HK1192226B - Solid salt forms of a pyrrole substituted 2-indolinone - Google Patents

Abstract

The present application generally relates to solid salt forms of a pyrrole substituted 2-indolinone. Specifically, the present invention relates to solid salt forms of the 3-pyrrole substituted 2-indolinone compound 5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pirrolidin-1-yl-ethyl)-amide. The invention also relates to polymorphs of the phosphate salt of the amide. The invention further relates to the use of the salts and polymorphs in the treatment of protein kinase related disorders.

Description

Solid salt forms of pyrrole substituted 2-indolones

This application is a divisional application of chinese patent application No.200680034505.3 entitled "solid salt form of pyrrole substituted 2-indolinone" filed on 8.9.2006.

Technical Field

The present invention relates to 3-pyrrole substituted 2-indolinone compounds, 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide. The aforementioned compounds are capable of modulating the activity of protein kinases ("PKs"). The compounds of the invention are therefore useful in the treatment of disorders associated with abnormal PK activity. Pharmaceutical compositions comprising salts of the compounds and processes for preparing them are disclosed. The invention also relates to a polymorph (polymorph) of the phosphate form of the amide.

Background

The following is provided merely as background information and is not admitted to be prior art to the present invention.

Solids, including drugs, often have more than one crystalline form, which is a known phenomenon of polymorphism. When a compound crystallizes in a plurality of solid phases which differ in crystal packing, polymorphism occurs. Many examples are mentioned In standard references for The Solid State properties of Drugs, such as Byrn, S.R., Solid-State Chemistry of Drugs, New York, Academic Press (1982), Kuhnert-Brandstatter, M., Thermosroscopy In The Analysis of pharmaceuticals, New York, Pergamon Press (1971) and Haleblian, J.K. and McCrone, W.pharmaceutical applications of polymorphism.J.Pharman.Sci., 58, 911 (1969). Byrn suggests that typically polymorphs exhibit different physical properties, including solubility and physical and chemical stability.

Because of differences in molecular packing, polymorphs may differ in ways that affect drug release, solid state stability, and drug manufacturing. The relative stability of polymorphs and the interconversion of polymorphs is particularly important for the selection of drug products on the market. The selection of a suitable polymorph may depend on physical stability. For example, the choice of drug product on the market may depend on the choice of and availability of a suitable polymorph having the desired characteristics (e.g., excellent physical stability or ability to be mass-produced). The performance of the solid dosage form should not be limited by the polymorph transition over the shelf life of the product. Of particular note are: there is no reliable method to predict the visible crystal structure of a given drug product or to predict the presence of polymorphs having desirable physical properties.

PK is an enzyme that catalyzes the phosphorylation of hydroxyl groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are astonishing because virtually all aspects of cell life (e.g., cell growth, differentiation, and proliferation) are dependent on PK activity in one way or another. In addition, abnormal PK activity has been linked to a number of conditions ranging from relatively non-life threatening diseases such as psoriasis to very fatal diseases such as glioblastoma (brain cancer).

Receptor Tyrosine Kinases (RTKs) are a class of PKs that are excellent candidates for molecular targeted therapeutic techniques because they play a key role in controlling cell proliferation and survival and are often dysregulated in a variety of malignancies. Mechanisms of dysregulation include overexpression (Her 2/neu in breast cancer, epidermal growth factor receptor in non-small cell lung cancer), activating mutations (KIT in gastrointestinal stromal tumors, fms-associated tyrosine kinase 3/Flk2(FLT3) in acute myeloid leukemia), and activated autocrine loops (vascular endothelial growth factor/VEGF receptor in melanoma (VEGF/VEGFR), platelet-derived growth factor/PDGF receptor in sarcoma (PDGF/PDGFR)).

Dysregulated RTKs have been described in comparable human and canine cancers. For example, aberrant expression of the Met oncogene occurs in both human meat and canine osteosarcomas. Interestingly, comparable activating mutations of the membrane-proximal (JM) domain of c-kit were found in 50% -90% of human gastrointestinal stromal tumors (GIST) and 30% -50% of advanced (advanced) canine MCTs (mast cell tumors). Although mutations in human GIST consist of deletions in this JM domain, whereas mutations in canine MCT consist of endogenous tandem duplications (ITDs) in this JM domain, both produce constitutive phosphorylation of KIT in the absence of ligand binding. RTKs and their ligands, VEGF, PDGF and FGF, regulate neovasculature in parenchymatous tumors (known as angiogenesis). Thus, new blood vessel growth into tumors can be inhibited by inhibiting RTKs.

Anti-angiogenic agents are molecules that inhibit the growth of blood vessels into tumors, which are much less toxic to the body than traditional anti-cancer drugs. U.S. patent 6,573,293, which is incorporated herein by reference, discloses compounds such as 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide (hereinafter "compound I"). It has the following structure:

compound I

Compound I is a small molecule that exhibits PK modulating ability. The compounds are therefore useful in the treatment of disorders associated with abnormal PK activity. It is an inhibitor of RTK, PDGFR, VEGFR, KIT and FLT 3. Compound I has been shown to inhibit KIT phosphorylation, prevent cell proliferation, and induce cell cycle arrest and apoptosis in vitro in malignant mast cell lines expressing various mutant forms of KIT. Compound I and related molecules were effective against tumor allografts (xenograft) caused by different human tumor-derived cell lines in preclinical models.

Compound I is useful for the treatment of cancer in companion animals, primarily dogs, and also in humans in particular. These cancers include, but are not limited to, leukemia, brain cancer, non-small cell lung cancer, squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, small cell lung cancer, glioma, colorectal cancer, urinary tract cancer and gastrointestinal stromal cancer. Compound I is useful for the treatment of diseases associated with mast cell overexpression, including but not limited to mastocytosis in humans and mastocytoma in dogs.

Compound I has recently been shown to be clinically effective against a variety of malignancies that arise spontaneously in dogs. In this study, 11 of 22 canine MCTs showed sustainable objective responses (partial and complete) to compound I treatment; 9 of these MCTs had ITDs in the JM domain of c-kit.

Compound I is readily crystallized. Its solubility in phosphate buffer at pH6 at 25 ℃ is about 10. mu.g/ml. When the compound is synthesized, very fine particles precipitate out of solution in the final step of the synthesis. Subsequent separation of these fine particles by filtration is slow and produces a hard residue after filtration. There is a need for salts of compound I that have physical stability and desirable physical properties.

Disclosure of Invention

The present invention encompasses salt forms of compound I. Five different salt forms of compound I are synthesized and described herein (see table 1). Which includes the hydrochloride, fumarate, citrate, phosphate and ascorbate salts of compound I. Based on the characteristics of these salts, the 1: 1 phosphate, the phosphate of compound I, was identified as a salt form with highly desirable characteristics. Polymorph screening revealed the presence of 10 polymorphs of compound I phosphate, designated herein as form I through form X.

In one aspect, this invention provides two salt forms of compound I, wherein the salt forms are selected from citrate and phosphate, and solvates and polymorphs thereof. In one embodiment, C is selected₂₂H₂₅FN₄O₂·H₃PO₄In the form of the phosphate salt of formula (I). In another embodiment, a phosphate form having a melting point of from about 285 ℃ to about 290 ℃ is selected. Compound I phosphate has the following structure:

phosphate salts of Compound I

In another embodiment, C is optionally present₂₂H₂₅FN₄O₂·C₆H₈O₇Citrate salt of formula (compound I citrate salt). In yet another embodiment, a citrate salt form having a melting point of from about 178 ℃ to about 183 ℃ is selected. Compound I citrate has the following structure:

compound I citrate salt

A second aspect of the invention is a pharmaceutical composition comprising compound I phosphate or compound I citrate, or a solvate or polymorph thereof, and a pharmaceutically acceptable carrier or excipient.

A third aspect of the invention is a method for modulating the catalytic activity of a protein kinase, the method comprising: contacting the protein kinase with compound I phosphate or compound I citrate, or a solvate or polymorph thereof. The protein kinase may be selected from the group consisting of receptor tyrosine kinases, non-receptor protein tyrosine kinases, and serine/threonine protein kinases.

A fourth aspect of the present invention is a method for preventing or treating a protein kinase-associated disorder in an organism, the method comprising: administering to the organism a therapeutically effective amount of a pharmaceutical composition comprising compound I phosphate or compound I citrate, or a solvate or polymorph thereof, and a pharmaceutically acceptable carrier or excipient. In one embodiment, the organism is a human. In another embodiment, the organism is a companion animal. In yet another embodiment, the companion animal is a cat or dog. The protein kinase related disorder may be selected from the group consisting of a receptor tyrosine kinase related disorder, a non-receptor protein tyrosine kinase related disorder, and a serine/threonine protein kinase related disorder. The protein kinase related disorder may be selected from the group consisting of an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder, a c-kit related disorder and a FLK related disorder. These disorders include, by way of example and not limitation, leukemia, brain cancer, non-small cell lung cancer, squamous cell tumor, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head cancer, neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small cell lung cancer, glioma, mastocytosis, mast cell tumor, colorectal cancer, urinary tract cancer, gastrointestinal cancer, diabetes, autoimmune disorders, hyperproliferative (hyper-proliferation) disorder, restenosis, fibrosis, psoriasis, von Heppel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, inflammatory disorders, immune disorders, and cardiovascular disorders.

A fifth aspect of the invention is a process for preparing a crystalline phosphate salt of the base 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide, which process comprises: introducing a stoichiometric amount (stoichiometric amount) of phosphoric acid to the base in a solution comprising a solvent or mixture of solvents, crystallizing the phosphate in solution, separating phosphate crystals from the solvent solution, and drying the crystals. Phosphoric acid may be introduced in a 40% molar excess relative to the base. The solvent may comprise isopropanol. The step of separating the crystals from the solvent solution may comprise adding acetonitrile to the solution and rotary evaporating (rotovapping) the solution. The step of separating the crystals from the solvent solution may also comprise filtration.

A sixth aspect of the invention is a process for preparing a crystalline citrate salt of the base 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide comprising: the process comprises the steps of introducing a stoichiometric amount of citric acid into the base in a solution comprising a solvent or mixture of solvents, crystallizing the citrate salt in the solution, separating citrate salt crystals from the solution, and drying the crystals. Citric acid may be introduced in a 40% molar excess relative to the base. The solvent may comprise methanol. The step of separating the crystals from the solvent solution may comprise adding acetonitrile to the solution and rotary evaporating the solution. The step of separating the crystals from the solvent solution may comprise filtration.

In a seventh aspect, the present invention provides a polymorph form I-X (as described herein) of the phosphate salt of compound I. In one embodiment, form I is provided.

An eighth aspect of the invention is a pharmaceutical composition comprising form I polymorph of compound I phosphate and a pharmaceutically acceptable carrier or excipient.

A ninth aspect of the invention is a method for modulating the catalytic activity of a protein kinase, said method comprising contacting said protein kinase with form I polymorph of compound I phosphate.

A tenth aspect of the invention is a method for the prevention or treatment of a protein kinase-associated disorder in an organism, comprising administering to said organism a therapeutically effective amount of form I polymorph of the phosphate salt of compound I. In one embodiment, the organism is a human or a companion animal. In another embodiment, the companion animal is a cat or dog. These conditions include, by way of example and not limitation, mast cell tumors and mastocytosis.

An eleventh aspect of the present invention is a method of preparing a polymorph of compound I phosphate, the method comprising: the phosphate salt is introduced into a solution comprising a solvent or mixture of solvents, optionally a bridging solvent is added to the solution, and the polymorph crystals are isolated from the solvent solution. The solution may comprise water plus acetonitrile. The solution may comprise methanol. The bridging solvent may be methanol.

A twelfth aspect of the invention is the use of compound I phosphate or compound I citrate or phosphate form I polymorph in the manufacture of a medicament for the treatment of a disease mediated by abnormal PK activity.

Drawings

Figure 1 is water gas sorption (sorption) data for a salt of compound I.

FIG. 2 is a powder X-ray diffraction pattern of Compound I citrate and Compound I phosphate.

Figure 3 is an X-ray diffraction pattern of ten unique solid powders from a polymorph screening study (see example 5). Forms I to X named in table 5 and 6 are shown.

FIG. 4 is from CH₂Cl₂TGA profile of solids (form VI, performed immediately after precipitation), hexane (form VII, after standing overnight) and acetonitrile (form VIII, after 3 days of standing).

FIG. 5 is the result of agarose gel electrophoresis of PCR products from MCT evaluated in example 7. Lanes 1-5 correspond to patients 1-5 in Table 8; lanes 6-14 correspond to patients 6-14 in Table 8. Control groups consisting of PCR products were generated from the C2 canine mast cell line containing 48-bp ITD (lane 15) and from normal canine cerebellum (wild type; lane 16).

Figure 6 is the reduction in MCT phosphorylated KIT and phosphorylated extracellular signal-regulated kinase (ERK)1/2 following a single dose of compound I phosphate.

Detailed Description

Defining: unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

when referring to temperature, the term "C" means either celsius or celsius.

The term "catalytic activity" refers to the phosphorylation rate of tyrosine under the direct or indirect influence of RTK and/or CTK, or the phosphorylation rates of serine and threonine under the direct or indirect influence of STK.

The term "companion animal" refers to domesticated animals including but not limited to cats and dogs that provide companion animals to humans.

The term "contacting" refers to bringing a compound of the invention and a target PK together in a manner such that the compound is able to directly affect (i.e., through interaction with the kinase itself) or indirectly affect (through interaction with another molecule upon which the catalytic activity of the kinase depends) the catalytic activity of the PK.

The term "IC₅₀"refers to the concentration of test compound that achieves half the maximum inhibition of the PK activity.

The term "modulation" or "modulating" refers to altering the catalytic activity of RTKs, CTKs, and STKs. In particular, modulation refers to activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, preferably, activation of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTKs, CTKs and STKs are exposed, or more preferably, inhibition of the catalytic activity of the RTKs, CTKs and STKs.

The term "PK" refers to receptor protein tyrosine kinases (RTKs), non-receptor or "cellular" tyrosine kinases (CTKs) and serine-threonine kinases (STKs).

The term "polymorph" refers to a solid phase of a substance that exists in several distinct forms due to the different arrangement and/or conformation (conformation) of the molecules in the crystal lattice. Typically, polymorphs have different chemical and physical properties.

The term "pharmaceutically acceptable excipient" refers to any substance other than a compound of the present invention that is added to a pharmaceutical composition.

The term "pharmaceutical composition" refers to a mixture of one or more salts of the invention or polymorphs of such salts, as described herein, and other chemical ingredients (e.g., physiologically/pharmaceutically acceptable carriers and excipients). The purpose of the pharmaceutical composition is to assist in the administration of the compound to an organism.

The term "physiologically/pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound to be administered.

The term "polymorph" can also be defined as different unsolvated crystalline forms of a compound. The term also includes solvates (i.e., forms containing solvent or water), amorphous forms (i.e., non-crystalline forms), and desolvated solvates (i.e., forms produced by simply removing the solvent from the solvate).

The term "solvate" is used to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term used is "hydrate".

The term "substantially free," with respect to the amount of a given polymorph in a sample, means that the other polymorph is present in an amount less than about 15 wt%. In another embodiment, "substantially free" means less than about 10 wt%. In another embodiment, "substantially free" means less than about 5 wt%. In yet another embodiment, "substantially free" means less than about 1 wt%. The ordinarily skilled artisan will appreciate that the phrase "present in an amount less than about 15 wt%" means that the polymorph of interest is present in an amount greater than about 85 wt%. Likewise, the phrase "less than about 10 wt%" means that the polymorph of interest is present in an amount greater than about 90 wt%, and so on.

The term "therapeutically effective amount" refers to an amount of a compound to be administered that prevents, reduces or ameliorates one or more symptoms of the condition being treated, or prolongs the survival of the subject being treated. In terms of cancer treatment, a therapeutically effective amount refers to an amount that has the following effects:

(1) reducing the size of the tumor;

(2) inhibit (i.e., slow to some extent, or stop) tumor metastasis;

(3) inhibit (i.e., slow to some extent, or stop) tumor growth; and/or

(4) One or more symptoms associated with the cancer are reduced to some extent (or eliminated).

To obtain a form with better physical properties, different salt forms of compound I can be synthesized. The base compound may be in solution. The solution is typically a solvent. In one embodiment, the solution is an alcohol. In another embodiment, the solvent may be isopropanol, methanol, acetonitrile or water plus acetonitrile. The solution may also comprise a mixture of solvents.

The salt may be crystallized using stoichiometric addition/crystallization techniques. A stoichiometric amount of counter ion (counter) is directed to the base in solution. In one embodiment, the relative ion amounts are in a 1: 1 ratio for the base. In another embodiment, the amount of counter ion is 0% to about 60% molar excess relative to the base. In another embodiment, the amount of counter ion is in excess of 10% to about 50% molar relative to the base. In another embodiment, the amount of counter ion is about 40% molar excess relative to the base. Counter ions may include hydrochloride, fumarate, citrate, phosphate, and ascorbate ions. In one embodiment, the counter ion is a phosphate ion. In another embodiment, the counter ion is a citrate ion.

The salt in solution can be crystallized by a variety of common techniques known to those skilled in the art, including cooling, evaporation, wetting, and the like. Excess solvent may be removed from the sample by methods known to those skilled in the art. In one embodiment, the solvent is removed from the solution by adding Acetonitrile (ACN) and rotary evaporation of the solution. The solution may be rotary evaporated at about 40 ℃ to about 60 ℃. In another embodiment, additional solvents (e.g., isopropanol and methyl ethyl ketone) may be added to the solution prior to rotary evaporation. To prevent light-induced isomerization, crystallization can be carried out in the dark. In one embodiment, the crystals are removed by filtration. In another embodiment, the filtration may be performed in an ambient laboratory atmosphere.

By these methods, the ascorbate, citrate, fumarate, hydrochloride and phosphate salts of compound I are crystallized. Specific examples of the crystallization method are provided below. HPLC analysis can be used to measure the purity of the resulting samples. Physical properties of the compounds can be measured by tests known to those skilled in the art, including melting point determination, powder X-ray diffraction, and dynamic moisture sorption gravimetric measurement. The parameters of these tests are described below.

Five salt forms are described herein (see table 1). These salts of compound I are often hygroscopic. For example, as shown in Table 1, at 80% humidity, the hydrochloride salt contains about 20% water, the fumarate salt contains about 9% water, and the ascorbate salt contains about 6.5% water. These characteristics make the use of salts in pharmaceutical formulations difficult and shorten the shelf life of the formulations. However, there are two salts, phosphate and citrate, which have been unexpectedly found to have low water absorption-containing about 1% water and about 3.8% water, respectively, at 80% relative humidity.

Based on the characteristics of these salts, the 1: 1 phosphate, compound I phosphate, was identified as a salt form with highly desirable characteristics including: good crystallinity, low water absorption, easy crystallization, good purity and lack of hydrates. Ten polymorphs of compound I phosphate, designated herein as forms I to X, are also described. Citrate salts also exhibit desirable characteristics such as low water absorption and good crystallinity.

Polymorphs of a compound of the invention are desirable because a particular polymorph of a compound may have better physical and chemical properties than other polymorph forms of the same compound. For example, one polymorph has increased solubility in certain solvents. This increased solubility may make the formulation or administration of the compounds of the invention easier. Different polymorphs may also have different mechanical properties (e.g. different compressibility, compactibility and tabletability) which may affect the tablettability of the drug product and thus the formulation of the drug product. A particular polymorph may also exhibit a dissolution rate in the same solvent that is different from other polymorphs. Different polymorphs may also have different physical (solid state transition from metastable polymorph to more stable polymorph) and chemical (reactive) stabilities. One embodiment of the present invention contemplates the form I polymorph of the phosphate salt of compound I, as described herein.

In some embodiments of the invention, pure, single polymorphs and mixtures comprising two or more different polymorphs are contemplated. A pure, single polymorph may be substantially free of other polymorphs.

Some embodiments of the present invention contemplate pharmaceutical compositions comprising one or more salts of compound I or polymorphs of such salts, as described herein, and a pharmaceutically acceptable carrier or excipient.

Polymorphs were produced from a concentrated solution of compound I phosphate. The concentrated solution may contain in the range of 60mg to 100mg of compound I phosphate per mL of solution. In one embodiment, about 70mg of compound I is dissolved in 1mL of phosphoric acid.

Crystals of the polymorph can be precipitated from the solvent by a variety of methods known to those skilled in the art, including, for example, slow evaporation, cooling of the supersaturated solution, precipitation from an anti-solvent (anti-solvent), and the like. In one embodiment, crystals of the polymorph are produced by adding the solution to an anti-solvent. The anti-solvent may be water plus Acetonitrile (ANC), ethanol, methanol, acetone, acetonitrile, THF, ethyl acetate, hexane, dichloromethane (CH)₂Cl₂) Isopropanol (IPA), Methyl Ethyl Ketone (MEK) and dioxane. In one embodiment, additional solvent (e.g., methanol) may be added. In another embodiment, the sample is allowed to stand overnight before removing the crystals. In yet another embodiment, the sample is allowed to stand for three days before removing the crystals. Standard methods known to those skilled in the art (including PXRD dynamic water vapor sorption gravimetric measurement, differential scanning calorimetry, thermogravimetric analysis, and optical microscopy) can be used to characterize the crystals. These techniques are described below.

Pharmaceutical compositions suitable for delivery of the compounds of the invention and methods for preparing them will be apparent to those skilled in the art. Such compositions and methods for preparing them can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

The choice of pharmaceutically acceptable excipient will depend in large part on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Carriers and excipients used to formulate pharmaceutically acceptable compositions comprising compound I are known in the art and are disclosed, for example, in U.S. patent No.6,573,293, which is incorporated herein by reference in its entirety. Methods of administration for this are also known in the art and are also described, for example, in U.S. patent No.6,573,293. Similar methods may also be used to formulate and administer pharmaceutically acceptable compositions comprised of the salts of compound I of the present invention or polymorphs of such salts.

The appropriate formulation depends on the route of administration chosen. For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hanks 'solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants (pendants) appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For parenteral administration (e.g., by bolus injection or continuous infusion), the formulation may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, which may contain formulatory agents such as suspending, stabilizing or dispersing agents.

The compounds of the invention may be administered directly into the bloodstream, into muscles or into internal organs. Suitable means for parenteral administration include: intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous. Suitable devices for parenteral administration include: needle (including micro-needles) injectors, needleless injectors, and infusion techniques. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably adjusted to a pH of from 3 to 9), but for some applications they may be more suitably formulated as sterile nonaqueous solutions or as dried forms for use with a suitable vehicle such as sterile, pyrogen-free (pyrogen) water. Furthermore, suspensions of the compounds of the invention may be prepared in a lipophilic vehicle. Suitable lipophilic carriers include fatty oils (e.g. sesame oil), synthetic fatty acid esters (e.g. ethyl oleate and triglycerides) or materials such as liposomes.

The compounds of the invention may be administered orally. Oral administration may include swallowing (whereby the compound enters the gastrointestinal tract) and/or buccal, lingual or sublingual administration (whereby the compound enters the blood stream directly from the mouth). For oral administration, the compounds may be formulated by combining the compounds of the invention with pharmaceutically acceptable carriers well known in the art. Formulations suitable for oral administration include solid, semi-solid and liquid systems, such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids or powders; lozenges (including liquid filled); chewable tablets; gelling; a rapidly dispersing dosage form; a film; beads (ovules); spraying; and buccal/mucosal patches.

The compounds of the invention may also be applied topically, dermally (intradermally), or transdermally to the skin or mucosa. Typical formulations for this use include gels, hydrogels, lotions, solutions, emulsions, ointments, dusting powders, dressings, foams, films, skin patches, wafer dressings, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohols, water, mineral oil, liquid paraffin, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Transdermal enhancers may be included, see, for example, J Pharm Sci, 88(10), 955 Across 958, by Finnin and Morgan (October 1999). Other topical application modes include by electroporation, electrophoresis, sonophoresis, and microneedle or needle-free (e.g., Powderject)^TM、Bioject^TMEtc.) delivery of the injection.

The compounds of the invention may be formulated for rectal administration, for example as a suppository or as a closed urine enema using a conventional suppository base such as cocoa butter or other glycerides.

The compounds of the present invention may also exist in unsolvated as well as solvated forms.

Embodiments of the invention also include methods for modulating PK catalytic activity, comprising: contacting the PK with one or more salts of compound I of the invention or a polymorph of these salts. Such "contacting" may be achieved "in vitro", i.e. in test tubes, petri dishes, etc. In vitro, the contact may involve only the compound and PK of interest, or may involve the entire cell. It is also possible to maintain or grow cells in a cell culture dish and to contact the cells with the compound in that environment. In this context, the ability of a particular compound, as defined below, to affect a PK related disorder, i.e. the IC of the compound₅₀The compound may be measured before it is attempted to be used in vivo in a complex living organism. For cells in vitro, there are a variety of methods for obtaining PK for contact with a compound, which are well known to those skilled in the art, including, but not limited to, direct cell microinjection and a variety of transmembrane vector techniques.

Embodiments of the present invention include methods for treating and preventing protein kinase-associated disorders in an organism (e.g., a companion animal or a human) comprising administering a therapeutically effective amount of a pharmaceutical composition comprising one or more salts of compound I of the present invention or polymorphs of such salts, and a pharmaceutically acceptable carrier or excipient for the organism.

In one embodiment of the invention, the protein kinase related disorder is selected from the group consisting of a receptor tyrosine kinase related disorder, a non-receptor tyrosine kinase related disorder and a serine-threonine kinase related disorder. In another embodiment of the invention, the protein kinase related disorder is selected from the group consisting of an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder and an FLK related disorder.

The receptor protein kinases whose catalytic activity is modulated by the compounds of the invention are selected from the group consisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFR α, PDGFR β, CSFIR, C-kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. The cellular tyrosine kinase whose catalytic activity is modulated by a compound of the invention is selected from the group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and YRk. Serine-threonine protein kinases whose catalytic activity is modulated by the compounds of the invention are selected from the group consisting of CDK2 and Raf.

In yet another embodiment of the invention, the protein kinase associated disorder is selected from the group consisting of squamous cell tumor, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small cell lung cancer, glioma, colorectal cancer, urinary tract cancer, gastrointestinal cancer, mastocytosis, and mast cell tumor. In one embodiment of the invention, the protein kinase associated disorder is selected from the group consisting of diabetes, autoimmune disorders, hyperproliferative disorders, restenosis, fibrosis, psoriasis, von Heppel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, inflammatory disorders, immune disorders and cardiovascular disorders.

Pharmaceutical compositions suitable for use in the present invention include compositions comprising an active ingredient in an amount sufficient to achieve the desired purpose, e.g., modulation of PK activity or treatment or prevention of PK related disorders.

Determination of a therapeutically effective amount is known to those skilled in the art, especially in the light of the detailed disclosure provided herein. For any compound used in the methods of the invention, a therapeutically effective amount or dose can be estimated initially from cell culture assays. Thus, the dose can be formulated for use in animal models to obtainIncluding IC measured in cell culture₅₀The circulating concentration range within. This information can then be used to more accurately determine the dosage used in a human or companion animal.

In practice, the amount of the compound administered is from about 0.001mg to about 100mg per kg body weight, and such total doses may be administered at once or in divided doses. The amount of the composition administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician or veterinarian, and the like. In the case of topical administration or selective uptake, the effective local concentration of the drug product may be independent of plasma concentration, and other procedures known in the art may be used to determine the correct dose amount and time interval.

Embodiments of the invention also include methods of treating cancer in a companion animal, comprising: administering a pharmaceutical composition comprising one or more salts of compound I of the present invention, or polymorphs of such salts, and a pharmaceutically acceptable carrier or excipient.

Furthermore, the invention allows for: salts of compound I or polymorphs of such salts (as described herein) may be metabolized by enzymes in the body of an organism (e.g., a companion animal or human) to produce metabolites that modulate the activity of protein kinases. These metabolites are also within the scope of the present invention.

The compounds of the present invention may be administered alone or in combination with one or more other compounds of the present invention, or in combination with one or more other pharmaceutical products (or any combination thereof). It is also contemplated that salts of compound I or polymorphs of these salts as described herein may be combined with other chemotherapeutic agents useful in the treatment of the diseases and disorders discussed above. For example, the compounds of the present invention may be combined with fluorouracil alone, or in further combination with chrysanthemic acid or other alkylating agents. The compounds of the present invention may be used in combination with other antimetabolite chemotherapeutic agents such as, but not limited to, folic acid analogs or purine analogs. The compounds may also be used in combination with natural product-based chemotherapeutic agents, antibiotic chemotherapeutic agents, enzyme chemotherapeutic agents, platinum coordination complexes, and hormone antagonists. It is also contemplated that the compounds of the present invention may be used in combination with mitoxantrone (mitoxantrone) or paclitaxel (paclitaxel) for the treatment of parenchymal cancers or leukemias.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following detailed description describes how to make the various compounds of the present invention and/or how to carry out the various processes of the present invention, and is to be construed as illustrative only and not limiting in any way of the foregoing disclosure. Those skilled in the art will quickly know the appropriate changes in the schemes with respect to the reactants and with respect to the reaction conditions and techniques.

Examples

EXAMPLE 1 Synthesis of Compound I, i.e., 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl]- 2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide

Condensation of 5-fluoro-1, 3-dihydro-indol-2-one with 5-formyl-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide as described in us patent 6,574,293 (example 129) gives compound I.

And (3) a large-scale expansion process: 5-formyl-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (61g), 5-fluoro-1, 3-dihydro-indol-2-one (79g), ethanol (300mL), and pyrrolidine (32mL) were refluxed for 4.5 hours. Acetic acid (24mL) was added to the mixture and reflux continued for 30 minutes. The mixture was cooled to room temperature and the solid was collected by vacuum filtration and washed twice with ethanol. The solid was stirred in 40% aqueous acetone (400mL) containing 12N hydrochloric acid (6.5mL) for 130 min. The solid was collected by vacuum filtration and washed twice with 40% aqueous acetone. The solid was dried under vacuum to give 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (86g, 79% yield) as an orange solid.

5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (100g) and dimethylformamide (500mL) were stirred and benzotriazol-1-yloxytris (dimethylamino) phosphine hexafluorophosphate (221g), 1- (2-aminoethyl) pyrrolidine (45.6g) and triethylamine (93mL) were added. The mixture was stirred at ambient temperature for 2 hours. The solid product was collected by vacuum filtration and washed with ethanol. The solid was slurry-washed by stirring in ethanol (500mL) at 64 ℃ for one hour, which was cooled to room temperature. The solid was collected by vacuum filtration, washed with ethanol and dried under vacuum to give 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide (101.5g, 77% yield).

EXAMPLE 2 Synthesis of salts of Compound I

Example 2A. Compound I phosphate

2.67 mmoles of Compound I with 40mL of 0.092M phosphoric acid (about 40% molar excess relative to the hypothetical 1: 1 salt) and 40mL of isopropanol were added to the flask. Acetonitrile was then added to the aqueous solution in 30mL aliquots and the solution was rotary evaporated at 60 ℃ to remove water. A total of 120mL of acetonitrile was used to remove the water from the solution. The crystals were filtered and air dried. The crystals were free flowing, appearing orange; 1.09g was collected, yielding 83% yield.

Example 2B. Compound I citrate salt

2.64 mmol of Compound I with 34mL of 0.1M citric acid (3.4 mmol) and 35mL of methanol were added to the flask. The solution was rotary evaporated at 50 ℃. The volume of the solution was reduced, resulting in crystals with poor crystallinity, so 20mL isopropanol and 10mL methyl ethyl ketone were added to dissolve the solid. The mixture was rotary evaporated at 60 ℃ to give orange crystals. The crystals were filtered and air dried. The yield of the process is about 60%, and the process can be improved by further reducing the solvent volume prior to filtration.

Example 3 physical Properties of salts of Compound I

The method comprises the following steps: tests used to measure the physical properties of the salts of compound I include melting point determination, HPLC purity, powder X-ray diffraction and dynamic water gas sorption gravimetric measurement.

Powder X-ray diffraction (PXRD): powder XRD was performed using the Scintag X2Advanced diffraction System (lab259-1088, controlled by Scintag DMS/NT1.30a and Microsoft Windows NT4.0 software). The system uses a copper X-ray source (45kV and 40mA) to provideUsing tube divergence and anti-scatter slits of 2mm and 4mm and detector anti-scatter and receiving slits of 0.5mm and 0.2mm width the data was collected from 2 theta angles of 2 ° to 35 ° using a step scan of 0.03 °/step and a count time of 1 second per step the experiment used a Scintag round, top-loaded stainless steel sample holder (insert with 9mm diameter) the powder was loaded into the holder, it was pressed gently, typically by a glass slide, to ensure coplanarity between the sample surface and the holder surface.

Dynamic water gas sorption gravimetric measurement (DMSG): DMSG isotherms were collected on a temperature-controlled atmospheric microbalance. About 10mg of sample was placed on the sample pan of the balance. Humidity was sequentially changed from indoor Relative Humidity (RH) to 0% RH in steps of 3% RH, increasing to 90% RH, followed by RH decreasing to 0% again. The mass was then measured every two minutes. When the change in sample mass was less than 0.5 μ g in 10 minutes, the RH was switched to the next target value. Data collection was controlled using Visual Basic program dmsgscn2.exe, exporting the information to Excel spreadsheet.

As a result: table 1 shows a summary of the data for ascorbate, citrate, fumarate and phosphate salts of compound I. HPLC analysis showed that these salts were of relatively high purity and that the formation of the salts did not cause significant changes in purity.

TABLE 1 summary of salts synthesized for Compound I

Area percentage under HPLC peak

The hydrochloride, fumarate and ascorbate salts are very hygroscopic (see figure 1). The other two salts (citrate and phosphate) have a lower moisture sorption profile, absorbing only less than 3% of water at 70% relative humidity.

The powder X-ray patterns show that the phosphate and citrate salts have relatively high crystallinity (see tables 2 and 3; and figure 2).

TABLE 2 PXRD peaks for Compound I phosphate

*：±0.1°

**: the relative intensity of each peak was determined by normalizing its intensity to the intensity of its most intense peak at an angle of 27.0 deg. (as 100).

TABLE 3 PXRD peaks for Compound I citrate

*：±0.1°

**: the relative intensity of each peak was determined by normalizing its intensity to the intensity of its most intense peak at an angle of 25.5 deg. (as 100).

EXAMPLE 4 preparation and analysis of Compound I phosphate

Example 4A preparation of Compound I phosphate

The phosphate salt was prepared from compound I free base. A sample of Compound I phosphate was prepared as described above (batch No. 35282-CS-51). 4mL of 0.977M phosphoric acid was added to 1.059g of free base in the flask, followed immediately by 4mL of acetonitrile. A suspension is obtained. The suspension was heated gently on a hot plate. Addition of 40mL of water and heating while stirring for about one hour did not completely dissolve the solid. The solid was filtered and washed with 10mL acetonitrile. PXRD showed this to be the phosphate salt of compound I.

Example 4B analysis of Compound I phosphate

Batch No. 35282-CS-51 was designated as polymorph form I of the phosphate salt of Compound I. It has high crystallinity, good flowability and large crystal size. No melting occurred at the melting temperature of compound I free base (free base polymorph form a, 256 ℃; free base polymorph form B, 259 ℃), and the presence of a high melting point of the solid (281 ℃ to 297 ℃) showed: the crystals of run No. 35282-CS-51 are in different salt forms, which are not the free base compound I. The purity of the batch was 99.6% as tested by HPLC.

Example 4℃ estimation of phosphate solubility of Compound I

A sample of 1mg-2mg of Compound I phosphate (batch No. 35282-CS-51) was transferred to a 10mL glass vial (tared) and weighed (to the nearest 0.1 mg). The vials were charged with solvent in a stepwise manner (one solvent per vial) with 0.5mL solvent per step. The solvents used were buffer (pH 2), buffer (pH 5), water, methanol, Tetrahydrofuran (THF), acetonitrile and acetone. After each addition, the vial was capped and shaken. Dissolution of the solid was visually observed. If no significant dissolution was observed, more solvent was added immediately. If dissolution is evident, the vial is placed on the table for at least 30 minutes before the next addition of solvent. This procedure was repeated until no crystals were visible against both the black and white background. The solubility then equals the weight of the compound divided by the final volume and the volume before the last addition. If the solid is still present after addition of 10mL of solvent, the solubility is expressed as: less than the quotient of the weight divided by the final volume. If the solid is completely dissolved after the first addition of solvent, the solubility is expressed as the quotient of more than the weight divided by the volume of solvent. All experiments were performed at room temperature.

The estimated solubility of the phosphate salt of compound I, along with the solubility of the free base, is shown in table 4 in mg/mL. The solubility of compound I phosphate in the same solvent is lower than the solubility of compound I free base except in water (at different pH levels). The solubility of the phosphate salt of compound I depends on the pH value of the solution, which becomes quite high (3mg/mL) when the pH is 2 or lower. The melting point of the phosphate of Compound I (batch No. 35282-CS-51) was about 281-297 ℃ which is considerably higher than that of the free base of Compound I (free base polymorph form A, 256 ℃ C.; free base polymorph form B, 260 ℃ C.). An important result is that compound I phosphate is more wettable to water than compound I free base.

Table 4.estimated solubilities of compound I free base and compound I phosphate in different solvents at 23 ℃

^aThe pH 2 buffer is composed of HCl and KCl

^bThe pH 5 buffer was composed of potassium phthalate and sodium hydroxide

^cCannot be obtained, but is expected<0.005mg/mL

^d1g of Compound I phosphate corresponds to 0.802g of Compound I free base

^eThe pH of the final solution was 4.91.

EXAMPLE 5 Generation of phosphate polymorphs of Compound I

The low solubility of compound I phosphate seen in example 4C indicates that a highly concentrated solution of compound I phosphate (60-100mg/mL, deep orange red) will help precipitate a polymorph of compound I phosphate from different solvents. Such concentrated solutions are prepared by dissolving compound I as the free base in about 1M phosphoric acid. For example, about 70mg of compound I as the free base can be dissolved in 1mL of 1M phosphoric acid. The amount of compound I and phosphoric acid used as free base may depend on the desired solution concentration and batch size of the solution. In the example where the precipitate was vacuum filtered immediately after precipitation, about 1mL of the desired solution was then dropped into ten of about 10mL of the antisolvents to precipitate crystals of the saltAnd (3) a body. These solvents are water plus Acetonitrile (ACN), ethanol, methanol, acetone, acetonitrile, THF, ethyl acetate, dichloromethane (CH)₂Cl₂) And isopropyl alcohol (IPA). In an example where the precipitate was vacuum filtered after standing overnight or three days, an additional solvent of Methyl Ethyl Ketone (MEK) and dioxane was used. Some organic solvents (e.g., ethyl acetate, hexane, CH)₂Cl₂) Not easily mixed with water and two layers of solvent were observed. Even within a few minutes after the addition, only a small amount of precipitate was visible at the interface. In such a case, about 1mL of methanol was added as a bridging solvent to increase the miscibility between the two layers. Methanol appears to increase miscibility well because the colorless organic layer starts to yellow as soon as methanol is added. The vial was then vigorously shaken by hand for about one minute. The solid precipitated from the organic solvent was vacuum filtered immediately after precipitation (within 20 minutes) and for the isolation of metastable and stable polymorphs, the solid precipitated from the organic solvent was vacuum filtered after standing overnight or three days. The powder was then analyzed. The different solids are numbered in the order of discovery.

Example 6 analysis of Compound I phosphate polymorphs

Example 6A. analytical methods

All powders resulting from the above polymorph screening step were analyzed by PXRD as described in example 3 above. When a new PXRD pattern is observed, supplementary techniques are used to characterize the solids, including dynamic water gas sorption gravimetric measurement (also described in example 3), differential scanning calorimetry, thermogravimetric analysis (when necessary), and optical microscopy.

Differential Scanning Calorimetry (DSC): DSC data were obtained using a DSC calorimeter (TA Instruments 2920). The aluminum DSC pan was charged with powder (1-5 mg). An aluminum lid was placed on top of the pan and allowed to curl. The crimped disk is placed in the sample tube along with an empty disk as a reference. Unless otherwise indicated, the temperature was increased from 30 ℃ to 300 ℃ or 350 ℃ at a rate of 10 ℃/min.

Thermogravimetric analysis (TGA): TGA experiments were performed using a high resolution analyzer (TA Instruments model 2950). Using TA Instruments Thermal Solutions^TMfor NT (version 1.3L) data collection with Universal Analysis^TMfor NT (version 2.4F) for data analysis. Samples (5-10mg) were placed on aluminum pans, which were also placed on platinum weighing pans before being heated. Before loading the sample, the tare weight of the aluminum and platinum disks was subtracted. The temperature was increased linearly from 30 ℃ to 300 ℃ at a rate of 10 ℃/min. A dry nitrogen purge was used.

Polarizing microscopy: microscopy was performed on an Olympus BHSP polarization microscope. The powder was suspended in silicone oil and dispersed between a microscope slide and a cover slip. Prior to observation, the coverslip was gently rubbed against the slide to disperse the particles well.

Example 6B analysis performed immediately after precipitation

The results are summarized in table 5. Precipitation occurs upon reaction of the acidic solution with the anti-solvent. First, the precipitate is a loose floc. Typically the color is yellow or light orange. The resulting solid was viscous. Microscopic observation of these solids revealed that they consisted of very small crystals with good birefringence under polarized light. At least six different PXRD patterns were observed on the solids from the nine solvent systems (see figure 3). The amide side chains on this molecule are flexible, adopting different conformations in the free base form B and its hydrochloride form. Thus, the molecule may be conformationally homogeneous polymorphs in different solid forms. The PXRD pattern for solids precipitated from ethyl acetate, hexane, and IPA appeared the same. However, because the solids from these three solvents have low diffraction signals, it is difficult to compare them in detail with other PXRD patterns.Therefore, they are not specified as new forms. The precipitate from methanol was identical to reference batch 35282-CS-51 (designated as form I). TGA data for all precipitates indicated residual solvent at a level of 1.7% to 4.7%. Among these solids, those derived from CH₂Cl₂Appears to be a solid with solvent retained in the crystals. The TGA profile shows a sharp decrease in sample weight at a temperature of about 125 ℃ (see figure 4). The DSC recorded the result as an endotherm at about the same temperature. Furthermore, the powder consists of crystals with a well-defined morphology and is free-flowing, a very different property with respect to the other batches of precipitate. The powder showed medium crystallinity by PXRD, but it showed good crystallinity as observed by a polarizing microscope. The other batches consisted of very fine crystals. On the DSC curve of these powders, a broad and shallow endotherm is immediately visible once the sample is loaded into the sample tube. This observation is reflected by TGA as a gradual weight loss from the heating of the TGA. Thus, with respect to these batches, the remaining solvent may be the solvent adsorbed by the surface, rather than the solvent in the crystal lattice.

(blank pages)

Table 5 physical analysis of solids isolated immediately after precipitation

^aTo a weight loss of 160 ℃.^bFor purposes of clarity, the temperatures listed in the table are peak temperatures only.^cPeak temperature of broad endothermic event from the onset temperature of DSC run. These events are also reflected by the gradual weight loss over the TGA. This type of thermal event is characteristic of loss of solvent adsorbed on the surface.^dPeak temperature of relatively sharp endothermic events on DSC. This event is also recorded by TGA as a sudden weight loss at the corresponding peak temperature. This type of thermal event is characteristic of desolvation of solvates.^eAggregates consist of very fine particles.^fThe ND representation cannot be assigned to a distinct form because the diffraction peak signal of the corresponding PXRD pattern is too low to be compared in detail with other forms of PXRD patterns.^gForm VI, is a solid comprising a solvent.

Example 6℃ precipitate after three days of standing in solvent

These results are summarized in table 7. After standing in the solvent for up to three days, a new non-villiated orange-red solid phase appeared. Apart from the precipitate obtained from methanol, the fluffy precipitate obtained from all organic solvents underwent conversion. Clearly, the solids that precipitate immediately after precipitation are metastable in these cases because they convert to a more stable solid form (form I) over time. This conversion appears to be complete within hours in most solvent systems. However, to ensure completion of the process, they were allowed to stand for a longer period of time to avoid obtaining a mixture of the two solid forms. The TGA profile shows that the solids obtained from ethane and acetonitrile, respectively, show sudden weight loss at about 124 ℃ and 153 ℃, which correlates with an endotherm at a similar temperature on DSC. Thus, they also appear to contain a limited amount of solvent in the crystal lattice. The stoichiometry of residual solvent is about 0.6 for acetonitrile and about 0.14 for hexane. Needle crystals were grown from acetonitrile after three days of standing. The PXRD pattern of the solid with acetonitrile remaining is unique, while the PXRD pattern of the hexane solvate is similar to that of the earlier identified solid with CH remaining₂Cl₂Of (2) (fig. 3). Both solids with residual solvent (hexane and acetonitrile) underwent weight loss on the TGA tray. After the corresponding residual solvent was removed by heating (table 7, fig. 3), unique PXRD patterns were observed for both solids, indicating that removal of solvent molecules from the solids caused a structural change in the solvate crystals (thus, solvent molecules in the crystal lattice were not only on the crystal surface). However, the PXRD pattern signal intensity for acetonitrile desolvation is low. The DSC case of acetonitrile desolvation was shown to be 74 ℃ andtwo additional thermal events at 174 ℃ were absent, whereas the desolvation event at 153 ℃ was absent. Cooling of the sample after desolvation may have altered the solids that underwent an energy change at 174 ℃.

When other organic solvents were used, the longer standing period of the precipitate produced a solid with the same PXRD pattern as form I (lot 35282-CS-51), although the morphology of the crystals was different (table 6). The same PXRD pattern indicates that these solids have the same lattice structure. The different morphology should then be due to the influence of the solvent. It is clear that form I is the most stable solid phase of all the non-solvated polymorphs reported herein. Other solid forms that are solvent-free are metastable and rapidly convert to form I when contacted with a solvent. From CH₂Cl₂The resulting solid appeared to flow more readily than that obtained from hexane. TGA, morphology and flowability indicate that they are two distinct solids.

Table 6 physical analysis of solids separated after various times of standing after precipitation

^aTo a weight loss of 160 ℃.^bFor purposes of clarity, the temperatures listed in the table are peak temperatures only.^cPeak temperature of relatively sharp thermal events on DSC. This event is also recorded by TGA as a sudden weight loss at the corresponding peak temperature.^dThe transition was complete within 5 hours (from yellow to orange-red).^eThe transition was not evident within 5 hours, but was essentially complete within 3 days (yellow loosely precipitated to orange-red well-formed needle crystals).^fThe conversion was completed within 5 hours. The color change was not noticeable (from yellow to light orange).^gForms VII and VIII are solids containing solvents.

TABLE 7 physical analysis of the solids produced after desolvation

Example 7 inhibition of KIT phosphorylation in canine mast cell tumors

The purpose is as follows: the development of target therapies for cancer offers the opportunity to directly evaluate the effects of drugs on molecular targets and to find associations between these effects and the tumor biology and pharmacokinetics of drugs. This may contribute to tumor drug development as it establishes a pharmacodynamic/pharmacokinetic relationship and provides critical information about the therapeutic effect of the target agent. The objective of this study was to evaluate the effect of a single dose of phosphate of the receptor tyrosine kinase inhibitor compound I on its molecular target KIT activity in canine mast cell tumors, using KIT phosphorylation as a marker for direct target inhibition in canine patients with advanced MCT. Phosphorylation of ERK1/2 (mitogen activated protein kinase (MAPK) downstream of KIT signaling), compound I phosphate plasma concentration and c-KIT mutations were also studied to measure how these parameters correlate with KIT phosphorylation following compound I phosphate treatment.

Study drug: compound I phosphate in the form of 20-mg tablets was used.

Research and design: this study was performed to verify target modulation in dogs with relapsing or metastatic grade II/III MCT. Patients received a single oral dose of compound I phosphate of 3.25 mg/kg. Samples were obtained from tumors using a 6-mm needle biopsy device, before administration of compound I phosphate and 8 hours after treatment. Multiple biopsy samples were taken if possible. Each sample was frozen in liquid nitrogen and stored at-70 ℃ prior to analysis. The blood sample used for plasma level analysis of compound I phosphate was obtained at the same time as the tumor biopsy (see below).

Plasma levels of compound I phosphate: blood samples were drawn from the jugular vein and placed in red-cap serum collection vacuum glass tubes. The samples were placed at room temperature, allowed to clot, centrifuged at 1500rmp for 10 min at 4 ℃, transferred to a cryovial, and the plasma frozen at-70 ℃ for subsequent analysis. Briefly, phosphate standards of compound I in plasma samples (20 μ Ι) or canine plasma were mixed with methanol (200 μ Ι) containing DL-chloroquinuclein hydrochloride (internal standard) on 96-well polypropylene plates (orochemttechnology, Wesrmont, IL). The plate was mixed by rotary shaking for 1 minute and the samples were centrifuged at 4000rmp for 10 minutes. 10 microliters of the supernatant was taken and injected onto an LC/MS/MS system, where separation was performed on a BataBasic C-18(5 μm, 100X 4.6mm) reverse phase high performance liquid chromatography column (Keystone Scientific, Foster City, Calif.). Quantification of the amount of internal standard and compound I phosphate in each canine plasma sample was performed based on a standard curve generated using known compound amounts from 0.2ng/ml to 500 ng/ml.

c-kit regulatory analysis: for most samples, TRIzol (Invitrogen, Carlsbad, Calif.) was used to extract RNA according to the manufacturer's instructions. cDNA was then generated from this RNA using dNTPs, random primers, 5 Xfirst strand buffer, 0.1M DTT, and Superscript Taq polymerase (all from Promega, Madison, Wis.). The amount of cDNA was determined for each sample. For the remaining samples, genomic DNA was prepared as described previously (Down, S., Chien, M.B., Kass, P.H., Moore, P.F., and London, C.A. Presence and reliability of internal standards functionalities in the experiments 11and12of c-kit in the test cells of the standards, am. J.Vet.Res., 63: 1718-1723, 2002, which is incorporated herein by reference in its entirety). For both reactions, the PCR was run for 40 cycles, each consisting of 94 deg.C (1 min), 59 deg.C (1 min) and 72 deg.C (1 min), with a 5 min 72 deg.C extension at the end of the reaction. The C-kit cDNA generated from the canine C2 mast cell line and cDNA generated from normal canine cerebellum were used as controls.

Separating the PCR products by electrophoresis on a 4% agarose gel; the expected size of the wild type c-kit PCR product is 196bp for PCR from cDNA and 190bp for PCR from genomic DNA. For cases where ITDs were not evident (only a single band appeared), the PCR products were gel purified using the Promega PCR Wizard Clean-up kit (Promega) and sequenced on the University of California-Davis core sequencing instrument using P1 (forward) and P5 or P2 (reverse) primers to exclude the presence, absence or point mutations of very small ITDs. DNASIS sequence analysis programs were used to perform sequence alignments and comparisons.

Analysis of KIT and ERK phosphorylation: tumor biopsies were frozen in liquid nitrogen and then ground to powder using a liquid nitrogen cooled mortar (cryomortar) and pestle, then stored at-70 ℃ until use. For analysis of KIT, the ground tumors were homogenized, lysed, and immunoprecipitated from 1mg of the starting tumor lysate using agarose conjugated antibodies against KIT (SC-1493 AC; Santa Cruz Biotechnology, Santa Cruz, CA), as described previously (Abrams, t.j., Lee, l.b., Murray, l.j., Pryer, n.k., Cherrington, j.m.11248inhibitors KIT and plant-derived growth factor receptor beta inhibiting modules of human cell lung cancer. 2: 471 478, 2003; which is incorporated herein by reference in its entirety). When multiple biopsy samples are available, repeated immunoprecipitation/Western blot analyses are performed on different biopsy samples. The amount of phosphorylated KIT in each sample was determined by Western blot using an antibody against the phosphorylated tyrosine 719 of murine KIT (3391; Cell Signaling Technology, Beverly, MA), which corresponds to canine KIT tyrosine 721, which is an autophosphorylation site, and thus a surrogate for KIT kinase activity. For analysis of total KIT, blot bands were excised, resealed, and hybridized again with an antibody directed against KIT (A-4542; DAKO Corp., Carpinteria, Calif.). For analysis of p42/44ERK, the same tumor lysates as used for KIT analysis were probed by Western blot with antibodies against phospho-Thr 202/Tyr204ERK1/2 (9101B; Cell Signaling Technology), followed by cutting out the bands and hybridization again with antibodies against total ERK (9102; Cell Signaling Technology). Evaluable tumor biopsy pairs for KIT and ERK1/2 were considered: wherein detectable total protein is present in both biopsy samples of the pair. Target modulation was scored by the naked eye of three observers, regardless of JM status and plasma concentration. After treatment, a decrease of ≥ 50% in phospho-protein signal in relation to total protein signal in biopsy samples compared to biopsy samples before treatment was assessed as positive (with respect to target modulation), whereas a decrease of < 50% was assessed as negative.

As a result: fourteen dogs participated in this clinical study, the primary objective of which was to determine whether a reduction in KIT tyrosine phosphorylation occurred following oral administration of a single dose of compound I phosphate. Phosphorylation of KIT tyrosine was assessed using a phosphorus-specific antibody directed against autophosphorylation sites in KIT as a surrogate for KIT kinase activity. Furthermore, c-KIT JM mutation status (ITD + or ITD-) was determined from baseline tumor biopsy samples and plasma concentrations of compound I phosphate were measured 8 hours after dose administration to correlate these parameters with inhibition of KIT phosphorylation. 11 of 14 dogs were only evaluated for KIT target modulation. Three dogs considered unevaluable had undetectable or greatly reduced total KIT protein in one or two biopsy samples and therefore could not be used to score for target modulation. Data for all dogs participating in the study are summarized in table 8.

TABLE 8 summary data for all participating patients

^aNE, unassassassable, P-KIT, phosphorus-Tyr 721KIT, P-ERK1/2, phosphorus-Thr 202/Tyr204ERK 1/2.

Of the 14 dogs analyzed, 5 (36%) were found by PCR analysis (figure 5, lanes 1-5) to have ITD evidence for all five tumors. Interestingly, patient 2 clearly lost the wild type c-kit allele. The PCR products from the remaining nine dogs had no evidence of ITD (fig. 5, lanes 6-14), and the PCR products were directly sequenced, which did not show any kind of mutation (insertion, deletion or point mutation). For lanes 3, 6, 8 and 9, PCR reactions were performed using genomic DNA to give slightly smaller (190bp) wild-type products.

The levels of total and phosphorylated KIT expressed as baseline in MCT varied from animal to animal. Higher KIT expression correlates with higher tumor grade. One of six grade II tumors had high KIT expression and four of eight grade III tumors had high KIT expression (fig. 6). For example, total KIT expression in patient 2 (grade III) tumors was significantly higher than in patient 11 (grade II) tumors. Dogs with grade III tumors also had a higher incidence of baseline high levels of phosphorylated KIT than those with grade II tumors, consistent with increased c-KIT mutation frequency and thus increased levels of ligand-independent phosphorylated KIT in advanced tumors. Five of seven dogs with grade III tumors that could be assessed had high levels of baseline phosphorylated KIT; four of these were positive for the presence of ITD in c-kit. Only 1 class II tumor had significant phosphorylated KIT; the animal also expressed the ITD mutant c-kit.

Eight out of eleven dogs that could be assessed were assessed positive for target modulation using a > 50% reduction in phosphorylated KIT (relative to total KIT in biopsy samples) as an assessment criterion after compound I phosphate treatment compared to pre-treatment samples. Examples of phosphorylated and total KIT collected from tumor or immunoprecipitates of test samples before and after treatment with compound I phosphate are shown in fig. 6. Five tumors (fig. 6, left) were assessed as positive for target modulation, while two tumors (fig. 6, right) were assessed as negative. Biopsy samples evaluated negative for inhibition of KIT phosphorylation after compound I phosphate treatment all had significantly less baseline phosphorylated KIT than those evaluated positive (fig. 6).

To assess the effect of compound I phosphate inhibition on downstream signaling pathways for KIT phosphorylation control, the level of phosphorylated MAPK ERK1/2 was assessed by Western blot analysis of duplicate test samples for KIT analysis. Eleven out of fourteen tumors were evaluable for phospho-ERK 1/2 target modulation (two of these were also evaluable for KIT target modulation). In an evaluable ten cases, 7 showed a decrease in the ratio of phospho-ERK 1/2 to total ERK1/2 in the tumor sample after compound I phosphate administration compared to the baseline tumor sample (see fig. 6). ERK target modulation was more often detected in MCTs with relatively high baseline ERK expression and phosphorylation than those with low ERK.

Based on preclinical work in rodent models, the therapeutic range of compound I for target inhibition was considered to be 50-100ng/mL over 12 hours of the 24 hour dosing cycle. After a single dose of 3.25mg/kg, the plasma concentrations of compound I phosphate ranged from 33.2 to 186ng/mL at 8 hours (approximately Cmax), with an average of 105 ± 9ng/mL (table 8). In one animal, the plasma concentration of compound I phosphate was outside the range of the other samples (0.3 ng/mL). Twelve of the fourteen dogs had plasma levels that were considered to be within the therapeutic range established by phase I clinical studies for compound (London, c.a., Hannah, a.l., Zadovoskaya, r., Chien m.b., Kollias-Baker, c., Rosenberg, m., Downing, s., Post, g., Boucher, j., Shenoy, n., Mendel, d.b., and chemington, j.m. phase identity-assessing study of SU11654, a receptor 11 monomer epsilon kinase identity bit, in dogs with nanoparticles metallic chemicals. The mean plasma concentrations (79.2 ± 41ng/ml) of dogs with evidence of KIT target modulation and those not assessed as KIT target modulation (137 ± 36ng/ml) were quite different (P ═ 0.08).

Discussion: the present correlation study was designed to investigate target modulation in comparable populations by studying the effect of a single clinically effective dose of compound I phosphate on KIT phosphorylation in canine MCT, and subsequently on signal transduction via MAPK. Plasma concentrations of compound I phosphate obtained in this study were measured near the predicted Cmax (based on the preclinical pharmacokinetic study) consistent with drug levels measured in phase I clinical studies investigating effective doses of compound I and therapy (table 8).

MCT biopsy samples had detectable inhibition of KIT activation for eight of the evaluable eleven (73%), as measured by a decrease in phosphorylated KIT following a single oral dose of compound I phosphate. Three patients who did not show detectable KIT target modulation after treatment had MCTs expressing low levels of KIT and phospho-KIT at baseline. The lack of significant target modulation in these patients may be due to technical limitations of the detection method; the sensitivity of phosphorus-specific antibodies to phosphorylated KIT (relative to non-phosphorylated KIT) may be insufficient in samples with low baseline KIT expression. Inhibition of KIT activity is more closely associated with low baseline KIT expression (than with c-KIT ITD genotype). Based on cellular assays, it was predicted that both wild-type KIT and ITD mutant KIT would be inhibited by compound I phosphate in vivo, since compound I could block phosphorylation of wild-type KIT and ITD mutant KIT in vitro with comparable potency.

Compound I phosphate also affects signal transduction pathways downstream of KIT. Mutations in c-KIT have been reported to activate distinct signaling pathways from each other and from wild-type KIT in GIST and hematopoietic malignancies. In canine MCT, all but one tumor sample had detectable baseline phosphorylated ERK 1/2. In seven of the eleven evaluable pairs of tumor biopsies, ERK1/2 was inhibited as measured by a decrease in phosphorylated ERK1/2 after treatment. Tumors evaluated positive for ERK1/2 inhibition are not all positive for KIT phosphorylation. ERK1/2 target modulation was not associated with tumor grade or the presence or absence of c-kit ITD mutations. In the case of KIT target modulation, more frequent ERK1/2 target modulation could be detected in tumors expressing high levels of ERK1/2 and baseline phosphorylated ERK 1/2.

Detection of inhibition of molecular targets of compound I phosphate following MCT treatment was used as evidence of target modulation against compound I phosphate in this setting. The clinical relevance of this finding is supported by the correlation between inhibition of molecular targets and plasma drug concentrations in the therapeutic range, and the previously reported clinical objective response to compound I in canine patients with MCTs expressing activating mutations in the target genes, providing proof of concept for the use of compound I phosphate in this patient population. Because dogs with other malignancies (including breast cancer, soft tissue sarcoma, and multiple myeloma) also experienced sustained objective responses to treatment with compound I, inhibition of KIT at this plasma concentration could reasonably be extrapolated to successful inhibition of other closely related compound I receptor tyrosine kinase targets expressed by these tumors, based on their potency in vitro and in vivo, providing a molecular mechanism for guest responses in these tumors. For example, canine breast tumors express VEGFR, which is inhibited in vitro assays by indolone tyrosine kinase inhibitors at concentrations comparable to concentrations directed against intracellular KIT (Liao, a.t., Chien, m.b., Shenoy, n., Mendel, d.b., McMahon, g., Cherrington, j.m., and London, c.a.inhibition of regulatory activity for the presence of a mutant KIT by mutant immobilized indole tyrosine kinase inhibitors blood, 100: 585-. Inhibition of both wild-type c-kit and ITD mutant c-kit in MCT by compound I phosphate can therefore be used as a surrogate for inhibition of the compound I phosphate's associated RTK targets, VEGFR and PDGFR (which are aberrantly expressed and/or modulated by a number of different tumor types). Finally, molecular target inhibition associated with clinical guest responses in canine tumors has guided the process of developing related compounds in human cancers against clinical populations expressing activated KIT, VEGFR and PDGFR.

Example 8 oral administration of multiple doses of Compound I phosphate in treatment of dogs with recurrent mast cell tumors Heart, placebo-controlled, double-blind, randomized study

The purpose is as follows: compound I phosphate oral tablets were evaluated for their effectiveness in treating mast cell tumors in client-owned animals with postoperative recurrence-detectable disease in masked, negative control studies. The study evaluated the response to disease of a dose of compound I phosphate administered every other day at 3.25mg Free Base Equivalent (FBE)/kg body weight using a modified (RECIST) response standard. In this study, the presence or absence of c-kit mutations in mast cell tumors was evaluated as a covariate (covariate). For decision making purposes, the study lasted 6 weeks.

One hundred fifty-three (153) dogs were randomized into two treatment groups at a ratio of 4: 3: t01 (placebo, where n-65) and T02 (compound I phosphate, where n-88). Ten veterinary oncology practices (practices) in the united states were selected and the situation registered. For enrollment, dogs must have recurrent mast cell tumors (at least one target lesion must have a diameter of a minimum length of 20 mm) ± regional lymph node metastasis. The maximum of three target lesions (measurable mast cell tumors) and all non-target lesions (all remaining lesions, measurable or unmeasurable) was determined as baseline by two evaluators. Efficacy is based on guest response (complete response or partial response) revisited at week 6, where the Mean of the Sum of the longest diameters of the target lesions (Mean Sum LD) obtained by both evaluators was compared to the baseline Mean Sum LD to calculate a percentage increase or decrease. The assessment of non-target lesions is subjective. Complete Response (CR) is defined as the disappearance of all target and non-target lesions and the absence of appearance of new lesions; partial Response (PR) is defined as at least a 30% reduction in Mean Sum LD of target lesions compared to baseline Mean Sum LD, no worsening of non-target lesions, and no new lesions appearing. Tissue samples from tumor and distant normal skin were collected prior to randomization and used to assess the c-kit mutation status.

Eighty-six (86) T02 and 65T 01 animals were included in the efficacy analysis. Data analysis indicated that the primary endpoint had a statistically significant increase (objective response) with compound I phosphate (T02) compared to placebo (T01). Compared to T01 animals (7.9%; 5/63), T02 animals had significantly greater efficiency (38.3%; 33/86) (p < 0.001). The disease progression in T01 animals (66.7%; 42/63) was nearly twice that of T02 animals (33.7%; 29/86). Dogs in the T02 group that were positive for the c-kit mutation were likely to have nearly twice the proportion of objective responses (60%, 12/20 vs 32.8%, 21/64, respectively) as those for which the c-kit mutation was negative.

In summary, this study demonstrates the effectiveness of compound I phosphate oral tablets for treatment of recurrent mast cell tumors in consumer-owned dogs.

Many modifications and variations of the present invention as shown in the above-described illustrative embodiments can be expected by those skilled in the art. Accordingly, only those limitations are placed upon the invention which appear in the appended claims.

Claims (2)

1. A polymorph of the citrate salt of 5- [ 5-fluoro-2-oxo-1, 2-dihydro-indol- (3Z) -ylidenemethyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amide, wherein the polymorph has a powder X-ray diffraction pattern obtained using CuK alpha 1 emission at a wavelength of 1.5406 angstroms, the diffractogram comprises peaks expressed in degrees ± 0.1 degrees of 2 theta angles of 3.2, 7.3, 9.2, 9.5, 10.8, 12.7, 13.2, 14.3, 14.8, 15.9, 17.2, 17.8, 18.2, 18.3, 19.6, 20.9, 21.6, 21.7, 21.8, 23.2, 24.2, 24.9, 25.5, 26.4, 26.8, 27.1, 32.0 and 34.4 and wherein the polymorph has a melting point from 178 ℃ to 183 ℃.
2. 2. A pharmaceutical composition comprising the polymorph of claim 1, and a pharmaceutically acceptable carrier or excipient.