HK1159097B - Phenylamino isonicotinamide compounds - Google Patents

Description

Phenylaminoisonicotinamide compounds

Technical Field

The present invention relates to a series of substituted phenylamino isonicotinamide compounds useful for the treatment of proliferative diseases, such as cancer and inflammation, in mammals. The use of such compounds in the treatment of proliferative diseases in mammals, particularly humans, and pharmaceutical compositions comprising said compounds are also disclosed.

Background

The Ras/Raf/MEK/ERK pathway is a central signaling pathway that signals from a variety of cell surface receptors to nuclear transcription factors that regulate gene expression. This pathway is commonly referred to as the MAP kinase pathway, while MAPK represents a mitogen-activated protein kinase, suggesting that mitogens, cytokines, and growth factors may stimulate this pathway (Steelman et al, Leukemia (Leukemia)2004, 18, 189-. Depending on the stimulus and cell type, this pathway may propagate signals that cause prevention or induction of apoptosis or cell cycle progression. The Ras/Raf/MEK/ERK pathway has been shown to play an important role in the prevention of cell proliferation and apoptosis. Aberrant activation of this pathway is commonly observed in malignantly transformed cells. Amplification of the Ras proto-oncogene and active mutations leading to expression of the constitutively active Ras protein is observed in approximately 30% of all human cancers (Stirewalt et al, Blood (Blood)2001, 97, 3589-95).

Mutant, oncogenic forms of Ras are present in 50% of colon cancers and > 90% of pancreatic cancers, as well as in many other types of cancers (Kohl et al, Science 1993, 260, 1834-1837). The effects of Ras on proliferation and tumorigenesis have been documented in immortal cell lines (McCubrey et al, Int J Oncol 1995, 7, 295310). The bRaf mutation has been found to be present in more than 60% of malignant melanomas (Davies, H et al, Nature 2002, 417, 949-.

This pathway is often considered a key target for therapeutic intervention due to the high level of mutations detected in Ras (Chang et al, Leukemia (Leukemia)2003, 77, 1263-93).

The Ras/Raf/MEK/ERK signaling pathway can pass through downstream transcription factor targets (including NF-_κ ^κB. CREB, Ets-1, AP-1 and c-Myc) regulate proliferation. ERKs can directly phosphorylate Ets-1, AP-1 and c-Myc, leading to their activation. Alternatively, ERKs can phosphorylate and activate the downstream kinase target RSK, which in turn phosphorylates and activates transcription factors (e.g., CREB). These transcription factors induce gene expression, which plays an important role in cell cycle progression (e.g., Cdk's, cyclins, growth factors) and prevention of apoptosis (e.g., anti-apoptotic Bcl-2 and cytokines). In general, treatment of cells with growth factors leads to activation of ERK, which leads to proliferative effects and in some cases differentiation (Lewis et al, adv. cancer Res, 1998, 74, 49-139).

The MEK family of genes contains five genes: MEK1, MEK2, MEK3, MEK4 and MEK 5. The dual specificity kinase family contains both serine/threonine and tyrosine kinase activities. The structure of MEK contains an amino-terminal negative regulatory domain and a carboxy-terminal MAP kinase-binding domain, which are required for ERK binding and activation. Deletion of the regulatory MEK1 domain results in activation of the constitutive MEK1 and ERK (Steelman et al, Leukemia (Leukemia)2004, 18, 189-.

MEK1 is a 393-amino acid protein with a molecular weight of 44kDa (Crews et al Science 1992, 258, 478-80). MEK1 was moderately expressed in embryonic development, highly expressed in adult tissues, and the highest levels were detected in brain tissues. MEK1 requires phosphorylation of S218 and S222 for activation, and substitution of these residues with aspartic acid (D) or glutamic acid (E) results in increased activity and lesion formation in NIH3T3 cells (Huang et al, Mol Biol Cell, 1995, 6, 237-45). Constitutive activity of MEK1 in primary cell cultures promotes senescence and induces p53 and p16^INK4aIn immortalized cells or in the absence of p53 or p16^INK4aThe opposite was observed in cells of (Lin et al, Genes Dev, 1998, 12, 3008-3019). Constitutive activity of MEK1 inhibition of NF-via negative regulation of p38MAPK activity_κ ^κTranscription of B (Carter et al, J Biol Chem 2000, 275, 27858-64). The main physiological substrates of MEK are members of the ERK (extracellular signal-regulated kinase) or MAPK (mitogen-activated protein kinase) protein family. Aberrant expression of MEK1 was detected in a number of different types of cancer, and mutant forms of MEK1 were able to transform fibroblasts, hematopoietic cells and other cell types.

Constitutive activation of MEK1 leads to cell transformation. MEK1 therefore represents a possible target for pharmaceutical intervention in proliferative and inflammatory diseases (Lee et al, Nature 1994, 372, 739-) -746; Dudley et al, Proc. Natl. Acad. Sci. U.S.A.1995, 92, 7686-) -7689).

Useful MEK inhibitors have been developed that show potential therapeutic benefit in several studies. For example, small molecule MEK inhibitors are capable of inhibiting human tumor growth in nude mouse xenografts (Yeh, T. et al, Proceedings of the American Association of Cancer Research, 2004, 45, Abs 3889; Lee, P. et al, Proceedings of the American Association of Cancer Research, 2004, 45, Abs 3890). MEK inhibitors have also entered the Clinical trial phase, namely ARRY142886(Wallace, E. et al, Proceedings of the American Association of Cancer Research, 2004, 45, Abs 3891; Adjei, A.A. et al, Journal of Clinical Oncology (Journal of Clinical Oncology)2008, 26, 2139. Thinopathy 2146; Shannon, A.M. et al, Molecular Cancer Therapeutics (Molecular Cancer Therapeutics)2007, 6, 3414S-3415S Part 2), PD-5901 (Swanton C, Johnson S IDDB MEETING REPORT 2003, February 13-1; Haura, E.B. et al, Molecular Cancer Therapeutics (Cancer Therapeutics)2007, 3468S-3568, Ab.25, Molecular Cancer Therapeutics (Cancer Therapeutics)2007, 3435, Ab.S-03269, Molecular Cancer Therapeutics (Cancer of Molecular Oncology) 2007, Molecular Cancer Therapeutics (Cancer of 3482, Ab. PT 35, Ab. 25, Ab.S-25, Abs 389 1; Molecular Cancer Therapeutics, Molecular Cancer of Molecular Cancer (Cancer of Molecular Oncology) 2007, Molecular Cancer of 3435, Ab. PD-3435, Molecular Cancer Therapeutics, Ab. Ab, Molecular Cancer Therapeutics (Molecular Cancer of Molecular Cancer) (Molecular Research, Ab. No. 35, Ab. No. 35, Ab. No. 35, s., Molecular Cancer Therapeutics 2007, 6, 3595S-3595S Part 2, RDEA-119(2007 press release) and RDEA-436(2008 press).

In US 5,525,625; WO 98/43960; WO 99/01421; WO 99/01426; WO 00/41505; WO 00/42002; WO 00/42003; WO 00/41994; WO 00/42022; WO 00/42029; WO 00/68201; WO 01/68619; WO 02/06213; WO 03/077855; WO 03/077914; WO 2004/005284; compounds suitable as MEK inhibitors are also disclosed in WO 2004/056789, WO 2006/045514, WO 2008/076415, WO 2007/121269 and WO 2007/121481.

Disclosure of Invention

It is an object of the present invention to provide novel MEK inhibitors for the treatment of hyperproliferative diseases associated with potent MEK activity, as well as diseases modulated by the MEK cascade, such as cancer and inflammation, in mammals, which inhibitors have superior pharmacological properties, including their activity as well as their solubility, metabolic clearance and bioavailability characteristics.

Accordingly, the present invention provides novel, substituted phenylamino isonicotinamide compounds, which are MEK inhibitors, useful in the treatment of the above-mentioned diseases, as well as pharmaceutically acceptable salts, solvates or prodrugs thereof.

The compounds are defined by formula (I):

formula (I)

And pharmaceutically acceptable salts, solvates or prodrugs thereof, wherein:

x is NH or O, and the compound is,

r1 is hydrogen, methyl, ethyl, n-propyl, i-propyl, SH or Hal,

r2 is hydrogen, methoxy, ethoxy, ethynyl (ethynylene), cyano, SH or Hal,

r3, R4 are independently selected from hydrogen, SH or Hal,

R⁵、R⁶independently selected from OH, SH or NH₂And is

Hal is F, Cl, Br or I.

Preferred are compounds of formula (I) subformulae IA, IB, IC, ID, IE, IF, IG and IH and pharmaceutically acceptable salts, solvates or prodrugs thereof, wherein:

in subformula IA:

x is NH, and the catalyst is a catalyst,

R¹is Hal, methyl or ethyl,

R²is hydrogen, Hal, methoxy or ethynyl,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

R⁵、R⁶is an OH group, and the molecular weight of the compound is,

hal is F, Cl, Br or I;

in subformula IB:

x is NH, and the catalyst is a catalyst,

R¹in order to be Hal, the catalyst is,

R²is hydrogen or Hal, and is,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

R⁵、R⁶is an OH group, and the molecular weight of the compound is,

hal is F, Cl, Br or I;

in subformular IC:

x is NH, and the catalyst is a catalyst,

R¹is F, Cl, a methyl group or an ethyl group,

R²is hydrogen, I, Br, methoxy or ethynyl,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

R⁵、R⁶is an OH group, and the molecular weight of the compound is,

hal is F, Cl, Br or I;

in subformula ID:

x is NH, and the catalyst is a catalyst,

R¹is F, Cl, a methyl group or an ethyl group,

R²is hydrogen, I, Br, methoxy or ethynyl,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl, and the like,

R⁵、R⁶is OH;

in subformula IE:

x is NH, and the catalyst is a catalyst,

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl, and the like,

R⁵、R⁶is OH;

in subformula IF:

x is NH, and the catalyst is a catalyst,

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl, and the like,

R⁵、R⁶is OH;

in subformula IG:

x is NH, and the catalyst is a catalyst,

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is a hydrogen atom, and is,

R⁴is a hydrogen atom, and is,

R⁵、R⁶is OH;

in subformula IH:

x is NH, and the catalyst is a catalyst,

R¹in the case of F, the content of the compound,

R²the compound is shown as I, and the compound is,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl, and the like,

R⁵、R⁶is OH.

More preferred compounds of formula (I) and subformulae IA, IB, IC, ID, IE, IF, IG and IH thereof are compounds of formula (II):

wherein R is¹、R²、R³And R⁴Having the meaning indicated for formula (I) or a preferred subformula IA, IB, IC, ID, IE, IF, IG or IH thereof.

Particularly preferred compounds of formula (I) and/or formula (II) include compounds of the formula:

when the symbol "+" is shown between two structures, a 1: 1 racemic mixture of the two enantiomers is indicated.

The compounds of the present invention may be in the form of prodrug compounds. "prodrug compound" means a derivative that can be converted into the biologically active compound of the present invention in an organism under physiological conditions, for example, by oxidation, reduction, hydrolysis, and the like, each of which can be carried out with or without the participation of an enzyme. Examples of prodrugs are the compounds of the following cases: wherein the amino group in the compounds of the invention is acylated, alkylated or phosphorylated, e.g., eicosanoylamino, alanylamino, pivaloyloxymethylamino; or wherein the hydroxyl group is acylated, alkylated, phosphorylated or converted to a boronic ester, for example acetyloxy, palmitoyloxy, pivaloyloxy, succinoyloxy, fumaroyloxy, alanoyloxy; or wherein the carboxyl group is esterified or amidated; or wherein the thiol group forms a disulfide bridge with a carrier molecule (e.g., a peptide) that selectively delivers the drug to the target and/or to the cytosol. These compounds can be prepared from the compounds of the present invention according to known methods. Other examples of prodrugs are compounds wherein the carboxylic acid ester in the compounds of the invention is for example converted into alkyl-, aryl-, choline, amino, acyloxymethyl, linolenoyl-ester. Metabolites of the compounds of the invention are also within the scope of the invention.

When tautomerism (e.g., keto-enol tautomerism) of the compounds of the present invention or prodrugs thereof exists, it is intended to claim both their individual forms (e.g., keto or enol forms), respectively, and mixtures thereof in any proportion. The same applies to their stereoisomers, e.g., enantiomers, cis/trans isomers, conformational isomers, and the like.

If desired, isomers can be separated according to methods known in the art (e.g., liquid chromatography). The same applies to their enantiomers, for example, using chiral stationary phase separation. Furthermore, enantiomers can be separated by conversion to diastereomers, i.e. coupling with enantiomerically pure auxiliary compounds, followed by separation of the resulting diastereomers and cleavage of the auxiliary residues. Alternatively, any enantiomer of a compound of the invention may be obtained by stereoselective synthesis using optically pure starting materials.

The compounds of the present invention may be in the form of pharmaceutically acceptable salts or solvates. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. Where the compounds of the invention contain one or more acidic or basic groups, the invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, especially their pharmaceutically acceptable salts. The compounds according to the invention which contain acidic groups can therefore be present in the form of salts and, according to the invention, can be used, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include: sodium, potassium, calcium, magnesium or salts containing ammonia or organic amines (e.g., ethylamine, ethanolamine, triethanolamine or amino acids). The compounds of the invention containing one or more basic groups, i.e. groups which can be protonated, can be present in the form of salts and, according to the invention, in the form of addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric, hydrobromic, phosphoric, sulfuric, nitric, methanesulfonic, p-toluenesulfonic, napadisylic, oxalic, acetic, tartaric, lactic, salicylic, benzoic, formic, propionic, pivalic, diethylacetic, malonic, succinic, pimelic, fumaric, maleic, malic, sulfamic, phenylpropionic, gluconic, ascorbic, isonicotinic, citric, adipic and other acids known to those skilled in the art. If the compounds of the invention contain both acidic and basic groups in the molecule, then, in addition to the salt forms, the invention likewise comprises internal salts or betaines (zwitterions). The salts can be prepared by conventional methods known to those skilled in the art, for example by contacting them with organic or inorganic acids or bases in solvents or dispersants, or by exchanging anions or cations with other salts. The invention likewise comprises all salts of the compounds of the invention which, owing to their low physiological compatibility, are not suitable for direct use in medicaments but can be used, for example, as chemical reaction intermediates or in the preparation of pharmaceutically acceptable salts.

In addition, the present invention relates to a pharmaceutical composition containing the compound of the present invention or a prodrug compound thereof or a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient and a pharmaceutically acceptable carrier.

"pharmaceutical composition" means one or more active ingredients and one or more inert ingredients constituting a carrier, as well as any product obtained directly or indirectly from: any two or more components are combined, complexed, aggregated, or one or more components are dissociated, or one or more components are otherwise reacted or interacted with. Thus, the pharmaceutical compositions of the present invention include any composition made by combining a compound of the present invention and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may additionally contain one or more other compounds as active ingredients, for example one or more additional compounds of the present invention, prodrug compounds, or other MEK inhibitors. The pharmaceutical compositions include suitable compositions for oral, rectal, topical, parenteral (including subcutaneous, intramuscular and intravenous), ocular (ocular), pulmonary (nasal or buccal inhalation) or nasal administration, the most suitable route in any given case depending on the nature and severity of the condition being treated and on the nature of the active ingredient. They may conveniently be presented in unit dosage form and prepared by any of the methods well known in the pharmaceutical art.

In one embodiment, the compounds and pharmaceutical compositions are used to treat the following conditions: cancers, such as brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, kidney, renal, ovarian, prostate, colorectal, esophageal, testicular, gynecological, thyroid; melanoma; hematological malignancies, such as acute myeloleukemia, multiple myeloma, chronic myeloleukemia, myelocytic leukemia; or any other type of solid or liquid tumor. In another embodiment, the pharmaceutical composition may be used to treat non-cancerous hyperproliferative diseases, such as benign skin hyperplasia (e.g. psoriasis), restenosis, polycystic kidney disease or prostate diseases (e.g. Benign Prostatic Hypertrophy (BPH)), as well as inflammatory and autoimmune diseases: such as rheumatoid arthritis, Crohn's disease, asthma, ulcerative colitis, irritable colon syndrome, multiple sclerosis, lupus erythematosus and other conditions. In another embodiment, the pharmaceutical composition may be used to treat a genetic disorder characterized by upregulation of the MEK/ERK pathway, such as Costello syndrome, noonan syndrome, pericardial syndrome, and others.

The invention also relates to the use of a compound of the invention in the manufacture of a medicament for the treatment of hyperproliferative diseases associated with excess activity of MEK, as well as diseases modulated by the MEK cascade, or for the treatment of conditions mediated by aberrant proliferation, such as cancer and inflammation, in a mammal.

The present invention also relates to compounds or pharmaceutical compositions for treating pancreatitis or kidney disease (including proliferative glomerulonephritis and diabetes-induced nephropathy) or pain in a mammal, comprising a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, and a pharmaceutically acceptable carrier.

The present invention also relates to a compound or pharmaceutical composition for preventing implantation of a blastocyst in a mammal, which comprises a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, and a pharmaceutically acceptable carrier.

The present invention also relates to a compound or pharmaceutical composition for treating a disease associated with angiogenesis or vasculogenesis in a mammal comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, prodrug or hydrate thereof and a pharmaceutically acceptable carrier.

In one embodiment, the compound or pharmaceutical composition may be used to treat the following diseases: angiogenic tumors, chronic inflammation (e.g., rheumatoid arthritis, inflammatory bowel disease), atherosclerosis, skin disorders (e.g., psoriasis, eczema, and scleroderma), diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangiomas, gliomas, melanomas, kaposi's sarcoma and ovarian, breast, lung, pancreatic, prostate, colon, and epidermal cancers.

The present invention also relates to the use for treating a hyperproliferative disorder in a mammal comprising administering to said mammal a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof. In one embodiment, the use relates to the treatment of cancer, such as brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, kidney, ovary, prostate, colorectal, esophageal, testicular, gynecological, thyroid cancer. In another embodiment, the use relates to the treatment of a non-cancerous hyperproliferative disease, such as benign skin hyperplasia (e.g., psoriasis), restenosis, or prostate disease (e.g., Benign Prostatic Hypertrophy (BPH)).

The present invention also relates to the use of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for the treatment of hyperproliferative disorders in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, and an antineoplastic agent in combination therewith, said agent being selected from the group consisting of: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, anti-angiogenic agents, cell cycle inhibitors, enzyme inhibitors, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens or immunomodulators.

The present invention also relates to the use of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for the treatment of pancreatitis or kidney disease or pain in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof. The present invention also relates to the use of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for the manufacture of a medicament for the prevention of blastocyst implantation in a mammal in need thereof.

The present invention also relates to the use of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for the treatment of a disease associated with angiogenesis or vasculogenesis in a mammal in need thereof, which comprises administering to said mammal a therapeutically effective amount of the compound. In one embodiment, the use is for the treatment of: tumor angiogenesis, chronic inflammation such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases (e.g., psoriasis, eczema, and scleroderma), diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangiomas, gliomas, melanomas, kaposi's sarcoma and ovarian, breast, lung, pancreatic, prostate, colon, and epidermal cancers. Patients treatable according to the methods of the invention with a compound of the invention, or pharmaceutically acceptable salts, prodrugs and hydrates of said compounds, include patients diagnosed with: for example, psoriasis, restenosis, atherosclerosis, BPH, lung cancer, bone cancer, chronic myelomonocytic leukemia, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular cancer, gynecological tumors (e.g., uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, or vulval cancer), hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system (e.g., thyroid cancer, parathyroid cancer, or adrenal cancer), soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, bladder cancer, cancer of the kidney or ureter (e.g., renal cell cancer, renal pelvis cancer), or tumors of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, and other tumors, Brain stem glioma or pituitary tumor).

The present invention also relates to a compound or pharmaceutical composition for inhibiting abnormal cell growth in a mammal, which comprises an amount of a compound of the present invention, or a pharmaceutically acceptable salt or solvate or prodrug thereof, in combination with an amount of another anti-cancer therapy, wherein the amounts of the compound, salt, solvate or prodrug, and the amount of the chemotherapy are together effective to inhibit abnormal cell growth. Many anti-cancer treatments are known in the art. In one embodiment, the anti-cancer treatment comprises chemotherapy selected from the group consisting of: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens. In another embodiment, the anti-cancer treatment is an antibody selected from the group consisting of: bevacizumab, CD 40-specific antibody, chTNT-1/B, denosumab, zanolimumab, IGF 1R-specific antibody, lintuzumab, edrecolomab, WX G250, rituximab, ticilimumab, trastuzumab and cetuximab (cetuximab).

The present invention also relates to a method of inhibiting abnormal cell growth or treating a hyperproliferative disorder in a mammal, comprising administering to the mammal an amount of a compound of the present invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in combination with radiation therapy, wherein the amount of the compound, salt, solvate or prodrug is effective to inhibit abnormal cell growth or treat the hyperproliferative disorder in the mammal when used in combination with the radiation therapy. Techniques for administering radiation therapy are known in the art, and these techniques may be combined with the therapies described herein. In such combination therapy, administration of a compound of the invention can be determined as described herein. It is believed that the compounds of the present invention may render abnormal cells more susceptible to radiation therapy, thereby killing and/or inhibiting the growth of such cells.

Accordingly, the present invention also relates to a method of sensitizing abnormal cells in a mammal to radiation therapy comprising administering to the mammal an amount of a compound of the present invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, effective to sensitize the abnormal cells to radiation therapy. The amount of a compound, salt or solvate in the present methods can be determined according to the methods described herein for determining an effective amount of such compounds. The present invention also relates to a method of inhibiting abnormal cell growth in a mammal comprising administering to the mammal an amount of a compound of the present invention, or a pharmaceutically acceptable salt, solvate, prodrug or isotopically labeled derivative thereof, and one or more agents selected from the group consisting of an anti-angiogenic agent, a signaling inhibitor, and an anti-proliferative agent.

In practical use, the compounds of the present invention as active ingredients may be combined in intimate admixture with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. In the case of oral liquid preparations, any conventional pharmaceutical media can be used, for example, suspensions, elixirs and solvents; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granules, lubricants, binders, disintegrating agents, and the like. In the case of oral solid preparations, the compositions may be presented as, for example, powders, hard and soft capsules, and tablets, with solid oral dosage forms being preferred over liquid dosage forms.

Because of their ease in administration, tablets and capsules represent the most advantageous oral unit dosage form in which case solid pharmaceutical carriers are obviously employed. If desired, the tablets may be coated by standard aqueous or non-aqueous techniques. The compositions and formulations should contain at least 0.1% of the active compound. Of course, the percentage of active compound in these compositions may vary and may advantageously range from about 2% to about 60% by weight. The amount of active compound in the therapeutically useful composition is such that an effective dose is obtained. The active compounds can likewise be administered intranasally, for example, as liquid drops or as sprays.

Tablets, pills, capsules, and the like may likewise comprise: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents, such as corn starch, potato starch, alginic acid; lubricants, such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin. Where the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the unit dose. For example, tablets may be coated with shellac, sugar coating or both. In addition to the active ingredient, a syrup or elixir may contain: sucrose as a sweetening agent, methyl and propyl p-hydroxybenzoate as preservatives, colouring and flavouring agents (e.g. cherry or orange flavour).

The compounds of the invention may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared by appropriate mixing in water with a surfactant, such as hydroxy-propylcellulose. Dispersions can be prepared from glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include sterile injectable aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the forms must be sterile and must be sufficiently fluid to allow them to be easily injected. In the case of preparation and storage, the form must be stable and must be capable of resisting contamination by microorganisms, such as bacteria and fungi. The carrier can include solvents or dispersion media such as water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may provide a mammalian, especially human, effective dose of a compound of the invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. administration is possible. Dosage forms include tablets, lozenges, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably, the compounds of the invention are administered orally.

The effective amount of the active ingredient administered may depend on the particular compound being administered, the mode of administration, the condition being treated, and the severity of the condition being treated. The dosage can be readily determined by one skilled in the art.

In the treatment or prevention of cancer, inflammation or other proliferative diseases for which the compounds of the invention are indicated, substantially satisfactory results are obtained when a daily dose of from about 0.01mg to about 100mg per kg of animal body weight is administered, preferably a single daily dose. For large mammals, the total daily dosage is from about 0.1mg to about 1000mg, preferably from about 0.2mg to about 50 mg. In the case of a 70kg adult, the total daily dose is approximately about 0.2mg to 200 mg. The dosage regimen may be adjusted to provide the optimum therapeutic response.

The invention also relates to a kit comprising the following individual packages:

a) an effective amount of a compound of the present invention or a physiologically acceptable salt, solvate or prodrug thereof; and

b) an effective amount of another pharmaceutically active ingredient.

The kit comprises a suitable container, such as a box, a separate bottle, a bag or an ampoule. Kits may comprise, for example, separate ampoules, each containing an effective amount of a compound of the invention and/or pharmaceutically acceptable derivatives, solvates and stereoisomers thereof, including mixtures thereof in all ratios, and an effective amount of another active ingredient, either in dissolved or lyophilized form.

Some abbreviations that may appear in this application are as follows:

abbreviations

The compounds of the present invention may be prepared according to the procedures set forth in the schemes and examples below, using appropriate materials, as further illustrated in the specific examples below.

In addition, other compounds claimed herein can be readily prepared by using the methods described herein, in conjunction with conventional techniques in the art. However, the compounds illustrated in the examples do not form the only category contemplated by the present invention. The examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand known variations of the conditions and methods of the following preparative procedures for preparing these compounds.

The compounds of the invention are typically isolated in the form of their pharmaceutically acceptable salts, such as those described above. The free amine base (amine-free base) corresponding to the isolated salt can be prepared by neutralization with a suitable base, such as aqueous sodium bicarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and the free amine base liberated is extracted with an organic solvent and then evaporated. The free amine base isolated in this manner can be further converted to other pharmaceutically acceptable salts by dissolution in an organic solvent, followed by addition of the appropriate acid, and finally evaporation, precipitation or crystallization.

The preparation of the compounds of the invention is illustrated in the schemes below. Unless otherwise stated in the flow chart, any variables have the same meaning as described above.

The present invention also relates to methods of preparing the compounds disclosed in formulas (I) and (II), subformulae IA-IH, and Table 1, as described in the schemes and working examples below.

Scheme 1

Scheme 1 shows a general synthetic route for all examples 2.1-2.20 of the synthesis of formula (I). The first step is a condensation reaction between a substituted aniline (11) and 3-fluoro-isonicotinic acid or its derivative (12) to yield the respective acid intermediate (13). The resulting acid intermediate is then reacted with a suitable amine or alcohol to form the amide or ester (14), respectively. An appropriate deprotection step is carried out in the presence of a protecting group (e.g. an acetonide group or other group) to finally obtain compound (15).

Scheme 2

Scheme 2 shows the synthesis of a preferred amine diol intermediate or diol protected analog thereof. The synthesis reaction starts first with the acetylation of racemic cyclohex-2-ol (16). The acetyl group of (17) is substituted in a stereospecific manner in a palladium catalysed reaction with potassium phthalimide (potassium phthalimide) in the presence of a Trost ligand (as described in Trost et al, j.am. chem. soc.1994, 116, 4089-. The double bond is then syn-dihydroxylated by treatment with osmium tetroxide to give the diol (19) which is then freed directly from phthalimido groups (to give the amine 27), or the diol is first protected as acetonide (20) and the phthalimido groups are subsequently freed to give the amine (21).

Scheme 3

The synthesis of racemic aminodiols is shown in scheme 3. Such as (1) Nishikawa, N; asai, M; ohuabu, N; isobe, m.j.org.chem.1988, 188-; blades, K; moore, p.r.; waring, m.j.; WinteR, j.j.g.; helliwell, m.; newcombe, n.j.; stemp, G.J.org.chem.2002, 7946-7956, which begins with an Overman rearrangement of a racemic cyclohex-2-enol (16) (via a trichloroacetimidate intermediate) to form trichloroacetamide (22), which is then syn-dihydroxylated by treatment with osmium tetroxide to form diols 23-26. The resulting mixture of four diastereoisomers of aminodiols (23-26) was separated by chiral chromatography into two pairs of racemates (23/24 and 25/26) which were then deprotected to yield the corresponding racemic aminodiols (27/28 and 29/30).

Examples

The examples described below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the specification or claims in any way.

1. Analysis of

The LC/MS analysis was performed according to the following two methods:

the method A comprises the following steps: discovery C was used at a flow rate of 400. mu.L/min¹⁸5 μm, 3 × 30mm column, 5 μ L of quantitation loop (sample loop), mobile phase: (A) 0.1% formic acid in water; mobile phase (B): 0.1% formic acid in methanol; the retention time is expressed in minutes. Details of the method: (I) UV/Vis diode radiometry on a Quaternary Pump G1311A (Agilent) or UV-detection in ESI + mode at 254 and 280nm with G1315B (Agilent) and Finnigan LCQ Duo MS detectors in a linear gradient from 15% to 95% of (B) within 3.2 min; (II) 95% of (B) was held for 1.4 minutes; (III) decreasing from 95% to 15% of (B) in a linear gradient within 0.1 min; (IV), 15% of (B) was kept for 2.3 min.

The method B comprises the following steps: waters Symmetry C¹⁸3.5 μm, 4.6X 75mm column, flow rate 1mL/min, quantitation loop 10 μ L, mobile phase (A) is 0.05% TFA in water, mobile phase (B) is 0.05% TFA in CAN; the retention time is expressed in minutes. The specific method comprises the following steps: (I) in ESI + mode on Binary Pump G1312A (Agilent) equipped with UV/Vis diode array detector G1315B (Agilent) and Agilent G1956B (SL) MS detectors, UV-detection at 254 and 280nm, 20-85% elution with (B) linear gradient for 10 min; (II) keep elution with 85% of (B) for 1min, (III) decrease from 85% to 20% of (B) in a linear gradient within 0.2 min; (IV) 20% of (B) was used to keep elution for 3.8 min.

Chiral HPLC analysis

Chiral HPLC analysis was performed on an Agilent 1100Series system using a ChiralPak AD-H column (250 × 4.6mm) manufactured by Daicel Chemical Industries, ltd. The method was performed with 5.0. mu.L of 100% methanol at an injection rate of 1mL/min for 15 minutes at 25 ℃ and UV-detection at 254 and 280 nm.

Preparative HPLC

Using Waters Atlantis dC₁₈ OBD^TM10 μ M (30X 250mm) column or using Waters Sunfire Prep C₁₈OBD 10. mu.M (30X 250mm) column was subjected to preparative HPLC. The column was used in a Waters Prep LC 4000 system equipped with a dosing ring (10mL) and an ISCO UA-6UV/Vis detector at a flow rate of 60 mL/min. The mobile phase was extracted from two solvent pools containing (a) water and (B) HPLC-grade acetonitrile. A typical preparative analysis was performed using a linear gradient (e.g., 0-60% solvent B, 60 minutes).

2. Chemical synthesis

2.1 Cyclohex-2-enyl acetate (17)

To a solution of 2-cyclohex-1-enol (16) (45.5mmol, 5.02mL) in pyridine (0.27mol, 22mL) was added acetic anhydride dropwise at 0 deg.C, and the reaction mixture was stirred at 0 deg.C for 1/2 hours. The ice bath was removed and the mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc and diluted with HCl (1N), saturated NaHCO₃Washed with brine and concentrated in vacuo to give 6g (94%) of crude product 17, which was used in the next step without further purification. Thin layer chromatography with 20% EtOAc in hexanes with 5% H₂SO₄EtOH staining, Rf of (16): 0.4 Rf of (17): 0.8.

2.22- [ (1R) -cyclohex-2-en-1-yl ] -1H-isoindole-1, 3(2H) -dione (18)

To potassium phthalimide (160mmol, 29.6g), N' - (1S, 2S) -cyclohexane-1, 2-diylbis [2- (diphenylphosphino) benzamide](Trost ligand) (6mmol, 4.14g), [ Pd (p-allyl) Cl]2(2mmol, 0.73g) and tetrahexylammonium bromide (216mmol, 94g) in dry DCM (40mL) was added a solution of 2-cyclohexen-1-yl acetate (17) (13g crude) in 40m L dry DCM. The mixture was stirred at ambient temperature overnight. The reaction was then quenched with water (250mL) and extracted with DCM (3X 250 mL). The organic layers were combined and washed with brine (400mL) and Na₂SO₄Drying and concentration gave the crude product as a yellow oil. Recrystallization from MeOH afforded 9.024g of product (purity: 100% by HPLC analysis, 50% yield). The mother liquor was purified by flash chromatography (10% EtOAc/hexanes) to provide 8.8g of 18 (86% purity by HPLC analysis, 48% yield). NMR and MS data were in agreement with published: trost, b.m.; bunt, R.C.J.Am.chem.Soc.1994, 4089-4090.

2.32- [ (1R, 2S, 3R) -2, 3-dihydroxycyclohexyl ] -1H-isoindole-1, 3(2H) -dione (19)

To a suspension of 2- [ (1R) -cyclohex-2-en-1-yl ] -1H-isoindole-1, 3(2H) -dione (18) (19.9mmol, 4.53g) and 4-methylmorpholine-N-oxide (60mmol, 7.01g) in acetone/water (4: 1, 62.5mL) was added osmium tetroxide (0.2mmol, 50 mg). The reaction was stirred for 4 hours. The mixture was concentrated and saturated sodium sulfite (40mL) was added. Then immediately extracted three times with EtOAc. The combined organic layers were washed with water and brine, dried over magnesium sulfate and concentrated to give the crude product as a white solid (4.94g, 95% yield). Chiral HPLC [8.53min ]. The analytical data were consistent with published data (Donohoe, J.T.; et al, J.org.chem., 2002, 67, 7946-

42- [ (3aS, 4R, 7aR) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-yl ] -1H-isoindole-1, 3(2H) -dione (20)

To a solution of 2- [ (1R, 2S, 3R) -2, 3-dihydroxycyclohexyl ] -1H-isoindole-1, 3(2H) -dione (19) (50.0g, 191.4mmol) and 2, 2-methoxypropane (500mL, 4.07mol) in acetone (500mL) was added a catalytic amount of p-toluenesulfonic acid, and the resulting reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adjusting to pH 10 with saturated sodium carbonate solution and removing the solvent. Water (750mL) was added to the residue, which was extracted with ethyl acetate (2X 750, 500 mL). The organic phases were combined, dried over sodium sulfate and concentrated to give the title product as a white solid (55.8g, 96.8% yield). LC/MS [ method B: rt: 6.16 min; m/z: 302(M +1) ].

2.5(3aS, 4R, 7aR) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-amine (21)

A mixture of 2- [ (3aS, 4R, 7aR) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-yl ] -1H-isoindole-1, 3(2H) -dione (20) (191.4mmol, 50.00g), hydrazine (308.4mmol, 15.0mL) in ethanol (500mL) was refluxed for 4 hours. The reaction was monitored by HPLC. HPLC analysis showed the reaction was not complete (86% conversion). To the reaction mixture was added 3mL more hydrazine and refluxed for 2 hours. The reaction was cooled to 0 ℃ and filtered. The filter cake was washed with IPA and dried to obtain the desired product 21(9.52g, yield 21%, purity by weight calculated according to NMR: 84%). The filtrate was concentrated and the residue was dissolved in IPA (ca. 200 mL). The by-product crystallized and was filtered off, volatiles were removed from the filtrate and dried to yield a second crop of desired product 21(28.46g, 64% yield). TLC (80% MeOH/EtOAc with 1 drop acetic acid, ninhydrin dip stain).

2.63- [ (2-fluoro-4-iodophenyl) amino ] isonicotinic acid

A mixture of 2-fluoro-4-iodo-phenylamine (20.0g, 84.38mmol) in dry tetrahydrofuran (80mL) was cooled to-65 deg.C under an inert atmosphere, and 1.0M lithium bis (trimethylsilyl) amide (255mL, 255mmol) was added slowly at a rate that maintained the internal temperature below-55 deg.C. After the final addition was complete, the viscous slurry was stirred for 30 minutes and then treated with 3-fluoro-isonicotinic acid (8.0g, 56.69 mmol). The mixture was stirred at room temperature for 4 days, then poured into 2.0N aqueous sodium hydroxide (1000mL) and ethyl acetate (250 mL). The layers were separated and the organic phase was extracted again with aqueous sodium hydroxide (2X 1000 mL). The pH of the combined aqueous phase components was adjusted to 2 with concentrated hydrochloric acid, which helped the precipitation of solids. The product was filtered, washed with water (300mL), dried at 40 ℃ for 18h under high vacuum to give the product (19.05g, 53.19mmol, 94%) as a yellow solid.

2.7N- [ (3aS, 4R, 7aR) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-yl ] -3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (31)

A suspension of 3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinic acid (10.46g, 29.2mmol), (3aS, 4R, 7aR) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-amine (21) (5.00g, 29.2mmol), 1-hydroxybenzotriazole (4.41g, 29.2mmol) and EDCI (5.6g, 29.2mmol) in DMF (100mL) was stirred at room temperature overnight. The reaction was quenched with water (150mL) and extracted with ethyl acetate (150 mL). An emulsion was formed and collected, filtered, and the filtrate was combined to the organic layer. The organic layer was washed with saturated sodium bicarbonate solution (150mL) and water (2 × 150mL), brine, dried over sodium sulfate and concentrated to give a brown foam. The crude product was purified by crystallization from IPA. The mother liquor was concentrated and purified by flash chromatography to afford the desired product (12g, yield 80%). LC/MS [ method A: rt: 7.35 min; m/z: 512(M +1) ].

2.8(N- [ (1R, 2S, 3R) -2, 3-dihydroxycyclohexyl ] -3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide) (8)

17.62mL of 2M HCl in ether was added to a brown solution of N- [ (3aR, 4S, 7aS) -2, 2-dimethylhexahydro-1, 3-benzodioxol-4-yl ] -3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (31) (6.65mmol, 3.15g) in MeOH (64mL) and the resulting reaction mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated to about 30mL to form a yellow solid, which was filtered to obtain the product as the hydrochloride salt. 10mL of MeOH was added to the salt and ammonium hydroxide was added until the pH was 10. A white solid formed and the solid was collected by filtration (1.83g, 58% yield) and washed with water. The filtrate was concentrated to give a second crop of product as a white solid (0.56g, 18% yield). LC/MS [ method B: rt: 5.83 min; m/z: 472(M +1) ]. Chiral HPLC [7.06min ].

2.92, 2, 2-trichloro-N-cyclohex-2-en-1-yl acetamide (22)

To a solution of 2-cyclohexen-1-ol (16) (18g, 0.183mol) in anhydrous dichloromethane (275mL) was added DBU (41.88g, 0.275mol) and the mixture was cooled to-20 ℃. To this solution was added dropwise trichloroacetonitrile (47)66g, 0.330 mol). The reaction was stirred at-20 ℃ for 3h and quenched with aqueous ammonium chloride. The organic phase is separated and dried over potassium carbonate. The solvent was removed in vacuo to give the intermediate cyclohex-2-en-1-yl 2, 2, 2-trichloroacetimidate (ethanomidate). This was dissolved in toluene (100mL) and treated with potassium carbonate (30 g). The mixture was refluxed for 12 hours. After cooling, the mixture was filtered through celite and the filtrate was evaporated to give 9g (20%) of (22) as a solid. LC/MS: found mass spectra (m/z, MS, 242.9), method: a-0.1% HCOOH, B-ACN (70%), flow-0.8 ml/min, column: GENESIS C1850X 4.6mm 3U, Rt (min): 1.645,¹H NMR(CDCl₃，400MHz)δ1.64-1.74(3H，m)，1.96-2.12(3H，m)，4.46-4.47(1H，m)，5.64-5.67(1H，m)，5.97-5.6.01(1H，m)，6.59(1H，bs)。

2.102, 2, 2-trichloro-N- [ (1RS, 2SR, 3RS) -2, 3-dihydroxycyclohexyl ] acetamide (racemic mixture of 23 and 24)

To a solution of 2, 2, 2-trichloro-N-cyclohex-2-en-1-yl acetamide (22) (4.5g, 0.0182mol) and N-methylmorpholine-N-oxide (7.5g, 0.055mol) in acetone (100mL) was added water (25mL) followed by a catalytic amount of osmium tetroxide (0.1g, solid). The reaction mixture was stirred at room temperature for 14h and quenched with a saturated solution of sodium sulfite (20 mL). The mixture was stirred for a further 20min, then the solvent was removed in vacuo and the residue was purified by chromatography using petroleum ether/ethyl acetate (3/7) as eluent to give 3.3g (60%) of the racemic diol as a solid.

LC/MS: found mass spectrum (m/z, -MS, 275.8), method: a-0.1% HCOOH, B-CAN (70%), flow rate-0.8 mL/min; column: GENESIS C1850X 4.6mm 3U; rt (min): 0.695,¹H NMR(CDCl₃，400MHz)δ1.31-1.70(6H，m)，3.87-3.92(2H，m)，4.00-4.10(1H，m)，7.68(1H，bs)。

2.11(1RS, 2SR, 3RS) -3-aminocyclohexane-1, 2-diol HCl (racemic mixture of 27 and 28)

2, 2, 2-trichloro-N- [ (1RS, 2SR, 3RS) -2, 3-dihydroxycyclohexyl]A mixture of acetamide (23 and 24) (2.3g) and 5M aqueous HCl (30mL) was refluxed for 10h and evaporated under reduced pressure to give 1.2g (86%) of the title compound as a viscous liquid. MS: mass spectrum (m/z, MS, 131.9), HPLC > 98%, method: a-water, B-CAN, flow-0.8 mL/min, column: c18XDB, 250X 4.6mm, SC \ 276. Rt (min), 2.715, in the form of a solid solution,¹H NMR(CD₃OD 400MHz)δ1.43-1.91(6H，m)，2.98-3.02(1H，m)，3.31-3.42(1H，m)，3.84(1H，m)。

2.12N- [ (1SR, 2RS, 3SR) -2, 3-dihydroxycyclohexyl ] -3-fluoro-5- [ (2-fluoro-4-iodophenyl) isonicotinamide (2)

3-fluoro-5- (2-fluoro-4-iodo-phenylamino) -isonicotinic acid (119mg, 0.32mmol) and 1, 1' -carbonylbis (1H-imidazole) (67mg, 0.41mmol) were suspended in DMSO (2 mL). The mixture was stirred at room temperature overnight (12 hr). 3-aminocyclohexane-1, 2-diol (racemic 27/28) (40mg, 0.31mmol) and triethylamine (0.07mL, 0.49mmol) were then added. The mixture was stirred at room temperature for a further 6 h. After completion of the reaction, the reaction mixture was treated with aqueous sodium hydroxide (1mL, 1.0M) and stirred at room temperature for 4 hours. The solution was neutralized to pH 7 with concentrated hydrochloric acid. The mixture was then rotary evaporated to remove most of the water. The obtained mixture was purified by preparative HPLC to obtain the product (2). LC/MS [ method A: rt: 4.89 min; m/z: 490(M +1) ].

2.13N- [ (1SR, 2RS, 3SR) -2, 3-dihydroxycyclohexyl ] -3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide (3)

3- (2-fluoro-4-iodo-phenylamino) -isonicotinic acid (300mg, 0.84mmol) and 1, 1' -carbonylbis (1H-imidazole) (177mg, 1.1mmol) were suspended in DMSO (4 mL). The mixture was stirred at room temperature overnight (12 hr). 3-aminocyclohexane-1, 2-diol (racemic 27/28) (110mg, 0.84mmol) was then added. The mixture was stirred at room temperature for a further 12 h. The mixture was separated by preparative HPLC to give two products, racemic amide (3) and racemic ester (4). LC/MS [ method A: rt: 6.01 min; m/z: 472(M +1) ]. Chiral HPLC [7.10min, 13.12min ].

2.143-fluoro-5- (2-fluoro-4-iodo-phenylamino) -isonicotinic acid (1RS, 2SR, 3RS) -3-amino-2-hydroxy-cyclohexyl ester (4)

See example 2.13. LC/MS [ method A: rt: 4.53 min; m/z: 472(M +1) ].

2.152-chloro-N- ((1RS, 2SR, 3RS) -2, 3-dihydroxy-cyclohexyl) -5- (2-fluoro-4-iodo-phenylamino) -isonicotinamide (1)

2-chloro-5- (2-fluoro-4-iodo-phenylamino) -isonicotinic acid (75mg, 0.19mmol) and 1, 1' -carbonylbis (1H-imidazole) (40mg, 0.25mmol) were suspended in DMSO (2.5 mL). The mixture was stirred at rt overnight (12 h). Then 3-aminocyclohexane-1, 2-diol (rac 27/28) (32mg, 0.19mmol) and triethylamine (0.05mL, 0.38mmol) were added. The mixture was stirred at room temperature overnight. The mixture was purified by preparative HPLC to give the product. LC/MS [ method B: rt: 5.359 min; m/z: 506(M +1) ].

163- (4-bromo-2-fluoro-phenylamino) -N- ((1RS, 2SR, 3RS) -2, 3-dihydroxy-cyclohexyl) -isonicotinamide (5)

Prepared using the general procedure of (1), LC/MS [ method B: rt: 5.602 min; m/z: 425(M +1) ].

2.173- (4-bromo-2-chloro-phenylamino) -N- ((1RS, 2SR, 3RS) -2, 3-dihydroxy-cyclohexyl-isonicotinamide (6)

Prepared using the general procedure of (1), LC/MS [ method B: rt: 6.163 min; m/z: 441(M +1) ].

2.18N- ((1R, 2S, 3R) -2, 3-dihydroxy-cyclohexyl) -3- (2-fluoro-phenylamino) -isonicotinamide (10)

To a sealed tube was added (N- [ (1R, 2S, 3R) -2, 3-dihydroxycyclohexyl ] -3- [ (2-fluoro-4-iodophenyl) amino ] isonicotinamide) (8) (42mg, 0.09mmol), sodium borohydride (34mg, 0.89mmol), palladium dichloride (7.9mg, 0.04mmol), sodium hydroxide (17.8mg, 0.45mmol), THF-water (1: 1, 3 mL). The mixture was stirred at room temperature for 2 days. The mixture was filtered and the filtrate was concentrated. The residue obtained was purified by flash chromatography to obtain the product. LC/MS [ method A: rt: 0.43 min; m/z: 346(M +1) ].

193- (4-bromo-2-fluorophenylamino) -N- ((1R, 2S, 3R) -2, 3-dihydroxycyclohexyl) isonicotinamide (9)

Prepared by the general procedure of (1) but using chirally pure 3-aminocyclohexane-1, 2-diol (27) instead of the racemic mixture of diols. LC/MS [ method B: rt: 5.19 min; m/z: 426.1(M +1) ].

2.20N- ((1S, 2R, 3S) -2, 3-dihydroxycyclohexyl) -3- (2-fluoro-4-iodophenylamino) isonicotinamide (7)

Prepared by the general procedure of (1) but using chirally pure 3-aminocyclohexane-1, 2-diol (28) instead of the racemic mixture of diols. LC/MS [ method A: rt: 4.54 min; m/z: 472.3(M +1) ].

3. Biological activity

3.1MEK-1 enzyme assay (LANCE-HTRF)

The activity of the compounds of the invention can be determined by the following method: human MEK1 kinase activity was monitored using homogeneous fluorescence assay (fluorogenic, fluorescent based assay). This assay uses time-resolved fluorescence resonance energy transfer to probe phosphorylation of ERK1 by MEK 1. The assay was performed in low volume 96 well microtiter plates. In a total volume of 15. mu.l, 10mM MgCl containing 20mM Tris/HCl was used₂、100μM NaVO₄Compounds were incubated with 100nM MEK1, 15 μ M ATP, 300nM ERK2 in a buffer of 1mM DTT and 0.005% Tween20(pH 7.4). After 2 hours, 5nM europium-anti-PY 20(Perkin Elmer) and 50nM buffer of anti-GST-allophycocyanin (CisBio) were addedSolution containing 50mM EDTA and 0.05% BSA, the reaction was incubated for 1 hour in the dark. Time-resolved fluorescence was measured using LJL-analyzers (molecular devices) with an excitation wavelength of 340nm and an emission wavelength of 665 nm. The final concentration of DMSO was 2%. To evaluate the inhibitory properties of the compounds, IC was determined₅₀Values, as shown in table 1.

3.2 tumor cell proliferation assay (ATP Lite)

Mouse colon C26, human melanoma A375, and human pancreas MiaPaCa-2 cells plated in 96-well Corning white plates (1500 cells/well C26, 2000 cells/well A375 and MiaPaCa-2) at 37 deg.C with 5% CO₂Incubate overnight in ambient. Inhibitors were serially diluted in 100% DMSO and subsequently added to the cells to a final concentration of 0.25% DMSO. Cells were incubated in cell growth medium (DMEM with 10% fetal bovine serum, 2mM glutamine for C26 and MiaPaCa-2, RPMI with 10% fetal bovine serum, 2mM glutamine for A375) for 4 days in the presence of the test compound. Cell proliferation was quantitatively determined using the ATP lite cell proliferation kit (Packard). Inhibition of cell proliferation is shown in table 1. Columns 2-5 show the concentration of compound required to induce 50% cell death of human endometrial cells (IC)₅₀Expressed in μ M).

TABLE 1

3.3 in vivo potency Studies (mouse xenograft model)

Male nude (nu/nu) mice are injected subcutaneously on the right forelimb with a number of human tumor cell line cells, such as Colo-205, a375, or MiaPaCa 2. Tumors were measured 1 week after cell implantation using a vernier caliper. Measuring the length (I) and width (W) of the tumor using the equation I W²The tumor volume was calculated 2. Animals were grouped such that the average tumor volume of each group was 150-200mm³Starting with the compoundTreatment (unit labeled day 0). Tumor volume and body weight were determined for each animal on days 0, 4, 6, 8, 10, 12 and 14. Tumor volume and percent body weight were analyzed by analysis of variance with two-way repeated measures (RM-ANOVA), followed by Fisher's post-hoc multiple pair-wise comparisons of the mean values of the treatment groups. In this embodiment, the compounds are effective in these tumor models, and can result in dose-dependent tumor growth inhibition, including tumor regression or tumor disappearance. For example, compound (9) was able to inhibit tumor growth by 98.5% in the Colo-205 xenograft model when administered by the oral route at a daily dose of 1.5 mg/kg. Compound (8) was able to achieve 100.69% inhibition of Tumor Growth (TGI) in the same model when administered by the oral route at 50 μ g/kg/day, and 115.33% inhibition of tumor growth when administered at 150 μ g/kg/day. In the MiaPaCa2 model, compound (8) had a TGI of 100.97% at a dose of 33. mu.g/kg/day and a TGI of 110.74% at a dose of 50. mu.g/kg/day.

Claims (12)

1. A compound of formula (II):

wherein:

R¹hydrogen, methyl, ethyl, n-propyl, i-propyl or Hal,

R²is hydrogen, methoxy, ethoxy or Hal,

R³、R⁴independently selected from hydrogen or Hal, and

hal is F, Cl, Br or I.

2. A compound according to claim 1, wherein the groups not specified in more detail are as defined in formula (II) of claim 1, but wherein:

in subformula IA:

R¹is Hal, methyl or ethyl,

R²is hydrogen, Hal or methoxy,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

hal is F, Cl, Br or I;

in subformula IB:

R¹in order to be Hal, the catalyst is,

R²is hydrogen or Hal, and is,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

hal is F, Cl, Br or I;

in subformular IC:

R¹is F, Cl, a methyl group or an ethyl group,

R²is hydrogen, I, Br or a methoxy group,

R³is hydrogen or Hal, and is,

R⁴is hydrogen or Hal, and is,

hal is F, Cl, Br or I;

in subformula ID:

R¹is F, Cl, a methyl group or an ethyl group,

R²is hydrogen, I, Br or a methoxy group,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl;

in subformula IE:

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl;

in subformula IF:

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl;

in subformula IG:

R¹is F or Cl, and the like,

R²is the compound I or the compound Br,

R³is a hydrogen atom, and is,

R⁴is hydrogen;

in subformula IH:

R¹in the case of F, the content of the compound,

R²the compound is shown as I, and the compound is,

R³is hydrogen or F, and the compound is,

R⁴is hydrogen or Cl.

3. A compound and pharmaceutically acceptable salts thereof, wherein the compound is selected from the group consisting of:

。

4. a pharmaceutical composition comprising as an active ingredient a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

5. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for use as a medicament.

6. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for use in the treatment of hyperproliferative diseases associated with excessive MEK activity, as well as diseases modulated by the MEK cascade, in a mammal.

7. A compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein the disease is selected from cancer, inflammation, pancreatitis or kidney disease, pain, benign hyperplasia of the skin, restenosis, prostate disease, diseases associated with angiogenesis or vasculogenesis, tumor angiogenesis, skin diseases selected from psoriasis, eczema and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma and Kaposi's sarcoma.

8. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein the disease is cancer.

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the cancer is a cancer of the brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, kidney, ovary, prostate, colorectal, esophageal, testicular, gynecological, or thyroid; melanoma; acute myeloid leukemia, multiple myeloma, chronic myeloid leukemia, or myeloid cell leukemia.

10. The use of a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of hyperproliferative diseases associated with excess MEK activity, and diseases modulated by the MEK cascade, in a mammal.

11. The use according to claim 10, wherein the disease is selected from the group consisting of cancer, inflammation, pancreatitis or kidney disease, pain, benign hyperplasia of the skin, restenosis, prostate disease, diseases associated with angiogenesis or vasculogenesis, tumor angiogenesis, skin diseases selected from psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, and kaposi's sarcoma.

12. A kit comprising the following individually packaged products:

a) an effective amount of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and

b) an effective amount of another pharmaceutically active ingredient.