# Patent

## Title
Substituted pyridazine carboxamide compounds

## Abstract

The new pyridazine derivatives have unexpected drug properties as inhibitors of protein kinases especially against ALK and are useful in treating disorders related to abnormal protein kinase activities such as cancer, neurological and psychiatric diseases.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/US2011/055428, filed Oct. 7, 2011, which claims the benefit of U.S. Provisional Patent Application No. 61/391,423, filed Oct. 8, 2010, the entire contents of the aforementioned applications are hereby incorporated herein by reference.


TECHNICAL FIELD OF THE INVENTION
This invention relates to novel pyridazine derivatives, their salts, solvates, hydrates and polymorphs thereof. The invention also provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions associated with protein kinase modulation.
BACKGROUND OF THE INVENTION
Protein kinases are enzymes that catalyze the phosphorylation of hydroxyl groups of tyrosine, serine, and threonine residues of proteins. Many aspects of cell life (for example, cell growth, differentiation, proliferation, cell cycle and survival) depend on protein kinase activities. Furthermore, abnormal protein kinase activity has been related to a host of disorders such as cancer and inflammation. Therefore, considerable effort has been directed to identifying ways to modulate protein kinase activities. In particular, many attempts have been made to identify small molecules that act as protein kinase inhibitors.
The c-Met proto-oncogene encodes the Met receptor tyrosine kinase. The Met receptor is a 190 kDa glycosylated dimeric complex composed of a 50 kDa alpha chain disulfide-linked to a 145 kDa beta chain. The alpha chain is found extracellularly while the beta chain contains transmembrane and cytosolic domains. Met is synthesized as a precursor and is proteolytically cleaved to yield mature alpha and beta subunits. It displays structural similarities to semaphorins and plexins, a ligand-receptor family that is involved in cell-cell interaction. The ligand for Met is hepatocyte growth factor (HGF), a member of the scatter factor family and has some homology to plasminogen (Longati, P. et al., Curr. Drug Targets 2001, 2, 41-55); Trusolino, L. and Comoglio, P. Nature Rev. Cancer 2002, 2, 289-300].
Met functions in tumorigenesis and tumor metastasis. Expression of Met along with its ligand HGF is transforming, tumorigenic, and metastatic (Jeffers, M. et al., Oncogene 1996, 13, 853-856; Michieli, P. et al., Oncogene 1999, 18, 5221-5231). MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. Numerous studies have correlated the expression of c-MET and/or HGF/SF with the state of disease progression of different types of cancer (including lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovaries, stomach, skin, and bone cancers). Furthermore, the overexpression of c-MET or HGF have been shown to correlate with poor prognosis and disease outcome in a number of major human cancers including lung, liver, gastric, and breast. c-MET has also been directly implicated in cancers without a successful treatment regimen such as pancreatic cancer, glioma, and hepatocellular carcinoma.
Met mutants exhibiting enhanced kinase activity have been identified in both hereditary and sporadic forms of papillary renal carcinoma (Schmidt, L. et al., Nat. Genet. 1997, 16, 68-73; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1997, 94, 11445-11500). HGF/Met has been shown to inhibit anoikis, suspension-induced programmed cell death (apoptosis), in head and neck squamous cell carcinoma cells. Anoikis resistance or anchorage-independent survival is a hallmark of oncogenic transformation of epithelial cells (Zeng, Q. et al., J. Biol. Chem. 2002, 277, 25203-25208).
Increased expression of Met/HGF is seen in many metastatic tumors including colon (Fazekas, K. et al., Clin. Exp. Metastasis 2000, 18, 639-649), breast (Elliott, B. E. et al., 2002, Can. J. Physiol. Pharmacol. 80, 91-102), prostate (Knudsen, B. S. et al., Urology 2002, 60, 1113-1117), lung (Siegfried, J. M. et al., Ann. Thorac. Surg. 1998, 66, 1915-1918), and gastric (Amemiya, H. et al., Oncology 2002, 63, 286-296). HGF-Met signaling has also been associated with increased risk of atherosclerosis (Yamamoto, Y. et al., J. Hypertens. 2001, 19, 1975-1979; Morishita, R. et al., Endocr. J. 2002, 49, 273-284) and increased fibrosis of the lung (Crestani, B. et al., Lab. Invest. 2002, 82, 1015-1022).
Ax1 belongs to the subfamily of receptor tyrosine kinases (RTKs) that also includes Tyro3 and Mer. It was originally cloned from patients with chronic myelogenous leukemia and, when overexpressed, it exhibits transforming potential. Ax1 overexpression has been reported in a variety of human cancers, and is associated with invasiveness and metastasis in lung, prostate, breast, and gastric cancers as well as in renal cell carcinoma and glioblastoma. A recent study showed that Ax1 overexpression via a “tyrosine kinase switch” leads to resistance to imatinib in gastrointestinal stromal tumors. Ax1 expression is induced by chemotherapy drugs and overexpression of Ax1 confers drug resistance in acute myeloid leukemia. Ax1 has also been shown to regulate endothelial cell migration and tube formation. These findings suggest that Ax1 may be involved in the regulation of multiple aspects of tumorigenesis (for review, see Li et at, Oncogene 2009, 28: 3442).
Anaplastic lymphoma kinase (ALK) belongs to the receptor tyrosine kinase (RTK) superfamily of protein kinases. ALK expression in normal adult human tissues is restricted to endothelial cells, pericytes, and rare neural cells. Oncogenic, constitutively active ALK fusion proteins are expressed in anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumors (IMT) due to t2; chromosomal translocations. ALK has also recently been implicated as an oncogene in a small fraction of non-small-cell lung cancers and neuroblastomas (Choi et al, Cancer Res 2008; 68: (13); Webb et al, Expert Rev. Anticancer Ther. 9(3), 331-356, 2009).
Anaplastic large-cell lymphomas (ALCLs) are a subtype of the high-grade non-Hodgkin's family of lymphomas with distinct morphology, immunophenotype, and prognosis. ALCLs are postulated to arise from T cells and, in rare cases, can also exhibit a B cell phenotype. In addition, there are 40% of cases for which the cell of origin remains unknown and that are classified as “null”. First described as a histological entity by Stein et al. based on the expression of CD30 (Ki-1), ALCL presents as a systemic disease afflicting skin, bone, soft tissues, and other organs, with or without the involvement of lymph nodes. ALCL can be subdivided into at least two subtypes, characterized by the presence or absence of chromosomal rearrangements between the anaplastic lymphoma kinase (ALK) gene locus and various fusion partners such as nucleophosmin (NPM). Approximately 50-60% of cases of ALCL are associated with the t(2;5;)(p23;q35) chromosomal translocation, which generates a hybrid gene consisting of the intracellular domain of the ALK tyrosine kinase receptor juxtaposed with NPM. The resulting fusion protein, NPM-ALK has constitutive tyrosine kinase activity and has been shown to transform various hematopoietic cell types in vitro and support tumor formation in vivo. Other less frequent ALK fusion partners, e.g., tropomyosin-3 and clathrin heavy chain, have also been identified in ALCL as well as in CD30-negative diffuse large-cell lymphoma. Despite subtle differences in signaling and some biological functions, all fusions appear to be transforming to fibroblasts and hematopoietic cells. Extensive analysis of the leukemogenic potential of NPM-ALK in animal models has further corroborated the importance of NPM-ALK and other ALK rearrangements in the development of ALK-positive ALCL and other diseases.
ALK fusion proteins have also been detected in cell lines and/or primary specimens representing a variety of other tumors including inflammatory myofibroblastic tumor (IMT), neuroectodermal tumors, glioblastomas, melanoma, rhabdomyosarcoma tumors, and esophageal squamous cell carcinomas (see review by Webb T R, Slavish J, et al. Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy. Expert Rev Anticancer Ther. 2009; 9(3): 331-356). Recently, ALK is also implicated in small percent of breast and colorectal cancers (Lin E, Li L, et al. Exon Array Profiling Detects EML4-ALK Fusion in Breast, Colorectal, and Non-Small Cell Lung Cancers. Mol Cancer Res 2009; 7(9):1466-76).
ALK is further implicated in neurological diseases. Evaluation of adult ALK homozygotes, where the ALK kinase domain was deleted, revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain (Bilsland J G, Wheeldon A, Mead A, et al. Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications. Neuropsychopharmacology 2008; 33:685-700). These ALK knockout animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. These results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia, depression, and substance (cocaine) addiction.
2-amino-pyridines, such as PF-2341066, have been reported as potent inhibitors of the HGF receptor tyrosine kinase (c-Met) and ALK (J. G. Christensen, et al. Abstract LB-271, AACR 2006 meeting; H. Y. Zou et al. Cancer Res 2007; 67: 4408; patent disclosures: WO 2004076412, WO 2006021881, WO 2006021886).




In the phase I trial of PF-02341066 (crizotinib), a dramatic 60% radiographic response rate has been observed in NSCLC patients with EML4-ALK (Kwak E L, Camidge D R, Clark J, et al. Clinical activity observed in a phase I dose escalation trial of an oral c-met and ALK inhibitor, PF-02341066. J Clin Oncol 2009; 27:15s abstract 3509). Subsequent expansion trial confirmed its activity: out of 50 evaluable patients, 64% achieved objective response (ORR) by RECIST, and 90% had disease control (Bang Y, Kwak K L, Shaw D R, et al. Clinical activity of the oral ALK inhibitor, PF-02341066, in ALK-positive patients with non-small cell lung cancer (NSCLC). J Clin Oncol 2010; 28:7s, suppl; abstr 3). It is remarkable that the dramatic response was observed in patients who were refractory to chemotherapies and EGFR inhibitors. Furthermore, most patients had only minor side effects, suggesting that inhibition of ALK is well tolerated. These results firmly validated ALK as a safe and effective target for this subset of NSCLC patients who has no effective alternative systemic treatment options.
Unfortunately, the dramatic response to PF-02341066 is only transient. Most patients develop resistance and the disease progresses after about 6-18 months of treatment. In particular, a significant portion of the patients develop brain metastasis which PF-02341066 is unable to treat.
Previously, we described the substituted pyridazine carboxamide compounds as protein kinase inhibitors (WO 2009/154769). Most of these compounds potently inhibit c-Met and ALK with IC50 of <100 nM. As there is still unmet need in treatment options for kinase mediated diseases such as NSCLC with ALK fusion proteins as described above, herein we further optimized the pyridazine compounds to address the unmet medical needs.
SUMMARY OF THE INVENTION
The invention relates to pyridazine derivative compounds (e.g., compounds of the formulae herein), compositions comprising the compounds, and methods of using the compounds and compound compositions. The compounds and compositions comprising them are useful for treating or preventing disease or disease symptoms, including those mediated by or associated with protein kinase modulation activity.
The present invention solves the problems set forth above by providing an isolated compound of Formula I




or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof; wherein:

R1, R2, R3, R4, R5 each are independently H, alkyl or Z1;
R6 is alkyl, OR7, NR7R8, aryl or heteroaryl, saturated or unsaturated heterocyclyl, wherein R6 is optionally substituted by 1-3 groups, independently selected from alkyl, cycloalkyl, heterocyclyl, alkoxy, hydroxyalkyl, and Z1;
R7 and R8 are each independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heterocyclyl, heteroaryl, or R7 and R8 together with nitrogen form a mono- or bi-cyclic heterocyclyl or heteroaryl;
Each Z1 is halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where each m is independently 0-6;
Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each R16 independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl; and
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl;
with the proviso that R6 is not methylpiperazinyl, pyrrolidinyl, methanamino, or piperazinyl.
The compounds of this invention, and compositions comprising them, are useful for treating or lessening the severity of protein kinase modulated diseases, disorders, or symptoms thereof, i.e., disorders effectively treated by inhibitors of protein kinases, e.g., c-met, ron, Ax1, ALK and its fusion proteins such as EML4-ALK and NPM-ALK.
In another aspect, the invention relates to a method of treating a disease or disease symptom in a subject in need thereof including administering to the subject an effective amount of a compound of any formulae herein, or pharmaceutical salt, solvate or hydrate thereof (or composition thereof). The disease or disease symptom can be any of those modulated by a protein kinase (e.g., c-met, ron, Ax1, ALK and its fusion proteins such as EML4-ALK and NPM-ALK). The disease or disease symptom can be, for example, cancer or proliferation disease or disorder (e.g., including those delineated herein).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts apoptosis of H3122 cells treated with either PF-2341066 or the compound of Example 1 herein.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The terms “ameliorate” and “treat” are used interchangeably and both mean decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a disease or disorder delineated herein).
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
By “marker” is meant any alteration that is associated with a disease or disorder. For example, any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
The term “compound” as used herein, is also intended to include salts, prodrugs, and prodrug salts of a compound of formulae herein. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of “prodrug,” “prodrug salt,” “solvate,” “hydrate,” or “polymorph” in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term “compound” is used without recitation of these other forms.
A salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another preferred embodiment, the compound is a pharmaceutically acceptable acid addition salt.
As used herein and unless otherwise indicated, the term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise biohydrolyzable moieties such as amides, esters, carbamates, carbonates, and phosphate analogues. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, “Biotransformation of Drugs”.
As used herein and unless otherwise indicated, the term “biohydrolyzable moiety” means a functional group (e.g., amide, ester, carbamate, carbonate, or phosphate analogue, that either: 1) does not destroy the biological activity of the compound and confers upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is itself biologically inactive but is converted in vivo to a biologically active compound.
A prodrug salt is a compound formed between an acid and a basic group of the prodrug, such as an amino functional group, or a base and an acidic group of the prodrug, such as a carboxyl functional group. In a one embodiment, the prodrug salt is a pharmaceutically acceptable salt.
Particularly favored prodrugs and prodrug salts are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or central nervous system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. See, e.g., Alexander, J. et al. Journal of Medicinal Chemistry 1988, 31, 318-322; Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985; pp 1-92; Bundgaard, H.; Nielsen, N. M. Journal of Medicinal Chemistry 1987, 30, 451-454; Bundgaard, H. A Textbook of Drug Design and Development; Harwood Academic Publ.: Switzerland, 1991; pp 113-191; Digenis, G. A. et al. Handbook of Experimental Pharmacology 1975, 28, 86-112; Friis, G. J.; Bundgaard, H. A Textbook of Drug Design and Development; 2 ed.; Overseas Publ.: Amsterdam, 1996; pp 351-385; Pitman, I. H. Medicinal Research Reviews 1981, 1, 189-214.
The term “pharmaceutically acceptable,” as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A “pharmaceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention.
Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like salts. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.
Suitable bases for forming pharmaceutically acceptable salts with acidic functional groups of prodrugs of this invention include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.
As used herein, the term “hydrate” means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
As used herein, the term “solvate” means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
As used herein, the term “polymorph” means solid crystalline forms of a compound or complex thereof which may be characterized by physical means such as, for instance, X-ray powder diffraction patterns or infrared spectroscopy. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat, light or moisture), compressibility and density (important in formulation and product manufacturing), hygroscopicity, solubility, and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing. For example, one polymorph might be more likely to form solvates or might be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution of particles of it.
The term “substantially free of other stereoisomers” as used herein means less than 25% of other stereoisomers, preferably less than 10% of other stereoisomers, more preferably less than 5% of other stereoisomers and most preferably less than 2% of other stereoisomers, or less than “X”% of other stereoisomers (wherein X is a number between 0 and 100, inclusive) are present. Methods of obtaining or synthesizing diastereomers are well known in the art and may be applied as practicable to final compounds or to starting material or intermediates. Other embodiments are those wherein the compound is an isolated compound. The term “at least X % enantiomerically enriched” as used herein means that at least X % of the compound is a single enantiomeric form, wherein X is a number between 0 and 100, inclusive.
The term “stable compounds”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents).
“Stereoisomer” refers to both enantiomers and diastereomers.
As used herein, the term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.
The terms “alk” or “alkyl” refer to straight or branched chain hydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms. The expression “lower alkyl” refers to alkyl groups of 1 to 4 carbon atoms (inclusive).
The term “arylalkyl” refers to a moiety in which an alkyl hydrogen atom is replaced by an aryl group.
The term “alkenyl” refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one double bond. Where an alkenyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a double bond.
The term “alkoxy” refers to an —O-alkyl radical. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.
The term “alkynyl” refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one triple bond. Where an alkynyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a triple bond.
The term “alkylene” refers to a divalent straight chain bridge of 1 to 5 carbon atoms connected by single bonds (e.g., —(CH2)x—, wherein x is 1 to 5), which may be substituted with 1 to 3 lower alkyl groups.
The term “alkenylene” refers to a straight chain bridge of 2 to 5 carbon atoms having one or two double bonds that is connected by single bonds and may be substituted with 1 to 3 lower alkyl groups. Exemplary alkenylene groups are —CH═CH—CH═CH—, —CH2—CH═CH—, —CH2—CH═CH—CH2—, —C(CH3)2CH═CH— and —CH(C2H5)—CH═CH—.
The term “alkynylene” refers to a straight chain bridge of 2 to 5 carbon atoms that has a triple bond therein, is connected by single bonds, and may be substituted with 1 to 3 lower alkyl groups. Exemplary alkynylene groups are —C≡C—, —CH2—C≡C—, —CH(CH3)C≡C— and —C≡C—CH(C2H5)CH2—.
The terms “cycloalkyl” and “cycloalkenyl” as employed herein includes saturated and partially unsaturated cyclic, respectively, hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbon.
The terms “Ar” or “aryl” refer to aromatic cyclic groups (for example 6 membered monocyclic, 10 membered bicyclic or 14 membered tricyclic ring systems) which contain 6 to 14 carbon atoms. Exemplary aryl groups include phenyl, naphthyl, biphenyl and anthracene.
“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, and, in addition, having a completely conjugated pi-electron system, wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples, without limitation, of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, quinazoline, isoquinoline, purine and carbazole.
The terms “heterocycle”, “heterocyclic” or “heterocyclo” refer to fully saturated or partially unsaturated cyclic groups, for example, 3 to 7 membered monocyclic, 7 to 12 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one ring, wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system.
The term “heterocyclyl” refers to fully saturated or partially unsaturated cyclic groups, for example, 3 to 7 membered monocyclic, 7 to 12 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one ring, wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Each ring of the heterocyclyl group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclyl group may be attached at any heteroatom or carbon atom of the ring or ring system.
The term “substituents” refers to a group “substituted” on any functional group delineated herein, e.g., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)nOH, (CH2)nOR15, (CH2)nC(O)NR15R16, NR15S(O)2R17, where n is independently 0-6 inclusive. Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl. Each R16 is independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl. Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl. Each C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl and C1-C4 alkyl in each R15, R16 and R17 can optionally be substituted with halogen, CN, C1-C4 alkyl, OH, C1-C4 alkoxy, NH2, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, or 1,2-methylenedioxy.
The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
As used herein, the term protein “alterations” are defined as changes from normal physiological conditions. Example alterations include mutation(s), truncation(s), fusion(s) with other protein(s), over-expression, or down-expression.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein. All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
Compounds of the Invention
In one aspect, the present invention provides a compound of Formula I:




or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof; wherein:

R1, R2, R3, R4, R5 each are independently H, alkyl or Z1;
R6 is alkyl, OR7, NR7R8, aryl or heteroaryl, saturated or unsaturated heterocyclyl, wherein R6 is optionally substituted by 1-3 groups, independently selected from alkyl, cycloalkyl, heterocyclyl, alkoxy, hydroxyalkyl, and Z1;
R7 and R8 are each independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heterocyclyl, heteroaryl, or R7 and R8 together with nitrogen form a mono- or bi-cyclic heterocyclyl or heteroaryl;
Each Z1 is halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where each m is independently 0-6;
Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each R16 is independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl; and
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl;
with the proviso that R6 is not methylpiperazinyl, pyrrolidinyl, methanamino, or piperazinyl.
In one embodiment, the invention provides a compound of Formula I wherein R7 and R8 together with nitrogen form a bi-cyclic heterocyclyl or heteroaryl optionally substituted by 1-3 groups of Z1.
In another embodiment, the present invention provides a compound of Formula II:




or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof; wherein:

R1, R2, R3, R4, R5 each are independently H, alkyl or Z1;
R6 is alkyl, OR7, NR7R8, aryl or heteroaryl, saturated or unsaturated heterocyclyl, wherein R6 is optionally substituted by 1-3 groups, independently selected from alkyl, cycloalkyl, heterocyclyl, alkoxy, hydroxyalkyl, and Z1;
R7 and R8 are each independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heterocyclyl, heteroaryl, or R7 and R8 together with nitrogen form a mono- or bi-cyclic heterocyclyl or heteroaryl;
Each Z1 is halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where each m is independently 0-6;
Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each R16 is independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl; and
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl.
In a further embodiment, the invention provides for a compound of formula III:




or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof; wherein n is 0-2; R5, R11 each are independently H, alkyl or Z1; R9 is alkyl or Z1; R10 is H, alkyl, or Z1; and each Z1 is independently halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where each m is independently 0-6;

Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each R16 is independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl; and
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl.
In another embodiment, the invention provides for a compound of formula III above, wherein n is 2; R5, R9, R10, R11, each are independently H, alkyl or Z1;
each Z1 is independently halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, oxo, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where m is independently 0-6 inclusive;
Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each leis independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl;
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl.
Representative compounds of the invention are depicted in Tables 1 and 2. In these examples the stereochemistry at the chiral carbon atoms is independently either RS, R, or S, unless specified. The structures depicted herein, including the Table 1 and 2 structures, may contain certain —NH—, —NH2 (amino) and —OH (hydroxyl) groups where the corresponding hydrogen atom(s) do not explicitly appear; however they are to be read as —NH—, —NH2 or —OH as the case may be. In certain structures, a stick bond is drawn and is meant to depict a methyl group.








TABLE 1











2


 






3


 






4


 






5


 






6


 






7


 






8


 






9

















TABLE 2











1


 






10


 






11


 






12


 






13


 






14


 






15


 






16


 






17


 






18


 






19


 






20









Representative compounds of the invention are listed below:

    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide;
    {5-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide;
    {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-[4-(morpholin-4-ylcarbonyl)phenyl]carboxamide;
    {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-(4-{[4-(2-hydroxyethyl)piperazinyl]carbonyl}phenyl)carboxamide;
    {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-pyrrolidinylpiperidyl)carbonyl]phenyl}carboxamide;
    {6-Amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-ethylpiperazinyl)carbonyl]phenyl}carboxamide;
    {6-Amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-cyclopropylpiperazinyl)carbonyl]phenyl}carboxamide;
    N-(4-{[(2R)-2-(pyrrolidinylmethyl)pyrrolidinyl]carbonyl}phenyl){6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}carboxamide;
    N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}{6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{3-fluoro-4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-(4-{[4-(4-methylpiperazinyl)piperidyl]carbonyl}phenyl)carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-[4-(4-pyridylcarbonyl)phenyl]carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3,6-diazabicyclo[4.3.0]non-3-yl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3-oxopiperazinyl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methyl(1,4-diazaperhydroepinyl))carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-[4-(1,4-diazaperhydroepinylcarbonyl)phenyl]carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3,3-dimethylpiperazinyl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-hydroxypiperidyl)carbonyl]phenyl}carboxamide;
    {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-(4-{N-[2-(diethylamino)ethyl]carbamoyl}phenyl)carboxamide.


The synthesis of compounds of the formulae herein can be readily effected by synthetic chemists of ordinary skill Relevant procedures and intermediates are disclosed, for instance, herein. Each of the patents, patent applications, and publications, whether in traditional journals or available only through the internet, referred to herein, is incorporated in its entirety by reference.
Other approaches to synthesizing compounds of the formulae herein can readily be adapted from references cited herein. Variations of these procedures and their optimization are within the skill of the ordinary practitioner.
The specific approaches and compounds shown above are not intended to be limiting. The chemical structures in the schemes herein depict variables that are hereby defined commensurately with chemical group definitions (moieties, atoms, etc.) of the corresponding position in the compound formulae herein, whether identified by the same variable name (e.g., R1, R2, R, R′, X, etc.) or not. The suitability of a chemical group in a compound structure for use in synthesis of another compound structure is within the knowledge of one of ordinary skill in the art. Additional methods of synthesizing compounds of the formulae herein and their synthetic precursors, including those within routes not explicitly shown in schemes herein, are within the means of chemists of ordinary skill in the art. Methods for optimizing reaction conditions, if necessary minimizing competing by-products, are known in the art. The methods described herein may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compounds herein. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
The methods delineated herein contemplate converting compounds of one formula to compounds of another formula. The process of converting refers to one or more chemical transformations, which can be performed in situ, or with isolation of intermediate compounds. The transformations can include reacting the starting compounds or intermediates with additional reagents using techniques and protocols known in the art, including those in the references cited herein. Intermediates can be used with or without purification (e.g., filtration, distillation, sublimation, crystallization, trituration, solid phase extraction, and chromatography).
Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.
The invention also provides compositions comprising an effective amount of a compound of any of the formulae herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph or prodrug, if applicable, of said compound; and an acceptable carrier. Preferably, a composition of this invention is formulated for pharmaceutical use (“a pharmaceutical composition”), wherein the carrier is a pharmaceutically acceptable carrier. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in amounts typically used in medicaments.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In certain embodiments, the compound of the formulae herein is administered transdermally (e.g., using a transdermal patch). Other formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa. (17th ed. 1985).
Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.
In certain preferred embodiments, the compound is administered orally. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. Methods of formulating such slow or controlled release compositions of pharmaceutically active ingredients, such as those herein and other compounds known in the art, are known in the art and described in several issued US Patents, some of which include, but are not limited to, U.S. Pat. Nos. 4,369,172; and 4,842,866, and references cited therein. Coatings can be used for delivery of compounds to the intestine (see, e.g., U.S. Pat. Nos. 6,638,534, 5,217,720, and 6,569,457, 6,461,631, 6,528,080, 6,800,663, and references cited therein). A useful formulation for the compounds of this invention is the form of enteric pellets of which the enteric layer comprises hydroxypropylmethylcellulose acetate succinate.
In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
The pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and iontophoretic administration are also included in this invention.
Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or central nervous system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. See, e.g., Alexander, J. et al. Journal of Medicinal Chemistry 1988, 31, 318-322; Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985; pp 1-92; Bundgaard, H.; Nielsen, N. M. Journal of Medicinal Chemistry 1987, 30, 451-454; Bundgaard, H. A Textbook of Drug Design and Development; Harwood Academic Publ.: Switzerland, 1991; pp 113-191; Digenis, G. A. et al. Handbook of Experimental Pharmacology 1975, 28, 86-112; Friis, G. J.; Bundgaard, H. A Textbook of Drug Design and Development; 2 ed.; Overseas Publ.: Amsterdam, 1996; pp 351-385; Pitman, I. H. Medicinal Research Reviews 1981, 1, 189-214.
Application of the subject therapeutics may be local, so as to be administered at the site of interest. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access.
According to another embodiment, the invention provides a method of impregnating an implantable drug release device comprising the step of contacting said drug release device with a compound or composition of this invention. Implantable drug release devices include, but are not limited to, biodegradable polymer capsules or bullets, non-degradable, diffusible polymer capsules and biodegradable polymer wafers.
According to another embodiment, the invention provides an implantable medical device coated with a compound or a composition comprising a compound of this invention, such that said compound is therapeutically active.
In another embodiment, a composition of the present invention further comprises a second therapeutic agent. The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered alone or with a compound of any of the formulae herein. Drugs that could be usefully combined with these compounds include other kinase inhibitors and/or other chemotherapeutic agents for the treatment of the diseases and disorders discussed above.
Such agents are described in detail in the art. Preferably, the second therapeutic agent is an agent useful in the treatment or prevention of a disease or condition selected from cancer.
Even more preferably the second therapeutic agent co-formulated with a compound of this invention is an agent useful in the treatment of c-met, ron, Ax1, or ALK and its fusion proteins such as EML4-ALK and NPM-ALK mediated disease/disorders.
In another embodiment, the invention provides separate dosage forms of a compound of this invention and a second therapeutic agent that are associated with one another. The term “associated with one another” as used herein means that the separate dosage forms are packaged together or otherwise attached to one another such that it is readily apparent that the separate dosage forms are intended to be sold and administered together (within less than 24 hours of one another, consecutively or simultaneously).
In the pharmaceutical compositions of the invention, the compound of the present invention is present in an effective amount. As used herein, the term “effective amount” refers to an amount which, when administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration or progression of the disorder being treated, prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a compound of this invention can range from about 0.001 mg/kg to about 500 mg/kg, more preferably 0.01 mg/kg to about 50 mg/kg, more preferably 0.1 mg/kg to about 2.5 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, the severity of the disease, the route of administration, the sex, age and general health condition of the patient, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.
For pharmaceutical compositions that comprise a second therapeutic agent, an effective amount of the second therapeutic agent is between about 20% and 100% of the dosage normally utilized in a monotherapy regime using just that agent. Preferably, an effective amount is between about 70% and 100% of the normal monotherapeutic dose. The normal monotherapeutic dosages of these second therapeutic agents are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which references are entirely incorporated herein by reference.
It is expected that some of the second therapeutic agents referenced above will act synergistically with the compounds of this invention. When this occurs, its will allow the effective dosage of the second therapeutic agent and/or the compound of this invention to be reduced from that required in a monotherapy. This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention, synergistic improvements in efficacy, improved ease of administration or use and/or reduced overall expense of compound preparation or formulation.
Methods of Treatment
According to another embodiment, the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof (e.g., those delineated herein) comprising the step of administering to said subject an effective amount of a compound or a composition of this invention. Such diseases are well known in the art and are also disclosed herein.
In one aspect, the method of treating involves treatment of a disorder that is mediated by the protein kinase, e.g., c-met, ron. In another aspect, the method of treating involves treatment of a disorder that is mediated by the c-met, ron, Ax1, or ALK or its fusion proteins such as EML4-ALK and NPM-ALK kinases. In certain embodiments, the disease is mediated by the c-met, ron, Ax1, or ALK or its fusion proteins such as EML4-ALK and NPM-ALK kinases, or the alterations of one or more of the c-met, ron, Ax1, and ALK kinases.
In another aspect, the invention provides a method of treating a disease in a subject comprising administering to the subject a compound of any of the formulae herein.
In another aspect, invention provides a method of treating a disease in a subject comprising administering to the subject a composition comprising a compound of any of the formulae herein.
In certain embodiments, the disease is mediated by the c-met or ron kinases.
In another embodiment, the disease is cancer or a proliferation disease.
In yet another embodiment, the disease is lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovaries, stomach, skin, and bone cancers, gastric, breast, pancreatic cancer, glioma, and hepatocellular carcinoma, papillary renal carcinoma, or head and neck squamous cell carcinoma.
In yet another embodiment, the disease is non-small cell lung cancer (NSCLC) resistant to the treatment by PF-2341066.
In yet another embodiment, the disease is non-small cell lung cancer (NSCLC) with brain metastasis.
In yet another embodiment, the disease is a neurological disease, psychiatric disease such as schizophrenia, depression, and substance (e.g. cocaine, tobacco, or alcohol) addiction or abuse, or related diseases (e.g. obesity, diabetes, cardiovascular diseases).
In a one embodiment, the method of this invention is used to treat a subject suffering from or susceptible to a disease or condition. Such diseases, disorders or symptoms thereof include, for example, those modulated by a protein kinase (e.g., c-met, ron, Ax1, ALK and its fusion proteins such as EML4-ALK and NPM-ALK). The disease or disease symptom can be, for example, cancer or proliferation disease or disorder. The disease or disease symptom can be lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovaries, stomach, skin, and bone cancers, gastric, breast, pancreatic cancer, glioma, and hepatocellular carcinoma, papillary renal carcinoma, or head and neck squamous cell carcinoma. Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
In yet another embodiment, the compounds of the formulae herein (and compositions thereof) can be used to treat subjects having a disease or disorder who have been treated with and developed resistance to other therapeutic agents (e.g., anticancer agent, neurological disease agent, psychiatric disease agent, cardiovascular disease agent, antiobesity or diabetes agent. In one aspect, the methods herein include those where a subject resistant to treatment with PF-2341066 (or identified as having developed resistance to PF-2341066) is administered a compound of the formulae herein (or composition thereof). In other aspects, the subject thereby responds to such treatment such that the disorder is modulated or ameliorated relative to prior to treatment with the compound of the formulae herein.
In another embodiment, the invention provides a method of modulating the activity of a protein kinase, (e.g. protein tyrosine kinase, kinases listed herein) in a cell comprising contacting a cell with one or more compounds of any of the formulae herein.
In another embodiment, the above method of treatment comprises the further step of co-administering to said patient one or more second therapeutic agents. The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for indications herein. Additional therapeutic agents include but are not limited to agents for treatment of diseases, disorders or symptoms thereof including for example, anticancer agents, antiproliferative agents, antineoplastic agents, antitumor agents, antimetabolite-type/thymidilate synthase inhibitor antineoplastic agents, alkylating-type antineoplastic agents, antibiotic-type antineoplastic agents, or, any other agent typically administered as a primary or adjuvant agent in cancer treatment protocols (e.g., antinausea, antianemia, etc.), including for example, vinblastine sulfate, vincristine, vindesine, vinestramide, vinorelbine, vintriptol, vinzolidine, tamoxifen, toremifen, raloxifene, droloxifene, iodoxyfene, megestrol acetate, anastrozole, letrazole, borazole, exemestane, flutamide, nilutamide, bicalutamide, cyproterone acetate, goserelin acetate, luprolide, finasteride, herceptin, methotrexate, 5-fluorouracil, cytosine arabinoside, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, cisplatin, carboplatin, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotephan, vincristine, taxol, taxotere, etoposide, teniposide, amsacrine, irinotecan, topotecan, an epothilone, Iressa, Avastin, OSI-774, angiogenesis inhibitors, EGFR inhibitors, MEK inhibitors, VEGFR inhibitors, CDK inhibitors, Her1 and Her2 inhibitors and monoclonal antibodies.
The term “co-administered” as used herein means that the second therapeutic agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms. Alternatively, the additional agent may be administered prior to, consecutively with, or following the administration of a compound of this invention. In such combination therapy treatment, both the compounds of this invention and the second therapeutic agent(s) are administered by conventional methods. The administration of a composition of this invention comprising both a compound of the invention and a second therapeutic agent to a subject does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent or any compound of this invention to said subject at another time during a course of treatment.
Effective amounts of these second therapeutic agents are well known to those skilled in the art and guidance for dosing may be found in patents and published patent applications referenced herein, as well as in Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), and other medical texts. However, it is well within the skilled artisan's purview to determine the second therapeutic agent's optimal effective-amount range.
In one embodiment of the invention where a second therapeutic agent is administered to a subject, the effective amount of the compound of this invention is less than its effective amount would be where the second therapeutic agent is not administered. In another embodiment, the effective amount of the second therapeutic agent is less than its effective amount would be where the compound of this invention is not administered. In this way, undesired side effects associated with high doses of either agent may be minimized. Other potential advantages (including without limitation improved dosing regimens and/or reduced drug cost) will be apparent to those of skill in the art.
In yet another aspect, the invention provides the use of a compound of any of the formulae herein alone or together with one or more of the above-described second therapeutic agents in the manufacture of a medicament, either as a single composition or as separate dosage forms, for treatment or prevention in a subject of a disease, disorder or symptom set forth above. Another aspect of the invention is a compound of the formulae herein for use in the treatment or prevention in a subject of a disease, disorder or symptom thereof delineated herein.
In other aspects, the methods herein include those further comprising monitoring subject response to the treatment administrations. Such monitoring may include periodic sampling of subject tissue, fluids, specimens, cells, proteins, chemical markers, genetic materials, etc. as markers or indicators of the treatment regimen. In other methods, the subject is prescreened or identified as in need of such treatment by assessment for a relevant marker or indicator of suitability for such treatment.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target or cell type delineated herein modulated by a compound herein) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof delineated herein, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
In certain method embodiments, a level of Marker or Marker activity in a subject is determined at least once. Comparison of Marker levels, e.g., to another measurement of Marker level obtained previously or subsequently from the same patient, another patient, or a normal subject, may be useful in determining whether therapy according to the invention is having the desired effect, and thereby permitting adjustment of dosage levels as appropriate. Determination of Marker levels may be performed using any suitable sampling/expression assay method known in the art or described herein. Preferably, a tissue or fluid sample is first removed from a subject. Examples of suitable samples include blood, urine, tissue, mouth or cheek cells, and hair samples containing roots. Other suitable samples would be known to the person skilled in the art. Determination of protein levels and/or mRNA levels (e.g., Marker levels) in the sample can be performed using any suitable technique known in the art, including, but not limited to, enzyme immunoassay, ELISA, radiolabelling/assay techniques, blotting/chemiluminescence methods, real-time PCR, and the like.
The present invention also provides kits for use to treat diseases, disorders, or symptoms thereof, including those delineated herein. These kits comprise: a) a pharmaceutical composition comprising a compound of any of the formula herein or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof, wherein said pharmaceutical composition is in a container; and b) instructions describing a method of using the pharmaceutical composition to treat the disease, disorder, or symptoms thereof, including those delineated herein.
The container may be any vessel or other sealed or sealable apparatus that can hold said pharmaceutical composition. Examples include bottles, divided or multi-chambered holders bottles, wherein each division or chamber comprises a single dose of said composition, a divided foil packet wherein each division comprises a single dose of said composition, or a dispenser that dispenses single doses of said composition. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a “refill” of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle, which is in turn contained within a box. Preferably, the container is a blister pack.
The kit may additionally comprising information and/or instructions for the physician, pharmacist or subject. Such memory aids include numbers printed on each chamber or division containing a dosage that corresponds with the days of the regimen which the tablets or capsules so specified should be ingested, or days of the week printed on each chamber or division, or a card which contains the same type of information.
The compounds delineated herein can be assessed for their biological activity using protocols known in the art, including for example, those delineated herein. Certain of the compounds herein demonstrate unexpectedly superior attributes (e.g., inhibition of P450, metabolic stability, Met, Ron, etc.; pharmacokinetic properties, etc.) making them superior candidates as potential therapeutic agents.
All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, technical data sheets, internet web sites, databases, patents, patent applications, and patent publications.
EXAMPLES
Synthesis of 5-[(2,6-dichloro-3-fluorophenyl)ethoxy]-6-{(tert-butoxy)-N-[(tert-butyl)oxycarbonyl]carbonylamino}pyridazine-3-carboxylic acid (A)




Step 1: A suspension of A1 (400 g, 2.68 mol) in 25% ammonium hydroxide (3 L) was heated at 130° C. for 12 h in a sealed tube. After the tube was cooled to 0° C., the mixture was filtered. The resulting solid was washed with water for several times and dried under vacuo to provide A2 (284 g, 82%).
Step 2: To a solution of A2 (284 g, 2.19 mol) in methanol (3.5 L) was added NaHCO3 (368.4 g, 4.38 mol) at room temperature, followed by bromine (350 g, 2.19 mol) dropwise. After the addition was complete, the mixture was stirred for 20 h, then filtered and washed by methanol for several times. The filtrate was concentrated and the residue was dissolved in water (2 L) and extracted with ethyl acetate (2 L×3). The combined organic phase was washed with 10% sodium thiosulfate aq. (2 L), sat. sodium bicarbonate aq. (2 L) and brine (2 L), dried over anhydrous magnesium sulfate and evaporated. The residue was purified by column chromatography (EA:PE=2:1) to provide A3 (159.8 g, 35%).
Step 3: To a solution of A4 (150 g, 0.72 mol) in methanol (800 mL) cooled to 0° C., was added NaBH4 (66 g, 1.74 mol) in portions. The resulting mixture was stirred at r.t. for about 1 h and evaporated. Water (1 L) was added to the residue at 0° C., followed by 3N HCl until pH=6. The resulting mixture was extracted with ethyl acetate (400 mL×4). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give A5 (148.6 g, 98%).
Step 4: To a solution of A5 (147.6 g, 0.71 mol) in THF (3 L) was added 60% NaH (28.4 g, 0.71 mol) at 0° C., the resulting mixture was stirred at that temperature for 30 min, was then added A3 (147 g, 0.71 mmol) quickly. The resulting mixture was heated under reflux overnight and evaporated. The residue was purified by column chromatography (PE:EA=4:1) to provide the advanced intermediate A6 (89.3 g, 37.6%).
Step 5: To a solution of A6 (97 g, 0.288 mol) in DMF (1 L) was added Boc2O (113 g, 0.519 mol) and DMAP (7 g, 58 mmol). The mixture was stirred at r.t. overnight and evaporated. The residue was purified by column chromatography (PE:EA=10:1) to afford A7 (136 g, 88%).
Step 6: Sodium acetate (41 g, 0.50 mol) was added to a solution of A7 (136 g, 0.25 mol) in ethanol/DMF [(5:1) (1200 mL)]. The mixture was degassed, then added Pd(dppf)Cl2.CH2Cl2 (18.63 g, 22.5 mmol). The resulting mixture was heated at CO atmosphere at 90° C. for 1.5 h, then evaporated. The residue was purified by column chromatography (PE:EA=1:4) to afford A8 (141 g, 97%).
Step 7: To the solution of A8 (141 g, 0.246 mol) in THF (650 mL) was added 1N LiOH aq. (390 mL). The resulting mixture was stirred at r.t. over weekend, then acidified by 2N HCl to pH=5, extracted with ethyl acetate (300 mL×5). The combined organic phase was dried over Na2SO4, filtrated and concentrated to give A (134 g, 99%).
Synthesis of 6-[bis(tert-butoxycarbonyl)amino]-5-[(1R)-1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]pyridazine-3-carboxylic acid (B)




Step 1: To a solution of A5 (219 g, 1.05 mol) in 1,2-dichloroethane (3500 mL) was added Boc-D-Pro (141 g, 0.65 mol) followed by EDCI (163 g, 0.85 mol) and DMAP (21.57 g, 0.18 mol) at 0° C. The resulting mixture was stirred at r.t. overnight and then water (3500 mL) was added and separated, the water phase was extracted with DCM(1500 mL×3), dried over MgSO4, concentrated and purified by column chromatography to (PE:EA=30:1) to give B1 (55.96 g, yield: 51.1%)
Step 2: To a solution of B1 (59.96 g, 268 mmol) in THF (1200 mL) was added 60% NaH (10.71 g, 268 mmol) at 0° C., the resulting mixture was stirred at that temperature for 30 min, was then added A3 (55.82 g, 268 mmol) quickly. The resulting mixture was heated under reflux overnight and evaporated. The residue was purified by column chromatography (PE:EA=4:1) to provide the advanced intermediate B2 (33.95 g, 37.7%). 1H-NMR (300 MHz, CDCl3): δ=1.87 (d, 3H), 5.08 (s, 2H), 6.03-6.09 (m, 1H), 6.42 (s, 1H), 7.14 (t, 1H), 7.35 (dd, 1H). LC-MS [M+H]+: 336.0.
Step 3: To a solution of B2 (33.95 g, 101 mmol) in DMF (400 mL) was added BOC2O (39.59 g, 182 mmol) and DMAP (2.46 g, 20.2 mmol). The mixture was stirred at r.t. overnight and evaporated. The residue was purified by column chromatography (PE:EA=10:1) and the residue was treated with PE:EA=10:1 to afford B3 (46.9 g, 86.7%).
Step 4: Sodium acetate (14.34 g, 175 mmol) was added to a solution of B3 (46.9 g, 87.4 mmol) in ethanol/DMF [(5:1) (480 mL)]. The mixture was degassed, then added Pd(dppf)Cl2.CH2Cl2 (7.14 g, 8.74 mmol). The resulting mixture was heated at CO atmosphere at 90° C. overnight, then evaporated. The residue was purified by column chromatography (PE:EA=4:1) to afford B4 (47.1 g, 94.0%). 1H-NMR (300 MHz, CDCl3): δ=1.38 (s, 18H), 1.46 (t, 3H), 1.88 (d, 3H), 4.45-4.53 (m, 2H), 6.18 (q, 1H), 7.13 (t, 1H), 7.34 (dd, 1H), 7.57 (s, 1H). LC-MS [M+H]+: 574.0.
Step 5: To the solution of B4 (47.1 g, 82.1 mmol) in THF (400 mL) was added 1N LiOH aq. (98.5 mL). The resulting mixture was stirred at r.t. over weekend, then acidified by 2N HCl to pH=5, extracted with ethyl acetate (400 mL×3). The combined organic phase was dried over Na2SO4, filtrated and concentrated to give B (45.94 g,  ̃100%).
Synthesis of 6-[bis(tert-butoxycarbonyl)amino]-5-[(1S)-1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]pyridazine-3-carboxylic acid (C)




Step 1: To a solution of A5 (41.8 g, 200 mmol) in 1,2-dichloroethane (800 mL) was added Boc-L-Pro (26.9 g, 125 mmol) followed by EDCI (31.1 g, 163 mmol) and DMAP (4.12 g, 33.8 mmol) at 0° C. The resulting mixture was stirred at r.t. overnight and then water (350 mL) was added and separated, the water phase was extracted with DCM(150 mL×3), dried over MgSO4, concentrated and purified by column chromatography to (PE:EA=30:1) to give C1 (13.72 g, yield: 65.6%).
Step 2: The procedure from C1 to C was similar to that of B1 to B (9.46 g, yield: 26.4% from C1).
Example 1
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide




Step 1: The mixture of B (10.92 g, 20.0 mmol)), HATU (9.12 g, 24.0 mmol) and DIEA (3.87 g, 30.0 mmol) in DMF (100 mL) was stirred at room temperature for 0.5 h, then was added 1a (5.26 g, 24.0 mmol). The resulting mixture was stirred at room temperature for 0.5 h and evaporated. The residue was purified by column chromatography (EA:MeOH=5:1) to provide 1b (12.43 g, 83.2%).
Step 2: 1b (12.43 g, 16.7 mmol) was dissolved in a mixture of DCM (90 mL) and TFA (30 mL), stirred at r.t. for 2 hours and evaporated. The residue was adjusted by sat. Na2CO3 to pH=8 and extracted with DCM (150 mL×5). The combined organic phase was dried over MgSO4 and concentrated. The residue was triturated with methanol and filtered, then the solid was dissolved in DCM and a solution of HCl in Et2O was added, the mixture was stirred at r.t. overnight, then concentrated and dried over oil pump to afford 1 (8.31 g, 80.5%). 1H-NMR (300 MHz, DMSO-d6): δ=1.84 (d, 3H), 2.76 (d, 3H), 3.02-3.10 (m, 2H), 3.37-3.53 (m, 5H), 3.40-4.26 (m, 1H), 6.27 (q, 1H), 7.11 (s, 1H), 7.42-7.51 (m, 3H), 7.58-7.62 (m, 1H), 7.86-7.88 (m, 2H). LC-MS [M+H]+:547.2.
Example 2
Synthesis of {5-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide




The synthesis was similar to that of Example 1 (1.30 g). 1H-NMR (300 MHz, DMSO-d6): δ=1.86 (d, 3H), 2.48-2.52 (m, 2H), 2.76 (d, 3H), 3.02-3.12 (m, 2H), 3.33-3.47 (m, 4H) 6.31 (q, 1H), 7.12 (s, 1H), 7.43-7.66 (m, 4H), 7.91 (d, 2H), 8.22 (brs, 2H), 10.81 (s, 1H), 11.22 (brs, 1H). LC-MS [M+H]+: 547.1
Example 3
Synthesis of {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-[4-(morpholin-4-ylcarbonyl)phenyl]carboxamide




The synthesis was similar to that of Example 1 (60 mg, 87% for the final step). 1H-NMR (300 MHz, CDCl3): δ=1.90 (d, 3H), 3.58-3.72 (m, 8H), 5.40 (s, 2H), 6.25 (q, 1H), 7.07-7.12 (m, 1H), 7.32-7.37 (m, 1H), 7.40-7.45 (m, 3H), 7.73-7.76 (m, 2H), 9.90 (s, 1H). LC-MS [M+H]+: 534.0.
Example 4
Synthesis of {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-(4-{[4-(2-hydroxyethyl)piperazinyl]carbonyl}phenyl)carboxamide




Step 1: A mixture of 4a (0.5 g, 2.7 mmol), 4b (0.34 g, 2.7 mmol) and K2CO3 (0.74 g, 5.36 mmol) in CH3CN (25 mL) was heated under reflux for 2.5 h. The solid was filtered off and the filtrate was evaporated under vacuum. The residue was purified by column chromatography to provide 4c (0.4 g, 65%).
Step 2: 4c (400 mg, 1.74 mmol) was dissolved in a mixture of DCM (5 mL) and TFA (1.5 mL). The mixture was stirred at r.t. for 2 hours and evaporated to give 4d.
Step 3: To a solution of 4e (500 mg, 3 mmol), HATU (1.71 g, 4.5 mmol) and DIEA (1.16 g, 9 mmol) in DMF was added 4d (320 mg, 4.5 mmol). The mixture was stirred overnight at rt. After evaporated, the residue was purified by column chromatography (EA:MeOH=4:1) to afford 4f (0.52 g, 79%).
Step 4: To a solution of 4f (370 mg) in MeOH was added 10% Pd/C (200 mg). The mixture was hydrogenated at rt for 1 h. The reaction mixture was filtered and the filtrate was evaporated to give 4 g (276 mg, 86.5%).
Step 5: The procedure from 4g to 4 was similar to that in Example 1 (45 mg, 25% from 4d). 1H-NMR (300 MHz, CDCl3): δ=1.90 (d, 3H), 2.85-2.89 (m, 6H), 3.82 (t, 6H), 5.53 (s, 2H), 6.25 (q, 1H), 7.07-7.12 (m, 1H), 7.32-7.37 (m, 1H), 7.40-7.45 (m, 3H), 7.75-7.77 (m, 2H), 9.90 (s, 1H). LC-MS [M+H]+: 577.0.
Example 5
Synthesis of {6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-pyrrolidinylpiperidyl)carbonyl]phenyl}carboxamide




The synthesis was similar to that of Example 1 (67 mg, 68% for the final step). 1H-NMR (300 MHz, CDCl3): δ=1.50-1.58 (m, 3H), 1.77-2.00 (m, 7H), 1.90 (d, 3H), 2.22-2.29 (m, 1H), 2.55-2.59 (m, 4H), 2.94-3.02 (m, 2H), 5.38 (s, 2H), 6.25 (q, 1H), 7.06-7.12 (m, 1H), 7.32-7.41 (m, 4H), 7.71-7.74 (m, 2H), 9.87 (s, 1H). LC-MS [M+H]+: 601.0.
Example 6
Synthesis of {6-Amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-ethylpiperazinyl)carbonyl]phenyl}carboxamide




The synthesis was similar to that of Example 1 which provided 6 (305 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.25 (t, 3H), 1.87 (d, 3H), 2.48-2.51 (m, 2H), 2.98-3.15 (m, 4H), 3.37-3.47 (m, 4H), 6.27 (q, 1H), 7.10 (s, 1H), 7.43-7.64 (m, 4H), 7.90 (d, 3H), 10.76 (s, 1H), 11.07 (brs, 1H). LC-MS [M+H]+: 560.8.
Example 7
Synthesis of {6-Amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}-N-{4-[(4-cyclopropylpiperazinyl)carbonyl]phenyl}carboxamide




The synthesis was similar to that of Example 1 which provided 7 (280 mg). 1H-NMR (300 MHz, DMSO-d6): δ=0.79 (d, 2H), 1.17 (t, 3H), 1.86 (d, 3H), 2.78-2.86 (m, 1H), 3.27-3.55 (m, 6H), 4.03-4.30 (m, 1H), 6.29 (q, 1H), 7.13 (s, 1H), 7.44-7.54 (m, 2H), 7.60-7.65 (m, 1H), 7.90 (d, 2H), 8.22 (brs, 1H), 10.79 (s, 1H), 11.50 (brs, 1H). LC-MS [M+H]+:572.7.
Example 8
Synthesis of N-(4-{[(2R)-2-(pyrrolidinylmethyl)pyrrolidinyl]carbonyl}phenyl){6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}carboxamide




The synthesis was similar to that of Example 1 which provided 8 (547 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.69-1.79 (m, 1H), 1.85-1.98 (m, 9H), 2.09-2.16 (m, 1H), 3.04-3.22 (m, 2H), 3.30-3.47 (m, 3H), 3.54-3.74 (m, 3H), 4.42-4.50 (m, 1H), 6.30 (q, 1H), 7.13 (s, 1H), 7.48-7.65 (m, 4H), 7.87-7.90 (m, 2H), 8.40 (brs, 2H), 10.45 (brs, 1H), 10.79 (s, 1H). LC-MS [M+H]+: 600.7.
Example 9
Synthesis of N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}{6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}carboxamide




Step 1: A solution of 4e (732 mg, 4.38 mmol), HATU (2.50 g, 6.57 mmol) and DIEA (1.13 g, 8.8 mmol) in DMF (20 mL) was stirred at r.t. for 0.5 hour, then added dropwise to a solution of 9a (1.0 g, 8.8 mmol) in DMF (50 mL) under 0° C. for 1 hour. After the reaction was complete, the mixture was evaporated and the residue was dissolved in DCM (50 mL) and washed with sat. Na2CO3. The organic layer was separated, dried over anhydrous MgSO4 and evaporated to give 9b (808 mg, 70%).
Step 2: The procedure from 9b to 9c was similar to that of 4f to 4g which provided 9c which was used for next step without purification.
Step 3: The synthesis of 9 from 9c was similar to that of Example 1 which provided 9 (125 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.25 (d, 6H), 1.86 (d, 3H), 2.95-3.16 (m, 2H), 3.46-3.60 (m, 4H), 6.30 (q, 1H), 7.13 (s, 1H), 7.48-7.65 (m, 4H), 7.87 (s, 2H). LC-MS [M+H]+: 560.8.
Example 10
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{3-fluoro-4-[(4-methylpiperazinyl)carbonyl]phenyl}carboxamide




The synthesis was similar to that of Example 1 which provided 10 (265 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.82 (d, 3H), 2.79 (d, 3H), 2.92-3.09 (m, 2H), 3.16-3.49 (m, 4H), 3.60-3.65 (m, 1H), 4.48-4.59 (m, 1H), 6.22 (q, 1H), 7.05 (s, 1H), 7.37-7.50 (m, 2H), 7.56-7.61 (m, 1H), 7.74-7.87 (m, 2H). LC-MS [M+H]+:565.2.
Example 11
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-(4-{[4-(4-methylpiperazinyl)piperidyl]carbonyl}phenyl)carboxamide




The synthesis was similar to that of Example 1 which provided 11 (425 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.63-1.71 (m, 2H), 1.82 (d, 3H), 2.03-2.25 (m, 2H), 2.69-3.08 (m, 6H), 3.46-3.82 (m, 8H), 4.40-4.60 (m, 1H), 6.24 (q, 1H), 7.10 (s, 1H), 7.36-7.59 (m, 4H), 7.81 (d, 2H). LC-MS [M+H]+:630.3.
Example 12
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-[4-(4-pyridylcarbonyl)phenyl]carboxamide




Step 1: The mixture of B (567 mg, 1.04 mmol), HATU (475 mg, 1.25 mmol) and DIEA (216 mg, 2.08 mmol) in DMF (15 mL) was stirred at room temperature for 0.5 h, then was added 12a (247 mg, 1.25 mmol). The resulting mixture was stirred at room temperature for 0.5 h and evaporated. The residue was purified by column chromatography (EA:MeOH=5:1) to provide 12b (260 mg, 34.4%).
Step 2: The solution of 12b (260 mg, 0.36 mmol) in a mixture of DCM (10 mL) and TFA (3 mL) was stirred at r.t. for 2 hours and evaporated. The residue was adjusted by sat. Na2CO3 to pH=8 and extracted with DCM (10 mL×5). The combined organic phase was dried over MgSO4 and concentrated. The residue was triturated with methanol and filtered to afford 12 (120 mg, 63.7%). 1H-NMR (300 MHz, DMSO-d6): δ=1.82 (d, 3H), 3.41 (s, 3H), 6.21 (q, 1H), 7.02 (s, 2H), 7.47 (t, 1H), 7.58-7.63 (m, 3H), 7.77 (d, 2H), 8.09 (d, 2H), 8.80 (d, 2H), 10.95 (s, 1H). LC-MS [M+H]+: 526.1
Example 13
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3,6-diazabicyclo[4.3.0]non-3-yl)carbonyl]phenyl}carboxamide




The procedure was similar to Example 1 (35 mg). 1H-NMR (300 MHz, DMSO-d6): δ=1.86 (d, 3H), 1.65-2.44 (m, 5H), 3.21-3.53 (m, 8H), 5.00 (brs, 2H) 6.29 (q, 1H), 7.11 (s, 1H), 7.43-7.54 (m, 3H), 7.60-7.65 (m, 1H), 7.90 (d, 2H), 8.27 (brs, 2H), 10.81 (s, 1H) 11.64 (d, 1H). LC-MS [M+H]+: 573.2.
Example 14
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3-oxopiperazinyl)carbonyl]phenyl}carboxamide




The procedure was similar to Example 1 (23.3 mg). 1H-NMR (300 MHz, CDCl3): δ=1.86 (d, 3H), 3.47 (t, 2H), 3.79-3.88 (m, 2H), 4.26 (s, 2H), 5.55 (brs, 2H), 6.25 (q, 1H), 6.56 (q, 1H), 7.09 (t, 1H), 7.32-7.47 (m, 4H), 7.78 (d, 2H), 9.92 (s, 1H). LC-MS [M+H]+: 546.1.
Example 15
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-methyl(1,4-diazaperhydroepinyl))carbonyl]phenyl}carboxamide




Step 1: The mixture of B (1.09 g, 2.0 mmol), HATU (0.99 g, 2.6 mmol) and DIEA (516 mg, 4.0 mmol) in DMF (15 mL) was stirred at room temperature for 0.5 h, then was added 15a (939 mg, 2.6 mmol). The resulting mixture was stirred at room temperature for 0.5 h and evaporated. The residue was purified by column chromatography (PE/EA=10:1) to provide 15b (1.05 g, 77.6%).
Step 2: To a solution of 15b (1.05 g, 1.55 mmol) in THF was added 1N LiOH (1.86 mL, 1.86 mmol.). After stirred at room temperature for 5 h, the reaction mixture was acidified by 1N HCl until Ph=5 ̃6. The organic solvent was evaporated and the residue was extracted with DCM (30 mL×3). The combined the organic phase was dried over anhydrous MgSO4, filtrated and concentrated to provide 15c (0.95 g, 92.2%).
The procedure from 15c to 15 was similar to that of 12a to 12 (30 mg, yield: 35.7% from 15c). 1H-NMR (300 MHz, CDCl3): δ=1.90 (d, 3H), 2.02-2.08 (m, 2H), 2.38-2.39 (m, 1H), 2.49-2.55 (m, 2H), 2.58-2.62 (m, 1H), 2.67-2.76 (m, 3H), 2.91-2.93 (m, 1H), 3.51-3.59 (m, 2H), 3.77-3.87 (m, 2H), 5.51 (s, 2H), 6.23 (q, 1H), 7.10 (t, 1H), 7.32-7.45 (m, 4H), 7.75 (d, 2H), 9.89 (brs, 1H). LC-MS [M+H]+: 561.2
Example 16
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-[4-(1,4-diazaperhydroepinylcarbonyl)phenyl]carboxamide




The procedure from 15c to 16 was similar to that of 12a to 12 (86 mg, yield: 93.9% from 15c). 1H-NMR (300 MHz, DMSO-d6): δ=1.83 (d, 3H), 1.90-2.07 (m, 2H), 3.13-3.24 (m, 4H), 3.36-3.46 (m, 1H), 3.63-3.66 (m, 1H), 3.78-3.82 (m, 1H), 4.66 (brs, 3H), 6.28 (q, 1H), 7.11 (s, 1H), 7.41-7.54 (m, 3H), 7.60-7.64 (m, 1H), 7.87 (d, 2H), 8.18 (brs, 1H), 9.35 (brs, 2H), 10.75 (s, 1H). LC-MS [M+H]+: 547.1
Example 17
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3,3-dimethylpiperazinyl)carbonyl]phenyl}carboxamide




The procedure from 15c to 17 was similar to that of 12a to 12 to give 17 (37 mg, yield: 28.7% from 15c). 1H-NMR (300 MHz, DMSO-d6): δ=1.41-1.70 (m, 6H), 1.89 (d, 3H), 3.17-3.21 (m, 2H), 3.68-4.01 (m, 4H), 5.43 (s, 2H), 6.26 (q, 1H), 7.10 (t, 1H), 7.32-7.43 (m, 4H), 7.78 (d, 2H), 9.93 (s, 1H). LC-MS [M+H]+: 561.2.
Example 18
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}carboxamide




The procedure from 15c to 18 was similar to that of 12a to 12 to give 18 (31 mg, 24.6% from 15c). 1H-NMR (300 MHz, CDCl3): δ=1.25-1.43 (m, 6H), 1.91 (d, 3H), 3.15-3.48 (m, 4H), 3.66-3.89 (m, 0.5H), 4.55-4.78 (m, 0.5H), 5.49 (s, 2H), 6.26 (q, 1H), 7.10 (t, 1H), 7.33-7.44 (m, 4H), 7.78 (d, 2H), 9.93 (s, 1H). LC-MS [M+H]+: 561.2.
Example 19
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(4-hydroxypiperidyl)carbonyl]phenyl}carboxamide




The procedure from 15c to 19 was similar to that of 12a to 12 to give 19 (30.3 mg, yield: 30.3% from 15c). 1H-NMR (300 MHz, DMSO-d6): δ=1.53-1.59 (m, 2H), 1.88-1.94 (m, 5H), 3.21-3.33 (m, 2H), 3.66-3.75 (m, 1H), 3.93-3.99 (m, 1H), 5.89 (brs, 2H) 6.25 (q, 1H), 7.06-7.09 (m, 1H), 7.32-7.41 (m, 4H), 7.73 (d, 2H), 9.80 (s, 1H). LC-MS [M+H]+: 548.1
Example 20
Synthesis of {5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-(4-{N-[2-(diethylamino)ethyl]carbamoyl}phenyl)carboxamide




The procedure from 15c to 20 was similar to that of 12a to 12 to give 20 (35.4 mg, yield: 35.5% from 15c). 1H-NMR (300 MHz, CD3OD): δ=1.25-1.39 (m, 6H), 1.89 (d, 3H), 3.26-3.38 (m, 6H), 3.74 (t, 2H) 6.26 (q, 1H), 7.18 (s, 1H), 7.22-7.27 (m, 1H), 7.43-7.47 (m, 1H), 7.82-7.90 (m, 4H). LC-MS [M+H]+: 563.2.
Biological Data
Example 21
Met, ALK, Ax1 Biochemical Assays
Kinase Assays.
Assays were performed as described in Fabian et al. (2005) Nature Biotechnology, vol. 23, p. 329 and in Karaman et al. (2008) Nature Biotechnology, vol. 26, p. 127.
For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection 0.1) and incubated with shaking at 32° C. until lysis ( ̃90 minutes). The lysates were centrifuged (6,000×g) and filtered (0.2 mm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 mM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Most compounds in this invention are very potent ALK inhibitors with IC50 values of <10 nM, and some at <1 nM. Some compounds also showed potent inhibition of c-Met and Ax1 with IC50 of <20 nM. In contrast, similar compounds without the carbonylphenyl substituted carboxamide, e.g a (pyridine substituted carboxamide), b (methoxyphenyl substituted carboxamide) or c (morpholinophenyl substituted carboximide), showed much weaker ALK inhibitory activities (IC50 being >10 nM), though they are still potent c-Met inhibitors (IC50 being <20 nM).




It was also discovered that the R-enantiomer is the active isomer against ALK. While Example 1 showed IC50 of less than 1 nM, its S-isomer (Example 2) was inactive at up to 50 nM against ALK in this assay.
Some compounds in this invention (e.g. Examples 1, 13, 15, 16, 17, 18) were more potent ALK inhibitors than PF-2341066, and thus they have the potential of overcoming resistance to PF-2341066 (Table 3).







TABLE 3







ALK inhibitory activities in the Ambit assay (IC50 in nM)














Cpd
PF-2341066
Ex 1
Ex 13
Ex 15
Ex 16
Ex 17
Ex 18





IC50
>2
<1
<1
<1
<1
<1
<1









Numerous compounds of the current invention demonstrate very good potency. Furthermore, some compounds (e.g. Ex 16 and 18) are more potent than compounds disclosed in WO 2009/154769. For example, Example 6 of WO 2009/154769 showed IC50 of 17 nM in this assay, compared to <1 nM for Examples 16 and 18 in this invention.
Example 22
Cell Viability Assay of Tumor Cells with ALK Fusion Proteins
For viability experiments, tumor cells with ALK fusion proteins (H3122, H2228) were seeded in 96-well plates at 25%-33% confluency and exposed to drugs alone or in combination the following day. At 72 hours post drug addition, Cell Titer Blue Reagent (Promega, Madison, Wis.) was added and fluorescence was measured on a Spectramax spectrophotometer (Molecular Devices, Sunnyvale, Calif.) according to the manufacturer's instructions. All experimental points were set up in hextuplicate replicates and were performed at least two independent times. IC50's were calculated using GraphPad Prism version 5 for Windows. The curves were fit using a nonlinear regression model with a log (inhibitor) vs. response formula.
In the cell viability assay, some compounds in this invention (e.g. Examples 1, 16, 18) were more potent in inhibiting the H3122 and H2228 tumor cell growth than PF-2341066, and thus they have the potential of overcoming resistance to PF-2341066 (Table 4).







TABLE 4







Inhibition of H3122 and H2228 tumor cell growth (IC50 in nM)











Compound
PF-2341066
Ex 1
Ex 16
Ex 18





H3122
>150
 <50
 <30
 <30


H2228
>150
<100
<100
<100









Numerous compounds of the current invention demonstrate very good potency. Furthermore, Ex 16 and 18 of this invention are more potent than Examples 25 and 26 disclosed in WO 2009/154769 which showed IC50 of >50 nM in this assay.
Example 23
Cell Apoptosis Assay of Tumor Cells with ALK Fusion Proteins
For apoptosis experiments, cells were seeded in 12 well plates at 25% confluency and treated in triplicate with ALK TKI. At 72 h post drug addition, cells were collected, washed in PBS, and stained with annexin V and propidium iodide according to the manufacturer's instructions (Vybrant Apooptosis Assay Kit, Invitrogen, Carlsbad, Calif.). Data were collected on a FACSCanto II (BD Biosciences, San Jose, Calif.) and processed using WinList flow cytometry analysis software (Verity Software, Topsham, Me.).
In the apoptosis assay, Example 1 was more effective in inducing apoptosis in H3122 cells than PF-2341066 at the same concentration (FIG. 1).
Example 24
c-Met Receptor Phosphorylation Assay
A549 cells are used in this assay. Cells are seeded at a density of 40,000 cells/well in the growth media (RPMI+10% FBS) into 24-well plates and cultured overnight at 37° C. for attachment. Cells are exposed to the starvation media (RPMI+1% BSA). Dilutions of the test compounds are added to the plates and incubated at 37° C. for 1 hour. Cells are then cool down to room temperature for 15 min followed by stimulation with 40 ng/ml HGF for 15 minutes. Cells are washed once with ice-cold PBS and then lysed with 110 ul/well lysis buffer (Cell Signaling #9803 +0.2% protease inhibitor, Sigma P1860) for 1 hour at 4° C. Cell lysates are transferred to microcentrifuge tubes and are spun at 10000 rpm for 10 min at 4° C. and phosphorylated HGFR is quantitated by Human Phospho-HGF R/c-Met ELISA kit (R&D, DYC2480) according to the manufacture's instructions.
Example 25
Human Microsomal Stability
Pooled human liver microsomes (1 mg/mL) are incubated at 37° C. in phosphate buffer (pH 7.4) with the test compound (1 μM). The reaction is started with the addition of NADPH. (1 mM final concentration). Incubations are stopped after 0, 5, 10, 20, 40, 60 minutes with the addition of organic solvent. Quenched samples are analyzed for unchanged test compound by LC/MS/MS detection. Percent turnover is determined by the ratio of the amount (peak area) of unchanged test compound remaining in incubated samples to the amount of unchanged test compound in non-incubated samples (0 minutes). Half-life is calculated based on the percent turnover data.
In this assay, Example 16 (a compound of Formula III with n=2) and Example 18 (a compound of Formula III with R9 and R10 being methyl) had longer half-life than the analogous compound, Example 25 as disclosed in WO 2009/154769.
Example 26
Brain Penetration
To determine if a compound can cross the blood-brain-barrier (BBB), mice are dosed with the test compound. Two hours after dosing, mice are sacrificed and blood and brain tissues are collected and analyzed for test compound concentration. Brain penetration is defined as ratio of compound concentration in the brain tissues to that in plasma.
In this assay, Example 18 (a compound of Formula III with R9 and R10 being methyl and R11 being hydrogen) had higher brain penetration than Example 1 with R9 and R10 being hydrogen and R11 being methyl, suggesting that Example 18 is more likely to inhibit ALK in the brain than Example 1.
Example 27
In Vivo Anti-Tumor Efficacy of Crizotinib (PF-1066) and Example 18 in the SH-SY5Y Intracranial Tumor Model
Method:
Balb/C nude mice were anesthetized by i.p. injection of pentobarbital sodium (100 mg/kg body weight). One million SH-SY5Y cells in a volume of 10 μl full-culture medium were injected into the caudate putamen region using a small animal stereotactic apparatus. On the 7th day after implantation, the tumor-bearing mice were assigned into 4 groups, PF-1066, Example 18 low dose, Example 18 high dose, and vehicle were given twice daily (BID) by oral gavage, treatment was stopped until the animal is dead. Animal survival and body weight were observed and recorded.
Result:
After continuous treatment, the median survival time (MST) of the vehicle Control group, PF-1066 at 50 mg/kg twice daily (BID), Example 18 at 25 and 50 mg/kg BID are 25 days, 28 days, 25 days and 34.5 days (P<0.01), respectively. Thus, Example 18 prolonged life of these tumor bearing mice at 50 mg/kg BID with statistical significance, while crizotinib slightly prolonged life at the same dose but without statistical significance.
While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.
The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

## Claims

1. A compound of formula III:




or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof; wherein;
n is 0-2;
R5 and R11 each are independently H, alkyl or Z1;
R9 is alkyl or Z1;
R10 is H, alkyl, or Z1;
each Z1 is independently halogen, CN, NO2, OR15, SR15, S(O)2OR15, NR15R16, C1-C2 perfluoroalkyl, C1-C2 perfluoroalkoxy, 1,2-methylenedioxy, C(O)OR15, C(O)NR15R16, OC(O)NR15R16, NR15C(O)NR15R16, C(NR16)NR15R16, NR15C(NR16)NR15R16, S(O)2NR15R16, R17, C(O)R17, NR15C(O)R17, S(O)R17, S(O)2R17, R16, OXO, C(O)R16, C(O)(CH2)mOH, (CH2)mOR15, (CH2)mC(O)NR15R16, NR15S(O)2R17, where each m is independently 0-6;
Each R15 is independently hydrogen, C1-C4 alkyl or C3-C6 cycloalkyl;
Each R16 is independently hydrogen, alkenyl, alkynyl, C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl; and
Each R17 is independently C3-C6 cycloalkyl, aryl, heterocyclyl, heteroaryl, C1-C4 alkyl or C1-C4 alkyl substituted with C3-C6 cycloalkyl, aryl, heterocyclyl or heteroaryl.

2. The compound of claim 1, wherein R11 is hydrogen.

3. A compound of claim 1 wherein the compound is:
N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}{6-amino-5-[(2,6-dichloro-3-fluorophenyl)ethoxy]pyridazin-3-yl}carboxamide.

4. The compound of claim 1, wherein the compound is selected from the following:
{5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3-oxopiperazinyl)carbonyl]phenyl}carboxamide;
{5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[(3,3-dimethylpiperazinyl)carbonyl]phenyl}carboxamide; and
{5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-6-aminopyridazin-3-yl}-N-{4-[((3S,5R)-3,5-dimethylpiperazinyl)carbonyl]phenyl}carboxamide.

5. A composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.