OSTAT SERIES


Links


1 PANOBINOSTAT
2. BELINOSTAT
3' VORINOSTAT
4 MOCETINOSTAT
5 EZATIOSTAT
6 DACINOSTAT
7 GIVINOSTAT
8 TOPIROXOSTAT
9
10




WILL BE UPDATED SOON  FEBUXOSTAT,

Tazemetostat





1 PANOBINOSTAT



Panobinostat
HDAC inhibitors, orphan drug
cas 404950-80-7 
2E)-N-hydroxy-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide (alternatively, N-hydroxy-3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-acrylamide)
Molecular Formula: C21H23N3O2   Molecular Weight: 349.42622
  • Faridak
  • LBH 589
  • LBH589
  • Panobinostat
  • UNII-9647FM7Y3Z
A hydroxamic acid analog histone deacetylase inhibitor from Novartis.
NOVARTIS, innovator
Histone deacetylase inhibitors
Is currently being examined in cutaneous T-cell lymphoma, CML and breast cancer.
clinical trials click here  phase 3
DRUG SUBSTANCE–LACTATE AS IN  http://www.google.com/patents/US7989639  SEE EG 31
Panobinostat (LBH-589) is an experimental drug developed by Novartis for the treatment of various cancers. It is a hydroxamic acid[1] and acts as a non-selective histone deacetylase inhibitor (HDAC inhibitor).[2]
panobinostat
Panobinostat is a cinnamic hydroxamic acid analogue with potential antineoplastic activity. Panobinostat selectively inhibits histone deacetylase (HDAC), inducing hyperacetylation of core histone proteins, which may result in modulation of cell cycle protein expression, cell cycle arrest in the G2/M phase and apoptosis. In addition, this agent appears to modulate the expression of angiogenesis-related genes, such as hypoxia-inducible factor-1alpha (HIF-1a) and vascular endothelial growth factor (VEGF), thus impairing endothelial cell chemotaxis and invasion. HDAC is an enzyme that deacetylates chromatin histone proteins. Check for
As of August 2012, it is being tested against Hodgkin’s Lymphomacutaneous T cell lymphoma (CTCL)[3] and other types of malignant disease in Phase III clinical trials, against myelodysplastic syndromesbreast cancer and prostate cancer in Phase II trials, and against chronic myelomonocytic leukemia (CMML) in a Phase I trial.[4][5]
Panobinostat is a histone deacetylase (HDAC) inhibitor which was filed for approval in the U.S. in 2010 for the oral treatment of relapsed/refractory classical Hodgkin’s lymphoma in adult patients. The company is conducting phase II/III clinical trials for the oral treatment of multiple myeloma, chronic myeloid leukemia and myelodysplasia. Phase II trials are also in progress for the treatment of primary myelofibrosis, post-polycythemia Vera, post-essential thrombocytopenia, Waldenstrom’s macroglobulinemia, recurrent glioblastoma (GBM) and for the treatment of pancreatic cancer progressing on gemcitabine therapy. Additional trials are under way for the treatment of hematological neoplasms, prostate cancer, colorectal cancer, renal cell carcinoma, non-small cell lung cancer (NSCLC), malignant mesothelioma, acute lymphoblastic leukemia, acute myeloid leukemia, head and neck cancer and gastrointestinal neuroendocrine tumors. Early clinical studies are also ongoing for the treatment of HER2 positive metastatic breast cancer. Additionally, phase II clinical trials are ongoing at Novartis as well as Neurological Surgery for the treatment of recurrent malignant gliomas as are phase I/II initiated for the treatment of acute graft versus host disease. The National Cancer Institute had been conducting early clinical trials for the treatment of metastatic hepatocellular carcinoma; however, these trials were terminated due to observed dose-limiting toxicity. In 2009, Novartis terminated its program to develop panobinostat for the treatment of cutaneous T-cell lymphoma. A program for the treatment of small cell lung cancer was terminated in 2012. Phase I clinical trials are ongoing for the treatment of metastatic and/or malignant melanoma and for the treatment of sickle cell anemia. The University of Virginia is conducting phase I clinical trials for the treatment of newly diagnosed and recurrent chordoma in combination with imatinib. Novartis is evaluating panobinostat for its potential to re-activate HIV transcription in latently infected CD4+ T-cells among HIV-infected patients on stable antiretroviral therapy.
Mechanistic evaluations revealed that panobinostat-mediated tumor suppression involved blocking cell-cycle progression and gene transcription induced by the interleukin IL-2 promoter, accompanied by an upregulation of p21, p53 and p57, and subsequent cell death resulted from the stimulation of caspase-dependent and -independent apoptotic pathways and an increase in the mitochondrial outer membrane permeability. In 2007, the compound received orphan drug designation in the U.S. for the treatment of cutaneous T-cell lymphoma and in 2009 and 2010, orphan drug designation was received in the U.S. and the E.U., respectively, for the treatment of Hodgkin’s lymphoma. This designation was also assigned in 2012 in the U.S. and the E.U. for the treatment of multiple myeloma.
Cardiovascular disease is the leading cause of morbidity and mortality in the western world and during the last decades it has also become a rapidly increasing problem in developing countries. An estimated 80 million American adults (one in three) have one or more expressions of cardiovascular disease (CVD) such as hypertension, coronary heart disease, heart failure, or stroke. Mortality data show that CVD was the underlying cause of death in 35% of all deaths in 2005 in the United States, with the majority related to myocardial infarction, stroke, or complications thereof. The vast majority of patients suffering acute cardiovascular events have prior exposure to at least one major risk factor such as cigarette smoking, abnormal blood lipid levels, hypertension, diabetes, abdominal obesity, and low-grade inflammation.
Pathophysiologically, the major events of myocardial infarction and ischemic stroke are caused by a sudden arrest of nutritive blood supply due to a blood clot formation within the lumen of the arterial blood vessel. In most cases, formation of the thrombus is precipitated by rupture of a vulnerable atherosclerotic plaque, which exposes chemical agents that activate platelets and the plasma coagulation system. The activated platelets form a platelet plug that is armed by coagulation-generated fibrin to form a biood clot that expands within the vessel lumen until it obstructs or blocks blood flow, which results in hypoxic tissue damage (so-called infarction). Thus, thrombotic cardiovascular events occur as a result of two distinct processes, i.e. a slowly progressing long-term vascular atherosclerosis of the vessel wall, on the one hand, and a sudden acute clot formation that rapidly causes flow arrest, on the other. This invention solely relates to the latter process.
Recently, inflammation has been recognized as an important risk factor for thrombotic events. Vascular inflammation is a characteristic feature of the atherosclerotic vessel wall, and inflammatory activity is a strong determinant of the susceptibility of the atherosclerotic plaque to rupture and initiate intravascular clotting. Also, autoimmune conditions with systemic inflammation, such as rheumatoid arthritis, systemic lupus erythematosus and different forms of vasculitides, markedly increase the risk of myocardial infarction and stroke.
Traditional approaches to prevent and treat cardiovascular events are either targeted 1) to slow down the progression of the underlying atherosclerotic process, 2) to prevent clot formation in case of a plaque rupture, or 3) to direct removal of an acute thrombotic flow obstruction. In brief, antiatherosclerotic treatment aims at modulating the impact of general risk factors and includes dietary recommendations, weight loss, physical exercise, smoking cessation, cholesterol- and blood pressure treatment etc. Prevention of clot formation mainly relies on the use of antiplatelet drugs that inhibit platelet activation and/or aggregation, but also in some cases includes thromboembolic prevention with oral anticoagulants such as warfarin. Post-hoc treatment of acute atherothrombotic events requires either direct pharmacological lysis of the clot by thrombolytic agents such as recombinant tissue-type plasminogen activator or percutaneous mechanical dilation of the obstructed vessel.
Despite the fact that multiple-target antiatherosclerotic therapy and clot prevention by antiplatelet agents have lowered the incidence of myocardial infarction and ischemic stroke, such events still remain a major population health problem. This shows that in patients with cardiovascular risk factors these prophylactic measures are insufficient to completely prevent the occurrence of atherothrombotic events.
Likewise, thrombotic conditions on the venous side of the circulation, as well as embolic complications thereof such as pulmonary embolism, still cause substantial morbidity and mortality. Venous thrombosis has a different clinical presentation and the relative importance of platelet activation versus plasma coagulation are somewhat different with an preponderance for the latter in venous thrombosis, However, despite these differences, the major underlying mechanisms that cause thrombotic vessel occlusions are similar to those operating on the arterial circulation. Although unrelated to atherosclerosis as such, the risk of venous thrombosis is related to general cardiovascular risk factors such as inflammation and metabolic aberrations.
Panobinostat can be synthesized as follows: Reduction of 2-methylindole-3-glyoxylamide (I) with LiAlH4 affords 2-methyltryptamine (II). 4-Formylcinnamic acid (III) is esterified with methanolic HCl, and the resulting aldehyde ester (IV) is reductively aminated with 2-methyltryptamine (II) in the presence of NaBH3CN (1) or NaBH4 (2) to give (V). The title hydroxamic acid is then obtained by treatment of ester (V) with aqueous hydroxylamine under basic conditions.
Panobinostat is currently being used in a Phase I/II clinical trial that aims at curing AIDS in patients on highly active antiretroviral therapy (HAART). In this technique panobinostat is used to drive the HI virus’s DNA out of the patient’s DNA, in the expectation that the patient’s immune system in combination with HAART will destroy it.[6][7]

panobinostat

Panobinostat has been found to synergistically act with sirolimus to kill pancreatic cancer cells in the laboratory in a Mayo Clinic study. In the study, investigators found that this combination destroyed up to 65 percent of cultured pancreatic tumor cells. The finding is significant because the three cell lines studied were all resistant to the effects of chemotherapy – as are many pancreatic tumors.[8]
Panobinostat has also been found to significantly increase in vitro the survival of motor neuron (SMN) protein levels in cells of patients suffering fromspinal muscular atrophy.[9]
Panobinostat was able to selectively target triple negative breast cancer (TNBC) cells by inducing hyperacetylation and cell cycle arrest at the G2-M DNA damage checkpoint; partially reversing the morphological changes characteristic of breast cancer cells.[10]
Panobinostat, along with other HDAC inhibitors, is also being studied for potential to induce virus HIV-1 expression in latently infected cells and disrupt latency. These resting cells are not recognized by the immune system as harboring the virus and do not respond to antiretroviral drugs.[11]

Panobinostat inhibits multiple histone deacetylase enzymes, a mechanism leading to apoptosis of malignant cells via multiple pathways.[1]
The compound N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide (alternatively, N-hydroxy-3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-acrylamide) has the formula

Figure US07989639-20110802-C00001

as described in WO 02/22577. Valuable pharmacological properties are attributed to this compound; thus, it can be used, for example, as a histone deacetylase inhibitor useful in therapy for diseases which respond to inhibition of histone deacetylase activity. WO 02/22577 does not disclose any specific salts or salt hydrates or solvates of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide.
The compounds described above are often used in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, when appropriate, pharmaceutically acceptable base addition salts and acid addition salts, for example, metal salts, such as alkali and alkaline earth metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts, and sulfonate salts. Acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as alkyl sulfonate, arylsulfonate, acetate, maleate, fumarate, tartrate, citrate and lactate. Examples of metal salts are alkali metal salts, such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of ammonium salts are ammonium salt and tetramethylammonium salt. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. Sulfonate salts include mesylate, tosylate and benzene sulfonic acid salts.
……………………………..
GENERAL METHOD OF SYNTHESIS
ADD YOUR METHYL AT RIGHT PLACE

As is evident to those skilled in the art, the many of the deacetylase inhibitor compounds of the present invention contain asymmetric carbon atoms. It should be understood, therefore, that the individual stereoisomers are contemplated as being included within the scope of this invention.
The hydroxamate compounds of the present invention can be produced by known organic synthesis methods. For example, the hydroxamate compounds can be produced by reacting methyl 4-formyl cinnamate with tryptamine and then converting the reactant to the hydroxamate compounds. As an example, methyl 4-formyl cinnamate 2, is prepared by acid catalyzed esterification of 4-formylcinnamic acid 3 (Bull. Chem. Soc. Jpn. 1995; 68:2355-2362). An alternate preparation of methyl 4-formyl cinnamate 2 is by a Pd- catalyzed coupling of methyl acrylate 4 with 4-bromobenzaldehyde 5.
CHO

Figure imgf000020_0001
Additional starting materials can be prepared from 4-carboxybenzaldehyde 6, and an exemplary method is illustrated for the preparation of aldehyde 9, shown below. The carboxylic acid in 4-carboxybenzaldehyde 6 can be protected as a silyl ester (e.g., the t- butyldimethylsilyl ester) by treatment with a silyl chloride (e.g., f-butyldimethylsilyl chloride) and a base (e.g. triethylamine) in an appropriate solvent (e.g., dichloromethane). The resulting silyl ester 7 can undergo an olefination reaction (e.g., a Horner-Emmons olefination) with a phosphonate ester (e.g., triethyl 2-phosphonopropionate) in the presence of a base (e.g., sodium hydride) in an appropriate solvent (e.g., tetrahydrofuran (THF)). Treatment of the resulting diester with acid (e.g., aqueous hydrochloric acid) results in the hydrolysis of the silyl ester providing acid 8. Selective reduction of the carboxylic acid of 8 using, for example, borane-dimethylsuflide complex in a solvent (e.g., THF) provides an intermediate alcohol. This intermediate alcohol could be oxidized to aldehyde 9 by a number of known methods, including, but not limited to, Swern oxidation, Dess-Martin periodinane oxidation, Moffatt oxidation and the like.

Figure imgf000020_0002
The aldehyde starting materials 2 or 9 can be reductively aminated to provide secondary or tertiary amines. This is illustrated by the reaction of methyl 4-formyl cinnamate 2 with tryptamine 10 using sodium triacetoxyborohydride (NaBH(OAc)3) as the reducing agent in dichloroethane (DCE) as solvent to provide amine 11. Other reducing agents can be used, e.g., sodium borohydride (NaBH ) and sodium cyanoborohydride (NaBH3CN), in other solvents or solvent mixtures in the presence or absence of acid catalysts (e.g., acetic acid and trifluoroacetic acid). Amine 11 can be converted directly to hydroxamic acid 12 by treatment with 50% aqueous hydroxylamine in a suitable solvent (e.g., THF in the presence of a base, e.g., NaOH). Other methods of hydroxamate formation are known and include reaction of an ester with hydroxylamine hydrochloride and a base (e.g., sodium hydroxide or sodium methoxide) in a suitable solvent or solvent mixture (e.g., methanol, ethanol or methanol/THF).

Figure imgf000021_0001

NOTE ….METHYL SUBSTITUENT ON 10 WILL GIVE YOU PANOBINOSTAT

……………………………….
Journal of Medicinal Chemistry, 2011 ,  vol. 54,  13  pg. 4694 – 4720
(E)-N-Hydroxy-3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-acrylamide
lactate
(34, panobinostat, LBH589)
for str see above link
α-methyl-β-(β-bromoethyl)indole (29) was made according to method reported by Grandberg et al.(2. Grandberg, I. I.; Kost, A. N.; Terent’ev, A. P. Reactions of hydrazine derivatives. XVII. New synthesis of α-methyltryptophol. Zhurnal Obshchei Khimii 1957, 27, 3342–3345. )
The bromide 29 was converted to amine 30 by using similar method used by Sletzinger et al.(3. Sletzinger, M.; Ruyle, W. V.; Waiter, A. G. (Merck & Co., Inc.). Preparation of tryptamine
derivatives. U.S. Patent US 2,995,566, Aug 8, 1961.)
To a 500 mL flask, crude 2-methyltryptamine 30 (HPLC purity 75%, 1.74 g, 7.29 mmol) and 3-(4-
formyl-phenyl)-acrylic acid methyl ester 31 (HPLC purity 84%, 1.65 g, 7.28 mmol) were added,
followed by DCM (100 mL) and MeOH (30 mL). The clear solution was stirred at room temp for 30
min, then NaBH3CN (0.439 g, 6.99 mmol) was added in small portions. The reaction mixture was
stirred at room temp overnight. After removal of the solvents, the residue was diluted with DCM and
added saturated NaHCO3 aqueous solution, extracted with DCM twice. The DCM layer was dried
and concentrated, and the resulting residue was purified by flash chromatography (silica, 0–10%
MeOH in DCM) to afford 33 as orange solid (1.52 g, 60%). LC–MS m/z 349.2 ([M + H]+). 33 was
converted to hydroxamic acid 34 according to procedure D (Experimental Section), and the freebase
34 was treated with 1 equiv of lactic acid in MeOH–water (7:3) to form lactic acid salt which was
further recrystallized in MeOH–EtOAc to afford the lactic acid salt of 34as pale yellow solid. LC–MS m/z 350.2 ([M + H − lactate]+).
= DELTA
1H NMR (DMSO-d6)  10.72 (s, 1H, NH), 7.54 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 16 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 6.97 (td, J = 7.8 Hz, 1H), 7.44 (d, J = 15.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 2H), 7.08 (d, J = 7.8Hz, 2H), 7.01 (t, J = 7.4, 0.9 Hz, 1H), 6.91 (td, J = 7.4, 0.9 Hz, 1H), 6.47 (d, J = 15.2 Hz, 1H), 3.94(q, J = 6.8 Hz, 1H, lactate CH), 3.92 (s, 2H), 2.88 and 2.81 (m, each, 4H, AB system, CH2CH2),2.31 (s, 3H), 1.21 (d, J = 6.8 Hz, 3H).;
13C NMR (DMSO-d6)  176.7 (lactate C=O), 162.7, 139.0,
137.9, 135.2, 134.0, 132.1, 129.1, 128.1, 127.4, 119.9, 119.0, 118.1, 117.2, 110.4, 107.0, 66.0, 51.3,
48.5, 22.9, 20.7, 11.2.

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PANOBINOSTAT DRUG SUBSTANCE SYNTHESIS AND DATA
Figure US07989639-20110802-C00002

A flow diagram for the synthesis of LBH589 lactate is provided in FIG. A. A nomenclature reference index of the intermediates is provided below in the Nomenclature Reference Index:

Nomenclature reference index
CompoundChemical name
14-Bromo-benzaldehyde
2Methyl acrylate
3(2E)-3-(formylphenyl)-2-propenoic acid, methyl ester
43-[4-[[[2-(2-Methyl-1H-indol-3-
yl)ethyl]amino]methyl]phenyl]-2-
propenoic acid, methyl ester, monohydrochloride
5(2E)-N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-
yl)ethyl]amino]methyl]phenyl]-2-propenamide
62-hydroxypropanoic acid, compd. with 2(E)-N-
hydroxy-3-[4-[[[2-(2-methyl-1H-
indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide
Z3a2-Methyl-1H-indole-3-ethanamine
Z3b5-Chloro-2-pentanone
Z3cPhenylhydrazine
The manufacture of LBH589 lactate (6) drug substance is via a convergent synthesis; the point of convergence is the condensation of indole-amine Z3a with aldehyde 3.
The synthesis of indole-amine Z3a involves reaction of 5-chloro-2 pentanone (Z3b) with phenylhydrazine (Z3c) in ethanol at reflux (variation of Fischer indole synthesis).
Product isolation is by an extractive work-up followed by crystallization. Preparation of aldehyde 3 is by palladium catalyzed vinylation (Heck-type reaction; Pd(OAc)2/P(o-Tol)3/Bu3N in refluxing CH3CN) of 4-bromo-benzyladehyde (1) with methyl acrylate (2) with product isolation via precipitation from dilute HCl solution. Intermediates Z3a and 3 are then condensed to an imine intermediate, which is reduced using sodium borohydride in methanol below 0° C. (reductive amination). The product indole-ester 4, isolated by precipitation from dilute HCl, is recrystallized from methanol/water, if necessary. The indole ester 4 is converted to crude LBH589 free base 5 via reaction with hydroxylamine and sodium hydroxide in water/methanol below 0° C. The crude LBH589 free base 5 is then purified by recrystallization from hot ethanol/water, if necessary. LBH589 free base 5 is treated with 85% aqueous racemic lactic acid and water at ambient temperature. After seeding, the mixture is heated to approximately 65° C., stirred at this temperature and slowly cooled to 45-50° C. The resulting slurry is filtered and washed with water and dried to afford LBH589 lactate (6).
If necessary the LBH589 lactate 6 may be recrystallised once again from water in the presence of 30 mol % racemic lactic acid. Finally the LBH589 lactate is delumped to give the drug substance. If a rework of the LBH589 lactate drug substance 6 is required, the LBH589 lactate salt is treated with sodium hydroxide in ethanol/water to liberate the LBH589 free base 5 followed by lactate salt formation and delumping as described above.
All starting materials, reagents and solvents used in the synthesis of LBH589 lactate are tested according to internal specifications or are purchased from established suppliers against a certificate of analysis.

EXAMPLE 7 Formation of Monohydrate Lactate Salt
About 40 to 50 mg of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide free base was suspended in 1 ml of a solvent as listed in Table 7. A stoichiometric amount of lactic acid was subsequently added to the suspension. The mixture was stirred at ambient temperature and when a clear solution formed, stirring continued at 4° C. Solids were collected by filtration and analyzed by XRPD, TGA and 1H-NMR.

TABLE 7
LOD, %
PhysicalCrystallinity(Tdesolvation)
SolventT, ° C.Appear.and FormTdecomposit.1H-NMR
IPA4FFPexcellent4.3 (79.3)
HA156.3
Acetone4FFPexcellent4.5 (77.8)4.18 (Hbz)
HA149.5

The salt forming reaction in isopropyl alcohol and acetone at 4° C. produced a stoichiometric (1:1) lactate salt, a monohydrate. The salt is crystalline, begins to dehydrate above 77° C., and decomposes above 150° C.
EXAMPLE 18 Formation of Anhydrous Lactate Salt
DL-lactic acid (4.0 g, 85% solution in water, corresponding to 3.4 g pure DL-lactic acid) is diluted with water (27.2 g), and the solution is heated to 90° C. (inner temperature) for 15 hours. The solution is allowed to cool down to room temperature and is used as lactic acid solution for the following salt formation step.
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide free base (10.0 g) is placed in a 4-necked reaction flask with mechanical stirrer. Demineralized water (110.5 g) is added, and the suspension is heated to 65° C. (inner temperature) within 30 minutes. The DL-lactic acid solution is added to this suspension during 30 min at 65° C. During the addition of the lactate salt solution, the suspension converted into a solution. The addition funnel is rinsed with demineralized water (9.1 g), and the solution is stirred at 65° C. for an additional 30 minutes. The solution is cooled down to 45° C. (inner temperature) and seed crystals (10 mg N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate monohydrate) are added at this temperature. The suspension is cooled down to 33° C. and is stirred for additional 20 hours at this temperature. The suspension is re-heated to 65° C., stirred for 1 hour at this temperature and is cooled to 33° C. within 1 hour. After additional stirring for 3 hours at 33° C., the product is isolated by filtration, and the filter cake is washed with demineralized water (2×20 g). The wet filter-cake is dried in vacuo at 50° C. to obtain the anhydrous N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt as a crystalline product. The product is identical to the monohydrate salt (form HA) in HPLC and in 1H-NMR, with the exception of the integrals of water signals in the 1H-NMR spectra.
In additional salt formation experiments carried out according to the procedure described above, the product solution was filtered at 65° C. before cooling to 45° C., seeding and crystallization. In all cases, form A (anhydrate form) was obtained as product.
EXAMPLE 19 Formation of Anhydrous Lactate Salt
DL-lactic acid (2.0 g, 85% solution in water, corresponding to 1.7 g pure DL-lactic acid) is diluted with water (13.6 g), and the solution is heated to 90° C. (inner temperature) for 15 hours. The solution was allowed to cool down to room temperature and is used as lactic acid solution for the following salt formation step.
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide free base (5.0 g) is placed in a 4-necked reaction flask with mechanical stirrer. Demineralized water (54.85 g) is added, and the suspension is heated to 48° C. (inner temperature) within 30 minutes. The DL-lactic acid solution is added to this suspension during 30 minutes at 48° C. A solution is formed. Seed crystals are added (as a suspension of 5 mg N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt, anhydrate form A, in 0.25 g of water) and stirring is continued for 2 additional hours at 48° C. The temperature is raised to 65° C. (inner temperature) within 30 minutes, and the suspension is stirred for additional 2.5 hours at this temperature. Then the temperature is cooled down to 48° C. within 2 hours, and stirring is continued at this temperature for additional 22 hours. The product is isolated by filtration and the filter cake is washed with demineralized water (2×10 g). The wet filter-cake is dried in vacuo at 50° C. to obtain anhydrous N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt (form A) as a crystalline product.
EXAMPLE 20 Conversion of Monohydrate Lactate Salt to Anhydrous Lactate Salt
DL-lactic acid (0.59 g, 85% solution in water, corresponding to 0.5 g pure DL-lactic acid) is diluted with water (4.1 g), and the solution is heated to 90° C. (inner temperature) for 15 hours. The solution is allowed to cool down to room temperature and is used as lactic acid solution for the following salt formation step.
10 g of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt monohydrate is placed in a 4-necked reaction flask. Water (110.9 g) is added, followed by the addition of the lactic acid solution. The addition funnel of the lactic acid is rinsed with water (15.65 g). The suspension is heated to 82° C. (inner temperature) to obtain a solution. The solution is stirred for 15 minutes at 82° C. and is hot filtered into another reaction flask to obtain a clear solution. The temperature is cooled down to 50° C., and seed crystals are added (as a suspension of 10 mg N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt, anhydrate form, in 0.5 g of water). The temperature is cooled down to 33° C. and stirring is continued for additional 19 hours at this temperature. The formed suspension is heated again to 65° C. (inner temperature) within 45 minutes, stirred at 65° C. for 1 hour and cooled down to 33° C. within 1 hour. After stirring at 33° C. for additional 3 hours, the product is isolated by filtration and the wet filter cake is washed with water (50 g). The product is dried in vacuo at 50° C. to obtain crystalline anhydrous N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl) ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt (form A).
EXAMPLE 21 Formation of Anhydrous Lactate Salt
DL-lactic acid (8.0 g, 85% solution in water, corresponding to 6.8 g pure DL-lactic acid) was diluted with water (54.4 g), and the solution was heated to 90° C. (inner temperature) for 15 hours. The solution was allowed to cool down to room temperature and was used as lactic acid solution for the following salt formation step.
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide (20 g) is placed in a 1 L glass reactor, and ethanol/water (209.4 g of a 1:1 w/w mixture) is added. The light yellow suspension is heated to 60° C. (inner temperature) within 30 minutes, and the lactic acid solution is added during 30 minutes at this temperature. The addition funnel is rinsed with water (10 g). The solution is cooled to 38° C. within 2 hours, and seed crystals (20 mg of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt, anhydrate form) are added at 38° C. After stirring at 38° C. for additional 2 hours, the mixture is cooled down to 25° C. within 6 hours. Cooling is continued from 25° C. to 10° C. within 5 hours, from 10° C. to 5° C. within 4 hours and from 5° C. to 2° C. within 1 hour. The suspension is stirred for additional 2 hours at 2° C., and the product is isolated by filtration. The wet filter cake is washed with water (2×30 g), and the product is dried in vacuo at 45° C. to obtain crystalline anhydrous N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide lactate salt (form A).
EXAMPLE 28 Formation of Lactate Monohydrate Salt
3.67 g (10 mmol) of the free base monohydrate (N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl) ethyl]amino]methyl]phenyl]-2E-2-propenamide) and 75 ml of acetone were charged in a 250 ml 3-neck flask equipped with a magnetic stirrer and an addition funnel. To the stirred suspension were added dropwise 10 ml of 1 M lactic acid in water (10 mmol) dissolved in 20 ml acetone, affording a clear solution. Stirring continued at ambient and a white solid precipitated out after approximately 1 hour. The mixture was cooled in an ice bath and stirred for an additional hour. The white solid was recovered by filtration and washed once with cold acetone (15 ml). It was subsequently dried under vacuum to yield 3.94 g of the lactate monohydrate salt of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide (86.2%).


References

  1. Revill, P; Mealy, N; Serradell, N; Bolos, J; Rosa, E (2007). “Panobinostat”Drugs of the Future 32 (4): 315. doi:10.1358/dof.2007.032.04.1094476ISSN 0377-8282.
  2.  Table 3: Select epigenetic inhibitors in various stages of development from Mack, G. S. (2010). “To selectivity and beyond”. Nature Biotechnology 28 (12): 1259–1266.doi:10.1038/nbt.1724PMID 21139608edit
  3.  ClinicalTrials.gov NCT00425555 Study of Oral LBH589 in Adult Patients With Refractory Cutaneous T-Cell Lymphoma
  4.  ClinicalTrials.gov: LBH-589
  5.  Prince, HM; M Bishton (2009). “Panobinostat (LBH589): a novel pan-deacetylase inhibitor with activity in T cell lymphoma”Hematology Meeting Reports (Parkville, Australia: Peter MacCallum Cancer Centre and University of Melbourne) 3 (1): 33–38.
  6.  Simons, J (27 April 2013). “Scientists on brink of HIV cure”. The Telegraph.
  7.  ClinicalTrials.gov NCT01680094 Safety and Effect of The HDAC Inhibitor Panobinostat on HIV-1 Expression in Patients on Suppressive HAART (CLEAR)
  8.  Mayo Clinic Researchers Formulate Treatment Combination Lethal To Pancreatic Cancer Cells
  9.  Garbes, L; Riessland, M; Hölker, I; Heller, R; Hauke, J; Tränkle, Ch; Coras, R; Blümcke, I; Hahnen, E; Wirth, B (2009). “LBH589 induces up to 10-fold SMN protein levels by several independent mechanisms and is effective even in cells from SMA patients non-responsive to valproate”Human Molecular Genetics 18 (19): 3645–3658. doi:10.1093/hmg/ddp313.PMID 19584083.
  10.  Tate, CR; Rhodes, LV; Segar, HC; Driver, JL; Pounder, FN; Burow, ME; and Collins-Burow, BM (2012). “Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat”Breast Cancer Research 14 (3).
  11.  TA Rasmussen, et al. Comparison of HDAC inhibitors in clinical development: Effect on HIV production in latently infected cells and T-cell activation. Human Vaccines & Immunotherapeutics 9:5, 1-9, May 2013.
  12. Drugs of the Future 32(4): 315-322 (2007)
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  22. 2300920311-26-2012Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth.Journal of medicinal chemistry
  23. 216344307-14-2011Discovery of (2E)-3-{2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl}-N-hydroxyacrylamide (SB939), an orally active histone deacetylase inhibitor with a superior preclinical profile.Journal of medicinal chemistry
  24. 214174194-28-2011Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.Journal of medicinal chemistry
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  26. 156509311-1-2005The American Society of Hematology–46th Annual Meeting and Exposition. HDAC, Flt and farnesyl transferase inhibitors.IDrugs : the investigational drugs journal
  27. US79896398-3-2011PROCESS FOR MAKING SALTS OF N-HYDROXY-3-[4-[[[2-(2-METHYL-1H-INDOL-3-YL)ETHYL]AMINO]METHYL]PHENYL]-2E-2-PROPENAMIDE
    US201028640911-12-2010SALTS OF N-HYDROXY-3-[4-[[[2-(2-METHYL-1H-INDOL-3-YL)ETHYL]AMINO]METHYL]PHENYL]-2E-2-PROPENAMIDE
    US20101792087-16-2010Use of HDAC Inhibitors for the Treatment of Bone Destruction
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    US20101373986-4-2010USE OF HDAC INHIBITORS FOR THE TREATMENT OF GASTROINTESTINAL CANCERS
    US200930640512-11-2009PROCESS FOR MAKING N-HYDROXY-3-[4-[[[2-(2-METHYL-1H-INDOL-3-YL)ETHYL]AMINO]METHYL]PHENYL]-2E-2-PROPENAMIDE AND STARTING MATERIALS THEREFOR
    US200928115911-13-2009USE OF HDAC INHIBITORS FOR THE TREATMENT OF LYMPHOMAS
    US200926443910-23-2009Combination of a) N–4-(3-pyridyl)-2-pyrimidine-amine and b) a histone deacetylase inhibitor for the treatment of leukemia
    US20091979368-7-2009SALTS OF N-HYDROXY-3-[4-[[[2-(2-METHYL-1H-INDOL-3-YL)ETHYL]AMINO]METHYL]PHENYL]-2E-2-PROPENAMIDE
    US20090120661-9-2009Method of Use of Deacetylase Inhibitors
US200831904512-26-2008Combination of Histone Deacetylase Inhibitors and Radiation
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WO2006021397A1Aug 22, 2005Mar 2, 2006Recordati Ireland LtdLercanidipine salts


................................................................................
2 BELINOSTAT
File:Belinostat.svg

Belinostat (PXD101)
PHASE 2, FAST TRACK FDA , ORPHAN STATUS
  • PDX101
  • PX 105684
  • PXD-101
  • PXD101
  • UNII-F4H96P17NZ
Belinostat (PXD101) is a novel HDAC inhibitor with IC50 of 27 nM, with activity demonstrated in cisplatin-resistant tumors.
Belinostat inhibits the growth of tumor cells (A2780, HCT116, HT29, WIL, CALU-3, MCF7, PC3 and HS852) with IC50 from 0.2-0.66 μM. PD101 shows low activity in A2780/cp70 and 2780AD cells. Belinostat inhibits bladder cancer cell growth, especially in 5637 cells, which shows accumulation of G0-G1 phase, decrease in S phase, and increase in G2-M phase. Belinostat also shows enhanced tubulin acetylation in ovarian cancer cell lines. A recent study shows that Belinostat activates protein kinase A in a TGF-β signaling-dependent mechanism and decreases survivin mRNA.
MW 318.07
MFC15H14N2O4S
414864-00-9  cas no
866323-14-0
(2E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]acrylamide
A novel HDAC inhibitor
…………………………
BELINOSTAT
Belinostat (PXD101) is experimental drug candidate under development byTopoTarget for the treatment of hematological malignancies and solid tumors. It is a histone deacetylase inhibitor.[1]
A hydroxamate-type inhibitor of histone deacetylase.
NCI: A novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antineoplastic activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cellular differentiation, and inhibiting angiogenesis. This agent may sensitize drug-resistant tumor cells to other antineoplastic agents, possibly through a mechanism involving the down-regulation of thymidylate synthase
In 2007 preliminary results were released from the Phase II clinical trial of intravenous belinostat in combination with carboplatin and paclitaxel for relapsedovarian cancer.[2] Final results in late 2009 of a phase II trial for T cell lymphomawere encouraging.[3] Belinostat has been granted orphan drug and fast trackdesignation by the FDA.[4]

 

The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).
Trichostatin A (TSA)

Figure imgf000005_0001
Suberoylanilide Hydroxamic Acid (SAHA)

Figure imgf000005_0002
Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa et al., 1994), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki et al., 1999). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999). Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g., liver fibrosis and liver cirrhosis. See, e.g., Geerts et al., 1998.
Recently, certain compounds that induce differentiation have been reported to inhibit histone deacetylases. Several experimental antitumour compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been reported to act, at least in part, by inhibiting histone deacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998; Kijima et al., 1993). Additionally, diallyl sulfide and related molecules (see, e.g., Lea et al., 1999), oxamflatin (see, e.g., Kim et al., 1999), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito et al., 1999; Suzuki et al., 1999; note that MS-27-275 was later re-named as MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995), FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwon et al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g., Richon et al., 1998) have been reported to inhibit histone deacetylases. In vitro, some of these compounds are reported to inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases, and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (see, e.g., Richon et al, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu, 1988). In vivo, phenybutyrate is reported to be effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (see, e.g., Warrell et al., 1998). SAHA is reported to be effective in preventing the formation of mammary tumours in rats, and lung tumours in mice (see, e.g., Desai et al., 1999).
The clear involvement of HDACs in the control of cell proliferation and differentiation suggest that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukaemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARσ (retinoic acid receptor). In some cases, a different translocation (t(11 ;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA- inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).
BELINOSTAT
Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, coloreetal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).
Psoriasis is a common chronic disfiguring skin disease which is characterised by well-demarcated, red, hardened scaly plaques: these may be limited or widespread. The prevalence rate of psoriasis is approximately 2%, i.e., 12.5 million sufferers in the triad countries (US/Europe/Japan). While the disease is rarely fatal, it clearly has serious detrimental effects upon the quality of life of the patient: this is further compounded by the lack of effective therapies. Present treatments are either ineffective, cosmetically unacceptable, or possess undesired side effects. There is therefore a large unmet clinical need for effective and safe drugs for this condition. Psoriasis is a disease of complex etiology. Whilst there is clearly a genetic component, with a number of gene loci being involved, there are also undefined environmental triggers. Whatever the ultimate cause of psoriasis, at the cellular level, it is characterised by local T-cell mediated inflammation, by keratinocyte hyperproliferation, and by localised angiogenesis. These are all processes in which histone deacetylases have been implicated (see, e.g., Saunders et al., 1999; Bernhard et al, 1999; Takahashi et al, 1996; Kim et al , 2001 ). Therefore HDAC inhibitors may be of use in therapy for psoriasis. Candidate drugs may be screened, for example, using proliferation assays with T-cells and/or keratinocytes.
 ………………………………………………………………………..

PXD101/Belinostat®
(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide, also known as PXD101 and Belinostat®, shown below, is a well known histone deacetylate (HDAC) inhibitor. It is being developed for treatment of a range of disorders mediated by HDAC, including proliferative conditions (such as cancer and psoriasis), malaria, etc.
Figure US20100286279A1-20101111-C00001
PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.
Figure US20100286279A1-20101111-C00002
Figure US20100286279A1-20101111-C00003
…………………………………..
GENERAL SYNTHESIS
IGNORE 10
Figure imgf000060_0002
ENTRY 45 IS BELINOSTAT
Scheme 1

Figure imgf000101_0001
By using amines instead of aniline, the corresponding products may be obtained. The use of aniline, 4-methoxyaniline, 4-methylaniline, 4-bromoaniline, 4-chloroaniline, 4-benzylamine, and 4-phenethyamine, among others, is described in the Examples below.
In another method, a suitable amino acid (e.g., ω-amino acid) having a protected carboxylic acid (e.g., as an ester) and an unprotected amino group is reacted with a sulfonyl chloride compound (e.g., RSO2CI) to give the corresponding sulfonamide having a protected carboxylic acid. The protected carboxylic acid is then deprotected using base to give the free carboxylic acid, which is then reacted with, for example, hydroxylamine 2-chlorotrityl resin followed by acid (e.g., trifluoroacetic acid), to give the desired carbamic acid.
One example of this approach is illustrated below, in Scheme 2, wherein the reaction conditions are as follows: (i) RSO2CI, pyridine, DCM, room temperature, 12 hours; (ii) 1 M LiOH or 1 M NaOH, dioxane, room temperature, 3-48 hours; (iii) hydroxylamine 2-chlorotrityl resin, HOAt, HATU, DIPEA, DCM, room temperature, 16 hours; and (iv) TFA/DCM (5:95, v/v), room temperature, 1.5 hours.
Scheme 2

Figure imgf000102_0001
Additional methods for the synthesis of compounds of the present invention are illustrated below and are exemplified in the examples below.
Scheme 3A

Figure imgf000102_0002
Scheme 3B

Figure imgf000103_0001
Scheme 4

Figure imgf000104_0001
Figure imgf000105_0001


Scheme 8

Figure imgf000108_0002
Scheme 9

Figure imgf000109_0001
……………………………………………………………………..
SYNTHESIS
Example 1
3-Formylbenzenesulfonic acid, sodium salt (1)

Figure imgf000123_0001
Oleum (5 ml) was placed in a reaction vessel and benzaldehyde (2.00 g, 18.84 mmol) was slowly added not exceeding the temperature of the reaction mixture more than 30°C. The obtained solution was stirred at 40°C for ten hours and at ambient temperature overnight. The reaction mixture was poured into ice and extracted with ethyl acetate. The aqueous phase was treated with CaC03 until the evolution of C02 ceased (pH~6-7), then the precipitated CaSO4was filtered off and washed with water. The filtrate was treated with Na2CO3 until the pH of the reaction medium increased to pH 8, obtained CaCO3 was filtered off and water solution was evaporated in vacuum. The residue was washed with methanol, the washings were evaporated and the residue was dried in desiccator over P2Oβ affording the title compound (2.00 g, 51%). 1H NMR (D20), δ: 7.56-8.40 (4H, m); 10.04 ppm (1 H, s).
Example 2 3-(3-Sulfophenyl)acrylic acid methyl ester, sodium salt (2)

Figure imgf000124_0001
Sodium salt of 3-formylbenzenesulfonic acid (1) (1.00 g, 4.80 mmol), potassium carbonate (1.32 g, 9.56 mmol), trimethyl phosphonoacetate (1.05 g, 5.77 mmol) and water (2 ml) were stirred at ambient temperature for 30 min., precipitated solid was filtered and washed with methanol. The filtrate was evaporated and the title compound (2) was obtained as a white solid (0.70 g, 55%). 1H NMR (DMSO- dβl HMDSO), δ: 3.68 (3H, s); 6.51 (1 H, d, J=16.0 Hz); 7.30-7.88 (5H, m).
Example 3 3-(3-Chlorosulfonylphenyl)acrylic acid methyl ester (3)

Figure imgf000124_0002
To the sodium salt of 3-(3-sulfophenyl)acrylic acid methyl ester (2) (0.670 g, 2.53 mmol) benzene (2 ml), thionyl chloride (1.508 g, 0.9 ml, 12.67 mmol) and 3 drops of dimethylformamide were added and the resultant suspension was stirred at reflux for one hour. The reaction mixture was evaporated, the residue was dissolved in benzene (3 ml), filtered and the filtrate was evaporated to give the title compound (0.6’40 g, 97%).
Example 4 3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a)

Figure imgf000125_0001
A solution of 3-(3-chlorosulfonylphenyl)acrylic acid methyl ester (3) (0.640 g, 2.45 mmol) in dichloromethane (2 ml) was added to a mixture of aniline (0.465 g, 4.99 mmol) and pyridine (1 ml), and the resultant solution was stirred at 50°C for one hour. The reaction mixture was evaporated and the residue was partitioned between ethyl acetate and 10% HCI. The organic layer was washed successively with water, saturated NaCl, and dried (Na2S0 ). The solvent was removed and the residue was chromatographed on silica gel with chloroform-ethyl acetate (7:1 , v/v) as eluent. The obtained product was washed with diethyl ether to give the title compound (0.226 g, 29%). 1H NMR (CDCI3, HMDSO), δ: 3.72 (3H, s); 6.34 (1H, d, J=16.0 Hz); 6.68 (1 H, br s); 6.92-7.89 (10H, m).
Example 5 3-(3-Phenylsulfamoylphenyl)acrylic acid (5a)

Figure imgf000125_0002
3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a) (0.220 g, 0.69 mmol) was dissolved in methanol (3 ml), 1N NaOH (2.08 ml, 2.08 mmol) was added and the resultant solution was stirred at ambient temperature overnight. The reaction mixture was partitioned between ethyl acetate and water. The aqueous layer was acidified with 10% HCI and stirred for 30 min. The precipitated solid was filtered, washed with water and dried in desiccator over P2Os to give the title compound as a white solid (0.173 g, 82%). Example 6 3-(3-Phenylsulfamoylphenyl)acryloyl chloride (6a)

Figure imgf000126_0001
To a suspension of 3-(3-phenylsulfamoylphenyl)acrylic acid (5a) (0.173 g, 0.57 mmol) in dichloromethane (2.3 ml) oxalyl chloride (0.17 ml, 1.95 mmol) and one drop of dimethylformamide were added. The reaction mixture was stirred at 40°C for one hour and concentrated under reduced pressure to give crude title compound (0.185 g).
Example 7
N-Hydroxy-3-(3-phenylsulfamoylphenyl)acrylamide (7a) (PX105684) BELINOSTAT

Figure imgf000126_0002
To a suspension of hydroxylamine hydrochloride (0.200 g, 2.87 mmol) in tetrahydrofuran (3.5 ml) a saturated NaHCOβ solution (2.5 ml) was added and the resultant mixture was stirred at ambient temperature for 10 min. To the reaction mixture a 3-(3-phenylsulfamoylphenyl)acryloyl chloride (6a) (0.185 g) solution in tetrahydrofuran (2.3 ml) was added and stirred at ambient temperature for one hour. The reaction mixture was partitioned between ethyl acetate and 2N HCI. The organic layer was washed successively with water and saturated NaCl, the solvent was removed and the residue was washed with acetonitrile and diethyl ether.
The title compound was obtained as a white solid (0.066 g, 36%), m.p. 172°C. BELINOSTAT
1H NMR (DMSO-d6, HMDSO), δ: 6.49 (1 H, d, J=16.0 Hz); 7.18-8.05 (10H, m); 9.16 (1 H, br s); 10.34 (1 H, s); 10.85 ppm (1 H, br s).
HPLC analysis on Symmetry C18column: impurities 4% (column size 3.9×150 mm; mobile phase acetonitrile – 0.1 M phosphate buffer (pH 2.5), 40:60; sample concentration 1 mg/ml; flow rate 0.8 ml/ min; detector UV 220 nm).
Anal. Calcd for C154N204S, %: C 56.59, H 4.43, N 8.80. Found, %: C 56.28, H 4.44, N 8.56.
……………………………………………………………………….
SYNTHESIS
US20100286279
Figure US20100286279A1-20101111-C00034


…………………………………………………….
SYNTHESIS AND SPECTRAL DATA
Journal of Medicinal Chemistry, 2011 ,  vol. 54,  13  pg. 4694 – 4720
(E)-N-Hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (28, belinostat, PXD101).
The methyl ester (27) (8.0 g) was prepared according to reported synthetic route,
(Watkins, C. J.; Romero-Martin, M.-R.; Moore, K. G.; Ritchie, J.; Finn, P. W.; Kalvinsh, I.;
Loza, E.; Dikvoska, K.; Gailite, V.; Vorona, M.; Piskunova, I.; Starchenkov, I.; Harris, C. J.;
Duffy, J. E. S. Carbamic acid compounds comprising a sulfonamide linkage as HDAC
inhibitors. PCT Int. Appl. WO200230879A2, April 18, 2002.)
but using procedure D (Experimental Section) or method described for 26 to convert the methyl ester to crude
hydroxamic acid which was further purified by chromatography (silica, MeOH/DCM = 1:10) to
afford 28 (PXD101) as off-white or pale yellow powder (2.5 g, 31%).
LC–MS m/z 319.0 ([M +H]+).
1H NMR (DMSO-d6)  12–9 (very broad, 2H), 7.90 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.70 (d, J
= 7.8 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 15.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 2H), 7.08 (d,
J = 7.8 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.50 (d, J = 15.8 Hz, 1H);
13C NMR (DMSO-d6)  162.1,
140.6, 138.0, 136.5, 135.9, 131.8, 130.0, 129.2, 127.1, 124.8, 124.1, 121.3, 120.4.
Anal.
(C15H14N2O4S) C, H, N
………………………………………………..
SYNTHESIS
PXDIOI / Belinostat®
(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide, also known as PXD101 and Belinostat®, shown below, is a well known histone deacetylate (HDAC) inhibitor. It is being developed for treatment of a range of disorders mediated by HDAC, including proliferative conditions (such as cancer and psoriasis), malaria, etc.

Figure imgf000003_0001
PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.
Scheme 1
Not isolated
Figure imgf000003_0002
ed on (A)
on (D)
Figure imgf000003_0003
d on (H)
Figure imgf000004_0001
There is a need for alternative methods for the synthesis of PXD101 and related compounds for example, methods which are simpler and/or employ fewer steps and/or permit higher yields and/or higher purity product.
Scheme 5

Figure imgf000052_0001
DMAP, toluene
Figure imgf000052_0003
Figure imgf000052_0002

Figure imgf000052_0004
Synthesis 1 3-Bromo-N-phenyl-benzenesulfonamide (3)

Figure imgf000052_0005
To a 30 gallon (-136 L) reactor was charged aniline (2) (4.01 kg; 93.13 g/mol; 43 mol), toluene (25 L), and 4-(dimethylamino)pyridine (DMAP) (12 g), and the mixture was heated to 50-600C. 3-Bromobenzenesulfonyl chloride (1) (5 kg; 255.52 g/mol; 19.6 mol) was charged into the reactor over 30 minutes at 50-600C and progress of the reaction was monitored by HPLC. After 19 hours, toluene (5 L) was added due to losses overnight through the vent line and the reaction was deemed to be complete with no compound (1) being detected by HPLC. The reaction mixture was diluted with toluene (10 L) and then quenched with 2 M aqueous hydrochloric acid (20 L). The organic and aqueous layers were separated, the aqueous layer was discarded, and the organic layer was washed with water (20 L), and then 5% (w/w) sodium bicarbonate solution (20 L), while maintaining the batch temperature at 45-55°C. The batch was then used in the next synthesis.
Synthesis 2 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrylic acid ethyl ester (5)

Figure imgf000053_0001
To the batch containing 3-bromo-N-phenyl-benzenesulfonamide (3) (the treated organic layer obtained in the previous synthesis) was added triethylamine (2.97 kg; 101.19 g/mol; 29.4 mol), tri(o-tolyl)phosphine (119 g; 304.37 g/mol; 0.4 mol), and palladium (II) acetate (44 g; 224.51 g/mol; 0.2 mol), and the resulting mixture was degassed four times with a vacuum/nitrogen purge at 45-55°C. Catalytic palladium (0) was formed in situ. The batch was then heated to 80-900C and ethyl acrylate (4) (2.16 kg; 100.12 g/mol; 21.6 mol) was slowly added over 2.75 hours. The batch was sampled after a further 2 hours and was deemed to be complete with no compound (3) being detected by HPLC. The batch was cooled to 45-55°C and for convenience was left at this temperature overnight.
The batch was then reduced in volume under vacuum to 20-25 L, at a batch temperature of 45-55°C, and ethyl acetate (20 L) was added. The batch was filtered and the residue washed with ethyl acetate (3.5 L). The residue was discarded and the filtrates were sent to a 100 gallon (-454 L) reactor, which had been pre-heated to 600C. The 30 gallon (-136 L) reactor was then cleaned to remove any residual Pd, while the batch in the 100 gallon (-454 L) reactor was washed with 2 M aqueous hydrochloric acid and water at 45-55°C. Once the washes were complete and the 30 gallon (-136 L) reactor was clean, the batch was transferred from the 100 gallon (-454 L) reactor back to the 30 gallon (-136 L) reactor and the solvent was swapped under vacuum from ethyl acetate/toluene to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it. The batch was then cooled to 0-100C and held at this temperature over the weekend in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). A sample of the wet-cake was taken for Pd analysis. The Pd content of the crude product (5) was determined to be 12.9 ppm.
The wet-cake was then charged back into the 30 gallon (-136 L) reactor along with ethyl acetate (50 L) and heated to 40-500C in order to obtain a solution. A sparkler filter loaded with 12 impregnated Darco G60® carbon pads was then connected to the reactor and the solution was pumped around in a loop through the sparkler filter. After 1 hour, a sample was taken and evaporated to dryness and analysed for Pd content. The amount of Pd was found to be 1.4 ppm. A second sample was taken after 2 hours and evaporated to dryness and analysed for Pd content. The amount of Pd had been reduced to 0.6 ppm. The batch was blown back into the reactor and held at 40-500C overnight before the solvent was swapped under vacuum from ethyl acetate to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it and the batch was cooled to 0-100C and held at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum for 25 hours. A first lot of the title compound (5) was obtained as an off-white solid (4.48 kg, 69% overall yield from 3-bromobenzenesulfonyl chloride (1)) with a Pd content of 0.4 ppm and a purity of 99.22% (AUC) by HPLC.
Synthesis 3 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrvlic acid (6)

Figure imgf000054_0001
To the 30 gallon (-136 L) reactor was charged the (E)-3-(3-phenylsulfamoyl-phenyl)- acrylic acid ethyl ester (5) (4.48 kg; 331.39 g/mol; 13.5 mol) along with 2 M aqueous sodium hydroxide (17.76 L; -35 mol). The mixture was heated to 40-50°C and held at this temperature for 2 hours before sampling, at which point the reaction was deemed to be complete with no compound (5) being detected by HPLC. The batch was adjusted to pH 2.2 using 1 M aqueous hydrochloric acid while maintaining the batch temperature between 40-500C. The product had precipitated and the batch was cooled to 20-300C and held at this temperature for 1 hour before filtering and washing the cake with water (8.9 L). The filtrate was discarded. The batch was allowed to condition on the filter overnight before being charged back into the reactor and slurried in water (44.4 L) at 40-500C for 2 hours. The batch was cooled to 15-20°C, held for 1 hour, and then filtered and the residue washed with water (8.9 L). The filtrate was discarded. The crude title compound (6) was transferred to an oven for drying at 45-55°C under vacuum with a slight nitrogen bleed for 5 days (this was done for convenience) to give a white solid (3.93 kg, 97% yield). The moisture content of the crude material was measured using Karl Fischer (KF) titration and found to be <0.1% (w/w). To the 30 gallon (-136 L) reactor was charged the crude compound (6) along with acetonitrile (47.2 L). The batch was heated to reflux (about 80°C) and held at reflux for 2 hours before cooling to 0-10°C and holding at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with cold acetonitrile (7.9 L). The filtrate was discarded and the residue was dried under vacuum at 45-55°C for 21.5 hours. The title compound (6) was obtained as a fluffy white solid (3.37 kg, 84% yield with respect to compound (5)) with a purity of 99.89% (AUC) by HPLC.
Synthesis 4 (E)-N-Hvdroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (PXD101) BELINOSTAT

Figure imgf000055_0001
To the 30 gallon (-136 L) reactor was charged (E)-3-(3-phenylsulfamoyl-phenyl)-acrylic acid (6) (3.37 kg; 303.34 g/mol; 11.1 mol) and a pre-mixed solution of 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in isopropyl acetate (IPAc) (27 g in 30 L; 152.24 g/mol; 0.18 mol). The slurry was stirred and thionyl chloride (SOCI2) (960 mL; density ~1.631 g/mL; 118.97 g/mol; -13 mol) was added to the reaction mixture and the batch was stirred at 20-300C overnight. After 18.5 hours, the batch was sampled and deemed to be complete with no compound (6) being detected by HPLC. The resulting solution was transferred to a 100 L Schott reactor for temporary storage while the
30 gallon (-136 L) reactor was rinsed with isopropyl acetate (IPAc) and water. Deionized water (28.9 L) was then added to the 30 gallon (-136 L) reactor followed by 50% (w/w) hydroxylamine (6.57 L; -1.078 g/mL; 33.03 g/mol; -214 mol) and another charge of deionized water (1.66 L) to rinse the lines free of hydroxylamine to make a 10% (w/w) hydroxylamine solution. Tetrahydrofuran (THF) (6.64 L) was then charged to the
30 gallon (-136 L) reactor and the mixture was stirred and cooled to 0-100C. The acid chloride solution (from the 100 L Schott reactor) was then slowly charged into the hydroxylamine solution over 1 hour maintaining a batch temperature of 0-10°C during the addition. The batch was then allowed to warm to 20-300C. The aqueous layer was separated and discarded. The organic layer was then reduced in volume under vacuum while maintaining a batch temperature of less than 300C. The intention was to distill out 10-13 L of solvent, but this level was overshot. A larger volume of isopropyl acetate (IPAc) (16.6 L) was added and about 6 L of solvent was distilled out. The batch had precipitated and heptanes (24.9 L) were added and the batch was held at 20-30°C overnight. The batch was filtered and the residue was washed with heptanes (6.64 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum with a slight nitrogen bleed over the weekend. The title compound (PXD101) was obtained as a light orange solid (3.11 kg, 89% yield with respect to compound (6)) with a purity of 99.25% (AUC) by HPLC.
The title compound (PXD101) (1.2 kg, 3.77 mol) was dissolved in 8 volumes of 1:1 (EtOH/water) at 600C. Sodium bicarbonate (15.8 g, 5 mol%) was added to the solution. Water (HPLC grade) was then added at a rate of 65 mL/min while keeping the internal temperature >57°C. After water (6.6 L) had been added, crystals started to form and the water addition was stopped. The reaction mixture was then cooled at a rate of 10°C/90 min to a temperature of 0-10cC and then stirred at ambient temperature overnight. The crystals were then filtered and collected. The filter cake was washed by slurrying in water (2 x 1.2 L) and then dried in an oven at 45°C for 60 hours with a slight nitrogen bleed. 1.048 kg (87% recovery) of a light orange solid was recovered. Microscopy and XRPD data showed a conglomerate of irregularly shaped birefringant crystalline particles. The compound was found to contain 0.02% water.
As discussed above: the yield of compound (5) with respect to compound (1) was 69%. the yield of compound (6) with respect to compound (5) was 84%. the yield of PXD101 with respect to compound (6) was 89%.
……………….
FORMULATION
Formulation Studies
These studies demonstrate a substantial enhancement of HDACi solubility (on the order of a 500-fold increase for PXD-101) using one or more of: cyclodextrin, arginine, and meglumine. The resulting compositions are stable and can be diluted to the desired target concentration without the risk of precipitation. Furthermore, the compositions have a pH that, while higher than ideal, is acceptable for use.

Figure imgf000047_0001
UV Absorbance
The ultraviolet (UV absorbance E\ value for PXD-101 was determined by plotting a calibration curve of PXD-101 concentration in 50:50 methanol/water at the λmax for the material, 269 nm. Using this method, the E1i value was determined as 715.7.
Methanol/water was selected as the subsequent diluting medium for solubility studies rather than neat methanol (or other organic solvent) to reduce the risk of precipitation of the cyclodextrin.
Solubility in Demineralised Water
The solubility of PXD-101 was determined to be 0.14 mg/mL for demineralised water. Solubility Enhancement with Cvclodextrins
Saturated samples of PXD-101 were prepared in aqueous solutions of two natural cyclodextrins (α-CD and γ-CD) and hydroxypropyl derivatives of the α, β and Y cyclodextrins (HP-α-CD, HP-β-CD and HP-γ-CD). All experiments were completed with cyclodextrin concentrations of 250 mg/mL, except for α-CD, where the solubility of the cyclodextrin was not sufficient to achieve this concentration. The data are summarised in the following table. HP-β-CD offers the best solubility enhancement for PXD-101.

Figure imgf000048_0001
Phase Solubility Determination of HP-β-CD
The phase solubility diagram for HP-β-CD was prepared for concentrations of cyclodextrin between 50 and 500 mg/mL (5-50% w/v). The calculated saturated solubilities of the complexed HDACi were plotted against the concentration of cyclodextrin. See Figure 1.
………………………..
  1.  Plumb, Jane A.; Finn, Paul W.; Williams, Robert J.; Bandara, Morwenna J.; Romero, M. Rosario; Watkins, Claire J.; La Thangue, Nicholas B.; Brown, Robert (2003). “Pharmacodynamic Response and Inhibition of Growth of Human Tumor Xenografts by the Novel Histone Deacetylase Inhibitor PXD101″. Molecular Cancer Therapeutics 2 (8): 721–728. PMID 12939461.
  2.  “CuraGen Corporation (CRGN) and TopoTarget A/S Announce Presentation of Belinostat Clinical Trial Results at AACR-NCI-EORTC International Conference”. October 2007.
  3. Final Results of a Phase II Trial of Belinostat (PXD101) in Patients with Recurrent or Refractory Peripheral or Cutaneous T-Cell Lymphoma, December 2009
  4.  “Spectrum adds to cancer pipeline with $350M deal.”. February 2010.
  5. Helvetica Chimica Acta, 2005 ,  vol. 88,  7  PG. 1630 – 1657, MP 172
  6. WO2009/40517 A2, ….
  7. WO2006/120456 A1, …..
  8. Synthetic Communications, 2010 ,  vol. 40,  17  PG. 2520 – 2524, MP 172
  9. Journal of Medicinal Chemistry, 2011 ,  vol. 54,   13  PG. 4694 – 4720, NMR IN SUP INFO

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………………………..
SPECTRUM
Tiny Biotech With Three Cancer Drugs Is More Alluring Takeover Bet Now
Forbes
The drug is one of Spectrum’s two drugs undergoing phase 3 clinical trials. Allergan paid Spectrum $41.5 million and will make additional payments of up to $304 million based on achieving certain milestones. So far, Raj Shrotriya, Spectrum’s chairman, 
……………………………..
Copenhagen, December 10, 2013
Topotarget announces the submission of a New Drug Application (NDA) for belinostat for the treatment of relapsed or refractory (R/R) peripheral T-cell lymphoma (PTCL) to the US Food and Drug Administration (FDA). The NDA has been filed for Accelerated Approval with a request for Priority Review. Response from the FDA regarding acceptance to file is expected within 60 days from the FDA receipt date.
read all this here
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3 VORINOSTAT
Vorinostat
Zolinza, SAHA, suberoylanilide hydroxamic acid, Suberanilohydroxamic acid, N-hydroxy-N'-phenyloctanediamide
US patent 5369108, PDT PATENT
For the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma who have progressive, persistent or recurrent disease on or following two systemic therapies. Inhibits histone deacetylase I & 3.
  • CCRIS 8456
  • HSDB 7930
  • M344
  • N-Hydroxy-N'-phenyloctanediamide
  • SAHA
  • SAHA cpd
  • Suberanilohydroxamic acid
  • suberoylanilide hydroxamic acid
  • UNII-58IFB293JI
N-hydroxy-N'-phenyl-octanediamide
Trade namesZolinza, 100 MG, CAPSULE, ORAL
 ZOLINZA (VORINOSTAT) [Merck Sharp & Dohme Corp.]
MedlinePlusa607050
Licence dataUS FDA:link
 LAUNCHED 2006 MERCKhttp://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021991s002lbl.pdf
Legal status-only (US)
RoutesOral
Pharmacokinetic data
Protein binding71%
MetabolismHepatic glucuronidation andoxidation
CYP system not involved
Half-life2 hours
ExcretionRenal (negligible)
Identifiers
CAS number149647-78-9 
ATC codeL01XX38
Chemical data
FormulaC14H20N2O3 
Mol. mass264.32 g/mol
Vorinostat (rINN) also known as suberanilohydroxamic acid (suberoyl+anilide+hydroxamic acid abbreviated as SAHA) is a member of a larger class of compounds that inhibit histone deacetylases (HDAC). Histone deacetylase inhibitors (HDI) have a broad spectrum of epigenetic activities.
Vorinostat is marketed under the name Zolinza for the treatment of cutaneous T cell lymphoma (CTCL) when the disease persists, gets worse, or comes back during or after treatment with other medicines.[1] The compound was developed by Columbia University chemist, Ronald Breslow.
VORINOSTAT
Vorinostat was the first histone deacetylase inhibitor[2] approved by the U.S. Food and Drug Administration (FDA) for the treatment of CTCL on October 6, 2006. It is manufactured by Patheon, Inc., in MississaugaOntarioCanada, for Merck & Co., Inc.White House Station, New Jersey.[3]
ZOLINZA contains vorinostat, which is described chemically as N-hydroxy-N'-phenyloctanediamide. The empirical formula is C14H20N2O3. The molecular weight is 264.32 and the structural formula is:
ZOLINZA® (vorinostat) Structural Formula Illustration
Vorinostat is a white to light orange powder. It is very slightly soluble in water, slightly soluble in ethanol, isopropanol and acetone, freely soluble in dimethyl sulfoxide and insoluble in methylene chloride. It has no chiral centers and is non-hygroscopic. The differential scanning calorimetry ranged from 161.7 (endotherm) to 163.9°C. The pH of saturated water solutions of vorinostat drug substance was 6.6. The pKa of vorinostat was determined to be 9.2.
Each 100 mg ZOLINZA capsule for oral administration contains 100 mg vorinostat and the following inactive ingredients: microcrystalline cellulose, sodium croscarmellose and magnesium stearate. The capsule shell excipients are titanium dioxide, gelatin and sodium lauryl sulfate.
Vorinostat has been shown to bind to the active site of histone deacetylases and act as a chelator for Zinc ions also found in the active site of histone deacetylases [4] Vorinostat's inhibition of histone deacetylases results in the accumulation of acetylated histones and acetylated proteins, including transcription factors crucial for the expression of genes needed to induce cell differentiation. [4]
SAHA inhibits class I and class II HDACs at nanomolar concentrations and arrests cell growth in a wide variety of transformed cells in culture at 2.5-5.0 µM. This compound efficiently suppressed MES-SA cell growth at a low dosage (3 µM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 µM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours.
Vorinostat has also been used to treat Sézary syndrome, another type of lymphoma closely related to CTCL.[5]
A recent study suggested that vorinostat also possesses some activity against recurrent glioblastoma multiforme, resulting in a median overall survival of 5.7 months (compared to 4 - 4.4 months in earlier studies).[6] Further brain tumor trials are planned in which vorinostat will be combined with other drugs.
Including vorinostat in treatment of advanced non-small-cell lung cancer (NSCLC) showed improved response rates and increased median progression free survival and overall survival (although the survival improvements were not significant at the P=0.05 level).[7]
It has given encouraging results in a phase II trial for myelodysplastic syndromes in combination with Idarubicin and Cytarabine.[8]

Vorinostat is an interesting target for scientists interested in eradicating HIV from infected persons.[9] Vorinostat was recently shown to have both in vitro and in vivo effects against latently HIV infected T-cells.[10][11]
Vorinostat, represented by structural formula (I) and chemically named as N-hydroxy-N'- phenyl-octanediamide or suberoylanilide hydroxamic acid (SAElA), is a member of a larger class of compounds that inhibit histone deacetylases (HDAC). Histone deacetylase inhibitors (HDI) have a broad spectrum of epigenetic activities and vorinostat is marketed, under the brand name Zolinza®, for the treatment of a type of skin cancer called cutaneous T-cell lymphoma (CTCL). Vorinostat is approved to be used when the disease persists, gets worse, or comes back during or after treatment with other medicines. Vorinostat has also been used to treat Sέzary's disease and, in addition, possesses some activity against recurrent glioblastoma multiforme.
Figure imgf000002_0001
Vorinostat was first described in US patent 5369108, wherein four different synthetic routes for the preparation of vorinostat are disclosed (Schemes 1 to 4).
The single step process illustrated in Scheme 1 involves coupling of the diacid chloride of suberic acid with aniline and hydiOxylamine hydrochloride. However, the yield of this reaction is only 15-30%.
Figure imgf000003_0001
Scheme 1
The multistep process illustrated in Scheme 2 begins with the monomethyl ester of suberic acid, which undergoes conversion to the corresponding acid chloride. Further coupling with aniline gives the methyl ester of suberanilic acid. Hydrolysis of the ester and further coupling with benzyl protected hydroxylamine gives benzyl protected vorinostat which on deprotection gives vorinostat.
HO. (CH2J6 OMe . ,OOMM e
O O
Figure imgf000003_0002
Figure imgf000003_0003
Figure imgf000003_0004
Scheme 2
In addition to the disadvantage of being a five-step process with overall yields reported as 35-65%, this process suffers from further disadvantages such as the use of the expensive monomethyl ester of suberic acid.
Figure imgf000004_0001
Scheme 3
The two step process illustrated in Scheme 3 involves coupling of the diacid chloride of suberic acid with aniline and O-benzyl hydroxylamine and then deprotection. However, the overall yield of this reaction is only 20-35%.
Figure imgf000004_0002
Scheme 4
The process illustrated in Scheme 4 is similar to that illustrated in Scheme 3, with the exception that O-trimethylsilyl hydroxylamine was used instead of O-benzyl hydroxylamine. The overall yield of this reaction is reported as 20-33%.
Another process for the preparation of vorinostat has been reported in J. Med. Chem.,
1995, vol. 38(8), pages 1411-1413. The reported process, illustrated in Scheme 5, begins with the conversion of suberic acid to suberanilic acid by a high temperature melt reaction.
Suberanilic acid is further converted to the corresponding methyl ester using Dowex resin and the methyl ester of suberanilic acid thus formed is converted to vorinostat by treatment with hydroxylamine hydrochloride. However, this process employs high temperatures (1900C) in the preparation of vorinostat which adds to the inefficiency and high processing costs on commercial scale. The high temperatures also increase the likelihood of impurities being formed during manufacture and safety concerns. The overall yield reported was a poor 35%.
Figure imgf000005_0001
MeOH, Dowex, 22 hours
Figure imgf000005_0002
Figure imgf000005_0003
Scheme 5
Another process for the preparation of vorinostat has been reported in OPPI Briefs, 2001, vol. 33(4), pages 391-394. The reported process, illustrated in Scheme 6, involves conversion of suberic acid to suberic anhydride, which on treatment with aniline gives suberanilic acid. Coupling of this suberanilic acid with ethyl chloroformate gives a mixed anhydride which upon treatment with hydroxylamine gives vorinostat in an overall yield of 58%. In the first step, there is competition between the formation of suberic anhydride and the linear anhydride and consequently isolation of pure suberic anhydride from the reaction mixture is very difficult. This process step is also hindered by the formation of process impurities and competitive reactions. In the second step, there is formation of dianilide by reaction of two moles of aniline with the linear anhydride. In the third step, suberanilic acid is an inconvenient by-product as the suberanilic acid is converted to a mixed anhydride with ethyl chloroformate, which is highly unstable and is converted back into suberanilic acid. Consequently, it is very difficult to obtain pure vorinostat from the reaction mixture. Although the reported yield was claimed to be 58%, when repeated a yield of only 38% was obtained.
Figure imgf000006_0001
Scheme 6
A further process for the preparation of vorinostat has been reported in J. Med. Chem., 2005, vol. 48(15), pages 5047-5051. The reported process, illustrated in Scheme 7, involves conversion of monomethyl suberate to monomethyl suberanilic acid, followed by coupling with hydroxylamine hydrochloride to afford vorinostat in an overall yield of 79%. However, the process uses the expensive monomethyl ester of suberic acid as starting material.
HOBt, DCC, DMF, RT, 4 hours
Figure imgf000006_0002
Figure imgf000006_0003
Figure imgf000006_0004


Processes for the preparation of vorinostat, and its form 1 crystalline polymorph, have been disclosed in patent applications US 2004/0122101 and WO 2006/127319. However, the disclosed processes, comprising the preparation of vorinostat from suberic acid, are a cumbersome three step process comprising the sequential steps of amidation of suberic acid with aniline, esterification of the mono-amide product with methanol, and finally reaction with hydroxylamine hydrochloride and sodium methoxide to afford vorinostat. This process is not very convenient as it involves elevated temperatures, lengthy reaction times and has a low overall yield of around 23%. In addition, the intermediate products and final product are not very pure and require exhaustive purification steps.

.........................
VORINOSTAT
A preferred embodiment of the first aspect of the present invention is illustrated in Scheme
Figure imgf000016_0001
suberic acid subefanilic acid      NH2OHHCl, CDI
Figure imgf000016_0002
suberoylanilide hydroxamic acid (T)
Scheme 8
Optionally, an activating agent can be used in step (a) and/ or step (b) to afford products with high yields and purity. Preferably, the activating agent is selected from cyanuric chloride, cyanuric fluoride, catecholborane, or a mixture thereof. The activating agent is preferably used in combination with the coupling agent. A preferred embodiment of the process according to the first aspect of the present invention comprises the following steps:
(i) taking a mixture of THF, CDI and DCC;
(ii) adding suberic acid; (iii) adding aniline in THF to the solution from step (ii);
(iv) stirring at 25-30°C;
(v) filtering off the solid dicyclohexyl urea formed in the reaction;
(vi) concentrating the filtrate in vacuo;
(vii) adding a solution of KOH in water; (vϋi) filtering off the solid by-product;
(ix) heating the filtrate;
(x) adding aq. HCl;
(xi) isolating suberanilic acid;
(xii) mixing the suberanilic acid and CDI in DMF; (xiii) adding hydroxylamine hydrochloride as solid to the mixture from step (xii);
(xiv) isolating vorinostat from the mixture obtained in step (xiii);
(xv) adding acetonitrile and aq. ammonia to the vorinostat from step (xiv);
(xvi) heating the mixture;
(xvii) cooling the mixture to 20-27°C; and (xvϋi) isolating pure vorinostat from the mixture obtained in step (xvii).
Preferably, by utilising the same organic solvent in steps (a) and (b), pure vorinostat can be obtained without isolation of any synthetic intermediate^).
A preferred embodiment of the second aspect of the present invention is illustrated in Scheme 9.
Figure imgf000018_0001
suberic acid N-hydtoxy-7-carboxy-heptanamide
Figure imgf000018_0002
Example 1
Stage 1 : Conversion of suberic acid to suberanilic acid
A mixture of CDI (0.5eq) and DCC (0.8eq) in THF (15 vol) was stirred for 1 hour at 25- 3O0C. Suberic acid (leq) and aniline (leq) in THF (1 vol) was added and the mixture stirred for a further 16-20 hours. The solid by-product was removed by filtration and the filtrate was concentrated in vacuo at 5O0C. The solid residue obtained was treated with a solution of KOH (2eq) in water (10 vol) and stirred for 30 minutes at 25-300C and any solid byproduct formed was removed by filtration. The filtrate obtained was heated at 6O0C for 3-4 hours and cooled to 200C before addition of an aqueous solution of HCl (17.5%, 3 vol). The mixture was stirred for 30 minutes and the solid filtered, washed with water (2x5 vol) and dried under vacuum at 60-650C. Molar Yield = 60-65% Purity by HPLC = 99.5%
Stage 2: Conversion of suberanilic acid to crude vorinostat The suberanilic acid (leq) obtained in stage 1 was dissolved in DMF (5 vol) and CDI (2eq) was added at 25-3O0C and maintained for 30 minutes under stirring. Hydroxylamine hydrochloride (4eq) was added and stirring continued for 30 minutes. Water (25 vol) was then added and the mixture stirred for 2 hours. The precipitated solid was filtered, washed with water (2x5 vol) and dried under vacuum at 500C. Molar Yield = 70-75% Purity by HPLC = 99% Stage 3: Purification of crude vorinostat
Aqueous ammonia (2.5 vol) was added to the crude vorinostat (leq) in acetonitrile (15 vol) at 25-30°C. The mixture was then maintained at 55-60°C for 1 hour before being cooled to 20-25°C and being stirred for a further hour. The resulting solid was filtered, washed with acetonitrile (2x0.5 vol) and dried under vacuum at 45-5O0C for 5 hours. Molar Yield = 55-60% Purity by HPLC > 99.8%
Example 2
Stage 1 : Conversion of suberic acid to crude vorinostat
A mixture of CDI (0.5eq) and DCC (0.8eq) in THF (15 vol) was stirred for 1 hour at 25- 30°C. Suberic acid (leq) and hydroxylamine (leq) in THF (1 vol) was added and the mixture stirred for a further 1 hour. Then CDI (0.5eq), DCC (0.8eq) and aniline (leq) were added to the mixture and the mixture was stirred for a further 16-20 hours. The solid byproduct was removed by filtration and the filtrate was concentrated in vacuo at 50°C to obtain crude vorinostat. Molar Yield = 55-60% Purity by HPLC > 95.8%
Stage 2: Purification of crude vorinostat
Aqueous ammonia (2.5 vol) was added to the crude vorinostat (leq) in acetonitrile (15 vol) at 25-3O0C. The mixture was then maintained at 55-600C for 1 hour before being cooled to 20-250C and being stirred for a further hour. The resulting solid was filtered, washed with acetonitrile (2x0.5 vol) and dried under vacuum at 45-500C for 5 hours. Molar Yield = 35-40% Purity by HPLC > 99.8%
...........................................
SYNTHESIS
Scheme V. - -
Figure imgf000012_0001

Vorinostat
Suberic acid (l.Oeq) was dissolved in tetrahydrofuran (15vol) and the clear solution was chilled to 0-5°C. Methyl chloro formate (l.leq) and triethylamine (1.1 eq) were added to the solution at the same temperature and the mixture was stirred for 15 minutes. The triethylamine.HCl salt formed was filtered off, then aniline (leq) was added to the reaction mixture at 0-50C and stirring was continued for 15 minutes. Methyl chloroformate (l.leq) and triethylamine (l.leq) were added to the clear solution and stirring was continued for a further 15 minutes at 0-5°C. This chilled reaction mixture was added to a freshly prepared hydroxylamine solution in methanol (*see below) chilled to 0-5°C and stirred for 15 minutes at 0-5°C. The solvent was removed under vacuum at 40°C and the residue obtained was taken in methylene dichloride and the organic solution was washed with water and dried over anhydrous sodium sulfate. Methylene dichloride was removed under vacuum at 40°C and acetonitrile was added to the residue. This mixture was stirred for 15 minutes before the solid was filtered under vacuum and dried under vacuum at 60°C to afford the product as a white solid. Molar yield = 35-41%; HPLC purity = 99.90%.
VORINOSTAT
1H-NMR (DMSO-d6): 1.27 (m, 4H, 2 x -CH2-), 1.53 (m, 4H, 2 x -CH2-), 1.94 (t, J = 7.3 Hz, 2H, -CH2-), 2.29 (t, J = 7.4 Hz, 2H, -CH2-), 7.03 (t, J = 7.35 Hz, IH, aromatic para position), 7.27 (t, J = 7.90 Hz, 2H, aromatic meta position), 7.58 (t, J = 7.65 Hz, 2H, aromatic ortho position), 8.66 (s, IH, -OH, D2O exchangeable), 9.85 (s, IH, amide -NH-, D2O exchangeable), 10.33 (s, IH, -NH-OH, D2O exchangeable).
13C-NMR (DMSO-d6): 25.04 (2C, 2 x -CH2-), 28.43 (2C, 2 x -CH2-), 32.24 (1C, -CH2-), 36.34 (1C, -CH2-), 119.01 (2C, Ar-C), 122.96 (1C, Ar-C), 128.68 (2C, Ar-C), 139.24 (1C, Ar- C, =CNH-), 169.23 (1C, -CO-), 171.50 (1C, -CO-).
*Preparation of hydroxylamine solution:
Potassium hydroxide (l.leq) was added to methanol (8vol) and the solution was chilled to 0-5°C. Similarly hydroxylamine hydrochloride (l.leq) was added to methanol (8vol) and chilled to 0-5°C. The chilled amine solution was added to the chilled alkali solution and stirred for 15 minutes at 0-50C. The white potassium chloride salt was filtered off and the filtrate was used as such.
..............................................................
POLYMORPHS


The present invention is directed to a Form I polymorph of SAHA characterized by an X-ray diffraction pattern substantially similar to that set forth in FIG. 13A. SAHA Form I is also characterized by an X-ray diffraction pattern including characteristic peaks at about at about 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0, and 43.3 degrees 2θ. SAHA Form I is further characterized by an X-ray diffraction pattern including characteristic peaks at about 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0, 43.3 degrees 20, and lacking at least one peak at about <8.7, 10.0-10.2, 13.4-14.0, 15.0-15.2, 17.5-19.0, 20.1-20.3, 21.1-21.3, 22.0-22.22, 22.7-23.0, 25.0-25.5, 26.0-26.2, and 27.4-27.6 degrees 2θ.
...............................................................
SPECTRAL DATA AND SYNTHESIS
Journal of Medicinal Chemistry, 2011 ,  vol. 54,  13  pg. 4694 - 4720
for structures see above link
Suberoylanilide hydroxamic acid (26, SAHA, vorinostat).
Suberic acid monomethyl ester (23) (15.09 g, 80.2 mmol) and DMF (0.10 mL) in anhydrous
DCM (300 mL) was added SOCl2 (34.6 mL, 0.481 mol), and the reaction mixture was refluxed for 3
h. The mixture was then concentrated. Toluene (300 mL) was added to the residue and evaporated
to afford crude acid chloride 24. Crude 24 was dissolved in DCM (240 mL), and followed by
addition of aniline (7.3 mL, 80.2 mmol) and Et3N (16.9 mL, 0.120 mol). The reaction mixture was
stirred for 90 min at room temp. The course of reaction was monitored by TLC (30% EtOAc in
hexanes) and LC–MS. DCM was removed, and ethyl acetate (500 mL) was added to dissolve the
residue. The organic layer was washed with aqueous NaHCO3 (500 mL × 2), 1 N HCl (400 mL × 2),
water, dried (Na2SO4), and evaporated to dryness under reduced pressure. The residue was purified
by vacuum liquid chromatography (silica, 20% EtOAc in hexanes) to afford compound 25as white crystalline solids (20.15 g, 96 %). NaOMe in MeOH solution (5.4 M, 106 mL, 0.573 mol) was added to a solution of compound 25 (10.05 g, 38.2 mmol) and NH2OH·HCl (26.54 g, 0.382 mol) in
dry MeOH (375 mL). The reaction mixture was stirred for 40 min at room temp. The reaction was
quenched by adding of 1 N HCl to pH 7–8. MeOH was removed under reduced pressure and water
(1 L) was added to the residue. The precipitated solid was filtered and washed with water (300 mL)
and EtOAc (150 mL) to afford crude 26 which was further purified by recrystallization. MeOH (200
mL) was added to crude 26 (5 g) and warmed to dissolve all solids. The MeOH solution was filtered,
and deionized water (400 mL) was added to the filtrate, the resulting solution was placed at 4 oC
overnight. Crystals obtained were filtered and washed with deionized water (100 mL) to afford pure
26 (vorinostat, SAHA) as off-white crystals. Overall yield: 80–85% from compound 23. Compound
26,
LC–MS m/z 265.1 ([M + H]+).
1H NMR (DMSO-d6)  10.35 (1H, s), 9.86 (1H, s), 8.68 (1H, s),
7.58 (2H, d, J = 7.6 Hz), 7.28 (2H, t, J = 7.5 Hz), 7.02 (1H, t, J = 7.4 Hz), 2.29 (2H, t, J = 7.4 Hz),
1.94 (2H, t, J = 7.4 Hz), 1.57 (2H, m), 1.49 (2H, m), 1.33 - 1.20 (2H, m); 13C NMR (DMSO-d6) 
171.2, 169.1, 139.3, 128.6, 122.9, 119.0, 36.3, 32.2, 28.4, 28.3, 25.0. Anal. (C10H20N2O3) C, H, N.


...................................................................

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.....................................................................


4  MOCETINOSTAT

Mocetinostat.png
Mocetinostat
 CAS  726169-73-9;
MGCD0103; MGCD-0103; MGCD 0103;
N-(2-AMINOPHENYL)-4-([[4-(PYRIDIN-3-YL)PYRIMIDIN-2-YL]AMINO]METHYL)BENZAMIDE
N-(2-Amino-phenyl)-4-[(4-pyridin-3-pyrimidin-2-ylamino)-methyl]-benzamide
Molecular Formula: C23H20N6O
Molecular Weight: 396.4445
SAN DIEGOAug. 11, 2014 /PRNewswire/ -- Mirati Therapeutics, Inc. (NASDAQ: MRTX) today announced that the U.S. FDA has granted Orphan Drug Designation to mocetinostat, a spectrum selective HDAC inhibitor, for diffuse large B-cell lymphoma (DLBCL). In June, mocetinostat was granted Orphan Drug Designation as a treatment for myelodysplastic syndrome (MDS).  Orphan drug designation is also being sought for bladder cancer patients with specific genetic alterations.
Identifiers
CAS number726169-73-9
PubChem9865515
ChemSpider8041206
ChEMBLCHEMBL272980
Jmol-3D imagesImage 1
Properties
Molecular formulaC23H20N6O
Molar mass396.44 g mol−1
Chemical structure for Mocetinostat
Mocetinostat (MGCD0103) is a benzamide histone deacetylase inhibitor undergoing clinical trials for treatment of various cancers including follicular lymphomaHodgkin's lymphoma and acute myelogenous leukemia.[1][2][3]
One clinical trial (for refractory follicular lymphoma) was temporarily put on hold due to cardiac problems but resumed recruiting in 2009.[4]
In 2010 favourable results were announced from the phase II trial for Hodgkin's lymphoma.[5]
MGCD0103 has also been used as a research reagent where blockage of members of the HDAC-family of histone deacetylases is required.[6]

Mechanism of action

It works by inhibiting mainly histone deacetylase 1 (HDAC1), but also HDAC2HDAC3, and HDAC11.[7]
About Mocetinostat
Mocetinostat is an orally-bioavailable, spectrum-selective HDAC inhibitor. Mocetinostat is enrolling patients in a Phase 2 dose confirmation study in combination with Vidaza as treatment for intermediate and high-risk MDS. Mirati also plans to initiate Phase 2 studies of mocetinostat as a single agent in patients with mutations in histone acetyl transferases in bladder cancer and DLBCL. Initial data from the Phase 2 studies is expected by the end of 2014. In addition to the ongoing Phase 2 clinical trials, mocetinostat has completed 13 clinical trials in more than 400 patients with a variety of hematologic malignancies and solid tumors.
About Mirati Therapeutics
Mirati Therapeutics is a targeted oncology company developing an advanced pipeline of breakthrough medicines for precisely defined patient populations. Mirati's approach combines the three most important factors in oncology drug development - drug candidates with complementary and compelling targets, creative and agile clinical development, and a highly accomplished precision medicine leadership team. The Mirati team is using a proven blueprint for developing targeted oncology medicines to advance and maximize the value of its pipeline of drug candidates, including MGCD265 and MGCD516, which are orally bioavailable, multi-targeted kinase inhibitors with distinct target profiles, and mocetinostat, an orally bioavailable, spectrum-selective histone deacetylase inhibitor. More information is available at www.mirati.com.
In eukaryotic cells, nuclear DNA associates with histones to form a compact complex called chromatin. The histones constitute a family of basic proteins which are generally highly conserved across eukaryotic species. The core histones, termed H2A, H2B, H3, and H4, associate to form a protein core. DNA winds around this protein core, with the basic amino acids of the histones interacting with the negatively charged phosphate groups of the DNA. Approximately 146 base pairs of DNA wrap around a histone core to make up a nucleosome particle, the repeating structural motif of chromatin.
Csordas, Biochem. J., 286: 23-38 (1990) teaches that histones are subject to posttranslational acetylation of the α,ε-amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1). Acetylation neutralizes the positive charge of the lysine side chain, and is thought to impact chromatin structure. Indeed, Taunton et al., Science, 272: 408-411 (1996), teaches that access of transcription factors to chromatin templates is enhanced by histone hyperacetylation. Taunton et al. further teaches that an enrichment in underacetylated histone H4 has been found in transcriptionally silent regions of the genome.
Histone acetylation is a reversible modification, with deacetylation being catalyzed by a family of enzymes termed histone deacetylases (HDACs). Grozinger et al., Proc. Natl. Acad. Sci. USA, 96: 4868-4873 (1999), teaches that HDACs are divided into two classes, the first represented by yeast Rpd3-like proteins, and the second represented by yeast Hda1-like proteins. Grozinger et al. also teaches that the human HDAC1, HDAC2, and HDAC3 proteins are members of the first class of HDACs, and discloses new proteins, named HDAC4, HDAC5, and HDAC6, which are members of the second class of HDACs. Kao et al., Genes Dev., 14: 55-66 (2000), discloses HDAC7, a new member of the second class of HDACs. More recently, Hu et al. J. Bio. Chem. 275:15254-13264 (2000) and Van den Wyngaert, FEBS, 478: 77-83 (2000) disclose HDAC8, a new member of the first class of HDACs.
Richon et al., Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998), discloses that HDAC activity is inhibited by trichostatin A (TSA), a natural product isolated from Streptomyces hygroscopicus, and by a synthetic compound, suberoylanilide hydroxamic acid (SAHA). Yoshida and Beppu, Exper. Cell Res., 177: 122-131 (1988), teaches that TSA causes arrest of rat fibroblasts at the Gand Gphases of the cell cycle, implicating HDAC in cell cycle regulation. Indeed, Finnin et al., Nature, 401: 188-193 (1999), teaches that TSA and SAHA inhibit cell growth, induce terminal differentiation, and prevent the formation of tumors in mice. Suzuki et al., U.S. Pat. No. 6,174,905, EP 0847992, JP 258863/96, and Japanese Application No. 10138957, disclose benzamide derivatives that induce cell differentiation and inhibit HDAC. Delorme et al., WO 01/38322 and PCT/IB01/00683, disclose additional compounds that serve as HDAC inhibitors.
The molecular cloning of gene sequences encoding proteins with HDAC activity has established the existence of a set of discrete HDAC enzyme isoforms. Some isoforms have been shown to possess specific functions, for example, it has been shown that HDAC-6 is involved in modulation of microtubule activity. However, the role of the other individual HDAC enzymes has remained unclear.
These findings suggest that inhibition of HDAC activity represents a novel approach for intervening in cell cycle regulation and that HDAC inhibitors have great therapeutic potential in the treatment of cell proliferative diseases or conditions. To date, few inhibitors of histone deacetylase are known in the art.
....................
Figure imgf000015_0002
Mocetinostat (MGCD-0103)
N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl^^
..............................
Example 426 Synthesis of N-(2-Amino-phenyl)-4-[(4-pyridin-3-pyrimidin-2-ylamino)-methyl]-benzamide
Figure US06897220-20050524-C00802
Step 1: Synthesis of 4-Guanidinomethyl-benzoic acid methyl ester Intermediate 1
The mixture of 4-Aminomethyl-benzoic acid methyl ester HCl (15.7 g, 77.8 mmol) in DMF (85.6 mL) and DIPEA (29.5 mL, 171.2 mmol) was stirred at rt for 10 min. Pyrazole-1-carboxamidine HCl (12.55 g, 85.6 mmol) was added to the reaction mixture and then stirred at rt for 4 h to give clear solution. The reaction mixture was evaporated to dryness under vacuum. Saturated NaHCOsolution (35 mL) was added to give nice suspension. The suspension was filtered and the filter cake was washed with cold water. The mother liquid was evaporated to dryness and then filtered. The two solids were combined and re-suspended over distilled H2O (50 ml). The filter cake was then washed with minimum quantities of cold H2O and ether to give 12.32 g white crystalline solid intermediate 1 (77% yield, M+1: 208 on MS).
Step 2: Synthesis of 3-Dimethylamino-1-pyridin-3-yl-propenone Intermediate 2
3-Acetyl-pyridine (30.0 g, 247.6 mmol) and DMF dimethyl acetal (65.8 mL, 495.2 mmol) were mixed together and then heated to reflux for 4 h. The reaction mixture was evaporated to dryness and then 50 mL diethyl ether was added to give brown suspension. The suspension was filtered to give 36.97 g orange color crystalline product (85% yield, M+1: 177 on MS).
Step 3: Synthesis of 4-[(4Pyridin-3-pyrimidin-2-ylamino)-methyl]benzoic acid methyl ester Intermediate 3
Intermediate 1 (0.394 g, 1.9 mmol) and intermediate 2 (0.402 g, 2.3 mmol) and molecular sieves (0.2 g, 4A, powder, >5 micron) were mixed with isopropyl alcohol (3.8 mL). The reaction mixture was heated to reflux for 5 h. MeOH (50 mL) was added and then heated to reflux. The cloudy solution was filtrated over a pad of celite. The mother liquid was evaporated to dryness and the residue was triturated with 3 mL EtOAc. The suspension was filtrated to give 0.317 g white crystalline solid Intermediate 3 (52%, M+1: 321 on MS).
Step 4: Synthesis of N-(2-Amino-phenyl)-4-[(4-pyrymidin-2-ylamino)-methyl]-benzamide
Intermediate 3 (3.68 g, 11.5 mmol) was mixed with THF (23 mL), MeOH (23 mL) and H2O (11.5 mL) at rt. LiOH (1.06 g, 25.3 mmol) was added to reaction mixture. The resulting reaction mixture was warmed up to 40° C. overnight. HCl solution (12.8 mL, 2N) was added to adjust pH=3 when the mixture was cooled down to rt. The mixture was evaporated to dryness and then the solid was washed with minimum quantity of H2O upon filtration. The filter cake was dried over freeze dryer to give 3.44 g acid of the title compound (95%, M+1: 307 on MS).
Acid (3.39 g, 11.1 mmol) of the title compound, BOP (5.679 g, 12.84 mmol) and o-Ph(NH2)(2.314 g, 21.4 mmol) were dissolved in the mixture of DMF (107 mL) and Et3N (2.98 mL, 21.4 mmol). The reaction mixture was stirred at rt for 5 h and then evaporated to dryness. The residue was purified by flash column (pure EtOAc to 5% MeOH/EtOAc) and then interested fractions were concentrated. The final product was triturated with EtOAc to give 2.80 g of title product
(66%, MS+1: 397 on MS).
 1H NMR (400 MHz, DMSO-D6) δ (ppm): 9.57 (s, 1H), 9.22 (s, 1H), 8.66 (d, J=3.5 Hz, 1H), 8.39 (d, J=5.1 Hz, 2H), 8.00 (t, J=6.5 Hz, 1H), 7.90 (d, J=8.2 Hz, 2H), 7.50 (m, 3H), 7.25 (d, J=5.1 Hz, 1H), 7.12 (d, J=7.4 Hz, 1H), 6.94 (dd, J=7.0, 7.8 Hz, 1H), 6.75 (d, J=8.2 Hz, 1H), 6.57 (dd, J=7.0, 7.8 Hz, 1H), 4.86 (s, 2H), 4.64 (d, J=5.9 Hz, 2H).

References

  1.  "Pharmion Corporation (PHRM) Release: Clinical Data On Oncology HDAC Inhibitor MGCD0103, Presented At The American Society of Clinical Oncology 42nd Annual Meeting" (Press release). Colorado, United States: BioSpace. June 6, 2006.
  2. Gelmon, K.; Tolcher, A.; Carducci, M.; Reid, G. K.; Li, Z.; Kalita, A.; Callejas, V.; Longstreth, J. et al. (2005). "Phase I trials of the oral histone deacetylase (HDAC) inhibitor MGCD0103 given either daily or 3x weekly for 14 days every 3 weeks in patients (pts) with advanced solid tumors"J. Clin. Oncol. 2005 ASCO Annual Meeting. 23 (16S). 3147.
  3.  MethylGene to Resume Development of its HDAC Inhibitor, MGCD0103 (Mocetinostat), Sept 2009
  4.  "METHYLGENE TO RESUME DEVELOPMENT OF ITS HDAC INHIBITOR, MGCD0103 (MOCETINOSTAT)". 21 Sep 2009.
  5.  "Final Phase 2 Clinical Data for Mocetinostat (MGCD0103) in Relapsed/Refractory Hodgkin Lymphoma Patients". 6 Dec 2010.
  6. Pfefferli, Catherine; Müller, Fritz; Ja¿wi¿ska, Anna; Wicky, Chantal (2014). "Specific NuRD components are required for fin regeneration in zebrafish". BMC Biol. 12 (30). doi:10.1186/1741-7007-12-30PMID 24779377.open access publication - free to read
  7.  MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo


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THERAPEUTIC COMBINATIONS AND METHODS FOR CARDIOVASCULAR IMPROVEMENT AND TREATING CARDIOVASCULAR DISEASE
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COMBINATION OF ERa+ LIGANDS AND HISTONE DEACETYLASE INHIBITORS FOR THE TREATMENT OF CANCER
12-21-2007
Assay for efficacy of histone deacetylase inhibitors
5-25-2005
Inhibitors of histone deacetylase

2-8-2012
HDAC INHIBITORS AND HORMONE TARGETED DRUGS FOR THE TREATMENT OF CANCER
6-3-2011
Sequential Administration of Chemotherapeutic Agents for Treatment of Cancer
5-6-2011
METHODS FOR TREATING OR PREVENTING COLORECTAL CANCER
1-12-2011
Inhibitors of histone deacetylase
1-12-2011
Inhibitors of Histone Deacetylase
11-24-2010
Inhibitors of histone deacetylase
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6-12-2009
Administration of an Inhibitor of HDAC and an mTOR Inhibitor
5-22-2009
Combinations of HDAC Inhibitors and Proteasome Inhibitors
5-15-2009
Combination Therapy

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5 EZATIOSTAT


Ezatiostat structure
Ezatiostat
168682-53-9 (Ezatiostat); 286942-97-0 (Ezatiostat HCl salt)
gamma-Glu-S-BzCys-PhGly diethyl ester
 HCl; salt, D08917, 

Ezatiostat hydrochloride

Target: glutathione S-transferase P1-1 (GSTP1-1) inhibitor
Pathway: hsa00480 Glutathione metabolism
Activity: Treatment of disorders of bone marrow cellular growth and differentiation
see http://www.ncbi.nlm.nih.gov/pubmed?term=TLK-199&cmd=search

Telik, Inc.
innovator
TLK199; TLK-199; TLK 199; Brand name: TELINTRA®ethyl (2R)-[(4S)-4-amino-5-ethoxy-5-oxopentanoyl]-S-benzyl-L-cysteinyl-2- phenylglycinate. 
ethyl (2S)-2-amino-5-[[(2R)-3-benzylsulfanyl-1-[[(1R)-2-ethoxy-2-oxo-1-phenylethyl]amino]-1-oxopropan-2-yl]amino]-5-oxopentanoate.
IUPAC/Chemical name: 
(S)-ethyl 2-amino-5-(((R)-3-(benzylthio)-1-(((S)-2-ethoxy-2-oxo-1-phenylethyl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoate
C27H35N3O6S
Exact Mass: 529.2246
nmr.http://www.medkoo.com/Product-Data/Ezatiostat/ezatiostat-QC-CRB40225web.pdf
Telintra is a small molecule product candidate designed to stimulate the production of blood cells in the bone marrow. Many conditions are characterized by depleted bone marrow, including myelodysplastic syndrome, a form of pre-leukemia in which the bone marrow produces insufficient levels of one or more of the 3 major blood elements (white blood cells, red blood cells and platelets). A reduction in blood cell levels is also a common, toxic effect of many standard chemotherapeutic drugs.
Ezatiostat is a liposomal small-molecule glutathione analog inhibitor of glutathione S-transferase (GST) P1-1 with hematopoiesis-stimulating activity. After intracellular de-esterification, the active form of ezatiostat binds to and inhibits GST P1-1, thereby restoring Jun kinase and MAPK pathway activities and promoting MAPK-mediated cellular proliferation and differentiation pathways. This agent promotes the proliferation and maturation of hematopoietic precursor cells, granulocytes, monocytes, erythrocytes and platelets
Phase II trial myelodysplastic syndrome (MDS): Cancer. 2012 Apr 15;118(8):2138-47.
Phase I trial myelodysplastic syndrome (MDS): J Hematol Oncol. 2012 Apr 30;5:18. doi: 10.1186/1756-8722-5-18; Blood. 2009 Jun 25;113(26):6533-40; J Hematol Oncol. 2009 May 13;2:20.
Ezatiostat hydrochloride is the hydrochloride acid addition salt of ezatiostat. Ezatiostat, also known as TLK199 or TER 199, is a compound of the formula:

Ezatiostat has been shown to induce the differentiation of HL-60 promyelocyte leukemia cells in vitro, to potentiate the activity of cytotoxic agents both in vitro and in vivo, and to stimulate colony formation of all three lineages of hematopoietic progenitor cells in normal human peripheral blood.
In preclinical testing, ezatiostat has been shown to increase white blood cell production in normal animals, as well as in animals in which white blood cells were depleted by treatment with cisplatin or fluorouracil. Similar effects may provide a new approach to treating myelodysplastic syndrome (MDS).
Many conditions, including MDS, a form of pre-leukemia in which the bone marrow produces insufficient levels of one or more of the three major blood elements (white blood cells, red blood cells, and platelets), are characterized by depleted bone marrow. Myelosuppression, which is characterized by a reduction in blood cell levels and in a reduction of new blood cell generation in the bone marrow, is also a common, toxic effect of many standard chemotherapeutic drugs.
Ezatiostat hydrochloride in a liposomal injectable formulation was studied in a clinical trial for the treatment of MDS, and results from this trial, reported by Raza et al., J Hem. One, 2:20 (published online 13 May 2009), demonstrated that administration of TLK199 was well tolerated and resulted in multi-lineage hematologic improvement.
Ezatiostat hydrochloride in a tablet formulation has been evaluated in a clinical trial for the treatment of MDS, as reported by Raza et al., Blood, 113:6533-6540 (prepublished online 27 April 2009) and a single-patient report by Quddus et al., J Hem. One, 3:16 (published online 23 April 2010), and is currently being evaluated in clinical trials for the treatment of MDS and for severe chronic idiopathic neutropenia.
When used for treating humans, it is important that a crystalline therapeutic agent like ezatiostat hydrochloride retains its polymorphic and chemical stability, solubility, and other physicochemical properties over time and among various manufactured batches of the agent. If the physicochemical properties vary with time and among batches, the administration of a therapeutically effective dose becomes problematic and may lead to toxic side effects or to ineffective therapy, particularly if a given polymorph decomposes prior to use, to a less active, inactive, or toxic compound.
Therefore, it is important to choose a form of the crystalline agent that is stable, is manufactured reproducibly, and has physicochemical properties favorable for its use as a therapeutic agent.
Ezatiostat hydrochloride (USAN) has the molecular weight of 566.1, the trademark of Telintra®, and the CAS registry number of 286942-97-0. Ezatiostat hydrochloride has been evaluated for the treatment of myelodysplastic syndrome (MDS), in a Phase I-IIa study using a liposomal formulation (U.S. Pat. No. 7,029,695), as reported at the 2005 Annual Meeting of the American Society for Hematology (Abstract #2250) and by Raza et al. in Journal of Hematology & Oncology, 2:20 (published online on 13 May 2009); and in a Phase I study using a tablet formulation, as reported at the 2007 Annual Meeting of the American Society for Hematology (Abstract #1454) and by Raza et al. in Blood, 113:6533-6540 (prepublished online on 27 Apr. 2009), and in a single patient case report by Quddus et al. in Journal of Hematology & Oncology, 3:16 (published online on 23 Apr. 2010).
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http://www.google.com/patents/US20110301376

Preparation of Ezatiostat Hydrochloride
In another aspect, this invention provides a process comprising the steps of contacting a compound of formula:


or a salt thereof with a compound of formula:


or a salt thereof and an activating agent under conditions which provide a compound of formula:


In one embodiment, the process further comprises deprotecting the compound of formula:


under conditions which provide a compound of formula:


or a salt thereof. In another embodiment, the compound provided is ezatiostat hydrochloride.
In another aspect, this invention provides a process comprising contacting a compound of formula:


or a salt thereof with an ethylating agent under conditions which provide a compound of formula:


In another embodiment, the process further comprises debenzylating the compound of formula:


under conditions which provide a compound of formula:


or a salt thereof.
In another aspect, this invention provides a process comprising the steps of contacting a compound of formula:


or a salt thereof having a t-butoxycarbonyl group with an activating agent and a compound of formula:


or a salt thereof under conditions which provide a compound of formula:


In another embodiment, the process further comprises deprotecting the tertiarybutyloxycarboyl (Boc) group under conditions to provide a compound of formula:


or a salt thereof.
Certain preferred embodiments of this invention are illustrated in the reaction scheme and described below. In the peptide coupling the amino acid reagents are used generally at a 1:1 molar ratio, and the activating reagent (isobutyl chloroformate) and the base (NMM) are used in slight excess over the amino acid reagents; while in the esterification of the N-BOC-L-glutamic acid γ-benzyl ester the esterifying agent (diethyl sulfate) and base are used in about 1.4-fold excess.

EXAMPLESAs relevant and unless otherwise noted, all operations were conducted under nitrogen purge and with stirring. Water was osmosis purified, and solvents were filtered. Unless otherwise stated, all temperatures are in degrees Celcius (° C.) and the following abbreviations have the following definitions:Et EthylHCl(g) HCl gasN-BOC or N-Boc N-tertiarybutyloxycarbonylL LiterKg KilogramNMM N-methylmorpholineMol Molew/w weight by weightExample 1Preparation of S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester hydrochloride (3)Without stirring, 45.1 Kg N-BOC-S-benzyl-L-cysteine (1) was added to a 600 L jacketed glass-lined reactor, followed by 45 L ethyl acetate. Stirring was started and the temperature was reduced to 13° C. NMM, 15.3 Kg, was added over 50 minutes, and rinsed in with 6 L ethyl acetate, and stirring stopped. Ethyl acetate, 315 L, was added to an 800 L cooled jacketed glass-lined reactor, followed by 20.7 Kg isobutyl chloroformate, rinsed in with 11 L ethyl acetate, and the mixture cooled to −10° C. The N-BOC-S-benzyl-L-cysteine NMM salt solution was added to the 800 L reactor over 5 hours, its reactor rinsed with 11 L ethyl acetate, and the rinse solution added to the 800 L reactor, while maintaining the temperature at (−10˜−7)° C. D-Phenylglycine ethyl ester hydrochloride, 31.2 Kg, was added in 8 portions over 50 minutes, followed by 15.3 Kg NMM in 8 portions over 1.3 hours, rinsed in with 2×5 L portions of ethyl acetate, allowing the mixture to warm to −1° C. by the end of the addition. The mixture was gradually warmed to 1° C. for 30 minutes, then to 20° C. over 2 hours, and maintained at (20˜25)° C. for 5 hours. The reaction mixture was washed twice with water: the first time adding 66 L water, stirring at room temperature for 40 minutes, allowing the phases to separate for 30 minutes, then removing the aqueous phase; the second time adding 68 L water, bringing the pH to 1.9 with the addition of 0.45 L 36% hydrochloric acid, stirring at room temperature for 35 minutes, allowing the phases to separate for 1 hour, then removing the aqueous phase. The organic phase was then heated to 38° C., and the pressure reduced to about 0.25 bar until no further gas was released, then to about (0.07-0.1) bar and solvents removed by distillation until 266 L of distillate had been removed. Four cycles of addition of 45 L ethyl acetate and removal of 45 L solvent by distillation were performed, and the water content of the remaining mixture was checked to ensure that it was below 0.1%. With the mixture at 36° C., 194 L heptanes was added, maintaining the temperature about 36° C., and held at that temperature for 2.3 hours. A further 194 L heptanes was added, allowing the temperature to cool to 30° C., and the temperature then reduced to −1° C. over 2.3 hours and then to −5° C. over 1 hour, and N-BOC-S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester recovered by filtration, washing twice with 30 L each of heptanes at −5° C., giving 85 Kg (63 Kg dry basis) N-BOC-S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester. Without stirring, the damp N-BOC-S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester was loaded into an 800 L jacketed glass-lined reactor, followed by 257 L ethyl acetate. Stirring was started and the temperature brought to 22° C., then the nitrogen purge stopped and 12.2 Kg hydrogen chloride gas was added through an immersion tube over 1.8 hours, allowing the temperature to increase to 38° C. The temperature was increased to 41° C., and the mixture held at that temperature for 9 hours. About 280 L of solvents were removed by distillation at that temperature and a pressure of (0.2˜0.1) bar over about 2 hours. Two cycles of addition of ethyl acetate and removal of solvent by distillation were performed, using 52 L in the first cycle and 77 L in the second cycle, and the viscous solution of S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester hydrochloride (3) in ethyl acetate, 148 Kg, was cooled to room temperature and filtered into a storage drum.Example 2Preparation of N-BOC-L-glutamic acid α-ethyl ester (6)Without stirring, 41 Kg N-BOC-L-glutamic acid γ-benzyl ester (4) was added to an 800 L jacketed glass-lined reactor, followed by 2.5 L water and 123 L ethyl acetate. The mixture was then stirred until the N-BOC-L-glutamic acid γ-benzyl ester completely dissolved, keeping the temperature below 15° C. Potassium carbonate fine powder, 23.4 Kg, was added in five batches, and the mixture then heated to 55° C. and maintained at that temperature for 40 minutes, giving a heterogeneous and completely fluid mixture. Diethyl sulfate, 26.2 Kg, was added over 2 hours, and rinsed in with 5 L ethyl acetate, with the temperature remaining at about 52° C. The nitrogen purge was stopped and a solution of 20 Kg ammonium chloride in 73 L water at room temperature added over 2 hours to the mixture, maintaining the temperature near 50° C., then rinsing in with 10 L water. Nitrogen purging was resumed, and the mixture was maintained at about 50° C. for 3 hours, then lowered to about 45° C., the stirring stopped, the phases allowed to separate for 30 minutes, and the lower, aqueous, phase removed. The organic phase, containing N-BOC-L-glutamic acid γ-benzyl α-ethyl ester (5), was washed three times with water, each time adding 41 L water, stirring at room temperature for 30 minutes, allowing the phases to separate for 30 minutes, then removing the aqueous phase. The organic phase was heated to 35° C., and the pressure reduced, starting at about 0.2 bar and reducing as necessary until 82 Kg solvent had been removed by distillation, leaving about (70˜80) L of slightly opalescent solution. This solution was heated to 53° C., and 102 L heptanes was added, maintaining the same temperature. The solution was then filtered, rinsing with a further 13 L heptanes, then cooled to 32° C. to cause crystallization and maintained at that temperature for 1 hour. A further 66 L heptanes was added, and the mixture cooled to 22° C. and held for 1 hour, then cooled to −5° C. and held for another 1 hour. The mixture was then filtered to isolate the N-BOC-L-glutamic acid γ-benzyl α-ethyl ester (5), which was washed twice, each time with 25 L heptanes cooled to (−5˜0)° C., and dried under vacuum at 40° C., giving 39.3 Kg N-BOC-L-glutamic acid γ-benzyl α-ethyl ester (5).A 4000 L hydrogenator was purged with nitrogen, then under nitrogen sweep and no stirring loaded with 39.2 Kg N-BOC-L-glutamic acid γ-benzyl α-ethyl ester (5), 2.0 Kg 5% palladium on carbon, and 432 L ethyl acetate, and purged (3 bar) and decompressed (0.2 bar) twice with nitrogen and twice with hydrogen. Stirring was begun and the mixture heated to (37±2)° C., hydrogenated at that temperature under 2.8 bar hydrogen pressure until no further hydrogen absorption occurred, then held under 2.8 bar hydrogen pressure for 12 hours. Completion of hydrogenation was confirmed by thin-layer chromatography of a sample. The mixture was cooled to 28° C., the hydrogen purged from the hydrogenator, and the hydrogenator purged (2 bar) and decompressed (0.2 bar) twice with nitrogen. The mixture was filtered through a filter precoated with 10 Kg powdered cellulose in 200 L ethyl acetate, then the filter washed with the ethyl acetate used to form the precoat, giving a total of 626 Kg of a dilute ethyl acetate solution containing 29.5 Kg N-BOC-L-glutamic acid α-ethyl ester (6). This was distilled at (35˜40)° C. and (0.16˜0.18) bar to give 67 L of concentrated solution, then 29 L of ethyl acetate added and the solution redistilled to again give 67 L of concentrated solution.Example 3Preparation of Ezatiostat HydrochlorideThe concentrated solution of N-BOC-L-glutamic acid α-ethyl ester (6), 61.2 Kg (containing 27.8 Kg N-BOC-L-glutamic acid α-ethyl ester), was added to a 600 L jacketed glass-lined reactor, rinsed in with 5 L ethyl acetate, then cooled to 14° C. NMM, 10.8 Kg, was added over 50 minutes and rinsed in with 5 L ethyl acetate, then stirring stopped, giving an ethyl acetate solution of N-BOC-L-glutamic acid α-ethyl ester NMM salt. Ethyl acetate, 475 L, was added to a 1300 L cooled jacketed glass-lined reactor, followed by 14.5 Kg isobutyl chloroformate, rinsed in with 2×10 L ethyl acetate, and the mixture cooled to −11° C. The N-BOC-L-glutamic acid α-ethyl ester NMM salt solution was added to the 1300 L reactor over 1.3 hours, its reactor rinsed with 10 L ethyl acetate, and the rinse solution added to the 1300 L reactor, then stirred for an additional 30 minutes, while maintaining the temperature at about −13° C.S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester hydrochloride (3) in ethyl acetate, 112 Kg (containing 41.3 Kg S-benzyl-L-cysteinyl-D-phenylglycine ethyl ester hydrochloride) was added in 4 portions over 45 minutes, and rinsed in with 5 L ethyl acetate, followed by 10.8 Kg NMM in 8 portions over 1.3 hours, rinsed in with 2×5 L portions of ethyl acetate, allowing the mixture to warm to −4° C. by the end of the addition. The mixture was gradually warmed to 30° C. over 2 hours, and maintained at (30˜35)° C. for 2 hours. The reaction mixture was washed twice with water: the first time adding 100 L water, heating to 41° C., allowing the phases to separate for 30 minutes, then removing the aqueous phase; the second time adding 100 L water, bringing the pH to 2.0 with the addition of 0.8 L 36% hydrochloric acid, stirring at 43° C. for 30 minutes, allowing the phases to separate for 1 hour, then removing the aqueous phase. The organic phase was then heated to 42° C., and the pressure reduced to about 0.25 bar until no further gas was released and solvents removed by distillation until 495 L of distillate had been removed. Four cycles of addition of 120 L ethyl acetate and removal of 120 L solvent by distillation were performed, and the water content of the remaining mixture was checked to ensure that it was below 0.1%. With the mixture at 42° C., 610 L of ethyl acetate was added, maintaining the temperature about 41° C., then heating to 58° C. to ensure dissolution. The solution was filtered, rinsing the filter with 18 L ethyl acetate, and the solution allowed to cool to 22° C. The nitrogen purge was stopped and 22.2 Kg hydrogen chloride gas was added through an immersion tube over 2 hours, then the mixture held at that temperature for 2 hours. The mixture was heated to 31° C. over 1.5 hours, and held at about that temperature for 15.5 hours. Solvents were removed by distillation at 33° C. and a pressure of about 0.13 bar over about 1.5 hours to give a volume of concentrated solution of about 630 L. Ethyl acetate, 100 L, was added, and the mixture cooled to 25° C. and held at that temperature for 30 minutes. The crude ezatiostat hydrochloride was recovered by filtration and washed with 30 L ethyl acetate, giving 113 Kg damp crude ezatiostat hydrochloride, which was dried at 40° C. under vacuum for 24 hours to give 52.8 Kg dry crude ezatiostat hydrochloride.Example 4Crystallization of Ezatiostat Hydrochloride to Form Pure Crystalline Ezatiostat Hydrochloride Ansolvate Form D61.5 Kg crude ezatiostat hydrochloride was added to a reactor at room temperature, followed by 399 liter (L) ethanol, and this mixture was heated to 68° C. to completely dissolve the ezatiostat hydrochloride, filtered, then allowed to cool to 65° C. and checked for clarity and the absence of crystallization. About 1.3 Kg of ezatiostat hydrochloride ansolvate form D was suspended in 9 L of ethyl acetate, and about one-half of this suspension was added to the ethanol solution. The mixture was cooled to 63° C. and the second half of the suspension added to the mixture. The resulting mixture was cooled gradually to 45° C., 928 L ethyl acetate was added, and the mixture was cooled to 26° C. and held at about that temperature for about 5 hours, then cooled to −2° C. The mixture, containing crystalline ezatiostat hydrochloride ansolvate, was filtered, and the residue washed twice with 65 L of chilled (0-5° C.) ethyl acetate. The crystalline ezatiostat hydrochloride ansolvate was dried at 30° C. for 48 hours, then cooled to room temperature and sieved. Analysis of the material by DSC and XRPD confirmed its identity as crystalline ezatiostat hydrochloride ansolvate, and Karl Fischer analysis showed a water content of 0.1%.Example 5Purifying Ezatiostat Hydrochloride Crystals to Form Pure Crystalline Ezatiostat Hydrochloride Ansolvate Form DCrude ezatiostat hydrochloride, 51.4 Kg, was added to a 600 L jacketed glass-lined reactor at room temperature, followed by 334 L of ethanol. The mixture was heated to 68° C. to completely dissolve the ezatiostat hydrochloride. The resulting solution was filtered into a 1300 L jacketed glass-lined reactor, and an additional 27 L ethanol warmed to 66° C. used to rinse the first reactor into the second reactor through the filter. The resulting solution in the second reactor was cooled to 63° C. and checked for complete dissolution; then 4 L of a seeding suspension of crystalline ezatiostat hydrochloride ansolvate in ethyl acetate was added, and the mixture cooled to 60° C. The remaining 4 L of the seeding suspension was added, and the mixture cooled to 47° C. over 2 hours. The solids in the mixture were shown by DSC to contain more than one form of ezatiostat hydrochloride, so the stages of heating to dissolution, cooling, and adding seeding suspension (this time 2×2 L), were repeated, then the mixture cooled to 41° C. This time the solids in the mixture were confirmed by DSC to be crystalline ezatiostat hydrochloride ansolvate. Ethyl acetate, 776 L, was added, and the mixture was cooled to 25° C. over 1.3 hours and further to 20° C. over an additional 5 hours, then cooled to −3° C. The mixture, containing crystalline ezatiostat hydrochloride ansolvate, was filtered and the solids washed twice with 54 L each of chilled (−5˜0)° C. ethyl acetate. The damp solids of crystalline ezatiostat hydrochloride ansolvate, 70 Kg, were dried in a vacuum oven at 25° C. for 16 hours, 35° C. for 7 hours, then at room temperature for 1 hour, then sieved. The crystalline ezatiostat hydrochloride ansolvate, 44.2 Kg, had a loss on drying at 40° C. under vacuum for 2 hours of 0.09%, and a water content by Karl Fischer analysis of 0.09%.
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U.S. Pat. No. 5,763,570
 https://www.google.com/patents/US5763570
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http://www.google.com/patents/WO2011156025A1?cl=en
Example 1. Preparation Of Ezatiostat Hydrochloride Ansolvate By Slurrying
[0082] Ezatiostat hydrochloride monohydrate was added to methyl tert-butyl ether at room temperature in excess, so that undissolved solids were present. The mixture was then agitated in a sealed vial at room temperature for 4 days, and the solids were then isolated by suction filtration. XRPD analysis of the solids established that the isolated solids were ezatiostat hydrochloride ansolvate.
[0083] Ezatiostat hydrochloride monohydrate was added to hexanes at 60 °C in excess, so that undissolved solids were present. The mixture was then agitated in a sealed vial at 60 °C for 4 days, and the solids were then isolated by suction filtration. XRPD analysis of the solids established that the isolated solids were ezatiostat hydrochloride ansolvate.
Example 2. Preparation Of Crystalline Ezatiostat Hydrochloride Ansolvate By Heating
[0084] DSC of crystalline ezatiostat hydrochloride monohydrate showed the pattern in FIG. 1, as discussed in paragraph above. Hot stage microscopy showed an initial melt followed by a recrystallization at 153 °C and a final melt at 166 °C. VT-XRPD, where XRPD patterns were obtained at 28 °C, 90 °C, and 160 °C during heating, and 28 °C after cooling of the formerly heated material, showed the presence of ezatiostat hydrochloride monohydrate at 28 °C and 90 °C during heating and of crystalline ezatiostat hydrochloride ansolvate at 160 °C and 28 °C after cooling of the formerly heated material. This confirmed that the transition at around 153/156 °C was a conversion of ezatiostat hydrochloride monohydrate form A to crystalline ezatiostat hydrochloride ansolvate form D and that the final DSC endothermic peak at about 177 °C (166 °C in the hot stage microscopy) was due to the melting of crystalline ezatiostat hydrochloride ansolvate. This was further confirmed by XRPD of the TG-IR material, where XRPD patterns obtained at room temperature both before and after heating to about 160 °C showed that the material before heating was form A and that the material after heating was form D ansolvate. DSC of crystalline ezatiostat hydrochloride ansolvate prepared by recrystallization showed the pattern in FIG. 5, with only the endothermic peak at about
177 °C followed by a broad endotherm at about (205 – 215) °C. Accordingly, the presence of the DSC endothermic peak at about 177 °C, for example at (177±2) °C, when measured under the conditions described above, is considered characteristic of crystalline ezatiostat hydrochloride ansolvate, and the substantial absence of thermal events at temperatures below this is considered indicative of the absence of other forms of ezatiostat hydrochloride
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http://www.google.com/patents/WO2013082462A1?cl=en
Ezatiostat, also known as TLK199 or TER 199, is a compound of the formula:

[0003] Ezatiostat has been shown to induce the differentiation of HL-60 promyelocytic leukemia cells in vitro, to potentiate the activity of cytotoxic agents both in vitro and in vivo, and to stimulate colony formation of all three lineages of hematopoietic progenitor cells in normal human peripheral blood. In preclinical testing, ezatiostat has been shown to increase white blood cell production in normal animals, as well as in animals in which white blood cells were depleted by treatment with cisplatin or fluorouracil. Similar effects may provide a new approach to treating myelodysplasia syndrome (MDS).
[0004] Many conditions, including MDS, a form of pre-leukemia in which the bone marrow produces insufficient levels of one or more of the three major blood elements (white blood cells, red blood cells, and platelets), are characterized by depleted bone marrow.
Myelosuppression, which is characterized by a reduction in blood cell levels and in a reduction of new blood cell generation in the bone marrow, is also a common, toxic effect of many standard chemotherapeutic drugs.
[0005] Ezatiostat hydrochloride is the hydrochloride acid addition salt of ezatiostat.
Ezatiostat hydrochloride in a liposomal injectable formulation was studied in a clinical trial for the treatment of MDS, and results from this trial, reported by Raza et al., J. Hem. One, 2:20 (published online 13 May 2009), demonstrated that administration of TLK199 was well tolerated and resulted in multi-lineage hematologic improvement. Ezatiostat hydrochloride in a tablet formulation has been evaluated in a clinical trial for the treatment of MDS, as reported by Raza et al, Blood, 113:6533-6540 (prepublished online 27 April 2009) and a single-patient report by Quddus et al, J. Hem. One, 3:16 (published online 23 April 2010), and is currently being evaluated in clinical trials for the treatment of MDS and for severe chronic idiopathic neutropenia.
…………………………………..
http://www.google.com/patents/WO2013082462A1?cl=en
Example 1
[0048] 80 mg of crystalline ezatiostat hydrochloride was placed in a round bottom flask and dissolved in 25 mL of methanol. The solvent was then evaporated on a rotary evaporation apparatus under reduced pressure at 30 °C. After 30 minutes, the solid sample was removed from the round bottom flask and stored in a sealed vial at 2 °C in a refrigerator. Analysis of this sample was carried out within 24 hours of removing it from the rotary evaporation apparatus.
[0049] The resulting amorphous material was analyzed by 1H NMR, 13C NMR, DSC, and X-Ray powder diffraction experiments. The DSC conditions were 30 to 300 °C at 10 °C/ min using 7 mg of the amorphous material. The X-Ray powder diffraction was taken at 0-60 of 2theta. The crystalline ezatiostat hydrochloride was also analyzed.

Phase II trials: Ezatiostat is the first GSTP1-1 inhibitor shown to cause clinically significant and sustained reduction in RBC transfusions, transfusion independence, and multilineage responses in MDS patients. The tolerability and activity profile of ezatiostat may offer a new treatment option for patients with MDS. (source:  Cancer. 2012 Apr 15;118(8):2138-47.)
 
References
1: Galili N, Tamayo P, Botvinnik OB, Mesirov JP, Brooks MR, Brown G, Raza A. Prediction of response to therapy with ezatiostat in lower risk myelodysplastic syndrome. J Hematol Oncol. 2012 May 6;5:20. doi: 10.1186/1756-8722-5-20. PubMed PMID: 22559819; PubMed Central PMCID: PMC3407785.
2: Raza A, Galili N, Mulford D, Smith SE, Brown GL, Steensma DP, Lyons RM, Boccia R, Sekeres MA, Garcia-Manero G, Mesa RA. Phase 1 dose-ranging study of ezatiostat hydrochloride in combination with lenalidomide in patients with non-deletion (5q) low to intermediate-1 risk myelodysplastic syndrome (MDS). J Hematol Oncol. 2012 Apr 30;5:18. doi: 10.1186/1756-8722-5-18. PubMed PMID: 22546242; PubMed Central PMCID: PMC3416694.
3: Lyons RM, Wilks ST, Young S, Brown GL. Oral ezatiostat HCl (Telintra®, TLK199) and idiopathic chronic neutropenia (ICN): a case report of complete response of a patient with G-CSF resistant ICN following treatment with ezatiostat, a glutathione S-transferase P1-1 (GSTP1-1) inhibitor. J Hematol Oncol. 2011 Nov 2;4:43. doi: 10.1186/1756-8722-4-43. PubMed PMID: 22047626; PubMed Central PMCID: PMC3235963.
4: Raza A, Galili N, Smith SE, Godwin J, Boccia RV, Myint H, Mahadevan D, Mulford D, Rarick M, Brown GL, Schaar D, Faderl S, Komrokji RS, List AF, Sekeres M. A phase 2 randomized multicenter study of 2 extended dosing schedules of oral ezatiostat in low to intermediate-1 risk myelodysplastic syndrome. Cancer. 2012 Apr 15;118(8):2138-47. doi: 10.1002/cncr.26469. Epub 2011 Sep 1. PubMed PMID: 21887679.
5: Quddus F, Clima J, Seedham H, Sajjad G, Galili N, Raza A. Oral Ezatiostat HCl (TLK199) and Myelodysplastic syndrome: a case report of sustained hematologic response following an abbreviated exposure. J Hematol Oncol. 2010 Apr 23;3:16. doi: 10.1186/1756-8722-3-16. PubMed PMID: 20416051; PubMed Central PMCID: PMC2873355.
6: Steensma DP. Novel therapies for myelodysplastic syndromes. Hematol Oncol Clin North Am. 2010 Apr;24(2):423-41. doi: 10.1016/j.hoc.2010.02.010. Review. PubMed PMID: 20359635.
7: D’Alò F, Greco M, Criscuolo M, Voso MT. New treatments for myelodysplastic syndromes. Mediterr J Hematol Infect Dis. 2010 Aug 11;2(2):e2010021. doi: 10.4084/MJHID.2010.021. PubMed PMID: 21415972; PubMed Central PMCID: PMC3033133.
8: Raza A, Galili N, Callander N, Ochoa L, Piro L, Emanuel P, Williams S, Burris H 3rd, Faderl S, Estrov Z, Curtin P, Larson RA, Keck JG, Jones M, Meng L, Brown GL. Phase 1-2a multicenter dose-escalation study of ezatiostat hydrochloride liposomes for injection (Telintra, TLK199), a novel glutathione analog prodrug in patients with myelodysplastic syndrome. J Hematol Oncol. 2009 May 13;2:20. doi: 10.1186/1756-8722-2-20. PubMed PMID: 19439093; PubMed Central PMCID: PMC2694211.
9: Raza A, Galili N, Smith S, Godwin J, Lancet J, Melchert M, Jones M, Keck JG, Meng L, Brown GL, List A. Phase 1 multicenter dose-escalation study of ezatiostat hydrochloride (TLK199 tablets), a novel glutathione analog prodrug, in patients with myelodysplastic syndrome. Blood. 2009 Jun 25;113(26):6533-40. doi: 10.1182/blood-2009-01-176032. Epub 2009 Apr 27. PubMed PMID: 19398716.

Author Unknown, “Dose-Ranging Study of Telintra® Tablets + Revlimid® in Patients with Non-Deletion (5q) Low to Intermediate-1 Risk Myelodysplastic Syndrome (MDS)“, Clinical Trials, 2010, Retrieved from the Internet: URL:http://clinicaltrials.gov/ct2/show/NCT01062152?term=ezatiostat&rank=2.
2
Author Unknown, “Phase 2 Study Comparing Two Dose Schedules of Telintra(TM) in Myelodysplastic Syndrome (MDS)“, 2008, Retrieved from the Internet: URL: http://clinicaltrials.gov/ct2/show/NCT00700206?term=ezatiostat&rank=3.
3
Author Unknown, “Telik initiates phase I trial of ezatiostat in patients with myelodysplastic syndrome“, Thomson Reuters Integrity, 2010, Retrieved from the Internet: URL:https://integrity.thomson-pharma.com/integrity/xmlxsl/pk-ref-list.xml-show-ficha-ref? p-re-id=1444034.
4
Author Unknown, “Telik initiates Telintra Phase 2 trial in Revlimid refractory or resistant, del 5q MDS“, 2011, Abstract, retrieved from Internet: URL:http://www.new-medical.net/new/20110608/Telik-initiates-Telintra-Phase-2-trial-in-Revlimid-refractory-or-resistant-del-5q-MDS.aspx.
5
Author Unknown, “Telik reports phase II data on ezatiostat in MDS“, Thomson Reuters Integrity, 2010, Retrieved from the Internet: URL:https://integrity.thomson-pharma.com/integrity/xmlxsl/pk-ref -list.xml-show-ficha-ref?p-ref-id=1513842.
6
Author Unknown, “Phase 2 Study Comparing Two Dose Schedules of Telintra™ in Myelodysplastic Syndrome (MDS)“, 2008, Retrieved from the Internet: URL: http://clinicaltrials.gov/ct2/show/NCT00700206?term=ezatiostat&rank=3.
7
Author Unknown, “Telik initiates phase I trial of ezatiostat in patients with myelodysplastic syndrome“, Thomson Reuters Integrity, 2010, Retrieved from the Internet: URL:https://integrity.thomson-pharma.com/integrity/xmlxsl/pk—ref—list.xml—show—ficha—ref? p—re—id=1444034.
8
Author Unknown, “Telik reports phase II data on ezatiostat in MDS“, Thomson Reuters Integrity, 2010, Retrieved from the Internet: URL:https://integrity.thomson-pharma.com/integrity/xmlxsl/pk—ref —list.xml—show—ficha—ref?p—ref—id=1513842.
9 * Beckmann (Eng. Life Sci. 2003, 3, 113-120).
10
International Search Report dated Apr. 19, 2012 for PCT/US2011/030376 filed Mar. 29, 2011.
11
Kibbe, A. Croscarmellose Sodium. Handbook for Pharmaceutical Excipients, American Pharmaceutical Association, Third Edition, 2000, pp. 160-162.
12
Lyttle et al. “Isozyme-specific Glutathione-S-Transferase Inhibitors: Design and Synthesis,” Journal of Medicinal Chemistry, American Chemical Society, 1994, 37:189-194.
13
Quddus et al. “Oral Ezatiostat HCI (TLK199) and Myelodysplastic syndrome: A case report of sustained hematologic response following an abbreviated exposure“, Journal of Hematology & Oncology, 2010, 3:16.
14
Raza et al. “Multilineage Hematologic Improvement (HI) by TLK199 (TELINTRA(TM)), A Novel Glutathione Analog, in Myelodysplastic Syndrome: Phase 2 Study Results.” Poster Presentation, 2005, American Society of Hematology.
15
Raza et al. “Phase 1 Dose Escalation Study of TLK199 Tablets (Ezatiostat HCI, TELINTRA®), a Novel Glutathione Analog, in Myelodysplastic Syndrome.” Poster Presentation, 2007, American Society of Hematology.
16
Raza et al. “Phase 1 Dose Escalation Study of TLK199 Tablets (Telintra), a Novel Glutathione Analog, in Myelodysplastic Syndrome,” Abstract #1454 appears in Blood, vol. 100, issue 11, Nov. 16, 2007.
17
Raza et al. “Phase 1 multicenter dose-escalation study of ezatiostat hydrochloride (TLKI99 tablets), a novel glutathione analog prodrug, in patients with myelodysplastic syndrome“, BLOOD, 2009, 113(26):6533-6540.
18
Raza et al. “Phase 1-2a multicenter dose-escalation study of ezatiostat hydrochloride liposomes for injection (Telintra®, TLKI99), a novel glutathione analog prodrug in patients with myelodysplastic syndrome,” Journal of Hematology & Oncology, 2009, 2:20.
19
Raza et al. “Phase 2 Randomized Multicenter Study of Extended Dosing Schedules of Oral Ezatiostat HCI (Telintra), a Glutathione Analog Prodrug GSTP1-1 Inhibitor, In Low to Intermediate-1 Risk Myelodysplastic Syndrome (MDS)“, Myelodysplastic Syndromes: Poster II, Abstract 2910, Blood 2010; 116:2910a.
20
Raza et al. “Multilineage Hematologic Improvement (HI) by TLK199 (TELINTRA™), A Novel Glutathione Analog, in Myelodysplastic Syndrome: Phase 2 Study Results.” Poster Presentation, 2005, American Society of Hematology.
21
Rowe, et al. Hypromellose. Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 2003, pp. 297-300.
22
Rowe, et al. Mannitol. Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 2009, pp. 1-3.
23
Rowe, et al. Sucrose; Magnesium Stearate. Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 2006, 744, pp. 430-431.
24
U.S. Appl. No. 13/041,136, filed Mar. 4, 2011, Parent et al.
25
U.S. Appl. No. 13/094,693, filed Apr. 26, 2011, Leclerc et al.
26
U.S. Appl. No. 13/108,752, filed May 16, 2011, Brown et al.
27
U.S. Appl. No. 13/108,754, filed May 16, 2011, Brown et al.
28
U.S. Appl. No. 13/108,756, filed May 16, 2011, Brown et al.
29
Yoshioka et al . “Crystalline State and Polymorphism in Solid Drugs,” In: “Stability of drugs and dosage forms,” Kluwer Academic, 2000, ISBN: 0-306-46404-7, Chaper 2.2.11, pp. 107-108.

WO2013082462A1 * Nov 30, 2012 Jun 6, 2013 Telik, Inc. Amorphous ezatiostat ansolvate
US20120251496 * Mar 20, 2012 Oct 4, 2012 Telik, Inc. Ezatiostat for treating multiple myeloma
……….
Full-size image (11 K)
Figure 1.
Known Yes1 kinase inhibitors, dasatinib and saracatinib.

Table 1. Select Yes1 kinase inhibitors from a HTS and their corresponding clinical phase, known targets, and IC50 values
Compound name and NCGC ID Structure Clinical phase Known targets Yes1 IC50 (nM)
Dasatinib (1)
NCGC00181129
Approved Lyn, PDGFR, KIT, Lck, BTK, Bcr–Abl,
Fyn, Yes1, c-Src
0.5 (<1.0)a
Saracatinib (2)
NCGC00241099
Phase II/III c-Src, Bcr–Abl, Yes1, Lck 6.2 (0.70)a
AEE-788 (3)
NCGC00263149
Phase I/II EGFR, HER-2, VEGFR-2 17.5 (13.1)a
Dovitinib (4)
NCGC00249685
Phase III FGFR, EGFR, PDGFR, VEGFR-1,2 31 (1.4)a
DCC-2036 (5)
NCGC00263172
Phase I/II Bcr–Abl, Tie-2, Lyn, FLT3, VEGFR-2 2.5 (1.5)a
SGI-1776 (6)
NCGC00263186
Discontinued Pim-1, FLT3 2670 (240)a
AMG-Tie-2-1 (7)
NCGC00263199
Preclinical Tie-2 8.7 (22.0)a
AZ-23 (8)
NCGC00250381
Preclinical Trk 39.1 (3.0)a
Dorsomorphin (9)
NCGC00165869
Preclinical AMPK, BMPR, TGFβ Receptor 195.9 (29.8)a
AZ-628 (10)
NCGC00250380
Preclinical Raf Kinase B,C 348.3 (51.2)a
http://www.sciencedirect.com/science/article/pii/S0960894X13006677
Data in parentheses were gathered by Reaction Biology Corp. using a [γ-33P]-ATP radiolabeled enzyme activity assay at an ATP concentration of 10 μM (www.reactionbiology.com).



6





DACINOSTAT



Dacinostat (LAQ-824, NVP-LAQ824,)
((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1 H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide
(2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide
404951-53-7
C22H25N3O3
Exact Mass: 379.18959
Molecular Weight: 379.45
Novartis (Originator)
Dacinostat, also known as LAQ824, is a hydroxamate histone deacetylase inhibitor with potential anticancer activity. LAQ824 sensitized nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. LAQ824 reduced clonogenic survival of the H23 and H460 cell lines five-fold compared with controls and four-fold compared with either agent alone (P<0.001). In phase I trials,  LAQ824 was well tolerated at doses that induced accumulation of histone acetylation, with higher doses inducing changes consistent with HSP90 inhibition.
NVP-LAQ824 inhibits histone deacetylase enzymatic activities in vitro and transcriptionally activated the p21 promoter in reporter gene assays. When tested on a variety of solid tumour cell lines, NVP-LAQ824 exhibited selective anti-proliferative effects, inducing cell growth inhibition in some, while inducing cell death in others. To induce cell death, a minimum of 16 h exposure to NVP-LAQ824 is required. Flow cytometry studies revealed that both tumour cell lines and normal diploid fibroblasts arrested in the G2/M phase of the cell cycle after compound treatment. However, an increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was only observed in the cancer cell lines.
Annexin V staining data confirmed that NVP-LAQ824 induced apoptosis in tumour cells, but not in normal cells. To relate HDAC inhibition to the anti-proliferative effects of NVP-LAQ824, expression of HDAC 1 was inhibited using antisense and this was sufficient to activate p21 expression, hypophosphorylate Rb and inhibit cell growth. Furthermore, tumour cells treated with NVP-LAQ824 caused acetylation of HSP90 and degradation of its cargo oncoproteins. Finally, NVP-LAQ824 exhibited antitumour effects in a xenograft animal model.
To determine if NVP-LAQ824 inhibited histone deacetylases in vivo, tumours treated with the drug were immunoblotted with an antibody specific for acetylated histones H3 and H4 and the results indicated increased histone H3 and 114 acetylation levels in NVP-LAQ824 treated cancer cells. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel HDAC inhibitor is currently in clinical trials as an anticancer agent. see: http://www.ncbi.nlm.nih.gov/pubmed/15171259.
Reversible acetylation of histones is a major regulator of gene expression that acts by altering accessibility of transcription factors to DNA. In normal cells, histone deacetylase (HDA) and histone acetyltrasferase together control the level of acetylation of histones to maintain a balance. Inhibition of HDA results in the accumulation of hyperacetylated histones, which results in a variety of cellular responses.
Inhibitors of HDA have been studied for their therapeutic effects on cancer cells. For example, butyric acid and its derivatives, including sodium phenylbutyrate, have been reported to induce apoptosis in vitro in human colon carcinoma, leukemia and retinoblastoma cell lines. However, butyric acid and its derivatives are not useful pharmacological agents because they tend to be metabolized rapidly and have a very short half-life in vivo. Other inhibitors of HDA that have been widely studied for their anti-cancer activities are trichostatin A and trapoxin. Trichostatin A is an antifungal and antibiotic and is a reversible inhibitor of mammalian HDA. Trapoxin is a cyclic tetrapeptide, which is an irreversible inhibitor of mammalian HDA.
Although trichostatin and trapoxin have been studied for their anti-cancer activities, the in vivo instability of the compounds makes them less suitable as anti-cancer drugs. There remains a need for an active compound that is suitable for treating tumors, including cancerous tumors, that is highly efficacious and stable
……………………….
PATENT
WO 200222577
Proc Am Assoc Cancer Res 2002,43Abst 3671
The esterification of 4-formylcinnamic acid (I) with methanol and HCl gives the methyl ester (II), which can be obtained by Heck coupling of 4-bromobenzaldehyde (III) with methyl acrylate (IV). The reductocondensation of (II) with tryptamine (V) by means of NaBH(OAc)3 in dichloroethane yields the secondary amine (VI), which is alkylated with 2-(tert-butyldimethylsilyloxy)ethyl bromide (VII) by means of DIEA in DMSO to afford the tertiary amine (VIII). The reaction of the methyl ester group of (VIII) with KOH and hydroxylamine in methanol provides the silylated hydroxamic acid (IX), which is finally deprotected with TFA in water.
References
1: Wang H, Cheng F, Woan K, Sahakian E, Merino O, Rock-Klotz J, Vicente-Suarez I, Pinilla-Ibarz J, Wright KL, Seto E, Bhalla K, Villagra A, Sotomayor EM. Histone deacetylase inhibitor LAQ824 augments inflammatory responses in macrophages through transcriptional regulation of IL-10. J Immunol. 2011 Apr 1;186(7):3986-96. doi: 10.4049/jimmunol.1001101. Epub 2011 Mar 2. PubMed PMID: 21368229.
2: Schwarz K, Romanski A, Puccetti E, Wietbrauk S, Vogel A, Keller M, Scott JW, Serve H, Bug G. The deacetylase inhibitor LAQ824 induces notch signalling in haematopoietic progenitor cells. Leuk Res. 2011 Jan;35(1):119-25. doi: 10.1016/j.leukres.2010.06.024. Epub 2010 Jul 31. PubMed PMID: 20674020.
3: Cho YS, Whitehead L, Li J, Chen CH, Jiang L, Vögtle M, Francotte E, Richert P, Wagner T, Traebert M, Lu Q, Cao X, Dumotier B, Fejzo J, Rajan S, Wang P, Yan-Neale Y, Shao W, Atadja P, Shultz M. Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824). J Med Chem. 2010 Apr 8;53(7):2952-63. doi: 10.1021/jm100007m. PubMed PMID: 20205394.
4: Vo DD, Prins RM, Begley JL, Donahue TR, Morris LF, Bruhn KW, de la Rocha P, Yang MY, Mok S, Garban HJ, Craft N, Economou JS, Marincola FM, Wang E, Ribas A. Enhanced antitumor activity induced by adoptive T-cell transfer and adjunctive use of the histone deacetylase inhibitor LAQ824. Cancer Res. 2009 Nov 15;69(22):8693-9. doi: 10.1158/0008-5472.CAN-09-1456. Epub 2009 Oct 27. PubMed PMID: 19861533; PubMed Central PMCID: PMC2779578.
5: Ellis L, Bots M, Lindemann RK, Bolden JE, Newbold A, Cluse LA, Scott CL, Strasser A, Atadja P, Lowe SW, Johnstone RW. The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy. Blood. 2009 Jul 9;114(2):380-93. doi: 10.1182/blood-2008-10-182758. Epub 2009 Apr 21. PubMed PMID: 19383971.
6: de Bono JS, Kristeleit R, Tolcher A, Fong P, Pacey S, Karavasilis V, Mita M, Shaw H, Workman P, Kaye S, Rowinsky EK, Aherne W, Atadja P, Scott JW, Patnaik A. Phase I pharmacokinetic and pharmacodynamic study of LAQ824, a hydroxamate histone deacetylase inhibitor with a heat shock protein-90 inhibitory profile, in patients with advanced solid tumors. Clin Cancer Res. 2008 Oct 15;14(20):6663-73. doi: 10.1158/1078-0432.CCR-08-0376. PubMed PMID: 18927309.
7: Chung YL, Troy H, Kristeleit R, Aherne W, Jackson LE, Atadja P, Griffiths JR, Judson IR, Workman P, Leach MO, Beloueche-Babari M. Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of a novel histone deacetylase inhibitor, LAQ824, in human colon carcinoma cells and xenografts. Neoplasia. 2008 Apr;10(4):303-13. PubMed PMID: 18392140; PubMed Central PMCID: PMC2288545.
8: Cuneo KC, Fu A, Osusky K, Huamani J, Hallahan DE, Geng L. Histone deacetylase inhibitor NVP-LAQ824 sensitizes human nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. Anticancer Drugs. 2007 Aug;18(7):793-800. PubMed PMID: 17581301.
9: Kato Y, Salumbides BC, Wang XF, Qian DZ, Williams S, Wei Y, Sanni TB, Atadja P, Pili R. Antitumor effect of the histone deacetylase inhibitor LAQ824 in combination with 13-cis-retinoic acid in human malignant melanoma. Mol Cancer Ther. 2007 Jan;6(1):70-81. PubMed PMID: 17237267.
10: Leyton J, Alao JP, Da Costa M, Stavropoulou AV, Latigo JR, Perumal M, Pillai R, He Q, Atadja P, Lam EW, Workman P, Vigushin DM, Aboagye EO. In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3′-deoxy-3′-[18F]fluorothymidine positron emission tomography. Cancer Res. 2006 Aug 1;66(15):7621-9. PubMed PMID: 16885362.
 
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 7


1 Vote

Givinostat structure.svg

ITF2357, UNII-5P60F84FBH, ITF-2357, Gavinostat,
[6-(diethylaminomethyl)naphthalen-2-yl]methyl N-[4-(hydroxycarbamoyl)phenyl]carbamate,
diethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-amine
4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]benzohydroxamic acid
CAS 497833-27-9 FREE BASE
199657-29-9 HCL SALT
Molecular Formula: C24H27N3O4
Molecular Weight: 421.48888 g/mol
PHASE 2  Italfarmaco (INNOVATOR)
DESCRIBED IN U.S. Pat. No. 6,034,096 or in U.S. Pat. No. 7,329,689.
Givinostat.png
Givinostat (INN[1]) or gavinostat (originally ITF2357) is a histone deacetylase inhibitor with potential anti-inflammatory, anti-angiogenic, and antineoplastic activities.[2] It is a hydroxamate used in the form of its hydrochloride.
Givinostat is in numerous phase II clinical trials (including for relapsed leukemias and myelomas),[3] and has been granted orphan drug designation in the European Union for the treatment of systemic juvenile idiopathic arthritis[4] and polycythaemia vera.[5]
In 2010, orphan drug designation was assigned in the E.U. for the treatment of systemic-onset juvenile idiopathic arthritis and for the treatment of polycythemia vera. In 2013, this designation was assigned by the FDA for the treatment of Duchenne’s muscular dystrophy and for the treatment of Becker’s muscular dystrophy.
ITF2357 was discovered at Italfarmaco of Milan, Italy. It was patented in 1997 and first described in the scientific literature in 2005.[6][7]
Givinostat hydrochloride, an orally active, synthetic inhibitor of histone deacetylase, is being evaluated in several early clinical studies at Italfarmaco, including studies for the treatment of myeloproliferative diseases, polycythemia vera, Duchenne’s muscular dystrophy and periodic fever syndrome. The company was also conducting clinical trials for the treatment of Crohn’s disease and chronic lymphocytic leukemia; however, the trials were terminated.
No recent development has been reported for research into the treatment of juvenile rheumatoid arthritis, for the treatment of multiple myeloma and for the treatment of Hodgkin’s lymphoma.
Patent Submitted Granted
Monohydrate hydrochloride of the 4-hydroxycarbamoyl-phenyl)-carbamic acid (6-diethylaminomethyl-naphtalen-2-yl) ester [US7329689] 2005-11-03 2008-02-12

Adverse effects

In clinical trials of givinostat as a salvage therapy for advanced Hodgkin’s lymphoma, the most common adverse reactions were fatigue (seen in 50% of participants), mild diarrhea or abdominal pain (40% of participants), moderate thrombocytopenia (decreased platelet counts, seen in one third of patients), and mild leukopenia (a decrease in white blood cell levels, seen in 30% of patients). One-fifth of patients experienced prolongation of the QT interval, a measure of electrical conduction in the heart, severe enough to warrant temporary suspension of treatment.[8]

Mechanism of action

Givinostat inhibits class I and class II histone deacetylases (HDACs) and several pro-inflammatory cytokines. This reduces expression of tumour necrosis factor (TNF), interleukin 1α and β, and interleukin 6.[7]
It also has activity against cells expressing JAK2(V617F), a mutated form of the janus kinase 2 (JAK2) enzyme that is implicated in the pathophysiology of many myeloproliferative diseases, including polycythaemia vera.[9][10] In patients with polycythaemia, the reduction of mutant JAK2 concentrations by givinostat is believed to slow down the abnormal growth of erythrocytes and ameliorate the symptoms of the disease.[5]
………………….
PATENT
http://www.google.co.in/patents/US7329689
Hydrochloride of (6-diethylaminomethyl-naphthalen-2-yl)-methyl ester of (4-hydroxycarbamoylphenyl)-carbamic acid (II)
has been described in U.S. Pat. No. 6,034,096 as a derivative of hydroxamic acid having anti-inflammatory and immunosuppressive activity, probably owing to the ability thereof to inhibit the production of pro-inflammatory cytokines. This compound is obtained according to Example 12 of the above-mentioned patent as an anhydrous, amorphous, hygroscopic, deliquescent solid which is difficult to handle.
The 4-(6-diethylaminomethyl-naphthalen-2-ylmethoxycarbonylamino)-benzoic acid can be prepared as described in Example 12, point C, of U.S. Pat. No. 6,034,096.
The acid (1.22 kg, 3 moles) was suspended in THF (19 l) and the mixture was agitated under nitrogen over night at ambient temperature. The mixture was then cooled to 0° C. and thionyl chloride (0.657 l, 9 moles) was added slowly, still under nitrogen, with the temperature being maintained below 10° C. The reaction mixture was heated under reflux for 60 minutes, DMF (26 ml) was added and the mixture was further heated under reflux for 60 minutes.
The solvent was evaporated under vacuum, toluene was added to the residue and was then evaporated. This operation was repeated twice, then the residue was suspended in THF (11.5 l) and the mixture was cooled to 0° C.
The mixture was then poured into a cold solution of hydroxylamine (50% aq., 1.6 l, 264 moles) in 5.7 l of water. The mixture was then cooled to ambient temperature and agitated for 30 minutes. 6M HCl was added until pH 2 was reached and the mixture was partially evaporated under vacuum in order to eliminate most of the THF. The solid was filtered, washed repeatedly with water and dissolved in a solution of sodium bicarbonate (2.5%, 12.2 l). The solution was extracted with 18.6 l of a mixture of THF and ethyl acetate (2:1 v/v). 37% HCl (130 ml) were added to the organic layer in order to precipitate the monohydrate of the (6-diethylaminomethyl-naphthalen-2-yl)-methyl ester hydrochloride of the (4-hydroxycarbamoyl-phenyl)-carbamic acid. If necessary, this operation can be repeated several times to remove any residues of the original acid.
Finally, the solid was dried under vacuum (approximately 30 mbar, 50° C.), producing 0.85 kg (60%) of compound (I).
HPLC purity: 99.5%; water content (Karl Fischer method): 3.8%; (argentometric) assay: 99.8%.



Elemental analysis

C % H % Cl % N %



Calculated for 60.56 6.35 7.45 8.83

C24H30ClN3O5

Found 61.06 6.48 7.48 8.90

…..

PATENT
http://www.google.co.in/patents/US20120302633

…………..

…………………..
http://www.google.com/patents/US6034096

EXAMPLE 12

4-[6-(Diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]-benzohydroxamic acid hydrochloride

A. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (22.2 g, 115 mmol) was added to a solution of 2,6-naphthalenedicarboxylic acid (25 g, 115 mmol) and hydroxybenzotriazole (15.6 g, 115 mmol) in dimethylformamide (1800 ml) and the mixture was stirred at room temperature for 2 hours. Diethyl amine (34.3 ml, 345 mmol) was added and the solution was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure and the crude was treated with 1N HCl (500 ml) and ethyl acetate (500 ml), insoluble compounds were filtered off and the phases were separated. The organic phase was extracted with 5% sodium carbonate (3×200 ml) and the combined aqueous solutions were acidified with concentrated HCl and extracted with ethyl acetate (3×200 ml). The organic solution was then washed with 1N HCl (6×100 ml), dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure yielding 18.5 g (Yield 60%) of pure 6-(diethylaminocarbonyl)-2-naphthalenecarboxylic acid; m.p.=122-124° C.
1 H-NMR d 8.67 (s, 1H), 8.25-8.00 (m, 4H), 7.56 (d, 1H), 3.60-3.20 (m, 4H), 1.30-1.00 (m, 6H).
B. A solution of 6-(diethylaminocarbonyl)-2- naphthalenecarboxylic acid (18 g, 66 mmol) in THF (200 ml) was slowly added to a refluxing suspension of lithium aluminium hydride (7.5 g, 199 mmol) in THF (500 ml). The mixture was refluxed for an hour, then cooled at room temperature and treated with a mixture of THF (25 ml) and water (3.5 ml), with 20% sodium hydroxide (8.5 ml) and finally with water (33 ml). The white solid was filtered off and the solvent was removed under reduced pressure. Crude was dissolved in diethyl ether (200 ml) and extracted with 1N HCl (3×100 ml). The aqueous solution was treated with 32% sodium hydroxide and extracted with diethyl ether (3×100 ml). The organic solution was dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure yielding 12.7 g (79% yield) of pure 6-(diethylaminomethyl)-2-naphthalenemethanol as thick oil.
1 H-NMR d 7.90-7.74 (m, 4H), 7.49 (m, 2H), 5.32 (t, 1H, exchange with D2 O), 4.68 (d, 2H), 3.69 (s, 2H), 2.52 (q, 4H), 1.01 (t, 6H).
C. A solution of 6-(diethylaminomethyl)-2-naphthalene-methanol (12.5 g, 51 mmol) and N,N’-disuccinimidyl carbonate (13.2 g, 51 mmol) in acetonitrile (250 ml) was stirred at room temperature for 3 hours, then the solvent was removed and the crude was dissolved in THF (110 ml). This solution was added to a solution of 4-amino benzoic acid (7.1 g, 51 mmol) and sodium carbonate (5.5 g, 51 mmol) in water (200 ml) and THF (100 ml). The mixture was stirred overnight at room temperature, then THF was removed under reduced pressure and the solution was treated with 1N HCl (102 ml, 102 mmol). The precipitate was filtered, dried under reduced pressure, tritured in diethyl ether and filtered yielding 13.2 g (yield 64%) of pure 4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]-benzoic acid; m.p.=201-205° C. (dec.)
1 H-NMR d 10.26 (s, 1H), 8.13 (s, 1H), 8.05-7.75 (m, 6H), 7.63 (m, 3H), 5.40 (s, 2H), 4.32 (s, 2H), 2.98 (q, 4H), 1.24 (t, 6H).
D. A solution of 4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]benzoic acid (13.1 g, 32 mmol) and thionyl chloride (7 ml, 96 mmol) in chloroform (300 ml) was refluxed for 4 hours, then the solvent and thionyl chloride were evaporated. Crude was dissolved in chloroform (100 ml) and evaporated to dryness three times. Crude was added as solid to a solution of hydroxylamine hydrochloride (2.7 g, 39 mmol) and sodium bicarbonate (5.4 g, 64 mmol) and 1N sodium hydroxide (39 ml, 39 mmol) in water (150 ml) and THF (50 ml). The mixture was stirred overnight at room temperature, then THF was removed under reduced pressure and the aqueous phase was extracted with ethyl acetate (3×100 ml). The combined organic phases were dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure. Crude was dissolved in THF and treated with a 1.5 N etheric solution of HCl. The solid product was filtered and dried yielding 6 g (yield 41%) of pure 4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]benzohydroxamic acid hydrochloride as white solid; m.p.=162-165° C., (dec.)
1 H-NMR d 11.24 (s, 1H, exchange with D2 O), 10.88 (s, 1H, exchange with D2 O), 10.16 (s, 1H), 8.98 (bs, 1H, exchange with D2 O), 8.21 (s, 1H), 8.10-7.97 (m, 3H), 7.89 (d, 1H), 7.80-7.55 (m, 5H), 5.39 (s, 2H), 4.48 (d, 2H), 3.09 (m, 4H), 1.30 (t, 6H).

References

 1


  1. Guerini V, Barbui V, Spinelli O, et al. (April 2008). “The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F)”. Leukemia 22 (4): 740–7. doi:10.1038/sj.leu.2405049. PMID 18079739.

Further reading

US6034096 12 May 1997 7 Mar 2000 Italfarmaco S.P.A. Compounds with anti-inflammatory and immunosuppressive activities

Citing Patent Filing date Publication date Applicant Title
US8518988 * 3 Dec 2010 27 Aug 2013 Chemi Spa Polymorph of the hydrochloride of the (4-hydroxycarbamoyl-phenyl)-carbamic acid (6-dimethylamino methyl-2-naphthalenyl) ester
US20120302633 * 3 Dec 2010 29 Nov 2012 Chemi Spa Novel polymorph of the hydrochloride of the (4-hydroxycarbamoyl-phenyl)-carbamic acid (6-dimethylamino methyl-2-naphthalenyl) ester
WO2011092556A1 3 Dec 2010 4 Aug 2011 Chemi Spa Novel polymorph of the hydrochloride of the (4-hydroxycarbamoyl-phenyl)-carbamic acid (6-dimethylamino methyl-2-naphtalenyl) ester





 8 TOPIROXOSTAT


str1

Figure JPOXMLDOC01-appb-C000001
Topiroxostat
托匹司他
FUJI YAKUHIN  ........INNOVATOR
Approved in japan PMDA JUNE 28 2013
Xanthine oxidase inhibitor
FOR GOUT AND HYPERURICEMIA
Launched - 2013, Fuji YakuhinSanwa, Topiloric  Uriadec
IUPAC Name: 4-(5-pyridin-4-yl-1H-1,2,4-triazol-3-yl)pyridine-2-carbonitrile
CAS Registry Number: 577778-58-6
4 - [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 - carbonitrile (1)
5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole
Synonyms: 4-(5-PYRIDIN-4-YL-1H-1,2,4-TRIAZOL-3-YL)PYRIDINE-2-CARBONITRILE,
AC1NRB9T, Topiroxostat (JAN/INN),  DB01685, D09786, FYX-051
SK-0910
4-[5-PYRIDIN-4-YL-1H-[1,2,4]TRIAZOL-3-YL]-PYRIDINE-2-CARBONITRILE,
C13H8N6 MF,248.2482 MW
TOPIROXOSTAT
托匹司他
A xanthine oxidase inhibitor used to treat gout and hyperuricemia.
PATENT EXP 3/12/22, US /EU/CN

str1
FYX-051, TOPIROXOSTAT is a xanthine oxidase inhibitor. This agent was approved in Japan by Fuji Yakuhin and Sanwa for the treatment of gout and hyperuricemia in 2013 and launched at the same year. In 2009, the compound was licensed to Sanwa by Fuji Yakuhin in Japan for the codevelopment and commercialization of gout.
The number of patients with hyperuricemia in Japan is reported to be 1.25 million and the number suffering from asymptomatic hyperuricemia is estimated to reach several millions. Hyperuricemia is becoming a popular disease.
Presently, hyperuricemia and gout due to hyperuricemia are treated by improving the living environment and administering various drug therapies for each period when an attack of gout is predicted to occur (presymptomatic period), when an attack of gout occurs, or when an attack of gout subsides. That is, preventive therapy is conducted in the presymptomatic period by administering colchicines as well as controlling the daily living environment. When an attack occurs, drug therapy using non-steroidal or steroidal anti-inflammatory agents is mainly conducted. After the attack subsides, patients are given guidance to improve their lifestyle. When improvement is judged insufficient, an assessment is made as to whether hyperuricemia is caused by reduced excretion of uric acid or by increased production of uric acid followed by treatment with drugs, which exhibit a uricosuric effect, such as probenecid and benzbromarone, those which inhibit resorption of uric acid, such as sulfinpyrazone, those which improve acidurea conditions, such as citrates, and xanthine oxidase inhibitors which inhibit production of uric acid, such as allopurinol. Colchicine is said to be able to prevent about 90% of attacks through inhibiting chemotaxis and phagocytosis of leukocytes, such as neutrophils, if administration thereof has been completed within a few hours before the attack. Since colchicine has various adverse effects, however, the use thereof is limited to the minimum and it is therefore difficult to timely administer it.
Accordingly, drug therapies are mainly adopted, but only allopurinol is available for the treatment of a disease caused by increased production of uric acid. However, a metabolite of allopurinol, oxypurinol, tends to accumulate and may cause calculi formation. Furthermore, this drug has been reported to induce adverse events such as rash, a decreased renal function and hepatitis, and it is not easy to administer.
Examples of compounds having xanthine oxidase inhibiting activity that can be used for treating gout caused by increased production of uric acid and that are effective for hyperuricemia and gout due to hyperuricemia have been described in J. Medicinal Chemistry, 1975, Vol. 18, No. 9, pp. 895–900, Japanese Patent Publication No. 49-46622 and Japanese Patent Publication No. 50-24315, which disclose some 1,3,5-substituted or 3,5-substituted 1,2,4-triazole compounds.
4 - [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 - carbonitrile (1) has a xanthine oxidase inhibitory activity and serum uric acid level known as the agent that reduces (Patent Document 1).
Figure JPOXMLDOC01-appb-C000001
The method for producing the compound (1), for example, 2 by Reissert Henze reaction isonicotinic acid methyl N-oxide - is a cyano isonicotinate, and the hydrazide which is then, 4 - this condensed cyanopyridine After obtaining a hydrazide of isonicotinic acid N-oxide (Patent Document 1, Example 12) and method, a cyano group after introduction, 4 by Reissert Henze reaction - method of condensing a cyano pyridine is known (Patent Document 1, Example 39).Further, 4 - as a starting material cyano-N-oxide, a triazole ring after construction (Patent Document 3), Reissert Henze unprotected or (Patent Document 2) to protect the ring condensed with isonicotinic acid hydrazide method of obtaining the compound (1) by introducing a cyano group by the reaction have also been reported.
The crystalline polymorph, yet the same molecule with the same chemical composition, the molecular arrangement in the crystal are different, and are different crystalline states. The pharmaceutical compounds having crystal polymorphism such the differences in physicochemical properties, affect pharmacological activity, solubility, bioavailability, stability and the like are known.Therefore, when the crystal polymorphism is present in a pharmaceutically useful compound, producing compounds of the crystalline form highly useful from polymorphs thereof is desirable.
WO 2003/064410 discloses WO 2005/009991 discloses Japanese Patent Publication No. 2005-41802
However, 4 of the above Patent Document - no description about the presence of crystalline polymorph on carbonitrile - pyridine-2-[yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazol] It has not been, to these manufacturing methods, it is disclosed a method for the purpose of improving the chemical purity and yield, there is no description of the crystallographic plane.
Method of producing topiroxostat, useful for preventing or treating gout; and its intermediates. Picks up from WO2012060308, claiming the use of this topiroxostat for treating renal dysfunction. Along with the concurrently published WO2014017515, claiming crystalline Forms I and II of this compound, which, Fuji Yakuhin, in collaboration with Sanwa Kagaku, has developed and launched for the treatment of gout and hyperuricemia.WO-2014017516
Crystalline Forms I and II of topiroxostat, useful for preventing or treating gout. Along with the concurrently published WO2014017516, claiming a method of producing this compound. Picks up from WO2012060308, claiming a method of treating renal dysfunction using topiroxostat, which Fuji Yakuhin, in collaboration with Sanwa Kagaku, has developed and launched for the treatment of gout and hyperuricemia.WO-2014017515
novel 1,2,4-triazole compounds having an optionally substituted 2-cyanopyridin-4-yl group at 3-position and an optionally substituted aromatic group at 5-position inhibit a xanthine oxidase and are useful for treatment of gout and hyperuricemia, and have previously filed a patent application (Patent Document 1). The compounds can be prepared according to a method shown by the following reaction scheme:
  • Figure imgb0001
    wherein TMS represents trimethylsilyl group and Ar represents an aromatic group
    Although this method can achieve the object in a small-scale production, there were such problems that the process for production of a substituted or unsubstituted 2-cyanoisonicotinic acid hydrazide is complicated, and a reaction solvent must be selected in compliance with the physical property of the product compound in each step, and isolation of a product is required in each step. Furthermore, the overall yield is not sufficiently high, and therefore there is a problem in the production on an industrial scale.
    Patent Document 1: JP-A-2002-017825
    • A compound represented by formula (1) which is a starting material may be prepared by a method described in, for example, JP-A-47-7120, JP-A-61-152661A, JP-A-62-149673, JP-A-2002-528447, or European Patent Application No. 559363 specification. However, it is preferable to prepare compound (1) according to the following reaction scheme:
    • Figure imgb0004

SYNTHESIS
















PATENT
EP1650204A1
    Example 2
      Preparation of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole p-toluenesulfonate
    • To the toluene solution obtained in Example 1 (2) was added 2-propanol (700 mL), and the mixture was stirred. To the resulting solution was added p-toluenesulfonic acid monohydrate (151.16 g) and the resulting mixture was stirred for 8 hours at an internal temperature of 80°C. The mixture was brought to room temperature, and the precipitated crystals were taken out and washed with 2-propanol (210 mL×2). The white crystals were dried under reduced pressure at 60°C for 15 hours to give 106.0 g of the captioned compound as white crystals. Subsequently, 90.0 g of the crystals was suspended in a mixture of 2-butanol (49 mL) and water (491 mL) and heated to an internal temperature of 80°C for 1 hour. The internal temperature was brought to room temperature, and the crystals were filtered and washed with a mixture of 2-butanol and water (1:10) (270 mL×3). The resulting crystals were dried under reduced pressure at 60°C for 15 hours to give 75.7 g of the captioned compound in a high purity.
    • 1H―NMR(DMSO-d6)δppm:2.29(s,3H), 7.11 (m,2H), 7.48 (dd, 2H, J=6.48, 1.62Hz) , 8.32-8.35(m, 3H) , 8.57(dd, 1H, J=1.62, 0.81Hz) , 8.94-8.98(m, 3H)
Example 3
Preparation of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
  • To the white crystals (50.5g) obtained in Example 2 was added 2-propanol (937.5 mL) and water (312.5 mL), and the resulting mixture was heated and dissolved at an internal temperature of 80°C. Immediately thereafter, the solution was filtered and the filtrate was cooled to an internal temperature of 20°C. To the resulting suspension was added dropwise 0.52 mol/l of an aqueous sodium hydrogen carbonate solution (250 mL), and the mixture was stirred at room temperature for 2 hours. Then the crystals were filtered and washed with water (150 mL×3) and 2-butanol (150 mL×2). The crystals were dried under reduced pressure at 80°C for 15 hours to give 29.4 g of the captioned compound as pale yellow crystals.
  • 1H―NMR(DMSO-d6)δppm:8.02(dd, 2H, J=4.59, 1.62Hz),8.32(dd, 1H, J=5.13, 1.62Hz), 8.55(dd, 1H, J=1.62, 1.08Hz), 8.80(dd, 2H, J=4.59, 1.62Hz), 8.93 (dd, 1H, J=5.13, 1.08Hz)

SYNTHESIS
US7074816
Example 12
5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
1) Production of methyl isonicotinate N-oxide
13.9 g of isonicotinic acid N-oxide was added to 209 ml of methylene chloride, 29.7 g of 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline was further added thereto, and the mixture was stirred under argon atmosphere at room temperature for one hour. 32.1 g of methanol was added to this mixture, which was stirred at room temperature for 17 hours. After the solvent was evaporated under reduced pressure, the residue was subjected to silica gel column chromatography. Chloroform-acetone (3:1) was used as an eluent to yield 11.1 g of a white powder.
1H-NMR (CDCl3) δppm: 3.95 (3H, s), 7.88 (2H, d, J=7.25 Hz), 8.22 (2H, J=7.25 Hz)
2) Production of Methyl 2-cyanoisonicotinate
11.1 g of the crystal obtained in 1) was dissolved in 170 ml of acetonitrile, 14.6 g of triethylamine and 21.5 g of trimethylsilylnitrile were added thereto, and the mixture was refluxed under argon atmosphere for 16 hours. After the solvent was evaporated under reduced pressure, the residue was subjected to silica gel column chromatography. Chloroform-acetone (95:5) was used as an eluent to yield 8.44 g of a pale yellow powder.
1H-NMR (CDCl3) δppm: 4.01 (3H, s), 8.08 (1H, d, J=5.45 Hz), 8.24 (1H, s), 8.90 (1H, d, J=5.45 Hz)
3) Production of 2-cyanoisonicotinic acid hydrazide
8.44 g of the crystal obtained in 2) was added to 85 ml of methanol, 1.84 g of hydrazine was further added thereto, and the mixture was stirred under argon temperature for 2 hours. After the solvent was evaporated under reduced pressure, chloroform was added to the residue, which was stirred at room temperature for one hour. The precipitated crystal was filtered, washed with chloroform and dried with a vacuum pump to yield 4.15 g of a pale yellow powder.
1H-NMR (DMSO-d6) δppm: 4.72 (2H, s), 8.05 (1H, d, J=5.12 Hz), 8.31 (1H, s),8.90 (1H, d, J=5.12 Hz), 10.23 (1H, s)
4) Production of the Object Compound
2.67 g of 4-cyanopyridine was dissolved in 40 ml of methanol, 0.83 g of sodium methoxide was added thereto, and the mixture was stirred at room temperature for one hour. Then 4.15 g of the crystal obtained in 3) was added and the mixture was refluxed for 37 hours. After the reaction completed, the precipitated solid was filtered, washed with methanol and dried with a vacuum pump to yield 3.66 g of the object compound as a yellow powder.
1H-NMR (DMSO-d6) δppm: 8.01 (2H, dd, J=4.54, 1.57 Hz), 8.31 (1H, dd, J=5.11, 1.65 Hz), 8.53 (1H, dd, J=1.65, 0.50 Hz), 8.80 (2H, dd, J=4.54, 1.57 Hz), 8.93 (1H, dd, J=5.11, 0.50 Hz)
Example 39
5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
1) Production of isonicotinic acid (N-2-tert-butoxycarbonyl)hydrazide-1-oxide
585 ml of methylene chloride was added to 39.0 g of isonicotinic acid N-oxide, and after 34.0 g of triethylamine was further added thereto, the mixture was cooled under argon atmosphere to −15° C. 33.5 g of ethyl chlorocarbonate in 117 ml of methylene chloride was added dropwise to this mixture, which was stirred at a temperature from −5 to −10° C. for one hour. Then 44.4 g of tert-butyl ester of carbamic acid in 117 ml of methylene chloride was added dropwise to this mixture and it was allowed to slowly rise to room temperature while it was stirred. The precipitated solid was filtered after 15 hours, washed with methylene chloride, and dried with a vacuum pump to yield 49.7 g of white crystal.
1H-NMR (DMSO-d6) δppm: 1.42 (9H, s), 7.82 (2H, d, J=7.09 Hz), 8.33 (2H, d, J=7.09 Hz), 9.02 (1H, s), 10.44 (1H, s)
Production of 2-cyanoisonicotinic acid hydrazine 1½ P-Toluenesulfonic acid salt
228 ml of dioxane was added to 30.4 g of the crystal obtained in 1), and after 13.1 g of trimethylsilyl cyanide and 38.8 g of N,N-dimethylcarbamoyl chloride were further added thereto, the mixture was stirred under argon atmosphere at 60° C. for 5 hours. After the solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate and subsequently washed with 1.5 M sodium carbonate aqueous solution and a saturated saline solution and dried over magnesium sulfate. After the magnesium sulfate was filtered off, the solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue, 68.5 g of p-toluenesulfonic acid monohydrate was added thereto, and the mixture was stirred at room temperature for 22 hours. The precipitated crystal was filtered, washed with ethyl acetate, and dried with a vacuum pump to yield 40.3 g of white crystal 2).
1H-NMR (DMSO-d6) δppm: 2.28 (4.5H, s), 7.12 (3H, dd, J=7.92 & 0.66 Hz), 7.48 (3H, dd, J=7.92 & 0.66 Hz), 8.10 (1H, dd, J=5.11 & 1.81 Hz), 8.39 (1H, dd, J=1.81 & 0.33 Hz), 8.99 (1H, dd, J=5.11 & 0.33 Hz)
3) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
9.98 g of 4-cyanopyridine was dissolved in 250 ml of methanol, and after 7.77 g of sodium methoxide was added thereto, the mixture was stirred at room temperature for one hour. Then 40.3 g of the crystal obtained in 2) was added and the mixture was refluxed for 24 hours. After the reaction completed, the precipitated crystal was filtered, washed with methanol, and dried with a vacuum pump to yield 16.3 g of yellow crystal.
1H-NMR (DMSO-d6) δppm: 8.01 (2H, dd, J=4.54 & 1.57 Hz), 8.31 (1H, dd, J=5.11 & 1.65 Hz), 8.53 (1H, dd, J=1.65 & 0.50 Hz), 8.80 (2H, dd, J=4.54 & 1.57 Hz), 8.93 (1H, dd, J=5.11 & 0.50 Hz)
4) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole
45 ml of ethanol and 15 ml of 1-methyl-2-pyrrolidone were added to 3.0 g of the crystal obtained in 3), and the mixture was heated and stirred at 80° C. for 19 hours. The crystal was filtered, subsequently washed with a mixture of ethanol and 1-methyl-2-pyrrolidone (3:1) and ethanol, and dried with a vacuum pump to yield 2.71 g of yellow crystal.
5) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole p-toluenesulfonic acid salt
5 ml of ethanol and 30 ml of water were added to 2.48 g of the crystal obtained in 4), and after 3.8 g of p-toluenesulfonic acid monohydrate was further added thereto, the mixture was stirred at room temperature for 5 hours. The precipitated crystal was filtered, subsequently washed with a mixture of ethanol and water (1:6), water and then ethanol, and dried with a vacuum pump to yield 3.5 g of white crystal.
1H-NMR (DMSO-d6) δppm: 2.28 (3H, s), 7.12 (2H, dd, J=7.75 & 0.50 Hz), 7.48 (2H, dd, J=7.75 & 0.50 Hz), 8.33 (1H, dd, J=5.12 & 1.65 Hz), 8.45 (2H, d, J=6.11 Hz), 8.57 (1H, dd, J=1.65 & 0.66 Hz), 8.96˜9.02 (3H, m)
6) Production of the object compound
17 ml of ethanol and 17 ml of water were added to 3.36 g of the crystal obtained in 5), and the mixture was stirred at room temperature for 30 minutes. A solution of sodium carbonate (0.74 g of sodium carbonate in 17 ml of water) was further added, and the mixture was stirred at room temperature for 2 hours. The precipitated crystal was filtered, subsequently washed with water and ethanol, and dried with a vacuum pump to yield 1.89 g of the object compound as a pale yellow crystal.

2D image of a chemical structureTOPIROXOSTAT
SYNTHESIS
WO2014017516A1
Figure JPOXMLDOC01-appb-C000020
(First step)
The first step, 4 - is a step of obtaining a compound (3) is reacted in the presence of an alkali metal alkoxide, cyano-N-oxide and (2), and isonicotinic acid hydrazide.
4 used in this reaction - isonicotinic acid hydrazide and (2) a cyano-N-oxide is a known compound both, I can be prepared by known means.
The alkali metal alkoxide is used, 6 alkoxide alkali metal C 1-C are preferred, sodium methylate, sodium ethylate and the like can be given as specific examples. The reaction is preferably carried out in a solvent, as the solvent, alcohol solvents such as methanol, ethanol and the like are preferable.
The reaction is preferably first in a solvent, is treated with an alkali metal alkoxide compound (2) and then to react the isonicotinic acid hydrazide. First, heated to reflux under cooling, at 80 ℃ from 15 ℃ preferably, 30 minutes and 12 hours in general, the reaction temperature in the reaction with an alkali metal alkoxide (2) with the compound is reacted 1-4 hours, preferably about. Under the temperature conditions, using an excess amount or one equivalent of 30 minutes to 12 hours usually, reaction with isonicotinic acid hydrazide Subsequent to reaction for 1 to 5 hours, preferably.
Example 1:
Synthesis 4 oxide (3) - - - (4 - pyridin-carbonyl) -4 - N "pyridine hydrazide imide -1 was suspended in 40mL of methanol cyanopyridine-N-oxide and (2) 5.00g, sodium was added to methylate 22.4mg, and the mixture was stirred for 2 hours under 40 ℃ nitrogen atmosphere. was cooled to room temperature. reaction solution was stirred for 4 hours at 40 ℃ was added isonicotinic acid hydrazide 5.71g at the same temperature, precipitated The filtrated crystals were, washed with methanol 15mL, and dried 15 hours at 80 ℃, N "- to give (3) 9.60g oxide - (4 - pyridin) -4 - pyridine-hydrazide imide -1.
1 H-NMR (DMSO-d 6) δ (ppm): 6.98 (br, 2H), 7.81 (d, 2H, J = 5.77Hz), 7.85 (d, 2H, J = 7 .09 Hz), 8.29 (d, 2H, J = 7.09Hz), 8.73 (d, 2H, J = 5.77Hz), 10.37 (br, 1H)
MS m / z: 256 [M-H] -
(Second step)
The second step is a step of obtaining compound (4) by cyanation agent cyano compound (3).
As the cyanation agent used, trialkyl cyanide alkali metal cyanide, sodium cyanide, potassium cyanide and the like, zinc cyanide, trimethylsilyl cyanide and the like.
The cyanation reaction is preferably, for example, be carried out (Heterocycles, Vol.22, No.5, 1994) by Reissert Henze reaction. This reaction, for example, to give compound (4) by an organic solvent in the compound (3), and after activation with carbamoyl halide, and reacting the cyano agent. The alkylcarbamoyl halide used in the carbamoylation is a first step in Reissert Henze reaction, 6 alkylcarbamoyl halide di C 1-C dimethylcarbamoyl chloride, and di-propyl carbamoyl chloride can be used, preferably, dimethylcarbamoyl is chloride. The solvent used in this reaction, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, tetrahydrofuran and acetonitrile can be used, however, N, N-dimethylformamide is preferred. Further, 15 ~ 60 ℃, more preferably 30 ~ 50 ℃ reaction temperature. The reaction time is preferably 1 to 24 hours, more preferably 1 to 3 hours. As the cyanation agent used in the cyanation reaction followed, cyano agents above can be used, sodium cyanide, potassium cyanide, zinc cyanide, and trimethylsilyl cyanide, and more preferably, it is sodium cyanide . -20 ~ 60 ℃ is preferred, more preferably -10 ~ 40 ℃, reaction temperature is 1-4 hours.
Is a novel compound (4) The compound obtained in this second step, it is useful as an intermediate for the production of compound (1). If through Compound (4) can be synthesized in good yield and easily without the need for purification in the second step is also possible, and can be produced (1) Compound industrially efficiently compound (4).
Synthetic N "hydrazide (4) - (4 - pyridine carbonyl) -4 - pyridine carboxylic acid N'-(carboxylic imidoyloxy - 2 - - cyano-4)
Example 2
4 pyridine hydrazide imide -1 - oxide ( was suspended in N, N-dimethylformamide 48mL and 3) 10.0g, under nitrogen atmosphere, followed by stirring for 1 hour was added dimethylcarbamoyl chloride 9.20g at 40 ℃. was added sodium cyanide 2.48g at the same temperature, After cooling to 5 ℃ below. reaction mixture was stirred for 1 hour, the crystals were collected by filtration. precipitate was successively added dropwise a 5% aqueous sodium bicarbonate solution 100mL, and 100mL water, and washed with water 100mL, at 80 ℃ for 15 h and dried under reduced pressure to give 4 - hydrazide (4) 9.28g of pyridine-carboxylic acid N'-(carboxylic imide yl - 2 - cyano-4).
1 H-NMR (DMSO-d 6) δ (ppm): 7.15 (br, 2H), 7.82 (d, 2H, J = 5.61Hz), 8.14 (d, 1H, J = 5 .11 Hz), 8.37 (s, 1H), 8.75 (d, 2H, J = 5.61Hz), 8.86 (d, 1H, J = 5.11Hz), 10.47 (br, 1H )
MS m / z: 265 [M-H] -
Figure JPOXMLDOC01-appb-C000019
(Third step)
The third step is a step of obtaining a compound (1) by the presence of an acid catalyst, the cyclization reaction of the compound (4).
As the acid, organic phosphoric acid, p-toluenesulfonic acid, such as hydrochloric acid, inorganic acids can be used, inorganic acids, phosphoric acid is particularly preferable. As the reaction solvent, water, 2 - butanol, 2 - mixed solvent of alcohol and water or alcohol, propanol, ethanol and the like can be used, but water and 2 - I was mixed 5:1 to 10:1 butanol solvent. The reaction temperature and time, 60 ~ 100 ℃, preferably 2 to 12 hours at 70 ~ 90 ℃, I want to 8-10 hours, preferably.
Intermediates and compounds of the present invention the method (1) can be isolated and purified from the washed reaction mixture, recrystallization, by means of various conventional chromatography.
Example 3:
4 - [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 - carbonitrile 4 Synthesis of (1) - pyridine-carboxylic acid N'- (2 - cyano-4 - carboxylic imide yl) water 82mL, 2 hydrazide (4) 9.25g - butanol was added 8.2mL, phosphate 4.00g, was stirred for 8 h at 80 ℃. After cooling to room temperature, the reaction mixture was precipitated crystals were collected by filtration, water: 2 - were washed with a mixed solution of 92.5mL butanol = 10:1. The 13 h and dried under reduced pressure at 80 ℃ crystals obtained 4 - [5 - (pyridin-4 - yl) - 1 H-1, 2,4 - triazol-3 - yl] pyridine-2 - carbonitrile (1 I got a) 7.89g.
Topiroxostat

1 H-NMR (DMSO-d 6) δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
MS m / z: 247 [M-H] -
PATENT
WO2014017515A1
Synthetic water-carbonitrile p-toluenesulfonate - pyridine Example 1: 4 - [yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazol]: 2 - butanol = was added monohydrate 6.62g p-toluenesulfonic acid in a mixed solution of 55mL of 10:1, 4 at 80 ℃ - [5 - (pyridin-4 - yl)-1H-1, 2,4 - yl] pyridine-2 - - triazol-3 was added carbonitrile 7.85g, and the mixture was stirred at the same temperature for 1 hour. After cooling to room temperature, the reaction mixture, and the precipitated crystals were collected by filtration, and water: 2 - were washed with a mixed solution of 40mL of butanol = 10:1. The dried under reduced pressure for 10 hours at 80 ℃ crystals obtained 4 - [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 - carbonitrile p-toluene I got a sulfonate 12.6g.
1 H-NMR (DMSO-d 6) δ (ppm): 2.29 (s, 3H), 7.11 (m, 2H), 7.48 (dd, 2H, J = 6.48,1.62 Hz ) ,8.32-8 .35 (m, 3H), 8.57 (dd, 1H, J = 1.62,0.81 Hz) ,8.94-8 .98 (m, 3H)
- [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazole and potassium carbonate 8.22g, 4 in a mixed solution of 80mL of ethanol = 9:1: preparation water of crystal form I: Example 2 I was dissolved carbonitrile p-toluenesulfonate 10.0g - -3 - yl] pyridine-2. After stirring for 5 hours plus 15mL 6M hydrochloric acid at 20 ℃, was the precipitated crystals were collected by filtration, and washed with water 100mL. The 23 h and dried under reduced pressure at 80 ℃, 4 - to obtain carbonitrile 5.78g - pyridin-2 [yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazole. Having a DSC as shown in FIG 4 and the powder X-ray diffraction pattern shown in FIG 1, the resulting crystals were type-I crystals.
1 H-NMR (DMSO-d 6) δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃
N, N carbonitrile 40.0g - preparation of 4 Form II - [5 - (pyridin-4 - yl)-1H-1, 2,4 - yl - triazol-3]-2: Example 3 - dimethylformamide was added 300mL, and stirred for 25 min at 150 ℃. After cooling to room temperature the solution, and the precipitated crystals were collected by filtration, and washed twice with water 200mL, 4 and dried under reduced pressure overnight at 80 ℃ the crystal - [5 - (pyridin-4 - yl)-1H-1 , 2,4 - I got carbonitrile 30.4g - yl] pyridine-2 - triazole-3. Having a DSC as shown in FIG 5 and powder X-ray diffraction pattern shown in FIG 2, the resulting crystals were type II crystals.
1 H-NMR (DMSO-d 6) δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃
The 25 ℃, about 2g carbonitrile, - preparation of the hydrate 4 - [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2: Example 4 I was stored for 14 days under conditions of relative humidity 97%. Having a DSC as shown in FIG 7 and the powder X-ray diffraction pattern shown in FIG 3, the obtained crystal was a hydrate.
1 H-NMR (DMSO-d 6) δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃
Test Example: solubility test Type I crystal by crystal form, II-type crystal, and water solubility of the hydrate was calculated by absorbance measurement method, a saturated solution concentration of each sample. I Figure 8 shows the results.Whereas the 6.2μg/mL water solubility of crystalline Form I, II type crystal 4.2μg/mL, hydrate was 1.9μg/mL.
From Figure 8, the water solubility of Form II and Form I crystals is good, water-soluble type I crystal is particularly good.

NMR
BMCL Volume 19, Issue 21, 1 November 2009, Pages 6225–6229
http://www.sciencedirect.com/science/article/pii/S0960894X09012372?np=y
view compd 39 and ignore rest
Full-size image (3 K)TOPIROXOSTAT, FYX O51
view compd 39 and ignore rest

SUPP INFO.......https://docs.google.com/viewer?url=http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0960894X09012372/1-s2.0-S0960894X09012372-mmc1.doc/271398/FULL/S0960894X09012372/50d911fe734c16dfb94912d481cb466a/mmc1.doc
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Nucleosides, Nucleotides and Nucleic Acids, 2008 ,  vol. 27,  6-7  pg. 888 - 893
Inoue, Tsutomu; Sato, Takahiro; Ashizawa, Naoki; Iwanaga, Takashi; Matsumoto, Koji; Nagata, Osamu; Nakamura, Hiroshi
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Topiroxostat
Topiroxostat.svg
Systematic (IUPAC) name
4-[5-(4-Pyridinyl)-1H-1,2,4-triazol-3-yl]-2-pyridinecarbonitrile
Clinical data
Trade namesTopiloric, Uriadec
Legal status
  • Approved in Japan
Identifiers
CAS Number577778-58-6
ATC codeNone
PubChemCID: 5288320
ChemSpider4450517
Chemical data
FormulaC13H8N6
Molecular mass248.24 g/mol
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C1=CN=CC=C1C2=NC(=NN2)C3=CC(=NC=C3)C#N





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