HK1188999A

HK1188999A - Substituted phenoxypyridines

Info

Publication number: HK1188999A
Application number: HK14102137.4A
Authority: HK
Inventors: M.希契科克
Original assignee: 拜耳知识产权有限责任公司
Priority date: 2010-10-29
Filing date: 2011-10-27
Publication date: 2014-05-23

Description

Substituted phenoxypyridines

Technical Field

The present invention relates to substituted phenoxypyridines (hereinafter referred to as "compounds of general formula (I)") as described and defined herein, to processes for preparing said compounds, to intermediates used for preparing said compounds, to pharmaceutical compositions and combinations comprising said compounds and to the use of said compounds for manufacturing a pharmaceutical composition for the treatment or prophylaxis of a disease, in particular of a hyper-proliferative and/or angiogenesis disorder, as a sole agent or in combination with other active ingredients.

Background

Cancer is a disease caused by abnormal growth of tissues. Some cancers have the potential to invade local tissues and can also metastasize to distant organs. The disease can develop in a wide variety of different organs, tissues and cell types. Thus, the term "cancer" refers to a collection of more than one thousand different diseases.

In 2002, over four hundred and forty thousand people worldwide were diagnosed with breast, colon, ovarian, lung or prostate cancer, and over two hundred and fifty thousand people died of these devastating diseases (Globocan2002 Report). In the united states alone, over one hundred and twenty-fifty thousand new cases and over 500000 deaths from cancer are expected in 2005. The majority of these new cases are expected to be colon (100000), lung (170000), breast (210000) and prostate (230000). The incidence and prevalence of cancer is expected to increase by about 15% over the next 10 years, reflecting an average increase of 1.4% [1 ].

There is increasing evidence that cancer can be viewed as a "signal transduction disease" in which alterations in the cellular genome that affect the expression and/or function of oncogenes and tumor suppressor genes ultimately affect the transduction of signals that normally regulate cell growth, differentiation and programmed cell death (apoptosis). Elucidation of the deregulated signal transduction pathways in human cancer has led to an increasing design of mechanism-based therapeutics [2 ]. Recently, signal transduction inhibition as a therapeutic strategy for human malignancies has been significantly successful, such as the development of Gleevec for the treatment of Chronic Myelogenous Leukemia (CML) and gastrointestinal stromal tumors, promising a new era of "molecular targeted" therapies [3-5 ].

The mitogen-activated protein kinase (MAPK) module is a key integration point of the signal transduction cascade that links various extracellular stimuli to proliferation, differentiation and survival. Scientific research over the past two decades has created a very detailed molecular anatomy of this pathway, which has now evolved to include five distinct MAPK subfamilies with distinct molecular and functional characteristics [ extracellular signal-regulated kinase ERK-1/2, c-Jun-N-terminal kinase (JNK), p38 kinase, ERK-3/4, and ERK-5] [6-8 ]. Although certain subfamilies, such as the p38 family, are becoming therapeutic targets for inflammatory and degenerative diseases, the MAPK cascade (the major mitogen pathway initiated by peptide growth factors) from Ras to ERK-1/2 has begun to emerge as a major target for molecular therapy of different types of human cancer [9-11 ]. In many human tumors, MAPK pathways are abnormally activated due to genetic and epigenetic changes, resulting in increased proliferation and resistance to apoptotic stimuli. In particular, mutated oncogenic forms of Ras are found in 50% of colon cancers and >90% of pancreatic cancers. Recently, BRAF mutations have been found in >60% of malignant melanomas [13 ]. These mutations result in constitutively activated MAPK pathways. Furthermore, overexpression or mutational activation of certain receptor tyrosine kinases can also lead to increased activation of the Raf-MEK-ERK pathway.

At the crossover point regulated by MEK, the modular nature of the Raf/MEK/ERK cascade becomes less pleiotropic [14 ]. No MEK substrate other than ERK-1/2 has been identified. Phosphorylated ERK is a product of MEK activity, and its detection in cancer cells and tumor tissues therefore provides a direct measure of MEK inhibition. The selectivity of MEK towards ERK1/2, linked to the availability of antibodies specific for the doubly phosphorylated and activated form of ERK, makes MEK an interesting target for anticancer drug development. In addition, it has recently been shown that MEK activation modulates matrix mineralization (Blood2007,40,68), and that modulation of MEK activity may thus also be useful in the treatment of diseases caused by or accompanied by dysregulation of tissue mineralization, more particularly in the treatment of diseases caused by or accompanied by dysregulation of bone mineralization.

First generation MEK inhibitor PD98059[15 ]]And U0126[16 ]]Do not appear to compete with ATP and therefore may have different MEK binding sites; these compounds have been widely used in vitro and in vivo model systems to attribute biological activity to ERK 1/2. IC of the second generation MEK1/2 inhibitor PD184352 (now referred to as CI-1040)₅₀In the low nanomolar range, has improved bioavailability and also appears to act through an allosteric non-ATP competitive mechanism [17]. Oral treatment with CI-1040 has been shown to inhibit colon cancer growth in vivo in a mouse model [18]Moreover, the compound has been evaluated in a phase I/II clinical trial in which it eventually fails due to insufficient efficacy [19]. Recently, other allosteric MEK inhibitors have entered the clinic, but have been found to have limitations such as poor exposure profile, limited efficacy, and/or tissue toxicity. Small molecule MEK inhibitors have been disclosed, including in U.S. patent application publications 2003/0232869, 2004/0116710, 2003/0216420 and in U.S. patent applications 10/654580 and 10/929295, each of which is incorporated herein by reference. Many other patent applications have emerged over the past few years, including U.S. patent 55256625; WO 98/43960; WO 99/01421; WO 99/01426; WO 00/41505; WO 00/41994; WO 00/42002; WO 00/42003; WO 00/42022; WO 00/42029; WO 00/68201; WO 01/68619; WO 02/06213; WO 03/077914; WO 03/077855; WO 04/083167; WO 05/0281126; WO 05/051301; WO 05/121142; WO 06/114466; WO 98/37881; WO 00/35435; WO 00/35436; WO 00/40235; WO 00/40237; WO 01/05390; WO 01/05391; WO 01/05392; WO 01/05393; WO 03/062189; WO 03/062191; WO 04/056789; WO 05/000818; WO 05/007616; WO 05/009975; WO 05/051300; WO 05/051302; WO 05/028426; WO06/056427; WO 03/035626; and WO 06/029862.

Despite advances in the art, there remains a need for cancer therapies and anti-cancer compounds. More specifically, there remains a need for new structurally novel MEK inhibitors with a balanced potency-property profile. It is particularly desirable to identify new MEK inhibitors that contain structural motifs that have not previously been demonstrated to be compatible with effective MEK inhibition. It would be particularly advantageous if these structural motifs also allowed for improved MEK potency and/or modulation of compound properties, including physicochemical, pharmacodynamic and pharmacokinetic properties.

WO2006/045514A1(Applied Research Systems ARS Holding N.V.) relates to 3-arylaminopyridine derivatives. Such compounds are MEK inhibitors and are useful in the treatment of hyperproliferative diseases, such as cancer, restenosis and inflammation.

WO2008/138639(Bayer Schering Pharma Aktiengesellschaft) relates to substituted phenylaminobenzene compounds, pharmaceutical compositions comprising such compounds, and the use of such compounds or compositions for the treatment of hyperproliferative and/or angiogenic disorders. The compounds were found to be potent and selective MEK inhibitors. The compounds are derived from a 1-substituted-2-phenylaminophenyl backbone having a further specifically substituted side chain at the 6-position of the phenyl backbone. This finding is surprising because a survey of published phenyl backbone-derived MEK inhibitors and previous structure-activity relationship analyses (see, e.g., Haile Tecle/Pfizer Global Research: "MEK inhibitors", published on Drew University at 6/15/2006) indicate that, in phenyl backbone-based MEK inhibitors, the larger 6-substituent is detrimental to achieving high MEK inhibition potency. The compounds are potent inhibitors of MEK and inhibit the activation of the MEK-ERK pathway.

However, the above-mentioned background art does not specifically describe the compounds of the general formula (I) of the present invention, stereoisomers, tautomers, N-oxides, hydrates, solvates or salts thereof, as described and defined herein and hereinafter referred to as "compounds of the present inventionOr a mixture thereof, or a pharmacological activity thereof, said compound carrying a specific-C (= O) NHR at the 4-position of the depicted pyridine ring₃A substituent and a specific substituted oxygen atom at the 3- (5) -position of the depicted pyridine ring.

It has now been found that said compounds of the invention have surprisingly advantageous properties, which form the basis of the present invention.

In particular, said compounds of the invention have been shown to have a very high activity in the a375 proliferation assay, as indicated by the biological results provided in the tables at the end of the present description. It will be apparent to those skilled in the art of pharmaceutical chemistry that the compounds of the invention will have such levels of activity: the compounds of the present invention are indeed effective in strongly inhibiting cancer cell proliferation.

Moreover, the compounds may have significantly reduced affinity for human carbonic anhydrase. It is known to the person skilled in the art that affinity for human carbonic anhydrases leads to an undesired accumulation of the corresponding compounds in human erythrocytes.

In addition, the compounds may have a significantly improved CYP inhibitory profile, to the extent that CYP is not detectable.

In view of this, the compounds of general formula (I) according to the invention are therefore useful for the treatment or prevention of diseases caused by or accompanied by uncontrolled cell growth, proliferation and/or survival, an inappropriate cellular immune response or an inappropriate cellular inflammatory response, in particular wherein the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response or inappropriate cellular inflammatory response is mediated by the mitogen-activated protein kinase (MEK-ERK) pathway, such as hematological tumors, solid tumors and/or their metastases, such as leukemia and myelodysplastic syndrome, malignant lymphomas, head and neck tumors including brain tumors and brain metastases, diseases with inappropriate cellular immune response or inappropriate cellular inflammatory response, and in particular wherein the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response or inappropriate cellular inflammatory response is mediated by the mitogen-activated protein kinase (MEK-ERK) pathway, such as, Breast, gastrointestinal, endocrine, breast and other gynaecological tumours including non-small cell and small cell lung tumours, urological tumours including renal, bladder and prostate tumours, skin tumours and sarcomas, and/or metastases thereof.

Disclosure of Invention

According to a first aspect, the present invention comprises a compound of general formula (I), or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof:

wherein:

r1 is aryl, heteroaryl, C₁-C₆Alkyl radical, C₂-C₆-alkenyl, C₃-C₆-cycloalkyl or 3-to 7-membered heterocycloalkyl group,

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₆-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R,-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R’，-N(R)C(=O)N(R’)R’’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -n (r) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R’，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

r2 is a halogen atom, C₂-C₆-alkynyl or-S-C₁-C₆-an alkyl group;

r3 is selected from hydrogen atom and C₁-C₆Alkyl radical, C₃-C₆-a cycloalkyl, 3-to 7-membered heterocycloalkyl, aryl or heteroaryl group, said C₁-C₆Alkyl radical, C₃-C₆-cycloalkyl, aryl or heteroaryl optionally substituted by-OH, -NH₂-N (H) R, -N (R) R', halogen atom, cyano or C₁-C₆-alkoxy is substituted one or more times in the same or different ways;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

Definition of

The terms mentioned herein preferably have the following meanings:

the term "halogen atom" or "halogen/halo" is understood to mean a fluorine, chlorine, bromine or iodine atom.

The term "C₁-C₆Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3,4, 5 or 6 carbon atoms, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl or 1, 2-dimethylbutyl or isomers thereof. In particular, the radicals have 1,2 or3 carbon atoms ("C)₁-C₃-alkyl "), methyl, ethyl, n-propyl or isopropyl.

The term "halo-C₁-C₆Alkyl is understood to mean preferably a straight-chain or branched, saturated monovalent hydrocarbon radical, where the term "C" is₁-C₆Alkyl "is as defined above and wherein one or more hydrogen atoms are replaced by halogen atoms in the same or different manner, i.e. independently of each other. In particular, the halogen atom is F. Said halo-C₁-C₆Alkyl is, for example, -CF₃、-CHF₂、-CH₂F、-CF₂CF₃or-CH₂CF₃。

The term "C₁-C₆Alkoxy is to be understood as meaning preferably a straight-chain or branched, saturated monovalent hydrocarbon radical of the formula-O-alkyl, where the term "alkyl" is as defined above, for example methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentoxy, isopentoxy or n-hexoxy, or their derivativesIsomers.

The term "halo-C₁-C₆Alkoxy is understood as preferably meaning a straight-chain or branched, saturated, monovalent C radical as defined above in which one or more hydrogen atoms are replaced by halogen atoms in the same or different manner₁-C₆-alkoxy groups. In particular, the halogen atom is F. Said halo-C₁-C₆Alkoxy is, for example, -OCF₃、-OCHF₂、-OCH₂F、-OCF₂CF₃or-OCH₂CF₃。

The term "C₁-C₆-alkoxy-C₁-C₆Alkyl is understood to preferably denote-C in which one or more hydrogen atoms are defined as above, in the same or different manner₁-C₆Alkoxy substituted straight-chain or branched saturated monovalent alkyl radicals as defined above or their isomers, for example methoxyalkyl, ethoxyalkyl, propoxyalkyl, isopropoxyalkyl, butoxyalkyl, isobutoxyalkyl, tert-butoxyalkyl, sec-butoxyalkyl, pentoxyalkyl, isopentyloxyalkyl, hexyloxyalkyl, where the term "C" is used₁-C₆-alkyl "is as defined above.

The term "halo-C₁-C₆-alkoxy-C₁-C₆Alkyl is understood to preferably mean a straight-chain or branched, saturated, monovalent-C radical as defined above in which one or more hydrogen atoms are replaced by halogen atoms in the same or different manner₁-C₆-alkoxy-C₁-C₆-an alkyl group. In particular, the halogen atom is F. Said halo-C₁-C₆-alkoxy-C₁-C₆Alkyl is, for example, -CH₂CH₂OCF₃、-CH₂CH₂OCHF₂、-CH₂CH₂OCH₂F、-CH₂CH₂OCF₂CF₃or-CH₂CH₂OCH₂CF₃。

The term "C₂-C₆Alkenyl "is understood to preferably mean a straight-chain or branched monovalent hydrocarbon radical which contains one or more double bonds and has 2,3,4, 5 or 6 carbon atoms, in particular 2 or3 carbon atoms (" C)₂-C₃-alkenyl "), it being understood that in case the alkenyl group comprises more than one double bond, the double bonds may be separated from each other or conjugated. The alkenyl group is, for example, vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl, homoallyl, (E) -but-2-enyl, (Z) -but-2-enyl, (E) -but-1-enyl, (Z) -but-1-enyl, pent-4-enyl, (E) -pent-3-enyl, (Z) -pent-3-enyl, (E) -pent-2-enyl, (Z) -pent-2-enyl, (E) -pent-1-enyl, (Z) -pent-1-enyl, hex-5-enyl, (E) -hex-4-enyl, m, (Z) -hex-4-enyl, (E) -hex-3-enyl, (Z) -hex-3-enyl, (E) -hex-2-enyl, (Z) -hex-2-enyl, (E) -hex-1-enyl, (Z) -hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E) -1-methylprop-1-enyl, (Z) -1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E) -2-methylbut-2-enyl, (Z) -2-methylbut-2-enyl, (E) -1-methylbut-2-enyl, (Z) -1-methylbut-2-enyl, (E) -3-methylbut-1-enyl, (Z) -3-methylbut-1-enyl, (E) -2-methylbut-1-enyl, (Z) -2-methylbut-1-enyl, (E) -1-methylbut-1-enyl, (Z) -1-methylbut-1-enyl, 1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl, 4-methylpent-4-enyl, 3-methylpent-4-enyl, 2-methylpent-4-enyl, 1-methylpent-4-enyl, 4-methylpent-3-enyl, (E) -3-methylpent-3-enyl, (Z) -3-methylpent-3-enyl, (E) -2-methylpent-3-enyl, (Z) -2-methylpent-3-enyl, (E) -1-methylpent-3-enyl, (Z) -1-methylpent-3-enyl, isopropylvinyl, 4-methylpent-4-enyl, 3-methylpent-3-enyl, methyl-pentenyl, methyl-3-enyl, methyl-pentenyl, ethyl-1-, (E) -4-methylpent-2-enyl, (Z) -4-methylpent-2-enyl, (E) -3-methylpent-2-enyl, (Z) -3-methylpent-2-enyl, (E) -2-methylpent-2-enyl, (Z) -2-methylpent-2-enyl, (E) -1-methylpent-2-enyl, (Z) -1-methylpent-2-enyl, (E) -4-methylpent-1-enyl, (Z) -4-methylpent-1-enyl, (E) -3-methylpent-1-enyl, (Z) -3-methylpent-1-enyl, methyl-2-enyl, methyl-4, (E) -2-methylpent-1-enyl,(Z) -2-methylpent-1-enyl, (E) -1-methylpent-1-enyl, (Z) -1-methylpent-1-enyl, 3-ethylbut-3-enyl, 2-ethylbut-3-enyl, 1-ethylbut-3-enyl, (E) -3-ethylbut-2-enyl, (Z) -3-ethylbut-2-enyl, (E) -2-ethylbut-2-enyl, (Z) -2-ethylbut-2-enyl, (E) -1-ethylbut-2-enyl, (Z) -1-ethylbut-2-enyl, methyl-1-enyl, ethyl-3-enyl, ethyl-but-2-enyl, ethyl, (E) -3-ethylbut-1-enyl, (Z) -3-ethylbut-1-enyl, 2-ethylbut-1-enyl, (E) -1-ethylbut-1-enyl, (Z) -1-ethylbut-1-enyl, 2-propylprop-2-enyl, 1-propylprop-2-enyl, 2-isopropylprop-2-enyl, 1-isopropylprop-2-enyl, (E) -2-propylprop-1-enyl, (Z) -2-propylprop-1-enyl, (E) -1-propylprop-1-enyl, (Z) -1-propylprop-1-enyl, (E) -2-isopropylprop-1-enyl, (Z) -2-isopropylprop-1-enyl, (E) -1-isopropylprop-1-enyl, (Z) -1-isopropylprop-1-enyl, (E) -3, 3-dimethylprop-1-enyl, (Z) -3, 3-dimethylprop-1-enyl, 1- (1, 1-dimethylethyl) vinyl, but-1, 3-dienyl, penta-1, 4-dienyl, hex-1, 5-dienyl or methylhexadienyl. In particular, the group is vinyl or allyl.

The term "C₂-C₆Alkynyl is understood as preferably meaning a straight-chain or branched, monovalent hydrocarbon radical which comprises one or more triple bonds and comprises 2,3,4, 5 or 6 carbon atoms, in particular 2 or3 carbon atoms ("C)₂-C₃-alkynyl "). Said C is₂-C₆Alkynyl is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl, 1-ethylprop-2-ynyl, prop-2-ynyl, but-3-methylbut-1-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl, 1-methylpent-4-ynyl, 2-methylpent-3-ynyl, 1-methylpent-3-ynyl, 4-methylpent-2-ynyl, 1-methylpent-2-ynyl, 4-methylpent-1-ynyl, 3-methylpent-1-ynyl, 2-ethylbut-3-ynyl, 1-ethylbut-2-ynyl, 1-propylprop-2-ynyl, 1-isopropylprop-2-ynyl, 2-di-alkynylMethyl but-3-alkynyl, 1-dimethyl but-2-alkynyl or3, 3-dimethyl but-1-alkynyl. In particular, the alkynyl group is ethynyl, prop-1-ynyl or prop-2-ynyl.

The term "C₃-C₆Cycloalkyl "is understood to mean preferably a saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 3,4, 5 or 6 carbon atoms. Said C is₃-C₆Cycloalkyl is, for example, a monocyclic hydrocarbon ring such as a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group, or a bicyclic hydrocarbon ring such as a perhydropentalene or decahydronaphthalene ring. The cycloalkyl ring may optionally contain one or more double bonds, for example a cycloalkenyl group such as cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl, wherein the bond between the ring and the rest of the molecule (whether saturated or unsaturated) may be on any carbon atom of the ring.

The term "alkylene" is understood to mean preferably an optionally substituted hydrocarbon chain (or "chain") having 1,2, 3,4, 5 or 6 carbon atoms, i.e. -CH-which is optionally substituted₂- ("methylene" or "monobasic chain" or such as-C (Me))₂-）、-CH₂-CH₂- ("ethylene", "dimethylene" or "dibasic chain"), -CH₂-CH₂-CH₂- ("propylene", "trimethylene" or "triad"), -CH₂-CH₂-CH₂-CH₂- ("butylene", "tetramethylene" or "tetrabasic"), -CH₂-CH₂-CH₂-CH₂-CH₂- ("Pentylene", "pentamethylene" or "five-membered chain") or-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂- ("hexamethylene", "hexamethylene" or "six-membered chain"). In particular, the alkylene chain has 1,2, 3,4 or5 carbon atoms, more particularly 1 or 2 carbon atoms.

The term "3-to 7-membered heterocycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 2,3,4, 5 or 6Carbon atoms and one or more heteroatom-containing groups selected from: c (= O), O, S, S (= O), S (= O)₂、NR^aWherein R is^aRepresents a hydrogen atom, C₁-C₆-alkyl-, or halo-C₁-C₆-alkyl-; it is to be understood that the heterocycloalkyl group may be attached to the rest of the molecule through any of the carbon atoms or the optionally present nitrogen atom.

In particular, the 3-to 7-membered heterocycloalkyl group can comprise 2,3,4, or5 carbon atoms and one or more of the above-mentioned heteroatom-containing groups ("3-to 7-membered heterocycloalkyl"), more particularly, the heterocycloalkyl group can comprise 4 or5 carbon atoms and one or more of the above-mentioned heteroatom-containing groups ("5-to 7-membered heterocycloalkyl").

In particular, the heterocycloalkyl group may be, for example, but not limited to, a 4-membered ring such as azetidinyl, epoxypropyl (oxyethanyl), or a 5-membered ring such as tetrahydrofuranyl, dioxolyl (dioxolanyl), pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl, or a 7-membered ring such as diazepanyl (diazepanyl) ring. Optionally, the heterocycloalkyl ring may be benzo-fused.

The heterocyclyl group may be bicyclic, such as, but not limited to, a5, 5-membered ring, for example, a hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl) ring, or a5, 6-membered bicyclic ring, for example, a hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring, or for example.

As mentioned above, the nitrogen atom containing ring may be partially unsaturated, i.e. it may contain one or more double bonds, such as, but not limited to, a2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydrooxazolyl or 4H- [1,4] thiazinyl ring, or it may be benzo-fused, such as, but not limited to, a dihydroisoquinolinyl ring.

The term "aryl" is understood to mean preferablyA monovalent, aromatic or partially aromatic, monocyclic, bicyclic or tricyclic hydrocarbon ring having 6,7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms ("C)₆-C₁₄Aryl "), in particular a ring having 6 carbon atoms (" C)₆-aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C₉-aryl "), such as indanyl or indenyl, or a ring having 10 carbon atoms (" C₁₀Aryl), such as tetralinyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C₁₃Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C)₁₄Aryl "), for example anthracenyl. A specific example of an aryl group is one of the following possible structures:

wherein z represents O, S, NH or N (C)₁-C₆-alkyl) and indicates the point of attachment of the aryl group to the rest of the molecule.

The term "heteroaryl" is understood as preferably meaning a monovalent monocyclic, bicyclic or tricyclic aromatic ring system having 5,6, 7,8, 9, 10, 11, 12, 13 or 14 ring atoms ("5-to 14-membered heteroaryl"), in particular 5 or 6 or 9 or 10 carbon atoms, and which comprises at least one heteroatom which may be identical or different (such as oxygen, nitrogen or sulfur), in addition which may be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinylAnd the like, and benzo derivatives thereof such as quinolyl, quinazolinyl, isoquinolyl and the like; or azocinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, xanthenyl or azanylAnd the like.

In general and unless otherwise indicated, the heteroaryl or heteroarylene includes all possible isomeric forms thereof, e.g., positional isomers thereof. Thus, for some illustrative, non-limiting examples, the term pyridyl or pyridinylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl and pyridinylene-4-yl; alternatively, the term thienyl or thienylene includes thien-2-yl, thien-3-yl, and thien-3-yl.

As used throughout this document, the term "C₁-C₆At C₁-C₆-alkyl group "," C₁-C₆-haloalkyl "," C₁-C₆-alkoxy "or" C₁-C₆-haloalkoxy "is understood in the context of the definition to mean an alkyl group having from 1 to 6 limited number of carbon atoms, i.e. 1,2, 3,4, 5 or 6 carbon atoms. It is also understood that the term "C" refers to₁-C₆"is to be understood as meaning any subrange comprised therein, such as C₁-C₆、C₂-C₅、C₃-C₄、C₁-C₂、C₁-C₃、C₁-C₄、C₁-C₅、C₁-C₆(ii) a In particular C₁-C₂、C₁-C₃、C₁-C₄、C₁-C₅、C₁-C₆(ii) a More particularly C₁-C₄(ii) a In "C₁-C₆-haloalkyl "or" C₁-C₆In the case of a haloalkoxy group, more particularly C₁-C₂。

Similarly, the term "C" as used herein₂-C₆", as used throughout this document, e.g., at" C₂-C₆-alkenyl "and" C₂-C₆-alkynyl "is understood in the context of its definition to mean alkenyl or alkynyl groups having a limited number of carbon atoms from 2 to 6, i.e. 2,3,4, 5 or 6 carbon atoms. It is also understood that the term "C" refers to₂-C₆"is to be understood as meaning any subrange comprised therein, such as C₂-C₆、C₃-C₅、C₃-C₄、C₂-C₃、C₂-C₄、C₂-C₅(ii) a In particular C₂-C₃。

In addition, the term "C" as used herein₃-C₆", as used throughout this document, e.g., at" C₃-C₆-cycloalkyl "is understood in the context of the definition of" cycloalkyl "to mean cycloalkyl having 3 to 6 limited numbers of carbon atoms, i.e. 3,4, 5 or 6 carbon atoms. It is also understood that the term "C" refers to₃-C₆"is to be understood as meaning any subrange comprised therein, such as C₃-C₆、C₄-C₅、C₃-C₅、C₃-C₄、C₄-C₆、C₅-C₆(ii) a In particular C₃-C₆。

The term "substituted" means that one or more hydrogens of the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency at the present time is not exceeded and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The term "optionally substituted" means optionally substituted with a specified group, radical or moiety.

Substituents of a ring system refer to substituents attached to an aromatic or non-aromatic ring system, e.g., the substituents replace available hydrogens on the ring system.

The term "one or more times" as used herein, for example in the definition of a substituent of a compound of the general formula according to the invention, is to be understood as meaning "one, two, three, four or five times, in particular one, two, three or four times, more particularly one, two or three times, even more particularly one or two times".

When the plural form of the words compound, salt, polymorph, hydrate, solvate and the like are used herein, it is to be understood that reference to a compound, salt, polymorph, isomer, hydrate, solvate and the like in the singular is also intended.

"stable compound" or "stable structure" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an effective therapeutic agent.

The compounds of the present invention may contain one or more asymmetric centers, depending on the location and nature of the various substituents desired. Asymmetric carbon atoms may exist in either the (R) or (S) configuration, resulting in a racemic mixture in the case of one asymmetric center and a diastereomeric mixture in the case of multiple asymmetric centers. In some cases, asymmetry may also exist due to hindered rotation about a particular bond, for example, the central bond connects two substituted aromatic rings of a particular compound.

The ring substituents may also be present in cis or trans form. All such configurations (including enantiomers and diastereomers) are intended to be included within the scope of the present invention.

Preferred compounds are those that produce a more desirable biological activity. Isolated, pure or partially purified isomers and stereoisomers, or racemic or diastereomeric mixtures of the compounds of the invention are included within the scope of the invention. Purification and isolation of such materials can be accomplished by standard techniques known in the art.

Optical isomers may be obtained by resolution of the racemic mixture according to conventional methods, for example by formation of diastereomeric salts using an optically active acid or base, or by formation of covalent diastereomers. Examples of suitable acids are tartaric acid, diacetyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Mixtures of diastereomers may be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, e.g., by chromatography or fractional crystallization. The optically active base or acid is then released from the separated diastereomeric salt. Another different method of separating optical isomers involves the use of chiral chromatography (e.g., a chiral HPLC column) with or without conventional derivatization, which can be optimally selected to maximize separation of the enantiomers. Suitable chiral HPLC columns are produced by Diacel, such as chiralel OD and chiralel OJ, all of which are routinely selected. Enzymatic separation may also be used with or without derivatization. Likewise, the optically active compounds of the present invention can be obtained by chiral synthesis using optically active starting materials.

To distinguish the different types of isomers from each other, reference is made to IUPAC Rules Section E (Pure Appl Chem45,11-30,1976).

The present invention includes all possible stereoisomers of the compounds of the invention, either as single stereoisomers or as any mixture of said isomers in any proportion. The separation of single stereoisomers, such as single enantiomers or single diastereomers, of the compounds of the invention may be achieved by any suitable prior art method, such as chromatography, particularly, for example, chiral chromatography.

In addition, the compounds of the present invention may exist in tautomeric forms. For example, any compound of the invention comprising a pyrazole moiety as heteroaryl may, for example, exist in the form of a 1H tautomer or a 2H tautomer or even in the form of a mixture of any amount of the two tautomers, or any compound of the invention comprising a triazole moiety as heteroaryl may, for example, exist in the form of a 1H tautomer, a 2H tautomer or a 4H tautomer or even in the form of a mixture of any amount of the 1H, 2H and 4H tautomers:

the present invention includes all possible tautomers of the compounds of the invention, either as single tautomers or as any mixtures of said tautomers, in any ratio.

In addition, the compounds of the present invention may exist in the form of N-oxides, which are defined as compounds of the present invention in which at least one nitrogen is oxidized. The present invention includes all such possible N-oxides.

The invention also relates to useful forms of the compounds as disclosed herein, such as metabolites, hydrates, solvates, prodrugs, salts, especially pharmaceutically acceptable salts, and co-precipitates.

The compounds of the invention may be present in the form of hydrates or solvates, wherein the compounds of the invention comprise as structural element of the crystal lattice of the compound a polar solvent, such as in particular water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric proportions. In the case of stoichiometric solvates, such as hydrates, there may be semi- (hemi-) solvates or hydrates, (semi- (hemi-) solvates or hydrates, mono-, sesqui-, di-, tri-, tetra-, penta-, etc. solvates or hydrates, respectively. The present invention includes all such hydrates or solvates.

In addition, the compounds of the invention may be present in free form, for example in the form of a free base, a free acid or a zwitterion, or in the form of a salt. The salt may be any salt, which may be an organic or inorganic addition salt, in particular any pharmaceutically acceptable organic or inorganic addition salt commonly used in pharmacy.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, for example, S.M.Berge et al, "Pharmaceutical Salts," J.pharm.Sci.1977,66, 1-19.

Suitable pharmaceutically acceptable salts of the compounds of the invention may be, for example, acid addition salts of the compounds of the invention which carry a nitrogen atom in the chain or ring and which are sufficiently basic, for example with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid (disufuric acid), phosphoric acid or nitric acid, or acid addition salts with organic acids such as: such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, Mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid (hemisulfuric acid), or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with: n-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris (hydroxymethyl) aminomethane, aminopropanediol, sovak base, 1-amino-2, 3, 4-butanetriol. In addition, the basic nitrogen-containing groups may be quaternized with the following agents: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromide, and the like.

Those skilled in the art will also recognize that acid addition salts of the claimed compounds can be prepared by reacting the compounds with the appropriate inorganic or organic acid by any of a variety of known methods. Alternatively, the alkali metal salts and alkaline earth metal salts of the acidic compounds of the present invention are prepared by reacting the compounds of the present invention with an appropriate base by various known methods.

The present invention includes all possible salts of the compounds of the invention, which may be single salts or any mixture of said salts in any proportion.

The term "in vivo hydrolysable ester" as used herein is understood to mean an in vivo hydrolysable ester of a compound of the invention which comprises a carboxy or hydroxy group, for example a pharmaceutically acceptable ester which can be hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for the carboxyl group include, for example, alkyl esters, cycloalkyl esters and optionally substituted phenylalkyl esters, in particular benzyl esters, C₁-C₆Alkoxymethyl esters, e.g. methoxymethyl ester, C₁-C₆Alkanoyloxymethyl esters, e.g. pivaloyloxymethyl ester, phthalidyl ester, C₃-C₈cycloalkoxy-carbonyloxy-C₁-C₆Alkyl esters such as 1-cyclohexylcarbonyloxyethyl ester; 1, 3-dioxole-2-carbonylmethyl(1,3-dioxolen-2-onylmethyl ester), such as 5-methyl-1, 3-dioxole-2-carbonylmethyl ester; and C₁-C₆Alkoxycarbonyloxyethyl esters, such as 1-methoxycarbonyloxyethyl ester, and the esters may be formed on any of the carboxyl groups of the compounds of the invention.

In vivo hydrolysable esters of compounds of the invention which contain a hydroxy group include inorganic acid esters (e.g. phosphate esters), [ α ] acyloxyalkyl ethers and related compounds which are cleaved by in vivo hydrolysis of the ester to form the parent hydroxy group. Examples of [ α ] acyloxyalkyl ethers include acetoxymethyl ether (acetoxymethyloxy) and 2, 2-dimethylpropionyloxymethyl ether (2, 2-dimethylpropionyloxymethyloxy). The selection of groups which form in vivo hydrolysable esters with hydroxyl groups include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, alkoxycarbonyl groups (to form alkyl carbonates), dialkylcarbamoyl and N- (dialkylaminoethyl) -N-alkylcarbamoyl groups (to form carbamates), dialkylaminoacetyl and carboxyacetyl groups. The present invention includes all such esters.

In addition, the present invention includes all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

According to a second aspect, the present invention includes a compound of formula (I) as described above, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₁₀-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R,-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R，-N(R)C(=O)N(R)R’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C3-C6-cycloalkyl, -N (R) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

r2 is a halogen atom, C₂-C₆-alkynyl or-S-C₁-C₆-an alkyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

According to a third aspect, the present invention includes a compound of formula (I) as described above, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₁₀-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R,-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H， -N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R，-N(R)C(=O)N(R’)R’’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -n (r) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R’，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

r2 is a bromine atom, an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

According to a fourth aspect, the present invention comprises a compound of formula (I) as described above, or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate thereof, wherein:

r1 is aryl, C₁-C₆-alkyl or C₂-C₆-an alkenyl group,

said group being substituted with one or more substituents selected from:

C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl, -C (= O) NH₂，-C(=O)N(H)R,-C(=O)N(R)R’，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -OH, C₁-C₆-alkoxy-, -S (= O)₂NH₂，-S(=O)₂N (H) R or-S (= O)₂N (R) R' group;

r2 is a bromine atom, an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r and R' are independently of each other C₁-C₆-an alkyl group.

According to a fifth aspect, the present invention includes a compound of formula (I) as described above, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

r1 is aryl, C₁-C₆Alkyl radical, C₂-C₆-an alkenyl group,

said group being substituted with one or more substituents selected from:

C₁-C₆-alkyl-, C substituted by two OH₁-C₆-alkyl, -NH₂，-N(H)C(=O)R，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl or-OH group;

r2 is an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom;

r is C₁-C₆-an alkyl group.

In a further embodiment of the above aspects, the present invention relates to compounds of formula (I), wherein

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₆-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R,-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R’，-N(R)C(=O)N(R’)R’’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -n (r) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R’，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂， -SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

R1 is aryl, C₁-C₆-alkyl or C₂-C₆-an alkenyl group,

said group being substituted with one or more substituents selected from:

C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl, -C (= O) NH₂，-C(=O)N(H)R，-C(=O)N(R)R’，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -OH, C₁-C₆-alkoxy-, -S (= O)₂NH₂，-S(=O)₂N (H) R or-S (= O)₂N (R) R' group;

R1 is aryl, C₁-C₆Alkyl radical, C₂-C₆-an alkenyl group,

said group being substituted with one or more substituents selected from:

R1 is C₁-C₆-an alkyl group, which is substituted by one or more groups selected fromThe substituent (b):

R1 is aryl, substituted with one or more substituents selected from:

a halogen atom, C₁-C₆-alkyl-, -NH-, -₂，-N(H)C(=O)R，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl or-OH group;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R2 is a halogen atom, C₂-C₆-alkynyl or-S-C₁-C₆-an alkyl group;

in yet another embodiment of the above aspects, the present invention relates to a compound of formula (I) wherein R2 is a bromine atom, an iodine atom or C₂-an alkynyl group;

In a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R2 is an iodine atom or C₂-an alkynyl group;

in yet another embodiment of the above aspects, the present invention relates to a compound of formula (I) wherein R2 is an iodine atom;

in a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein R2 is C₂-an alkynyl group;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

in a further embodiment of the above aspects, the present invention relates to a compound of formula (I) wherein R3 is a hydrogen atom;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R, R' and R "are independently of each other C₁-C₆-an alkyl group;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R is C₁-C₆-an alkyl group;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R' is C₁-C₆-an alkyl group;

in a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R '' is C₁-C₆-an alkyl group;

it is to be understood that the present invention relates to any subcombination within the scope of any embodiment of the compounds of general formula (I) of the invention described hereinabove.

In a further aspect, the invention includes compounds of formula (I) as disclosed in the examples section hereinafter.

According to another aspect, the present invention relates to a process for preparing a compound of the present invention, said process comprising the steps described herein.

According to another aspect, the present invention relates to the preparation of compounds of general formula (I) useful in the present invention, in particular to intermediate compounds useful in the processes described herein. Specifically, the present invention includes compounds of general formula (2):

wherein R2 and R3 are as defined above for general formula (I).

According to a further aspect, the present invention relates to the use of an intermediate compound of formula (2) as described above for the preparation of a compound of the invention of formula (I) as described above.

Detailed description of the experiments and general methods

The following table lists the abbreviations used in this section and in the examples section.

General procedure

General methods for the synthesis of key intermediates and compounds of the invention are described in the following paragraphs.

The routes and methods described below illustrate general synthetic routes for the compounds of general formula (I) of the present invention and are not limiting. It will be apparent to those skilled in the art that the order of transformations illustrated in the schemes can be varied in a variety of ways. Thus, the order of transformations illustrated in the schemes is not limiting. Additionally, interconversion of any of substituents R1, R2, or R3 may be achieved before and/or after the exemplified transformations. These modifications may be, for example, the introduction of protecting groups, the removal of protecting groups, the reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to those skilled in the art. These transformations include those that introduce functionality that allows for further interconversion of substituents. Suitable protecting Groups and their introduction and removal are well known to those skilled in the art (see, e.g., t.w.greene and p.g.m.wuts in Protective Groups in Organic Synthesis, 3 rd edition, Wiley 1999). Specific examples are described in subsequent paragraphs.

A general route for the preparation of compounds of general formula (I) is described in scheme 1.

Route 1

Route 1. general route for the preparation of Compounds of general formula (I), wherein R¹、R²And R³Has the meaning described above for the general formula (I), and X represents a halogen atom. It will be appreciated by those skilled in the art that compounds A, B, C and D are commercially available or can be prepared according to methods available in the public domain.

Reacting 3, 5-dihaloisonicotinic acid of formula (A) with an appropriately substituted 2-fluoroaniline of formula (B) in a suitable solvent system such as THF in the presence of a suitable base such as lithium hexamethyldisilazane at a temperature of-78 ℃ to room temperature, preferably room temperature, to provide 3-halo-5- [ (2-fluoro-4-R) of general formula (1)²) Amino group]An isonicotinic acid intermediate.

The intermediate of formula (1) is then converted to an intermediate of formula (2) by reaction with a suitable amine of formula (C) in the presence of a suitable activating agent such as N, N-carbonyldiimidazole in a suitable solvent system such as N, N-dimethylformamide at a temperature from room temperature to the boiling point of the corresponding solvent, preferably at room temperature.

The intermediate of formula (2) is reacted with a suitable alcohol of formula (D), such as 5-hydroxy-1, 3-benzoxazol-2 (3H) -one, in the presence of a suitable base, such as cesium carbonate, in a suitable solvent, such as N, N-dimethylformamide, at a temperature from room temperature to the boiling point of the corresponding solvent, to provide the compound of formula (I).

The compounds of general formula (Ia) can be synthesized according to the methods described in scheme 2. Compounds of formula E are commercially available.

Route 2

Route 2. other general routes for the preparation of compounds of general formula (Ia), wherein R²And R³Have the meanings described above for the general formula (I). PG represents a "suitable protecting group", for example, a tert-butyloxycarbonyl (Boc) group. Y represents a carbon atom or a S = O group. R represents C₁-C₆-an alkyl group.

Intermediates of formula (2) are converted to intermediates of formula (3) by reaction with suitably protected substituted phenols of formula (E), such as tert-butyl (3-hydroxyphenyl) carbamate, in a suitable solvent system, such as N, N-dimethylformamide, in the presence of a suitable base, such as cesium carbonate, at a temperature from room temperature to the boiling point of the corresponding solvent.

The intermediate of general formula (3) is converted to the intermediate of general formula (4) by removal of the protecting group, e.g. removal of tert-butyloxycarbonyl (Boc), by methods known to the skilled person in the art in the presence of a suitable acid, such as TFA, in a suitable solvent, such as DCM, at a temperature between room temperature and the boiling point of the solvent.

The intermediate of formula (4) is then reacted with a suitable sulfonyl chloride or carbonyl chloride of formula (F), for example isopropyl sulfonyl chloride, in the presence of a suitable base such as pyridine in a suitable solvent such as pyridine at a temperature of from 0 ℃ to room temperature to provide the compound of formula (Ia).

The compounds of general formula (Ib) can be synthesized according to the procedures described in scheme 3. Compounds of formula G are commercially available.

Route 3

Route 3. more particular Process for preparing Compounds of formula (Ib) by dihydroxylation of intermediates of formula (5), wherein R²And R³Have the meanings described above for the general formula (I). Q, Q 'and Q' 'independently of one another represent a hydrogen atom, a methyl group, or 1-2 alkenyl groups forming a ring (it being understood that Q' and Q '' together may form a cycloalkenyl ring).

Dihydroxylation of the synthetic intermediates of general formula (5), which can be obtained by the methods described in schemes 1 and 2 and analogously to the detailed description (see below), gives exemplary compounds of general formula (Ib). Conditions for the dihydroxylation of olefins are well known in the art and include the use of stoichiometric amounts of osmium tetroxide or alkaline potassium permanganate. Alternatively, catalytic amounts of osmium tetroxide can be used in combination with stoichiometric oxidizing agents, such as hydrogen peroxide, tert-butyl hydroperoxide, N-methylmorpholine-N-oxide or potassium ferricyanide K₃Fe(CN)₆. Optionally, a promoter (promoter) such as DMAP may promote this conversion in a suitable solvent such as acetone. Whereas the conditions described provide the corresponding diol in racemic form, stereoselectivity of the olefin can be achieved using the Sharpless Asymmetric Dihydroxylation (SAD) reaction (see: chem. Rev.1994,94,2483) under conditions widely known in the artAnd (4) dihydroxylation.

The compounds of formula (I) can be converted into compounds of formula (Ic) according to the procedures described in scheme 4.

Route 4

Scheme 4. general procedure for converting Compounds of formula (I) to Compounds of formula (Ic), wherein R¹、R²And R³Has the meaning described above for the general formula (I), and R represents C₂-C₆-an alkenyl group or a hydrogen atom.

The compounds of formula (I) are converted into compounds of formula (Ic) by coupling reactions known to the person skilled in the art, preferably by Sonogashira coupling reaction or Sonogashira-type coupling reaction with acetylene or an acetylene equivalent (equivalent) (see below).

The iodine or bromine containing intermediates of formula (I), for example, can be reacted with acetylene in the presence of a catalytic amount of a Pd catalyst such as bis (triphenylphosphine) palladium (II) chloride, a catalytic amount of cuprous iodide in the presence of a solvent such as DMF, and optionally in the presence of a base such as a trialkylamine to form the corresponding alkyne derivative (Ib) or, alternatively, a mono-trialkylsilyl protected acetylene such as Trimethylsilyl (TMS) acetylene can be used for such Sonogashira type coupling reaction under the conditions described above, and then the trialkylsilyl group can be removed by treatment with tetrabutylammonium fluoride or potassium carbonate, for example in methanol For example, (a) Chinchilla, R.; Najera, C.Chem.Rev.2007,107,874, (b) Negishi, E. -i., Anastasia, L.Chem.Rev.2003,103,1979; see also (c) Eur.J.org.Chem.2005,20,4256, (d) J.org.Chem.2006,71,2535 and references therein, (e) Chem.Commun.2004,17,1934). Various palladium catalyst/cocatalyst/ligand/base/solvent combinations are disclosed in the scientific literature that allow fine tuning of the desired reaction conditions to provide numerous other functional groups on both coupling partners (see literature in the above review). Furthermore, recently developed methods using, for example, zinc acetylide, alkynyl magnesium salts or alkynyl trifluoroborate broaden the scope of the method.

Detailed description of the experiments

The NMR peak patterns in the following detailed experimental description are described in terms of their behavior in the spectra, without taking into account possible higher order effects. The Name of the compound was generated using ACD/Name Batch version 12.01. In some cases, commonly accepted names for commercially available reagents are used. Biotage optionally equipped with a robotic unit may be usedThe reaction using microwave irradiation is performed in a microwave oven. The reported reaction times using microwave heating are understood to be the defined reaction times after the indicated temperatures have been reached. The compounds and intermediates prepared according to the process of the invention may require purification. Purification of organic compounds is well known to those skilled in the art, and several purification methods may exist for the same compound. In some cases, purification may not be necessary. In some cases, the compound may be purified by crystallization. In some cases, the impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, in particular flash column chromatography: using, for example, pre-filled silica gel columns, e.g. from Separtis, e.g.Quick action silica gel orFast NH₂Silica gel with Isolera autopurifier (Biotage) combination, and eluent, e.g. hexane/ethyl acetate or DCM/methanol gradient. In some cases, the compound may be purified by preparative HPLC: using, for example, an online electrospray ionization mass spectrometer equipped with a diode array detector Waters autopurifier and/or in combination with a suitable pre-packed reverse phase column, and an eluent, such as a gradient of water and acetonitrile (which may contain additives such as trifluoroacetic acid, formic acid or ammonia). In some cases, the purification methods described above can provide those compounds of the invention having a sufficiently basic or acidic functionality in the form of a salt, for example, in the case of a sufficiently basic compound of the invention, for example, in the form of a trifluoroacetate or formate salt, or, in the case of a sufficiently acidic compound of the invention, for example, in the form of an ammonium salt. Such salts can be converted to their free base or free acid forms, respectively, by various methods known to those skilled in the art, or can be used in the form of a salt for subsequent biological assays. It is to be understood that the particular form of the compounds of the invention isolated as described herein (e.g., salts, free bases, etc.) is not necessarily the only form in which the compounds may be used in biological assays to quantify a particular biological activity.

The percent yields reported in the following examples are based on the starting components used in the minimum molar amounts. Air and moisture sensitive liquids and solutions were transferred with a syringe or pipette and introduced into the reaction vessel through a rubber septum. Commercial grade reagents and solvents were used without further purification. The term "vacuum concentration" means that a Buchi rotary evaporator is used at a minimum pressure of about 15mm Hg. All temperatures reported are uncorrected, in degrees Celsius (° C).

In order that the invention may be better understood, the following examples are set forth. These examples are for illustration only and should not be construed as limiting the scope of the invention in any way. Any publications mentioned herein are incorporated by reference in their entirety.

Analytical LC-MS conditions:

the LC-MS data provided in the following detailed experimental description refer (unless otherwise indicated) to the following conditions:

preparative HPLC conditions:

"purification by preparative HPLC" in the detailed experimental description below refers (unless otherwise indicated) to the following conditions:

and (3) analysis:

preparation:

chiral HPLC conditions:

the chiral HPLC data provided in the detailed experimental description below refer to the following conditions:

and (3) analysis:

the system comprises the following steps:	Dionex:Pump680,ASI100,Waters:UV-Detektor2487
		column:	Chiralpak IC5μm150x4.6mm
solvent:	hexane/ethanol 80:20+0.1% diethylamine
		Flow rate:	1.0mL/min
temperature:	25℃
		solution:	1.0mg/mL EtOH/MeOH1:1
sample introduction:	5.0μl
		and (3) detection:	UV280nm

preparation:

flash column chromatography conditions:

"purification by (flash) column chromatography" as described in the detailed experimental description below refers to the use of the Biotage Flashmaster II or Isolera (SP4) purification system. For technical specifications see "Biotage product catalog" at www.biotage.com.

Optical rotation measurement conditions:

the optical rotation was measured in DMSO at a wavelength of 589nm, at 20 ℃, at a concentration of 1.0000g/100mL, for an integration time of 10s, and at a film thickness of 100.00 mm.

Synthesis of intermediates

Intermediate 1.A

Preparation of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinic acid

25g of 3, 5-difluoroisonicotinic acid (157.141mmol,1eq.) and 37,245g of 2-fluoro-4-iodoaniline (157.141mmol.1eq.) were dissolved in 1275,5mL of anhydrous THF and placed under a nitrogen atmosphere. The mixture was cooled to +3 ℃ with an ice bath, after which 471.422mL of lithium hexamethyldisilazide (LiHMDS) solution (1M in THF; 471.422mmol,3.eq.) were slowly added. After the base addition was complete, the reaction mixture was warmed to rt and stirring was continued for 18 h. The reaction mixture was concentrated in vacuo and then partitioned between aqueous sodium hydroxide (2N,800ml) and dichloromethane. The separated organic phase was washed twice with sodium hydroxide solution (2N, 300ml each). The combined aqueous layers were cooled to 0 ℃ and treated with HCl (concentrated hydrochloric acid, 222ml) until ph =2 was reached. The resulting yellow suspension was stirred at room temperature for 18h, then the precipitate was filtered off to give 5.15g (13.01mmol,8%) of the analytically pure target compound as a brown solid. The organic filtrate also formed a suspension. The precipitate was also filtered off and washed twice with dichloromethane. The precipitate was suspended in water (400ml), cooled to 0 ℃ and treated with HCl (concentrated hydrochloric acid, 20ml) until ph =1 was reached. The resulting yellow suspension was stirred at room temperature for 18h, then the precipitate was filtered off to give 28.27g (71.41mmol,45%) of the analytically pure title compound as a yellow solid.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=7.15(t,1H),7.47(dbr,1H),7.66(dd,1H),8.04-8.17(m,2H),8.68(sbr,1H)。

LC-MS retention time: 1.11min

MS ES⁺:376.9[M+H]⁺

Intermediate 2.A

Preparation of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide

5.000g 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinic acid (13.294mmol,1.eq.) and 4.980g1, 1' -carbonyldiimidazole (30.710mmol, 2.31eq) were weighed into a round bottom flask. 650mL of anhydrous DMF was added under nitrogen and the resulting mixture was stirred at 60 ℃ for 1.5 hours. The mixture was cooled to 3 ℃ in an ice bath, then ammonia (25 wt% in water) was added dropwise, and the mixture was stirred at room temperature for 18 hours. The resulting slurry was filtered off, the precipitate washed with water and then dried in vacuo to give 2.54g (6.36mmol,48%) of the analytically pure title compound.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=7.13(dt,1H),7.44(dbr,1H),7.63(dd,1H),8.05-8.18(m,3H),8.34(sbr,1H)。

LC-MS retention time 1.13min

MS ES⁺:375.9[M+H]⁺

EXAMPLES Compounds

Example 1

Preparation of 3- [ (2-fluoro-4-iodophenyl) amino ] -5- [ (2-oxo-2, 3-dihydro-1, 3-benzoxazol-5-yl) oxy ] isonicotinamide.

Under nitrogen 100mg of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino]Isonicotinamide (0.251mmol,1.eq.) was dissolved in DMF, followed by the addition of 245mg cesium carbonate (0.752mmol,3eq.) and 45mg 5-hydroxy-1, 3-benzoxazol-2 (3H) -one (0.301mmol,1.2 eq.). The resulting mixture was stirred at a bath temperature of 50 ℃ for 3 days. Then, the mixture is placed in NH₄Aqueous Cl (30ml) and dichloromethane (30ml) were partitioned. The phases were separated and the aqueous layer was extracted twice more with dichloromethane/isopropanol (4:1, 30ml each). The combined organic layers were washed with brine (30ml), dried on a silicone filter and concentrated in vacuo. The residue was purified by flash chromatography to yield 43mg (0.08mmol,33%) of the analytically pure target compound.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=6.81(dd,1H),6.87(d,1H),7.11(br.dd,1H),7.28(d,1H),7.43(br.d,1H),7.62(dd,1H),7.66(s,1H),7.95(br.s,1H),8.05(br.s,1H),8.06–8.15(m,2H),11.71(br.s,1H)。

LC-MS retention time 1.16min

MS ES⁺:506.9[M+H]⁺

Example 2

Preparation of tert-butyl [3- ({ 4-carbamoyl-5- [ (2-fluoro-4-iodophenyl) amino ] pyridin-3-yl } oxy) phenyl ] carbamate.

31mg of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino]Isonicotinamide (0.083mmol,1.eq.) and 17mg (3-Hydroxynicotinamide)Phenylphenyl) carbamic acid tert-butyl ester (0.083mmol,1eq.) was dissolved in 1ml DMF followed by addition of 81mg cesium carbonate (0.248mmol,3 eq.). The resulting mixture was stirred at room temperature for 18 hours. Due to incomplete reaction, the mixture was stirred at 50 ℃ bath temperature for another 3 days. Then, the mixture is placed in NH₄Aqueous Cl (10ml) and dichloromethane (10ml) were partitioned. The phases were separated and the aqueous layer was extracted twice more with dichloromethane (10ml each). The combined organic layers were washed with brine (20ml), dried on a silicone filter and concentrated in vacuo. The residue was purified by flash chromatography to yield 17mg (0.03mmol,30%) of the analytically pure target compound.

LC-MS retention time 1.39min

MS ES⁺:565.43[M+H]⁺

Example 3

Preparation of 3- (3-aminophenoxy) -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide.

840mg of tert-butyl [3- ({ 4-carbamoyl-5- [ (2-fluoro-4-iodophenyl) amino ] pyridin-3-yl } oxy) phenyl ] carbamate (1,488mmol,1eq.) are dissolved in 8mL of dichloromethane under a nitrogen atmosphere. Then 1.6ml TFA was added and the brown solution was stirred at room temperature for 18 hours. 4ml of saturated aqueous NaHCO3 solution were then added and the mixture was stirred for 4 hours while forming a suspension which was filtered. The precipitate was dried in vacuo to give 615mg of analytically pure target compound (1.488mmol, yield 87%) as a yellow solid.

1H-NMR(400MHz,DMSO-d6):δ[ppm]=5.24(m,2H),6.15(m,1H),6.19(m,1H),6.31(m,1H),6.97(t,1H),7.12(m,1H),7.43(br.d,1H),7.62(dd,1H),7.72(m,1H),7.94(br.s,2H),8.08–8.12(m,1H),8.24(br.s,1H)。

LC-MS retention time 1.17min

MS ES⁺:464.7[M+H]⁺

Example 4

Preparation of 3- [ (2-fluoro-4-iodophenyl) amino ] -5- {3- [ (isopropylsulfonyl) amino ] phenoxy } isonicotinamide.

150mg of 3- (3-aminophenoxy) -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (0.323mmol,1eq.) was dissolved in 2mL of pyridine under nitrogen, then 69mg of isopropylsulfonyl chloride (0.485mmol,1.5eq.) was added and the mixture was stirred at room temperature for 18 hours. The reaction residue was partitioned between dichloromethane (20ml) and water (10 ml). The phases were separated and the aqueous phase was extracted twice more with dichloromethane (20ml each). The combined organic layers were washed with brine (20ml), dried on a silicone filter and concentrated in vacuo. The liquid residue is dissolved in 4ml of toluene and concentrated again in vacuo. Purification by preparative HPLC afforded 45mg of the analytically pure target compound (0.07mmol, yield 21%).

1H-NMR(300MHz,DMSO-d6):δ[ppm]=1.20(d,6H),2.38–2.56(m,1H),6.67(dd,1H),6.91-7.00(m,2H),7.12(dt,1H),7.27(t,1H),7.42(br.d,1H),7.62(dd,1H),7.76(d,1H),7.92(br.s,1H),8.03(br.s,1H),8.12-8.16(m,2H),9.88(br.s,1H)。

LC-MS retention time 1.23min

MS ES⁺:571.1[M+H]⁺

In analogy to general procedure 1, the following example compounds 5-10 were prepared by treatment of 3- (3-aminophenoxy) -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide with the corresponding commercially available sulfonyl chloride and phosgene in the presence of pyridine.

Example 11

Preparation of 3- [ (2-fluoro-4-iodophenyl) amino ] -5- [ (4-methylpent-3-en-1-yl) oxy ] isonicotinamide.

500mg of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino]Isonicotinamide (0.253mmol,1.eq.) was dissolved in 5ml of DMF and then 1225mg cesium carbonate (3.759mmol,3eq.) and 125mg 4-methyl-3-penten-1-ol (0.253mmol,1eq.) were added. The resulting mixture was stirred at a bath temperature of 70 ℃ for 24 hours. Due to incomplete reaction, the mixture was stirred at a bath temperature of 70 ℃ for a further 2 days. Due to incomplete reaction, an additional 125mg of 4-methyl-3-penten-1-ol (0.253mmol,1eq.) and 408mg of cesium carbonate (0.253mmol,1eq.) were added and the mixture was stirred at a bath temperature of 70 ℃ for an additional 2 days. The mixture obtained is reacted in NH₄Aqueous Cl (50ml) and dichloromethane (50ml) were partitioned. The phases were separated and the aqueous layer was extracted twice more with dichloromethane (50ml each). The combined organic layers were washed with brine (50ml), dried on a silicone filter and concentrated in vacuo. The residue was suspended in ethyl acetate and stirred for 1h, after which the precipitate was filtered off and dried in vacuo to yield 252mg (0.52mmol,42%) of the analytically pure title compound.

LC-MS retention time 1.42min

MS ES⁺:456.0[M+H]⁺

Example 12

Preparation of 3- [ (3, 4-dihydroxy-4-methylpentyl) oxy ] -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide.

252mg3- [ (2-fluoro-4-iodophenyl) amino ] -5- [ (4-methylpent-3-en-1-yl) oxy ] isonicotinamide (0.554mmol,1eq.) are dissolved in 36ml acetone and 6ml water are added to form a suspension. Then 454mg of N-methylmorpholino-N-oxide (3.875,7eq.) and 555. mu.l of osmium tetroxide solution (2.5% by weight in tert-butanol, 0.044mmol,0.08eq.) were added and the resulting suspension was stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the residue was partitioned between 50ml each of water and dichloromethane/isopropanol (4: 1). The aqueous layer was separated and extracted twice more with dichloromethane/isopropanol (4: 1). The combined organic layers were washed with brine, dried on a silica gel filter and concentrated to give 272mg (0.55mmol,100%) of the analytically pure target compound without further purification.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=1.00(s,3H),1.05(s,3H),1.58(m,1H),2.04(m,1H),3.25-3.36(m,1H),4.17(s,1H),4.23(m,2H),4.62(d,1H),7.12(t,1H),7.41(br.d,1H),7.61(dd,1H),7.87(br.s,1H),7.95(br.s,1H),7.99-8.06(m,2H),8.78(s,1H)。

LC-MS retention time 1.05min

MS ES⁺:490.0[M+H]⁺

Example 13

Preparation of 3- {3- [ (ethylsulfonyl) amino ] phenoxy } -5- ({ 2-fluoro-4- [ (trimethylsilyl) ethynyl ] phenyl } amino) isonicotinamide.

745mg of 3- [ (2-fluoro-4-iodophenyl) amino ] -5- [3- (propionylamino) phenoxy ] isonicotinamide (1.339mmol;1eq.) are suspended in 13.5ml of triethylamine under nitrogen. 70mg of triphenylphosphine (0.268mmol,0.2eq.) and 31mg of bis (dibenzylideneacetone) palladium (0) (0.054mmol,0.04eq.) and 10mg of cuprous iodide (0.054mmol,0.04eq.) were then added successively. Stirring was carried out for 5min, then 1.1ml of trimethylsilylacetylene (8.034mmol,6eq.) was added, and the resulting mixture was stirred at a bath temperature of 60 ℃ for 18 h. The suspension was filtered and the residue was concentrated in vacuo. The residue was purified by flash chromatography to yield 262mg (0.47mmol,35%) of the analytically pure target compound.

LC-MS retention time 1.46min

MS ES⁺:527.1[M+H]⁺

Example 14

Preparation of 3- {3- [ (ethylsulfonyl) amino ] phenoxy } -5- [ (4-ethynyl-2-fluorophenyl) amino ] isonicotinamide.

258mg of 3- {3- [ (ethylsulfonyl) amino ] phenoxy } -5- ({ 2-fluoro-4- [ (trifluoromethylsilyl) ethynyl ] phenyl } amino) isonicotinamide (0.490mmol,1eq.) was dissolved in 4mL of tetrahydrofuran under a nitrogen atmosphere. Then 0.5ml of tetra-n-butylammonium fluoride (0.490mmol,1eq.) was added and the mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between ethyl acetate and half-saturated (half-saturated) aqueous sodium bicarbonate. The organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash chromatography to yield 101mg (0.22mmol,45%) of the analytically pure target compound.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=1.15(t,3H),3.09(q,2H),4.14(s,1H),6.70(dd,1H),6.90–6.98(m,2H),7.16–7.39(m,4H),7.83(s,1H),7.93(br.s,1H),8.04(br.s,1H),8.22-8.28(m,2H),9.92(s,1H)。

LC-MS retention time 1.13min

MS ES⁺:455.0[M+H]⁺

Example 15

Preparation of 3- [ (2-fluoro-4-iodophenyl) amino ] -5-methoxyisonicotinamide.

140mg of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (0.340mmol,1eq.) are dissolved in 3mL of tetrahydrofuran and cooled to a bath temperature of-75 ℃, after which 18mg of sodium methoxide (0.340mmol,1eq.) are added and the mixture is brought to room temperature. The resulting mixture was then stirred at a bath temperature of 80 ℃ for 3 days. Due to incomplete reaction, an additional 18mg of sodium methoxide (0.340mmol,1eq.) was added and the mixture was stirred at 80 ℃ bath temperature for an additional 1 day. The reaction mixture was concentrated in vacuo and the residue was partitioned between 20ml each of water and dichloromethane. The aqueous layer was separated and extracted twice more with dichloromethane (20ml each). The combined organic layers were washed with brine (30ml), dried on a silicone filter and concentrated. The residue was purified by flash chromatography to yield 67mg (0.17mmol,51%) of the analytically pure target compound.

1H-NMR(300MHz,DMSO-d6):δ[ppm]=3.89(s,3H),7.09(t,1H),7.40(br.d,1H),7.60(dd,1H),7.87(br.s,1H),7.89(br.s,1H),8.00-8.05(m,2H),8.59(s,1H)。

LC-MS retention time 1.09min

MS ES⁺:387.9[M+H]⁺

Example 16

Preparation of N- (2-aminophenyl) -3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide.

100mg of 3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinic acid (0,266mmol,1eq.; intermediate 1A) was suspended in 2mL of DMF, then 28,754mg of 1, 2-diaminobenzene (0,266mmol,1eq.), 215,339mg of HATU (0,566mmol,2.13eq.) and 73,196mg of N, N-diisopropylethylamine (0,566mmol,2.13eq.) were added and stirring was continued at 50 ℃ for 18 h. The reaction mixture was partitioned between 20ml ethyl acetate and 20ml brine. The aqueous layer was extracted twice more with 20ml ethyl acetate each time. The combined organic layers were dried on a silicone filter and concentrated in vacuo. The residue was purified by flash chromatography to give 69mg of the title compound in a UV purity of 72%. This material was used in the next step without further purification.

LC-MS retention time 1.29min

MS ES⁺:466.9[M+H]⁺

Example 17

Preparation of N- (2-acetamidophenyl) -3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide.

69mg of N- (2-aminophenyl) -3-fluoro-5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (0,148mmol,1eq.) are dissolved in 1mL of tetrahydrofuran under nitrogen, then 0.592mL of a solution of hexamethyldisilazane (1M in THF, 4eq.) is added and stirring is continued at room temperature for 18 h. The reaction mixture was partitioned between 20ml ethyl acetate and 15ml aqueous HCL (1M). The aqueous layer was extracted twice more with 20ml ethyl acetate each time. The combined organic layers were washed with 20ml of brine, dried on a silicone filter and concentrated in vacuo. The residue was purified by flash chromatography to give 14mg (0.03mmol,19%) of the analytically pure target compound.

1H-NMR(400MHz,DMSO-d6):δ[ppm]=1.97(s,3H),7.10(td,1H),7.13–7.21(m,2H),7.39–7.49(m,3H),7.63(dd,1H),8.14(br.s,1H),8.20(s,1H),8.33(br.s,1H),9.48(s,1H),10.00(s,1H)。

LC-MS retention time 1.21min

MS ES⁺:509.17[M+H]⁺

In addition, the compounds of formula (I) of the present invention may be converted into any salt as described herein by any method known to those skilled in the art. Similarly, any salt of a compound of formula (I) of the present invention may be converted to the free compound by any method known to those skilled in the art.

Pharmaceutical compositions of the compounds of the invention

The invention also relates to pharmaceutical compositions comprising one or more compounds of the invention. These compositions can be used to achieve a desired pharmacological effect by administration to a patient in need thereof. For purposes of the present invention, a patient is a mammal, including a human, in need of treatment for a particular condition or disease. Accordingly, the present invention includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of the present invention or a salt thereof. A pharmaceutically acceptable carrier is preferably one that is relatively non-toxic and non-injurious to a patient at concentrations consistent with effective activity of the active ingredient, such that any side effects caused by the carrier do not destroy the beneficial effects of the active ingredient. A pharmaceutically effective amount of a compound is preferably an amount that results in or affects the particular condition being treated. The compounds of the present invention may be administered together with a pharmaceutically acceptable carrier in any effective conventional dosage unit form including immediate release, sustained release and timed release formulations in the following manner: oral, parenteral, topical, nasal, ocular (ophthalmic), sublingual, rectal, vaginal administration and the like.

For oral administration, the compounds may be formulated into solid or liquid preparations such as capsules, pills, tablets, troches (troche), lozenges (lozenge), melt gels (melt), powders, solutions, suspensions or emulsions and may be prepared according to methods known in the art for the preparation of pharmaceutical compositions. The solid unit dosage form may be a capsule, which may be of the ordinary hard or soft capsule type, comprising, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment, the compounds of the present invention can be compressed into tablets with conventional tablet bases (e.g., lactose, sucrose, and corn starch) and in combination with: binders such as acacia, corn starch or gelatin, disintegrating agents such as potato starch, alginic acid, corn starch and guar gum, tragacanth, acacia for assisting the disintegration and dissolution of the tablets after administration, lubricants such as talc, stearic acid or magnesium stearate, calcium stearate or zinc stearate for improving the flowability of the tablet granulation and preventing adhesion of the tablet materials to the surfaces of the tablet dies and punches, dyes, colorants and flavouring agents such as peppermint, oil of wintergreen or cherry flavouring for improving the organoleptic properties of the tablets and making them more acceptable to the patient. Suitable excipients for oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols (e.g., ethanol, benzyl alcohol, and polyvinyl alcohol), with or without the addition of pharmaceutically acceptable surfactants, suspending agents, or emulsifying agents. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both.

Dispersible powders and granules are suitable for use in the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, such as those described above, may also be present.

The pharmaceutical composition of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as liquid paraffin, or a mixture of vegetable oils. Suitable emulsifying agents may be (1) natural gums, for example gum acacia and gum tragacanth, (2) natural phosphatides, for example soya bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, (4) condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. The suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate; one or more colorants; one or more flavoring agents; and one or more sweetening agents, such as sucrose or saccharin.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent and a preservative such as methyl and propyl parabens as well as flavoring and coloring agents.

The compounds of the invention may also be administered parenterally, i.e., subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly or intraperitoneally, in injectable doses of the compounds, preferably in a physiologically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid or a mixture of liquids, such as water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol, isopropanol or cetyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2, 2-dimethyl-1, 1-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides or acetylated glycerides, with or without the addition of pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hypromellose or carboxymethylcellulose, or emulsifying agents and other pharmaceutically acceptable adjuvants.

Exemplary oils useful in the parenteral formulations of the invention are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal, ammonium and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, alkyl pyridinium halides and alkylamine acetates; anionic detergents such as alkyl sulfonates, aryl sulfonates and olefin sulfonates, alkyl sulfates and alkyl sulfosuccinates, olefin sulfates and olefin sulfosuccinates, ether sulfates and ether sulfosuccinates and monoglyceride sulfates and monoglycerides sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides, and poly (oxyethylene-oxypropylene), ethylene oxide copolymers or propylene oxide copolymers; and amphoteric detergents such as alkyl-beta-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and mixtures thereof.

The parenteral compositions of the invention will typically comprise from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be advantageously employed. To minimize or eliminate irritation at the injection site, such compositions may comprise a nonionic surfactant having a hydrophilic-lipophilic balance (HLB) of preferably from about 12 to about 17. The amount of surfactant in such formulations is preferably from about 5% to about 15% by weight. The surfactant may be a single component having the above HLB, or a mixture of two or more components having the desired HLB.

Exemplary surfactants for parenteral formulations are polyethylene sorbitan fatty acid esters such as sorbitan monoleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide and propylene glycol.

The pharmaceutical composition may be in the form of a sterile aqueous suspension for injection. Such suspensions may be formulated according to known methods using: suitable dispersing or wetting agents and suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hypromellose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally-occurring phosphatide, for example lecithin, condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.

The sterile injectable preparation may also be a sterile solution or suspension for injection in a non-toxic parenterally-acceptable diluent or solvent. Diluents and solvents which can be used are, for example, water, ringer's solution, isotonic sodium chloride solution and isotonic glucose solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. In this regard, any less irritating fixed oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are, for example, cocoa butter and polyethylene glycols.

Another formulation used in the methods of the invention utilizes a transdermal delivery device ("patch"). Such transdermal patches may be used to provide continuous or discontinuous delivery of a controlled amount of a compound of the present invention. The construction and use of transdermal patches for delivering agents is well known in the art (see, e.g., U.S. patent No.5,023,252 to 1991, published on 6/11, which is incorporated herein by reference). Such patches may be configured for continuous, pulsed, or on-demand delivery of the agent.

Controlled release formulations for parenteral administration include liposomal microspheres, polymeric microspheres, and polymeric gel formulations known in the art.

It may be desirable or necessary to deliver the pharmaceutical composition to a patient by a mechanical delivery device. The construction and use of mechanical delivery devices for delivering pharmaceutical agents is well known in the art. Direct techniques such as administering drugs directly to the brain typically involve placing a drug delivery catheter into the ventricular system of the patient to bypass the blood brain barrier. One such implantable delivery system for delivering agents to specific anatomical locations of the body is described in U.S. patent No. 5011472, published 4-30 1991.

The compositions of the present invention may also contain, as necessary or desired, other conventional pharmaceutically acceptable formulation ingredients, which are commonly referred to as carriers or diluents. Conventional procedures for preparing such compositions into suitable dosage forms may be used. Such ingredients and procedures include those described in the following references, all of which are incorporated herein by reference: powell, M.F. et al, "Complex of Excipients for particulate Formulations" PDA Journal of Pharmaceutical Science & Technology1998,52(5), 238-.

Common pharmaceutical ingredients that may be used to formulate the composition for the intended route of administration include:

acidulants (examples include, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid);

alkalizing agents (examples include, but are not limited to, ammonia, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine (triethanolamine), triethanolamine (trolamine));

adsorbents (examples include, but are not limited to, powdered cellulose and activated carbon);

aerosol propellants (examples include, but are not limited to, carbon dioxide, CCl₂F₂、F₂ClC-CClF₂And CClF₃)；

Air displacement agents (examples include, but are not limited to, nitrogen and argon);

antifungal preservatives (examples include, but are not limited to, benzoic acid, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, sodium benzoate);

antibacterial preservatives (examples include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, and thimerosal);

antioxidants (examples include, but are not limited to, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, thioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite);

adhesive substances (examples include, but are not limited to, block polymers, natural and synthetic rubbers, polyacrylates, polyurethanes, silicones, polysiloxanes, and styrene-butadiene copolymers);

buffering agents (examples include, but are not limited to, potassium metaphosphate, dipotassium hydrogen phosphate, sodium acetate, anhydrous sodium citrate, and sodium citrate dihydrate);

a carrier (examples include, but are not limited to, acacia syrup, flavoring elixir, cherry syrup, cocoa syrup, orange syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection, and bacteriostatic water for injection);

chelating agents (examples include, but are not limited to, sodium edetate and edetic acid);

coloring agents (examples include, but are not limited to FD & C Red No.3, FD & C Red No.20, FD & C Yellow No.6, FD & C Blue No.2, D & C Green No.5, D & C Orange No.5, D & C Red No.8, caramel, and Red iron oxide);

clarifying agents (examples include, but are not limited to, bentonite);

emulsifying agents (examples include, but are not limited to, acacia, cetomacrogol, cetyl alcohol, glycerol monostearate, lecithin, sorbitan monooleate, polyoxyethylene 50 monostearate);

encapsulating agents (examples include, but are not limited to, gelatin and cellulose acetate phthalate);

flavors (examples include, but are not limited to, anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil, and vanillin);

humectants (examples include, but are not limited to, glycerin, propylene glycol, and sorbitol);

abrasives (examples include, but are not limited to, mineral oil and glycerin);

oils (examples include, but are not limited to, peanut oil (arachis oil), mineral oil, olive oil, peanut oil (peanout oil), sesame oil, and vegetable oils);

ointment bases (examples include, but are not limited to, lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment);

penetration enhancers (transdermal delivery) (examples include, but are not limited to, mono-or polyhydric alcohols, mono-or polyvalent alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalins, terpenes, amides, ethers, ketones, and ureas);

plasticizers (examples include, but are not limited to, diethyl phthalate and glycerol);

solvents (examples include, but are not limited to, ethanol, corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection, and sterile water for rinsing);

hardening agents (examples include, but are not limited to, cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin, stearyl alcohol, white wax, and yellow wax);

suppository bases (examples include, but are not limited to, cocoa butter and polyethylene glycol (mixtures));

surfactants (examples include, but are not limited to, benzalkonium chloride, nonoxynol 10, octoxynol 9, polysorbate 80, sodium lauryl sulfate, and sorbitan monopalmitate);

suspending agents (examples include, but are not limited to, agar, bentonite, carbomer, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hypromellose, kaolin, methylcellulose, tragacanth and magnesium aluminum silicate);

sweetening agents (examples include, but are not limited to, aspartame, dextrose, glycerin, mannitol, propylene glycol, saccharin sodium, sorbitol, and sucrose);

tablet antiadherents (examples include, but are not limited to, magnesium stearate and talc);

tablet binders (examples include, but are not limited to, acacia, alginic acid, sodium carboxymethylcellulose, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, non-crosslinked polyvinylpyrrolidone, and pregelatinized starch);

tablet and capsule diluents (examples include, but are not limited to, dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium phosphate, sorbitol, and starch);

tablet coatings (examples include, but are not limited to, liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, methyl cellulose, ethyl cellulose, cellulose acetate phthalate, and shellac);

tablet direct compression excipients (examples include, but are not limited to, dibasic calcium phosphate);

tablet disintegrating agents (examples include, but are not limited to, alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin potassium, crospovidone, sodium alginate, sodium starch glycolate, and starch);

tablet glidants (examples include, but are not limited to, colloidal silicon dioxide, corn starch, and talc);

tablet lubricants (examples include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, stearic acid, and zinc stearate);

tablet/capsule opacifiers (examples include but are not limited to titanium dioxide);

tablet polishes (examples include, but are not limited to, carnauba wax and white wax);

thickening agents (examples include, but are not limited to, beeswax, cetyl alcohol, and paraffin wax);

tonicity agents (examples include, but are not limited to, glucose and sodium chloride);

viscosity increasing agents (examples include, but are not limited to, alginic acid, bentonite, carbomer, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, sodium alginate, and gum tragacanth); and

wetting agents (examples include, but are not limited to, heptadecaethyleneoxycetanol, lecithin, sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

The pharmaceutical composition of the present invention can be exemplified as follows:

sterile intravenous solution: sterile water for injection can be used to prepare a 5mg/mL solution of the desired compound of the invention, with the pH adjusted as necessary. The solution was diluted to 1-2mg/mL with sterile 5% glucose for administration and administered as an intravenous infusion over about 60 min.

Lyophilized powder for intravenous administration: sterile preparations can be prepared from (i)100-1000mg of the desired compound of the invention in the form of a lyophilized powder, (ii)32-327mg/mL sodium citrate, and (iii)300-3000mg dextran 40. The formulation is reconstituted to a concentration of 10-20mg/mL with sterile saline for injection or 5% glucose, then further diluted to 0.2-0.4mg/mL with saline or 5% glucose and administered as an intravenous bolus or intravenous infusion over 15-60 minutes.

Intramuscular injection suspension: the following solutions or suspensions can be prepared for intramuscular injection:

50mg/mL of the desired Water-insoluble Compound of the invention

5mg/mL sodium carboxymethylcellulose

4mg/mL TWEEN80

9mg/mL sodium chloride

9mg/mL benzyl alcohol

Hard capsule: a large number of unit capsules were prepared by filling standard two-piece hard capsules with 100mg of powdered active ingredient, 150mg of lactose, 50mg of cellulose and 6mg of magnesium stearate, respectively.

Soft capsule: preparation of active ingredient in digestible oil such as soybean oil, cotton seed oil or olive oilAnd injected into molten gelatin by a positive displacement pump to form soft capsules containing 100mg of the active ingredient. The capsules were washed and dried. The active ingredient may be dissolved in a mixture of polyethylene glycol, glycerol and sorbitol to prepare a water-miscible drug mixture.

Tablet formulation: a number of tablets were prepared by conventional procedures such that the dosage unit contained 100mg of active ingredient, 0.2mg of colloidal silicon dioxide, 5mg of magnesium stearate, 275mg of microcrystalline cellulose, 11mg of starch and 98.8mg of lactose. Suitable aqueous and non-aqueous coatings may be employed to increase palatability, improve appearance and stability, or delay absorption.

Immediate release tablet/capsule: these are solid oral dosage forms prepared by conventional and novel processes. These units are taken orally without water for immediate dissolution and delivery of the drug. The active ingredient is mixed in a liquid containing ingredients such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or caplets by freeze-drying and solid-state extraction techniques. The pharmaceutical compound can be tableted with a viscoelastic and thermoelastic sugar and a polymer or effervescent component to produce a porous matrix that is quick-releasing without the need for water.

Combination therapy

The compounds of the present invention may be administered as the sole agent or in combination with one or more other agents, wherein the combination does not cause unacceptable adverse effects. The invention also relates to such combinations. For example, the compounds of the present invention may be combined with known agents and the like that are resistant to hyperproliferative diseases or other indications, as well as mixtures and combinations thereof. Other indications include, but are not limited to, anti-angiogenic agents, mitotic inhibitors, alkylating agents, anti-metabolites, DNA-intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzyme inhibitors, topoisomerase inhibitors, biological response modifiers, or anti-hormones.

The additional agent may be aldesleukin, alendronic acid, alpha-interferon (alfaferone), alitretinoin, allopurinol, sodium allopurinol for injection (alprimem), palonosetron hydrochloride injection (aloxi), altretamine, aminoglutethimide, amifostine, amsacrine, anastrozole, dolasetron tablet (anzmet), alfa bepotastine injection (aranesp), arglabin, arsenic trioxide, exemestane tablet, 5-azacytidine, azathioprine, BAY80-6946, BCG or tide, amastatin, betamethasone acetate, betamethasone phosphate sodium, bexarotene, bleomycin sulfate, uridine bromide, bortezomib, busulfan, calcitonin, alendronide, alendronabine (calmutath), carboplatin, platinum, bipenemelamine, interleukins, cline, clindamycin sulfate, clindamycin, berrubine, clindamycin, berrubicin, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin liposome citrate (Daunoxome), dexamethasone sodium phosphate, estradiol valerate, dinebukin 2(denileukin diftox), methylprednisolone, deslorelin, dexrazoxane, diethylstilbestrol, fluconazole, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, leuprolide acetate (eligard), labyrine injection (elitek), epirubicin hydrochloride injection (ellence), aprepirubitan capsule (emide), epirubicin, alfafurtine (epoetin), alfafa (epogen), alfa (epogen), etaplatin, levamisole, estradiol (estrace), estradiol sodium phosphate, estramustine, ethinyl estradiol, amifostine, etidronic acid, etoposide, etofazole, farston, filgrastim, finasteride, filgrastim, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, formestane, fosetabine, fotemustine, fulvestrant, gamma-globulin (gammagard), gemcitabine, gemumab, imatinib mesylate (gleevec), carmustine wafer capsule (gliadel), goserelin, glatirelin hydrochloride, histrelin, topotecan (hydroxycamtin), hydrocortisone, erythrohydroxynonyladenine (eynyladenine), hydroxyurea, ibritumomab, idarubicin, ifosfamide, alpha interferon, alpha 2 interferon, alpha-2A interferon, alpha-2B interferon, alpha-n 1 interferon, alpha-3 interferon, beta interferon, gamma-1 a interferon, gamma-1 a interferon, and gamma-gamma, Interleukin-2, interferon alpha (intron A), gefitinib tablet (iressa), irinotecan, granisetron, lentinan sulfate, letrozole, leucovorin, leuprolide acetate, levamisole, calcium levofolinate (levofolinic acid calcium salt), levothyroxine sodium (levothroid), levothyroxine sodium (levoxyl), lomustine, lonidamine, dronabinol, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen tablet (menest), 6-mercaptopurine, mesna, methotrexate, metivox, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane (Modrenal), Myocoet, nedaplatin, filgrastim (neurin), recombinant interleukin 11 (nethea), glutethionine (netrux), milbexate, medetomidine (D), and mycophenolate, NSC-631570, OCT-43, octreotide, ondansetron hydrochloride, cefixime (orapirred), oxaliplatin, paclitaxel, prednisone sodium phosphate (pidiapred), pemetrexed, pyroxin, pentostatin, streptolysin (picibanil), pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, poinimustine, prednisolone, prednisone, equine estrogens, procarbazine, recombinant human erythropoietin alpha, raltitrexed, RDEA119, recombinant human interferon beta 1a injection (rebif), rhenium-186 hydroxyethylphosphonate, rituximab, roscovalen (roferon-A), romopeptide, pilocarpine hydrochloride (salagen), octreotide, sargrastim, semustine, xifuran, sobutyrazol, prednisolone, phosphoethanine, dry cell therapy, strontium 89, sodium thyroxine chloride, levofloxacin, and paclitaxel, Tamsulosin, tasolinamine, testolactone, docetaxel injection (taxotere), temocillin, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, troosulfan, tretinoin, methotrexate (trexal), trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, vilulizine, dexrazoxane, netostatin stimalamer, ondansetron, ABI-007, acolbifene, interferon gamma-1 b (actalimune), afafitfenak, aminopterin, acyclonidine, oxyprisperidone, alprostanil, astrisil, astrotrichub, Avenasfeldr 779, Avenasfeld-779, trex, tretinomycin, trexab, trexate, trexabexabin, trexabin, trexatrinosin, trexatrinexazine, trexase, trexatrinexazine, trexat, CDC-501, celecoxib, cetuximab, clinatropine, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, isoxatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implants, holmium-166 DOTMP, ibandronic acid, gamma interferon, pegylated interferon alpha-2 b (intron-PEG), ixabepilone (ixabepilone), keyhole limpet hemocyanin (keyhol lipped hemoanin), L-651582, lanreotide, lasofoxifene, libra, farnesol protein transferase inhibitor (lonafinaranfamib), mirhexifen, minophosphonic acid (minodronate), MS-209, MTP-PE liposomes, MX-6, nararelin, nevira, novalubicin, norvonospherol, trexostat, TCidosoxel, paclitaxel, disodium-S, dimeglumine, dimeglume, dimerate, medrycan, melphalan, paclobulin, PN-401, QS-21, quazepam, R-1549, raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, seocalcitol, T-138067, erlotinib hydrochloride tablets (tarceva), taxoprxin, alpha-1 thymosin, thiazolufrine, tipifarnib, tirapazamine, TLK-286, toremifene, TransMID-107R, valcephradine, vapreotide, vatalanib (vatalanib), verteporfin, vinflunine, Z-100, zoledronic acid, or combinations thereof.

Optional anti-hyperproliferative agents that may be added to the compositions include, but are not limited to, compounds listed in the cancer chemotherapeutic regimen of the Merck index 11 edition (1996) (incorporated by reference), such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, asparaginase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycin), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifene, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, and, Vinblastine and vindesine.

Other anti-hyperproliferative agents suitable for use with The compositions of The present invention include, but are not limited to, those compounds recognized in Goodman and Gilman's The Pharmacological Basis of Therapeutics (9 th edition), edited by Molinoff et al, McGraw-Hill, pages 1225-1287 (1996) (incorporated by reference) for use in The treatment of neoplastic disease, such as aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2' -difluorodeoxycytidine, docetaxel, erythrononyl adenine, ethinylestradiol, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone hexanoate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, medecan, Paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.

Other anti-hyperproliferative agents suitable for use with the compositions of the present invention include, but are not limited to, other anti-cancer agents such as epothilone and its derivatives, irinotecan, raloxifene and topotecan.

The compounds of the invention may also be administered in combination with a protein therapeutic. Such protein therapeutics suitable for use in the treatment of cancer or other angiogenic disorders and suitable for use with the compositions of the invention include, but are not limited to, interferons (e.g., alpha, beta, or gamma interferons), hyperactive monoclonal antibodies, Tuebingen, TRP-1 protein vaccines, Colostrinin, anti-FAP antibodies, YH-16, gimumab, infliximab, cetuximab, trastuzumab, dinil interleukin 2, rituximab, alpha 1 thymosin, bevacizumab, mecamylamine Rifafibate (mecamylin Rifabat), Ompur interleukin, natalizumab, rhMBL, MFE-CP1+ ZD-2767-P, ABT-828, ErbB 2-specific immunotoxins, SGN-35, MT-103, Rifamate (rinfabate), AS-1402, B43-genistein, L-19 series radioimmunotherapeutic, AC-9301, NY-ESO-1 vaccine, IMC-1C11, CT-322, rhCC10, r (m) CRP, MORAB-009, Avisuramine (aviscumine), MDX-1307, Her-2 vaccine, APC-8024, NGR-hTNF, rhH1.3, IGN-311, endostatin, Voloximab (volociximab), PRO-1762, lexatuzumab (lexatuzumab), SGN-40, pertuzumab (pertuzumab), EMD-273, L19-IL-2 fusion protein, PRX-321, CNTO-328, MDX-214, tegafur peptide (tigotide), CAT-3888, labetazumab (labetazumab), radiolabeled pertuzumab (Labetuzumab), radioisotope-crosslinked trastuzumab of eming particles, EM-Acuk-1421, interleukin (Hitachi), HPV-7, HPV-3516, HPV-30625, and, Javelin-melanoma, NY-ESO-1 vaccine, EGF vaccine, CYT-004-MelQbG10, WT1 peptide, agovacizumab (oregomab), ofatumumab, zalutumumab (zalutumumab), betheumatin interleukin (cindrekin bestudox), WX-G250, Albuferon, aflibercept, denosumab (denosumab), vaccine, CTP-37, efungumab (efungumab) or 131I-chTNT-1/B. Monoclonal antibodies useful as protein therapeutics include, but are not limited to, molobuzumab-CD 3, abciximab, edrecolomab, daclizumab, gemtuzumab (gentuzumab), alemtuzumab, ibritumomab tiuxetan (ibritumomab), cetuximab, bevacizumab, efalizumab (efalizumab), adalimumab (adalimumab), omalizumab, moelimumab-CD 3, rituximab, daclizumab, trastuzumab, palivizumab, basiliximab, and infliximab.

In general, the use of cytotoxic and/or cytostatic agents in combination with a compound or composition of the invention will serve the following functions:

(1) produces better efficacy in reducing tumor growth or even eliminating tumors than either agent administered alone,

(2) allowing for the administration of smaller amounts of the administered chemotherapeutic agent,

(3) providing a chemotherapeutic treatment that is well tolerated by patients and has fewer harmful pharmacological complications than observed with single agent chemotherapy and certain other combination therapies,

(4) allowing the treatment of a wider range of different cancer types in mammals, particularly humans,

(5) providing a higher response rate in the treated patient,

(6) provides longer survival in the treated patient compared to standard chemotherapy treatment,

(7) provide longer tumor progression time, and/or

(8) At least as good efficacy and tolerability as the agents used alone are obtained as compared to known cases where other cancer agents produce antagonistic effects in combination.

Method for sensitizing cells to radiation

In a different embodiment of the invention, the compounds of the invention can be used to sensitize cells to radiation. That is, treatment of cells with a compound of the invention prior to radiation therapy of the cells makes the cells more susceptible to DNA damage and cell death than they would be if the cells were not subjected to any treatment with a compound of the invention. In one aspect, a cell is treated with at least one compound of the invention.

Accordingly, the present invention also provides a method of killing cells, wherein one or more compounds of the invention are administered to the cells along with conventional radiation therapy.

The invention also provides methods of rendering a cell more susceptible to cell death, wherein the cell is treated with one or more compounds of the invention to cause or induce cell death prior to treating the cell. In one aspect, after treating the cells with one or more compounds of the invention, the cells are treated with at least one compound, at least one method, or a combination thereof to cause DNA damage for inhibiting the function of normal cells or killing the cells.

In one embodiment, the cells are killed by treating the cells with at least one DNA damaging agent. That is, after treating a cell with one or more compounds of the invention sensitizes the cell to cell death, the cell is treated with at least one DNA-damaging agent to kill the cell. DNA damaging agents useful in the present invention include, but are not limited to, chemotherapeutic agents (e.g., cisplatin), ionizing radiation (X-ray, ultraviolet radiation), carcinogens, and mutagenic agents.

In another embodiment, the cells are killed by treating the cells with at least one method to cause or induce DNA damage. Such methods include, but are not limited to: activating a cellular signal transduction pathway (which causes DNA damage when the pathway is activated), inhibiting a cellular signal transduction pathway (which causes DNA damage when the pathway is inhibited), and inducing a biochemical change in a cell (wherein the change causes DNA damage). By way of non-limiting example, DNA repair pathways in a cell may be inhibited, thereby preventing repair of DNA damage and resulting in abnormal accumulation of DNA damage in a cell.

In one aspect of the invention, the compounds of the invention are administered prior to irradiation or other induction that causes DNA damage in the cell. In another aspect of the invention, the compounds of the invention are administered concurrently with irradiation or other induction that causes DNA damage to cells. In yet another aspect of the invention, the compounds of the invention are administered immediately after the initiation of irradiation or other induction that causes DNA damage to the cells.

In another aspect, the cell is in vitro. In another embodiment, the cell is in vivo.

As described above, it has surprisingly been found that the compounds of the present invention effectively inhibit allo-MEK and are therefore useful in the treatment or prevention of diseases of, or accompanied by, uncontrolled cell growth, proliferation and/or survival, an inappropriate cellular immune response or an inappropriate cellular inflammatory response, in particular wherein said uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response or inappropriate cellular inflammatory response is mediated by allo-MEK, e.g. haematological tumours, solid tumours and/or metastases thereof, such as leukaemia and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and metastases, cancers of the brain, and cancers of the brain, Breast, gastrointestinal, endocrine, breast and other gynaecological tumours including non-small cell and small cell lung tumours, urological tumours including renal, bladder and prostate tumours, skin tumours and sarcomas, and/or metastases thereof.

Thus, according to another aspect, the present invention relates to a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described and defined herein, for use in the treatment or prevention of a disease, as described above.

Accordingly, another particular aspect of the present invention is the use of a compound of general formula (I) as described above for the preparation of a pharmaceutical composition for the treatment or prevention of a disease.

The diseases mentioned in the first two paragraphs are diseases with or accompanied by uncontrolled cell growth, proliferation and/or survival, an inappropriate cellular immune response or an inappropriate cellular inflammatory response, in particular wherein the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response or inappropriate cellular inflammatory response is mediated by Mps-1, such as hematological tumors, solid tumors and/or metastases thereof, e.g. leukemia and myelodysplastic syndrome, malignant lymphomas, head and neck tumors including brain tumors and brain metastases, breast tumors including non-small cell lung tumors and small cell lung tumors, gastrointestinal tumors, endocrine tumors, inflammatory cell responses, and the like, Breast and other gynaecological tumours, urological tumours including renal tumours, bladder tumours and prostate tumours, skin tumours and sarcomas, and/or metastases thereof.

In the context of the present invention, in particular in the context of an "inappropriate immune response or inappropriate cellular inflammatory response" as used herein, the term "inappropriate" is to be understood as preferably meaning a response which is weaker or stronger than the normal response and which is associated with, causes or leads to the pathology of the disease.

Preferably, the use is for the treatment or prevention of a disease, wherein the disease is a hematological tumor, a solid tumor and/or metastases thereof.

Methods of treating hyperproliferative disorders

The present invention relates to methods of treating hyperproliferative disorders in mammals using the compounds of the present invention and compositions thereof. The compounds may be used to inhibit, block, reduce, etc., cell proliferation and/or cell division and/or induce apoptosis. The method comprises administering to a mammal, including a human, in need thereof an amount of a compound of the present invention, a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof, and the like, effective to treat the condition. Hyperproliferative disorders include, but are not limited to, psoriasis, keloids and other hyperplasia affecting the skin, Benign Prostatic Hyperplasia (BPH), solid tumors such as breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer and their distant metastases. Such conditions also include lymphomas, sarcomas and leukemias.

Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer and non-small cell lung cancer as well as bronchial adenomas and pleural pneumococcal tumors.

Examples of brain cancers include, but are not limited to, brainstem and hypothalamic gliomas, cerebellum and brain astrocytomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors.

Tumors of the male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcomas.

Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland cancers.

Urinary tract tumors include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter cancer, urinary tract cancer, and human papillary renal cancer.

Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, kaposi's sarcoma, malignant melanoma, merkel cell skin cancer, and non-melanoma skin cancer.

Head and neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip cancer, oral cavity cancer, and squamous cell. Lymphomas include, but are not limited to, aids-related lymphoma, non-hodgkin's lymphoma, cutaneous T-cell lymphoma, burkitt's lymphoma, hodgkin's disease, and central nervous system lymphoma.

Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas, and rhabdomyosarcomas.

Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

These disorders have been well characterized in humans, but also exist in other mammals with similar etiologies, and can be treated by administering the pharmaceutical compositions of the present invention.

Reference throughout this document to the use of the term "treating" is conventional, e.g., for the purpose of counteracting, alleviating, reducing, alleviating, ameliorating the condition of a disease or disorder such as sarcoma, and the like.

Methods of treating kinase disorders

The invention also provides methods for treating disorders associated with abnormal mitogen extracellular kinase activity including, but not limited to, stroke, heart failure, hepatomegaly, cardiac enlargement, diabetes, alzheimer's disease, cystic fibrosis, symptoms of xenograft rejection, septic shock, or asthma.

An effective amount of a compound of the invention may be used to treat such disorders, including those diseases mentioned in the background section above (e.g., cancer). Moreover, such cancers and other diseases may be treated with the compounds of the present invention regardless of the mechanism of action and/or the relationship of the kinase to the condition.

The phrase "abnormal kinase activity" or "abnormal tyrosine kinase activity" includes any abnormal expression or activity of the gene encoding the kinase or the polypeptide encoded thereby. Examples of such aberrant activity include, but are not limited to, overexpression of the gene or polypeptide; gene amplification; mutations that produce constitutively active or highly active kinase activity; gene mutation, deletion, substitution, addition, and the like.

The present invention also provides methods of inhibiting kinase activity, particularly mitogen extracellular kinase activity, comprising administering an effective amount of a compound of the present invention, including salts, polymorphs, metabolites, hydrates, solvates, prodrugs (e.g., esters) and diastereomeric forms thereof. Kinase activity may be inhibited in cells (e.g., in vitro) or in cells of a mammalian subject, particularly a human patient in need of treatment.

Methods of treating angiogenic disorders

The invention also provides methods of treating conditions and diseases associated with excessive and/or abnormal angiogenesis.

Inappropriate and abnormal expression of angiogenesis can be harmful to an organism. Many pathological states are associated with growth of unrelated (extra) blood vessels. These include, for example, diabetic retinopathy, ischemic retinal vein occlusion, and retinopathy of prematurity [ Aiello et al, New engl.j.med.1994,331, 1480; peer et al, lab. invest.1995,72,638], age-related macular degeneration [ AMD; see Lopez et al invest, opthalmols, vis, 1996,37,855], neovascular glaucoma, psoriasis, retrolental fibroplasia, angiofibroma, inflammation, Rheumatoid Arthritis (RA), restenosis, in-stent restenosis, restenosis following vascular grafts, and the like. In addition, the increased blood supply associated with cancerous and tumor tissue promotes growth, resulting in rapid tumor enlargement and metastasis. In addition, the growth of new blood and lymph vessels in tumors provides an exit route for cancerous cells (renegade cells), promoting metastasis and leading to the spread of cancer. Thus, the compounds of the present invention may be used to treat and/or prevent any of the aforementioned angiogenic disorders, e.g., by inhibiting and/or reducing angiogenesis; inhibit, block, reduce, etc., endothelial cell proliferation or other types associated with angiogenesis, and cause cell death or apoptosis of such cells.

Dosage and administration

Effective dosages of the compounds of the present invention for the treatment of each of the desired indications can be readily determined based on standard laboratory techniques known to evaluate compounds for the treatment of hyperproliferative and angiogenic disorders, by standard toxicity tests, as well as by standard pharmacological tests for determining treatment of the disorders described hereinabove in mammals, and by comparing these results with those of known drugs used to treat these disorders. The amount of active ingredient administered in the treatment of one of these conditions may vary widely depending on the following considerations: the particular compound and dosage unit employed, the mode of administration, the course of treatment, the age and sex of the patient to be treated, and the nature and extent of the condition being treated.

The total amount of active ingredient to be administered is generally from about 0.001mg/kg to about 200mg/kg body weight/day, and preferably from about 0.01mg/kg to about 20mg/kg body weight/day. A clinically useful dosing regimen will be one to three times daily dosing to once every four weeks. In addition, a "drug withdrawal period" (where no drug is administered to the patient for a certain period of time) may be advantageous for the overall balance between pharmacological efficacy and tolerability. A unit dose may contain from about 0.5mg to about 1500mg of the active ingredient and may be administered one or more times per day, or less than once per day. The average daily dose administered by injection, including intravenous, intramuscular, subcutaneous and parenteral injection, and using infusion techniques, may preferably be from 0.01 to 200mg/kg of total body weight. The average daily rectal dosage regimen is preferably from 0.01 to 200mg/kg of total body weight. The average daily vaginal dosage regimen is preferably 0.01-200mg/kg total body weight. The average daily topical dosage regimen is preferably 0.1-200mg administered one to four times daily. The transdermal concentration is preferably the concentration required to maintain a daily dose of 0.01-200 mg/kg. The average daily inhaled dose regimen is preferably from 0.01 to 100mg/kg of total body weight.

The specific starting and maintenance dosage regimen for each patient will, of course, vary depending upon the following factors: the nature and severity of the condition as determined by the clinician, the activity of the particular compound used, the age and general health of the patient, the time of administration, the route of administration, the rate of excretion of the drug, the drug combination, and the like. Thus, the desired therapeutic regimen and the amount of a compound of the invention, pharmaceutically acceptable salt, ester or composition thereof to be administered can be determined by one skilled in the art using routine therapeutic testing.

Preferably, the disease to which the method is directed is a hematological tumor, a solid tumor and/or metastases thereof.

The compounds of the invention are particularly useful in the treatment and prevention (i.e. prevention) of tumor growth and metastasis, particularly of solid tumors of all indications and stages, with or without prior treatment of said tumor growth.

Methods for determining specific pharmacological or pharmaceutical properties are well known to those skilled in the art.

The example assay experiments described herein are intended to illustrate the invention and the invention is not limited to the examples provided.

Biological evaluation

The utility of the compounds of the invention can be demonstrated by their in vitro activity, for example, in the in vitro tumor cell proliferation assay described below. The link between activity in vitro tumor cell proliferation experiments and antitumor activity in a clinical setting is well established in the art. For example, the therapeutic effects of paclitaxel (Silvestrini et al, Stem Cells1993,11(6),528-35), Teedison (Bissery et al, Anti Cancer Drugs1995,6(3),339) and topoisomerase inhibitors (Edelman et al, Cancer Chemother. Pharmacol.1996,37(5),385-93)) are demonstrated by utility in vitro tumor proliferation assays.

The activity of the compounds of the invention can be demonstrated by in vitro, ex vivo and in vivo assays well known in the art. For example, to demonstrate the activity of the compounds of the present invention, the following assay can be used.

Biological assay

In vitro tumor cell proliferationAnd (3) determination:

proliferation assay

An adherent tumor Cell Proliferation assay for testing compounds of The invention includes what is known as The Cell Titer-Glo reader developed by Promega (Cunningham, BA "A growth Issue: Cell Proliferation assay. model kit origin quantification of Cell growth" The science 2001,15(13),26, and Crouch, S P et al, "The use of ATP biologins as a medium of Cell Proliferation and Cell Proliferation" Journal of Immunological methods1993,160, 81-88).

Measurement 1: HCT116Cell Titer Glo (CTG) proliferation assay:

HCT116 cells (human colorectal cell line, expressing mutant BRAF V600E)]96-well black-primed tissue culture plates (Costar3603 black/primed) incubated at 37 ℃ were seeded at a density of 3000 cells/well in 100 μ l DMEM medium (DMEM/Ham's F12) with 10% Fetal Bovine Serum (FBS) and stabilized glutamine per well. Sister wells were inoculated on separate plates for time zero assay. All plates were incubated overnight at 37 ℃. Taking down a time zero plate: add 100. mu.l/well of CTG solution (Promega Cell Titer Glo solution) to sister plate time zero wells; the plates were mixed for 2min on an orbital shaker to ensure cell lysis, incubated for 10min, and luminescence read on a VICTOR3(Perkin Elmer). 24 hours after cell inoculation, test compounds were diluted in 50 μ l of medium and added at final concentrations ranging from 10 μ M up to 300pM low, depending on the activity of serially diluted test compounds with a final DMSO concentration of 0.4%. After addition of test compound, wells were incubated at 37 ℃ for 72 hours. Then, Promega Cell Titer Glo was usedAssay kit, 100 μ l lysis buffer containing a mixture of luciferase and its substrate luciferin was added to each well and incubated in the dark at room temperature for 10min to stabilize the luminescent signal. Samples were read on a VICTOR3(Perkin Elmer) using a luminescence protocol. By passingThe measurements were normalized with respect to the extinction (=0%) of the zero-point plate and the extinction (=100%) of untreated (0 μ M) cells, and the percentage change in cell growth was calculated. IC50 values were determined by 4-parameter fitting using company's own software.

And (3) determination 2: a549Cell Titer Glo (CTG) proliferation assay:

a549 cells [ human non-small cell lung cancer cell line, expressing mutant K-Ras G12S ] were seeded at a density of 2000 cells/well in 96-well black-primed tissue culture plates (Costar3603 black/primed) incubated at 37 ℃,100 μ l per well of DMEM medium (DMEM/Ham's F12) with 10% Fetal Bovine Serum (FBS) and stable glutamine. Cell Titer Glo proliferation assays for a549 cells were performed using the same protocol as described above for HCT116 cells.

Measurement 3: colo205Cell Titer Glo (CTG) proliferation assay:

colo205 cells were seeded at 3000 cells/well in RPMI1640 growth medium supplemented with 10% FBS in 96-well tissue culture plates. Cells were incubated in a medium containing 5% CO₂Incubated overnight at 37 ℃ in a humidified incubator. On the following day, test compounds were added to wells, serially diluted in RPMI1640 medium containing 10% FBS and 0.03% DMSO, and the plates were incubated at 37 ℃ for 72 h. At different time points (0 and 72h post-dose), evaluation of Cell density was performed by adding Cell Titer Glo reagent (cat # G7572, Promega, Madison WI) to each well, then culturing the plates on a shaker at room temperature for 10min, then reading the luminescence on a Victor3 instrument. For IC₅₀Analysis, data analysis was performed using Analyze5 software.

Measurement 4: a375Cell Titer Glo (CTG) proliferation assay:

a375 cells [ human malignant melanoma cells, ATCC # CRL-1619, expression mutant BRAF V600E]The 96-well black-primed tissue culture plates incubated at 37 ℃ (Costar3603 black/primed) were seeded at 3000 cells/well density in 100 μ l DMEM medium with 10% Fetal Bovine Serum (FBS) and stabilized glutamine per well (Biochr)om, FG0435; +3,7g/L sodium bicarbonate; +4,5g/L D-glucose). Sister wells were inoculated on separate plates for time zero assay. The plates were incubated overnight at 37 ℃. Taking down a time zero plate: add 67. mu.l/well of CTG solution (Promega Cell Titer Glo solution) to sister plate time zero wells; the plates were mixed for 2min on an orbital shaker to ensure cell lysis, incubated for 10min, and luminescence read on a VICTOR3(Perkin Elmer). 24 hours after cell inoculation, test compounds diluted in 50 μ l of medium were added at final concentrations ranging from 10 μ M up to 300pM low, depending on the activity of serial dilutions of test compounds with a final DMSO concentration of 0.4%. After addition of test compound, wells were incubated at 37 ℃ for 72 hours. Then, Promega Cell Titer Glo was usedAssay kit, 100 μ l lysis buffer containing a mixture of luciferase and its substrate luciferin was added to each well and incubated in the dark at room temperature for 10min to stabilize the luminescent signal. Samples were read on a VICTOR3(Perkin Elmer) using a luminescence protocol. Percent change in cell growth was calculated by normalizing the measurements to the extinction (=0%) of the zero-point plate and the extinction (=100%) of untreated (0 μ M) cells. IC50 values were determined by 4-parameter fitting using company's own software.

Alternatively, cell proliferation was determined by Crystal Violet (CV) staining:

measurement 5: a375 Crystal Violet (CV) proliferation assay:

cell proliferation of a375 cells [ human melanoma cell line, mutant BRAF V600E ] was determined by Crystal Violet (CV) staining: cultured human A375 cells were seeded at a density of 1500 cells/measurement spot in 200 μ l growth medium (DMEM/HAMS F12 with 10% FBS and 2mM glutamine) in 96 well microtiter plates. After 24 hours, cells from the plate (zero plate) were stained with crystal violet (see below), while the medium in the other plates was replaced by fresh medium (200. mu.l) to which the test substance had been added at different concentrations (0. mu.M, and in the range 0.3 nM-30. mu.M; final concentration of solvent dimethyl sulfoxide 0.5%). Cells were cultured for 4 days in the presence of the test substance. Cell proliferation was determined by staining the cells with crystal violet: cells were fixed for 15min at room temperature by adding 20 μ l of 11% glutaraldehyde solution per measurement point. The fixed cells were washed three times with water, and then the plates were dried at room temperature. Cells were stained by adding 100 μ l of 0.1% crystal violet solution per measurement point (pH was adjusted to pH3 by adding acetic acid). Stained cells were washed three times with water, and then the plates were dried at room temperature. The dye was dissolved by adding 100. mu.l of 10% acetic acid solution per measurement point and the extinction was determined photometrically at a wavelength of 595 nm. Percent change in cell growth was calculated by normalizing the measurements to the extinction (=0%) of the zero-point plate and the extinction (=100%) of untreated (0 μ M) cells. IC50 values were determined by 4-parameter fitting using company's own software.

Alternatively, Crystal Violet (CV) staining assays can be performed as follows:

measurement 6: other conditions for a375 Crystal Violet (CV) proliferation assay:

cultured human A375 cells were seeded at a density of 1500 cells/measurement spot in 200 μ l growth medium (DMEM/HAMS F12(Biochrom; FG4815) with 10% FBS and 2mM glutamine) in 96-well microtiter plates. After 24 hours, cells from the plate (zero plate) were stained with crystal violet (see below), while the medium in the other plates was replaced by fresh medium (200. mu.l) to which the test substance had been added at different concentrations (0. mu.M, and in the range 0.3 nM-30. mu.M; final concentration of solvent dimethyl sulfoxide 0.5%). Cells were cultured for 4 days in the presence of the test substance. Cell proliferation was determined by staining the cells with crystal violet. Cells were fixed for 15min at room temperature by adding 20 μ l of 11% glutaraldehyde solution per measurement point. The fixed cells were washed three times with water, and then the plates were dried at room temperature. Cells were stained by adding 100 μ l of 0.1% crystal violet solution per measurement point (pH was adjusted to pH3 by adding acetic acid). Stained cells were washed three times with water, and then the plates were dried at room temperature. The dye was dissolved by adding 100. mu.l of 10% acetic acid solution per measurement point and the extinction was determined photometrically at a wavelength of 595 nm. Percent change in cell growth was calculated by normalizing the measurements to the extinction (=0%) of the zero-point plate and the extinction (=100%) of untreated (0 μ M) cells. IC50 values were determined by 4-parameter fitting using company's own software.

Similarly to the above methods, in vitro inhibition of proliferation of other cell lines can be determined. Details of exemplary other tumor cell lines are provided below:

in addition, the following assays can be used to evaluate the biological importance of the compounds of the present invention.

Measurement 7: inhibition of human carbonic anhydrase 1 and 2

The principle of the invention is based on the hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase, followed by photometric determination of the dye 4-nitrophenolate (Pocker & Stone, Biochemistry,1967,6, 668).

Mu.l of test compound dissolved in DMSO (100 Xfinal concentration) was pipetted (4 Xassay) into the wells of a 96-well microtiter plate at a concentration of 0.03-10. mu.M (final). Wells containing solvent but no test compound were used as reference values (1. wells without carbonic anhydrase were used for correction of non-enzymatic hydrolysis of substrate, and 2. wells with carbonic anhydrase were used to determine the activity of uninhibited enzyme).

188. mu.l assay buffer (10mM Tris/HCl, pH7.4,80mM NaCl) with or without 3 units/well of carbonic anhydrase I or II (Sigma-Aldrich # C4396, resp. Sigma-Adrich # C6165) were pipetted into the wells of a microtiter plate. The enzymatic reaction was started by adding 10. mu.l of a substrate solution (1mM 4-nitrophenylacetate (Fluka #4602) in anhydrous acetonitrile) (final concentration: 50. mu.M). The plates were incubated at room temperature for 60 minutes. The extinction was determined photometrically at a wavelength of 400 nm. Enzyme inhibition was calculated after normalizing the measured values to the extinction of the reaction in wells without enzyme (=100% inhibition) and wells without enzyme inhibition (=0% inhibition). IC50 values were determined by 4-parameter fitting using company's own software.

Measurement 8: determination of the Compound distribution between blood and plasma (blood/plasma ratio)

The concentration of the test compound (Cbl) in (human) blood relative to its plasma concentration (Cpl), i.e. the blood/plasma ratio, was evaluated using 0.5ml of fresh heparinized (human) blood to which different concentrations of the drug had been added (maximum solvent concentration in blood of 0.5%) and mixed well. After incubation for 15min at 37 ℃ in an overhead shaker, plasma was prepared by centrifugation at 1000 Xg. A calibration curve consisting of at least 5 concentration points was prepared by adding different amounts of drug to the plasma and serial dilutions. Calibration samples and triplicate plasma samples were pelleted with 4 volumes of methanol containing the appropriate amount of internal standard, incubated overnight at-20 ℃, and centrifuged at 2000xg for 20 min. The supernatant was analyzed by LC-MS and the drug concentration in plasma was estimated from the calibration curve.

The (human) blood/plasma ratio was calculated as: Cbl/Cpl = concentration of added drug in blood (nominal value)/plasma concentration (measured value).

Measurement 9

MEK biochemical assay: DELFIA

DELFIA MEK kinase assay was used to monitor the activity of MEK inhibitors. By first preparing 70. mu.L of kinase reaction buffer (50mM HEPES pH7.5,5mM NaF,5mM glycerol phosphate, 1mM sodium vanadate, 10mM MgCl₂1mM DTT and 1% (v/v) DMSO) were mixed with 20nM GST-MEK, 20nM His-Raf and 100nM biotinylated ERK1 (final concentration) and the kinase was performed in 96-well microtiter platesAnd (4) reacting. Then, compounds were added to final concentrations of 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.03. mu.M, 0.01. mu.M, 0.003. mu.M, 0.001. mu.M, 0.0003. mu.M and 0. mu.M to generate a dose-response inhibition curve. The kinase reaction was initiated by the addition of 20. mu.L ATP (final concentration 100. mu.M). After 2h incubation, the reaction was stopped by adding 20. mu.l of 0.5M EDTA. Then, 100 μ L of the reaction mixture was transferred to a 96-well streptavidin plate (cat #15120, Pierce inc. rockford, IL) and incubated for 2 h. The biotinylated substrate, ERK1, was collected and the plates were washed with TBST. An anti-phospho-p 44/42MAPK antibody (cat #91065, Cell Signaling Technologies, Danvers, MA) was added and bound to the phosphorylated substrate. Thereafter, incubation with europium-labeled anti-mouse antibody (cat # AD0124, Wallac Inc, Turku, Finland) was followed by a washing step. An enhancing solution is added to dissociate the europium ions into solution where they form highly fluorescent chelates with the components of the enhancing solution. The fluorescence of each sample was proportional to kinase activity and counted on a VICTOR5 instrument (Wallac Inc.). For IC₅₀Analysis, data analysis was performed using Analyze5 software.

Assay 10

MEK1 activated kinase assay

The kinase Cot1 activates MEK1 by phosphorylating its activation loop. The inhibitory activity of the compounds of the invention on this MEK1 activation was quantified using the HTRF assay described in the following paragraphs.

Human Cot 1N-terminal His 6-tagged recombinant kinase domain (amino acids 30-397, available from Millipore, cat. No. 14-703) expressed in insect cells (SF21) and purified by Ni-NTA affinity chromatography was used as kinase. An inactive C-terminal His 6-tagged GST-MEK1 fusion protein (Millipore cat. no14-420) was used as a substrate for the kinase reaction.

For the assay, 50nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black small-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 24nM GST-MEK1 and 166.7. mu.M Adenosine Triphosphate (ATP) in assay buffer were added[50mM TRIS/HCl pH7.5,5mM MgCl₂2mM dithiothreitol, 0.01% (v/v) Igepal CA630(Sigma),5mM glycerol beta-phosphate]And the mixture was incubated at 22 ℃ for 10min to pre-bind the test compound to GST-MEK1 before the kinase reaction started. The kinase reaction was then initiated by adding 2 μ l of Cot1 solution in assay buffer and the resulting mixture was incubated at 22 ℃ for a reaction time of 20 min. In this assay, the concentration of Cot1 was adjusted according to the activity of the enzyme batch, and the concentration of Cot1 was chosen appropriately to bring the assay into the linear range, with a typical enzyme concentration being about 2 ng/. mu.l (final concentration in a 5. mu.l assay volume). By adding HTRF detection reagent (13nM anti-GST-XL 665[ #61GSTXL LB, Fa. cis. Biointernational, Marcoule, France)]1nM Eu-cryptate-labeled anti-phospho-MEK 1/2(Ser217/221) [ #61P17KAZ, Fa]) The reaction was stopped in a solution (5. mu.l) of aqueous EDTA (100mM EDTA,500mM KF, 0.2% (w/v) bovine serum albumin pH7.5 in 100mM HEPES/NaOH).

The resulting mixture was incubated at 22 ℃ for 2h to allow phosphorylated GST-MEK1 to bind to anti-GST-XL 665 and Eu-cryptate labeled anti-phospho-MEK 1/2 antibody. Then, the amount of Ser217/Ser221 phosphorylated substrate was evaluated by measuring the resonance energy transfer from the anti-phospho-MEK 1/2 antibody labeled with Eu-cryptate to anti-GST-XL 665. Thus, the fluorescence emission at 620nm and 665nm after excitation at 350nm is determined in an HTRF reader, for example Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer). The ratio of the emission at 665nm to that at 622nm was taken as a measure of the amount of phosphorylated substrate. Data were normalized (enzyme reaction without inhibitor =0% inhibition, all other assay components except enzyme =100% inhibition). Typically, the values of test compounds at each concentration are determined in duplicate on the same microtiter plate at 10 different concentrations in the range of 20 μ M to 1nM (20 μ M,6.7 μ M,2.2 μ M,0.74 μ M,0.25 μ M,82nM,27nM,9.2nM,3.1nM and 1nM, the dilution series being prepared by 1:3 serial dilution at the level of stock solution concentrated 100-fold prior to measurement), and the IC is calculated using the software itself according to a 4-parameter fit₅₀The value is obtained.

Measurement 11

Determination of phospho-ERK mechanism

A375 and Colo205 cells were seeded 25000 cells/well in RPMI1640 growth medium supplemented with 10% FBS in 96-well tissue culture plates. Cells were incubated in a medium containing 5% CO₂Incubated overnight at 37 ℃ in a humidified incubator. On the following day, to prepare assay plates, anti-rabbit Meso-Scale Discovery (MSD) plates (cat # L41RA-1, Meso-Scale Discovery, Gaithersburg, MD) were blocked with 100 μ L5% MSD blocking buffer for 1h at room temperature and then washed 3 times with 200 μ L TBST buffer. anti-phospho-ERK rabbit polyclonal antibody diluted 1:200 in 2.5% MSD Blocker A-TBST was added (25. mu.l) to each well, and the plates were then incubated at room temperature and with shaking for 1 h. The plate was then washed once with Phosphate Buffered Saline (PBS) and ready to receive cell lysate. In carrying out the preparation of assay plates, test compounds were added to wells of cell-containing plates from the previous day, serially diluted in RPMI1640 medium containing 10% FBS, 0.1% Bovine Serum Albumin (BSA) and 0.03% DMSO, and the plates were incubated at 37 ℃ for 1.5 h. After this incubation, compound-treated plates were washed three times with PBS, solubilized in 30. mu.l Bio-Rad lysis buffer (cat #98601, Bio-Rad Laboratories, Hercules, Calif.), and then shaken on ice for 30 min. The lysate was then added to the phospho-ERK coated MSD plates and the plates were incubated overnight at 4 ℃. On the following day, the plates were washed three times with TBST and 25 μ l1:3000 diluted total ERK monoclonal antibody (Cat #610123, BD Biosciences, San Diego, CA) was added to the plates, which were then incubated at room temperature and with shaking for 1 h. After incubation, the plates were washed three times with TBST as before, and 25. mu.l of MSD sulfo-tag anti-mouse antibody (cat # R32AC-5) diluted at 1:1000 was added to each well. The plates were incubated at room temperature and with shaking for 1h and then washed four times with TBST. Before reading the plates, 150. mu.l of MSD Read buffer T was added and the plates were Read immediately on the MSD instrument. For IC₅₀Analysis, data analysis was performed using Analyze5 software.

Measurement 12

Other conditions for the mechanistic pERK assay

To determine ERK1/2 phosphorylation in tumor cell lines, Singleplex Mesoscale Discovery (MSD) was used. The assay was set up as a sandwich immunoassay. Cell lysates generated from different tumor cell lines treated with serially diluted MEK inhibitor compounds were added to MSD plates. Phosphorylated ERK1/2 present in the sample binds to the coated antibody immobilized on the surface of the working electrode. The sandwich was completed by binding the detection antibody to the immobilized phospho-ERK 1/2. The detection antibody is labeled with an electrochemiluminescent compound. Application of a voltage to the plate electrode causes luminescence of the label bound to the electrode surface by the antibody-phosphate ERK1/2 sandwich complex. Measurement of luminescence provides a quantitative determination of the amount of phosphorylated ERK1/2 present in the sample. In detail, for each cell line used for the assay, by gradually increasing the number of cells, the linear range of phosphoerk signal assays must be determined. For the final assay, the number of cells previously assayed was seeded in 96-well plates. 24h after seeding, cells were treated with serially diluted allosteric MEK inhibitor compounds for 1.5h, then the cells were lysed and the lysates were transferred to MSD assay plates. The manufacturer's protocol was changed because binding of phosphorylated ERK to coated antibody was performed overnight at 4 ℃ rather than 3h at room temperature, which resulted in better signal intensity.

A375 or Colo205 cells were seeded at 45000 cells/well in 50 μ L DMEM growth medium (Biochrom FG0435) (A375), supplemented with 10% FBS (Biochrom # S0410), RPMI growth medium (Biochrom FG1215) (Colo-205), supplemented with 10% FBS (Biochrom # S0410), 10mM HEPES (Biochrom L1613), 4.5g/L glucose and 1mM sodium pyruvate (Biochrom L0473), respectively, in 96-well tissue culture plates. Cells were incubated in a medium containing 5% CO₂Incubated overnight at 37 ℃ in a humidified incubator.

phospho-ERK assays were performed using Mesoscale Discovery (MSD) (# K111DWD) according to the manufacturer's recommendations. Briefly, the protocol is:

on the day after inoculation of the cells for preparation of the assay plates, MSD was blocked with 150 μ l MSD blocking buffer for 1h at room temperature, after which they were washed four times with 150 μ l Tris wash buffer. In performing the assay plate preparation, test compounds were added to wells of the cell-containing plates from the previous day, serially diluted in the corresponding growth medium containing 10% FBS and 0.1% DMSO, and the plates were incubated at 37 ℃ for 1.5-2 h. After this incubation, the medium was aerated, and the cells were lysed in 50 μ l lysis buffer and then shaken for 30min at 4 ℃. Then 25. mu.L lysate was added to the blocked MSD plate and the plate was incubated overnight at 4 ℃. On the following day, the plates were washed four times with Tris wash buffer and 25 μ Ι of detection antibody solution was added to the plates, which were then incubated for 1h at room temperature and with shaking. After incubation, the plates were washed four times with Tris wash buffer, 150 μ l MSD Read buffer T was added and the plates were Read immediately on the MSD instrument. For IC50 analysis, data analysis was performed using its own software.

Measurement 13

In vivo efficacy studies: staged human xenograft model

The in vivo antitumor activity of the lead compounds was evaluated in mice using a xenograft model of human BRAF mutant melanoma and colon cancer. Female athymic NCR nude mice were subcutaneously transplanted with human melanoma (LOX) or human colon cancer (Colo205) cell lines obtained from the american type culture collection (ATCC, Maryland). When the tumor size reached about 100mg, treatment was started. The compounds were administered orally and freshly formulated in PEG/water (80%/20%, respectively). The general health of the mice was monitored daily and mortality was recorded. Tumor size and body weight were recorded twice weekly starting on the first day of treatment. Animals were euthanized according to Bayer IACUC guidelines. Treatments that produce a lethality greater than 20% and/or a 20% net weight loss are considered "toxic".

Tumor growth was measured three times a week with an electronic caliper and tumor weight (mg) was calculated as follows: [ Length (mm) x Width (mm)²]/2. The antitumor efficacy was determined from tumor growth inhibition (% TGI). TGI for treatment days was calculated using the following formula: (100-mean treated tumor (T)/mean control weight (C) x100) =% T/C. The control used in the calculation was either "untreated control" or "vehicle", either of which provided the most conservative representation of the data. Compounds exhibiting a TGI greater than or equal to 50% are considered active. Statistical significance was determined using a single-or two-tailed T-test. The test compounds showed significant dose-dependent tumor growth inhibition in both LOX and Colo205 models.

Measurement 14

Brain penetration

In female NMRI mice, the penetration of the test compound into the brain was evaluated after intravenous administration. Test compounds were administered as a solution at a standard dose of 5mg/kg using a solubilizing agent such as PEG400 or ethanol in well tolerated amounts. Animals in each group (3 animals per group) were sacrificed at 5min, 15min, 30min, 1h and 3h post-dose, and blood and brain were sampled. Collecting blood into heparin lithium test tubeNeutralized and centrifuged at 3000rpm for 15 min. 100 μ L aliquots were taken from the supernatant (plasma), precipitated by addition of 400 μ L cold acetonitrile and frozen at-20 ℃ overnight. Brain samples were homogenized with 50mM Tris-HCl buffer (pH 7.5(1:5 w/v)), precipitated with acetonitrile (1:5, v/v) and frozen overnight at-20 ℃. Plasma and brain samples were then thawed and centrifuged at 3000rpm for 20 minutes at 4 ℃. An aliquot of the supernatant was taken and subjected to analytical testing using an Agilent1200HPLC system with LCMS/MS detection.

AUC (area under the concentration-time curve) in plasma and brain was calculated from the concentration-time curve, and the AUC brain/AUC plasma ratio was reported as brain-to-plasma ratio. The lower limit of the brain-to-plasma ratio using this method is about 1-2% due to residual blood in the non-perfused brain tissue.

Measurement 15

Pharmacokinetics in rats

For in vivo pharmacokinetic experiments, rats were dosed intravenously with 0.5-1mg/kg and gavage with 1-10mg/kg of test compound formulated as a solution (using a solubilizing agent such as PEG400 in well tolerated amounts).

For pharmacokinetics after intravenous administration, test compounds were administered as a bolus intravenous injection and blood and brain samples were taken at 2min, 8min, 15min, 30min, 45min, 1h, 2h, 4h, 6h, 8h and 24h post-administration. Additional samples were taken at later time points (e.g., 48h, 72h) depending on the expected half-life. For pharmacokinetics after gavage, test compounds were gavage administered to fasted rats and blood samples were taken at 5min, 15min, 30min, 45min, 1h, 2h, 4h, 6h, 8h and 24h post-administration. Additional samples were taken at later time points (e.g., 48h, 72h) depending on the expected half-life. Collecting blood into heparin lithium test tubeNeutralized and centrifuged at 3000rpm for 15 min. 100 μ L aliquots were taken from the supernatant (plasma), precipitated by addition of 400 μ L cold acetonitrile and frozen at-20 ℃ overnight. The samples were then thawed and centrifuged at 3000rpm for 20 minutes at 4 ℃. An aliquot of the supernatant was taken and subjected to analytical testing using an Agilent1200HPLC system with LCMS/MS detection. PK parameters were calculated by non-compartmental analysis using PK calculation software.

PK from concentration-time curve after i.v.: CL plasma of test compound: total plasma clearance (L/kg/h); CL blood of test compound: total blood clearance: CL plasma Cp/Cb (L/kg/h), where Cp/Cb is the ratio of the concentrations in plasma and blood. PK parameters calculated from concentration-time curves after i.g.: cmax: maximum plasma concentration (mg/L), Cmaxnor: cmax divided by the dose administered (kg/L), Tmax: time point (h) at which Cmax was observed. Parameters calculated from i.v. and i.g. concentration-time curves: AUCnorm: t =0h to infinity (extrapolated) area under the concentration-time curve divided by the dose administered (kg x h/L); AUC (0-tlast) norm: t =0h to the area under the concentration-time curve at the last time point at which plasma concentrations could be determined divided by the dose administered (kg x h/L); t 1/2: terminal half-life (h), F: oral bioavailability: aucnom after gavage divided by aucnom (%) after intravenous administration.

Assay 16

Method for determining thermodynamic (equilibrium) solubility-shaking flask

1. Introduction to the design reside in

In many research and development experiments, the solubility of New Chemical Entities (NCEs) is an important physicochemical characteristic that affects the performance of a compound.

Thermodynamic (or equilibrium) solubility studies the solubility of compounds as equilibrium saturated solutions.

2. Principle of method

Thermodynamic solubility of the compounds was determined by the flask method. Quantification was performed by HPLC and UV detection.

The starting material is a solid compound. The assay requires about 2-3mg of dry compound for sample preparation and calibration.

Depending on the problem, an aqueous buffer of any pH may be used as the solvent.

A saturated solution of the drug was prepared and the solution was mixed for 24h to ensure equilibrium was reached. The solution is then centrifuged or filtered to remove insoluble fractions and the concentration of the compound in the solution is determined using a standard calibration curve.

The measured solubility was measured in the range of about 0.1 to about 2000 mg/l.

3. Materials and instruments

Sample set-up (work up)

4ml screw cap glass bottle

Screw cap

1.1ml HPLC glass vial

Eppendorf tube

Syringe type filter

1ml syringe

Weighing boat

Chemicals and solvents

Water (Millipore)

Acetonitrile

NH4OH

Trifluoroacetic acid

Na₂HPO₄x2H₂O

KH₂PO₄

Phosphate buffer pH6.5

Riedel buffer, various pH

Instrument for measuring the position of a moving object

Stirrer

Centrifugal machine

HPLC

UV detector

Chromatographic conditions are as follows:

HPLC column: xterra MS C182.5. mu. m4.6x30mm

Sample introduction volume: sample preparation: 3X 5. mu.l and 3X 50. mu.l

Standard sample 5. mu.l, 10. mu.l, 20. mu.l

Flow rate: 1.5ml/min

Mobile phase: two gradients, depending on the nature of the test compound

Gradient 1 (acidic):

a: water/0.01% TFA

B acetonitrile/0.01% TFA

0min→95%A5%B

0-3min → 35% A65% B, linear gradient

3-5min → 35% A65% B, isocratic

5-6min → 95% A5% B, isocratic

Gradient 2 (basic):

a is water/0.025% NH4OH

B acetonitrile/0.025% NH4OH

0min→95%A5%B

0-3min → 35% A65% B, linear gradient

3-5min → 35% A65% B, isocratic

5-6min → 95% A5% B, isocratic

UV detector wavelength near absorption maximum (200-400nm)

4. Method of producing a composite material

Preparation of samples and standards

Preparation of samples

Weigh compound (ca. 1-2mg, exact weight) in a 4ml glass vial

Add 1.0ml buffer

The suspension was placed on a stirrer and stirred at room temperature for 24h (+ -2 h)

Filtration of sample solution through syringe filter into HPLC vial or centrifugation of sample

Preparation of the standard:

weigh compound (ca. 1-2mg, exact weight) in weigh boat

Dissolve the compound in acetonitrile/water 60:40 and dilute to 50ml

5. Analysis of

Samples and standards were analyzed by HPLC and UV detection.

For each sample, two injection volumes (5 and 50 μ Ι) were performed in triplicate. For the standard, three injection volumes were performed.

6. Interpretation and documentation

The areas of sample and standard injection were determined by suitable HPLC software. The theoretical solubility values (in mg/l) were automatically evaluated using Excel and processed by the LIM system. Results are reported as Pix

Measurement 17

CYP inhibition assay

It has been shown that the use of in vitro assays to evaluate the inhibitory potency of new drug candidates against CYP-mediated metabolism as part of a strategy can effectively minimize the likelihood of drug interaction with co-administered drugs.

The inhibitory potency of the test compounds against 5 human cytochrome P450 subtypes (CYP1a2, 2C8, 2C9, 2D6, and 3a4) was determined. In the case of CYP3a4, time-dependent inhibitory potency was also tested by applying a 30min pre-incubation time to the test compounds in a metabolically active incubation system.

Human liver microsomes (collected from >30 male and female donors) were incubated with a single CYP subtype selective standard probe (phenacetin, amodiaquine, diclofenac, dextromethorphan and midazolam) in the presence and absence of increasing concentrations of test compound to compare the extent of formation of the corresponding metabolites. In addition, a series of incubations performed in the absence of test compound served as negative controls. In addition, the inhibitory potency of the standard inhibitors was included as positive controls (fluvoxamine pre-incubation for CYP1a2, montelukast pre-incubation for CYP2C8, sulfaphenazole pre-incubation for CYP2C9, fluoxetine pre-incubation for CYP2D6, ketoconazole pre-incubation for CYP3a4, and mibefradil pre-incubation for CYP3a 4). The incubation conditions (protein and substrate concentrations, incubation time) were optimized for linearity and metabolite conversion. The incubation medium consisted of 50mM potassium phosphate buffer (pH7.4) containing 1mM EDTA, NADPH-producing system (1mM NADP,5mM glucose-6-phosphate, glucose-6-phosphate dehydrogenase (1.5U/mL)). Serial dilutions and incubations were performed on 96-well plates at 37 ℃ on a Genesis Workstation (Tecan, Crailsheim, FRG). A final incubation volume of 200. mu.L was used. The reaction was stopped by adding 100 μ L of acetonitrile containing the corresponding internal standard. Precipitated proteins were removed by centrifugation of 96-well plates, and supernatants were combined and analyzed by LC-MS/MS. LC-MS/MS quantification of the metabolites acetaminophen (CYP1A2), desethylamodiaquine (CYP2C8), 4-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6) and 1-hydroxymidazolam (CYP3A4) was performed with the PE SCIEX API3000LC/MS/MS system (Applied Biosystems, MDS Sciex, Concord, Ontario, Canada).

And (3) data analysis: CYP-mediated activity in the presence of inhibitor was expressed as a percentage of the corresponding control value. The enzyme inhibition parameter IC50 was calculated using sigmoidal curve fitting data and non-linear least squares regression analysis of the control activity percentage versus test inhibitor concentration curve.

The compounds of the invention are tested for activity using one or more of the assays described above. The results are shown in the following table:

watch (A)

In the above table, "NT" means "not tested".

It is believed that one skilled in the art can, using the preceding information and information available in the art, utilize the present invention to its fullest extent. Those skilled in the art will recognize that changes may be made in the structure, materials, compositions and methods disclosed in the present invention without departing from the spirit or scope of the invention, which is set forth herein, and that such changes are considered to be within the scope of the invention. The compounds described in the examples are intended to be representative of the invention, it being understood that the scope of the invention is not limited to the examples. The headings set forth above are intended to guide where certain information may be found in the present application, but are not to be construed as the only places in the invention where information on such subject matter may be found.

All publications and patents cited above are incorporated herein by reference.

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Claims

1.A compound of general formula (I), or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof:

wherein:

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₆-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R，-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R’，-N(R)C(=O)N(R’)R’’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -n (r) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R’，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

r2 is a halogen atom, C₂-C₆-alkynyl or-S-C₁-C₆-an alkyl group;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

2. The compound of claim 1, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₁₀-cycloalkyl-C₁-C₆-alkyl-, 3-to 10-membered, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R，-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R，-N(R)C(=O)N(R)R’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C3-C6-cycloalkyl, -N (R) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂An n (R) R 'or-S (= O) (= NR) R' group;

r2 is a halogen atom, C₂-C₆-alkynyl or-S-C₁-C₆-an alkyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

3. The compound of claim 1 or 2, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

said group being substituted with one or more substituents selected from:

a halogen atom, or-CN, C₁-C₆-alkyl-, halo-C₁-C₆-alkyl-, H₂N-C₁-C₆-alkyl-, R (R') N-C₁-C₆-alkyl-, HO-C₁-C₆Alkyl, C substituted by two OH₁-C₆-alkyl radical, C₁-C₆-alkoxy-C₁-C₆-alkyl-, halo-C₁-C₆-alkoxy-C₁-C₆-alkyl-, C₃-C₁₀-cycloalkyl-C₁-C₆-alkyl-, 3-to 7-membered heterocycloalkyl-C₁-C₆-alkyl-, aryl-C₁-C₆-alkyl-, heteroaryl-C₁-C₆-alkyl-, -C (= O) R, -C (= O) NH₂，-C(=O)N(H)R，-C(=O)N(R)R’，-C(=O)OH，-C(=O)OR，-NH₂，-N(H)R，-N(R)R’，-N(H)C(=O)H，-N(H)C(=O)R，-N(R)C(=O)R’，-N(H)C(=O)NH₂，-N(H)C(=O)N(H)R，-N(H)C(=O)N(R)R’，-N(R)C(=O)NH₂，-N(R)C(=O)N(H)R，-N(R)C(=O)N(R’)R’’，-N(H)C(=O)OR，-N(R)C(=O)OR’，-NO₂，-N(H)S(=O)R，-N(R)S(=O)R’，-N(H)S(=O)NH₂，-N(H)S(=O)N(H)R，-N(H)S(=O)N(R)R’，-N(R)S(=O)NH₂，-N(R)S(=O)N(H)R’，-N(R)S(=O)N(R’)R’’，-N(H)S(=O)₂R，-N(H)S(=O)₂-C₃-C₆-cycloalkyl, -n (r) S (= O)₂R’，-N(H)S(=O)₂NH₂，-N(H)S(=O)₂N(H)R，-N(H)S(=O)₂N(R)R’，-N(R)S(=O)₂NH₂，-N(R)S(=O)₂N(H)R’，-N(R)S(=O)₂N(R’)R’’，-N=S(=O)(R)R’，-OH，C₁-C₆-alkoxy-, -OC (= O) H, -OC (= O) R, -OC (= O) NH₂，-OC(=O)N(H)R，-OC(=O)N(R)R’，-OC(=O)OR，-SH，C₁-C₆-alkyl-S-, -SC (= O) NH₂，-SC(=O)N(H)R，-SC(=O)N(R)R’，-S(=O)₂R，-S(=O)₂NH₂，-S(=O)₂N(H)R，-S(=O)₂N (R) R 'or-S (= O) (= NR) R'A group;

r2 is a bromine atom, an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r, R ' and R ' ' are independently of each other C₁-C₆-an alkyl group.

4. The compound of claim 1 or 2, or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate, thereof, wherein:

r1 is aryl, C₁-C₆-alkyl or C₂-C₆-an alkenyl group,

said group being substituted with one or more substituents selected from:

r2 is a bromine atom, an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom, C₁-C₆-alkyl or C₃-C₆-a cycloalkyl group;

r and R' are independently of each other C₁-C₆-an alkyl group.

5. A compound according to any one of claims 1,2 or3, or a tautomer, stereoisomer, N-oxide, salt, hydrate, or solvate thereof, wherein:

r1 is aryl, C₁-C₆Alkyl radical, C₂-C₆-alkenesThe radical(s) of the group(s),

said group being substituted with one or more substituents selected from:

r2 is an iodine atom or C₂-an alkynyl group;

r3 is a hydrogen atom;

r is C₁-C₆-an alkyl group.

6. The compound of any one of claims 1-4, selected from:

3- [ (2-fluoro-4-iodophenyl) amino ] -5- [ (2-oxo-2, 3-dihydro-1, 3-benzoxazol-5-yl) oxy ] isonicotinamide;

tert-butyl [3- ({ 4-carbamoyl-5- [ (2-fluoro-4-iodophenyl) amino ] pyridin-3-yl } oxy) phenyl ] carbamate;

3- (3-aminophenoxy) -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide;

3- [ (2-fluoro-4-iodophenyl) amino ] -5- {3- [ (isopropylsulfonyl) amino ] phenoxy } isonicotinamide;

3- [ (2-fluoro-4-iodophenyl) amino ] -5- {3- [ (methylsulfonyl) amino ] phenoxy } isonicotinamide;

3- {3- [ (ethylsulfonyl) amino ] phenoxy } -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide;

3- {3- [ (cyclopropylsulfonyl) amino ] phenoxy } -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide;

3- (3-acetamidophenoxy) -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide;

3- [ (2-fluoro-4-iodophenyl) amino ] -5- [3- (propionylamino) phenoxy ] isonicotinamide;

3- [ (2-fluoro-4-iodophenyl) amino ] -5- [3- (isobutyrylamino) phenoxy ] isonicotinamide;

3- [ (2-fluoro-4-iodophenyl) amino ] -5- [ (4-methylpent-3-en-1-yl) oxy ] isonicotinamide;

3- [ (3, 4-dihydroxy-4-methylpentyl) oxy ] -5- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide;

3- {3- [ (ethylsulfonyl) amino ] phenoxy } -5- [ (4-ethynyl-2-fluorophenyl) amino ] isonicotinamide; and

3- [ (2-fluoro-4-iodophenyl) amino ] -5-methoxyisonicotinamide.

7. A process for the preparation of a compound of general formula (I) according to any one of claims 1 to 6, said process comprising the step of reacting an intermediate compound of general formula (2) with an alcohol of general formula D, thereby obtaining a compound of general formula (I):

wherein R2 and R3 are as defined for the general formula (I) in any one of claims 1 to 6,

wherein R1 is as defined for general formula (I) in any one of claims 1 to 6,

wherein R1, R2 and R3 are as defined for general formula (I) in any one of claims 1 to 6.

8. A compound of general formula (I), a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, according to any one of claims 1 to 6, for use in the treatment or prophylaxis of a disease.

9. A pharmaceutical composition comprising a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, according to any one of claims 1 to 6, and a pharmaceutically acceptable diluent or carrier.

10. A pharmaceutical combination comprising:

-one or more compounds of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, according to any one of claims 1 to 6; and

-one or more agents selected from: a taxane, such as docetaxel, paclitaxel or taxol; epothilones such as ixabepilone, paclitaxel (Patupilone) or salgopilone (Sagopilone); mitoxantrone; prednisolone; dexamethasone; estramustine; vinblastine; vincristine; doxorubicin; doxorubicin; idarubicin; daunorubicin; bleomycin; etoposide; cyclophosphamide; ifosfamide; procarbazine; melphalan; 5-fluorouracil; capecitabine; fludarabine; cytarabine; Ara-C; 2-chloro-2' -deoxyadenosine; thioguanine; antiandrogens, such as flutamide, cyproterone acetate or bicalutamide; bortezomib; platinum derivatives, such as cisplatin or carboplatin; chlorambucil; methotrexate and rituximab.

11. Use of a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, according to any one of claims 1 to 6, for the prophylaxis or treatment of a disease.

12. Use of a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, according to any one of claims 1 to 6, for the preparation of a medicament for the prophylaxis or treatment of a disease.

13. The use of claim 11 or 12, wherein the disease is a disease caused by uncontrolled cell growth, proliferation and/or survival, an inappropriate cellular immune response, or an inappropriate cellular inflammatory response, particularly wherein the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response, or inappropriate cellular inflammatory response is mediated by the mitogen-activated protein kinase (MEK-ERK) pathway, more particularly wherein the disease caused by uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response, or inappropriate cellular inflammatory response is a hematologic tumor, a solid tumor, and/or metastases thereof, such as leukemia and myelodysplastic syndrome, malignant lymphoma, head and neck tumors including brain tumors and brain metastases, breast tumors including non-small cell lung tumors and small cell lung tumors, Gastrointestinal tumors, endocrine tumors, breast tumors and other gynecological tumors, urological tumors including renal tumors, bladder tumors and prostate tumors, skin tumors and sarcomas, and/or metastases thereof.

14. A compound of the general formula (2):

wherein R2 and R3 are as defined for general formula (I) in any one of claims 1 to 6.

15. Use of a compound of general formula (2) according to claim 14 for the preparation of a compound of general formula (I) according to any one of claims 1 to 6.