RU2418790C2 - Novel hydrosulphate salt - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to hydrosulphate salt of compound 1

its solvates, crystalline forms and amorphous forms, as well as to method of obtaining them. In addition, invention relates to application of claimed salt in manufacturing of medication for treatment of MEK-mediated disease states and to methods of said state treatment.

EFFECT: elaboration of efficient method of obtaining hydrosulphate salt of compound.

16 cl, 3 ex, 3 tbl, 3 dwg

Description

BACKGROUND OF THE INVENTION

Related Application

This application claims the priority of provisional application US No. 60/752781, filed December 21, 2005, which is included in this description of the invention by this link in its entirety.

Field of Invention

The present invention relates to a new salt and, more specifically, to a new salt of (2-hydroxyethoxy) amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (hereinafter referred to as “Compound 1”), which is an MEK (mitogen-activated protein kinase kinase) inhibitor useful in the treatment and / or prevention of proliferative disease states, such as cancer, in a mammal. More specifically, the present invention relates to the hydrosulfate salt of compound 1 and methods for producing said salt. Also provided are pharmaceutical compositions comprising the hydrosulfate salt of compound 1, and the use of this salt in the manufacture of medicaments for the treatment and / or prophylaxis of proliferative disease states such as cancer in a human or animal body, and methods for treating proliferative disease conditions such as cancer, mammal by introducing a therapeutically effective amount of a hydrosulfate salt of compound 1.

Description of the state of the art

Signaling in cells through growth factor receptors and protein kinases is an important regulator of cell growth, proliferation and differentiation. In normal cell growth, growth factors through activation of receptors (i.e., PDGF (platelet-derived growth factor) or EGF (epidermal growth factor) or others) activate MAP (mitogen-activated protein kinase) kinase pathways of metabolism. One of the most important and best-studied MAP kinase pathways involved in normal and uncontrolled cell growth is the Ras / Raf kinase pathway of metabolism. Active GTP (guanosine triphosphate) -linked Ras leads to activation and indirect phosphorylation of Raf kinase. Raf then phosphorylates MEK1 and 2 at two serine residues (S218 and S222 for MEK1 and S222 and S226 for MEK2) (Ann et al., Methods in Enzymology, 2001, 332: 417-431). Activated MEK then phosphorylates the only known substrates, MAP kinases ERK1 and 2. Phosphorylation of ERK by MEK occurs at Y204 and T202 for ERK1 and Y185 and T183 for ERK2 (Ahn et al., Methods in Enzymology 2001, 332: 417-431). Phosphorylated ERK dimerizes and then translocates to the nucleus, where it accumulates (Khokhlatchev et al., Cell 1998, 93: 605-615). In the nucleus, ERK is involved in a number of important cellular functions, including, but not limited to, nuclear transport, signal transduction, DNA repair, assembly and translocation of nucleosomes, processing and translation of transport RNA (Ahn et al., Molecular Cell, 2000, 6: 1343- 1354). In general, treatment of cells with growth factors leads to activation of ERK1 and 2, which causes proliferation and, in some cases, differentiation (Lewis et al., Adv. Cancer Res. 1998, 74: 49-139).

In proliferative diseases, genetic mutations and / or overexpression of growth factor receptors, downstream signaling proteins or protein kinases involved in the ERK kinase pathway of metabolism leads to uncontrolled cell proliferation and, ultimately, to the formation of a tumor. For example, some types of cancer contain mutations that cause continuous activation of this pathway due to the continuous production of growth factors. Other mutations can cause defects in the deactivation of the activated complex of GTP-linked Ras, which again leads to the activation of the MAP kinase pathway. Mutated oncogenic forms of Ras are found in 50% of cases of colon cancer and more than 90% of cases of pancreatic cancer, as well as in many other types of cancer (Kohl et al., Science, 1993, 260: 1834-1837). Recently, bRaf mutations have been detected in more than 60% of cases of malignant melanoma (Davies, N., et al., Nature 2002, 417: 949-954). These mutations in bRaf lead to a constitutively active MAP kinase cascade. Studies of primary tumor samples and cell lines have also shown constitutive or increased activation of the MAP kinase pathway of metabolism in cancer of the pancreas, colon, lung, ovary and kidney (Hoshino, R. et al., Oncogene 1999, 18: 813-822). Therefore, there is a strict correlation between types of cancer and increased activation of the MAP kinase pathway of metabolism resulting from genetic mutations.

Since constitutive or increased activation of the MAP kinase cascade plays a crucial role in cell proliferation and differentiation, it is believed that inhibition of this pathway will have a beneficial effect in hyperproliferative diseases. MEK is a key player on this path, as it is located below Ras and Raf. In addition, it is an attractive therapeutic target because the only known MEK phosphorylation substrates are MAP kinases, ERK1, and 2. In separate studies, inhibition of MEK has shown a potential therapeutic beneficial effect. For example, it has been shown that MEK inhibitors, which are small molecules, inhibit the growth of human tumors in xenografts of nude mutant nude mice (Sebolt-Leopold et al., Nature-Medicine 1999, 5 (7): 810-816; Trachet et al ., AACR April 6-10, 2002, Poster # 5426; Tecle, H., IBC 2 ^nd International Conference of Protein Kinases, September 9-10, 2002), block static allodynia in animals (WO 01/05390) and inhibit growth cells in acute myeloid leukemia (Milella et al., J. Clin. Invest. 2001, 108 (6): 851-859).

MEK inhibitors are known to be small molecules. Over the past few years, there have been at least thirteen patent applications: US 5525625; 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 and WO 02/06213.

MEK inhibitors are also described in WO 03/077914. (2-Hydroxyethoxy) -amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid, or “compound 1”, is exemplified in WO 03 / 077914 and has the following structural formula:

compound 1

Compound 1 has been shown to have inhibitory activity against MEK and, therefore, is useful in the treatment of a hyperproliferative disease such as cancer.

WO 03/077914 discloses, in general terms, some pharmaceutically acceptable salts of the compounds described therein. In particular, WO 03/077914 states that pharmaceutically acceptable salts of the compounds disclosed therein having a sufficiently basic moiety and which can form acid addition salts contain pharmaceutically acceptable anions, and a number of such anions are listed. Similarly, suitable salts of compounds having an acid moiety should be prepared by treating the compound with a basic compound, in particular an inorganic base.

A convenient form of the pharmaceutically active compound used in drugs is a form that provides suitable processing properties to process and prepare the pharmaceutical composition. However, it is also necessary to ensure the optimal biological properties of the final composition, such as the dissolution rate of the tablets and the bioavailability of the active ingredient, and often when choosing a particular form that best meets all these different requirements, you have to compromise. However, in some cases, salts are not easily formed and / or are unstable, probably due to low pKa values. The pKa value expresses the strength of the acid and base, that is, the tendency of the acid to lose a proton or base to attach a proton (Bronsted J.N., Rec. Trav. Chim. (1923) 47: 718). In particular, this is true for compound 1.

SUMMARY OF THE INVENTION

The present invention provides a hydrosulfate salt (drug: H ₂ SO ₄ 1: 1) of compound 1 and its various forms, which are all included in the scope of the invention. The salt may be in various forms, which are all included in the scope of the invention. These forms include both anhydrous forms and solvates. An additional form can be obtained by desolvation of solvates. In a specific embodiment, the salt is in anhydrous form.

According to a further aspect of the present invention, there is provided a method for using the hydrosulfate salt of compound 1 as a medicament for treating a hyperproliferative disease or condition.

An additional aspect of the invention is the use of the hydrosulfate salt of compound 1 in the manufacture of a medicament for the treatment or prevention of a hyperproliferative disease or condition.

Additional advantages and new features of this invention will be partially set forth in the following description and will be partially apparent to those skilled in the art after studying the following detailed account, or may be understood by practical use of the invention. The advantages of this invention can be realized and achieved using the means, combinations, compositions and methods specifically indicated in the attached claims.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The accompanying drawings, which are incorporated herein and form part of the detailed presentation, illustrate non-limiting embodiments of this invention and, together with the description, serve to explain the principles of the present invention.

In graphic materials:

figure 1 shows the x-ray powder diffraction (XRPD) of the hydrosulfate salt of compound 1;

Figure 2 shows the infrared spectrum of the hydrosulfate salt of compound 1 obtained using the diffuse reflectance infrared spectroscopy Fourier transform (DRIFTS, Diffuse Reflectance Infrared Fourier Transform Spectroscopy) sampling method, and

figure 3 presents the results of the concentration levels of compound 1 in plasma after administration to dogs on an empty stomach, oral dispersed doses of compound 1 (×) and hydrosulfate salt (▲), equivalent to 150 mg of free base.

DETAILED DESCRIPTION OF THE INVENTION

Below will be presented a detailed link to some embodiments of this invention, examples of which are illustrated in the accompanying structures and formulas. Although the present invention will be described in combination with numbered embodiments, it should be understood that they are not intended to limit the invention to these embodiments. On the contrary, it is intended that the invention covers all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize a variety of methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the described methods and materials. In the event that one or more of the included literature, patent and similar material differs from this application or contradicts it, including specified terms, use of terms, described methods or the like, but not limited to, this application shall prevail.

The present invention provides a hydrosulfate salt (a medicine for H ₂ SO ₄ 1: 1) of compound 1 and its various forms, which are all included in the scope of the invention. The salt may be in various forms, which are all included in the scope of the invention. These forms include both anhydrous forms and solvates. An additional form can be obtained by desolvation of solvates. In a specific embodiment, the salt is an anhydrous hydrosulfate salt of compound 1. Additionally, the present invention provides a hydrosulfate salt form of compound 1, which exhibits unique physical and pharmaceutical properties, which makes it particularly suitable for use in pharmaceuticals.

In some embodiments, the salts of compound 1 are crystalline. It was found that crystalline salts are better than the free base based on their technological properties, from a production point of view, in particular their static and rheological properties. The formation of salts may provide a purification method, since it is possible to separate the impurities formed in the process, and salts are usually easier to isolate than the free base.

In one embodiment of the invention, the hydrosulfate salt of compound 1 is a crystalline salt, which was unexpectedly found to have improved pharmaceutical properties compared to the free base of compound 1 and some other salt forms of compound 1. In particular, the dissolution rate of this crystalline salt, as well as its bioavailability has been found to be especially high compared to the free base and other salts, as illustrated in the examples below. The increased bioavailability of the hydrosulfate salt of compound 1 compared to the free base, as shown, does not depend on the pharmaceutical composition used for administration. The bioavailability of the free base and hydrosulfate forms was compared in this description of the invention after dosing in the same dispersed compositions, but similar differences in bioavailability were also observed for simple tablet compositions.

If it is claimed that the present invention relates to a salt of compound 1, which is a crystalline salt, it is convenient that the degree of crystallinity is more than about 60%, more conveniently more than about 80%, preferably more than about 90% and more preferably more than approximately 95%. Most preferably, the degree of crystallinity is higher than about 98%.

The magnitude of the increased bioavailability offered by the hydrosulfate salt is unexpected and very useful since the free base of compound 1 is classified as a Class 4 compound of BCS (Biopharmaceutical Classification System). BCS Class 4 compounds usually have low bioavailability due to both low dissolution rate and permeability, and limiting absorption permeability means that salts of this type are usually not expected to exhibit a significant effect on absorption (See, for example, Dressman et al. (2001) Pharm Tech. July: 68).

Suitable solvates of the hydrosulfate salt of compound 1 are formed from a wide variety of solvents, in particular organic solvents such as tetrahydrofuran (THF), acetonitrile (ACN), ethanol (EtOH) and methanol (MeOH). Suitable organic solvents include esters, such as C _1-6 alkyl esters, for example ethyl acetate, and ketones, such as C _1-6 alkyl ketones, for example methyl ethyl ketone (2-butanone).

Obtaining salt can be carried out by reacting a suspension of compound 1 in an organic solvent and water with sulfuric acid. To obtain a 1: 1 salt, approximately 1 equivalent of sulfuric acid is used. Thus, in an additional aspect of the invention, a method for producing a hydrosulfate salt of compound 1, including:

1) the interaction of a suspension of compound 1 in an organic liquid and water with approximately 1 equivalent of sulfuric acid;

2) the allocation of salt from the resulting solution; and

3) after that, if desired or necessary, the formation of its solvate.

The molar ratio of the amount of sulfuric acid to the amount of compound 1 is suitable in the range from 1.00: 1 to 2: 1, for example in the range from 1.05: 1 to 1.15: 1. The use of sulfuric acid in the form of concentrated sulfuric acid is suitable. In a specific embodiment, the molar ratio of sulfuric acid to compound 1 is 1.10: 1.0.

A suitable amount of water added in step (1) is limited to the amount necessary to ensure the formation of salt. The exact amounts used will depend on the particular nature of the solvent, the concentration of sulfuric acid and so on, but usually water will be present in an amount of less than 20% (vol./about.) Of the total amount of liquid present, for example from 13-17% (vol. /about.).

In a specific embodiment, the organic solvent used in step (1) is 2-butanone (methyl ethyl ketone), water is about 15% of the liquid volume, and the total amount of liquid used relative to compound 1 is about 8 ml per gram of compound 1.

It is suitable to carry out the addition of sulfuric acid in step (1) in a controlled manner, for example at a temperature below 10 ° C, and the rest of step (1) is then carried out at an elevated temperature, for example at 30-90 ° C, as a further example in the range between 55-75 ° C and, as another additional example, at approximately 65 ° C.

Suitable organic liquids include organic solvents in which compound 1 and its salts are sparingly soluble. The expression “sparingly soluble” as used herein means having a solubility of less than 100 ml of solvent per gram of solute, for example between 30 and 100 ml of solvent per gram of solute. Such solvents include alkyl ketones, for example C _1-6 alkyl ketones, such as 2-butanone, alcohols, such as C _1-6 alcohols, such as methanol or ethanol, and esters, such as C _1-6 alkyl esters, for example ethyl acetate. In one embodiment, the organic solvent is methyl ethyl ketone (2-butanone).

It is suitable to filter the reaction mixture between steps (1) and (2) to remove any impurities. The residue is optionally washed, for example, with a mixture of organic liquid and water, and the desired salt is crystallized from the filtrate, which can optionally be combined with the washing solution.

In some embodiments, the hydrosulfate salt is isolated in step (2) by cooling the reaction mixture, possibly with the addition of additional organic liquid, so that the hydrosulfate salt precipitates. The additional organic liquid may be the same organic liquid that was used in step (1), or it may be another organic liquid provided that it acts on the hydrosulfate salt of compound 1 as an anti-solvent. Seeding of the solution with the hydrosulfate salt crystals of compound 1 can facilitate the precipitation process.

In one embodiment, prior to cooling, the filtrate is first subjected to a distillation step to remove water and ensure that the salt is separated in an acceptable yield. In a specific embodiment, the solvent is 2-butanone and the filtrate is distilled at atmospheric pressure.

After cooling, a salt can be isolated from the resulting suspension, for example, by filtration. The recovered material can then be dried, for example, at an elevated temperature, for example from 40-60 ° C, and as another example, at approximately 50 ° C, until a constant weight is achieved. If the product is a solvate with an organic liquid, such as methanol, then it can be desolvated by heating at this stage if desired.

The physical properties of the hydrosulfate salt were investigated and further described in the examples.

The present invention also includes compounds labeled with isotopes that are identical to those set forth in the present invention, but in which one or more than one atom is replaced by an atom having an atomic mass or atomic number different from the atomic mass or atomic number that is commonly found in nature. Examples of isotopes that can be included in the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ² N, ³ N, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl, respectively. The hydrosulfate salt of compound 1 and its polymorphic forms that contain the isotopes and / or other isotopes of other atoms mentioned above are within the scope of this invention. Some compounds of the present invention labeled with isotopes, for example, those which include radioactive isotopes, such as ³ H and ¹⁴ C, are useful in analyzes of the distribution of the drug and / or substrate in the tissues. Tritium isotopes, i.e. ³ N, and carbon-14, i.e. ¹⁴ C, are especially widely used due to the ease of their preparation and detection. In addition, substitution with heavier isotopes such as deuterium, i.e. ² N, may provide some therapeutic benefits resulting from increased metabolic stability, for example, an increased in vivo half-life or a need for a reduced dosage, and therefore may be used in some specific circumstances. . The salts of the present invention labeled with isotopes can usually be obtained by implementing the procedures disclosed in WO 03/077914 by replacing an unlabeled isotope reagent with an easily accessible isotope-labeled reagent in the salt process, or, if desired, using sulfuric isotope-labeled acid in salt production.

The composition may be in a form suitable for oral administration (for example, in the form of tablets, lozenges, hard or soft capsules, emulsions, dispersible powders or granules, syrups, elixirs or oily, or prepared for immediate administration of aqueous suspensions), for administration by inhalation ( for example, as a fine powder or liquid aerosol), for administration by insufflation (for example, as a fine powder), for parenteral injection (for example, as a sterile solution, suspension or emulsion for intravenous, subcutaneous, intramuscular, intravascular or infusion dosage), for local administration (for example, in the form of creams, ointments, gels, oily solutions or suspensions or prepared for immediate administration of aqueous suspensions) or for rectal administration (for example, in the form of a suppository) . In one embodiment, the hydrosulfate salt of compound 1 is administered orally. The compositions mentioned above can usually be obtained in a conventional manner using conventional excipients.

The amount of active compound administered will depend on the subject being treated, the severity of the disorder or condition, the rate of administration, the availability of the compound and the choice of the attending physician. However, an effective dosage is in the range of from about 0.01 to about 100 mg per kg of body weight per day, preferably from about 1 to about 35 mg / kg / day, in single or divided doses. For a person weighing 70 kg, this amount will be from about 0.7 to 7000 mg / day, preferably from about 70 to about 2500 mg / day. In some cases, dosage levels below the lower limit of the above range may be more than sufficient, while in other cases, higher doses can be used without any harmful side effect, provided that such increased doses are first divided into several small doses for introduction during the day. A unit dosage form, such as a tablet or capsule, will usually contain, for example, 1-1000 mg of the active ingredient, and preferably 5-420 mg of the active ingredient. A daily dose in the range of 0.03-6 mg / kg is preferably used.

According to a further aspect of the present invention, there is provided a hydrosulfate salt of compound 1, as defined herein, for use in a method of treating or preventing a human or animal organism through therapy. An additional feature of the present invention is the hydrosulfate salt of compound 1, as defined here, for use as a medicine. In a further aspect of the present invention, there is provided a hydrosulfate salt of a compound as defined herein for use as a medicament for treating disease conditions mediated by MEK, in particular proliferative disorders, or abnormal cell growth, such as cancer, in a warm-blooded mammal such as humans . Accordingly, an additional aspect of the invention provides the use of a hydrosulfate salt of compound 1, as defined herein, in the manufacture of a medicament for use in the treatment of disease conditions mediated by MEK, in particular proliferative disorders, or abnormal cell growth, such as cancer, in a warm-blooded mammal, such as a person.

According to a further feature of the invention, there is provided a method of treating disease conditions mediated by MEK, in particular proliferative disorders, or abnormal cell growth, such as cancer, in a warm-blooded mammal, such as a human in need of such treatment, comprising administering to said mammal an effective amount of a hydrosulfate salt of compound 1 as defined herein, or a pharmaceutical composition as defined herein.

Specific examples of proliferative disorders that can be treated using the salts or compositions of the invention include mammalian hyperproliferative disorders. Specific types of cancer are cancer of the brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, renal, kidney, ovary, prostate, colorectal, esophageal, testicular, gynecological or thyroid cancer .

However, the compounds and compositions of the invention can also be used in the treatment of noncancerous hyperproliferative disorder, such as benign skin hyperplasia (e.g., psoriasis), restenosis or prostatic hyperplasia (e.g., benign prostatic hypertrophy (BPH)).

Other examples of MEK-mediated diseases that can be treated using the compounds or compositions of the invention include pancreatitis or kidney disease (including proliferative glomerulonephritis and kidney disease caused by diabetes) or pain in a mammal.

The compounds and compositions can also be used to prevent implantation of blastocytes in a mammal or to treat a disease associated with the formation and development of blood vessels or angiogenesis in a mammal. Such diseases may include tumor angiogenesis, a chronic inflammatory disease such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema and scleroderma, diabetes, diabetic retinopathy, retrolental fibroplasia, age-related macular degeneration, hemangioma, melanoma, Kaposi’s sarcoma, and cancer of the ovary, breast, lung, pancreas, prostate, colon, and epidermoid cancer.

The terms “abnormal cell growth” and “hyperproliferative disorder” are used interchangeably herein and refer to cell growth that is independent of normal regulatory mechanisms (eg, loss of contact inhibition). It includes, for example, the abnormal growth of: (1) tumor cells (tumors) that proliferate by expression of a mutated tyrosine kinase or overexpression of receptor tyrosine kinase; (2) benign and malignant cells in other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumors that proliferate through receptor tyrosine kinases; (4) any tumors that proliferate by aberrant activation of serine / threonine kinase; and (5) benign and malignant cells of other proliferative diseases in which serine / threonine kinase activation occurs.

The term "treatment", as used herein, unless otherwise indicated, means a change in direction, alleviation, inhibition of progression, or the prevention of a disorder or condition to which the term applies, or one or more than one symptom of such a disorder or condition. The term "treatment" used in this description of the invention, unless otherwise indicated, also refers to the act of treatment, defined directly above as "treatment".

Thus, patients who can be treated with the compounds or compositions of the present invention include, for example, patients who have been diagnosed with psoriasis, restenosis, atherosclerosis, BPH, lung cancer, non-small cell lung cancer, bone cancer, CMML (chronic myelomonocytic leukemia), pancreatic cancer, colorectal cancer, skin cancer, head and neck cancer, melanoma (in particular skin or intraocular melanoma), uterine cancer, ovarian cancer, colorectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer , testicular cancer, gynecological tumors (e.g., uterine sarcomas, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma or vulvar carcinoma), ovarian cancer, multiple myeloma, hepatocellular carcinoma, Hodgkin's disease, cancer of the esophagus, endocrine cancer, cancer systems (e.g., thyroid, parathyroid, or adrenal cancer), soft tissue sarcomas, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, in particular acute myeloid leukemia, sal idiotic tumors in childhood, lymphocytic lymphomas, bladder cancer, cancer of the kidney or ureter (e.g., renal cell carcinoma, renal pelvis carcinoma) or neoplasm of the central nervous system (CNS) (e.g. primary CNS lymphoma, spinal axis tumors, gliomas of the brainstem brain or pituitary adenoma).

The hydrosulfate salt of compound 1 can be used as a single therapy, or it can include, in addition to the hydrosulfate salt of compound 1, one or more than one substance and / or treatment. Such co-treatment can be achieved by simultaneous, sequential or separate administration of the individual components of the treatment. In the field of medical oncology, it is normal practice to use a combination of different forms of treatment to treat each patient with cancer. In medical oncology, the other component (s) of such a joint treatment, in addition to the hydrosulfate salt of compound 1, may be surgery, radiotherapy or chemotherapy. Such chemotherapy may cover such categories of therapeutic agent as:

(1) anti-angiogenic agents, such as agents that inhibit the effects of vascular endothelial growth factor (VEGF) (for example, an antibody against vascular endothelial cell growth factor bevacizumab [Avastin ™] and VEGF receptor tyrosine kinase inhibitors, such as 4- (4-bromo- 2-fluoroanilino) -6-methoxy-7- (1-methylpiperidin-4-ylmethoxy) quinazoline (ZD6474; Example 2 in WO 01/32651), 4- (4-fluoro-2-methylindol-5-yloxy) -6 -methoxy-7- (3-pyrrolidin-1-ylpropoxy) quinazoline (AZD2171; Example 240 in WO 00/47212), vatalanib (PTK787; WO 98/35985) and SU 11248 (sunitinib; WO 01/60814), such compounds as disclosed in international patent applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354, and those that work by mechanisms different from the mechanisms described here (for example, linomide, integrin αvβ3 function inhibitors, angiostatin, razoxin , thalidomide, MMP-2 inhibitors (matrix metalloproteinases 2), MMP-9 inhibitors (matrix metalloproteinases 9) and COX-II inhibitors (cyclooxygenase II)), and

(2) vascular targeting agents (e.g., combretastatin phosphate and the compounds disclosed in WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213) and vascular damage agents described in the publication of the international patent application No. WO 99/02166 (e.g., N-acetylcolquinol-O-phosphate);

(3) cytostatic agents such as antiestrogens (e.g. tamoxifen, toremifene, raloxifene, droloxifene and iodoxifene), negative regulators of estrogen receptors (e.g. fulvestrant), progestogens (e.g. megestrol acetate), aromatase inhibitors (e.g. anastrol, ageraz borazole and exemestane), anti-progestogens, anti-androgens (e.g. flutamide, nilutamide, bicalutamide and cyproterone acetate), LHRH agonists and antagonists (luteinizing hormone releasing hormone) (e.g. goserelin acetate, leuprorelin and b) gibitory 5α-reductase inhibitor (e.g., finasteride);

(4) anti-invasive agents (for example, metalloproteinase inhibitors like marimastat and inhibitors of the functioning of receptors for the urokinase plasminogen activator or anti-Heparanese antibody;

(5) growth factor function inhibitors (such growth factors include, for example, platelet growth factor and hepatocyte growth factor), such inhibitors include growth factor antibodies, growth factor receptor antibodies (eg, anti-erbb2 antibody trastuzumab [Herceptin ™], an antibody to EGFR (epidermal growth factor receptors) panitumumab, an anti-erbB1 antibody cetuximab [C225]), as well as any antibodies to growth factors or growth factor receptors disclosed by Stern et al. in Critical reviews in oncology / haematology, 2005, Vol. 54, pp. 11-29); such inhibitors also include tyrosine kinase inhibitors, such as epidermal growth factor family inhibitors (e.g., EGFR family tyrosine kinase inhibitors, such as N- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) - quinazolin-4-amine (gefitinib, AZD1839), N- (3-ethynylphenyl) -6,7-bis (2-methoxymethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N- (3 -chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (Cl 1033)), and erbB2 tyrosine kinase inhibitors such as lapatinib, hepatocyte growth factor family inhibitors, family inhibitors platelet-derived growth factors, such as imatinib, serine / threonine kinase inhibitors (e.g., Ras / Raf signaling inhibitors, such as farnesyl transferase inhibitors, e.g. sorafenib (BAY 43-9006)), cell-signal inhibitors via MEK and / or ACT kinases, inhibitors c-kit, abl kinase inhibitors, IGF receptor kinase inhibitors (insulin-like growth factor); aurora kinase inhibitors (for example, AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459) and cyclin-dependent kinase inhibitors such as CDK2 and / or CDK4 inhibitors;

(6) antiproliferative / antineoplastic drugs and combinations thereof used in medical oncology, such as antimetabolites (e.g., antifolates, such as methotrexate, fluoropyrimidines, such as 5-fluorouracil, tegafur, purine, and analogues of adenosine, and cytosine arabinoside, hydroxymera for example, one of the antimetabolites specifically disclosed in European Patent Application No. 239362, such as N- (5- [N- (3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -N-methylamino -2-tenoyl) -L-glutamic acid]; antitumor antibiotics (eg measures, anthracyclines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin and mitramycin); platinum derivatives (e.g. cisplatin and carboplatin); alkylating agents (e.g., mustard, mustard (nitrogen) , chlorambucil, busulfan, cyclophosphamide, ifosfamide, nitrosoureas and thiotepa); anti-mitotic agents (e.g. vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine, and taxoids such as taxol and taxotere); topoisomerase inhibitors (for example, epidophyllotoxins, such as etoposide and teniposide, amsacrine, topotecan, camptothecin and irinotecan); enzymes (e.g. asparaginase) and thymidylate synthase inhibitors (e.g. raltitrexed);

and additional types of chemotherapeutic agent, including:

(7) biological response modifiers (e.g., interferon);

(8) antibodies (e.g. edrecolomab (edrecolomab));

(9) antisense therapies, such as those aimed at the targets listed above, such as ISIS 2503, and anti-ras antisense therapy;

(10) gene therapy approaches, including, for example, approaches for replacing aberrant genes, such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-targeted enzymatic prodrug therapy) approaches, such as those using cytosine deaminase, thymidine kinase, or bacterial enzyme nitrode approaches to increase patient tolerance to chemotherapy or radiation therapy, such as multidrug resistance gene therapy; and

(11) immunotherapeutic approaches, including, for example, ex-vivo and in vivo approaches to increase the immunogenicity of a patient’s tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to reduce T-cell anergy , approaches using transfected immune cells, such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines, and approaches using ant idiotypic antibodies.

For example, the hydrosulfate salt of compound 1 can be used in combination with an effective amount of one or more than one substance selected from antiangiogenic agents, signal transduction inhibitors and antiproliferative agents.

In a specific embodiment, antiangiogenic agents, such as MMP-2 inhibitors (matrix metalloproteinases 2), MMP-9 inhibitors (matrix metalloproteinases 9) and COX-II inhibitors (cyclooxygenase II), can be used in combination with the hydrosulfate salt of compound 1 of the present invention and the pharmaceutical compositions described herein. Examples of useful COX-II inhibitors include CELEBREX ™ (alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloprotein kinase inhibitors are described in WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90 / 05719, WO 99/52910, WO 99/52889, WO 99/29667, US patent 5863949 and US patent 5861510, all of which are incorporated herein by reference in their entirety. Suitable inhibitors of MMP-2 and MMP-9 are those that do not have inhibitory activity or have weak inhibitory activity to MMP-1. In particular, those are used which selectively inhibit MMP-2 and / or MMP-9 with respect to other matrix metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7 , MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Specific examples of MMP inhibitors useful in the present invention are AG-3340, RO 32-3555 and RS 13-0830.

Therefore, an additional aspect of the present invention is the hydrosulfate salt of compound 1 in combination with any of the antitumor agents listed above in (1) to (11) above. According to a further aspect of the present invention, there is provided a hydrosulfate salt of compound 1 in combination with one or more antitumor agents listed above in (1) to (11) above. According to an additional aspect of the present invention, a hydrosulfate salt of compound 1 is provided in combination with any of the classes of antitumor agents listed above in (1) to (11) above.

In this description, when the term "combination" is used, it should be understood that it relates to simultaneous, separate or sequential administration. In one aspect of the invention, “combination” refers to simultaneous administration. In another aspect of the invention, “combination” refers to separate administration. In a further aspect of the invention, “combination” refers to sequential administration. In cases where the administration is sequential or separate, the delay in the administration of the second component should not be such as to lose the effect of the benefits of the combination.

According to an additional aspect of the present invention, there is provided a kit comprising a hydrosulfate salt of a compound in combination with an antitumor agent selected from those listed above in (1) to (11).

According to a further aspect of the present invention, there is provided a kit comprising:

a) the hydrosulfate salt of compound 1 in a first unit dosage form;

b) an antitumor agent selected from those listed above in (1) to (11) in a second unit dosage form; and

c) a container intended for storing said first and second dosage forms.

The activity of compound 1 was detected in the following analysis. 6 His N-terminally labeled constitutively active MEK1 (2-393) is expressed in E. coli, and the protein is purified by conventional methods (Ahn et al., Science 1994, 265, 966-970). MEK1 activity is assessed by measuring the incorporation of γ- ³³ P-phosphate from γ- ³³ P-ATP into His-labeled N-terminus of ERK2, which is expressed in E. coli and purified by conventional methods in the presence of MEK1. The analysis is carried out in a 96-well polypropylene plate. The incubation mixture (100 μl) contains 25 mM Hepes, pH 7.4, 10 mM MgCl ₂ , 5 mM β-glycerophosphate, 100 μM sodium orthovanadate, 5 mM DTT (dithiothreitol), 5 nM MEK1 and 1 μM ERK2. Inhibitors are suspended in DMSO (dimethyl sulfoxide), and all reaction mixtures, including controls, are prepared at a final concentration of 1% DMSO. Reactions are initiated by the addition of 10 μM ATP (with 0.5 μCi γ- ³³ P-ATP / well) and incubated at ambient temperature for 45 minutes. To stop the reaction and precipitate the proteins add an equal volume of 25% TCA (trichloroacetic acid). The precipitated proteins are separated on type B fiberglass filter plates, and excess labeled ATP is washed using a Tomtec MACH III harvester. The plates were allowed to air dry, after which Packard Microscint 20 was added at a rate of 30 μl / well, and the plates were read using Packard TopCount. In this analysis, compound 1 showed an IC ₅₀ value of less than 50 micromolar.

EXAMPLES

The following examples are included to illustrate the invention. However, it should be understood that these examples do not limit the invention and are intended only to offer a method of carrying out the invention. The yields in the examples are given as they turned out, and can probably be increased after further improvement. As standard in the ¹ H NMR spectrum (400 MHz) using TMS (tetramethylsilane) (0.00 mn ^{-1), 13} C NMR spectrum (100 MHz) - NMR solvent (39.5 million ^-1), and ¹⁹ F NMR spectrum - trichlorofluoromethane (0.00 mn ^-1). FTIR spectra (Fourier transform infrared spectra) were obtained on a Nicolet Magna 860 ESP FTIR spectrometer in various ways, including from 2% w / w dispersion of this material in KBr powder using a DRIFTS sampling method of 4,000-400 cm ^-1 mid-infrared.

Example 1

Obtaining hydrosulfate salt of compound 1

To a stirred suspension of (2-hydroxyethoxy) -amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (100 g, 0.206 mol) (prepared as described in Example 10, from WO 03/077914, which is incorporated herein by reference and as described below) in 2-butanone (680 ml) and water (115 ml) at 0-5 ° C. sulfuric acid (12.3 ml, 0.226 mol) and then water (5 ml), maintaining a temperature of 10 ° C or lower. The stirred mixture was heated to 65 ° C and held for 30 minutes, after which it was filtered to remove any impurities. The filter was washed with a mixture of 2-butanone (85 ml) and water (15 ml). The combined filtrates were heated to 72 ° C, after which 2-butanone (500 ml) was added, maintaining the temperature between 60 and 72 ° C. The resulting mixture was distilled at atmospheric pressure (distillation temperature approximately 73-74 ° C) until then, until it was collected 500 ml of distillate.

A second aliquot of 2-butanone (500 ml) was added while maintaining the temperature of the mixture above 70 ° C. The resulting mixture was again distilled until 250 ml of distillate was collected. The mixture was cooled to 0-5 ° C. over about 1 hour. The resulting suspension was filtered, washed with 2-butanone (240 ml) and dried under reduced pressure at 50 ° C until a constant mass was obtained to obtain (2-hydroxyethoxy) -amide hydrosulfate 6- (4-bromo-2-chlorophenylamino) -7-fluoro -3-methyl-3H-benzoimidazole-5-carboxylic acid (103.5 g, 0.186 mol, 90% yield) as an off-white crystalline solid. ¹ H NMR (400 MHz, D ₆ DMSO) δ 3.58 (2H, t, CH ₂ OH), 3.89 (2H, t, CH ₂ ON), 3.99 (3H, s, CH ₃ ), 6.47 (1H, dd, ArH), 7.29 (1H, dd, ArH), 7.63 (1H, d, ArH), 7.91 (1H, s, ArH), 7.96 (3H, br, ROH, NH, SOH), 8.10 (1H, br, ArNH ), 8.94 (1H, s, NCHN), 11.79 (1H, s, ONH). ¹³ C NMR (100 MHz, D ₆ DMSO) δ 32.1 (CH ₃ ), 58.5 (CH ₂ OH), 77.3 (CH ₂ ON), 108.2 (CH), 109.6 (CBr), 115.8 (CH), 120.6 (CCl ), 122.0 (C), 125.0 (CC = O), 129.4 (C), 130.5 (CH), 131.1 (CH), 132.3 (C), 140.6 (C), 145.8 (CF), 146.5 (CH), 164.2 (C = O).

The results of infrared analysis are shown in FIG. 2. The assignments of spectrum frequencies are summarized in Table 1.

Table 1 Wave Attribution number (cm ^-1 ) 3,255 It includes OH stretching vibrations of the primary alcohol group and NH stretching vibrations of the secondary aromatic amine and secondary amide groups. 3,200-2,700 Includes = CH stretching vibrations of the aromatic ring and the benzimidazole group and aliphatic CH stretching vibrations. 2,700-2,300 Includes multiple NH ⁺ stretching vibrations of the benzimidazole 1: 1 sulfate salt group. 1,673 C = O stretching vibrations of the secondary amide group, where 1,653 the carbonyl group undergoes various environmental effects, such as a hydrogen bond. 1,640-1,370 It includes C = C stretching vibrations of the aromatic ring, C = C and C = N stretching vibrations of the benzimidazole group, OH stretching vibrations of the primary alcohol group, and aliphatic CH stretching vibrations. 1,570 The combined CNH band of the secondary amide group. 1,506 Includes CNH flexural vibrations of the secondary aromatic amino group. 1,213 Aryl C-F stretching vibration. 1,189 Asymmetric SO ₃ ^- stretching vibration of the benzimidazole 1: 1 sulfate salt group. 1,100-1,000 It includes the C — O stretching vibration of the primary alcohol group and the aryl C-Br stretching vibration. 1.011 Symmetric SO ₃ ^- stretching vibration of a benzimidazole 1: 1 sulfate salt group. 920-600 It includes CH fan (wag) vibrations and C = C ring bending vibrations of the 1,2,4-trisubstituted aromatic ring and the benzimidazole group. 888 Includes S-O (H) stretching vibration of a benzimidazole 1: 1 sulfate salt group.

Example 1A

Obtaining hydrosulfate salt of compound 1

Sulfuric acid (1.52 ml, 27.86 mmol) was added to a stirred suspension of (2-hydroxyethoxy) -amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzimidazole-5- carboxylic acid (10 g, 0.0214 mol) (obtained as described in Example 10 from WO 03/077914, which is incorporated herein by reference and as described below) in tetrahydrofuran (THF) (62 ml) and water (8 ml), while maintaining a temperature of 10 ° C or lower. The stirred mixture was heated to 65 ° C and held for 30 minutes, after which it was filtered to remove any impurities. Then, THF (150 ml) was added to the mixture, maintaining the temperature above 60 ° C. Then the mixture was cooled to 0-5 ° C for approximately 2 hours. The resulting suspension was filtered, washed with THF (30 ml) and dried under reduced pressure at 50 ° C until constant weight was obtained to obtain (2-hydroxyethoxy) -amide hydrosulfate 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3 -methyl-3H-benzoimidazole-5-carboxylic acid (9.81 g, 0.17 mol, 82% yield) as an off-white crystalline solid. The material was the same as that obtained in Example 1 above.

Example 1B

Preparation of (2-Hydroxyethoxy) -amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid

Stage A: 2,3,4-Trifluoro-5-nitrobenzoic acid: 125 ml of H ₂ SO _{4 were} charged into a three-neck round-bottom flask with a volume of 3 liters. Fuming nitric acid (8.4 ml, 199 mmol) was added and the mixture was gently mixed. In portions of 5 g in 90 minutes, 2,3,4-trifluorobenzoic acid (25 g, 142 mmol) was added. The dark brownish yellow solution was stirred for 60 minutes; during this time the reaction was completed. The reaction mixture was poured into 1 liter of ice: water and extracted with diethyl ether (3 × 600 ml). The combined extracts were dried (MgSO ₄ ) and concentrated under reduced pressure to give a yellow solid. The solid was suspended in hexanes and stirred for 30 minutes, after which it was filtered to obtain 29 g (92%) of the pure desired product as an off-white solid: MS (mass spectrum) CHIAD (chemical ionization at atmospheric pressure), detected : (-) m / z 220 (M-1).

Stage B: 4-amino-2,3-difluoro-5-nitrobenzoic acid: A solution of ammonium hydroxide (approximately 30% in water) (35 ml, 271 mmol) with stirring was added to the solution of 2,3,4-trifluoro- 5-nitrobenzoic acid (15 g, 67.8 mmol) in 30 ml of water at 0 ° C. After complete addition of the ammonium hydroxide solution, the reaction mixture was warmed to room temperature with stirring. After 2.5 hours, the reaction mixture was cooled to 0 ° C and concentrated HCl was carefully added until the pH of the reaction mixture was 0. The reaction mixture was diluted with water (30 ml) and extracted with diethyl ether (3 × 50 ml). The combined organic extracts were dried (MgSO ₄ ) and concentrated under reduced pressure to obtain 14 g (95%) of pure target product: MS HIAD (-), found m / z 217 (M-1).

Stage B: 4-amino-2,3-difluoro-5-nitrobenzoic acid methyl ester: 2M solution of tetramethylsilane (TMS) diazomethane in hexanes (6.88 ml, 13.75 mmol) was added to the suspension of 4-amino-2,3 -difluoro-5-nitrobenzoic acid (2.00 g, 9.17 mmol) in 25 ml of a mixture of tetrahydrofuran (THF): MeOH (4: 1) at 0 ° C in a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature. After 0.5 hours, by careful addition of acetic acid, the excess TMS of diazomethane was destroyed. Then the reaction mixture was concentrated under reduced pressure and dried in vacuo to give 1.95 g (92%) of pure target product: MS HIAD (-), found m / z 231 (M-1).

Stage D: 4-amino-3-fluoro-5-nitro-2-phenylaminobenzoic acid methyl ester: 4-amino-2,3-difluoro-5-nitrobenzoic acid methyl ester (23.48 g, 101.1 mmol) was suspended in xylenes (125 ml) and aniline (92 ml, 1011 mmol) was added. The reaction mixture was stirred at 125 ° C for 16 hours under N ₂ . The reaction mixture was cooled to room temperature, and solids precipitated from the solution. The solids were collected by filtration and washed with xylenes and then with diethyl ether. 22.22 g (72.78 mmol) of a yellow solid were isolated, which was a pure target product. The filtrate was concentrated under reduced pressure, dissolved in methylene chloride and passed through a pad of silica gel, eluting with methylene chloride. The desired fractions were concentrated under reduced pressure to obtain a brown solid, which was triturated with diethyl ether to obtain 5.47 g (17.91 mmol) of a yellow solid, which was a pure target product. The total yield of the product was 27.69 g (90%). MS KhIAD (-), found m / z 304 (M-1).

Stage D: 7-Fluoro-6-phenylamino-3H-benzoimidazole-5-carboxylic acid methyl ester: 4-amino-3-fluoro-5-nitro-2-phenylaminobenzoic acid methyl ester (16.70 g, 54.71 mmol ), formic acid (250 ml, 6.63 mol) and 20% Pd (OH) ₂ / C (9.00 g, 16.91 mmol) in ethanol (250 ml) were stirred at 40 ° C for 2 hours under N ₂ , and then at 95 ° C for 16 hours. The reaction mixture was cooled to room temperature and filtered through Celite, washing with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain a yellow solid. The solid was triturated with diethyl ether to give 13.47 g (86%) of the expected product as a tan solid. MS KhIAD, detected (+) m / z 286 (M + 1); MS KhIAD (-), found m / z 284 (M-1).

Stage E: 6- (4-Bromophenylamino) -7-fluoro-3H-benzoimidazole-5-carboxylic acid methyl ester: 7-fluoro-6-phenylamino-3H-benzoimidazole-5-carboxylic acid methyl ester (4.99 g, 17.51 mmol) was dissolved in N, N-dimethylformamide (275 ml). Solid N-bromosuccinimide (3.15 g, 17.70 mmol) was added, and the reaction mixture was stirred at room temperature under N ₂ . After 30 minutes, the reaction was quenched by the addition of an aqueous saturated sodium bisulfite solution. Then the reaction mixture was poured into a separatory funnel, diluted with water and ethyl acetate, and the layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed three times with water, once with brine and then dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to obtain 6.38 g (100%) of the pure desired product as a tan solid. MS IER (+) (electrospray ionization), found m / z 364, 366 (M + Br sample).

Stage G: 6- (4-Bromo-2-chlorophenylamino) -7-fluoro-3H-benzoimidazole-5-carboxylic acid methyl ester: 6- (4-bromophenylamino) -7-fluoro-3H-benzoimidazole-5- methyl ester carboxylic acid (6.38 g, 17.51 mmol) was dissolved in N, N-dimethylformamide (275 ml). Solid N-chlorosuccinimide (2.36 g, 17.70 mmol) was added, and the reaction mixture was stirred at room temperature under N ₂ until completion of the reaction (5-6 days). The reaction was quenched by the addition of an aqueous saturated sodium bisulfite solution to give a suspension. The resulting solids were collected by filtration, washed with water and diethyl ether, and dried under reduced pressure to obtain 6.07 g (87%) of the pure desired product as a beige solid. MS IER (+), found m / z 398, 400 (M + Br sample).

Stage 3: 6- (4-Bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid methyl ester and 6- (4-bromo-2-chlorophenylamino) -7- methyl ester fluoro-1-methyl-1H-benzoimidazole-5-carboxylic acid: 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3H-benzoimidazole-5-carboxylic acid methyl ester solution (150 mg, 0.38 mmol ), iodomethane (28 μl, 0.45 mmol) and potassium carbonate (78 mg, 0.56 mmol) in dimethylformamide (1.5 ml) were stirred at 75 ° C for one hour. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous potassium carbonate (2x), brine and dried (Na ₂ SO ₄ ). Flash column chromatography (methylene chloride / ethyl acetate, 20: 1) gave 56 mg (36%) of the more mobile 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5 methyl ester -carboxylic acid as a white solid. ¹⁹ F NMR (376 MHz, CD ₃ OD) - 133.5 (s). MS KhIAD (+), found m / z 412, 414 (M +, Br sample). 54 mg (35%) of 6-4 (-Bromo-2-chloro-phenylamino) -7-fluoro-1-methyl-1H-benzoimidazole-5-carboxylic acid methyl ester was also isolated as a white solid. ¹⁹ F NMR (376 MHz, CD ₃ OD) - 139.9 (s). MS KhIAD (+), found m / z 412, 414 (M +, Br sample).

Stage I: 6- (4-Bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid: 6- (4-bromo-2-chlorophenylamino) -7-fluoro- methyl ester 3-methyl-3H-benzoimidazole-5-carboxylic acid (56 mg, 0.14 mmol) was dissolved in a mixture of THF / water (2: 1, 3 ml) and NaOH (0.55 ml, 1.0 M aqueous solution, 0.55 mmol). After stirring for two hours, the volume of the reaction mixture was reduced to one quarter of the original volume by rotary evaporation, and the residue was diluted with 50 ml of water. The aqueous solution was acidified to pH 2 by adding 1.0 M aqueous HCl and extracted with tetrahydrofuran / ethyl acetate (1: 1) (3x), dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to obtain 43 mg (79%) of pure carbon acid in the form of an off-white solid. MS IER (+), found m / z 397, 398 (M + Br sample).

Stage K: (2-Vinyloxy-ethoxy) -amide 6- (4-bromo-2-chlorophenylamino-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid: 6- (4-Bromo-2-chlorophenylamino ) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2.00 g, 5.0 mmol), O- (2-vinyloxyethyl) -hydroxylamine (0.776 g, 7.5 mmol), HOBt (1-hydroxybenzotriazole, 0.88 g, 6.5 mmol), triethylamine (1.61 ml, 2.3 mmol) and EDCl (N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide, 1.3 g, 6 , 5 mmol) was dissolved in dimethylformamide (52 ml) and stirred at room temperature for 48 hours.The reaction mixture was diluted with ethyl acetate, washed with water (3x), saturated potassium carbonate (2x), saturated ammonium chloride (2x), brine, dried (Na ₂ SO ₄ ) and concentrated under reduced pressure to an off-white solid. After trituration of the solid with diethyl ether, 2.18 g (90%) ) of the desired product in the form of an off-white solid. MS IER (=), found m / z 483, 485 (M + Br sample).

Stage L: (2-Hydroxy-ethoxy) -amide 6- (4-bromo-2-chloro-phenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid: Hydrochloric acid (14 ml, 1 , 0 M aqueous solution, 14 mmol) was added to a suspension of (2 vinyloxyethoxy) -amide 6- (4-bromo-2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2.18 g, 4.50 mmol) in ethanol (50 ml), and the reaction mixture was allowed to stir for 24 hours. The reaction mixture was concentrated to dryness by rotary evaporation, and the solids were partitioned between ethyl acetate / tetrahydrofuran (3: 1) and saturated potassium carbonate. The aqueous phase was extracted with ethyl acetate / tetrahydrofuran (3: 1) (3x), the combined organic phases were dried (Na ₂ SO ₄ ) and concentrated to give 2.11 g (100%) of (2-hydroxyethoxy) -amide (4-bromo- 2-chlorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid as an off-white solid. MS IER (+), found m / z 457, 459 (M + Br sample). ¹ H NMR (400 MHz, MeOH-d ₄ ) δ 8.26 (s, 1H), 7.78 (s, 1H), 7.57 (d, 1H), 7.24 (dd, 1H), 6.40 (dd, 1H), 3.86 ( s, 3H), 3.79 (m, 2H), 3.49 (m, 2H). ¹⁹ F NMR (376 MHz, MeOH-d ₄ ) - 133.68 (s).

Example 2

Study of the physical properties of hydrosulfate salt

The product of example 1 to determine its physical properties was subjected to the following tests.

Powder X-Ray Diffraction (DLS)

All samples were tested on a D5000 diffractometer. Powder X-ray diffraction spectra were recorded by placing crystalline salt on substrates from a Siemens silicon single crystal crystal plate (SSC) and distributing the sample in a thin layer using a glass slide. The sample was rotated at a speed of 30 revolutions per minute (to improve the statistics of readings) and was irradiated with X-rays generated using a copper tube with a long-sharp focus with an operating voltage of 40 kV and a current of 40 mA and a wavelength of 1.5406 angstroms. A collimated x-ray source was passed through an automatic slit with variable divergence at V20, and the reflected radiation was directed through a 2 mm anti-scattering slit and a 0.2 mm detection slit. The sample was exposed for 1 second to a 0.02-degree increase in 2-theta (continuous scanning option) in the range from 2 degrees to 40 degrees 2-theta in the theta-theta version. The operating time was 31 minutes and 41 seconds. As a detector, the device was equipped with a scintillation counter. Data was monitored and collected using the Dell Optiplex 686 NT 4.0 Workstation with Diffract + software.

Data was collected in the 2-theta range of 2–40 ° with 2-theta increases of 0.02 °, from 4 s to increase and distributed in Table 2 containing the relative intensities obtained from diffractograms measured with fixed slits.

table 2 % relative intensity Definition 25-100 OS is very strong 10-25 C is strong 3-10 Wed - medium 1-3 SL - weak

The scan results are shown in FIG. 1, where the upper gray line is a powder X-ray diffraction (XRD) pattern of the hydrosulfate salt of compound 1, and the lower black line is a free form. The most intense lines, starting with the most intense, are shown in Table 3. The peak at 24.59 ° is especially strong.

Table 3 Scale 2 theta Relative intensity 24.59 OS 20.97 OS 23,99 C 27.65 C 12.24 C 23.49 C 24.30 C 17.02 C 25.91 C 22.50 Wed

Powder X-ray diffraction specialists will understand that relative peak intensities can be affected, for example, by granules larger than 30 microns and non-unitary characteristic ratios that can influence sample analysis. One skilled in the art will also understand that the position of reflections can be affected by the exact height at which the sample is located in the diffractometer and the zero calibration of the diffractometer. A slight effect can also have a flat surface of the sample. Therefore, the presented data of the diffraction pattern should not be regarded as absolute values (see Jenkins, R. & Snyder, R.L., "Introduction to X-Ray Powder Diffractometry", John Wiley & Sons, 1996 for more information).

Example 3

In vivo study: the study of salts compared to the free base in a dispersed composition

A study to measure plasma levels of compound 1 was carried out after administration of fasting dogs to oral doses equivalent to 150 mg of free base in 7.5 ml of various dispersed compositions in pharmaceutically acceptable dispersing agents with compound 1, both in free form and in the form of hydrosulfate salt.

A single dose of 150 mg was administered on an empty stomach orally to beagle dogs weighing 12-17 kg and aged about 2 to 6 years on each of the three days of dose administration. Between each day of the dose was a break of 1 week.

All pharmaceutical compositions were prepared for immediate administration immediately before the dose was added by adding a disposable syringe (10 ml) of 7.5 ml of the appropriate dispersed solution into vials containing the appropriate dosage form in an amount equivalent to 150 mg of free state, capping and vortex mixing for 30 seconds to form a dispersion.

The dispersion was removed from the vial using a disposable syringe and administered to the animal through a probe directed into the stomach. The vials were rinsed twice by adding a separate 15 ml aliquot of water for each of the two washes (total volume of washes 30 ml) with a 20 ml disposable syringe, capping, vortexing for 5 seconds, removing the washing solution from the vial using a disposable syringe and administering to the animal by a probe.

Dogs were fed 400 g of a special diet every day (Special Diet Services Laboratory Diet A) and were left ad libitum water. Directly before administration and at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 18, 24, 36, and 48 hours, whole blood (2 ml) was taken from the jugular vein into lithium heparinized tubes. Blood was centrifuged at 3000 rpm for 15 minutes, and the plasma was collected in ordinary blood tubes and stored until analysis at -20 ° C.

Plasma (50 μl) was analyzed in relation to the concentration of compound 1. Two dogs were excluded from the study because of their vomiting immediately after administration of the dose. The profiles of the average plasma concentration of compound 1 detected after oral administration of the dose are shown in FIG. 3, where the line composed of ▲ illustrates the composition comprising the hydrosulfate salt of compound 1 and the line composed of × shows the results for the free base of compound 1 in the same composition.

Changes in composition appear to have a relatively small effect on exposure (results not shown). However, in cases where compound 1 was administered as a hydrosulfate salt, an approximately 4–8-fold increase in exposure was obtained.

The above description is only intended to illustrate the principles of this invention. Moreover, since numerous modifications and changes will be apparent to those skilled in the art, it does not seem desirable to limit the invention to the precise construction and method described above. Accordingly, in order to fall within the scope of the invention defined by the following claims, it is possible to refer to all suitable modifications and equivalents.

The words “comprise,” “comprising,” “include,” “including,” and “include,” when used in this detailed description and in the following claims, are intended to concretize the presence of established features, single entities, components or steps, but they do not interfere with the presence or addition of one or more of the features, whole, components, stages or their groups.

Claims (16)

1. The hydrosulfate salt of compound 1

2. The hydrosulfate salt of compound 1 according to claim 1 in anhydrous form. 3. The hydrosulfate salt of compound 1 according to claim 1 in the form of a solvate. 4. The hydrosulfate salt of compound 1 according to claim 1, which is crystalline. 5. The hydrosulfate salt of compound 1 according to claim 4, wherein said hydrosulfate salt of compound 1 has a powder X-ray diffraction pattern with at least one specific peak at about 24.59 °. 6. The hydrosulfate salt of compound 1 according to claim 5, wherein said hydrosulfate salt of compound 1 has a powder X-ray diffraction pattern with specific peaks at 2-theta approximately equal to 24.59 °, 20.97 °, 23.99 °, 27 , 65 °, 12.24 °, 23.49 °, 24.30 °, 17.02 °, 25.91 ° and 22.50 °. 7. The hydrosulfate salt of compound 1 according to claim 1, which has a powder X-ray diffraction pattern substantially the same as the powder X-ray diffraction pattern shown in FIG. 8. The hydrosulfate salt of compound 1 according to claim 1 in amorphous form. 9. A method of producing a hydrosulfate salt of compound 1 according to any one of claims 1 to 8, including
1) the interaction of a suspension of compound 1 in an organic liquid, which is an organic solvent, in which compound 1 and its salts have a solubility of between 30 and 100 ml of solvent per gram of solute with at least a stoichiometric amount of sulfuric acid and water;
2) the allocation of salt from the resulting solution; and
3) after that, if desired or necessary, the formation of its solvate. 10. The method according to claim 9, where the amount of water added in stage (1) is limited by the amount necessary to ensure the formation of salt. 11. The method according to claim 9, where stage (1) is carried out at a temperature of from 40-80 ° C. 12. The method according to claim 9, where the organic liquid is a C _1-6 alkylketone. 13. The method according to any one of claims 9-12, wherein the hydrosulfate salt is isolated in step (2) by cooling the reaction mixture, possibly by adding additional organic liquid, so that the hydrosulfate salt precipitates. 14. The hydrosulfate salt of compound 1 according to any one of claims 1 to 8 for use as a medicine for the treatment of disease conditions mediated by MEK. 15. The use of the hydrosulfate salt of compound 1 according to any one of claims 1 to 8 in the manufacture of a medicament for use in the treatment of disease conditions mediated by MEK. 16. A method for treating disease conditions mediated by MEK in a warm-blooded mammal in need of such treatment, comprising administering to said mammal an effective amount of the hydrosulfate salt of compound 1 according to any one of claims 1 to 8.

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