal nucleotide base as G, A, T, or C.
• Unincorporated dye terminators and salts that may compete for capillary eletrophoretic injection are removed by washing.
• capillary electrophoresis the products of the cycle sequencing reaction migrate through capillaries filled with polymer.
• the negatively charged DNA fragments are separated by size as they move through the capillaries toward the positive electrode.
• data collection software creates a sample file of the raw data.
• further data analysis is performed to translate the collected color data images into the corresponding nucleotide bases.
• the method may include the use of microarray- based targeted region genomic DNA capture and/or sequencing. Kits, reagents, and methods for selecting appropriate PCR primers and performing resequencing are commercially available, for example, from Applied Biosystems, Agilent, and NimbleGen (Roche
• PCR primers may be selected so as to amplify, for example, at least a relevant portion of a nucleic acid sequence encoding a mutant TR polypeptide having one or more mutations at amino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1.
• a nucleic acid sequence encoding a mutant TR polypeptide may be detected using a Southern blot in accordance with methods well known in the art.
• the methods of the invention comprise the step of performing an assay to detect a mutant of TR in a sample from a subject.
• a sample from a subject refers to any suitable sample containing cells or components of cells obtained or derived from a subject.
• the sample is a blood sample.
• the sample is a biopsy sample obtained from, for example, the thyroid gland.
• the disclosure also provides a ligand- mutant TR complex comprising: a mutant TR polypeptide and a compound of Formula (IV).
• a mutant TR polypeptide for example, the mutant TR
• polypeptide forming the complex comprises one or more mutations at amino acid positions 234, 243, 316, and 317 of SEQ ID NO: 1.
• the compound forming the complex is Compound A.
• the disclosure provides a primer-nucleic acid complex comprising: a mutant TR nucleic acid sequence, and a PCR primer that is complementary to the mutant TR nucleic acid sequence, wherein the mutant nucleic acid sequence comprises an EZH2 gene mutation in a nucleic acid sequence as defined in SEQ ID NO: 2.
• the XRPD data were collected on X-Ray Powder Diffractometer (CubiX-Pro XRD) with Cu Ka radiation (45 kV, 40 mA) from 3 to 45 degrees 2-theta (2 ⁇ ) at a scanning rate of 0.12 degrees/min and step size of 0.020 degrees.
• X-ray tube Cu KV, 45 kV, 40 mA
• thermocouple, reflux condenser, and N 2 inlet/outlet was charged with 3,6-dichloropyridazine (100 g, 0.672 mol, 1 wt), 4-amino-2,6-dichlorophenol (122 g, 0.686 mol, 1.02 equiv), and DMAC (500 mL, 5 vol).
• the resulting solution was charged with cesium carbonate (251 g, 0.771 mol, 1.15 equiv) and the suspension was heated to 110 °C. After 3 h at that temperature, the batch temperature was lowered to 70 °C and stirred at that temperature for 16 h. NMR analysis (DMSO) showed nearly all the dichloropyridazine had been consumed and the reaction was deemed complete.
• the batch was cooled to room
• the crude 2 above was taken up in acetic acid (1.48 L, 7.5 vol) and benzoic anhydride (168 g, 0.741 mol, 1.1 equiv) was added. The resulting mixture was heated to 100 °C and after 35 min at that temperature, the amount of 2 was 0.8%. Sodium acetate (110 g, 2 equiv) was added and the temperature increased to 110 °C. After 14.5 h at that temperature, HPLC analysis of the reaction mixture showed no intermediate remaining, and the reaction was deemed complete. The batch was cooled to 75 °C and water (1.5 L, 7.7 vol) was added over a period of 1 hour while maintaining a batch temperature between 72-75 °C.
• thermocouple, N 2 inlet/outlet, and a reflux condenser was charged with 4 (95 g, 0.253 mol, 1 wt), THF (665 mL, 7 vol), and LiCl (32.3 g, 0.759 mol, 3 equiv).
• the resulting suspension was heated to 35 °C, and isopropenylmagnesium bromide solution (0.5 M in THF, 1.72 L, 0.859 mol, 3.4 equiv) was added over a period of 80 min while maintaining a batch temperature between 35-45 °C. After heating the resulting slurry at 40 °C for 3 h, HPLC analysis showed a conversion of 87%).
• thermocouple, and N 2 inlet/outlet was charged with crude Int. 8 (104.4 g, 1 wt) and acetic acid (522 mL, 5 vol).
• the resulting slurry was heated to 50 °C and held at that temperature for 1.5 h.
• the batch cooled naturally to 25 °C over 2 h and was filtered through Sharkskin filter paper.
• the reactor and cake were washed sequentially with water (522 mL, 5 vol) and the cake conditioned under vacuum for 1.75 h.
• the light orange solid was dried to constant weight in a 40 °C vacuum oven to provide 89.9 g (78% from Int. 7) of the desired product.
• 1 H NMR (DMSO) was consistent with the assigned structure.
• thermocouple, N 2 inlet/outlet, and reflux condenser was charged with Int. 8 (89.3 g, 0.185 mol, 1 wt), DMAC (446 mL, 5 vol), and KOAc (20.0 g, 0.204 mol, 1.1 equiv).
• the mixture was heated to 120 °C and held at that temperature for 2 h.
• HPLC analysis showed complete conversion to Compound A.
• the batch temperature was adjusted to 18 °C over 1 h, and acetic acid (22.3 mL, 0.25 vol) was added.
• the batch temperature was adjusted to 8 °C, and water (714 mL, 8 vol) was added over 1 h; an orange slurry formed.
• the batch was filtered through Sharkskin filter paper and the cake was allowed to condition overnight under N 2 without vacuum for convenience.
• a premixed solution of 1 : 1 acetone/water (445 mL, 5 vol) was charged to the flask and added to the cake as a rinse with vacuum applied. After 2 h of conditioning the cake under vacuum, it was transferred to a clean 1 L, three-neck, round- bottom flask equipped with overhead stirring, a thermocouple, and N 2 inlet/outlet. Ethanol (357 mL, 4 vol) and acetone (357 mL, 4 vol) were charged and the resulting slurry was heated to 60 °C; dissolution occurred.
• Example 5 Scaled up preparation of 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-l,6- dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-l,2,4-triazine-6-carbonitrile (Compound A)
• the batch was agitated for 5 minutes and then allowed to separate for 35 minutes. The interface was not visible, so the calculated 23 L of the lower aqueous phase was removed. 16.0 L of 15% Sodium chloride in H 2 0 was added to the batch. The batch was agitated for 6 minutes and then allowed to separate for 7 minutes. The interface was visible at -19 L and the lower aqueous phase was removed. 17.0 L of 15% Sodium chloride in H 2 0 was added to the batch. The batch was agitated for 7 minutes and then allowed to separate for 11 minutes. The lower aqueous phase was removed. The vessel was set up for vacuum distillation and the batch was concentrated from 17.0 L to 8.0 L over 2 hours 20 minutes with the batch temperature kept around 21 °C.
• Benzoic anhydride (3.19 kg) and acetic acid (18.0 L) were charged to the vessel.
• the vessel was set up for vacuum distillation and the batch was concentrated from 28.0 L to 12.0 L over 2 days (overnight hold at 20 °C) with the batch temperature kept between 20 and 55 °C.
• J H NMR analysis indicated a mol ratio of acetic acid to ethyl acetate of 1.0:0.015.
• Acetic acid (4.0 L) was charged to the batch and the batch was distilled to 12 L.
• J H NMR analysis indicated a mol ratio of acetic acid to ethyl acetate of 1.0:0.0036.
• Acetic acid (20.0 L) was charged to the batch and the batch temperature was adjusted to 70.0 °C.
• the batch was sampled for HPLC analysis and 2 was 0.16%. Sodium acetate (2,20 kg) was added to the batch and the batch temperature was adjusted from 72.4 °C to 110.0 °C. After 18.5 hours, HPLC analysis indicated no Int. B detected. The batch temperature was adjusted from 111.3 to 74.7 °C and DI water (30.0 L) was added to the batch over 2 hours. The batch temperature was adjusted to 20 .5 °C and then filtered using a 24" Haselloy Nutsche filter equipped with polypropylene filter cloth. A previously prepared solution of 1:1 acetic acid in DI H 2 0 (10.0 L) was charged to the vessel and agitated for 5 minutes.
• the wash was transferred to the filter and the batch was then manually re- suspended in the filter before re-applying vacuum.
• DI H 2 0 (10.0 L) was charged to the vessel and then transferred to the filter.
• the batch was manually re-suspended in the filter before re-applying vacuum.
• DI H 2 0 (10.0 L) was charged directly to the filter and the batch was then manually re-suspended in the filter before re-applying vacuum.
• the filter cake was allowed to condition for 18 hours to give 14.4 kg of 4. HPLC analysis indicated a purity of 93.7%. This wet cake was carried forward into the purification.
• a 100 L jacketed glass vessel (purged with N 2 ) was charged with crude 4 (wet cake 14.42 kg), acetic acid (48.8 L) and the agitator was started. DI H 2 0 (1.74 L) was charged. The batch (a slurry) temperature was adjusted from 18.1 to 100.1 °C over 4.25 hours. The batch was held at 100.1 to 106.1 °C for 1 hour and then adjusted to 73.1 °C. DI H 2 0 (28.0 L) was added to the batch over 1 hour keeping the batch temperature between 73.1 and 70.3 °C. The batch temperature was adjusted further from 70.3 °C to 25.0 °C overnight. The batch was filtered using a 24" Hastelloy Nutsche filter equipped with polypropylene filter cloth.
• the batch was agitated at 24.5 °C for 17 hours at which point HPLC analysis indicated 9% 4.
• a 2nd 100-L jacketed glass vessel (purged with N2) was charged with 3N hydrogen chloride (18.3 L).
• the batch was transferred to the vessel containing the 3N HC1 over 25 minutes keeping the batch temperature between 20 and 46 °C. A bi-phasic solution was observed.
• the quenched batch was transferred back to the 1 st 100-L vessel to quench the small amount of residue left behind. THF (2.00 L) was used as a rinse.
• the batch temperature was observed to be 40.9 ° C and was agitated at 318 RPM for 45 minutes.
• the batch temperature was adjusted to 21.8 ° C and the layers were allowed to separate. The separation took 10 minutes.
• the lower aqueous phase was removed (-26.0 L).
• a solution of sodium chloride (1.56 kg) in DI water (14.0 L) was prepared and added to the batch. This was agitated at 318 RPM for 10 minutes and agitator was stopped. The separation took 3 minutes.
• the lower aqueous phase was removed (-16.0 L).
• the batch was vacuum distilled from 58.0 L to 18.4 L using ⁇ 24'7Hg and a jacket temperature of 50 to 55 °C.
• a solution of potassium hydroxide (2.30 kg) in DI water (20.7 L) was prepared in a 72-L round bottom flask. The vessel was set up for atmospheric distillation using 2 distillation heads and the batch was transferred to the 72-L vessel. THF (0.75 L) was used as a rinse.
• the batch volume was -41.0 L, the temperature was adjusted to 64.1 °C and distillation started with the aid of a N2 sweep. Heating was continued to drive the batch temperature to 85.4 °C while distilling at which point the 72-L vessel was set up for reflux (batch volume was about 28.0 L at the end of the distillation). The batch was held at 85 °C for 13 hours at which point HPLC analysis indicated 0.3% compound 6A. Heating was stopped and the batch was transferred to a 100-L jacketed glass vessel. Solids were observed. The batch temperature was adjusted from 70.6 °C to 56.7 °C. A previously prepared solution of sodium hydrogen carbonate (2.82 kg) in DI water (35.0 L) was added over 80 minutes keeping the batch temperature between 56.7 and 46.7 °C. The batch pH at the end of the addition was 9.8. The batch was held at
• the batch was filtered using a 24" Hastelloy Nutsche filter equipped with tight-weave polypropylene filter cloth. The filtration took 30 minutes.
• the vessel was rinsed with 14.3 L of a 1 : 1 acidic acid / DI water. The orange residue on the reactor washed away with the rinse. The rinse was transferred to the filter where the batch was manually re-suspended. Vacuum was re-applied to remove the wash.
• a 2 nd 1 : 1 acidic acid / DI water wash was performed as above and the batch was conditioned on the filter for 26 hours. HPLC analysis of the wet filter cake indicated purity was 90.4%.
• the batch was dried to a constant weight of 3.97 kg (91% yield) in a vacuum oven at 45 °C and 287Hg.
• the batch was transferred back to the 100 L vessel using vacuum through a 10 ⁇ in-line filter and a DMAC rinse (1.15 L) was used. The filtration was fast at the beginning but slow at the end, plugging up the filter.
• the batch temperature was adjusted to 11.1 °C and DI water (35.1 L) was added over 2 h 20 min, keeping the batch temperature between 5-15 °C.
• the batch was held for 1 h and filtered, using an 18" Nutsche filter equipped with tight-weave
• Methyl isobutyl ketone (2.90 L) was charged to the filter cake and the cake was manually resuspended. The liquors were pulled through with vacuum and the cake was conditioned with vacuum and nitrogen for 15 h. The filter cake dried into a tan, hard 18" x 1 1 ⁇ 2" disc. This was manually broken up and run through coffee grinders to give a 76% yield (2.72 kg) of MGL-3196 MIBK solvate as a tan, powdery solid. No oven drying was necessary.
• the NMR spectrum was consistent with the assigned structure and Karl Fischer analysis indicated ⁇ 0.1 % H 2 0. XRPD indicated the expected form MIBK solvate. TGA indicated 17.3% weight loss. HPLC analysis indicated a purity of 98.5%.
• Form I was found to have a melting onset around 321 °C, followed by decomposition upon melting by DSC ( Figure 2).
• Example 7 Preparation of Compound A Form I: conversion of Compound A solvate to Form I
• Compound A DMAC solvate can be converted, via the dihydrate and the MIBK solvate, to Form I as described in Example 7.
• the DMAC solvate was converted directly to Form I in 75% yield (yield calculated from Intermediate 8) by heating it with 8 volumes of ethanol to 80 °C for 2 hours followed by cooling to room temperature and filtering.
• a sample of Compound A that was a mixture of the DMAC solvate and dihydrate was converted to Form I in 69% yield by heating it with 8 volumes of MIBK to 80°C followed by cooling to room temperature.
• Modeling of interaction between Compound A and thyroid hormone receptor Crystal structures were obtained from the RCSB protein data bank (ID numbers: 1N46, 1NQ0, INQl, 1NQ2 and INUO).
• the protein co-crystal structures were aligned using MacPymol for Mac OS X (Copyright 2006 DeLano Scientific LLC. ; now a product of

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